S. Dhein - Cardiac Gap Junctions. Physiology, Regulation, Pathophysiology and Pharmacology (1998)

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Cardiac Gap JunctionsPhysiology, Regulation, Pathophysiology and Pharmacology

S. Dhein, Cologne

23 figures and 3 tables, 1998

...........................Stefan DheinInstitute of PharmacologyUniversity of Cologne(Germany)

All rights reserved. No part of this publication may be translated into other languages, reproduced orutilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying,or by any information storage and retrieval system, without permission in writing from the publisher.

Ó Copyright 1998 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland)Printed in Switzerland on acid-free paper by Reinhardt Druck, BaselISBN 3–8055–6567–4

IV

Dedicated to Aida

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...........................Contents

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IXPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI

1 Introduction: Cellular Coupling, Cardiac Activation Patterns andArrhythmia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Structure and Diversity of Gap Junction Channels . . . . . . . . . . 133 Distribution of Gap Junctions in the Heart . . . . . . . . . . . . . . 254 Function and Physiology of Gap Junction Channels . . . . . . . . . 355 Regulation of Gap Junction Expression, Synthesis and Assembly . 636 Gap Junctions in Cardiac Disease . . . . . . . . . . . . . . . . . . . . 737 Pharmacological Interventions at Gap Junctions . . . . . . . . . . . 898 Methods for Investigation of Gap Junctions . . . . . . . . . . . . . . 106

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140List of Suppliers of Specialized Items . . . . . . . . . . . . . . . . . . 141Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

VII

...........................Foreword

When I started as a novice in the field of cardiac electrophysiology, thedogma was that gap junctions are specialized membrane structures present inthe cardiac and smooth muscle of vertebrates where they serve to propagatethe action potential from cell to cell. Purkinje fibers and muscular trabeculaewere the preferred cardiac preparations. These multicellular preparations weresuitable to perform cable analyses and diffusion studies. At that time, mymentor, Silvio Weidmann, had already accomplished his elegant functionalstudies.

The subsequent progress in the field was prompted largely by the develop-ment of novel experimental approaches. On the one hand, the introductionof the patch-clamp method and the use of cell pairs led to a detailed descriptionof the intercellular current flow. As a result, we nowadays have extensiveknowledge about the conductive and kinetic properties of gap junctions andgap junction channels. On the other hand, immunohistochemistry and molecu-lar biology made one aware of the diversity of gap junction proteins andtheir distribution in tissues of the cardiovascular system. This reductionisticapproach led to the accumulation of an enormous amount of functional andstructural details. The combination of electrophysiology and molecular biologywill culminate eventually in the elucidation of the structure-function relation-ship of a single channel. However, scientists soon should explore the reversepath and try to integrate the collected data in the context of an intact heart.In this way, the knowledge gathered may provide a basis for new strategiesagainst cardiovascular diseases. Hopefully, this monograph will contribute tothis process.

Robert WeingartBerne, June 23, 1997

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...........................Preface

‘Cells live together and die singly’ Engelmann wrote at the end of thepast century. With this simple sentence he summarized the key feature providedby cell coupling via gap junction channels: these channels provide exchangeof small molecules and electrical coupling in the intact heart, but in the courseof ischemia, for example, they close, the cell gets isolated and is no longeractivated by the surrounding tissue. This may help the cell to survive, or thecell dies but without influencing the adjacent cells. In the chronic phase ofcardiac disease the distribution of various gap junction isoforms can change,thereby altering the tissue’s biophysics. This behavior opens new perspectivesfor arrhythmia research, for drug research and for research directed towardischemia, cardiac protection and cardiac pathophysiology.

The following book is written to give an insight into a relatively new fieldof cardiovascular research to basic researchers, cardiologists, physiologists andpharmacologists who wish to obtain information on cellular coupling in theheart or who wish to enter this new field of research. Therefore, the firstchapter gives an introduction to the various aspects of activation propagationand coupling in the heart. This is followed by two chapters which review ourpresent knowledge on the structural aspects of gap junction channels includingamino acid sequences and species variability as known so far. Thereafter, thephysiology of gap junction channels and the regulation of expression is de-scribed in the subsequent two chapters. Since it is known today that gapjunction distribution can change in the course of cardiac disease, these changesand their implications are described in the sixth chapter. The seventh chapterthen gives insight into pharmacological approaches to the modulation of gapjunction channel conductivity and outlines possible new therapeutic strategies.The final chapter is especially written for people who are interested in enteringthis fascinating field of cardiovascular research and describes practicalapproaches to gap junction research. The concept of double-cell voltage clamp,immunohistochemistry, isolation procedures for gap junctions and dye-coup-ling assays are described with practical protocols. At the end of the book alist of suppliers of specialized items, such as certain amplifiers, antibodies etc.,is given.

Stefan DheinCologne, April 1997

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Introduction: Cellular Coupling, CardiacActivation Patterns and Arrhythmia

What is the reason for thinking about cardiac gap junctions? In the lastyears, after the cardiac arrhythmia suppression trial (CAST), the understand-ing of cardiac arrhythmia and antiarrhythmic drug therapy has completelychanged. In that study Echt et al. [1991] observed lethal arrhythmia in patientsunder antiarrhythmic treatment with class-Ic agents (flecainide) during thepostinfarction period. Until that study, proarrhythmic drug activity had beenwidely neglected although it was reported earlier [Brugada and Wellens, 1988;Podrid, 1985; Podrid et al., 1987]. According to these studies and CAST, itcan be concluded that prophylactic treatment with antiarrhythmic drugs canparadoxically induce arrhythmia or aggravate arrhythmia in therapeutic con-centrations. This has been defined as the proarrhythmic risk of antiarrhythmicdrugs. First, it was confined to class-I antiarrhythmic agents but in betweenit became evident that not only class-I antiarrhythmics but also class-IIIantiarrhythmics exhibit proarrhythmia, the latter especially as torsade depointes arrhythmia [Carlsson et al., 1990]. In in vitro studies using isolatedrabbit hearts, it was shown that all class-I antiarrhythmics induced significantalterations in the activation patterns and disturbed the normal excitationprocess under control conditions [Dhein et al., 1993b]. Thus, it was concludedby these investigators that prophylactic treatment with antiarrhythmic drugsmay disturb the normal excitation process thereby inducing alterations in thegeometry of the activation pattern, which finally lead to arrhythmia.

What were the consequences? Prophylactic antiarrhythmic drug treatmenthas changed to a therapy which is carried out with great caution and care.The surprising finding that antiarrhythmic drugs can provoke arrhythmia madeit evident that arrhythmia is not only a phenomenon of a single cell butinvolves the whole tissue and that more determinants are involved than onlythe transmembrane currents.

The term, arrhythmogenic substrate, became a matter of interest to manyresearchers. The arrhythmogenic substrate means the pathologic and anatomicpreconditions for the initiation of tachyarrhythmias such as myocardial fibrosis,aneurysm, the border zone between normal and ischemic or infarcted tissue,scars, diffuse myocardial injury in cardiomyopathy or the chronic alterationsinduced by myocarditis, and furthermore, accessory pathways or variations inthe specific cardiac conduction system. These anatomic or pathologic altera-

1

tions alone do not provoke arrhythmia, but if additional factors, such asvariations in the autonomous innervation, changes in the electrolyte balance,extrasystoles or changes in the pacing frequency, coincide, these factors workin concert with the pathological and anatomical alterations and finally causearrhythmia.

In consequence, the goal of many studies was to define the changesresponsible for arrhythmogenesis on the tissue or whole organ level. Manyinvestigators sought for new approaches to arrhythmogenesis and antiarrhyth-mic agents. Especially safe antiarrhythmic agents for use in prophylactic treat-ment are searched for.

In the course of this development focusing on the arrhythmogenic sub-strate on the basis of tissue alterations the question how myocardial cellsinteract with each other became the center of attention. The intercellularcommunication via intercellular low-resistance pathways (gap junctions), otherforms of coupling and the cardiac networking became an important subjectof research.

How are cardiac cells coupled? How do cardiac cells interact electrically?These are important questions to be addressed and they lead to the topic ofcardiac networking. Sperelakis [1979] distinguished three forms of transferof excitation: (1) mechanical transmission; (2) chemical transmission, and(3) electrical transmission. Mechanical transmission, i.e. the contraction ofthe pre-cell depolarizes the membrane of the post-cell via membranous stretch-ing, can be ruled out as an important mechanism in the heart because theelectromechanical coupling time in the heart is longer than the time availablefor the transfer. Chemical transmission has been postulated with K+ as trans-mitter, since the K+ effluxing during an action potential will diffuse rapidlyin the bulk interstitial fluid around the sarcolemma but can accumulate in thenarrow cleft of the intercalated disk thereby depolarizing the post-membrane.This might contribute to the transmission process [Macdonald et al., 1975].However, the most important transfer mechanism is electrical transmissionvia low-resistance pathways, which have been identified as gap junction chan-nels. An early argument in support of the concept of low-resistance pathwayswas that the length constant k (measured in cardiac muscle bundles by extracel-lular application of current) ranges between 0.5 and 2.0 mm and that theinput resistance (measured change in voltage at the site of current injectiondivided by the applied current) is comparably low indicating that currentpasses to neighboring cells. Besides this, capacitive coupling and electricalfield coupling have been proposed as alternative mechanisms of electricaltransmission. Capacitive coupling according to Sperelakis [1979] means thata capacitive current flows through a capacitance (the membrane is a capacitorand the action potential is an alternating current AC signal) that acts to couple

21 Introduction

both cells. For several physical reasons (junctional capacitive coupling wouldbe decreased by a factor of 2 since the junction resembles 2 capacitors inseries; next, if only a small portion of the intercalated disk is involved, thetotal capacitance would be accordingly smaller, and third there may be a shuntto ground if the two membranes are not close enough to each other), capacitivecoupling may not work in normal cardiac tissue [for a detailed discussion ofthat matter see, Sperelakis, 1979]. Electrical field coupling [Sperelakis andMann, 1977] means the induction of an action potential in the post-cell bythe electrical field arising from the action potential at the intercalated disk ofthe pre-cell. The authors showed that an accumulation of K+ in the cleft of theintercalated disk is an important contributory factor allowing the membrane ofthe pre-cell at the intercalated disk to fire a fraction of a millisecond earlier thanthe surface membrane, which was necessary for effective coupling. However, atpresent it is uncertain what the contribution of electrical field coupling toelectrical transmission is in normal tissue.

Weingart and Maurer [1988] showed that after manipulating two separatecardiac ventricular cells into intimate side-to-side contact initially, i.e. beforeforming gap junction channels, there was no transmission of electrotonicpotentials or action potentials from one cell to the other. This experiment isagainst the theory of ephaptic impulse transmission or of electrical field coup-ling [Sperelakis, 1979] as a non-gap junctional mechanism of intercellularaction potential spreading. However, it remains to be elucidated whether moretissue than only one cell is needed for electrical field coupling or capacitivecoupling. The mass of activated tissue might be a determining feature. Thecomposition of the interstitial fluid, the K+ concentration in the clefts, andthe geometry of the clefts between two adjacent cells may also contribute tothe local electrical properties. In addition, it can be imagined that in tissue,if gap junctions close and the low-resistance pathways are occluded, theseforms of coupling may become more important.

In summary, from the present point of view the most important mecha-nism for transmission of excitation is coupling via the gap junction channels.

Considering the passive electrical properties of the tissue means first ofall to consider the properties of a muscle bundle, i.e. the passive cable properties.Muscle fibers are classically considered cables consisting of cells coupled inseries via ohmic resistors with each cell representing a resistor with a parallelcapacitor [for review see, Weidmann, 1990]. The change in voltage is a functionof distance (x) according to Vx>Vx0(expÖx/k) with the length constantk>z(rm /ri ) (rm>membrane resistance, ri>internal longitudinal resistance);the input resistance at x>0 can be described as rinput>Vx0 /I>rik. Taking thefiber radius into account, the specific membrane resistance Rm equals 2parm

[Xcm2] and specific internal resistance Ri>pr2ri . With the specific membrane

31 Introduction

capacitance the time constant s is described as s>CmRm. In a multicellularpreparation with parallel running fibers the longitudinal resistance of theextracellular space ro also has to be considered. For these conditions k isreflected by k>z(rm /[ri+ro]) and the conduction velocity h depends onh>z(1/{TfootCm[ri+ro]}). However, this cable theory, originally formulated fornerve axons [Hodgkin and Rushton, 1946] and later on for Purkinje fibers[Weidmann, 1952], is based on the assumption of continuity of the cable. Inconsequence of these assumptions, passive membrane properties were consid-ered to be of minor importance for the pathophysiology of conduction distur-bances and the basic mechanisms were ascribed to membrane ionic propertiesand their regional differences.

However, in cardiac tissue the situation is a bit more complicated byanisotropy and non-uniformity. Basically, action potential propagation is fasteralong the longitudinal axis of the fibers than in the transverse direction dueto higher intercellular resistance perpendicular to the fiber axis. In addition,on a microscopic basis propagation is discontinuous and the inhomogeneousand anisotropic distribution of the cellular connections influence action poten-tial upstroke and the safety factor of propagation [Spach and Dolber, 1990].The difference between the two forms of anisotropy, i.e. uniform versus nonuni-form, has many consequences for the pathophysiology of arrhythmia. Firstof all, it was observed that the action potential upstroke velocity and amplitudewere greater during transverse propagation. This was accompanied by a fasterfoot potential and led to the hypothesis that longitudinal propagation is,although faster, more vulnerable to block because of its lower upstroke velocityand amplitude. This behavior can be explained on a theoretical basis: theupstroke velocity increases as a result of reduced coupling [Delmar et al.,1987] since the current can not pass to the neighboring cells.

In nonuniform anisotropic tissue fractionated extracellular waveforms areoften encountered. Such complex waveforms with multiphasic shape can beinterpreted as the reflection of discontinuous propagation and each of themultiple negative peaks represent the activation of a small group of fibers. Itshould be kept in mind that with aging there is a general change in thebiophysical properties of the cardiac tissue from uniform to nonuniform aniso-tropy due to predominant uncoupling of side-to-side connections with increas-ing age [Spach and Dolber, 1986, 1990]. What are the consequences of non-uniformity for action potential propagation? In fibers with tight electricalcoupling, simulated extrasystoles with the shortest interval leading to a propa-gated response lead to a progressive reduction in conduction velocity in alldirections (i.e. in parallel to the reduced sodium channel availability). In con-trast, in nonuniform tissue the earliest premature beat leads to dissociatedmicroscopic longitudinal propagation, i.e. the large biphasic waveform changes

41 Introduction

to a multiphasic fractionated one. Similar behavior in nonuniform anisotropictissue could be induced with use-dependent sodium channel-blocking agents(e.g. quinidine). Besides this, more importantly, early premature beats in non-uniform anisotropic tissue can induce conduction block in longitudinal direc-tion while transverse propagation is still possible indicating a lower safetyfactor for longitudinal propagation in this tissue. It has been shown that thissituation can initiate reentrant arrhythmia [Spach and Dolber, 1990; Spachet al., 1988]. What is the basis? According to the leading circle concept [Allessieet al., 1977] it could be argued that the premature beat encounters refractorytissue. However, the authors could demonstrate even lower refractory periodsat the site of conduction block, and conclude from their findings that themicroreentry was solely based on discontinuous anisotropic propagation. Thesafety factor for longitudinal propagation is reduced when sodium conductanceis decreased as the consequence of early premature beats. Under these condi-tions the discontinuities in nonuniform anisotropic tissue can cause longitud-inal conduction block, whereas in homogeneous, i.e. uniform, anisotropictissue block occurs in both directions [for a detailed discussion see, Spach andDolber, 1990]. It is important to stress the point that on a macroscopic scale(many millimeters) propagation may behave as in continuous tissue, but on amicroscopic scale discontinuous propagation occurs and this discontinuouspropagation can cause slow conduction of even normal action potentials.Thus, slow conduction does not necessarily mean depressed conduction [Spachand Dolber, 1990].

What are the effects of coupling itself on transverse and longitudinalpropagation? Delmar et al. [1987] investigated longitudinal and transversepropagation in thin layers of sheep cardiac muscle before and after superfusionwith heptanol, an agent which reduces gap junctional coupling (see chapter 7).They found out that transverse propagation is more sensitive to electricaluncoupling indicating a lower safety factor under these circumstances. Afterexposure to heptanol, conduction block in transverse direction occurred afterabout 28 min whereas longitudinal block was observed after about 44 min.With regard to the findings of Spach and coworkers considered above, theauthors suggested that uncoupling can have opposite directional effects tothose seen if sodium conductance is reduced. It might be speculated that thesmaller number of gap junctions at the side-to-side border as compared tothe intercalated disks might form the basis for a higher sensitivity of transversepropagation to uncoupling.

In summary, longitudinal propagation seems to be more sensitive to re-duced sodium channel availability especially in nonuniform anisotropic tissue,and under these conditions reentrant arrhythmia can be initiated due to discon-tinuous propagation, whereas transverse propagation is more sensitive to un-

51 Introduction

coupling. However, it should be taken into account that heptanol does notspecifically block gap junctions and that under most pathophysiological condi-tions, for example ischemia, complex changes occur with at least both reducedsodium channel availability and gap junctional uncoupling. In such complexsituations it is probably difficult to foresee whether longitudinal or transversepropagation will fail.

Anisotropy and nonuniformity are at least in part due to inhomogeneitiesin the distribution of gap junctions and the biophysical properties of the tissueare in fact influenced by the intercellular coupling. At least four features haveto be considered. (1) Cardiac cells express different gap junction proteins (so-called connexins; in the heart, connexin 40, connexin 43 and connexin 45 aremost abundantly found; for details see chapters 2 and 3). Channels formedby these connexins are different with regard to their biophysical properties.In various parts of the heart the content of each of these isoforms is different.(2) There is a highly distinctive three-dimensional spatial distribution of inter-cellular connections with different patterns in different parts of the heart.(3) Furthermore, it has been shown that a single cardiomyocyte can expressdifferent isoforms of connexins [Saffitz et al., 1995]. Thus, the formation ofheterotypic channels combining the biophysical properties of more than oneconnexin is possible. (4) In the course of cardiac disease the specific patternof gap junction distribution can be altered, thus inducing changes in theconduction properties of the tissue (see chapter 6). Taken together, there area large number of putative mechanisms regulating and modulating intercellularcommunication in the heart.

The anisotropic ratio varies between different parts of the heart. In thecrista terminalis for example the ratio between the longitudinal and transversepropagation velocity is 10:1, whereas in ventricles the ratio was found to be3:1. What is the basis of this phenomenon? Saffitz et al. [1994] showed thatin the crista terminalis each cell is connected to 6.4×1.7 other cells whereasin the ventricle each cell is connected to 11.3×2.2 other myocytes. In addition,in the crista terminalis the gap junctions were confined to the ends of thecells forming predominantly end-to-end-connections whereas in the ventriclesapproximately similar numbers of gap junctions occurred in end-to-end andside-to-side orientation. Thereby, the effective length to width ratio of a ventri-cular cell is reduced from 6:1 to 3.4:1 [Saffitz et al., 1995], thus lowering thedegree of anisotropy. Due to this complex architecture, in the ventricle awavefront encounters a broad spectrum of possibilities to propagate in alongitudinal or transverse direction. Moving in the transverse direction, how-ever, makes it necessary to pass over more intercellular connections, whichmeans a higher resistance in that direction, so that the wavefront is slowed.In some cardiac diseases, e.g. in the border zone of healed infarction, the gap

61 Introduction

junction distribution is changed, each cell is connected to a fewer number ofother cells and especially the side-to-side connections are reduced [Luke andSaffitz, 1991] (see chapter 6). As a consequence, a wavefront travelling trans-verse to the fiber axis becomes slowed specifically in this critical area, whereasit propagates with normal velocity in the surrounding normal tissue. In thisborder zone this transversely propagating wavefront has to follow the sparseside-to-side connections and, thus, is urged to zigzag through the zone. Thisenhances the possibility of the wavefront meeting postrefractory tissue andinitiating the next beat of tachycardia.

This demonstrates how the architecture and structure of the tissue, thepassive properties of the tissue and the intercellular coupling pattern cancontribute to both the physiological activation pattern and the pathophysiolog-ical alterations in the activation pattern, and thus are an important determiningfactor in arrhythmogenesis. These factors have long been neglected, cardiovas-cular research was mainly focused on the active membrane properties of singlecells.

At the microscopic level, cardiac myocytes are shaped irregularly and gapjunctions are distributed in a nonuniform manner (see chapter 3). Becausethe direction of propagation varies, Spach and Heidlage [1995] investigatedthe implications of these microscopic irregularities for the load variationswithin individual cells. They modelled a two-dimensional array of cells (eachcell consisting of a finite number of 10*10-lm segments) with plicate junctions(in the plicate segment of the intercalated disk), interplicate junctions (inregions close beside the plicate segments) and combined plicate junctions(small step-like irregularities at the cell border) assuming the gap junctionsto behave as ohmic resistors of 0.5 (plicate), 0.33 (interplicate) and 0.062 lS(combined plicate junction). They found that during longitudinal propagationthe upstroke velocity of the local action potential Vmax was lowest at theproximal end of the cell, increased to its maximum at the distal fourth anddecreased distally. During transverse propagation, higher Vmax as well as rapidintracellular conduction with variable intracellular pathways was observed.At the end of some myocytes higher Vmax was found for transverse propagation.The charge elicited by the fast sodium current was inversely related to Vmax.The surrounding cellular network exhibited a strong modulating influenceon gap junction delay, Vmax and sodium current. Coupling the cells to theirsurrounding resulted in a decrease in Vmax, an increase in gap junction delayand in sodium current. Discontinuities during longitudinal propagation wereobserved at the end-to-end connections of the cells and, during transversepropagation, ‘large lateral jumps’ were found which coincided with the lateralborders of the cells. In summary, they concluded that on a microscopic levelcardiac propagation is stochastic and not as uniform as it appears on a larger

71 Introduction

macroscopic scale. Thus, small input changes, may produce large changes inthe propagation process, i.e. a change in the direction of propagation canevoke considerable changes in the gap junction delay and in the microscopicspread of excitation. Spach and Heidlage [1995] suggested this stochasticnature of propagation, a natural antiarrhythmic factor, by reestablishing thegeneral feature of the wavefront after small variations.

Depending on the direction of propagation, similar changes in Vmax havebeen observed in anisotropically grown cardiomyocyte cultures if major discon-tinuities existed using voltage-sensitive dyes [Fast and Kleber, 1994; Fast et al.,1996; Rohr, 1995]. In additional computer simulations these authors couldshow that the difference between longitudinal and transverse depended on thedegree of anisotropy and the pattern of gap junctions. The difference wasabolished in normally grown cell cultures. Since in the ageing heart or aftermyocardial infarction cells become separated by connective tissue layers, Fastet al. [1996] investigated the effect of longitudinal clefts on action potentialpropagation. They found that such structures result in local differences in Vmax

and in the local action potential upstroke. In addition, they showed that lackof Cx43 led to local conduction block and to disturbance of the activationspreading. This may be of importance for the arrhythmogenesis in chronicheart diseases which are characterized by a reduction in Cx43 and changesin the Cx43 distribution pattern (see chapter 6).

In a high-resolution mapping of the cardiac activation in isolated rabbithearts, the stochastic nature of propagation mentioned above may be reflectedby the beat-to-beat variability observed by others [Dhein et al., 1990].

Tachycardic arrhythmia are often maintained by reentrant circuits. Thus,the initiation of reentry is still a focus of arrhythmia research. It is, however,not in the scope of this chapter to give a detailed complete overview on the topicof arrhythmogenesis [readers interested in this are referred to the literature, e.g.Janse and Wit, 1989; Spach and Josephson, 1994]. This chapter focusses onthe role of some biophysical properties of the tissue and the gap junctionalintercellular communication. Different mechanisms have been discussed anddemonstrated in various models. Intrinsic repolarization inhomogeneities asgenerated by two stimuli (extrasystoles) S1 and S2 at the same site can leadto reentry without the requirement of an anatomical obstacle [Moe et al.,1964]. As shown by Allessie et al. [1977], the circulating excitation waves cancreate a functional central obstacle in the form of a centrally nonexcited region.Following this concept, which is known as the ‘leading circle concept’, themembrane potential of these central fibers is held above threshold due to theelectrotonic influence of the circulating wavefront. As a result, centripetalwaves cannot shortcut the circuit and are extinguished in the center. For thistype of reentry a minimum area of 30–50 mm2 is required. In 1966 Krinsky

81 Introduction

demonstrated in a 2-dimensional isotropic model that localized delays ofconduction within the circuit reduce the minimum perimeter of the reverber-ator so that it may be less than the wavelength of refractoriness k (k>RP*V;RP>refractory period, V>average conduction velocity).

This is substantially different from the classical reentry model requiringa central obstacle with the reentrant circuit path length equalling the wave-length of refractoriness (k>RP*V) [Wiener and Rosenbluth, 1946]. Similarforms of reentry can be initiated by S1 and S2 at different sites, but requireconsiderable large areas [Davidenko et al., 1990; Van Capelle and Durrer,1980].

Focusing on the passive electrical properties of the tissue, Spach et al.[1981] demonstrated that cardiac tissue is a nonuniform anisotropic medium.This has many implications for the theory of initiation of reentry and at leastfor the involvement of gap junctions. First of all, anisotropic reentry can occurin tissue without depolarization inhomogeneities. Second, microreentry is pos-sible in small areas of =10–15 or even =2 mm2 [Spach et al., 1988]. Thisnonuniformity can be caused by either microfibrosis with connective tissueseptae separating the fibers or by changes in the cellular coupling due toinhomogeneities in the distribution of gap junctions (see above), especiallywith sparse side-to-side coupling. This can lead to two different pathways:one of fast longitudinal conduction with a longer refractory period and anotherwith a very slow conduction and shorter refractory period. As explained abovethe longitudinal conduction is more sensitive to premature stimuli, causingconduction failure in the longitudinal direction whereas transverse propagationis maintained. This situation can initiate microreentry as shown by Spachet al. [1988]. The vulnerable period of anisotropic reentry is confined to theinterval between the refractory periods of longitudinal and transverse propa-gated action potentials [for review see Spach and Josephson, 1994]. Suchmechanisms may also play an important role in atrioventricular (AV) reentrysince the transitional zone of the AV node was found to exhibit markedlynonuniform anisotropic properties.

What is the role of the gap junctions? By coupling the myocardial cellsin both directions (longitudinal and transverse) they are responsible for thebiophysical properties of the tissue. A reduction in gap junction distributionor a closure of the gap junction channels causes nonuniformities and discon-tinuities which alter the biophysical properties of the tissue and make it moreprone to nonuniform anisotropic reentry. According to the model proposedby Krinsky [1966], a reduction in gap junctions or a closure of gap junctionchannels will lead to local slowing of conduction, thereby allowing smallerperimeters of reentrant arrhythmia. In addition, slowing of conduction isgenerally believed to be a risk factor for initiation of reentry. Since in many

91 Introduction

cardiac diseases, including acute (e.g. regional ischemia) and chronic (healedinfarction) states, changes in cellular coupling have been observed (seechapter 6) , it is tempting to speculate that altered intercellular communicationtakes part in the formation of the arrhythmogenic substrate [Quan and Rudy,1990; Joyner, 1982] by introducing discontinuities.

In the following paragraphs several types of arrhythmia will be discussedwith regard to the underlying mechanisms. Since it would be out of the scopeof this book on gap junction channels to discuss all possible mechanisms ofarrhythmia in detail, readers interested in a complete detailed review of thepathophysiology and clinics of arrhythmia are referred to the reviews by Janseand Wit [1989] and Pogwizd and Corr [1987, 1990] and to the specializedliterature.

Regional ischemia in the course of atherosclerotic coronary artery diseaseis one of the most important causes of arrhythmia in the Western industrialworld. These arrhythmias start with or often degenerate into ventricular fibril-lation and are the main cause of sudden cardiac death in these countries.However, in the course of ischemia and infarction the mechanisms by whicharrhythmia is induced vary with the duration of ischemia. In the acute phaseof ischemia, i.e. within the first 2–4 h ventricular arrhythmias often occur.

Within the first 30 min of ischemia two types of arrhythmia can bedistinguished: type-1a arrhythmias occur after 2–10 min with a peak at 5–6min. They often originate from the subepicardium and the mechanism isassumed to be associated with diastolic bridging leading to reentrant ar-rhythmia. Besides this, non-reentrant type-1a arrhythmias can also occur whichmay be due to the flow of injury current across the ischemic border causingectopic activity [Janse and Wit, 1989]. Type-1b arrhythmias occur later at12–30 min with a peak between 15 and 20 min and are considered to be dueto a partial recovery of dU/dtmax and the action potential duration followingcatecholamine release from the sympathetic nerve terminals. Apart from this,at the time of the occurrence of type-1b arrhythmia, gap junctional uncouplingwith an increase in intercellular resistance has been described (for details seechapter 6) [for review of ischemia-related arrhythmia see, Janse and Wit, 1989].

The acute phase of ischemia is followed by 3–6 h of predominantly sinusrhythm. Thereafter, the number of ventricular ectopic beats increases. In thesubacute phase of infarction (12–24 h) ventricular arrhythmias often occur.One of the mechanisms involved is reinfarction. If there is no acute reinfarctioninvolved, these arrhythmias have been suggested to originate from survivingstrands of Purkinje fibers in the subendocardium. The predominant mecha-nism has been postulated to be abnormal automaticity in these fibers. Thesefibers exhibit an increased sensitivity for catecholamines. In some cases acombination of focal activity and reentry in these fibers may be possible.

101 Introduction

The arrhythmias mainly observed are ventricular tachycardias, ventricularpremature depolarizations and accelerated idioventricular rhythms as well asatrioventricular dissociation.

During the process of healing the biophysical properties of the tissuechange dramatically. The connexin 43 content is reduced especially in thehealed and border zone (see chapter 6 for details) and the necrotic tissue isreplaced by scars. This implies that the pathways of activation can changeduring this process. It has been found that during the chronic phase aftermyocardial infarction, ventricular arrhythmias and sudden cardiac death canoccur. Programmed stimulation of the ventricles can induce both sustainedand nonsustained ventricular tachycardia and fibrillation. Within the infarctedarea islets of surviving myocardial cells may be found. These cells show some-what altered electrophysiological properties with maximum diastolic potential,action potential amplitude and duration as well as maximum upstroke velocitybeing moderately reduced during the first week after the infarction. Thereafter,these parameters return to normal except the action potential duration. Con-duction velocity transverse to the fiber axis is reduced, while velocity parallelto the fiber is nearly normal, which may reflect the loss of side-to-side connec-tions and the incorporation of connective tissue (see chapter 6). The actionpotential characteristics of surviving cells return to normal at later timesduring the process of healing, i.e. after more than 1 month, whereas theirregularities in conduction persist and probably participate in forming thearrhythmogenic substrate. Reentrant arrhythmias have been observed duringthat phase due to epicardial reentry in the border zone and in some cases tointramural or subendocardial reentry [for a detailed review see, Janse and Wit,1989].

Because of the complex structure of the AV node involving highly special-ized interdigitating fibers with certain connexins forming the basis of intercellu-lar communication (see chapter 3) it is tempting to speculate (although notshown experimentally with certainty) that changes in the distribution of gapjunctions or alterations in the gap junction conductance may contributeto bradycardiac arrhythmia like AV conduction blocks or to tachycardicarrhythmia, e.g. AV reentry. Similarly, changes in the gap junction distributionor regulation of conductance may participate in sinuatrial block. However,experiments on this topic are still lacking.

At least three types of disturbance in the intercellular communicationhave to be distinguished: (1) separation of the cardiac muscle fibers by strandsof connective tissue as occurring in microfibrosis; (2) changes in the distribu-tion of gap junction channels, and (3) changes in the conductance of gapjunctional channels either by alteration of the open probability or of the singlechannel conductance.

111 Introduction

What is the role of gap junctions in the initiation and perpetuation ofarrhythmia? Cole et al. [1988] compared experimental data with computersimulations and analyzed the relationship of gap junction uncoupling anddiscontinuous propagation in the heart. In summary, they found that reducingthe gap junction coupling led to a decrease in propagation velocity as well asto an increase in Vmax and in the time constant of the action potential foot(sfoot). The phase-plane loops became nonlinear. Shaw and Rudy [1995] demon-strated in a cable model, using the Luo-Rudy membrane model, that thevulnerable window of unidirectional block depends on both membrane excit-ability and intercellular coupling. A uniform decrease (*0.25) in intercellularcoupling increased the time of the vulnerable window by a factor of 3.6,whereas a decrease in excitability (*0.25) led to an increase by the factor 0.4.When inhomogeneities were present in the modelled fiber, the model becamemore sensitive to inhomogeneities in membrane excitability. As shown experi-mentally by Delmar et al. [1987] during impairment of the intercellular gapjunctional communication (by 1.5 mmol/l heptanol) transverse propagationbecomes more vulnerable to conduction block. Such a spatial dissociation inpropagation can form the basis for the occurrence of reentry (see above).However, in the course of ischemia both conduction velocity in longitudinaland transverse direction are reduced (from 50 to 33 and from 21 to 13 cm/s,respectively), so that the ratio between longitudinal and transverse conductionwas only slightly changed [Kleber et al., 1986] (from 2.38 to 2.54). These resultsdemonstrate the changes associated with an alteration in the intercellularcommunication, which probably contributes to enhanced arrhythmogeneityin situations with a reduction in the number of gap junctions, e.g. ischemic heartdisease (see chapter 6) [Peters, 1996], or reduced gap junction conductance, e.g.acute ischemia, hypoxia and acidosis (see chapters 4 and 6).

121 Introduction

2...........................

Structure and Diversity of Gap JunctionChannels

The gap junctional channel has two main functions: (a) to allow transportof small molecules (MW=1,200–1,900 as established in Chironomus salivarygland gap junctions) [Schwarzmann et al., 1981; Simpson et al., 1977] suchas intracellular messengers, small peptides and proteins, nucleotides as wellas injected dyes as fluorescein or lucifer yellow, which are often used in dye-coupling studies (see chapter 8) from one cell to another thereby forming asyncytium, and (b) to provide electrical coupling between cells with or withoutrectifying properties thereby allowing the propagation of an action potentialfrom one cell to another. Thus, the pore of the channel has to exhibit propertiesas a transcellular transport pipe and as an electrical connector which can beturned on and off.

The structure of gap junction channels has been investigated employingelectron microscopy, X-ray diffraction methods and molecular biology. Bythese techniques it was possible to define a model of the channel which is nowwidely accepted. According to these investigations a gap junctional channelis a polymeric structure consisting of 12 proteins called connexins. Correspond-ing to their molecular weight these connexins are designated as connexin 38(Cx38; 38 kD), connexin 40 (Cx40; 40 kD), connexin 43 (Cx43; 43 kD) andso forth. The connexin family comprises at least 15 different isoforms (seefig. 8), among which the isoforms Cx37, Cx40, Cx43, Cx45 and Cx46 areexpressed in mammalian cardiovascular tissue. The whole pipe-like channelis made of two connexons which are contributed by the two adjacent cells.Such a connexon begins at the cytoplasmic surface of the plasma membrane,crosses the lipid bilayer and ends up in the extracellular space between twoadjacent cells. In the neighboring cell another connexon is connected up tothis structure and both connexons then build up the gap junctional channelpassing the cell membranes of the two adjacent cells. These hexameric con-nexons consist of 6 polypeptide subunits, the so-called connexins, which sur-round the inner core of the channel. These connexins have been intensivelyinvestigated in the last years so that today the amino acid sequences of aconsiderable number of them is clarified. A connexin has 4 transmembranedomains (M1, M2, M3, M4), 2 extracellular loops (E1, E2), 1 intracellularloop and the N terminus and C terminus both at the cytoplasmic side. Theextracellular loops and membrane-spanning domains are highly conserved

13

[Caterall, 1988] comparing different connexins and different species, whereasthe intracellular loop as well as the N terminus and C terminus exhibit ahigher variability. Within the M3 segment typically phenylalanines are presentand located next to the charged groups. This is probably an important featureallowing the twisting motion which is required for channel opening and closure.

The N-terminal region exhibits about 50% identity (except for Cx30 andCx32, which are rather closely related). The C terminus varies in length from18 amino acids (Cx26) to 156 amino acids (Cx43) or even 191 amino acids(Cx46). In these cytoplasmic parts of the connexin the target sequences recog-nized by regulatory protein kinases (see chapter 4) can be found. The extracellu-lar loops are thought to participate in the process forming a complete gapjunctional channel from two hemichannels. In this context it is an interestingfeature of both extracellular loops E1 and E2 that each contains three cysteinresidues spaced by either 6, 5 or 4 amino acids. These cysteins are found atthe identical position in all connexins (Cx26, Cx30, Cx32, Cx38, Cx43). Ithas been speculated that they play a role in channel formation by disulfidebonds, although so far there are no hints on intermolecular disulfide bondsfrom SDS-PAGE studies determining the molecular weight of connexins. Incontrary, in Cx43 channels it was shown that the connexon integrity is main-tained by noncovalent bonds and that there are no intermolecular disulfidebonds [John and Revel, 1991]. Only the possibility of intramolecular disulfidebonds has been suggested [Dupont et al., 1989; John and Revel, 1991]. Theforming process of the channel is not yet well understood.

The most abundant gap junction protein found in the heart is Cx43. Afterthe liver gap junction protein (Cx32) had been cloned by Paul [1986], Beyeret al. [1987] looked for cardiac mRNA from rat hearts which would cross-hybridize with the cDNA for rat liver Cx32. They were able to identify threecDNAs together spanning 2,768 base pairs with a single open reading frameof 1,146 base pairs coding 382 amino acids with a calculated molecular massof 43,036 kD. The isolated rat cDNA sequence has a first initiation codon(ATG) at base 202 followed by the 1,146-base pair reading frame and atermination codon at base 1,348. The coding region is followed by 1,218 basesof the 3�-untranslated sequence which includes several termination codons butlacks a polyadenylic acid tail. The predicted molecular mass fits well with thatobtained from biochemical isolation of cardiac gap junction proteins by Manju-nath and Page [1985, 1986] using SDS-PAGE (44–47 kD). The difference may bedue to uncertainties with the SDS-PAGE method or to a co- or posttranslationalphosphorylation as is suggested in the study by Crow et al. [1990]. Partial aminoacid sequencing studies of the isolated protein revealed considerably high homo-logy between the found and predicted sequence near the N terminus [Manjunathet al., 1987; Nicholson et al., 1985]. From the sequencing studies it has been

142 Structure and Diversity of Gap Junction Channels

Fig. 1. Amino acid sequence of chicken Cx43 according to Veenstra et al. [1993]. Possibletargets for phosphorylating enzymes are underlined.

concluded that the first methionine residue is removed posttranslationally sothat in the mature protein the first amino acid is a glycine.

Regarding Cx43, Beyer et al. [1987] predicted a pI at 10.19 indicating avery basic protein. According to this study Cx43 has 34.3% polar and 42.4nonpolar amino acids, 9.4% acidic and 13.9% basic residues at neutral pH.Because of the 53 basic amino acids which include 8 histidine residues facing36 acidic residues, a net positive charge of 17 would result. Within the molecule4 hydrophobic regions have been identified in alternation with hydrophilicregions using the hydropathicity plot of Kyte and Doolittle [1982]. From thesedata the 4 membrane-spanning domains were predicted and the structure forCx43 as given in figure 1.

According to Lau et al. (1996) and Delmar et al., (1995) phosphorylationmay occur at the following sites: at the serine residues 364, 368, 372 (R-X-Smotif for PKA, PKG and PKC), 296, 365, 369, 373 (R-X-X-S motif for PKA,PKG, PKC and CaMK II), 244, 306 (K-X-X-S motif for PKG, PKC), 364,368, 373 (R-X-S-X-R motif for PKC), 297, 364, 368, 372 (S-X-R motif forPKG, PKC), 262 (S-X-K motif for PKG, PKC and at threonine residue 290(K-X-X-T motif for PKG, PKC) at tyrosine residue 265 (vSRC tyrosine kinase)


Fig. 2. Phase contrast microscopy of rabbit cardiac muscle. Note the ‘Glanzstreifen’with the intercalated disk (arrow). ¶1,000.

as well as at the serine residues 279, 282 and 255 which can be phosphorylatedby MAP kinase. The numbers refer to rat Cx43 as given here (membranedomains 1-4 are given in italics):1 MGDWSALGKLLDKVQAYSTAGGK VWLSVLFIFRILLLGTAV ESAWGDEQS51 AFRCNTQQPGCENVCYDKSFPISHVR FWVLQIIFVSVPTLLYLA HVFYVM101 RKEEKLNKKEEELKVAQTDGVNVEMHLKQIEIKKFKYGIEEHGKVKMRGG151 LLRTYIISILFKSVFEVA FLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFL201 SRPTEKT IFIIFMLVVSLVSLALNI IELFYVFFKGVKDRVKGRSDPYHAT251 TGPLSPSKDCGSPKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRN301 YNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNAKKVAAG351 HELQPLAIVDQRPSSRASSRASSRPRPDDLEI

The spatial structure of the channel has been investigated for a longtime. In the beginning, light microscopists described intercalated disks whichappeared as bands transverse to the longitudinal axis of the cardiac musclefiber [Eberth, 1866]. With modern phase contrast microscopes they can easilybe seen as shown in figure 2.

These bands were a matter of discussion for a long time until in 1954, forthe first time, Sjostrand and Andersson used electron microscopy to investigateintercalated disks in ultrathin osmium tetroxide-fixed sections of the mouseheart revealing that the disks were indeed transverse cell boundaries. Sub-sequently, several investigators reproduced their finding [Lindner, 1957; Moore


and Ruska, 1957; Poche and Lindner, 1955] and some new methods wereused in the investigation of these cell boundaries. It became obvious that theintercalated disk contains three distinct structures: fascia adherens (the mainportion of the disk); macula adherens or desmosome, and nexus. The fasciaadherens is made of two parallel lipid bilayers separated by a distance of200–300 A, whereas the desmosome is a more complex and almost laminatedstructure built from the two adjacent membranes. The nexus is the zone ofclose contact between the cells containing the gap junction channels.

Our present image of a gap junction channel is based on the X-raydiffraction studies of Makowsky [1988], Makowsky et al. [1977, 1984] andTibitts et al. [1990] and the low irradiation electron microscopy [Gogol andUnwin, 1988; Sikewar and Unwin, 1988; Sosinsky et al., 1988] as well as theuse of antibodies directed against specific amino acid sequences in the subunitsin order to get information on the topology [Milks et al., 1988; Zimmer et al.,1987] and the cloning of cDNA [Kumar and Gilula, 1986; Paul, 1986]. Fromthese studies the hexameric character of the channel became evident and, asalready mentioned, 6 connexins together form a connexon with an inner pore.Via the extracellular loops 2 connexons are interconnected to each otherthereby constituting the full gap junction channel. This structure has beensubject to intensive X-ray diffraction studies and electron microscopy. Thesestudies revealed the spatial structure of the channel with a total length ofapproximately 100–150 A and about 52 A in the portion within the lipid bilayerof each side [Chen et al., 1989] and the two parallel membranes separated bya 20 A gap spanned by the channels subunits [Beyer et al., 1995]. The diameterof the channel ranges between 20 and 30 A and about 15 A in the extracellularhalf of the lipid bilayer. The inner pore appeared solvent filled, and is =2.5nm at its widest point [Severs, 1994a, b]. Transmission electron microscopy ofpositively stained cross-sectioned cardiac gap junctions of mammalianventricles or atria revealed a 7-layered structure: two 3-layered lipid membranesand the gap between them. More recently Perkins et al. [1997] developed athree-dimensional model of the connexon with 50 A height and 6 lobes pro-truding from the extracellular surface, that would dock with an opposingconnexon to form an intercellular channel.

Using the freeze fracture technique, electron microscopy and laser scan-ning confocal microscopy, it became obvious that these gap junctional channelsare arranged as a cluster of channels with about 50 channels within one diskas stated by Gourdie et al. [1990].

From these studies and results Makowsky et al. [1977] developed a three-dimensional model for the channel. According to these studies the gap junctionchannels are arranged in clusters as shown in figure 3. A model of a singlechannel is given in figure 4.


Fig. 3. Drawing of a cluster of gap junction channels.

Fig. 4. Model of a single gap junction channel and a connexon.

The next question to answer was the mechanism of closure of the channel.It is widely accepted now that the channel is closed by a rotational movementof the hexamer [Unwin and Ennis, 1984; Unwin and Zampighi, 1980] asillustrated in figure 5. This twisting motion closing the central channel ispossible since the a-helix of the connexins, which is the part located withinthe lipid bilayer, is inclined with respect to the axis of the whole connexon[Milks et al., 1988].


Fig. 5. Model of gap junction channel opening and closure by a slight twisting motionof the connexon (bent arrow) which opens the central channel. Note the inclination of thea-helical segments of the connexins with regard to the axis of the whole connexon as proposedby Unwin and Zampighi [1980].

The next issue to discuss is the diversity of connexins, i.e. the variousisoforms, and species variability. Gap junctional channels exist in a broadvariety of tissues including the heart, vascular system, brain, epithelial tissues,uterus, lens cells, pancreas and kidney. However, these tissues are connectedby different isoforms of gap junctional connexins which can be distinguishedwith regard to their molecular weight. These differences are mainly due tovarious lengths of the C-terminal loop.

The smallest connexin is Cx26 with an approximate molecular weight of21–26 kD as determined using SDS-PAGE. The C terminus consists of 18amino acids. The N-terminal region contains 22 amino acids. Rat Cx26 hasbeen found in liver hepatocytes [Traub et al., 1989; Zhang and Nicholson,1989], pinealocytes, leptomengineal cells [Dermietzel et al., 1989], pancreaticacinar cells [Traub et al., 1989], endometrium [Risek et al., 1990] and in variousother tissues including lung, kidney, spleen, intestine, stomach and testes[Zhang and Nicholson, 1989]. The amino acid sequence is given in figure 6.

A connexin with a molecular weight of 30 kD (Cx30) has been isolatedand cloned from xenopus liver and was also found in the lung, intestine,stomach and kidney of xenopus [Gimlich et al., 1988]. The C terminus isenlarged to 58 amino acids. The N terminus contains 22 amino acids. Theamino acid sequence is also given in figure 6.

Another connexin with a molecular weight of 32 kD, Cx32, was clonedfrom human liver [Kumar and Gilula, 1986], rat liver [Paul, 1986] and wasalso found in hepatocytes [Paul, 1986; Traub et al., 1989], stomach, brain andkidney [Paul, 1986] as well as in pancreatic acinar cells [Dermietzel et al.,


Fig. 6. Amino acid sequence of Cx26, Cx30 and Cx32. Amino acids are given by aone-letter code [according to Paul et al., 1986; Bennett et al., 1991; Gimlich et al., 1988].

1984] and oligodendrocytes [Dermietzel et al., 1989]. The C terminus consistsof 76 amino acids and the N terminus of 22 amino acids. The amino acidsequence is given in figure 6.

The second group of connexins, including Cx37, Cx38, Cx40, Cx43 andCx46, is characterized by a longer N terminus which is elongated by 1 aminoacid in position 3. It is believed that these two groups of connexins representtwo parts of a phylogenetic tree of the connexin family as pointed out byBennett et al. [1991] (see also fig. 8).

The first protein of this second group is Cx38 which has been clonedfrom xenopus oocytes and was also found in the embryo [Ebihara et al., 1989;Gimlich et al., 1990]. The C terminus consists of 120 amino acids. It has ahigh homology to mouse Cx37. Studies in a xenopus oocyte expression systemrevealed that Cx38 by itself exhibits only poor channel forming ability, but ishighly effective in forming hybrid channels with Cx43. Thus, it has beensuggested that the function of Cx38 is to form hybrid rather than symmetricalchannels [Werner et al., 1993]. The full amino acid sequence of Cx37 is givenin figure 7. Cx37 is an isoform belonging to the same branch of the Cx family


Fig. 7. Amino acid sequence of Cx37, Cx40, Cx43 and Cx45. Amino acids are givenby a one-letter code [according to Kanter et al., 1992; Reed et al., 1993; Fishman et al.,1990]. Note that the N terminus is enlarged by one amino acid in position 3 as comparedto Cx26, Cx30 and Cx32.

tree, and has been found in rodents (mice, rats) and is highly expressed inlung [Willecke et al. 1991].

One of the last connexins which has been characterized is Cx40 [Kanteret al., 1992], which is one of the connexins typically found in the heart. However,it is preferentially expressed in the lung [Hennemann et al., 1992b]. Within theheart it has been discovered in atria (human, but not in all species, e.g. rat atriadoes not express Cx40), in the conduction system and in the vascular endothe-lium [Bastide et al., 1993]. Chick Cx42 has been considered to be the homologueof mammalian Cx40 with 70% of the amino acids being identical [Kanter et al.,1992]. Thus, it is referred to as Cx40. The ratio between Cx40, Cx43 and Cx45in heart can be altered in the course of cardiac diseases (for a detailed discussionsee chapter 6). The full amino acid sequence is given in figure 7.


Table 1.

Residue Human Rat Xenopus

124 D E D16 I V V

234 K R K251 S T N253 A P A257 A S G263 Q P P344 S A M 1

347 L V G 1

1 For Xenopus the residue number for these amino acidsis smaller by 3.

Cx43 is the gap junction protein most abundantly found in the hearts ofvarious species including human, dog, chicken and rat. The molecular weightas determined by SDS-PAGE ranges from 42 to 45 kD. Besides in the heart,Cx43 has been found in uterine muscle, granulosa cells, smooth muscle cells,kidney, eye (cornea and lens), epithelium [Beyer et al., 1987, 1989; Risek et al.,1990], liver, spleen, ovary [Gimlich et al., 1990], in fibroblasts [Musil et al.,1990a], in astrocytes and leptomeningeal cells [Dermietzel et al., 1989; Yama-moto et al., 1990]. mRNA for Cx43 has been found in endothelial cells,pericytes and vascular smooth muscle cells [Larson et al., 1990]. Cx43 seemsto be highly conserved between the species. A homology of 92% between chickCx43 and rat Cx43 has been found [Musil et al., 1990a]. Its N terminus exhibits23 amino acids and its C-terminal loop 156 amino acids. The full amino acidsequence is given in figure 7.

Using a rat Cx43 probe and a 10-day chick embryo cDNA library, chickCx42 (see above) and chick Cx45 were identified [Beyer, 1990]. This connexinis developmentally regulated with higher levels of its mRNA in early embryosthan in more mature organisms [Beyer, 1990; Veenstra et al., 1993]. In theextracellular loops E1 and E2 the typical three cystein residues can befound. Mouse Cx45 consists of 396 amino acids and has a molecular weightof 45, 671 [Willecke et al., 1993]. There is a homology of 85% between chickCx45 and canine Cx45 [Kanter et al., 1992]. The amino acid sequence is givenin figure 7.

Finally, the longest connexin is Cx46, which is also expressed in rat heartand has been cloned from lens [Beyer et al., 1988]. This connexin exhibits the


Fig. 8. The phylogenetic ‘connexin family tree’ according to Bennett et al. [1995].Branching points with closed ovals represent gene duplications whereas branching pointswithout ovals represent speciation. X>xenopus; Ch>chicken; ms>mouse; r>rat; bov>bovine; c>canine; h>human.

longest C terminus known so far with 191 amino acids, the total proteinconsisting of 416 amino acids [Paul et al., 1991].

In addition, an eye lens cell protein known before as MP 70 has recentlybeen identified as Cx50 [White et al., 1992].

Regarding species variability there are some points to mention. First, avariability in the distribution pattern of a distinct connexin isoform is possible,for example with Cx40 which is normally found in atria of many species butnot in rat atria. The details for these species differences with regard to thedistribution of connexins in the heart are given in chapter 3.

Second, the amino acid sequence can be altered and it has been shown thatthere are indeed some single amino acids which vary depending on the species.

In Cx43 the following differences are reported [for review see, Bennettet al., 1991] (table 1).

In some cases such variability has consequences for the regulation of thegap junction channels. Thus, in rat Cx32 the serine residue at position 233 is


phosphorylated by a cAMP-dependent protein kinase, but this is unique torCx32.

Finally, a phylogenetic analysis of the connexin family has been carriedout restricting the analysis to the two major conserved regions in connexingenes, and a ‘family tree’ was proposed (fig. 8). Since the distance betweenamphibian and mammalian orthologues is considerably smaller than betweengroup I and II, it was suggested that the branching between the two groupsoccurred rather early in phylogenesis, i.e. in the early or even before vertebratedivergence. The two extra cellular loops E1 and E2 are the most conservedregions of the connexins with three invariant cysteins (figures 6 and 7). Thetransmembrane domains M1-M4 are somewhat less well conserved, while thecytoplasmic loop and the C-terminal are the most variant regions of themolecule.


3...........................

Distribution of Gap Junctions in the Heart

In the first part of this chapter the distribution of gap junctions withina cell is discussed. In the second part the connexin pattern in the heart isdescribed, and in the third part the expression of various connexins in thevasculature is outlined.

Heart muscle fibers are coupled by gap junctions. These intercellularchannels provide the exchange of small molecules (=1,000 D), like secondmessengers, between the cells and they allow electrical coupling. Thus, thesecells connected to each other form a syncytium. However, from mappingstudies it became evident that under certain conditions, e.g. regional ischemia,the ischemic region uncouples. In addition, mapping studies demonstratedthat there is a special activation pattern which accounts for a directed activationof the whole heart. This activation pattern exhibits a considerable similarityfrom beat to beat. It is well known that the conduction velocity varies between0.3 and 0.6 m/s in the ventricles and 1.0 m/s in the Purkinje system. On theother hand conduction is delayed in the AV node. In addition, the activationhas to be transduced from the sinuatrial node to the atria, and from theendings of the Purkinje fibers to the ventricular myocytes. Thus, the couplingwithin the tissue and between various cells becomes an important feature toprovide the normal impulse conduction. From the above-mentioned considera-tions an association of the different functions and demands with differenttypes of coupling can be concluded. Thus, in general, Cx40 can be found inthe conduction system whereas ventricular myocytes are coupled by Cx43. Inthis chapter, the distribution of the various connexins within the cardiac tissuewill be described. First, the distribution of the gap junction channels withina cell will be outlined.

In the foregoing chapter, it was found that the intercalated disks seenon light microscopy contain the gap junction channels. However, it remainsuncertain how the gap junction channels are distributed within a disk or howthe cell-to-cell boundary is shaped. This question has been resolved using thefreeze-fracture technique supplemented with image-processing systems. Briefly,to freeze-fracture cardiac muscle specimens, these specimens have to be fixedwith glutaraldehyde in order to crosslink the proteins within the tissue, andincubated with glycerol in order to prevent ice crystal formation in the courseof the freezing procedure. After freezing and fracturing in vacuum the speci-mens have to be unidirectionally stained in vacuo by exposure to platinum

25

Fig. 9. Distribution of the gap junctions, desmosomes and fascia adherens in an intercal-ated disk of a cardiomyocyte as assessed by electron microscopy of freeze-fractured rat andrabbit hearts according to Severs [1990].

and carbon vapor at an angle of 45º, followed by deposition of carbon at anangle of 90º. Details of the method and variations in this technique are de-scribed in the book edited by Rash and Hudson [1979] and the applicationfor gap junction research in the review by Severs [1989]. Using this technique[Severs, 1990] and others (transmission electron microscopy of positivelystained serial ultrathin sections and scanning electron microscopy) [Hoyt et al.,1989] helped to clarify the arrangement of the gap junctions within the intercal-ated disk.

It became obvious that the transverse cell boundary is not a plane diskbut consists of several processes which interdigitate with the correspondingprocesses of the adjacent cell. These interdigitating membranes were formerlydescribed as the ‘plicate segment’. Within this plicate segment the gap junctionswere found to be located in the finger-like processes and the interface betweenthe myocytes parallel to the fiber axis as shown in figure 9.

From figure 9 it becomes clear that the fascia adherens is located transverseto the fiber axis on the cell processes and at the side walls of these processesgap junctions are located in clusters and desmosomes. According to Hoytet al. [1989] the gap junctions are arranged in a more or less ribbon-like fashion

263 Distribution of Gap Junctions in the Heart

whereas according to the model of Severs the gap junctions are distributedover a wider range. From a biophysical point of view this makes an importantdifference because the electrical transfer from one cell to another will beinfluenced by this structural arrangement. This would affect the spatial distri-bution of current flowing from one cell to another and could possibly affectthe efficacy of coupling. However, presently it is not absolutely certain whetherthe model of Hoyt et al. [1989] or of Severs [1990] is correct. Hoyt et al. [1989]found that about 3.6 myocytes overlap so that each is connected to 9 othermyocytes.

The relatively new technique of laser scanning confocal microscopy en-abled the determination of the number of gap junctions within one disk tobe in the order of 50 or even more [Gourdie et al., 1990] with a diameter ofup to 1.3 lm/gap junction. Within the gap junction the channels themselvesare arranged as parallel pipe-like structures. From freeze-fractured junctionsit has been estimated that about 12.9 ·103 channels are located in 1 lm2 gapjunction (rat right ventricular myocardium) [Chen et al., 1989].

The surface of the intercalated disk is occupied to 5.7×0.6% by gapjunctions (canine atrium) [Spira, 1971], 3.3% (right bundle, calf ) [Arluk andRhodin, 1974], or even 12.7–15.1% in canine left ventricular subepicardialmyocardium [Hoyt et al., 1989]. The rest of the intercalated disk is made offascia adherens and desmosomes. In the crista terminalis of the canine heartthe gap junction profile length has been estimated to be in the order of3.2–3.8 lm/100 lm intercalated disk length with 11–12 gap junctions/100 lmintercalated disk length and a mean gap junction profile length of about 0.3 lm[Saffitz et al., 1994].

However, connexins do not seem to be restricted to the transverse cellboundaries, since they have also been detected in several specimens at thelateral cell side. For example Oosthoek et al. [1993b] demonstrated Cx43-positive staining at the lateral cell side of human and bovine hearts (ventricles).Figure 10 shows another example from the rabbit heart, using an anti-Cx43monoclonal antibody in cryostat sections of the rabbit left ventricle. Pleasenote the distribution of Cx43 positivity at the transverse cell boundaries andat the lateral cell sides.

In cardiac tissue mRNA for Cx37, Cx40, Cx43, Cx45 and Cx46 has beendetected in dog, mouse and rat heart [Haefliger et al., 1990; Hennemann et al.,1992a, b; Kanter et al., 1992; Paul et al., 1991; Willecke et al., 1991]. However,in the heart, Cx40 and low levels Cx45, Cx43 is found most abundantly. Thedistribution of the various connexins exhibits a specific pattern with somespecies variability which will be discussed in the next section.

Cx43 has been detected in many areas of the heart; however, only verylow levels were found in AV node and sinus node. Cx43 was absent in AV


Fig. 10. Photomicrograph demonstrating immunohistochemical staining for Cx43 incryostat sections of the left ventricle of the rat heart. Objective: ¶40. neofluor achroplan,Zeiss. Magnification:¶400.

bundle and the bundle branches [Gourdie et al., 1992; van Kempen et al.,1991]. However, there are some discrepancies in the newer literature regardingthe Cx43 distribution pattern: Bastide et al. [1993] could not detect Cx43 inthe atrioventricular bundle and bundle branches of the rat heart. Similarly,Oosthoek et al. [1993a, b] found expression of Cx43 in the AV bundle andbundle branches in bovine and human hearts but a lack of Cx43 expressionin the AV node and the center of the sinoatrial node of human and bovineheart. In the central sinus node of the rat heart these authors did not findCx43 staining either. In contrast, Anumonwo et al. [1992] reported Cx43 insinoatrial nodal cell pairs isolated from the rabbit heart, and Trabka-Janiket al. [1994] in the hamster sinoatrial node cells. However, these authors didnot double stain the cells with an anti-a-smooth muscle actin antibody whichis known to specifically stain sinoatrial node cells. Thus, it might be possiblethat the Cx43-positive cells were obtained from the border zone of the sinoatrialnode. If Cx43 is investigated in the conduction system, it has to be taken intoaccount that Cx43 is expressed in these tissues [van Kempen et al., 1995] onlyafter birth.


Cx40 was found in sinus node cells, atrium, AV node, AV bundle andbundle branches and Purkinje fibers. Cx45 was expressed at low levels inPurkinje fibers and ventricles of the canine heart [Kanter et al., 1993a, b, c].

Following the anatomy of the heart more systematically, in the sinoatrialnode a center zone has to be distinguished from the periphery. It was foundin the human and bovine heart that in the center zone no Cx43 was expressed,but that from this center wings extended toward the superior caval vein andtoward the atrium. Whereas the center zone of the sinoatrial node was com-posed of cells, small myocytes, negative for Cx43, the cell size was graduallyincreasing within these wings. These wings interdigitate with strands from theatrium which were positive for Cx43. Nodal cells (negative for Cx43) wereseparated by strands of connective tissue from the Cx43 positive cells [Oosthoeket al., 1993b]. Similarly, ten Velde et al. [1995] described an abrupt changefrom negative staining for Cx43 in the sinoatrial node (guinea pig) to positivestaining in the atrium. Cx43-negative strands from the sinoatrial node towardthe crista terminalis became smaller in size and alternated with Cx43-positivelayers becoming progressively broader in the direction of the crista. Lateralcontacts between the Cx43 and the a-smooth muscle actin-positive sinoatrialnode cells were found to be rather sparse. Thus, the authors concluded that theprimary pacemaker seemed to be shielded from the hyperpolarizing influence ofthe atrium (which has a more negative resting membrane potential than thenode) by gradually coupling due to geometric factors (interdigitating fibers)and not by a gradient in Cx43 density. In addition, these authors found thatendocardial strands at the crista terminalis side of the sinoatrial node wereCx40 positive, but the node itself was negative. However, in canine heart Cx40and Cx45 have been detected [Davis et al., 1994; Kanter et al., 1993c].

In the atrium gap junctions with Cx43 have been found immunohisto-chemically [Gros et al., 1994] in rat and guinea-pig hearts. Besides Cx43, Cx40is also expressed in the atrium of several species including guinea pig [Groset al., 1994], goat [van der Velden et al., 1996], dog crista terminalis [Saffitzet al., 1994], man [Davis et al., 1995], but not or only in some cases in the rat[Gros et al., 1994]. In human atrium moderate amounts of Cx40, Cx43 andCx45 were determined [Davis et al., 1995].

The AV node is a highly specialized structure of the conduction system,which is designed for delayed conduction (with a rate-dependent delay) of theaction potentials from the atrium to the ventricles. Thus, it as been hypothe-sized that in the AV node other gap junction proteins may occur than in theventricular myocardium. Indeed, only low expression of Cx43 has been ob-served in rat AV-nodal tissue [Gourdie et al., 1992; van Kempen et al., 1991].In accordance with this finding, Oosthoek et al. [1993a] could not detect Cx43expression in human or bovine AV-nodal tissues. However, besides Cx40 Davis


et al. [1995] did find some Cx43 and Cx45 in human AV node. The reasonfor this discrepancy is not yet known. Since, however, van Kempen et al.[1995] could show that the connexin distribution patterns are to a large extentcomparable between various mammalian species, these data taken togethershow that Cx40 is probably the predominant connexin in the AV node, andCx43 plays only a minor role.

Cells of the AV bundle also express Cx40 and are lacking in Cx43 (ratheart) [Bastide et al., 1993; Gros et al., 1994]. However, this seems to dependon the species investigated. Thus, in guinea-pig hearts only Cx43 but not Cx40could be detected in the AV bundle [Gros et al., 1994]. In the human heartCx43 was found at the end-to-end intercalated disks of the AV bundle [Oos-thoek et al., 1993a].

A similar pattern is seen in the bundle branches which exhibit high amountof Cx40, Cx43 and Cx45 in the human heart according to Davis et al. [1995].On the other hand, in rat bundle branches Cx43 is absent [Gourdie et al.,1992; Gros et al., 1994; van Kempen et al., 1991], whereas in the guinea pigCx43 immunoreactivity was detected but not Cx40 expression [Gros et al.,1994].

Regarding the Purkinje fibers cells are connected via Cx43 and Cx40. Athreefold higher expression of Cx40 mRNA as compared to Cx43 mRNAoccurs in the canine Purkinje fibers [Kanter et al., 1993b, c]. Only very lowlevels of Cx45 were found in this study in Northern blots. The immunostainingintensity corresponded to these findings. A slightly enhanced amount of Cx43mRNA in Purkinje fibers as compared to ventricular muscle has been demon-strated in the study mentioned above. This difference in connexin distributionand density may contribute to the well-known differences in conduction prop-erties [Purkinje fiber conduction velocity: up to 2–3 versus 0.3–0.4 m/s inventricles). In contrast, in the adult rat heart no Cx43 was observed in theproximal Purkinje system [van Kempen et al., 1991], whereas Gourdie et al.[1992] did observe Cx43 in the Purkinje fibers of the rat. Gros et al. [1994]also stated that gap junctions of rat Purkinje fibers contain Cx43 and Cx40.In the human and bovine heart Cx43 is expressed in Purkinje fibers. In thebovine heart Oosthoek et al. [1993a] found a characteristic distribution patternof Cx43 with positive staining along the entire plasma membrane facingother Purkinje fibers but not those facing connective tissue. This characteristicpattern was not seen in the human heart.

In the ventricular myocardium Cx40, Cx43 and Cx45 have been detected[Kanter et al., 1994; Verheule et al., 1997] but only Cx43 and Cx45 have beenfound in the human heart in considerable amounts, whereas only a very lowexpression of Cx40 was observed which was located at the subendocardiumand at endothelial layers [Davis et al., 1995].


In general, in various parts of the conduction system higher amounts ofconnexin were found especially in the fast conducting tissues as compared toventricular myocardium. Only minimal expression of Cx37 and Cx46 betweenoccasional atrial and ventricular myocytes has been observed [Davis et al.,1995].

Besides the myocardium and conduction system, connexins are also ex-pressed in the coronary vasculature [Christ et al., 1996]. The arterial smoothmuscles behave like a syncytium and up- or downstream conduction of aresponse has also been seen in the endothelial layer. In addition, bidirectionalsignalling is required for regulation of the vascular tone, i.e. signal transductionfrom the perivascular nerves (and nerve varicositites) toward the vessel lumenand vice versa. Thus, it could be assumed that the cells of the vascular wallmay be interconnected by some type of cell-to-cell contacts. Indeed, in vasculartissue three connexins are expressed: Cx37, Cx40 and Cx43. As an example,Cx40 was identified in the endothelial layer of cardiac blood vessels [Bastideet al., 1993], but was lacking in the smooth muscle cell layer of the arterialwalls as was characterized in frozen sections stained for immunohistology.However, immunohistologically Cx43 was detected in smooth muscle cells ofthe media of pig coronary arteries as a discrete punctuation. Further investiga-tion by transmission electron microscopy revealed a lack of the typical gapjunctions in this tissue, although dye coupling between the smooth musclecells was observed [Beny and Connat, 1992]. The authors concluded that thesearterial smooth muscles cells were coupled through isolated gap junctionchannels, and not, as in the myocardium, through clusters of channels whichcan be detected microscopically. However, in other vasculatures gap junctionsbetween smooth muscle cells could be identified: junctional plaques were seenin the human corpus cavernosum and in the rat aorta with diameters of0.2–0.5 lm [Campos de Carvalho et al., 1993; Christ et al., 1993], as well asin rat and hamster resistance arteries [Little et al., 1995]. Besides Cx43, Cx40was also found. The distribution of Cx43 in a coronary vessel and a schematicdiagram of the distribution of various connexins in the vessel wall are shownin figures 11 and 12, respectively. Furthermore, it was found that Cx43 isexpressed more extensively in synthetic phenotype cells but only a few gapjunctions were observed between contractile cells [Rennick et al., 1993]. Thus,gap junction formation and Cx43 expression may depend on the phenotypeof smooth muscle cells. This may also account for the differences observedby different investigators.

Gap junctions between endothelial cells in the vascular wall contain chan-nels formed by Cx40, Cx43 and, in contrast to the media, by Cx37 (rat aorta)[for review see, Christ et al., 1996] with a reduced Cx43 density as comparedto smooth muscle cells.


Fig. 11. Immunohistochemical localization of Cx43 in a rabbit coronary arteriolarvessel. The lower figure gives the corresponding phase contrast microphotograph.


Fig. 12. Drawing of the distribution of various connexin isoforms within the vascularwall.

Presently it is not certain whether gap junctions between endothelial andsmooth muscle cells exist in the vascular wall. Such myoendothelial interconnec-tions could be an interesting mechanism of signal transduction from the lumenor the endothelium to the media and might also contribute to upstreamregulation of vascular tone, but, although theoretically anticipated, they havenot been shown unequivocally until now. However, there is some ultrastructuralevidence for myoendothelial gap junctions between endothelial cells and pro-cesses of the smooth muscle cells passing through fenestrae of the elasticainterna [Beny and Pacicca, 1994].

In addition, myocytes and fibroblasts can form functional gap junctionchannels [Goshima, 1970] which has been experimentally investigated in gapjunctions formed from both cells cultured from neonatal rat hearts [Rooket al., 1989]. It was found that the conductance between myocytes was in theorder of 43 pS, between fibroblasts about 22 pS and between myocytes andfibroblasts in the range of 29 pS, indicating that a heterojunction may existbetween both cell lines. Such heterojunctions are presently one of the maininterests in gap junction research, since many physiological phenomena regard-ing crosstalk between various tissues and developmental phenomena may beinvolved.


An interesting phenomenon discovered by several investigators in recentstudies is the occurrence of coexpression of various connexins in the gapjunctions of one cell. Using double-label immunofluorescence on disaggregatedcanine ventricular myocytes, Kanter et al. [1993c] could demonstrate withlaser scanning confocal microscopy and electron microscopy that cardiac myo-cyte gap junctions contain multiple channel proteins with Cx40/Cx43 andCx43/Cx45 colocalization, i.e. an individual cell contains more than one con-nexin isoform. There are two principally different possibilities for coexpression:(a) a single hemichannel could contain multiple connexins, or (b) hemichannelsconsisting of only one isoform may join another hemichannel made of anotherisoform thereby constituting a heteromeric gap junction channel. Presently, itis not clear whether hemichannels of mixed composition naturally occur.However, pairs of Cx32/Cx43 have been shown to be functional [Swensonet al., 1989; Werner et al., 1989].

In addition, using polyclonal anti-Cx43 and anti-Cx40 antibodies in frozensections of the rat heart, coexpression of Cx43 and Cx40 in ventricular myo-cytes was seen immunohistologically [Bastide et al., 1993]. However, it isuncertain whether Cx43 and Cx40 form heterotypic channels in the heart,since in injected oocytes they only form homotypic channels [Bruzzone et al.,1993]. Such heterotypic channels would be expected to possess a single channelconductance of about 50 pS and should exhibit a sensitivity to transjunctionalvoltage, since channels formed by Cx40 exhibit a single channel conductanceof 86–236 pS and a great sensitivity to transjunctional voltage and those madefrom Cx43 28–67 pS (being rather insensitive to transjunctional voltage) andCx45 about 29 pS as characterized in embryonic chick heart cells [Veenstraet al., 1992].

A colocalization of Cx43 and Cx40 has also been observed in the intercal-ated disks of the guinea pig atrium by Gros et al. [1994]. In addition, it wasshown that Cx40, but not Cx43, can form heterotypic channels with Cx37(Bruzzone et al., 1993). It can be suggested, that, if adjacent cells expressincompatible connexins, this provides a mechanism for the formation of differ-ent compartments.


4...........................

Function and Physiology ofGap Junction Channels

In this chapter insight into the regulation and electrophysiology of gapjunctions will be given and the current-voltage relationships for various connex-ins will be described. In the first part a special focus is on the physiologicalregulation of gap junctional opening and closure by calcium, sodium, magne-sium, cAMP, ATP, pH, pCO2, leukotrienes, catecholamines and acetylcholine.In the second part of this chapter the functions of gap junctions in the heartand in the various regions of the heart and vasculature (endothelium andsmooth muscle) are detailed. In the third part of the chapter electrophysiologyand biophysics will be discussed. Thus, current-voltage relationships, singlechannel conductances, differences between findings with double-cell voltage-clamp and with the dye-coupling method will be pointed out.

4.1 Regulation of the Channels

Gap junctional channels, like many other ion channels, can be modulatedvia second messengers and via phosphorylation processes. Besides these, in-tracellular calcium and pH have been proven to be important regulators ofchannel function. In this chapter the short-term regulatory processes are con-sidered, i.e. processes on a time scale of minutes. Besides this, regulatoryprocesses are known which take place over a period of 30 min up to severalhours and which involve formation or synthesis of new gap junction channels.The latter processes are described in the following chapter.

Gap junction conductance (gj) of neonatal rat heart cells varies withtemperature (37 ºC, 48.3 nS; 14 ºC, 21.4 nS; –2 ºC, 17.5 nS) [Bukauskas andWeingart, 1993] so that gj has been assumed to be at least in part enzymaticallycontrolled. Several protein kinases are known to be involved in the regulationof the gap junction channels. However, the situation is rather complicated sincethe same protein kinase may enhance or reduce gap junctional conductance indifferent tissues or in different species. Thus, generalizations should be avoidedand the specific condition has to be taken into account. One of the first tobe described was protein kinase A (PKA), the cAMP-dependent protein kinase,which can enhance junctional conductance in hepatocytes coupled via Cx32and Cx26 [Saez et al., 1986, 1990]. Similarly, an increase in junctional conduc-

35

tance in response to cAMP has been found in cardiac myocytes coupled viaCx43 [Burt and Spray, 1988; De Mello, 1988]. The changes in conductanceare very rapid and occur in several minutes. The action of PKA on rat hepato-cyte Cx32 has been attributed to a phosphorylation of Ser-233 which is embed-ded in a motive (Lys-Arg-Gly-Ser) known to conform with the target sequencefor PKA or PKG, i.e. basic-basic-spacer-Ser. The serine can be replaced bythreonine [Saez et al., 1990]. This sequence cannot be found in Cx43 so thatit has been hypothesized that Cx43 is not subject to direct phosphorylationby PKA. In accordance with this, Kwak and Jongsma [1996] investigated theinfluence of 8-Br-cAMP, a direct activator of PKA, on dye coupling andelectrical coupling in pairs of neonatal rat cardiac myocytes. They did notobserve a change in coupling in response to 8-Br-cAMP. In other tissuesas myometrium and Sertoli cells a PKA-dependent decrease in junctionalconductance has been found (table 2).

Injection of cAMP into canine Purkinje fibers also increased gap junctioncoupling [De Mello, 1984] within 60–90 s after injection of the nucleotide.The role of cAMP and ATP is further elucidated by the finding that, in doublewhole-cell patch-clamp experiments, a rundown is normally observed whichcan be suppressed by addition of cAMP and ATP to the pipette solution,indicating that the spontaneous uncoupling is probably due to washout ofintracellular nucleotides [Neyton and Trautmann, 1985; Somogyi and Kolb,1988a, b]. However, no increase in coupling in neonatal rat cardiomyocyteswas seen by Kwak and Jongsma [1996], which is in line with the finding ofBerthoud et al. [1993] that 1-hour treatment with 0.5 mmol/l 8-Br-cAMP ofMDCK cells, which express Cx43, did not change the immunoblot pattern ofCx43 indicating that 8-Br-cAMP did not induce phosphorylation via PKA ofCx43 in this cell line. In addition, dye transfer through Cx45 gap junctionchannels and electrical coupling in SKHep1 cells is not influenced by PKAactivation [Kwak et al., 1995a]. In the same model, transfectants (SKHp1/Cx26 and SKHep1/Cx43) were investigated demonstrating that PKA did notinfluence conductance of Cx43 or Cx26 channels either. However, the possibil-ity that PKA alters the open probability of the channels could not be ruledout in this study since the effects were observed in the presence of uncouplingagents. Additionally, it cannot be fully excluded that in the transfected cellline proteins necessary for the full and normal function of PKA are notexpressed.

In horizontal cells of turtle and fish retinae, a dopamine-induced increasein intracellular cAMP levels is associated with cellular uncoupling [DeVriesand Schwartz, 1989; McMahon et al., 1989] (the connexin isoform involvedis not identified). Inhibition of phosphodiesterase with IBMX after stimulationof adenylate cyclase using forskolin resulted in an increase in intracellular

364 Function and Physiology of Gap Junction Channels

Table 2. Synopsis of the influence of different protein kinases on cell-to-cell-coupling

Protein kinase Connexin Tissue Permeability gj Reference

PKA 43 Rat cardiac myocytes 0 0 Kwak and Jongsma [1996]43 Cardiac myocytes C C DeMello [1988; 1991]

Burt and Spray [1988a]32/26 Rat hepatocytes C Saez et al. [1986, 1990]43 Myometrium B Cole and Garfield [1986]43 Sertoli cells B Grassi et al. [1986]45 SKHep1 cells 0 0 Kwak et al. [1995a]43 SKHep1/Cx43 cells 0 0 Kwak et al. [1995a]26 SKHep1/Cx26 cells 0 0 Kwak et al. [1995a]

PKC 43 Rat cardiac myocytes B C Kwak & Jongsma [1996]43 Cardiac myocytes C Spray and Burt [1990]43 Rat cardiac myocytes B Munster and Weingart [1993]43 Rat cardiac myocytes B Doble et al. [1996]43 Sertoli cells C Grassi et al. [1986]? Rat epidermal cells B Gainer and Murray [1985]45 SKHep1 cells 0 C1 Kwak et al. [1995a]43 SKHep1/Cx43 cells B C3,B4 Kwak et al. [1995a]26 SKHep1/Cx26 cells B B5 Kwak et al. [1995a]

PKG 43 Rat cardiac myocytes B B Kwak and Jongsma [1996]43 Cardiac myocytes B Burt and Spray [1988a]45 SKHep1 cells 0 0 Kwak et al. [1995a]43 SKHep1/Cx43 B B2 Kwak et al. [1995a]26 SKHep/Cx26 0 0 Kwak et al. [1995a]

Tyr-kinase 43 Mouse fibroblasts B Crow et al. [1990]

0>No effect; permeability > as assessed by dye coupling; gj>assessed by measurement of electricalcoupling; 1>additional conductance state observed; 2>shift to lower conductance values of the frequencydistribution of Cx43 conductances; 3>small conductances favored; 4>frequency of 61 and 89 pS conduc-tance reduced; 5>frequency of 140–150 pS conductance reduced.

cAMP and in a reduction in electrical coupling and dye transfer [Piccolinoet al., 1984]. From patch-clamp studies it became evident that single-channelconductance was unaffected, but that the reduced overall conductance can beascribed to a decreased open probability [McMahon et al., 1989].

In addition to the effect of cAMP or PKA activation alone, different andmore complex actions have been observed if such a manipulation is carriedout at elevated intracellular calcium concentrations [DeMello, 1991]. In thepresence of 6 mmol/l Ca2+, injection of cAMP resulted in a biphasic change


in gap junction conductance with first an increase followed by a decline in gj

which was reversible on application of EGTA [De Mello, 1986a].Besides this, long-term effects of PKA activation and cAMP are known

and are described in the following chapter. It is possible that some of thecontradictory findings reported in the literature are due to species or tissuedifferences, or to differences in the intra- and extracellular calcium concentra-tions or to different time courses, i.e. if effects after a longer period are observed(let us assume longer than 15 min), besides direct influence on gap junctionconductance, an involvement in protein synthesis can also contribute to thetotal effect. In such cases single-channel measurements can be of great advan-tage.

Another important regulator of gap junction function is protein kinaseC (PKC), which is activated via diacylglycerol (DAG). DAG results frominositol lipid hydrolysis by phospholipase C (PLC) and is accompanied byinositol triphosphate release. Activation of PLC-induced inositol hydrolysis isan effect of membrane-receptor activation. Receptors being linked to the PLCsystem include, among others, the adrenergic a1 receptor, the histamine H1

receptor and the vasopressin V1 receptor. However, it should be kept in mindthat noradrenaline, histamine and vasopressin can also activate adenylatecyclase via b, H2 or V2 receptors, respectively. Thus, the effects seen with anyof these mediators and probably with many other mediators can be a compositeeffect. In addition, while inositol triphosphate mediates a transient elevationof intracellular calcium, PKC phosphorylates seryl and threonyl residues atspecific sites [Berridge, 1984]. Thus, receptor activation of PLC-linked receptorsnormally causes a double effect: an increase in intracellular calcium and PKC-dependent phosphorylations, both of which can affect gap junction channels.To study the effects of PKC alone, PKC can experimentally be stimulatedusing phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) incomparison to the inactive phorbol ester 4a-phorbol 12,13-didecanoate. How-ever, a number of isoforms of PKC exist in cardiac tissue including PKCa,PKCb, PKCe, PKCn and PKCc (rabbit heart) [Rouet-Benizeb et al., 1996].Interestingly, only PKCc was found to be located close to the intercalateddisks in this study. TPA treatment is assumed to result in a rapid translocationof PKCa and PKCe in cultured neonatal rat cardiac myocytes [Kwak andJongsma, 1996]. Thus, it might be speculated that not all isoforms contributeto the gap junction regulation and that differences in the subtypes of PKCresponsible for the effect may contribute to some of the differences observedbetween various preparations.

Very early in gap junction research an effect of PKC on cellular couplingwas observed with TPA on epidermal 3T3 cells. Following TPA administrationmetabolic coupling between these cells was inhibited [Murray and Fitzgerald,


1979]. A similar result was obtained using a DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol, to activate PKC in rat epidermal cells [Gainer and Murray, 1985].In addition, it was shown that lens cell protein and the liver 27-kD gap junctionprotein can be phosphorylated by PKC [Dermietzel et al., 1984; Takeda et al.,1987, 1989]. However, there are other reports indicating that Cx26 has noconsensus sequence for phosphorylation and is not phosphorylated in isolatedliver gap junctions incubated with ATP and the catalytic subunit of cAMP-dependent protein kinase, PKC, or Ca2+/calmodulin-dependent protein kinaseII [Saez et al., 1990; Traub et al., 1989; Zhang and Nicholson, 1989], whereasCx32 [Saez et al., 1990; Takeda et al., 1987, 1989] Cx43 [Crow et al., 1990;Laird et al., 1991; Musil et al., 1990a] and Cx45 [Laing et al., 1994; Traubet al., 1994] are phosphoproteins.

With regard to cardiac myocytes, increases as well as decreases in gj havebeen observed in pairs of neonatal cardiomyocytes after application of TPA[Munster and Weingart, 1993; Spray and Burt, 1990]. In addition, Kwak et al.[1995a] found that TPA increases electrical conductance assessed by a double-cell voltage-clamp method, but decreases permeability as assessed by dye coup-ling in neonatal rat cardiomyocyte gap junction channels. Thus, permeabilityfor small molecules and electrical conductance do not seem to be related to eachother under all conditions. In order to investigate this phenomenon in moredetail, Kwak et al. [1995a], in a very elegant study, used an expression system toestablish the effect of PKC, PKA and protein kinase G (PKG) on single-channelconductance and permeability. The human hepatoma cell line SKHep1, whichendogenously expressed low levels of Cx45 and which was not capable of trans-fering molecules as lucifer yellow from one cell to another under control condi-tions, was transfected with Cx43 or Cx26. The absence of dye transfer in cellsonly expressing Cx45 was not influenced by 8-Br-cAMP (PKA activation), TPA(PKC activation) or 8-Br-cGMP (PKG activation). PKC activation by TPA,however, reduced the frequency of 140- to 150-pS conductances in Cx26 trans-fectant and favored the smaller conductance state of Cx43 channels along witha decrease in the relative frequency of 61- and 89-pS events. This complicatedbehavior may eventually account for the diversity of results being reported inthe literature. In parental nontransfected SKHep1 cells which were coupled viaCx45, activation of PKC induced an additional 16 pS conductance state (to-gether with the 22- and 36-pS conductances observed before). Thus, Cx43 maybe regulated posttranscriptionally via PKC-dependent phosphorylation. On theother hand, the influence of PKC on Cx26 channels is expected to depend onanother mechanism, yet unknown, since Cx26 is not phosphorylated accordingto the findings of Traub et al. [1989] and Saez et al. [1990].

The cGMP-dependent PKG is also involved in the regulation of gapjunction channels. Activation of PKG by cGMP or cGMP analogues, such


as 8-Br-cGMP or b-phenyl-1,N2-ethanoguanosine-3�,5�-cyclic monophosphate(PET-cGMP), was reported to result in reduced dye coupling and gap junctionconductivity in cardiac myocytes coupled via Cx43 [Burt and Spray, 1988a;Kwak and Jongsma, 1996]. In a study on SKHep1 cells and transfectedSKHep1 cells Kwak et al. [1995a] found no effect of PKG on Cx26 and Cx45channels, but a reduction in dye coupling and a shift to lower conductancevalues of the frequency distribution of Cx43 conductances.

As many receptors involved in the regulation of cellular growth are linkedto tyrosine kinases, the role of tyrosine kinase in gap junctional coupling andtranscellular communication has also been investigated. Since the gene productof the cellular and viral src gene, a 60-kD protein, expresses tyrosine kinaseactivity, expression of the src gene has been used to investigate the role oftyrosine kinase. Activation of the src gene was shown to reduce dye transferaccompanied by tyrosine phosphorylation within about 30 min [Azarnia andLoewenstein, 1984]. In addition, the stimulation of receptor tyrosine kinasesvia epidermal growth factor and platelet-derived growth factor in cultures ofrat kidney cells and BalbC3T3 cells induced a decrease in dye transfer and inelectrical coupling within several minutes [Maldonado et al., 1988]. Crow etal. [1990] described phosphorylation of Cx43 at tyrosine residues in mousefibroblasts after transfection with Rous sarcoma virus. It has been shown inthe developing avian heart as well that tyrosine phosphorylation inhibits Cx43channels [Veenstra et al., 1992].

Injection of alkaline phosphatase into cell pairs of neonatal rat cardiomyo-cytes resulted in an enhanced frequency of single-channel events of higherconductance, i.e. of 71.5 pS [Kwak and Jongsma, 1996]. Similarly, Morenoet al. [1994b] observed two conductance states in SKHep1 cells transfectedwith human Cx43 of 60–70 and 90–100 pS. Depending on the phosphorylation,either the state of smaller or greater conductance was favored. Intracellularinjection of alkaline phosphatase preferably led to channel conductances ofabout 100 pS, whereas inhibition of phosphatases with okadaic acid gavepriority to 60 pS events. In accordance with these findings the protein kinaseinhibitor, staurosporine (i.e. preventing phosphorylation), induced a higheroccurrence of the 100 pS events. These findings indicate that phosphorylationgoes along with the increased occurrence of 60- to 70 pS events and dephosphory-lation with 100 pS events [Moreno et al., 1992, 1994b].

Several ions are involved in the regulation of gap junctional conductanceincluding Ca2+, Mg2+, H+ and Na+. Very early Ca2+-induced reduction ofjunctional permeability has been described in Chironomus salivary glands [Roseand Loewenstein, 1975] and heart [De Mello, 1975]. Using the calcium-sensitivefluorescent dye, aequorin, Rose and Loewenstein [1975, 1976] demonstratedparallel changes in pCa and uncoupling, concluding that the actual pCai is


responsible for uncoupling. However, according to Noma and Tsuboi [1987]intracellular calcium concentrations exceeding about 1 lmol/l, i.e. pCa lowerthan 6, are needed to affect coupling in guinea-pig hearts. The correspondingpK�Ca values were 6.6, 6.4 and 5.6 at pH 7.4, 7.0 and 6.5, respectively. Ateach of these pH values calcium induced uncoupling with a Hill coefficientremaining constant at about 3.4. Lowering the pH shifted the gj-Ca2+ relation-ship to the right, i.e. higher calcium concentrations were required for halfmaximal depression of gj . Noma and Tsuboi [1987] concluded from theseexperiments that Ca2+ and proton compete for negatively charged bindingsites at the ‘Ca2+-receptor’ site involved in the control of gj. They hypothesizedthat the negative charges necessary for calcium binding may be neutralizedby the protons and proposed a cooperative receptor model:

H2nR ACCB R ACCB CanR,2n H n Ca

with R>Ca receptor and n>number of Ca-binding sites per receptor, dedu-cing from this the normalized junctional conductance gj to follow the equation:

gj>1Ö(1/{1+(KCa /[Ca])n · (1+ ([H]/KH)2n)})

with KCa being the [Ca2+] required for half-maximal depression of gj and KH

the [H+] necessary to induce 50% protonation of the receptor. The apparenthalf-maximum Ca2+ concentration (KE

Ca) obtained experimentally is describedby the equation:

(KECa)n>{Kn

Ca · (1+([H] /KH)2n)}.

With the assumption of n>3 Noma and Tsuboi [1987] calculated KCa and KH

to be 3.16·10Ö7 and 1.12·10–7 mol/l, respectively. Since on the other hand thepH-gj relationship was not influenced by Ca2+, Noma and Tsuboi [1987]suggested two types of binding: one for divalent cations, and another for H+.

A moderate increase in intracellular calcium concentration obviously doesnot affect gj in adult heart cells [Rudisuli and Weingart, 1989, 1991], but higherchanges in [Ca2+]i reduce gj in guinea-pig and rat hearts [Maurer and Weingart,1987]. Maurer and Weingart [1987] concluded from their experiments that re-duction in gj occurs if the intracellular calcium concentration exceeds therange of 320–560 nmol/l, which is below the value proposed by Noma andTsuboi [1987]. Maurer and Weingart [1987] argued that the difference might bedue to different stability constants for the calcium buffer used to calculate thecytosolic Ca2+ concentration. It has been suggested that the binding site for Ca2+

and H+ is located on the cytoplasmic loop of Cx43 [Spray and Burt, 1990]. Whiteet al. (1990) showed that rises in [Ca2+]i did not affect gj if pH was maintainedat 7.0.


The 17-kD protein calmodulin acts as an intracellular Ca2+ receptortransducing the Ca2+ signal. A considerable number of the effects are mediatedby the activation of Ca2+-calmodulin-dependent protein kinases [Blackshearet al., 1988; Cheung, 1982]. Since the first report on a possible involvementof calmodulin in the regulation of gap junction intercellular communication[Peracchia et al., 1983] calmodulin-binding sites have been identified in avariety of junctional connexins such as Cx43, Cx32 and Cx38 [Girsch andPeracchia, 1992; Peracchia, 1988; Peracchia and Shen, 1993]. With regard tothe role of calmodulin it has been shown by Peracchia et al. [1983] thatcalmodulin inhibitors can prevent cellular uncoupling. Thus, it might be specu-lated that at least in part the calcium effects may be transduced by Ca2+

calmodulin.As described, the intracellular pH is an important regulator of gj . In-

tracellular acidification is known to decrease junctional electrical coupling incardiomyocytes and in Purkinje fibers [Burt, 1987; Reber and Weingart, 1982].gj was nearly constant in a pH range from 7.4 to 6.5 and decreased sharplywhen pH was reduced to 5.4 [Noma and Tsuboi, 1987]. This pH-gj relationshipwas principally not affected by intracellular pCa. They found a Hill coefficientof about 2.4, indicating the number of proton-binding sites per receptor anda half-maximal concentration of 6.1 (pKH). In neonatal rat heart cells Firekand Weingart [1995] found a similar pKH with 5.85. One H+-binding site couldbe identified as histidine-95 in cardiac Cx43 by Ek et al. [1994]. Hermans et al.[1996] investigated the effects of site-directed mutations in Cx43-transfectedSKHep1 cells by exchange of His-126 and His-142 and found an uncouplingeffect of acidification related to the position of histidines in the cytoplasmicloop rather than to the total number of histidines. They reported that a fallin pHi caused a reduction in open-channel probability but not in channelconductance. Using the NH3 /NH+

4 pH-clamp method in Cx43-transfectedSKHep1 cells, Cx43 channels close at pH 5.8. The single-channel conductance,however, was not altered (40.8 pS at pH 7.0). In contrast, Cx45 channels inthe same expression system closed at pH 6.3. In Cx45 channels the single-channel conductance (17.8 pS at pH 7.0) did not exhibit pH sensitivity. Thus,the Cx45 channel was concluded to be far more sensitive to changes in pH[Hermans et al., 1995].

Regarding the pH sensor, the carboxy tail length has been demonstratedas a determinant of pH sensitivity [Liu et al., 1993]. Further investigations[Morley et al., 1996] revealed a new model of intramolecular interactions inwhich the carboxy terminal serves as an independent domain that, undercertain conditions, can bind to another separate domain of the connexinprotein (e.g. a region including His-95) and close the channel, comparable tothe ball-and-chain model for potassium channels. In this receptor (His-95),


article (COOH-terminal) model, the receptor seems to be conserved amongthe connexins. An important argument for a more complex mechanism ofpH-gating is the delay between intracellular acidification and uncoupling(about 8 min). Delmar et al. (1995) hypothized an action of H+ in associationwith calcium on the previously phosphorylated connexin.

According to Noma and Tsuboi [1987] the KH value on closing the cardiacgap junction is estimated to be in the order of 7.94 ·10–7 mol/l, which is differentfrom that describing the effect of H+ on the gj -pCa relationship (1.12·10–7 mol/l;see above) suggesting different binding sites.

In crayfish septate axon, a more complex action of lowering pH hasbeen described [Peracchia, 1991a]: superfusion with Na-acetate led to a rapidincrease in junctional resistance (Rj) with a concomitant fall in pHi , but therecovery curve for pHi was slower than that for Rj . A concomitant increasein intracellular [Ca2+] was observed so that it was concluded that the pHi

effect on cellular uncoupling in crayfish septate axon is mediated by calcium.Thus, generalizations of the various mechanisms should be avoided and thespecific experimental model has to be taken into account.

From today’s point of view these acidification experiments may be contam-inated by the effects of the pHi -regulating pumps, for example the Na+/H+

exchanger and the Na+/HCO–3 symport as well as other systems, e.g. the Na+/

Ca2+ exchanger [Doering et al., 1996] and possibly the Na+/Ca2+-ATPase.Thus, Yang et al. [1996] described that Na+/HCO–

3 cotransport and Na+/H+

exchange contribute to the rate of cell-to-cell electrical uncoupling in ischemicmyocardium potentially related to an attenuated Na-dependent calcium load-ing. In addition, inhibition of proton extrusion with 1 mmol/l amiloride wasreported to enhance the effects of changes in pH on gj [Firek and Weingart,1995].

As a decrease in pHi an elevation in pCO2 can cause a dramatic decreasein coupling in amphibian embryos [Turin and Warner, 1978] and other tissues[Spray et al., 1985]. This has been used experimentally to uncouple prepara-tions. The CO2-induced effect is reversible. The decrease in coupling afterexposure to CO2 has been ascribed to the consecutive fall in pHi [Kolb andSomogyi, 1991]. An effect of increasing CO2 in the ventilation air of anesthe-tized dogs on the cardiac activation pattern has been described and wasattributed to gap junctional uncoupling [Vorperian et al., 1994]. However, thiscannot be transferred easily on other pathophysiological conditions, since asingle increase in pCO2 is seldom, and is often (in pathophysiological situa-tions for e.g. myocardial infarction) accompanied by other changes includ-ing depolarization, potassium efflux and many others, which will also affectthe sodium channel availability, which will reduce longitudinal propaga-tion velocity. That means that, under pathophysiological conditions, affec-


tion of the gap junctional channel will probably not be mono- but multi-causal.

Increasing Mg2+ has also been reported to cause a fall in junctionalconductance in pairs of adult guinea-pig cardiomyocytes [Noma and Tsuboi,1987]. This effect was established for a pMg range from 2 to 3 correspondingto 1 to 10 mmol/l at pH 7.4 in the absence of calcium. The obtained datacould be described by the equation:

gj>1Ö(1/{1+(KEMg /[Mg])n}),

with KEMg>3.16·10–3 mol/l (pKE

Mg>2.5) and n>3. A Hill coefficient of 3.0 wascalculated. Since the slope of the pCa-gj and the pMg-gj relationships weresimilar, it was suggested that both divalent cations bind to the same receptorsite. The uncoupling effect of magnesium has been observed in other systemsas well as, for example insect salivary gland [Oliveira-Castro and Loewenstein,1971]. The cardiac cytosolic free Mg2+ has been measured in the range of0.48 mmol/l [Murphy et al., 1989] which is below the concentrations in whichmagnesium induces gap junctional uncoupling.

Another ion known to be involved in the regulation of gap junctionconductance is Na+. Na+ withdrawal in adult rat cardiomyocytes inducedelectrical uncoupling as indicated by a decrease in gj /gj·max occurring within3 min after exposure to 0 mmol/l Na+ [Maurer and Weingart, 1987]. This hasbeen ascribed to the fact that lowering [Na+]0 was reported to result in bothan increase in intracellular calcium and a decline in intracellular sodiumconcentration, which have been interpreted as an impairment in the Na+/Ca2+-exchange mechanism, since calcium extrusion via this mechanism requires thetransport of sodium [Weingart and Maurer, 1987]. Besides this, De Mello[1976] described an increase in intracellular sodium concentration to causeuncoupling within 500 ms in Purkinje fibers as indicated by an increase ininput resistance. It is uncertain whether this was a direct effect of sodium ormay be secondary to a rise in intracellular calcium via the Na+/Ca2+-exchangemechanism.

As described in the paragraphs above, conclusions on a direct effect ofany of these ions should be drawn with caution, since a change in the concentra-tion of any of these may activate a regulatory mechanism to compensate forthe change, for example the Na+/H+ exchanger, the Na+/HCO–

3 symport orthe Na+/Ca2+-exchange mechanism.

In addition to ions, other small molecules have been described to play animportant physiological and pathophysiological role in the regulation of gapjunctional resistance. Thus, ATP acts as an important regulator. In 1979Wojtcak described that hypoxia in glucose free solution resulted in a rise inRj in cow ventricular trabeculae indicating that the intracellular ATP content


may participate in the regulation of gap junctional conduction. Lowering theintracellular ATP concentration down to 0.5 mmol/l from the approximatelyphysiological concentration of 5.0 mmol/l led to a rapid decline in gj [Sugiuraet al., 1990] in pairs of adult guinea-pig cardiomyocytes. The investigatorskept intracellular calcium and magnesium concentrations at levels less than10–9 and 0.3 ·10–3 mol/l, respectively. The decrease was reversible upon additionof ATP. Similar to the method used by Noma and Tsuboi [1987], they deter-mined the Hill coefficient for ATP with 2.6 and the half maximum cytosolicATP concentration in the order of 0.68 mmol/l, suggesting a total uncoupling(gj>0) if ATP is reduced below 0.1 mmol/l. The decrease in gj induced bylowering [ATP] to 0.5 mmol/l was not reversible by adding ADP (10 mmol/l)or 50 lmol/l cAMP or 1 lmol/l of the catalytic subunit of cAMP-dependentprotein kinase in these experiments. Thus, at Ca2+, Mg2+ and H+ concentra-tions considered to be approximately in the physiological range, [ATP] actsas a regulator of gj independent of cAMP-dependent phosphorylation. Theauthors suggested a direct effect of ATP via a specific ligand-receptor inter-action with the gap junctional proteins. Using metabolic inhibition by 2,4-dinitrophenol (or decrease to 0.1 mmol/l ATP) in adult guinea-pig cardiomy-ocytes Morley et al. [1992] also described an increase in gap junction resistance6 min after addition of 2,4-dinitrophenol. However, the increase in Rj was toosmall to impair cell-to-cell propagation in this experimental system.

With regard to other possible regulators of gj , arachidonic acid, whichcan be released from membrane phospholipids by activation of phospholipaseA2 secondary to activation of a variety of receptors, has been investigated.It became obvious from these experiments that in very high concentrations(50–100 lmol/l) arachidonic acid can evoke cellular uncoupling within severalminutes in rat lacrimal gland cells [Giaume et al., 1989]. Since inhibitors ofarachidonic acid metabolism did not prevent the arachidonic acid effect, itwas suggested that, at least under certain conditions, arachidonic acid (as afatty acid) may interact directly with the gap junction proteins or their lipidenvironment. It could be imagined that it is incorporated in the lipid bilayerand alters the geometry of the lipid surrounding of the channels as wassuggested for the effect of other lipophilic agents, e.g. heptanol, octanol andhalothane. In neonatal rat heart cells, arachidonic acid has also been observedto induce uncoupling [Schmilinsky-Fluri et al., 1990]. This was further investi-gated by Massey et al. [1992], who described a concentration-dependent effectof arachidonic acid on gj in neonatal rat heart cells in a physiologically morerelevant concentration range from 2 to 20 lmol/l. This uncoupling effect couldbe antagonized by inhibition of lipoxygenase with U70344A, but not withindomethacine suggesting that the arachidonic acid effect at that concentrationis mediated by a lipoxygenase metabolite, e.g. a leukotriene, but not by a


cyclooxygenase metabolite. The incorporation of arachidonic acid in thatstudy was 0.695 mol min–1. Thus, at least two effects of arachidonic acid haveto be considered: (1) an unspecific effect in high concentration probably dueto physicochemical interaction within the gap junction surrounding, and(2) lipoxygenase metabolites inhibiting the channels in a yet unknownmechanism.

Acetylcholine is involved in many aspects of the regulation of the cardio-vascular system. Thus, it may also play a role in the control of intercellularcommunication. Very early in gap junction research the effect of acetylcholineas an important transmitter on gap junction conductance has been investigated.First, Petersen and Ueda [1976] demonstrated an increase in junctional resis-tance in pancreatic acinar cells following the application of acetylcholine.Concomitantly, the release of amylase was stimulated. A minimum concentra-tion of 1 lmol/l acetycholine was required to evoke uncoupling. The nextquestion was, how is the acetylcholine effect mediated? Calcium has beenconsidered to contribute to the mechanism of action [Iwatsuki and Pertersen,1978], but does not seem to be the sole mediator as Neyton and Trautmann[1986] demonstrated an uncoupling effect in a double whole-cell techniquealthough calcium was strongly buffered using 20 mmol/l EGTA in the pipettesolution. PKC stimulation has been discussed to participate in the transductionof the acetylcholine effect [for a review see, Kolb and Somogyi, 1991]. In ratsubmandibular gland cells Kanno et al. [1993] demonstrated a reduction indye coupling from 97.2% (percentage of dye-coupled cells) to 75% and finally22.7% after application of 10–6 and 10–4 mol/l acetylcholine, respectively. Theeffect occurred within 10 min. A similar result was found in rat pancreaticacinar cells following the administration of 5 lmol/l acetylcholine, which re-sulted in cellular uncoupling (dye coupling method) and a 4- to 5-fold increasein amylase release [Chanson and Meda, 1993]. This effect was independentof cycloxygenase, calcium and PKC, but could be inhibited by 1 lmol/l ocadaicacid, an inhibitor of serine-threonine phosphatases, indicating the involvementof a phosphatase in the acetylcholine action.

In neonatal heart cells Takens-Kwak and Jongsma [1992] investigated theeffect of acetylcholine. They reported a reduction in the intercellular current(Ij) in response to 100 lmol/l of the parasympathomimetic drug, carbachol,which could be mimicked by 8-Br-cGMP. The effect only occurred in thewhole cell patch configuration, but not in the perforated patch configuration,suggesting that a cytosolic enzyme is necessary for the effect which is washedout by the pipette in the whole cell patch. Since they found the carbacholeffect to be antagonized by alkaline phosphatase Takens-Kwak and Jongsma[1992] concluded that a cytosolic phosphatase is involved in the action ofcarbachol and, thus, probably of acetylcholine, too.


Fig. 13. Synopsis of the physiological regulators of gap junction channels. §>PKCchanges the substate of conductance, *>not found by all investigators as differences betweenthe various isoforms of connexins or species variabilities.

Another group of transmitters involved in the control of the cardiovascularsystem by the autonomous nervous system includes the catecholamines, adren-aline and noradrenaline. In acinar submandibular gland cells of the rat the ad-ministration of 10–4 mol/l adrenaline elicits a reduction in dye coupling from 97to 75.3% dye-coupled cells [Kanno et al., 1993]. This could not be mimicked withisoprenaline, but was inhibited with phenoxybenzamine. Thus, the uncouplingeffect of adrenaline in this preparation is mediated by stimulation of the a-adre-noceptor, whereas a stimulation of the b-adrenoceptor has no effect.

In contrast, stimulation of the b-adrenoceptor in the heart increases inter-cellular coupling [Veenstra, 1991b]. However, one has to be cautious withgeneralizations because, following adrenergic stimulation in the intact heart,intracellular calcium and heart rate will also be enhanced, so that a complexeffect will occur which is difficult to assess experimentally. Perhaps epicardialmapping experiments measuring the anisotropic ratio, i.e. the ratio betweenlongitudinal and transverse conduction velocity with regard to the fiber axis,will give insight into the global effect of such a manipulation. De Mello [1986b]reported that adrenaline increased the spread of electrotonic potentials during


diastolic depolarization in canine Purkinje fibers probably due to a rise inintracellular [cAMP].

Very recently an effect of the fibroblast growth factor-2 (FGF-2; a majormember of the heparin-binding family of growth factors) on Cx43 in cardiacmyocytes has been described [Doble et al., 1996] possibly involving PKC orMAP kinase and tyrosine phosphorylation. The authors showed that incuba-tion with 10 ng/ml FGF-2 for 30 min induces Cx43 phosphorylation on serineresidues, with a concomitant loss in intercellular dye coupling and maskingof Cx43 epitopes located in residues 261–270. In a previous study it was alreadyshown that basic FGF exists in close association with cardiac gap junctions,and it has been suggested that it, thus, may play a role in gap junctionalintercellular communication [Kardami et al., 1991]. FGF-2 can be released,for example, from cardiomyocytes during contraction and after stimulationwith catecholamines. The factor is upregulated in response to myocardialdamage. In contrast to these findings, FGF-2 induced an increase in Cx43accumulation and, as a result, an enhancement of coupling between cardiacfibroblasts and capillary endothelial cells, respectively [Doble and Kardami,1995; Pepper and Meda, 1992].

The mechanisms described so far are synoptically summarized in figure 13.An important point to mention is that, as already said, the affection of thegap junction conductance is not mono- but multicausal under the most physio-logical and pathophysiological conditions due to the interactions between theintracellular mediators. Thus, most processes will affect intracellular calciumand, on the other hand, a change in intracellular calcium will activate a varietyof intracellular mechanisms and affect the activity of many calcium-dependentenzymes.

4.2 Functions in Heart and Vasculature

Whatare the functionsof intercellularcommunicationchannels in the intactorgan? In spite of many details regarding single-channel conductance and theoverall conductivity or permeability of gap junctions, their role in the matureand developing heart is currently under intensive investigation and we are prob-ably only at the beginning of an understanding of the role of intercellular com-munication. Experimentally, it is not possible to measure the gap junctioncurrent in an intact heart. However, modern experimental setups will make itpossible to get a deeper understanding for the role of these channels in intacttissue. Such setups include mapping experiments using voltage-sensitive dyeslike di8-ANEPPS and epicardial potential mapping. With these techniques it ispossible to visualize the spread of activation and to measure its velocity.


An essential role for the normal cardiac development has recently beenshown [Reaume et al., 1995] in mouse lacking Cx43 (see chapter 6). Intercellularcoupling obviously is a prerequisite for the correct development.

Furthermore, synchronization of contraction is facilitated by gap junc-tional communication as well as synchronization of electrical activation. Theelectrical coupling between cardiomyocytes mitigates differences in the mem-brane potential between these cells, for example in the course of an actionpotential if both cells repolarize at different timepoints. This results in smallerdifferences in the repolarization times thereby causing a reduction in thedispersion of the action potential duration. Since increased dispersion is knownto make the heart more prone to reentrant arrhythmia, sufficient gap junctionalcommunication can be considered as an endogenous arrhythmia-preventingmechanism. For a detailed discussion of the role of gap junctional communica-tion in the biophysics of cardiac activation as related to anisotropy, nonuni-formity and stochastic phenomena, see chapter 1; for a discussion of their rolein arrhythmia, see chapter 6, and for a possible pharmacological intervention atthe gap junctions for suppression of arrhythmia, refer to chapter 7.

As already pointed out (see chapter 3), in some specialized areas of theheart, e.g. sinus node, there are special interdigitating patterns of gap junctions,providing some form of isolation from the hyperpolarizing influence of othercells, for example the surrounding atrium. Interestingly, these gap junctionsare made from Cx40, which is more sensitive to the transjunctional voltagethan Cx43 channels, thereby providing better isolation if higher differences inthe membrane potential occur. However, such a physiological function can beimagined but has not been shown directly, yet.

The communication between cells via gap junctions can also provideexchange of small molecules and has been shown to protect cells againstoxidative stress by exchange of glutathion [Nakamura et al., 1995]. Exchangeof small molecules may have more functions when considering signallingmolecules such as cAMP or Ca2+. However, there are no experiments availableat present on the role of gap junctions in intact organs.

Apart from providing communication, gap junctions can, on the contrary,have the function to isolate cells from their surrounding. Such isolation byclosure of gap junctions in certain pathophysiologic conditions occurs, forexample, in the course of hypoxia [Wojtcak, 1979] and loss of ATP or duringmyocardial ischemia. This may have the advantage that these cells no longercommunicate and participate in myocardial contraction, thereby saving energy,although this has not yet been demonstrated.

In the vasculature release of local mediators such as endothelin, prosta-cyclin and nitric oxide can only affect the local vasotone (at the site of release)or regulate downstream constriction or dilation. Besides this, upstream regula-


tion has been observed, but not well understood. Often this was ascribed tothe influence of the perivascular nerves. However, since Beny and Connat[1992] showed that smooth muscle cells of the media of pig coronary arteriesare dye coupled via single hydrophilic channels, upstream regulation of vasoac-tivity and transmural regulation (from the luminal side to the periphery andin the opposite direction from the perivascular nerves to the lumen) hasbeen also considered to be influenced or provided by gap junctional coupling(fig. 12).

4.3 Electrophysiology and Voltage-Dependent Gating

In general, with regard to the current-voltage relationship there are twotypes of gap junction channels to distinguish: (a) channels with rectifyingbehavior, and (b) nonrectifying channels. In addition, the gap junction channelscan be distinguished by their single-channel conductance. The single-channelconductance of a given gap junction channel made of one connexin isoform,however, may exhibit different substates of single-channel conductance. Ingeneral, the following states can be distinguished: open states with (a) mainopen state, (b) several substates, (c) residual state, and (d) the closed state.Furthermore, it is important to differentiate between channels exhibiting sensi-tivity to transjunctional voltage and channels being more or less insensitive.For investigation of the current-voltage relationship there are principally threepossibilities: (a) investigation of freshly dissociated cells with the advantagethat these cells are isolated from an intact tissue and should resemble theproperties of these postmitotic cells rather well, and with the disadvantagethat in the course of some hours many of the channels are internalized ordissociate so that the investigator observes a so-called ‘run-down’ with regardto the coupling in such cell pairs; (b) investigation of cultured cells, e.g.embryonic chick heart cells or neonatal rat cardiomyocytes or others, and (c)investigation of gap junctional channels in transfected cells, e.g. SkHep1 cells,tumor cell lines or xenopus oocytes. In such elegant transfection systems aproblem may occur if regulatory processes are to be investigated which involvepathways not present in the transfected cell.

To investigate the current-voltage relationship in any of the given systems,the standard method is the double-cell voltage-clamp technique [Spray etal., 1981, 1985; Weingart, 1986] carried out as a double whole-cell patch asdescribed by Giaume [1991]. The principle is that a transjunctional voltagedifference (by clamping the two cells to different potentials) is applied for ashort time to a pair of coupled cells and the current necessary for maintenanceof the voltage difference is measured. In order to achieve such an experimental


Fig. 14. Experimental setup for double whole-cell patch measurement of the gap junctioncurrent.

setup two voltage-clamp amplifiers are connected via a patch-clamp pipetteto either cell (fig. 14). While in one cell the membrane potential is kept forexample at –40 mV (in order to inactivate the sodium current), the membranepotential of the other cell is set for example to –30 mV, thereby applying atranscellular voltage of 10 mV. In this way transcellular voltages rangingfrom –50 to +50 mV or from –100 to +100 mV are applied and symmetryis controlled by alteration of the cell being kept at –40 mV. Since currentcan flow across the cell membrane and across the junction between bothcells, the current in cell 1 I1 can be described under these conditions by theequation:

I1>(V1 /rm1)+[(V1ÖV2)/rj]

and accordingly the current in cell 2 by

I2>(V2 /rm2)+[(V2ÖV1)/rj],

with V1 and V2 being the voltage relative to the holding potential VH (e.g.–40 mV in order to inactivate the sodium current) and rm1 and rm2 the membraneresistance in cell 1 and 2, respectively. If cell 2 is kept at VH , currents I1 andI2 can be described as:

I1>(V1 /rm1)+(V1 /rj)

I2>V1 /rj


so that I2 is a direct measure of the current flowing across the junctionalmembrane. Problems may arise from the series resistance of the pipettes and,in some preparations, eventually from the cytosolic resistance or from theratio between these different resistances (for more details see chapter 8). Thecurrent measured is then plotted against the transcellular voltage Vj . Linearregression reveals the total intercellular resistance. gj follows the equation:

gj>Ncj P0 ,

with N>number of channels capable of opening and closing, P0 being the openprobability and cj the single-channel conductance. If, under the experimentalconditions described above, cells are progressively uncoupled by applicationof, e.g., heptanol or halothane, it is possible with some types of amplifiersto observe single-channel openings shortly before total uncoupling occurs.Alternatively gap junction channels can be reconstituted in lipid bilayers forobservation of single-channel conductances. More details and protocols aregiven in chapter 8.

Looking at the current measured using such a protocol with a pulseduration of about 2 s, one can distinguish two components of the junctionalcurrent: (a) the instantaneous component, and (b) the steady-state component.Plotting the instantaneous current Ij versus Vj may reveal a linear relation asshown by Veenstra et al. [1993], which means that the instantaneous gapjunctional conductance is constant, i.e. gj>Ij /Vj>constant. A linear currentvoltage relation under such conditions means that the junctional channelbehaves like an ohmic resistor with a constant resistance which is insensitiveto the transjunctional voltage. However, not in all cases an ohmic behaviorwill be seen. If with increasing transcellular voltage the current does not followa linear relation, some kind of rectification is present. Rectification means thatthe channel resistance increases or decreases with increasing or decreasingtransjunctional voltage, i.e. the channel favors a current in one direction orat a special range of transcellular voltage. In other words it behaves comparableto some kinds of diodes.

Investigation of the steady-state current and the steady-state conductanceis normally carried out by fitting the normalized gss /Vj relationships with thetwo state Boltzmann distribution which follows the function:

gss /ginst>{(gmaxÖgmin)/(1+exp[A(VjÖV0)])}+gmin

according to Spray et al. [1981] and Veenstra et al. [1993] with gmax being themaximum conductance (>1, normalized to instantaneous gj) and gmin theminimum conductance. V0 is the half-inactivation voltage where gj is betweengmin and gmax and A>zq/kT (z>number of equivalent electron charges, q>voltage sensor, k>Boltzmann constant, T>temperature).


Fig. 15. Typical example of a measurement of junctional conductance. For details seetext. (Freshly isolated adult-guinea pig cardiomyocytes, holding potential –40 mV, seriesresistance was overcome by using switch-clamp amplifiers (SEC05).) For pipette solution,etc., see chapter 8.

a

b

Using the technique of double whole-cell patch reveals data as shownin figure 15. The holding potential of both cells is –40 mV. One cell is thenclamped to –50 mV while the other cell is kept at the holding potentialthereby applying a transcellular voltage of 10 mV. The other tracks showthe currents necessary to maintain these voltages. Transcellular voltages


ranging from –50 to +50 mV were applied. From these experiments the I/Vrelationship in figure 15b could be constructed. A critical problem with suchmeasurements is the compensation of the series resistance since in the case ofthe junctional resistance being in the order of the series resistance of one ofthe patch pipettes, distortion of the measurement of the intercellular currentwill result.

When considering the question whether gap junctional channels are regu-lated by transcellular voltage or not, the reader may be confused by the variousfindings of different groups in various preparations. However, as a generalrule considering cardiac gap junction channels coupled by Cx43 channels, incell pairs of adult cardiac cells, which are coupled by large numbers of gapjunction channels, the instantaneous and the steady-state current-voltage rela-tions have been demonstrated to be linear, i.e. gj is independent of transjunc-tional voltage and rectification is not observed [Noma and Tsuboi, 1987;Reverdin and Weingart, 1988; Weingart, 1986; White et al., 1985]. In contrast,in embryonic cells (or with some limitations in neonatal cells) which communi-cate by only a few gap junction channels, linear current-voltage relationshipsfor instantaneous and steady-state gj are observed in a discrete transcellularvoltage range: ×50 mV (neonatal rat cardiomyocytes) [Rook et al., 1988];×30 mV (embryonic chick heart cells) [Veenstra, 1990], and ×60 mV (neo-natal hamster cardiomyocytes) [Veenstra, 1990]. Outside this range rectificationis observed and the slope of gss declines progressively to values near zero [Rooket al., 1988; Veenstra, 1990, 1991a], whereas the instantaneous gj remainsconstant. This voltage-dependent behavior of gss can be fitted with the two-stateBoltzmann equation described above. What is the basis of this voltage-sensitivegating? According to Rook et al. [1988] it is not the result of a change in thesingle-channel conductance cj but in the ratio topen /tclosed , which is decreasedso that at a given time point more channels are in the closed state.

Spray et al. [1985] found gj to be unaffected by the transjunctional potentialgradient and by the membrane potential in dispersed and reaggregated ratventricle cells. Similarly, Kameyama [1983] reported that Rj is independent ofthe transjunctional potential gradient. While the instantaneous gj is insensitiveto the transjunctional voltage, in most cases gss can exhibit voltage dependencein some preparations [Veenstra et al., 1993] (fig. 16). Voltage dependence inneonatal cardiac myocytes can especially be observed with large transjunc-tional voltages in the order of 80 mV or more.

Rectifying behavior was observed in crayfish axons with depolarizationat the presynaptic side increasing the junctional conductance [Furshpan andPotter, 1959; Giaume et al., 1987] and in fish [Auerbach and Bennett, 1969].It has been hypothesised by Bennett et al. [1991] that this rectifying behaviormay arise from a heterotypic composition of the channel.


Fig. 16. Voltage-dependent gating in pairs of rat Cx43 transfected RIN cells (a) andpairs of mouse Cx40 transfected HeLa cells (b) [Banach and Weingart, 1996; Bukauskaset al., 1995].

a

b

Do all connexins exhibit the same sensitivity to the transjunctional volt-age? Trying to answer this question, Nicholson et al. [1993] have shown in anxenopus oocyte expression system that gss of gap junction channels constitutedof Cx37 are more voltage-sensitive than those made from Cx40. In comparisonto these, Cx32 was more insensitive and Cx26 channels exhibited the minimumsensitivity. In addition, Veenstra et al. [1993] showed that the voltage sensitivityof gss of embryonic chick heart cells decreased with the age of embryos. InCx40-transfected neuroblastoma cells (N2A) the Boltzmann half-inactivationvoltage was determined with –54 and +47 mV at negative or positive Vj,respectively, indicating a sensitivity of gss of the Cx40 channel to transjunctional


Table 3. Boltzmann equation parameters for various connexins

Connexin Gmin V0 [mV] Slope factor References

37 0.27 ×28 0.08 Reed et al. [1993]

40 0.33/0.28 –54/+47 0.13/0.11 Beblo et al. [1995]0.32 ×35 0.225 Ebihara [1995]

43 0.37a ×60 0.106 Moreno et al. [1995]

45 0.072 ×13.4 0.115 Moreno et al. [1995]0.17/0.16 –16/+22 0.126/0.115 Ebihara [1995]

If two values are given, the data on the left are mean values at negative Vj and on theright mean values at positive Vj . It becomes evident that Cx43 exhibits the lowest sensitivityto Vj since the half inactivation voltage V0 is highest, whereas according to these data Cx45and Cx37 exhibit the strongest Vj dependence.

a Gmin /Gmax .

voltage [Beblo et al., 1995]. The Cx43 channel has been shown to possess avoltage-sensitive component as well with Boltzmann half-inactivation voltagesat –69 and +61 mV [Wang et al., 1992] (table 3).

In a recent study on mouse Cx40 transfected HeLa cells, Bukauskas et al.[1995] determined V0 –45/49 mV and gmin 0.24/0.26. Valiunas et al. [1997]investigated the dependence on transjunctional voltage in neonatal ratcardiomyocytes, which are normally coupled via Cx43, and found, depend-ing on the pipette solution used, V0 –51/51 mV, gmin 0.28/0.25 and z 3.1/2.9(KCl solution), or V0 –59/59 mV, gmin 0.15/0.16 and z 2.0/2.3 (TEA-aspartatesolution). In another detailed study Banach and Weingart [1996] observedasymmetrical-gating properties in rat Cx43-transfected RIN cells with V0

–73.7/65.1 mV and gmin 0.34/0.29, if an asymmetric protocol was used (i.e. onecell is kept at the holding potential and the other is stepped to different voltagesthereby applying a transjunctional voltage), whereas if a symmetrical protocolwas used (both cells are clamped to the holding potential and then a certainvoltage clamp step is applied but of opposite polarity in both cells), the authorsobserved symmetrical gating with V0 –60.5/59.5 mV, gmin 0.27/0.29.

The problems arising from the various findings regarding the voltagesensitivity of gss initiated a very elegant study by Jongsma et al. [1993] answeringthe question ‘Are cardiac gap junction channels voltage sensitive?’. In a com-puter simulation they modelled two cardiomyocytes interconnected via a gapjunction and varied the number of gap junction channels within this intercon-


nection. The open probability of a single channel was assumed to follow theequation:

p0>1/{1+exp([Aa+Ab][dVjÖV0])},

with a>k exp(ÖAa[dVjÖV0]) and b>k exp(ÖAb[dVjÖV0]) and Aa

(>0.041 mV–1) and Ab (>0.021 mV–1) being the voltage sensitivities of a and b,k (>2.5 s–1), the rate at which a equals b and V0 (>74 mV), the transjunctionalvoltage at whichaequalsb (the data were obtained from single-channel measure-ments carried out on neonatal rat heart cells by Rook et al. [1988]). They founda relationship between the pipette series resistance and the appearance of voltageinsensitivity of gss . It was shown that gap junctions with intermediate numbersof channels (130 or more channels were assumed)appear to be voltage insensitiveif pipettes with 60 MX are used and gap junctions with 300 or more channels if20 MX pipettes are used. In real measurements the circuit is even more compli-cated by the fact that, part of the junctional current is shunted to ground throughthe membrane resistance. The higher the series resistance the more difficult it isto detect sensitivity. In addition, they demonstrated that, in cells well coupledby large numbers of gap junction channels (as in real experiments often in adultcells), voltage sensitivity cannot be detected or is only very moderately present,whereas in cells weakly coupled by only a few channels sensitivity to transjunc-tional voltage can be observed. Thus, Jongsma et al. [1993] concluded that the‘cardiac gap junctions are moderately voltage sensitive’ and that the decreasein voltage sensitivity in the course of embryonic development as described byVeenstra [1991b] does not reflect a change in the regulation of junctional resis-tance but rather an increase in mean gap junction size. Finally, they state that,in real experiments on well-coupled heart cell pairs, this voltage sensitivity of gss

cannot be observed mainly because of the presence of gap junction channelaccess resistance and pipette series resistance.

Much has been speculated about the possible role of a sensitivity fortransjunctional voltage in the heart. It can be assumed that such a behaviorwould protect a cell from the hyperpolarizing or depolarizing influence ofother cells provided the transjunctional voltage is high enough, i.e. exceedingfor example 50 mV in rat heart cells (see above). This would be necessary forsinusnodal cells which are in close vicinity to the atrium which is relativelyhyperpolarized with regard to the sinus node. Similarly, AV node cells adjacentto non-nodal tissue may be subject to such an influence. In these cells potentialgradients high enough to disturb for example the pacemaker function mayarise and, thus, such a disturbing influence may be prevented by transjunctionalvoltage-sensitive gating of the gap junctions. This fits with the general findingthat Cx40 and Cx45 are more sensitive to transjunctional voltage than Cx43.However, there is at present no clear evidence for these hypotheses.


What is the real range of gap junction resistance measured in varioussystems? Kameyama [1983] reported in reaggregated cell pairs of adult guinea-pig hearts Rj in the order of 1.4–2.1 MX (corresponding to 476–714 nS) andNoma and Tsuboi [1987] 0.25–11 MX (90–3.900 nS) in adult guinea-pig cardio-myocytes with a peak in the distribution around 1,000 nS (i.e. 1 MX). Weingart[1986] calculated the gap junction conductance in adult cardiomyocytes with204 nS (4.9 MX). In neonatal or embryonic heart cell pairs lower conductanceswere reported: 0–30 nS (?33 MX) in embryonic chick heart cells from 7-day-old embryos [Veenstra, 1990] and 0.05–35 nS (?28 MX) in neonatal rat heartcells [Burt and Spray, 1988a].

In cable preparations of various species resistance has been reported inthe order of 200–600 Xcm: 523 Xcm in bullfrog trabeculae [Haas et al., 1983];200–250 Xcm in guinea-pig trabecular muscle [Daut, 1982]; 350–530 Xcm inrabbit Purkinje fibers [Colatsky and Tsien, 1979] and 588 Xcm in frog ventricu-lar trabeculae [Chapman and Fry, 1978].

An interesting phenomenon is that some of the voltage-insensitive gapjunctional channels are gated, i.e. the channels open and close rather thanremain open all the time. The mechanisms underlying this gating behavior ofvoltage-insensitive channels found in avian and mammalian hearts and inseptate axons of earthworms are still unknown [Brink, 1991].

Since as pointed out above the overall gap junction conductance doesnot only depend on the number and the open probability of the channels butalso on the single-channel conductance, single-channel conductance of variousconnexins, including Cx37, Cx40, Cx43, Cx45, will be discussed.

From the channel geometry with a pore sink diameter of 1.5 nm, a poremouth diameter of 2.3 nm and a pore length of 15 nm (values according toMakowski [1985] and Zampighi [1987]), Rudisuli and Weingart [1989] pre-dicted the single-channel conductance. According to their considerations andto Hille [1992], RChannel is given by the equation:

RChannel>RPore+RAccess

with

RPore>q(1p(d/2)2)

and

RAccess>2q/pd.

Assuming the inner pore to be filled with a 130-mmol/l salt solution q equals100 Xcm and RChannel equals 10–20 GX. The single-channel conductance waspredicted with 50 to 100 pS. However, it has been suggested and shown byvarious investigators [Loewenstein et al., 1978; Neyton and Trautmann, 1985;


Rook et al., 1988] that the regulation of gap junction conductance is not theall or nothing and not quantal, but graduate so that different single-channelconductances or substates have been postulated [Page, 1991]. This is probablyreflected by various substates of single-channel conductance found in variousgap junctional channels which will be discussed as well.

The channel found most abundantly in cardiac tissue is the Cx43 channel.In guinea-pig hearts the single-channel conductance of Cx43 channels hasbeen measured with 37 pS and it was characterized as insensitive to the non-junctional membrane potential [Rudisuli and Weingart, 1989]. In contrast inthe neonatal rat heart Burt and Spray [1988a] found a single-channel conduc-tance of about 60 pS. The voltage-insensitive component of gj of Cx43 channelshas been ascribed to a voltage-insensitive substate by Moreno et al. [1994a]who observed a graded response in Cx43 transfected hepatoma cells. In anotherstudy [Moreno et al., 1994b] these authors defined two substates of this channel,i.e. 60–70 pS and a higher conductance state with 90–100 pS.

Kwak et al. [1995b] also characterized two substates in neonatal rat cardio-myocytes with 20 and 40–45 pS. However, when taking the regulation byprotein kinases into account Takens-Kwak and Jongsma [1992] discriminatedeven three substates of single-channel conductance in neonatal rat heart cells:21, 40–45 and 70 pS. Similarly, in Cx43-transfected SKHep1 cells they foundthree different substates: 30.5×9.1, 61.2×9.8 and 89.1×12 pS [Kwak et al.,1995a]. From these different findings one might conclude that at least threedifferent substates of single-channel conductance of Cx43 channels can bedistinguished. Depending on phosphorylation or dephosphorylation by vari-ous protein kinases the different substates seem to be favored (see chapter4.1). As investigated by Valiunas et al. [1997] gap junction channels of neonatalrat heart cells formed by Cx43 possess several conductance states: a mainstate, several substates, and a residual state as well as a closed state. Dependingon the pipette solution cj (main state) was determined 96 pS (KCl), 61 pS(Cs-aspartate) and 19 pS (TEA-aspartate) and cj (residual state) was deter-mined 23, 12 and 3 pS, respectively, revealing cj (main state)-cj (residual state),ratios of 4.2, 5.1 and 6.3, indicating that the residual state restricts ion move-ment more efficiently than the main state. Transitions between the severalopen states (main, substates and residual state) were fast (=2 ms) in contrastto the transitions between open states and closed state (15–65 ms). The differ-ences in the results of various investigators may perhaps be due to differentexperimental models and conditions. An example of single-channel recordingsis given in figure 17.

The next channel to be dealt with is the Cx40 channel. The uniqueconductance and gating of gap junction channels formed by Cx40 has beeninvestigated in Cx40-transfected mouse neuroblastoma cells (N2A cells). In


Fig. 17. Single-channel conductance of neonatal rat heart cells. Note the residual con-ductance [Valiunas et al., 1997]. The Vj applied was 50 mV.

the presence of potassium glutamate (120 mmol/l) Beblo et al. [1995] measuredthe slope conductance of single Cx40 channels in the order of 158 pS. Themacroscopic steady-state current exhibited dependence on transjunctionalvoltage with a Boltzmann half-inactivation voltage of ×50 mV. The authorsfound a residual voltage-insensitive normalized junctional conductance in theorder of 35% of the maximum and a gating charge valence of 3. In these Cx40channels substates have also been described [Beblo et al., 1995] equal to 21and 48% of the main open-state conductance (which was in the range of158 pS), although these substates were reported to occur only occasionally.Bukauskas et al. [1995] investigated mouse Cx40-transfected HeLa cells andmeasured single-channel conductance. They observed three open states: mainstate (198 pS); several substates, and an residual state (36 pS) besides a closedstate. Transition between the open states were fast (1–2 ms) in contrast to thetransitions between open and closed states (15–45 ms).

The third channel found in heart muscle is the Cx45 channel. This wasreported to exhibit an overall conductance of 3.1×0.4 nS in cell pairs [Kwaket al., 1995a] with a mean single-channel conductance of 36.5×6.5 pS or1.3 nS in human Cx45-transfected SKHep1 cells [Moreno et al., 1995] and cj

of 32×8 pS. At higher transjunctional voltages an additional conductancestate with 22.5×4.3 pS was observed. This channel strongly depends on trans-junctional voltage [Moreno et al., 1995] comparable to Cx38 channels. How-ever, single-channel conductance is not a function of transcellular voltage.

Besides Cx40, Cx43 and Cx45, Cx37 channels are expressed in the cardio-vascular tissue. These channels are frequently found in endothelium. Thesechannels were expressed in human Cx37-transfected neuroblastoma cells (N2Acells) and exhibited a pronounced voltage dependence and multiple conduct-ance states [Reed et al., 1993]. Several single-channel conductances were found:


219×22, 165×6, 123×13 and 53×1 pS at Vj>30–40 mV. At higher Vj thelarge conductance was no longer observed and cj of 94×4, 69×5 and39×10 pS were detected at Vj>80–90 mV.

Finally, Cx26 channels were also investigated in an expression system(SKHep1 cells) by Kwak et al. [1995a]. They observed a mean single-channelconductance of 140 to 150 pS with substates of 70 and 110 pS.

Gating of the channel can be modulated either by an alteration in thesingle-channel conductance or by a change in the open probability. The macro-scopic channel conductance is then either influenced by the single-channelconductance, by the mean open time of a channel or by the frequency ofchannel openings, this means by the number of channels in the open state atthe same time or at a given time interval.

Do the gap junctional channels exhibit some sort of selectivity? Do theyconduct cations and anions? Do they exhibit characteristics known from somany ion-selective transmembrane channels or are they distinct from these?As already said, the first important difference to other ionic channels is thatgap junction channels are permeable to small molecules up to a molecularweight of about 1,000 Daltons. Another difference is that they conduct bothanions and cations. In order to further elucidate these questions Brink [1991]investigated the selectivity of gap junctional channels in septate earthwormaxons using the double whole-cell patch-clamp technique. According to thisstudy this channel exhibits a single-channel conductance of about 100 pSand is sensitive to calcium and pH. There was no inhibition of the channelconductance with various blockers commonly used in electrophysiology: tetra-ethylammonium (TEA), 4-aminopyridine (4-AP), Zn2+, Co2+ and Ni2+. Therewas no selectivity for K+ over Cs+, but the 100-pS channel seemed to besomewhat selective for cations over anions with a chloride conductivity topotassium conductivity ratio of 0.53. This channel is also voltage-insensitive.Molecules not larger than 1 kD or not exceeding an ionic radius of 0.8 or1.0 nm can be transported through the channel [Brink, 1991; Spray et al.,1991].

The rat Cx40 channel exhibits a detectable chloride permeability of 0.29relative to potassium [Beblo et al., 1995]. This indicates some selectivity forcations over anions as well. These channels were also permeable to 2�,7�-dichlorofluorescein and to the more polar 6-carboxyfluorescein dye. Interes-tingly, the 2�,7�-dichlorofluorescein permeability did not increase with increas-ing junctional conductance in that study.

With regard to small molecules other than ions Tsien and Weingart [1976]reported that 3H-cAMP diffuses across gap junctions in calf and cow ventricle.Furthermore, Weingart [1974] has shown that 14C-TEA (molecular weight 130)diffuses transjunctionally in sheep ventricular muscle and he found the channel


diameter to be in the order of 10 A. A number of different dyes with molecularweights ranging up to 859 Daltons have been demonstrated to diffuse acrossthe gap junctions including procion yellow (MW 697) in sheep and calfPurkinje fibers [Imanaga, 1974], 6-carboxyfluorescein (MW 670; diffusioncoefficient 5.8 ·10–6 cm2/s), lucifer yellow (MW 457; diffusion coefficient3.0 ·10–6 cm2/s), lissamine rhodamine B200 (MW: 859; diffusion coefficient8.6 ·10–7 cm2/s), while Chicago blue (MW 1,000 Daltons) did not diffuse acrossthe channels [Imanaga, 1974, 1987; Imanaga et al., 1987]. From these datathe authors calculated an upper limit for the channel diameter of 1.2–1.3 nm.

In more physical terms the permeability Pj through a gap junction channelcan be described using the equation:

Pj>Vcell ki /Aj ,

with Aj being the area of gap junctional membrane, Vcell the cell volume andki the experimentally measured rate constant for transcellular diffusion.

Thus, the gap junction channel behaves like a gated pore exhibiting someselectivity for cations over anions, and acting as a diffusion barrier for mole-cules exceeding 1,000 Daltons. It does not show the high selectivity for any sortof ion as known from other ionic channels. With regard to voltage sensitivity, itbehaves like an ohmic resistor as far as the instantaneous Gj is concerned.The steady-state conductance Gss can exhibit a more or less stronger sensitivityto Vj depending on the connexin the channel consists of.


5...........................

Regulation of Gap Junction Expression,Synthesis and Assembly

We have considered the structure, diversity and the function of gap junc-tional channels. But, how are gap junctional channels formed, how are theydegraded? Or are they not subject to any turnover?

Little is known at present on the process of channel formation and assem-bly. And even less on the regulation of channel formation. A very interestingand important question is: how can two cells direct their hemichannels sothat they fit each other forming the intercellular pore? Research in this areais still at its beginning and many of the processes involved are not well under-stood. But nevertheless, in this chapter the present knowledge on the regulationof channel expression and turnover will be summarized.

Gap junctions and their channels are not static; as many other cell proteinsthey underlie a turnover. For example it has been shown in the human neonateand child that there is a progressive polarization of the gap junctions towardsthe positions of the mature intercalated disks reaching the adult pattern at anage of about 6 years [Peters, 1996]. Thus, the pattern of gap junction expressioncan change with time as has been shown in various diseases, for example in thecourse of chronic myocardial infarction and heart failure (see chapter 6). On theother hand there is a considerable change in the expression of gap junctionsduring development as shown, for example, in the developing avian embryonicheart [Veenstra, 1990]. The basis of such alternating patterns must be a turnoverprocess with assembly and degradation of gap junctional channels.

If cells come into contact the occurrence of cell coupling has been detectedusing dye transfer and electrical methods. This raises an important question: israpid de novo synthesis required for the formation of gap junctional channels?Answering this question Epstein et al. [1977] inhibited the protein synthesisby treatment with cycloheximide and subsequently brought cells into closecontact. They observed progressive cellular coupling indicating that for theformation of gap junction channels no rapid de novo synthesis is necessary.This implies that presynthesized channels must be stored within a cell whichthen can form the channels. An alternative hypothesis is that hemichannelsare incorporated into the membrane under normal conditions and that aftercoming into contact with another cell the hemichannels of both cells aretranslocated in order to form channels bridging the gap by ‘interlocking’ ofhemichannels.

63

It has been demonstrated that cell coupling is established within 3–30 minafter bringing disaggregated cells into contact. Interestingly, the resulting in-crease in cell-to-cell conductance proceeds in quantal steps [Loewenstein, 1981;Loewenstein et al., 1978] which may be interpreted by the progressive assemblyof channels increasing the conductance stepwise with the formation of everychannel. However, it is not certain whether this is the correct interpretationor whether this is an oversimplification. Loewenstein [1981] has elaboratedthis hypothesis to the ‘self-trap’ model: precursors of the gap junction channels,either as hemichannels or as polypeptide constituents of these hemichannels(so-called ‘protochannels’), are present in the plasma membrane of both cellsand can diffuse freely within the plane of the plasma membrane lipid bilayer.If cells come into contact, such hemichannels or their precursors can alsocome into close contact by chance. If they are close enough so that theirextracellular loops E1 and E2 can form noncovalent bonds (by van der Waalsforces) the extracellular domains of the protochannels interlock and form thecomplete gap junction channel.

If neonatal rat heart cells are manipulated into contact, Valiunas et al.[1997] observed new gap junction channel formation at a rate of 1.3 channels/min (the first opening occurred within 7–25 min after physical cell contact).They argued that this formation occurs by docking of preformed hemichannelsof adjacent cells.

How are gap junctions synthesized and incorporated into the plasmamembrane? Normal plasma membrane proteins are synthesized at the ribo-somes of the endoplasmatic reticulum (ER) and cotranslationally inserted intothe membrane. This is followed by posttranslational folding and eventualoligomerization. Thereafter, the molecules are transported through the Golgiapparatus and carried to their final position in the plasma membrane. Falket al. [1995] investigated this process for rat liver, dog pancreatic and babyhamster kidney gap junctions. They could identify nearly the same pathwayfor gap junction assembly as described above for other membrane proteins.However, the integration of the gap junctions into the ER membrane requiresan additional ‘assisting factor’, which is most likely a cytoplasmic chaperon-like protein. Chaperones are cytoplasmic proteins which are involved in proteinfolding and assembly. Together with the chaperonines they help to give thenewly synthesized proteins their final structure [for review see, Hartl, 1996].The binding of this putative assisting factor was suggested to occur at theNH2 terminus of the gap junction protein anchoring the NH2 terminus to thecytoplasmic site of the ER membrane. Using the metabolic inhibitor monensin,Puranam et al. [1993] found an intermediate form of Cx43 in the Golgi of ratcardiac myocytes. Cx43 entered and accumulated in the Golgi network ofmonensin-treated cells. Further investigation revealed that one accumulating

645 Regulation of Gap Junction Expression, Synthesis and Assembly

form was the phosphorylated state of the nascent 40-kD form of Cx43 sug-gesting an early phosphorylation of the Cx43 protein in the secretory pathway.

Furthermore, in vivo studies revealed that the further assembly of thehemichannels formed in the Golgi network depended on protein phosphory-lation and the presence of adhesion molecules [Musil and Goodenough, 1993].Using Cx43 in rat kidney cell cultures Musil and Goodenough [1995] foundthat the connexon formation occurs after transport through the cis, medialand trans Golgi cisternae, since the connexon assembly could be blocked bybrefeldin A, a specific blocker for assembly processes occurring before or atthese compartments. The authors concluded that the connexon assembly takesplace in the trans Golgi network in contrast to other integral membraneproteins.

Secondary to the formation of connexons as oligomeres from connexinsthe association of a connexon in the plasma membrane in one cell with aconnexon in an adjacent cell membrane to form the intercellular channel hasto be considered. Miner et al. [1995] could demonstrate a regulatory rolefor the cadherins in the gap junction assembly: calcium-dependent adhesionproteins (cadherins) have been shown to have a significant influence on gapjunction assembly, since in disaggregated cells the formation of intact intercellu-lar channels can be inhibited by Fab fragments of N-cadherin-specific antibod-ies. If cadherins are involved, a calcium dependence of the gap junctionassembly process should be detectable. In Novikoff hepatoma cells expressingCx43, Miner et al. [1995] investigated the sensitivity of the gap junction forma-tion on extracellular calcium by means of electron microscopy and a dye-transfer technique. It became obvious that the percentage of coupled cellsafter reaggregation was decreased with a reduction in extracellular calciumconcentration from 1.8 nmol/l to 40 nmol/l in a nonlinear fashion. Besidesthis, no change in phosphorylation was observable. Two conclusions fromthese findings were made by the authors: on the one hand one can suggest asimple approximation of the two plasma membranes as the prerequisite ofintercellular channel formation and on the other hand a signalling processbetween calcium, cadherins and gap junction proteins can be imagined. Thelatter is supported by the finding of a nonlinear relationship between calciumand gap junction formation. Fishman et al. [1991b] looked at the expressionof Cx43 in the developing heart and found accumulation of Cx43 mRNAduring embryonic and early neonatal stages accompanied by a temporallydelayed increase in the protein. With maturation of the heart these levelsdecline suggesting that increases in intercellular coupling characterizing car-diac development do not solely depend on modulation of Cx43 gene expressionbut also involve formation of functional gap junction channels within theintercalated disks. One might speculate from the above findings that such


processes might be regulated via cell adhesion molecule signalling. However,at present these possible interactions have not been investigated. Thinkingabout the signalling and the regulation of gap junction assembly one mightconsider the cytoskeleton to be involved but, at present, gap junctions havenot been reported to be attached to the cytoskeleton.

During the development of the human heart it has been found that thereis a close and increasing association between the gap junctions and the fasciaadherens junctions [Peters et al., 1994]. While in the neonate Cx43 exhibits apunctuate distribution over the entire surface of the cardiomyocytes, duringpostnatal development Cx43 gap junctions become progressively confined tothe transverse terminals of the cell, i.e. to the intercalated disks. Gap junctionsand adhering junctions are frequently not closely adjacent in the neonate butbecome so with growing age (investigation for the first 6 years of life).

It is presently still uncertain whether phosphorylation processes play arole in gap junction formation. However, a relation between phosphorylationof Cx43 and its insertion into the plasma membrane has been described [Musilet al., 1990b]. Furthermore, a correlation between the formation of functionalgap junctions and expression of a cell adhesion molecule (L-CAM) andE-cadherin was reported [Mege et al., 1988; Musil et al., 1990b]. Berthouldet al. [1993] showed that a reduction in extracellular calcium led to a loss ofintercellular contact associated with a decrease in gap junctional intercellularcommunication as seen from reduced dye coupling and decreased anti-Cx43immunofluorescence. Restoration of the extracellular calcium concentrationresulted in reapparation of Cx43 immunoreactivity indicating the crucial roleof calcium for the gap junction formation process. This was, however, unrelatedto changes in the phosphorylation of Cx43. Stimulation of PKC with thephorbol ester TPA over periods longer than 15 min decreased immunolabelingat appositional membranes and increased cytoplasmic labelling.

The extracellular loops E1 and E2 seem to determine the formation ofthe gap junctional channel. This was inferred from a study by Warner et al.[1995] using synthetic peptide analogues to extracellular loop segments inorder to disturb the establishment of cellular coupling in pairs of embryonicchick heart myoballs expressing Cx43 and Cx32. Peptides resembling conservedmotives from extracellular loops E1 and E2 delayed gap junction formationin micromolar concentrations. The motives QPG and SHVR in loop E1 andSRPTEK in loop E2 were critical for gap junction formation (one lettercode, see Appendix). Interestingly there was no synergism between the peptideanalogue to E1 and the analogue to E2, which were both about equi-effective.This means that it is critical if the formation is disturbed in only one loop.The processes involved in gap junction formation and assembly are summarisedin figure 18.


Fig. 18. Synthesis, posttranslational modification and assembly of gap junctions.

Structurally it has been found that large gap junctions are surrounded bysmall gap junctions (0.5 lm in diameter containing 12–100 connexons) whichare located in the plicate and interplicate region of the intercalated disk [Severs,1990]. It has been suggested that some of these gap junctions contain newlyformed connexons freshly inserted into the lipid bilayer. However, this hasnever been proved. Regarding the fate of gap junctions Mazet et al. [1985]suggested that gap junctions facing the extracellular surface of dissociatedmyocytes are progressively internalized and form cytoplasmic vesicles whichmigrate into the cell interior and are degraded by lysosomal enzymes. Theendocytotic internalization process was also confirmed by Severs et al. [1989],but over a period of 15–22 h neither degradation nor synthesis of new gapjunction was observed. They concluded degradation to be much slower thanpreviously assumed. However, this is probably relevant for freshly isolated ordisaggregated cells used in experimental research and may account for the


well-known ‘run-down’ of intercellular coupling in the course of such experi-ments, but may have less relevance for the situation in the intact tissue in vivo.

Degradation of gap junctions seems to involve removal of the gap junctionfrom the plasma membrane by internalization of the entire gap junction withinone of the adjacent cells [Larsen, 1983; Mazet et al., 1985]. The interdigitatingprocess is pinched off and a double-wall vesicle is formed, which is finallydegraded within a lysosome. Within a cell so-called ‘annular’ gap junctionshave been seen representing circular profiles which are cross-sections of aninterdigitation or vesicle. Although Cx43 can be degraded in both lysosomesand proteosomes Tadros et al. [1996] have recently shown evidence of a majorproteolytic degradation of Cx43 in lysosomes in the heart.

Chen et al. [1989] described so-called gap junction-associated vesicles(GJAVs) in mammalian atrial and ventricular muscle. These GJAVs werelocated in the extracellular space in close vicinity to the intercalated disks inthe interstitial space near gap junctions associated with plicate segments, withinsome t-tubular profiles and between the layers of the basal lamina coveringthe nonjunctional membrane close to the interplicate segment. Negativelystaining with La(NO3)3 revealed that these GJAVs contained laminar structureswhich have been identified as typical connexon arrays. Pairs of these GJAVsform typical junctional pentalaminar structures. It was suggested that GJAVsresemble reservoirs of Cx43 and connexons possibly involved in the formationand degradation of gap junctions. The authors suggested that they may repre-sent extracellular tracks for myocyte cell processes helping to meet or to retractfrom the neighboring cells. However, further studies on this subject are requiredto fully exclude artefacts from preparation methods.

What about the real turnover rate of gap junction proteins as the basisof changes in gap junction pattern in the course of cardiac disease? There areseveral reports on a considerably high and rapid turnover of connexins bothin vivo and in vitro.

Laird et al. [1991] determined the turnover and posttranslational modi-fication of Cx43 in neonatal cardiomyocytes. After labelling with 35S-Met,immunoprecipitation with anti-Cx43 antibodies followed by SDS-PAGE andfluorography revealed a phosphorylated and a non-phosphorylated form ofCx43. In pulse-chase experiments the half-life of Cx43 was determined with1–2 h, and, furthermore, the turnover rate of the phosphate groups was experi-mentally defined by the half-life of the protein, i.e. phosphate groups canremain with the protein throughout its whole life span. This means that, atleast in neonatal cardiomyocytes, there is a rather rapid turnover of Cx43.Valiunas et al. [1997] determined a formation rate of 1.3 channels/min afterbringing cells into contact. Similarly, a considerably rapid turnover has beenobserved for Cx43 in other cultured cells [Musil et al., 1990b]. In addition, a


half-life of 1.9 h for Cx43 was demonstrated in pulse-chase experiments incultured rat heart ventricular myocytes by Darrow et al. [1995]. In the samestudy the half-life for Cx45 was determined with 2.9 h, suggesting a rapidturnover for both connexin isoforms. With regard to phosphorylation of theconnexins these authors observed various phosphorylations of Cx43 on serineand threonine residues producing multiple forms of the protein but onlyphosphoserine in Cx45 which exhibited substantially less heterogeneity ofphosphorylation.

The role of connexin phosphorylation is uncertain at present. However,it is likely that phosphorylation, especially different phosphorylation (at vari-ous sites as in Cx43), may control various aspects of gap junction metabolismand function.

The findings of Darrow et al. [1996] suggest that there might be a precursorpool for Cx43 whereas Cx45 may be synthesized de novo (see below).

A short half-life of proteins is often associated with proline, glutamic acid,serine and threonine-rich regions (so-called PEST-rich regions) [Rechsteiner,1988]. Taking a close look at the amino acid sequence of Cx43 reveals thatthere is no classic PEST-rich region. However, residues 272–285 and 327–340on the C terminus may resemble PEST-like regions which may account forthe rapid turnover as suggested by Laird et al. [1991].

Regarding other connexins, the turnover of Cx32 and Cx26 in culturedliver cells has been determined to be in the order of only several hours [Traubet al., 1989] and thus to be similarly rapid. In former years it was believedthat this rapid turnover in hepatocytes is the fastest turnover of connexins,but in the mean time it is generally assumed that the connexins are probablyall subject to rapid turnover.

5.1 Regulation of Gap Junction Synthesis by Intracellular Mediators

The synthesis of gap junctions can also be regulated. An increase incAMP, for example, increases junctional conductance over several hours, whichcan be inhibited by blockers of the mRNA synthesis [Kessler et al., 1985] orprotein synthesis [Azarnia et al., 1981; Kessler et al., 1985; Traub et al., 1987]indicating an increased synthesis of the gap junction protein to be involvedin this kind of long-term regulation. Similarly, In’t Veld [1985] observed a risein gap junctional particles between rat pancreatic B cells following a risein intracellular cAMP. Interestingly, sequences corresponding to the cAMP-response elements are close to the transcription start site of Cx32, so that ithas been suggested that cAMP may enhance transcription of Cx32 [Milleret al., 1988]. Saez et al. [1989] reported that cAMP delayed the uncoupling


of gap junctions in rat hepatocytes. This was ascribed to a possible decreasein the removal of gap junction proteins from the plasma membrane, i.e. to aslowing of degradation. Recently, Darrow et al. [1996] showed an increasedexpression of Cx43 and Cx45 following dibutyryl-cAMP exposure of neonatalrat cardiomyocytes, which was accompanied by an increase in conductionvelocity assessed by optically mapped action potential propagation using volt-age-sensitive dyes. However, the molecular mechanisms of this enhanced ex-pression appeared to be different for both connexins. After 1 mmol/l db-cAMPthe authors observed an increase in Cx45 but not in Cx43 synthesis within a2-hour interval. In contrast, 24-hour exposure to db-cAMP resulted in anincrease in Cx43 mRNA but not Cx45 mRNA, normalized to the GAPDHtranscript. This increase was not attributable to synthesis of new protein factorsas was indicated by the insensitivity to cycloheximide treatment. It is uncertainwhether the increased Cx43 mRNA levels are due to enhanced transcriptionor to stabilization of the transcripts. The selectivity of the effect might indicatethe action of a specific promoter or enhancer sequence near the Cx43 gene.The lack of sensitivity to cycloheximide suggests a possible modification ofproteins already existing, for example, by a change in the phosphorylationstate as a signalling event in the cAMP cascade. From these results the authorsconcluded that Cx45 may be upregulated posttranscriptionally (no change inmRNA, but in protein synthesis). In conclusion, the increased total amountin Cx43 by immunoblotting and, parallel to it, the lack of change in the proteinsynthesis rate of Cx43 reported by the authors may possibly be indicative ofa precursor pool of Cx43.

In contrast to cAMP, a stimulation of PKC with phorbol esters (TPA)has been shown to play an important role in the downregulation of gapjunctional coupling [Yancey et al., 1982]. Since reestablishment of intercellularcoupling was not seen after wash out of TPA in the presence of the proteinsynthesis inhibitor, puromycin [Fitzgerald et al., 1983], the phorbol ester prob-ably induces the elimination of junctional channels under these conditions.Thus, it might be possible that PKC is involved in the regulation of channeldegradation.

Obviously, there is some kind of cross-talk between PKC and cAMP inthe modulation of intercellular coupling, since cAMP can inhibit the uncoup-ling effect of phorbol esters if cells are exposed to both agents from the startof the experiment [Kanno et al., 1984], but this protective effect can be abol-ished by the protein synthesis inhibitor cycloheximide [Enomoto et al., 1984]in Balb/c3T3 cells.

At least tyrosine kinases seem to be involved in the regulation of connexinexpression. As stated previously, tyrosine kinases are often linked to growthfactor receptors. A possible involvement of tyrosine kinases in the regulation


Fig. 19. The Cx43 promoter region. Partial analysis of the human Cx43 gene extendingfrom –360 to the site of fusion where De Leon et al. [1994] inserted the luciferase reportergene at position +143. The transcription start site is numbered –1 [redrawn from De Leonet al., 1994]. The TATA box and the putative AP-1-binding sequence are underlined.

of gap junctional coupling seems to be reasonable in the light of Loewenstein’s[1968] hypothesis on the role of gap junctional communication in cellularproliferation. It was reported by Pepper and Meda [1992] that basic FGFexposure of microvascular endothelial cells leads to increased expression ofCx43. In contrast to these results, Doble et al. [1996] found decreased metaboliccoupling in cardiac myocytes in response to FGF-2, the FGF which is believedto participate short- and long-term on cardiac responses to injury, within30 min. However, in these experiments FGF-2 did not affect Cx43 mRNA orprotein synthesis. With regard to the intracellular distribution of Cx43, FGF-2exposure decreased immunofluorescence-staining intensity at sites of intermy-ocyte contact and induced phosphorylation of Cx43 in serine and tyrosineresidues. In other cells (cardiac fibroblasts), however, the same authors demon-strated enhanced intercellular coupling by induction of Cx43 accumulation[Doble and Kardami, 1995].

5.2 Molecular Biology/Expression of Gap Junctions

At present, only little is known on the molecular genetics of the connexins.Fishman et al. [1990, 1991a–c] isolated the entire gene encoding Cx43 includingabout 5,000 base pairs of 5�-flanking sequence, a region which may determinethe transcriptional activity of the gene. Regarding the molecular biology ofthe gap junction channel, Cx43 has been investigated in detail and the promoter


region has been analyzed [De Leon et al., 1994] by construction of chimericluciferase reporter genes containing nested deletions from the human Cx43gene (–2,400 to –50 base pairs, position relative to the transcription initiation).The transcriptional activity of the chimeric genes was assayed in several celltypes. High levels of luciferase activity required at least 175 base pairs of 5�-flanking sequence, whereas constructs which included 2,400 base pairs of theupstream sequence increased activity twofold in vivo but failed to increaseactivity in vitro. It was concluded from the experiments with several chimericconstructs that the proximal promoter may also confer tissue specificity. Thesestudies begin to characterize the cis-acting elements of the Cx43 gene. Theseregulate the strength and specificity of transcription. The promoter includesa TATA box (TTTTAAAA) and a putative AP-1-binding site (TGAGTCA).The full sequence of the Cx43 promotor region is given in figure 19 accordingto De Leon et al. [1994].

Regulation of Cx40 expression has been suggested to be somewhat differ-ent from that of the other connexins: Darrow et al. [1995] suggested fromtheir turn-over experiments a translational regulatory mechanism for Cx40since they observed Cx40 mRNA, i.e. transcription took place, but they couldnot find the protein suggesting that either the protein is not translated or aftertranslation rapidly degraded.


6...........................

Gap Junctions in Cardiac Disease

In this chapter changes in the distribution of gap junctions within themyocardial tissue, alterations of the distribution of special isoforms in thecourse of heart disease are described. Thus, changes in gap junction patternfor Cx43 and for Cx40 in the border zone of a chronic infarction are pointedout. Changes with growing age and in the course of heart failure are discussedas well.

6.1 Gap Junctions in Acute Cardiac Disease

One of the most intriguing problems in cardiovascular medicine is theacute myocardial ischemia and infarction, which often leads to lethal arrhyth-mia. There are many factors involved in the pathophysiology of cardiac isch-emia and arrhythmogenesis [for review see, Janse and Wit, 1989; Katz, 1992].The lack in oxygen and glucose supply leads to a loss of intracellular ATPand consequently to a failure of the Na+/K+-ATPase [Coronel, 1988; Gettes,1987, Rosen et al., 1987]. This results in depolarization of the membranepotential [Kleber et al., 1978, Kramer and Corr, 1984] and influx of calciumwhich is further enhanced by reduced calcium elimination via Ca2+-ATPasesor by exchange of the accumulating sodium against calcium. The mechanismsof calcium overload are complex and currently under investigation. In addition,K+ channels open and the extracellular [K+] rises to values of about 30 mmol/lor even more [Hirche et al,. 1980] in the interstitium of the tissue. This isenhanced by the opening of IK.ATP channels if ATP is reduced to very lowconcentrations [Furukawa et al., 1991; Wilde et al., 1990]. It is not certain atpresent whether other factors may also contribute to this K+-efflux. However,this K+-efflux is of pathophysiological importance since it can lead to furtherdepolarization, to depolarization of surviving Purkinje strands [Lazzara andScherlag, 1984], to the so-called injury current which can depolarize otherfibers [Janse et al., 1980; Janse and van Capelle, 1982] and to action potentialshortening. Reduction in action potential duration and conduction velocityresults in a decrease in the local wave length which, thus, becomes heteroge-neous with regard to its local distribution between ischemic and nonischemicheart. Differences in wave length are known to cause reentrant arrhythmias[Allessie et al.,1973; Krinsky, 1981]. Slowing of conduction is assumed to be

73

a key factor in the initiation of reentrant arrhythmia [Janse and Wit, 1989;Pogwizd and Corr, 1987, 1990]. The situation becomes much more complexif the biochemical alterations, hemodynamics (especially the hypotension re-sulting from pump failure) and sympathetic activation with release of nor-adrenaline and subsequent tachycardia are also taken into account. Amongbiochemical alterations the accumulation of long-chain acylcarnitines has beendiscussed to play a role in gap junctional uncoupling. Normally, long-chainfatty acids can be transported into the mitochondrium by binding to carnitine(via acylcarnitine transferase I), passing the mitochondrial membrane as long-chain acylcarnitine. The acyl group is then transferred to intramitochondrialCoA. In the course of ischemia this mechanism is altered and long-chainacylcarnitines accumulate within 2 min after the onset of ischemia in vivo[DaTorre et al., 1991]. Interestingly, there was a sevenfold accumulation ofacylcarnitine in the junctional sarcolemma as compared to the nonjunctionalregions [Wu et al., 1993]. Exogenous application of long-chain acylcarnitinesresulted in rapid onset of cell-to-cell uncoupling [Wu et al., 1993]. Inhibitionof accumulation of long-chain acylcarnitines significantly reduced the inci-dence of arrhythmia induced by ischemia in vivo [Corr et al., 1989]. In addition,Purkinje fibers exposed to lysophosphatidylcholines, which may be the casein subendocardium adjacent to ischemic myocardium, have been shown togenerate early after-depolarizations [Arnsdorff and Sawicki, 1981]. Lysophos-phatidylglycerides in combination with acidosis and elevated [K+] can inducedelayed after-depolarizations and triggered activity in isolated Purkinje fibers[Pogwizd et al., 1986].

However, an enhanced extracellular potassium concentration and depolar-ization of the fibers besides the other factors lead to a reduced sodium channelavailability, to a reduced maximum depolarization velocity, shortened actionpotentials and to a slowing of conduction. These changes result in an alterationin the activation patterns [Dhein et al., 1994] and an increase in dispersion ofaction potential duration, which is even more pronounced in the presence ofneutrophilic leukocytes [Dhein et al., 1995a].

There are two forms of arrhythmia in acute myocardial ischemia. Type-1a arrhythmias occur 2–10 min after the onset of ischemia with a peak at5–6 min. These arrhythmias are often of the reentrant type and are caused bydiastolic bridging (details see chapter 1). It is also possible that prematureventricular depolarizations occur in this phase and initiate reentry.

Besides these, type-1b arrhythmia can occur at 12–30 min after the onsetof ischemia with a peak at 15–20 min. These type-1b arrhythmias are eitherdue to a partial recovery of the cell excitability (partial recovery of dU/dt andof the action potential duration), which may be ascribed to the release ofcatecholamines [for review see, Janse and Wit, 1989] or are due to gap junctional

746 Gap Junctions in Cardiac Disease

uncoupling and disturbed intercellular communication. From the considera-tions in chapter 1, it may be concluded that a partial recovery of excitabilityin concert with gap junctional uncoupling may cause inhomogeneities in thepassive electrical properties of the tissue which may favor reentrant circuits,although it is yet uncertain whether reentry is the underlying mechanism of1b arrhythmia (perhaps reentry in the ventricular wall outside the subepicard-ium) or other mechanisms, for example abnormal automaticity.

What is the role of the gap junctions? Which of these changes may altergap junctional gating? The loss of ATP, the fall in intracellular pH resultingfrom anaerobic glycolysis, the calcium overload, the rise in pCO2, the sodiumoverload of the fibers, fatty acids released in the ischemic tissue (see chapter 7),accumulation of long-chain acylcarnitines, leukotrienes from activated leuko-cytes, potential gradients between depolarized (ischemic) and normal tissue,all these changes will, as outlined in the previous chapters, result in gapjunctional uncoupling. It is difficult or not possible to ascribe this effect toonly one or two of these factors since they work in concert and cannot beseparated from each other.

But, is there really any evidence that gap junctions uncouple in the courseof ischemia? Wojtcak [1979] reported an increase in internal longitudinalresistance in cow ventricular muscle after hypoxia. Dhein et al. [1997b] foundan increase in the coupling time (time between stimulus and the propagatedaction potential>stimulus-response delay) 12 min after inducing hypoxiawith concomitant glucose-free superfusion in guinea-pig papillary muscles(figure 20).

In a more sophisticated setup Kleber et al. [1987] investigated the effectof ischemia on the propagation velocity and on internal longitudinal resistancein perfused rabbit papillary muscles. These muscles were perfused via a canulainserted in the coronary artery supplying the papillary muscle. Ischemia wasinduced by perfusion stop. In this setting about 15 min after induction ofischemia uncoupling occurred. Thus, gap junction uncoupling probably is notamong the earliest changes in the course of ischemia but in later phases, i.e.after ?12 min, gap junctional uncoupling can occur and contribute to thechanges in cardiac excitation spreading. In addition to intercellular coupling,Dekker et al. [1996] investigated the changes in intracellular calcium concentra-tion in the course of ischemia in perfused rabbit papillary muscles. With regardto the mechanism of uncoupling, these authors favored the hypothesis thatischemia leads to an increase in intracellular calcium which was observed after12.6 min of ischemia and was considered the main trigger for uncoupling. Ina similar setup, Yamada et al. [1994] demonstrated that the accumulation oflong-chain acylcarnitines contributed to cellular uncoupling in the course ofischemia and was delayed by inhibition of acylcarnitine transferase I. Interes-


Fig. 20. Stimulus-response interval in guinea pig papillary muscle under normoxicand hypoxic conditions. Hypoxia was concomitted with glucose-free superfusion. Note thesignificant increase in the stimulus-response interval after 12 minutes of hypoxia.

tingly, uncoupling occurred concomitantly with the secondary rise in extracel-lular potassium. This secondary rise was also delayed by inhibition ofacyltransferase I with 10 lmol/l 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-car-boxylate (POCA). Since it has been shown that long-chain acylcarnitines canelevate intracellular calcium [Fischbach et al., 1992; Meszaros and Papano,1990], although inhibiting the L-type calcium current [Wu and Corr, 1992],Yamada et al. [1994] concluded from their experiments that long-chain acylcar-nitine-induced uncoupling is due to an effect of the substance per se on gapjunctional conductance and secondary to a rise in intracellular calcium.

However, as outlined above, the alterations occurring in the course ofischemia, especially in the in vivo situation, are so complex that it may bedifficult to ascribe a phenomenon such as cellular uncoupling to only a singlefactor. Since other factors occurring in ischemia can also contribute to uncoup-ling this phenomenon may be a multicausal rather than monocausal processincluding a rise in intracellular calcium [De Mello, 1975; Maurer and Weingart,1987; Noma and Tsuboi, 1985], intracellular protons [Noma and Tsuboi,1985], long-chain acylcarnitines accumulating in the junctional sarcolemmaduring hypoxia [Wu et al., 1993; Yamada et al., 1994] and reduced ATP content[Sugiura et al., 1990]. In vivo the situation may be even more complicated by


the presence of activated leukocytes releasing lipoxygenase metabolites whichhave been suggested to be involved in gap junctional uncoupling and worseningof arrhythmogenesis [Dhein et al., 1995a, b; Gottwald et al. 1997; Masseyet al., 1992].

In an interesting study Kieval et al. [1992] investigated pairs of cardiomy-ocytes isolated from rabbit hearts which had previously undergone globalnormothermic ischemia followed by 30-min of reperfusion in a Langendorff

setup in comparison to cells isolated from hearts which were either perfusedaccording to the Langendorff technique for 75 min (without ischemia) orisolated directly after removal of the heart without Langendorff perfusion. Inall three groups of cells the action potential characteristics were normal. MeanGj was also almost normal in all three groups but was significantly morewidely distributed in the postischemic group with a greater population ofcells exhibiting only poor communication. The authors thus concluded thatpostischemic myocytes resemble a heterogeneous population with regard tocellular coupling.

Ultrastructural changes also occur in the course of acute ischemia. Ashrafand Halverson [1978] and McCallister et al. [1979] observed alterations in thegap junctional membranes after 20–30 min of ischemia. Hypoxia lasting forlonger than 30 min induced loss of lipid aisles and in consequence a condensa-tion of connexons in perfusion-fixed rat hearts with a subsequent rapid crystal-line densely packed pattern for the next 10 min, i.e. after 40 min of hypoxia[De Maziere and Scheuermann, 1990]. At that time widespread cell damagebecame obvious and researchers speculated that the crystalline gap junctionpattern may be associated with cell injury becoming irreversible. However,even very early in ischemia, i.e. 5 min after induction of ischemia, ultrastruc-tural changes have been identified. Frank et al. [1987] suggested that rearrange-ment of sarcolemmal P-face particles may occur after 5 min and may resemble anunspecific response to alterations in membrane fluidity accompaning ischemia.However, the gap junctional surface density, i.e. the gap junction profile lengthsperunitmyocytesectionalarea, isnotalteredat theonsetofuncouplingat30 minof hypoxia,although a reduction in the P-face center-to-centerdistance in freeze-fracture replicas has been observed. Since this reduction in P-face particles alsooccurs before the onset of uncoupling, it is considered not to play a primary rolein the process of uncoupling [Hoyt et al., 1990; Peters, 1995]. However, it shouldbe taken into account that other elements of the cytoskeleton are also importantfor the assembly of gap junctions as pointed out in the previous chapter and,thus, should also be investigated in the course of ischemia and infarction in orderto find out the primary processes of uncoupling.

What are the consequences of gap junctional uncoupling? Is it a benefitor a risk, or even both? Gap junctional uncoupling on the one hand will lead


to electrical and metabolic isolation of the ischemic tissue. If between thistissue and the surrounding cells differences in action potential duration exist,these will be enhanced since coupling of the cells would smooth these differ-ences as described by Dhein et al. [1994] and as suggested from computersimulations by Muller and Dhein [1993] and Lesh et al. [1989]. Such enhanceddifferences in action potential duration, mean enhanced dispersion, which isconsidered a risk factor for the occurrence of reentrant arrhythmia [Han andMoe, 1964; Kuo et al., 1983]. In addition, uncoupling also means slowing ofconduction, which has also been considered a key factor in the initiation ofreentrant arrhythmia [Janse and Wit, 1989; Pogwizd and Corr, 1987, 1990].

Uncoupling would also alter the activation pathways which may resultin fractionation of the activation wavefronts and thereby lead to arrhythmia.On the other hand, isolation of the ischemic tissue will protect the surroundingtissue from the depolarizing influence which might induce arrhythmia viadepolarization of Purkinje fibers. In addition this uncoupling may providesome kind of energy-saving effect for the tissue since the ischemic tissue isno longer activated and will, thus, stop contracting thereby reducing energyconsumption. Thus, both beneficial and disadvantageous effects can resultfrom gap junctional uncoupling. It probably depends on the local spatialdistribution of the electrophysiological changes with regard to the microana-tomy whether arrhythmia occurs resulting from altered pathways of excitationor not. However, the occurrence of late phase arrhythmias have been suggestedto be related to gap junctional uncoupling [Dekker et al., 1996].

Another acute disturbance of the heart is acute arrhythmia. How dogap junctions behave in acute arrhythmia? One could imagine that acutetachycardic arrhythmias are concommitted by an increase in intracellularcalcium and sodium as suggested [Bredikis et al., 1981] and possibly by afall in intracellular ATP both possibly leading to uncoupling. In order toclarify that question Bredikis et al. [1981] submitted rabbit atrial muscles tohigh-frequency stimulation (10–15 Hz) for 15 min and measured the inputresistance. They found an intercellular uncoupling in response to the rapidpacing with enhanced input resistance which recovered within 20–60 minafter cessation of the rapid pacing. This tachycardia-induced increase wasinsensitive to treatment with atropine, propranolol or phentolamine. It istempting to speculate that such an uncoupling induced by rapid heart ratemay be an endogenous antiarrhythmic mechanism like some kind of a ‘self-defibrillation’ mechanism, although this has not been shown unequivocallyin controlled experiments. On the other hand, such uncoupling may alsoworsen the situation and provoke a change in the type of arrhythmia byaltering the excitation pathways.


6.2 Gap Junctions in Chronic Ischemic Cardiac Disease

One of the most important chronic alterations in the heart is the chronicphase after myocardial infarction. The postinfarction period is known to beassociated with an increased risk for sudden cardiac death and for the occur-rence of cardiac arrhythmia. Changes in conduction properties have beenidentified [Dillon et al., 1988], although the cells exhibit normal or nearnormal action potential characteristics [Wit and Janse, 1992]. Thus, cellularelectrophysiology does not explain the complete pathophysiology of thearrhythmogenic substrate. Thus, other factors, for example structural changesand passive electrical properties, have to be taken into account.

Many factors contribute to this high risk of arrhythmia, especially thestructural changes in the geometry of the tissue network. After infarction localcontractility changes and the necrosis zone is replaced by connective tissue.FGF-2 can be released from cardiomyocytes during contraction and afterstimulation with catecholamines. This factor is upregulated in response tomyocardial damage [Doble and Kardami, 1995; Doble et al., 1996] and candecrease intercellular dye coupling. It induces Cx43 phosphorylation on serineresidues, tyrosine phosphorylation and a masking of Cx43 epitopes in car-diomyocytes, whereas in fibroblasts coupling was found to be increased inresponse to FGF-2 (Doble and Kardami, 1995]. However, presently it is un-clear whether this factor affects adult as well as neonatal cardiomyocytes.Myocardial injury which has been reported to cause increases in local FGF-2[Kardami, 1990; Padua et al., 1993] could thus affect the intercellular couplingof the non-injured myocytes near the lesion. It is tempting to speculate thatthese changes might somehow be linked to the arrhythmias observed aftermyocardial infarction originating from abnormal conduction of activation inthe vicinity of scar areas [Saffitz et al., 1992]. Experiments have been performedindicating that slowed anisotropic conduction may exist beyond the immediateinterface with the infarct [Dillon et al., 1988].

However, what are the changes in gap junction distribution observedafter myocardial infarction? Two major abnormalities regarding gap junctiondistribution have been observed in ischemic heart disease using laser-scanningconfocal microscopy of anti-Cx43-stained specimens: (1) loss of the commonordered (polarized) distribution of the gap junctions, which was found pre-dominantly in the border zone adjacent to infarct scars, and (2) reduction inthe quantity of Cx43 gap junctions in areas distant from the infarct zone[Severs, 1994a, b]. This and other factors may result in a heterogeneous aniso-tropic conduction and locally reduced conduction velocity forming a pro-arrhythmic substrate. The active properties of cells and resting membranepotential can be quite normal in the presence of manifest cardiac arrhythmia,


so that one can conclude that possibly the passive electrical properties maybe of importance [Dillon et al., 1988; Spach et al., 1988; Ursell et al., 1985].In the light of today’s research gap junctions are one of the most importantdeterminants of these passive conduction properties [Peters et al., 1993; Saffitzet al., 1992]. In hearts of patients suffering from end-stage ischemic heartdisease and in biopsies from patients 3 months after myocardial infarction, itwas found that the gap junction distribution in histologically normal areas isalmost normal. In contrast, within the border zone of healed infarcts (somehundred micrometers from the infarct scar) the pattern of gap junction distribu-tion is disturbed with a wide dispersion of the gap junctions over the wholecell surface instead of being confined to the intercalated disks at the cell poles.These border zone myocytes also exhibit substantial heterogeneity with regardto orientation and ultrastructure, and sometimes a disorganization of theintercalated disks. Myocytes were observed which communicated via cell pro-cesses with gap junctions in the absence of fasciae adherentes. Besides thesechanges, annular gap junction profiles were found indicating a possible in-ternalization of gap junctions. Between all these cells normal cells also occur[Severs, 1994a, b].

Following experimental infarction in the dog heart (10 weeks after occlu-sion of the left anterior descending coronary artery) [Luke and Saffitz, 1991],a more diffuse interstitial fibrosis was observed which was associated with areduction in the number of gap junctions per unit length of disk membraneand decreased gap junction size of long gap junctions at the transverse sectionplanes. A selective reduction in the larger gap junctions which are normallyfound at the circumference of the intercalated disk (this arrangement is thoughtto facilitate an efficient intercellular current transfer) [Green and Severs, 1993]was found so that a decrease resulted in the proportion of total gap junctionin the interplicate segments of the intercalated disk. The number of cells towhich a cardiomyocyte is connected was reduced from 11.2 in control tissueto 6.5 in the fibrotic infarct border zone. Intercalated disk zones were lessclearly defined, and groups of junctions maintaining the intercellular couplingwere displaced. In addition, a reduction in the frequency of intercalated disks ofthe side branches of the cells was seen, which provide side-to-side intercellularcontacts [Luke and Saffitz, 1991]. They found a reduction in connections ofcells in primarily side-to-side apposition by 75%, while connection of end-to-end apposed cells were reduced by only 22%. This should result in a dispropor-tionate increase in resistivity in the transverse direction thus enhancing aniso-tropy, potentially contributing to the development of reentrant arrhythmia.Especially such side-to-side contacts are necessary for homogeneous wavefrontpropagation as was demonstrated in neonatal rat heart cell cultures, whichwere grown in a patterned structure [Fast and Kleber, 1993].


Thus, it can be hypothesized that these changes in gap junction distributioncontribute to the alterations in activation pattern associated with chronicmyocardial infarction and to the enhanced arrhythmogeneity, e.g. to reentrantarrhythmias originating in the border zone of healed infarcts. However, thisis not the only factor since, for example, changes in the geometry by incorpora-tion of connective tissue will also alter the pathways of excitation and willcause inhomogeneity of anisotropy. Besides this, surviving strands of Purkinjefibers within the infarcted area can act as arrhythmogenic foci, or surviving‘peninsulas’ of myocytes can form excitable bridges from one side of theinfarcted zone to the other, thereby connecting two parts which otherwisewould be isolated from each other [Factor et al., 1978].

In patients with triple-vessel disease and recurrently ischemic myocardiumundergoing aortocoronary bypass operation, the gap junction surface areaper unit cell volume was reduced by 47% (0.0027 versus 0.0051 lm2/lm3)[Peters, 1995; Peters et al., 1993]. Taken together these results indicate thatpatterns of electrical coupling and electrical continuity may change betweenthe degenerated infarct zone and the ventricular myocardium adjacent to thisarea [Smith et al., 1991]. However, there is no widespread derangement ingap junction organization although there may be quantitative alterations inexpression in the noninfarcted myocardium of the ischemic heart.

An interesting question is: what happens to the coronary vessels? Do theyalso undergo alterations in cellular coupling in the course of atherosclerosis?Blackburn et al. [1995] investigated these questions in atherosclerotic lesionsrepresenting different stages of the disease, which were obtained from coronaryarteries of hearts removed from patients undergoing cardiac transplantation.They investigated the artery segments after staining with a specific anti-Cx43antibody for immunofluorescence using a laser scanning confocal microscope.The investigations were carried out with a double-labeling technique using asecond cell-specific antibody. They found a colocalization of Cx43 with smoothmuscle cells but not with macrophages, and confirmed this result by electronmicroscopy. In addition, regions of intimal thickening and early atheroscleroticlesions exhibited increased Cx43 expression between the smooth muscle cells,most prominent in regions of intimal thickening (?10-fold increase). Thequantity of Cx43-positive gap junctions was lower in early atheromatic lesionsthan in regions with intimal thickening but was higher than in normal vessels.With further progression of the disease the Cx43 expression was found to beprogressively reduced from enhanced levels in early and earliest stages towardsdecreased levels (as compared to undiseased vessels) in the most advancedatheromatous lesions. With this development the distribution of the gap junc-tions changed and they became more patchy with larger diameters of theindividual junctions.


6.3 Gap Junctions in Heart Failure

Another cardiac disease often associated with cardiac arrhythmias is heartfailure. Many factors including high catecholamine levels, dilated tissue geo-metry, changes in the b-adrenoceptor population, impairment of the regulationof the intracellular (diastolic) calcium concentration, possibly enhanced endo-thelin levels and many more contribute to altered cardiac function and makethe heart more prone to arrhythmia. However, the question was whether, inaddition to the well-known structural changes, gap junction alterations mayalso partially form the arrhythmogenic substrate. Thus, researchers were inter-ested in whether in the course of heart failure gap junctional alterations mayoccur.

In patients suffering from heart failure due to ischemic cardiomyopathySevers [1994a, b] described two main alterations (1) changes in the normalspatial distribution of gap junctions at the border zone of healed infarcts, and(2) a reduction in the quantity of Cx43 in regions distant from infarct scars.

In patients with cardiac hypertrophy from chronically pressure-loaded hu-man left ventricles due to aortic valve stenosis, a general reduction in gap junc-tion surface area per unit cell volume by about 40% (0.0031 versus 0.0051 lm2/lm3) has been observed [Peters et al., 1993]. The gap junctions in the pathologicaltissue were larger than normal. The estimated gap junction content per cell wasreduced [Peters et al., 1993]. A reduction by 30% in the gap junction surface percell was observed [Peters, 1996]. However, the number of intercalated disks permyocyte and the mean density of packing of connexons at freeze-fracture inthese hearts remained unchanged as compared to control hearts.

In contrast to these findings, in guinea pigs with cardiac hypertrophyfollowing renovascular hypertension Peters [1996] reported a substantial in-crease in Cx43 gap junction expression in the early phase. Gap junction surfacedensity was increased by 45% per cell and by 30% per volume unit, whichwas contrary to the findings in hypertrophied human myocardium. However,the different pathophysiology should be taken into account. It might be specu-lated that factors like angiotensin II can alter cardiac growth and possibly thearchitecture of the tissue, although at present there is no experimental evidencefor an alteration in connexin expression.

In cardiomyopathic hamsters Luque et al. [1994] stained for Cx43 usingconfocal microscopy and found that some of the cardiomyocytes stain normallybut others stain diffusely, with a pixel intensity distribution of the confocalimages showing a 90% increase in the number of pixels and a 60% decreasein pixel intensity in the cardiomyopathic hearts as compared to control hearts.Thus, Cx43 seemed to be present in the cells but did not become localized onthe membranes as in normal cells.


Another important cardiovascular disease affecting the heart and oftenassociated with the pathogenesis of heart failure is chronic hypertension. Suchhearts exhibit complex structural changes and it has been asked whether thereis also an alteration in the gap junction distribution. Thus, researchers haveinvestigated hearts from hypertensive animals. In hearts from hypertensive ratsBastide et al. [1993] found a reduced expression of Cx43 but an enhancedexpression of Cx40 involving myocytes from the working myocardium. Sinceboth connexins possess different electrophysiological properties especially withregard to their sensitivity to transcellular voltage, the conductive propertiesof the tissue may thereby be altered and a proarrhythmic substrate may beformed.

Taken together all these findings described in ischemic heart, heart failureand hypertension suggest that a reduction in Cx43 expression may be a generalfeature in heart disease and may contribute to the enhanced arrhythmogeneityin many cardiac disorders.

6.4 Gap Junctions in Arrhythmia

Gap junctions have often been discussed to play an important role ininitiation and maintenance of acute arrhythmia (see above and chapter 1).In summary, all states with reduced intracellular pH, enhanced intracellularcalcium, reduced ATP levels, sodium overload, etc., can induce cellular un-coupling, leading to alterations in the activation pathways and the geometryof excitation and may thus induce arrhythmia (for a detailed discussion ofthe role of gap junctions in acute arrhythmia see chapter 1). The main effectof gap junctional uncoupling is to introduce or enhance discontinuities in theanisotropic tissue, thereby setting the stage for microreentry as discussed inchapter 1. Another effect of gap junctional uncoupling is the slowing ofconduction which is also believed to be a prerequisite of reentrant arrhythmia.

Besides acute arrhythmia, chronic arrhythmia is a common and importantclinical problem and chronification of arrhythmia is only poorly understood,although this might be the basis for new antiarrhythmic treatments from amore pathophysiological viewpoint.

One of the most intriguing questions is whether chronification of arrhyth-mia may be related to changes in the underlying tissue structure and geometryof cellular coupling. One of the most common forms of arrhythmia is chronicatrial fibrillation and it is well known that the longer this arrhythmia enduresthe harder it is to convert the heart to sinus rhythm. It has been hypothesizedthat at least this form of arrhythmia may induce structural changes therebyforming the arrhythmogenic substrate of a chronic arrhythmia.


In chronic atrial fibrillation Van der Velden et al. [1996] recently showedchanges in the Cx40 distribution pattern in goat atria with chronic fibrillation.In the goat model used, atrial fibrillation was induced via chronic high-fre-quency pacing and they observed that, after switching the stimulus off fibrilla-tion persisted for a period depending on the time elapsed during high-frequencypacing. Additional results were found in the authors working group using arat model of atrial fibrillation. As a particularity rat atrial cells are coupledvia Cx43 (see previous chapters). Rat atria were bathed in an organ bath insaline solution (superfusion at 7 ml/min) for 24 h and stimulated at 10 Hzthereby inducing atrial fibrillation which persisted if stimulation was switchedoff. After this time the atria were frozen, processed for immunohistochemistryand stained for Cx43. It became obvious from the experiments that in atriaexcised and immediately frozen the typical distribution of Cx43 gap junctionsat the borders of the cells (at the cell poles in longitudinal direction) couldbe observed. After 24 h in organ bath and beating at their spontaneous rate,this pattern was not changed, but after 24 h of atrial fibrillation the Cx43distribution changed with a more disperse pattern without the typical polariza-tion (figure 21).

From these experiments in rats and goats described above it was concludedthat chronic arrhythmia may represent a state in which the distribution patternof gap junctions can be altered by a yet unknown mechansim. This changein the gap junction pattern may then form the basis for chronification of thearrhythmia.

Taken together, all these findings point to a new understanding of thearrhythmogenic substrate as a more structural change reflecting the electricalnetwork ‘heart’. As a main point, at least in some of the diseases alterationsin the intercellular coupling, as a main determinant of the network, contributeto the formation of the arrhythmogenic substrate. Substances interfering eitherwith the cellular coupling via gap junctions or with the regulation of expressionand distribution of gap junction proteins may, thus, represent a new antiar-rhythmic approach [Dhein and Tudyka, 1995].

6.5 Gap Junctions in Infective Heart Diseases

Conduction disturbances are frequently found in acute and chronicChagas disease. In cultures of neonatal rat hearts, changes in the gap junctiondistribution were studied to discover whether they were associated with theinfection. In cultured cardiomyocytes infected with the unicellular parasiteTrypanosoma cruzi responsible for Chagas disease, which is the most commoncause of heart disease in South America, reduced gap junctional conductance


Fig. 21. Cx43 immunostaining of rat atria either native (a) or after 24 h of atrialfibrillation (b) [Dhein et al., 1997a].

a

b

and decreased expression of Cx43 at the junctional membranes have beenobserved using immunohistochemistry [Campos de Carvalho et al., 1992,1994]. Similarly the lucifer yellow dye transfer between infected cells wassignificantly reduced. Synchronized spontaneous beating becomes less regularin infected cells. Interestingly, the total amount of Cx43 was found to benormal, but the intracellular distribution was altered with high levels of in-tracellular Cx43 and only little at the appositional membranes. In addition, thecellular electrophysiology is altered with shortened action potential, elevatedintracellular resting calcium levels and altered response to a-adrenergic stimuli.


The immunohistochemical findings were correlated with reduced intercellularcoupling indicating a possible role of disturbed Cx43 expression and gapjunction function in the pathogenesis of Chagas disease and the arrhythmiasassociated with that disease.

6.6 Gap Junctions in Defective Heart Development

Genetic evidence has grown in the last years showing that connexins canplay an important role in the regulation of specific development. It is nowknown that mutations in the gene encoding Cx32 causes X-linked Charcot-Marie-Tooth disease [Bergoffen et al., 1993; Paul, 1995], a demyelinatingperipheral neuropathy. It was tempting to speculate that at least some of thecardiac malformations may be linked to mutations in the connexin genes.

Thus Reaume et al. [1995] investigated the role of mutations in the Cx43gene on fetal development in a mouse model. They created a null mutationin mice in the Gja1 gene encoding Cx43. They generated a mutation in theCx43 gene by homologous recombination in 129 strain R1 embryonic stemcells with a construct replacing main parts of the coding sequence with the neor

gene which lacked a promotor. Homozygous cell lines were morphologicallynormal and differentiated to embryoid bodies exhibiting beating heart muscleand blood islets, but were completely lacking in Cx43. 82% of the cells were notdye coupled. From heterozygous cell lines germline chimeras were generatedby injection into C57BL/6 blastocysts and the homozygous offspring fromheterozygous crosses was analyzed. No viable homozygous offspring wasfound. The pups died shortly after birth with cyanosis and signs of failure ofpulmonary gas exchange, although the lungs became expanded and breathingwas initiated. As long as they were alive the pups exhibited labored breathing.No alterations in external morphology, gross anatomy were detected exceptan enlargement of the conus of the heart’s pulmonary outflow tract of theright ventricle. This region was filled with intraventricular septae dividing theoutflow tract into separate or blind-ended chambers. Filling of the right vent-ricle with methylacrylate for corrosion casts revealed no passage of the resinto the pulmonary arteries. Reaume et al. [1995] concluded that these rightventricular dysplasias caused death in the neonates when the circulationchanges and the lungs must become perfused. Other tissues normally ex-pressing Cx43 as lungs, kidneys, brain and gut remained histologically normal.There was no increase or change in Cx40 and Cx45 mRNA levels in theirexperiments.

In addition to these results, mutations in the COOH terminal of Cx43 maybe underlying cardiac malformations in visceroatrial heterotaxia syndromes as


reported by Britz-Cunningham et al. [1995]. These authors investigated Cx43DNA from 25 normal subjects and 30 children suffering from various congen-ital heart diseases (including hypoplastic left and right heart syndromes, Di-George syndrome, septal defects, trisomy 13 and others) using the polymerasechain reaction. They expressed the mutant DNA in cell culture and investigatedits effect on the regulation of intercellular communication. Within the children’sgroup 6 children were identified, all suffering from syndromes including com-plex heart malformations, with substitutions of one or more phosphorylatableserine or threonine residues. In the Cx43 DNA of 5 of these patients a substitu-tion of proline for serine at position 364 was seen. If cells were transfectedwith the Ser364Pro mutant Cx43 they exhibited abnormalities in the regulationof intercellular communication. The authors concluded from their findingsthat mutations in the Cx43 gene leading to cell-to-cell communication defi-ciencies are associated with visceroatrial heterotaxia.

Another group [Kass et al., 1994] reported on a possible involvement ofCx40 in cardiac malformations. They evaluated seven generations with aninherited conduction system defect and dilated cardiomyopathy. This defectexhibited autosomal dominant transmission and perturbed both AV conduc-tion and cardiac contractility. Genome-wide linkage analysis revealed thatpolymorphic loci near the centromere region of chromosome 1 (chromosome1p1–1q1) were linked to the disease locus with a maximum multipoint lodscore of 13.2 in the interval between D1S305 and D1S176. The authors specu-lated from these results that mutations of Cx40 may result in conductionsystem disease and dilated cardiomyopathy, since both Cx37 and Cx40 havebeen mapped to chromosome 1pter–q12. Because Cx45 does not map tochromosome 1 and Cx37 to the distal p arm of chromosome 1, Cx40 remainedas a candidate gene responsible for that disease.

The role of gap junctions during development has been investigated furtherin preimplantation mouse embryos by Becker and Davies [1995]. Besides thenormal expression pattern of gap junctions in these embryos, they studiedthe developmental and junctional organization in mice naturally exhibitingreduced cell-to-cell communication (DDK syndrome, defect located on chro-mosome 11, it has been sugested that in DDK syndrome the regulation ofintracellular pH is disturbed leading to lower pHi which may uncouple cells)and in normal mice with experimentally altered gap junction permeability. Inprinciple they found that gap junctional communication is critical for themaintenance of compaction and the differentiation of an organized epitheliumin the embryo and, thus, for the preimplantation development. The DDKembryos appeared to be phenotypically normal until reaching the morulastage. Thereafter, cells start to decompact and the embryo dies before reachingthe expanded blastocyst stage.


6.7 Gap Junctions in the Aging Heart

As discussed earlier in this book, it has been shown that there are consider-able changes in the distribution and expression pattern of gap junctionalchannels with increasing age. However, these investigations primarily includedthe developing heart until maturity. It is presently not known whether the gapjunction distribution or the expression of various types of connexins is chang-ing with increasing age as far as senium is concerned. It will be difficult toelucidate this question because it is necessary to differentiate between age perse and heart disease which is worsened with age. Every even small or minimalinfarction and increasing heart failure with age will cause changes in thedistribution pattern, which are primarily related to the disease and not to ageper se.

However, it is well known that with increasing age microfibrosis is observedwhich in turn will seperate the fibers from each other and thereby enhancethe degree of nonuniformity as discussed in the first chapter of this book.This is accompanied by a reduction in side-to-side connections [Spach andDolber, 1986]. Thus, with increasing age the intercellular communication canbe expected to be reduced probably due to structural changes in the tissuewith deposition of collagenous fibers. Concomitant changes in the gap junctiondistribution are probably secondary to cardiac diseases, although at presentan effect of age per se cannot be excluded.


7...........................

Pharmacological Interventions atGap Junctions

In the previous chapters the role of gap junctions in cellular communicationand in cardiac disease has been outlined. It became obvious that in manydiseases the intercellular communication is reduced via gap junctional uncoup-ling or via reduced or altered expression of gap junctional channels. On thisbackground a straightforward idea would be to simply enhance the gap junc-tional coupling by any agent. However, one has to consider that the uncoupling,as pointed out in the previous chapter, is confined to certain areas, the diseasedareas for example, in the heart. In addition, the uncoupling also has somepositive effects, for example a putative energy-saving effect in ischemia.

What would happen if coupling were enhanced? There are at least twoprincipal possibilities: on the one hand it can be imagined that if couplingwere enhanced unselectively in the whole heart the uncoupled area would thenbe coupled to the normal tissue and would behave more like this, i.e. theischemia-induced action potential shortening would be reduced. Electricalinactivation would be antagonized and energy consumption would be en-hanced. Since the channel is also permeable to small molecules it can beanticipated that molecules like ATP can flow to the ischemic zone and willbe degraded there thus enhancing the ATP depletion in an ischemic setting.Metabolites and ions from an ischemic zone (e.g. lactate, H+, Ca2+, K+) wouldbe able to diffuse to the nonischemic zone possibly exerting unfavorable effectsthere. Within the undiseased zone activation patterns probably may change.The transition zone between altered and normal electrophysiological behaviormay become broadened.

On the other hand arrhythmia due to uncoupling may be prevented. Ifcoupling is enhanced selectively in the previously uncoupled area only withinthat area cellular uncoupling would be antagonized, which means that thesurrounding tissue would not be affected in the way described above. Theremight be a similar effect in the close border zone between diseased and normaltissue, but the effect would be confined to that zone. Inhomogeneities withinthe diseased zone would be smoothened whereas the normal tissue behaviorwould probably be less affected. This could especially smooth differences inaction potential duration and thereby prevent, in some situations, from reent-rant arrhythmia, since this is often related to differences in action potentialduration, to dispersion. Another important factor in the initiation of reentry

89

is the slowing of conduction velocity [Janse and Wit, 1989; Pogwizd and Corr,1987, 1990], which would be favored by cellular uncoupling and prevented atleast in part by improving intercellular communication.

Thus, enhancing intercellular coupling may exert a prophylactic effectagainst arrhythmia if arrhythmia is due to uncoupling. However, if the couplingeffect is unselective, it would probably postpone an impairing effect as discussedabove for ischemia. From this theoretical point of view selective coupling-enhancing effects on the previously uncoupled tissue would be desirable ratherthan unselective.

In contrast to these considerations, another strategy to follow may be theuncoupling strategy. In certain situations it might be favorable to cut out apart of the tissue. In ischemia, it might be interesting to investigate whethera full direct uncoupling of the ischemic zone might exert a protective effectduring reperfusion due to energy saving. However, a hardly achievable prereq-uisite would be a selective effect on the ischemic zone. Otherwise, uncouplingin the whole organ will probably make the heart more prone to arrhythmiaas outlined in chapter 1. For example Rohr et al. [1997] showed that, indiscontinuous tissue under certain conditions (if there is a pronounced mis-match between the current source which was represented by strands of culturedcells of 55 lm width in their experiments and the current sink being representedby the expansion of the strand to a rectangular monolayer), failure of antero-grade activation from the small current source to the large current load oc-curred, whereas successful retrograde activation was seen in the oppositedirection. Application of an uncoupling agent (palmitoleic acid) transientlyled to successful anterograde propagation of electrical activation in the regionof unidirectional block.

While improving intercellular coupling may exert prophylactic antiar-rhythmic effects under certain conditions, in acute manifest arrhythmia areduction in intercellular coupling may stop the arrhythmia by slowing thevelocity on the reentrant pathway so that wavelength and anatomic reentrantpath length do not fit each other any longer, a prerequisite for reentry suggestedpreviously by Krinsky [1981].

However, it should be considered that in both cases one has to distinguishwhether a substance uncouples or couples the whole tissue or only those partswith altered intercellular communication. Thus, the question arises: what ispresently known about the pharmacology of gap junction channels?

In the following a survey is given of the substances which have been foundto alter intercellular coupling. First drugs will be considered which uncouplegap junctions. A number of lipophilic compounds have been described toreduce gap junctional coupling. These substances include alcohols like hep-tanol and octanol, saturated and unsaturated fatty acids, and alcohols and

907 Pharmacological Interventions at Gap Junctions

volatile anesthetics like halothane and ethrane (enflurane). Halothane is oftenused in double-cell voltage-clamp or dye-transfer experiments to uncouplecells in concentrations of about 1.5 mmol/l [Burt and Spray, 1989; Morenoet al., 1994a; Nedergaard et al., 1995]. Ethrane is effective in concentrationsof about 4 mmol/l [Burt and Spray, 1989]. Octanol can uncouple embryonicchick ventricle cells [Veenstra and DeHaan, 1988] and adult rat ventricularmyocytes [White et al., 1985]. It has been suggested that this effect may becaused by limiting the channels from opening to their largest configuration,i.e. by interference with the switching between various conductance states[Chen and DeHaan, 1993]. With regard to the mechanism there is no effectof octanol on single-channel conductance itself in neonatal rat cardiomyocytes[Burt and Spray, 1988b]. As octanol, heptanol also reduces gap junctionalconductance [Bastide et al., 1995; Kimura et al., 1995; Rudisuli and Weingart,1989]. For experimental approaches this may be interesting since after applica-tion of 3 mmol/l heptanol single-channel behavior can be observed. Accordingto Rudisuli and Weingart [1989] the effect is fully reversible within 2 minafter washout in their experimental system. The concentration-response curverevealed a steep S-shaped relationship with a threshold concentration of about10Ö4 mol/l and the maximum effect at a concentration of about 10Ö3 mol/lheptanol. Kd was determined to be 0.16 mmol/l and the Hill coefficient z>2.3for the equation Gj>(Kd)2/((Kd)2+[heptanol]2). Regarding the mechanism ofaction Haydon et al. [1984] and Niggli et al. [1989] suggest that heptanol actsby incorporation into the plasma membrane and the lipid bilayer. Rudisuliand Weingart [1989] concluded from their findings that heptanol uncoupledcardiac cells via impairment of the open probability po, the gap junctionconductance gj being described by the equation gj>N·cj ·po (N>number ofchannels). Expression, phosphorylation or localization of Cx43 are not alteredby brief exposure (5–20 min) to 2 mmol/l heptanol [Kimura et al., 1995]. Themechanism of action of heptanol was further clarified by Bastiaanse et al.[1993], who showed that the uncoupling effect was based on a decrease in thefluidity of membranous cholesterol-rich domains. Gap junctions are embeddedin such cholesterol-rich domains of the membrane. The unitary conductanceswere unaltered by heptanol, so that the authors concluded that heptanoldecreases the open probability as already shown in a previous study by thisgroup [Takens-Kwak et al., 1992].

However, some authors showed that heptanol and octanol can also inhibitthe cardiac sodium current [Nelson and Makielski, 1990] and that generalanesthetics like octanol and decanol can interfere with the cardiac Na+/Ca2+

exchange [Haworth et al., 1989] at concentrations below those required forgap junctional uncoupling. This action is considered to contribute to theirwell-known negative inotropic effect.


Another group of lipophilic substances which can uncouple cardiac gapjunctions comprises fatty acids and alcohols. However, it depends on the lengthof the acyl chain and on saturation of the carbon bonds whether the fattyacids uncouple or not. Burt et al. [1993] systematically investigated the influenceof various saturated and unsaturated fatty acids on junctional coupling. Theyfound saturated fatty alcohols with an acyl chain length of 7–12 but not higher,as well as unsaturated C18 cis 9 fatty alcohol to be effective in uncouplingneonatal rat heart cells. Saturated fatty acids with acyl groups of 10–14 carbonsbut not more and unsaturated cis 9 fatty acids with acyl chain lengths of14–18 exhibited an uncoupling effect as well. Decanoic acid in concentrationsof 2 mmol/l rapidly and fully reversibly uncoupled cardiac cells. As with hep-tanol no change in cj was observed so that the investigators concluded thatthe uncoupling effects were due to a reduction in the open probability ratherthan in cj. These drugs are supposed to incorporate in the lipid bilayer andto increase disorder in the interior of the membrane (C9–C18 region)[Goldstein, 1984; Gruber and Low, 1988; Klausner et al., 1980; Pringle et al.,1981] thus acting by their physical properties rather than by chemical inter-action. All drugs listed above found to be effective uncouplers exhibit highrotational and lateral mobility in the bilayer. The cis 9 acyl chains requiremore space for rotation than the straight-chain analogues. The short-chaincompounds including arachidonic acid (see below) incorporate in the exteriorvolume of the bilayer, whereas halothane dissolves in the interior of the mem-brane. Although diverse in structure these lipophilic agents share a commonphysical property: incorporation into the membrane, disordering its structureand inhibiting gap junctional channels [Burt et al., 1993]. Interestingly, de-canoic acid and palmitoleic acid can uncouple heart cells without affectingother transmembrane channels contributing to the action potential [Burt etal., 1991]. Another important feature is the finding that multiple lipophileshave additive effects [Burt et al., 1993].

Cells, however, differ with regard to their sensitivity to these lipophiliccompounds. Adult rat heart cells, for example, are relatively resistant to un-coupling by these lipophiles [Ovadia and Burt, 1991]. Neonatal rat heart cellsand A7r5 cells, a neonatal rat aortic smooth muscle cell line, seem to be moresensitive. The underlying mechanisms for diverse tissue sensitivity remain tobe elucidated. According to the mechanism of heptanol-induced uncoupling[Bastiaanse et al., 1993] it is tempting to speculate that the cells might differin their cholesterol-rich domains. In addition, at present it is not clear whetherthere are differences in the sensitivity of various connexins. Oleic acid forexample has been shown to differentially affect gap junctional coupling be-tween neonatal rat cardiomyocytes and A7r5 cells: low concentrations of oleicacid (up to 1 lmol/l) reduced dye coupling in A7r5 cells by 50%, but higher


concentrations had no further effect. In contrast, neonatal rat cardiomyocytesbecame uncoupled to zero levels in a linear fashion in concentrations rangingfrom 1 to 25 lmol/l [Hirschi et al., 1993].

Although often used for uncoupling, one should keep in mind that the effectof these lipophilic agents is a physical effect on membrane structure. This meansthat these drugs are valuable tools for investigation of single-channel behavior,but they are probably not suitable for inducing uncoupling in order to investigateeffects of putative coupling agents on the overall conductance gj if these agentsare supposed to act via some receptor-coupled regulatory mechanism.

Another agent uncoupling cardiac gap junctions is arachidonic acid andsome of its metabolites. Exposure to arachidonic acid produces uncoupling inneonatal rat cardiomyocytes [Schmilinsky-Fluri et al., 1990]. The concentrationresponse curve analysis revealed a Kd of 4 lmol/l and a Hill coefficient of0.75. The uncoupling was reversible gj returning back to 61% after 30 minwashout and recovery could be accelerated by addition of bovine serum albu-min to the bath solution (gj reaching 86% of the initial value after 10 min ofwashout). The effect was specific for arachidonic acid and could not be mim-icked with analogues like arachidic acid (100 lmol/l) or arachidonamide(10 lmol/l). The single-channel conductance cj (mean cj>33.5 pS) was notaffected by arachidonic acid at concentrations ranging from 1 to 100 lmol/l,so that the authors concluded that arachidonic acid might reduce the openprobability of the channel. In contrast, 100 lmol/l arachidonic acid did notaffect nonjunctional membrane current in these experiments. 100 lmol/l arach-idonic acid induced uncoupling starting after 90 s, and after 2.5 min junctionalcurrent was no longer detectable.

In addition to these findings, Massey et al. [1992] reported that the uncou-pling effect of arachidonic acid was not only dose- but also time-dependentand that the dose-response curve could be shifted to the right by pretreatmentwith 2.5 lmol/l U70344A, a 5-lipoxygenase inhibitor, whereas pretreatmentwith the cyclooxygenase inhibitor indomethacin (100 lmol/l) had no effecton the arachidonic acid concentration-response curve. Complete uncouplingoccurred at membrane concentrations of 3–4 mol%. Incorporation of arachi-donic acid into the lipid bilayer was not affected by the inhibitors. Completeuncoupling was achieved with 20 lmol/l arachidonic acid within about 3.5min, with 5 lmol/l within 4.5 min and with 2 lmol/l within 9.5 min. Inhibitionof 5-lipoxygenase delayed this uncoupling. It was suggested by the authorsthat arachidonic acid is metabolized via lipoxygenase to metabolites whichcontribute to the uncoupling effect. However, lipoxygenase products like leuko-trienes themselves have not been investigated directly. Thus, it remains unclearwhether 5-HPETE, 5-HETE or the leukotrienes act as uncoupling agents andwhether this is a receptor-mediated effect.


Long-chain acylcarnitines which increase rapidly within minutes after theonset of ischemia or hypoxia can also uncouple cardiac muscle and reducegap junctional conductance [DaTorre et al., 1991; Wu et al., 1993; Yamadaet al., 1994] (for details see chapter 6). Regarding pharmacological inter-ventions, it was interesting that inhibtion of acylcarnitine transferase I by10 lmol/l POCA or by 100 lmol/l oxfenicine completely prevented the accumu-lation of long-chain acylcarnitines even within 40 min of ischemia in arteriallyperfused rabbit papillary muscles and delayed the onset of and progressionof uncoupling and ischemic contracture [Yamada et al., 1994]. The inhibitorsdid not influence the loss of intracellular ATP or the initial rise in extracellularpotassium, whereas the secondary rise in extracellular potassium, concomitantwith cellular uncoupling, was delayed.

Is there any physiological or pathophysiological role for these findings?Corr et al. [1984] found that ischemia enhances lipid metabolism and therebyleads to the liberation of fatty acids. Besides this, it has been outlined aboveand in the previous chapter that ischemia results in the accumulation of long-chain acylcarnitines. According to the findings described above these fattyacids may incorporate into the plasma membrane, disorder the lipid bilayersurrounding the gap junctional channels and, thereby, reduce the open proba-bility of the channel and contribute to cellular uncoupling during ischemia.Cytosolic levels of arachidonic acid have also been reported to be increasedin response to hypoxia or ischemia [Chien et al., 1984]. The finding thatarachidonic acid can reduce the conduction velocity of the action potential[Bayer and Forster, 1979; Szekeres et al., 1976] may reflect its uncouplingaction on gj. Thus, the release of both fatty acids and arachidonic acid maycontribute to the enhanced arrhythmogenesis during ischemia.

Pharmacological approaches include the inhibition of release of arachi-donic acid by inhibition of phospholipase A2 and the inhibition of acylcarni-tine transferase I by POCA and oxfenicine, the latter of which has been shownto prevent or at least delay ischemia-induced uncoupling. There are at presentno data available on the possible effects of inhibitors of arachidonic acidrelease on ischemia-induced uncoupling.

Gap junctions can also be uncoupled by weak organic acids. Acetic acidfor example has been shown to effectively uncouple gap junctions in crayfishseptate axons lowering pHi to values around 6.2 [Peracchia, 1991a; Ramon etal., 1991]. The uncoupling effect exhibits rapid onset and reversibilty. Similarly,Nedergaard et al. [1995] reported on an uncoupling effect of 10 mmol/l lacticacid adjusting the extracellular pH to values ranging between 6.48 and 7.30in Hanks’ buffered saline solutions. Lactic acid facilitates the intracellularacidification under these conditions [Nedergaard et al., 1991]. Propionicacid can also be used for uncoupling experiments as shown by Gottwald and


Dhein [1997]. If considering the mechanisms of the induced uncoupling thereare two possibilities: first, intracellular acidification may directly induce cellularuncoupling via the pHi-uncoupling effect; second, the organic acid may enterthe cell in its undissociated form, then dissociate and the resulting H+ maybe exchanged against Na+ via the type-1 Na/H-exchanger as shown byGottwald and Dhein [1997], thus inducing an intracellular sodium overloadand eventually a secondary rise in calcium.

Another group of drugs influences the intracellular sodium and calciumconcentrations thereby modulating gap junctional coupling. These drugs willbe described in the following paragraph. Among the drugs often used incardiology the digitalis glycosides have been shown to uncouple cardiac myo-cytes. Using a silicon oil chamber Weingart [1977] demonstrated in cow heartsthat the exposure to 2 lmol/l ouabain for 90 min increased longitudinal resis-tance from 420 to 1,032 Xcm and concomitantly reduced the conductionvelocity from 50 to 29 cm/s. The increase in longitudinal resistance was associ-ated with an increase in diastolic tension suggesting a rise in intracellularcalcium as the underlying mechanism. Similarly, De Mello [1976] observedan uncoupling effect of 0.68 lmol/l ouabain in Purkinje fibers. He suggestedan increase in intracellular [Na+] and a secondary increase in intracellular[Ca2+] as the underlying mechanism. It is widely accepted that exposure tocardiac glycosides in at least toxic concentrations produces an increase inintracellular [Na+] via inhibition of the Na+/K+-ATPase [Hoffman and Bigger,1985] thereby decreasing the transmembrane sodium gradient which causes asecondary rise in intracellular [Ca2+] via the impairment of the Na+/Ca2+-exchange mechanism. Besides this an increase in the slow inward calciumcurrent Isi has been described [Gilman et al., 1985]. Weingart and Maurer[1987] studied the effects of exposing guinea-pig ventricular cell pairs to 2 and20 lmol/l strophanthidin. They found a dose- and time-dependent uncouplingeffect of strophanthidin on nexal resistance. 2 lmol/l produced uncouplingafter 20–25 min and 20 lmol/l after 10–15 min. This could be accelerated ifthe pulse frequency in these experiments was enhanced from 0.3 to 1.0 Hz.Nexus resistance was enhanced by 2 lmol/l strophanthidin from 19 to 295 MXin these experiments. Inhibiting the transmembrane calcium current Isi antago-nized the uncoupling effect of the cardiac glycoside indicating that extra Ca2+

influx via Isi contributes to the uncoupling action.It can be imagined that these uncoupling effects of the cardiac glycosides

may contribute to the arrhythmogenic risk associated with digitalis therapyand intoxication.

Another compound increasing the intracellular sodium concentration isthe aconitine, a drug found in monkshood (Aconitum napellus) which is oneof the most toxic plants in middle Europe. In cardiac muscle the alkaloid


causes a prolonged sodium current with slowed repolarization. It is used inexperimental pharmacology to produce ventricular arrhythmia. Although notyet shown directly, it can be imagined that such drugs, opening the fast sodiumcurrent, will increase intracellular sodium load and uncouple the cells accord-ing to the findings of De Mello [1976] who found a rapid uncoupling aftersodium injection.

Regarding manipulation of the intracellular calcium concentration,caffeine has been used in experiments on intercellular communication. Ifcardiac cells are exposed to methylxanthines such as caffeine a phasic releaseof calcium from the sarcoplasmic reticulum can be observed [Chapman,1979]. Maurer and Weingart [1987] investigated the effect of exposing adultguinea-pig cardiac ventricular cell pairs to 5–10 mmol/l caffeine. This inter-vention did not change the gap junction conductance. However, if caffeinewas applied after reduction of extracellular Na+ (to 15 mmol/l, which reducedgj by 20%), caffeine induced a rapid (within 90 s) decrease in gj (74%). Afterdecoupling canine Purkinje cells by injection of calcium De Mello [1975]found a slowed recovery in the presence of 6 mmol/l caffeine (extracellular).It is difficult at present to interpret these results from a pharmacologicalpoint of view. Experiments have to be performed on the influence of caffeineor other drugs altering intracellular calcium balance on intercellular couplingand arrhythmogenesis in previously uncoupled preparations (preferably withhigh calcium). It has been shown in crayfish septate axons that the uncouplingeffect of halothane could be enhanced by coadministration of caffeine, whichmight give a partial explanation for arrhythmias in patients treated withmethylxanthines, like theophylline, during halothane anesthesia [Peracchia,1991b].

According to the calmodulin hypothesis [Peracchia, 1988], it can be an-ticipated that calmodulin antagonists should exhibit an influence on gap junc-tional coupling. Indeed, Peracchia et al. [1983] and Peracchia [1987] dem-onstrated that calmodulin inhibitors were able to prevent cell uncoupling. Incrayfish septate axon for example electrical uncoupling could be inhibited bythe calmodulin inhibitor W7 [Peracchia, 1987]. On this background systematicstudies on the influence of calmodulin antagonists on gap junctional resistancein several models of uncoupling would be highly desirable.

Regarding calcium, there is one study dealing with the effect of the calciumchannel antagonist, verapamil, on gap junctional conductivity. In the acinusof the rat submandibular gland the uncoupling effect of the secretagogueacetylcholine as assessed in dye-coupling studies could be inhibited in thepresence of 10 lmol/l verapamil [Kanno et al., 1993]. This is probably due tothe antagonization of calcium influx. In control cells (without uncoupling byacetylcholine) verapamil did not influence cell coupling. Unfortunately, there


are no data on the verapamil action on cardiac cells previously uncoupled byany calcium agonistic process available.

Another possibility of influencing gap junctional coupling is the modula-tion of the activity of intracellular phosphatases and protein kinases usingokadaic acid, staurosporine and phorbol esters. It has been outlined in theprevious chapters that single-channel conductance of various connexins isregulated by phosphorylation and dephosphorylation processes. Thus, it canbe expected that drugs inhibiting or stimulating phosphatases or protein ki-nases can alter gap junctional conductance. Moreno et al. [1994b] found thattreatment of SKHep1 cells transfected with human Cx43 with okadaic acid(300 nmol/l), an inhibitor of phosphatases type 1 and 2A, changed the fre-quency distribution of unitary junctional conductance. Under the influenceof okadaic acid a shift in the single-channel conductances from higher tolower conductance favoring a 60-pS conductance state was observed. Underthese conditions the phosphorylation of human Cx43 was increased.

The reverse effect could be expected if phosphorylation of Cx43 wouldbe inhibited by an inhibitor of protein kinases. Such an inhibitor is staurospo-rine which inhibits PKC and cyclic nucleotide-dependent protein kinases. Mo-reno et al. [1994b] investigated the effect of 300 nmol/l staurosporine on single-channel conductance and observed a decrease in the frequency of 60-pS eventsand an increase in 100-pS events. Thus, the unitary conductance can be modu-lated pharmacologically. However, since the global gap junctional conductancedepends on single-channel conductance and open probability, the overall effecton coupling cannot be concluded from these experiments. Because PKC hasbeen shown to increase gj [Kwak & Jongsma, 1996; Spray and Burt, 1990], itcan be anticipated that staurosporine, as an inhibitor of this enzyme, mayexhibit a decreasing effect on gj. On the contrary, the overall effect of okadaicacid might consist of an increase in gj. However, more experiments will benecessary for a final statement.

In addition, it is possible to stimulate protein kinases directly by treatmentwith phorbol esters [Kwak and Jongsma, 1996; Moreno et al., 1994b; Munsterand Weingart, 1993]. Munster and Weingart [1993] reported that exposure ofneonatal rat heart cells to 100–160 nmol/l TPA, a stimulator of PKC, led toa rapid decrease in the gap junction conductance gj. The onset of uncouplingwas observed 2–9 min after TPA application and maximum uncoupling within2–4 min thereafter. They concluded that TPA may affect channel kineticsrather than the single-channel conductance cj. The TPA effect occurred onlyacutely; 24-hour exposure of the cells to TPA did not result in changes in gj

attributable to downregulation of PKC. The TPA-induced change in gj canbe prevented by pretreatment with the PKC inhibitor, staurosporine. In furtherexperiments Munster and Weingart [1993] showed that the TPA effect depends


on the free intracellular calcium concentration: at low intracellular calciumlevels (18 nmol/l) the uncoupling TPA effect can be observed, whereas at higherlevels (100 nmol/l) the effect becomes mitigated or is completely inhibited (160nmol/l). This finding is somewhat contradictory since it is well known thatPKC requires Ca2+ for its action. However, the calcium sensitivity of thevarious isoforms of PKC is different with PKC-b exhibiting substantial activityin the absence of calcium and an undefined isoform without calcium-sensitivity[Nishizuka, 1988]. In addition, the subcellular distribution of calcium underthese conditions is not clear. At present, it is not possible to interpret thiscalcium-PKC inhibitor interaction on a mechanistic level. In addition, it isnot clear what the effect of TPA or other phorbol esters on cellular couplingin intact tissue might be, since in intact cardiac tissue the resting intracellularcalcium concentration ranges to about 150 nmol/l [Wier et al., 1987].

Other investigators observed an increase in gj in response to TPA [Kwakand Jongsma, 1996; Spray and Burt, 1990]. Kwak and Jongsma [1996] foundan increase by 16×2% in gj in neonatal rat cardiomyocytes after applicationof 100 nmol/l TPA (intracellular calcium was buffered with 10 mmol/l EGTAin the pipette solution). TPA shifted the frequency distribution of unitaryconductances cj to lower sizes. However, TPA decreased dye coupling in theseand other experiments [Kwak et al., 1995a].

The uncoupling TPA effects can also be mimicked with synthetic diacylgly-cerol analogues such as 1-oleoyl-2-acetyl-glycerol [Munster and Weingart,1993] in concentrations of 250 lmol/l, which also activates PKC. Lower con-centrations are ineffective.

Besides these approaches which act at intracellular enzymes, modulationof the autonomous nervous system by receptor agonists can alter gap junctionconduction. The parasympathomimetic carbachol for example, a drug whichacts at muscarinic and nicotinic acetylcholine receptors and is not susceptibleto cholinesterases, raises intracellular cGMP and can thus be expected todecrease gap junctional coupling via cGMP-dependent protein kinase. Takens-Kwak and Jongsma [1992] investigated the influence of 100 lmol/l carbacholon gj in cultured neonatal rat cardiomyocytes. In the whole cell techniquecarbachol exposure decreased gj by 20%. The frequency distribution of unitarycurrents was shifted by carbachol from 43 pS to a lower cj of about 21 pS inheptanol-uncoupled cells. The authors argued that a cGMP-dependent proteinkinase phosphorylates the channel and thereby closes the 40- to 45-pS channelswithout affecting the other population of 20-pS channels. A similar decrease ingj was obtained using 1.5 mmol/l 8-bromo-cGMP. The carbachol effect was notseen in the perforated patch technique due probably to loss of an intracellularcytosolic phosphatase. In dye-coupling experiments Shibata et al. [1995] alsoobserved reduced coupling in response to 100 lmol/l carbachol in cultured adult


rat and guinea-pig cardiomyocytes. However, uncoupling occurred only in thepresence of calcium whereas in the absence of calcium carbachol did not repressdye coupling between the cells. The uncoupling effect of carbachol has also beenestablished in other cells, for example pancreatic acinar cells [Somogyi and Kolb,1989] or rat submandibular gland [Kanno et al., 1993]. In the latter the effect ofcarbachol could be prevented by coadministration of atropine and could bemimicked with the cholinomimetic natural alkaloid pilocarpine.

Sympathomimetics have also been studied with regard to their effects ongap junctional coupling. Epinephrine has been shown to increase the spreadof electrotonic potentials during diastolic depolarization [De Mello, 1986b]in canine Purkinje fibers. This was interpreted as an effect of the rise inintracellular cAMP resulting from b-adrenoceptor stimulation and subsequentformation of cAMP by adenylate cyclase which then activates PKA. Detailsregarding the regulation by cAMP and by PKA have been described in chapter4. Similarly, De Mello [1989] reported on an improvement in intercellular coup-ling by the b-adrenoceptor agonist isoproterenol in cardiac cell pairs. Thus,stimulation of b-adrenoceptors can be assumed to result in enhancement ofintercellular coupling, at least in some preparations. However, on the basis ofthe findings of Kwak and Jongsma [1996] on a lack of the effect of PKA to altergap junction conductance in rat cardiomyocytes, caution seems necessary andspecies variability or tissue variability seems to play an important role.

In other cells, i.e. in rat submandibular gland, adrenaline (100 lmol/l) hasbeen shown to decrease the percentage of dye-coupled cells [Kanno et al.,1993], whereas isoproterenol was ineffective, so that the authors concludedthat the mechanism was transmitted via action on the a-adrenoceptors. Thiswas supported since the adrenaline effect could be suppressed by coadministra-tion of 10 lmol/l phenoxybenzamine.

Similar to a b-adrenoceptor stimulation intracellular cAMP can be in-creased by inhibition of phosphodiesterase. Thus, in turtle retina cells, cAMPleads to uncoupling and this can be mimicked by stimulation of adenylatecyclase with forskolin and concomitant inhibition of phosphodiesterase byIBMX [Piccolino et al., 1984]. In cardiac cells inhibition of phosphodiesterasehas been investigated using methylxanthine derivates [De Mello, 1989], re-sulting in an enhancement of intercellular coupling.

It should be kept in mind that stimulation of a given protein kinase canincrease or decrease gap junctional conductance depending on the tissue andspecies studied. Thus, generalizations should be avoided.

Norepinephrine-dependent phosphorylation of connexins by PKC hasbeen described in liver cells expressing the 27-kD gap junction protein [Takedaet al., 1989]. It can be assumed that in cardiac cells this would lead to thesame effects as direct stimulation of PKC via phorbol esters. Indirect evidence


for an improvement in cellular coupling by norepinephrine via a1-adrenocep-tors has been found in isolated rabbit hearts perfused according to the Langen-dorff technique, exposed to increasing concentrations of norepinephrine in theabsence and presence of the b-blocker, propranolol, and the a1-adrenoceptorantagonist, prazosine [Dhein et al., 1993a]. It became obvious in that studythat norepinephrine decreased the dispersion of action potential durationmeasured at 256 ventricular electrodes after blockade of the b-adrenoceptorsby propranolol. The effect could be suppressed by prazosine. Since the enhance-ment of the dispersion of action potential duration can be the result of cellularuncoupling [Lesh et al., 1989], the authors interpreted this action as a possibleimprovement in intercellular coupling.

In addition to the modulators of the autonomic nervous system, angio-tensin II was found to be effective in regulating cardiac gap junction conduct-ance. In adult ventricular cell pairs De Mello [1992] observed a 55% reductionin gap junction conductance gj within 20 s following the administration of1 lg/ml angiotensin II (approximately 0.9 lmol/l) to the bath solution. Theangiotensin-II effect was reversible within 2.5–3 min. The concentration-re-sponse curve started at 10 nmol/l resulting in a decrease of 18%. The uncoup-ling angiotensin-II effect could be prevented by the PKC inhibitorstaurosporine and could be suppressed by DuP 753 (70 lg/ml), an angiotensinreceptor-blocking agent, whereas DuP 753 alone did not alter gj. In additionalexperiments De Mello [1992] investigated the influence of the angiotensin-converting enzyme inhibitor, enalapril (1 lg/ml). Enalapril was added to thebath solution and resulted in an increase of 106% within 4–5 min. The onsetof the effect took 1.5 min, probably the time needed in vitro for ester hydrolysisof enalapril to MK-422, the active metabolite. The enalapril effect was dose-dependent in the range from 0.25 to 1.25 lg/ml, reaching maximum effect at1.0 lg/ml. In further investigations De Mello [1994] examined the possibleexistence of an intracellular renin-angiotensin system. In this study using adultrat heart cells intracellular dialysis of 10 nmol/l angiotensin I resulted in adecrease in gj of 76% within 7 min. This could be completely inhibited byintracellular dialysis of 1 nmol/l enalaprilat. Intracellular dialysis of angio-tensin II led to a decrease in gj of 60% in 45 s and was sensitive to PKCinhibition. The author concluded that there is an intracellular synthesis ofangiotensin II and conversion of angiotensin I. Since the angiotensin-II effectcould be prevented by intracellular administration of the receptor antagonistDuP 753, De Mello [1994] concluded that there is an intracellular angiotensin-II receptor involved in the regulation of gj.

Taken together these investigations point to a possible influence of therenin-angiotensin system on cardiac cellular coupling. According to De Mello[1992, 1994] it can be argued that at least in parts the positive effects of


angiotensin-converting enzyme inhibitors and protective effects in regionalmyocardial ischemia can be attributed to this improvement in intercellularcommunication. In the light of the theory of arrhythmia formation focussingon the generation of reentry by slowing conduction and by enhanced disper-sion, this coupling effect of an angiotensin-converting enzyme inhibitor canbe seen as an antiarrhythmic action. In 11-month-old cardiomyopathic ham-sters the uncoupling action of angiotensin II was most pronounced in poorlycoupled cells and could be enhanced by enalaprilat [De Mello, 1996]. Theauthor concluded that the decrease in intercellular coupling in cardiomyopathymay in part be due to an activation of the cardiac renin-angiotensin system.Renin, angiotensin I, angiotensin II and angiotensin-converting enzyme havebeen found in cardiomyocytes using immunofluorescent staining [Dostal et al.,1992] with the enzyme being located in the perinuclear region.

Another group of drugs affecting gap junctional conductance are theantiarrhythmic peptides [for a detailed review see, Dhein and Tudyka, 1995].In 1980 a hexapeptide with a molecular weight of 470 was isolated from bovineatria by Aonuma et al. [1980b]. This peptide improved synchronization ofembryonic chick heart cell aggregates, and was thus proposed to possessantiarrhythmic actvity. From bovine atria 200 lg/kg wet tissue of the purepeptide were yielded. Its antiarrhythmic action was established in neonatalrat cardiomyocytes. Fibrillation induced by either ouabain, 3 mmol/l Ca2+ or0.7 mmol/l K+ was converted to regular beating by 0.1 mg/l of the peptide.If added to the cell culture medium it increased the number of beating centers,the relative content of spreading cells and protein synthesis [Aonuma et al.,1980a]. In later studies the antiarrhythmic peptide (10 mg/kg) was shown tobe effective in vivo against CaCl2-induced and aconitine-induced arrhythmiain mice [Kohama et al., 1987]. Moreover it prevented fibrillation in dogs andrats [Aonuma et al., 1983]. In an ouabain and an ADP model the time to theonset of arrhythmia was prolonged by the antiarrhythmic peptide, while itfailed to prevent epinephrine-induced arrhythmia. In subsequent investigationsthe amino acid sequence was determined as H2N-Gly-Pro-4Hyp-Gly-Ala-Gly-COOH [Aonuma et al., 1982] and tissue levels could be measured using aradioimmunoassay in the heart (203 pmol/g), kidney (165 pmol/g) and blood(3.8 pmol/g) [Kohama et al., 1985]. Interestingly, in the course of CaCl2- andaconitine-induced arrhythmias the tissue levels in the heart, but not the kidney,increased, whereas in epinephrine-induced arrhythmia the tissue level wasfound to have decreased [Kohama et al., 1986]. In contrast, plasma levels wereincreased 3-fold in all 3 forms of arrhythmia.

Until that point the mechanism of action of the peptide remained unclear.The first investigation directed toward the elucidation of the underlying mecha-nism of action revealed that the antiarrhythmic peptide did not alter depolar-


ization velocity, action potential amplitude, duration or shape and did notexhibit any action on muscarinic receptors in canine Purkinje fibers [Argentieriet al., 1989], so that the authors concluded that the mechanism consisted ofeffects on passive membrane properties or other actions rather than an actionon membrane ionic currents. From the experiments carried out by Dhein et al.[1994, 1995b, 1996] and Muller et al. [1997a, b] it was concluded that theantiarrhythmic peptide and a synthetic derivative improve cellular couplingby an increase in gap junctional conductance.

Dhein et al. [1994] assessed the effects of the antiarrythmic peptide andseveral synthetic derivatives, synthesized according to the Merrifield techniqueusing the Fmoc strategy, in isolated rabbit hearts submitted to regional isch-emia. The action of the antiarrhyhtmic peptides under normal conditions wasa reduction in the dispersion of the action potential duration measured at 256ventricular unipolar electrodes. The antiarrhythmic peptide AAP10 (H2N-Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH2), an amide, was found to be the most effectivewith an onset of action at 0.1 nmol/l and maximum effect at 10 nmol/l. Theaction consisted of a homogenization of the action potential duration so thatlocal differences became smoothed without affecting the mean action potentialduration as shown in figure 22. The peptide did not exhibit any other influenceon cardiac parameters (e.g. left ventricular pressure, coronary flow, QRS dura-tion, PQ time). If hearts were submitted to regional ischemia by LAD occlusionfor 30 min, pretreatment with 10 nmol/l AAP10 led to a significant reductionin the ischemia-induced alterations in the activation patterns and to a reductionin the incidence of ventricular fibrillation, especially of late phase VF (typeIb) [Dhein et al., 1994, 1995b, 1996].

In order to clarify the mechanism of action Dhein et al. [1994] investigatedthe effects of AAP10 on the transmembrane action potential in isolated papil-lary muscles of guinea-pig heart. They found no effect on action potentialduration and morphology, on action potential amplitude, on maximum up-stroke velocity or on resting membrane potential in concentrations up to1 lmol/l. However, they observed a reduction in the coupling time within1 min after application, i.e. in the interval between the stimulus and the propa-gated action potential. The effect was reversible on wash out. Because due tothe lack of effect on the maximum upstroke velocity an effect on the sodiumcurrent could be ruled out, the reduction in coupling time was a first stronghint of a possible action on the gap junctions. Thus, the authors decided toinvestigate the effect of the antiarrhythmic peptide AAP10 on gap junctionalcurrent in adult guinea-pig ventricular cardiomyocytes directly using thedouble-cell voltage clamp (whole cell patch configuration).

In these experiments they found an improvement in gj by 10 nmol/l AAP10.It took about 2 minutes until the onset of this effect. The effect could be


Fig. 22. Reduction in dispersion of the ventricular action potential duration by thesynthetic antiarrhythmic peptide AAP10. The distribution of the action potential duration(assessed as the epicardial activation-recovery interval, ARI) on the surface of an isolated rabbitheart before and after treatment with AAP10. Note the greater variability of the epicardialaction potential duration (ARI) before administration of AAP10 [Dhein et al., 1997c].


Fig. 23. Survey of the various pharmacological interventions at the gap junctionalcoupling. For details see text.

washed out within several minutes [Dhein et al., 1995b; Muller et al., 1997a, b].They showed that the spontaneous decline in gj by Ö2.5 nS/min was reversedby AAP10, so that an increase of 1 nS/min was seen. In subsequent experimentsguinea-pig papillary muscles were submitted to hypoxia and glucose-free perfu-sion, so that they uncoupled after 12 min. This could be prevented by pretreat-ment with 10 nmol/l AAP10 [Dhein et al., 1997b; Muller et al., 1997b]. Sincein such low concentrations only a very slight effect on coupling time wasseen under normoxic conditions, the authors concluded that AAP10 mightpreferentially act on uncoupled cells. With regard to the antiarrhythmic actionthey favored the hypothesis that improvement in gap junctional couplingreduces action potential dispersion and prevents slowing of conduction byuncoupling, thereby preventing arrhythmia. This is in line with a stabilization


of the activation patterns under ischemic conditions as observed [Dhein et al.,1994, 1996, 1997c].

What are the therapeutic implications? According to De Mello [1986b],cellular coupling is mainly influenced by the intercellular axial resistivity in thedirection of propagation (Ri) and is inversely dependent on the nonjunctionalsarcoplasmic membrane resistance (Rm). As was described in the foregoingchapters Ri can be altered by a large number of factors and diseases. Changesin cellular coupling can be expected to alter conduction velocity and synchro-nization of the cells. Because under normal conditions the cells are more orless well coupled, it can be assumed that in most pathological states (e.g.regional ischemia, heart failure, acidosis, hypoxia, Chagas disease and others)coupling will be reduced resulting in slowing of conduction and possibleenhancement of dispersion, which both can make the heart more prone toreentrant arrhythmia. Thus, it can be imagined that such drugs, enhancingcellular coupling, may prevent arrhythmia in these states characterized byreduced coupling. However, until now they have only been shown to exert aprophylactic effect and it is questionable whether enhancement of couplingduring manifest arrhythmia is more effective than the existing classic antiar-rhythmics. In situations with uncoupling being due to structural changes suchas fibrosis, drugs which can improve gap junctional coupling are probablyineffective since they can enhance coupling only in functional gap junctions.In these situations one can speculate whether enhanced expression of gapjunctions might be useful but it has not yet been shown experimentally. Theantiarrhythmic peptides have been shown to be effective in the prophylaxis ofischemia-associated ventricular fibrillation (type-Ib arrhythmia) [Dhein et al.,1994, 1996] and ouabain-induced arrhythmia [Aonuma et al., 1980b], a statewhich is known to be associated with uncoupling [De Mello, 1976]. Accordingto the considerations at the beginning of this chapter, it might be advantageousthat at least AAP10 seems to act preferentially in uncoupled cells.

For the problem of prophylactic antiarrhythmic treatment these antiar-rhythmic peptides are probably not the final solution. Because of their peptidenature they are not well suited for in vivo studies and they are probably onlyindicated for the prevention of arrhythmias due to reduced coupling, but theyare a first step in the direction of a new class of drugs influencing gap junctionalcoupling.

The various pharmacological approaches to the modulation of gap junc-tional coupling are summarized in figure 23.


8...........................

Methods for Investigation ofGap Junctions

In this chapter more detailed information on the double-cell voltage-clamp setup and protocols for assessing gap junctional conductivity is given,as well as a description of the cell-isolation procedure for this purpose andcell culture models. Information on immunocytochemical localization of gapjunctions and on the experimental procedure of preparing specimens and slidesfor immunohistology is given. A protocol for isolation of gap junction proteinsis also outlined. Readers interested in more details of the cell-culture techniqueregarding incubators, sterile technique, etc., and different isolation and cultureprotocols are referred to more specialized literature [Lindl and Bauer, 1994;Piper, 1990].

Many experiments on gap junctions have been carried out using theneonatal rat heart cells. Thus, the procedure of isolating and culturing thesecells (as used in the author’s laboratory) will be discussed below.

8.1 Culture of Neonatal Rat Cardiomyocytes

Prepare the following solutions:

Coating solutionM199 (with Earl’s salts)100 lg/ml penicillin100 lg/ml streptomycin10% fetal calf serum (FCS)

PBS/glucose solutionNaCl 137 mmol/lKCl 2.7 mmol/lNa2HPO4 8.3 mmol/lKH2PO4 1.5 mmol/lGlucose 20 mmol/lpH to be adjusted to 7.4

Desaggregation solutionPhosphate-buffered saline (PBS) 50 mlGlucose 200 mgBovine serum albumin (BSA) 500 mgCollagenase type II (Gibco) 50 mg (204 U/mg)

106

Medium for resuspension of supernatantsM199 (with Hanks’ salts and HEPES)100 lg/ml penicillin100 lg/ml streptomycin10% FCS25 mmol/l HEPES2 mmol/l L-glutamine

Culture medium for the 1st dayM199 (with Earl’s salts and HEPES)2 mmol/l L-glutamine5% FCS100 lg/ml penicillin100 lg/ml streptomycin

Culture medium after the 1st dayM199 (with Earl’s salts and HEPES)2 mmol/l L-glutamine1% FCS100 lg/ml penicillin100 lg/ml streptomycin

Dulbecco’s wash solution for the 2nd dayNaCl 137 mmol/lKCl 2.68 mmol/lNa2HPO4·2H2O 6.48 mmol/lKH2PO4 1.47 mmol/lMgCl2·6H2O 0.49 mmol/lMgSO4·7H2O 0.81 mmol/lCaCl2 0.9 mmol/lpH to be adjusted to 7.2

The culture dishes have to be prepared 24 h before use. They have to be coated withthe coating medium.

Neonatal rats (1–2 days old) are killed by decapitation and then sprayed with 70%ethanol for desinfection. After thoracotomy the pericard is opened and the heart removed.Bath the organ in ice-cold PBS/glucose solution in a Petri dish to remove blood. Removeatria, transfer the heart to another Petri dish, chop up the ventricles with two sterile scalpelsand incubate in 7 ml desaggregation solution and stir gently (140 rpm) at 37 ºC. Allowsedimentation of the tissue and remove the supernatant. Add fresh dissociation solution andrepeat this procedure 6 times. Suspend the supernatants in the medium for resuspension ofsupernatants (each in 8–9 ml, ice-cold).

Centrifuge these cell solutions for 5 min at 700 rpm and resuspend the pellet in culturemedium for the 1st day. Seed the cells in 25-cm2 plastic flasks and incubate at 37 ºC/5% CO2.After 2 h of preplating (this time is required for nonmuscular cells to attach) the supernatantof this flask is filtered through a nylon mesh (pore width 100 lm) and then seeded in Petri dishesat a density of about 10,000 to 100,000 cells/cm2 in culture medium for the 1st day. After 24 hthe Petri dishes are rinsed off with Dulbecco’s wash solution and culture medium for the 1st

1078 Methods for Investigation of Gap Junctions

day (5% FCS) is added. 72 h after preparation half of this medium is removed and replaced bythe 1% FCS medium. Thereafter (i.e. in the following days), cells are incubated with the 1%FCS medium only. One may add 10% horse serum in order to inhibit fibroblast growth.

8.2 Culture of Embryonic Chick Cardiomyocytes

Cultured embryonic chick heart cells often form coupled cell pairs or aggregates whichmay be studied using the double-cell voltage-clamp technique. In the following an isolationand culture protocol is given as used in the author’s laboratory. Besides this, other protocolsmay also suit.

Prepare the following solutions:

Glutamine solutionGlutamine 200 mmol/l

Hanks’ buffered saline solution (HBSS)NaCl 8,000 mg/lKCl 400 mg/lKH2PO4 60 mg/lNa2HPO4 47.5 mg/lNaHCO3 350 mg/lGlucose 1,000 mg/lpH 7.4

Trypsin solution (0.25%)Trypsin 2.5% in HBSS (+phenol red) 3 mlHBSS 27 ml

Culture mediumFCS 4%Horse serum 2%Glutamine solution 3.4 ml/lPenicillin 100 IU/mlStreptomycin 100 IU/mlM199 add 1 liter

All material used for isolation and culture of the cells must be sterile, all media haveto be autoclaved.

Take 10 eggs (5–7 days old; make sure that they are kept at the same temperature untilthe start of the procedure), wash with 70% ethanol and open at the egg’s pole under sterileconditions in laminar flow. Decapitate the embryo, isolate the heart from the embryo andremove the atria. Bath the ventricles in 5–10 ml HBSS at room temperature in a Petri dish.

Chop up the ventricle with sharp scalpels and transfer to a watch-glass. Add somemilliliters of the 0.25% trypsin solution at 37 ºC for 7 min. Thereafter mix the solution welland filter through gauze with a mesh size of 100 lm in a Falcon tube containing 10 ml HBSS(4 ºC) and 4% FCS. Centrifuge for 5 min at 1,500 rpm and 4 ºC. Resuspend the pellet withHBSS, centrifuge again and repeat the procedure once again. Dissolve the pellet in about 10 ml


culture solution (M199 with supplement as described) so that a cell density of nearly 500,000cells/ml is yielded. Seed in plastic cell culture flasks and incubate for at least 24 h. After thistime, aggregates can be observed which exhibit spontaneous activity and in the periphery ofthese aggregates cell pairs are often found. The medium has to be changed every 24 h.

8.3 Isolation of Adult Guinea-Pig Cardiomyocytes

If adult cardiomyocyctes are used for the double-cell voltage-clamp technique, it is desir-able to yield a high amount of cell pairs. Therefore, protocols using proteases such as trypsinshould notbe used,because proteases mayenhance separationof the cellsand disruptor destroythe gap junctions. Similarly, protocols using proteases are critical if ion channels or receptorswith large extracellular protein domains are to be investigated in freshly isolated cells. In theauthor’s laboratory a collagenase protocol is commonly used to isolate adult guinea-pig cardio-myocyte pairs, and is described below. It should be noted that this is surely not the ‘only true’protocol, but it is a suitable one. First of all the following solutions have to be prepared.

Solution A3 mol/l NaCl 21 ml3 mol/l KCl 783 ll0.1 mol/l KH2PO4 6 ml0.1 mol/l MgSO4 12.5 ml0.4 mol/l NaHCO3 31.1 ml0.5 mol/l HEPES/Na 5 mlGlucose 0.54 gH2O add 500 ml

Solution BPyrovate 44 mgBSA 200 mgSolution A add 200 mlEquilibrate with 95% O2 and 5% CO2, adjust pH to 7.4

Solution C1 vial collagenase Worthington type 100 U/ml12.5 ll 0.1 mol/l CaCl2

Solution B add 10 ml

Solution DSolution B 20 ml0.1 mol/l CaCl2 10 mlEquilibrate with 95% O2 and 5% CO2

Solution E (only for short-term culture of the cells)FCS 10% 1 mlPenicillin 2.4 mg (or 100 IU/ml)Streptomycin 4 mg (100 IU/ml)Glutamine 200 mmol/l 100 llM199 add 20 ml


After the guinea pig (250–350 g) has been killed, the heart is prepared according tothe Langendorff technique and transferred to a Langendorff apparatus and perfused at 37 ºCfor 10 min with solution B (i.e. Ca2+-free saline). Thereafter, the heart is perfused withsolution C in a recirculating manner for about 30 min. It is important to make sure thatboth solutions do not get mixed. While perfusing with the collagenase solution, the heartbecomes ‘slimy’ and a little transparent. During this phase the perfusion rate often has tobe reduced. At the end of this period the heart is removed from the apparatus, atria areremoved and the remaining heart is cut into small pieces using two scalpels. The tissue istransferred to a glass test tube and dissolved with the rest of the collagenase solution, slightlygassed with 95% O2 and 5% CO2 at 37 ºC.

Next, the solution containing the tissue is filtered through gauze with a mesh size of200 lm and centrifuged for 1 min at about 600 rpm. After removing the supernatant, thepellet is resuspended in 10 ml solution D (37 ºC; no longer gas the solution). Again thesolution is centrifuged for 1 min at 600 rpm and the pellet resuspended in solution D. After1 min the calcium concentration in the solution is gradually increased (this is a criticalphase for the cells, because during this procedure many cells are impaired) by adding 5 ll0.1 mol/l CaCl2, 7.5 ll after another minute, 7.5 ll after the next minute and 10 and 15 llin the following 2 min, thereby adjusting the calcium concentration finally to about 0.45mmol/l.

Following these steps, the solution is centrifuged a last time for 1 min at 600 rpm andthe resulting pellet is either resuspended in 20 ml solution B (with additional Ca2+) or inTyrode solution, if the cells are to be used immediately for an experiment, or the pellet isdissolved in solution E for cultivating the cells. For this purpose 2 ml of the cell-containingsolution are dissolved with 5 ml solution E in Petri dishes (3 cm diameter) and kept in theincubator for a maximum of 3 days. However, it should be noted that the content of cellpairs gradually declines with time and is maximum shortly after isolation.

8.4 Immunohistochemistry

To detect gap junction proteins and their distribution within the tissue, immunohisto-chemical methods are commonly used. The best results are obtained with frozen sections,since other methods such as paraffin embedding or acrylate embedding may affect theantigeneity of the proteins to be detected (this is because tissue fixation, for example, glutaral-dehyde or formaldehyde cross-link proteins), although several laboratories have also usedthese embedding techniques with success. If tissue has to be transported prior to freezing,for example from the clincal theater to the laboratory, it is recommended to keep the delayto freezing as short as possible and to transport the tissue in cooled (5 ºC) tissue culturemedium (such as RPMI) or in cooled saline.

To freeze the tissue the following protocol can be used.

Perfuse the heart with a mixture of saline-buffered solution (e.g. Tyrode’s solution) andglycerine (1:1)Let the fluid drainTransfer the heart to a methanol bath (pre-cooled to Ö70 ºC)Let the methanol drainTransfer the tissue to fluid N2


In the next step sections have to be prepared from the tissue block. Frozen sections shouldbe cut at 5-lm thickness and be picked on aminopropyltriethoxysilane (APES)-coated slides(alternative: Superfrost slides) using a cryostat at Ö25 ºC. If small tissue samples are used,these can be embedded in Tissue-Tek to facilitate sectioning. The sections have to be air-dryedovernight. Thereafter, sections are fixed in acetone for 20 min (alternatively in methanol,30 min). Thereafter, sections are incubated for 30 min in Triton X–100 (0.1%; this step is option-ally,butcanbeusedtopermeabilize the tissueorculturedcells if theantibodybinds to intracellu-lar-binding sites, and can facilitate access of the antibody to the binding site). This is followedby coating with 1% BSA (fatty acid free) in PBS at pH 7.5 for 20 min for blocking unspecificprotein-binding sites in the specimen. Next, the section is exposed to the primary antibody(e.g. monoclonal mouse anti-rat Cx43, epitope: amino acids 252–270; Biermann GmbH, BadNauheim, Germany) at a working dilution of 1:100 for 1 h. Thereafter, the specimen has to bewashed in order to remove antibody which was not bound by specific sites (wash with PBS and1% BSA for 20 min, change wash solution 3 times). In the next step the secondary antibodyhas to be coupled to the primary in order to make it visible. In our laboratory, we use FITC-coupled secondary antibodies or DTAF-coupled (DTAF has the advantage of less bleechingduring exposure to the excitation light; for the primary antibody mentioned above a FITC-labeled goat anti-mouse IgG antibody, Sigma was used in the author’s laboratory. Using fluor-escence microscopy is a very elegant procedure which should be preferred if possible. However,in some cases problems may arise from unspecific fluorescence or, for certain investigations,from the fact that the structure of the tissue cannot be visualized simultaneously. In such cases,alternatively, secondary antibodies coupled to peroxidase or to alkaline phosphatase can beused. However, these have the disadvantage that unspecific reactions with tissue componentsare possible. A possible alternative is the use of the streptavidin-biotin method (see below).Next, the specimens are incubated with the secondary antibody for another hour at workingdilutions of 1:300 to 1:1,000. Thereafter, the slides are washed with PBS at pH 7.5 for 20 min,embedded in karyon and covered with a coverslip, dryed at 4 ºC (overnight, keep in the dark)and sealed with acryl varnish (nail varnish is also suitable). FITC and DTAF are both excitedwith blue light (at k>470 nm; excitation filters: 2·SP 490+2 mm LP 455) and emit green fluor-escent light (at k>540 nm; emission filter; barrier filter; LP 515).

Protocol for immunostaining (as used in the author’s laboratory)30 min 0.1% Triton X-10020 min 1% BSA in PBS (pH 7.5)1 h primary antibody (1:100)20 min PBS+1% BSA (pH 7.5)1 h secondary antibody 1:300 to 1:1,00020 min PBS (pH 7.5)Embed in karyon, cover with coverslip, dry at 4 ºC and seal with acryl varnish

PBS: 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4, add H2O to a final volume of 1liter. Adjust pH as desired.

The antibody stock solution should contain 1% BSA in PBS.

In the following, protocols for alkaline phosphatase-coupled, peroxidase-coupled anti-bodies and for the streptavidin-biotin system are given [Jackson and Blythe, 1993; Ormerodand Imrie, 1989].


If a biotinylated secondary antibody is used for the streptavidin-biotin method thefollowing protocol is suitable for frozen sections:

Air-dryed frozen sections have to be rehydrated in Tris-buffered saline (TBS)Let the fluid drainIncubate with primary antibody in TBS for 1 hWash with TBS (20 min, 3 times)Let the fluid drainIncubate with the biotinylated secondary antibody (30 min, antibody dilutions of 1:200 areoften suitable)Wash with TBS (20 min, 3 times)Make streptavidin-biotin/horseradish peroxidase complex from: 20 ll streptavidin, 20 ll bio-tinylated horseradish peroxidase, 1 ml TBS (shake well and wait 30 min)Let the fluid drain from the sections and incubate with the streptavidin-biotin/horseradishperoxidase complex for 30–60 minWash with TBS (20 min, 3 times)Develop the peroxidase in 3,3-diaminobenzidine tetrahydrochloride (DAB) solution: 3 mlDAB stock solution and 12 drops hydrogen peroxide (30% w/v) are dissolved in 400 ml TBS;incubate the specimen with this solution for 10 min (perhaps longer, control with microscope)Wash with tap waterIncubate with copper sulfate solution for 5 minWash with tap waterCounterstain as desiredEmbed in karyon, cover with coverslip, dry and seal with acryl varnish

TBS (0.5 mol/l, pH 7.6): 60.5 g tris(hydroxyl)methylamine are dissolved in 750 ml H2O, pHis adjusted to 7.6 with HCl, 85 g NaCl is added with H2O to a final volume of 10 litersDAB stock solution: 7.5 g DAB in 300 ml Tris buffer at pH 7.6 (caution, DAB is carcino-genous)Copper sulfate solution: 4 g CuSO4, 7.2 g NaCl, add to 1 liter H2O

For peroxidase-coupled secondary antibody the following protocol may be used:Air-dryed frozen sections have to be rehydrated in TBSLet the fluid drainIncubate with primary antibody in TBS for 1 hWash with TBS (20 min, 3 times)Let the fluid drainBlock the endogenous enzyme with 0.3% H2O2 for 30 min (alternatively, incubate in 2.3%periodic acid for 5 min, wash with water, rinse in 0.03% potassium borohydride and wash),or incubate for 5 min in 0.1% phenylhydrazine in PBS)Incubate with the peroxidase-conjugated secondary antibody (30 min)Wash with TBS (20 min, 3 times)Develop the peroxidase in DAB solution: 3 ml DAB stock solution and 12 drops hydrogenperoxide (30% w/v) are dissolved in 400 ml TBS, incubate the specimen with that solutionfor 10 min (perhaps longer, control with microscope)Wash with tap waterIncubate with copper sulfate solution for 5 minWash with tap water


Counterstain as desiredEmbed in karyon, cover with coverslip, dry and seal with acryl varnish

Alternatively alkaline phosphatase-coupled secondary antibody can be used with an anti-alkaline phosphatase antibody (APAAP technique). For this method the following protocolmay be used:Air-dryed frozen sections have to be rehydrated in TBSBlock the endogenous enzyme with 20% acetic acid for 5 minWash well with waterLet the fluid drainIncubate with primary antibody in TBS for 1 hWash with TBS (20 min, 3 times)Let the fluid drainIncubate with the alkaline phosphatase-conjugated secondary antibody (30 min)Wash with TBS (20 min, 3 times)Wash with waterDevelop the alkaline phosphatase with fast red: dissolve 5 mg sodium naphthol AS BIphosphate in dimethylformamide (few drops) and add to 5 mg fast red TR salt in 10 mlveronal acetate buffer (pH>9.2), incubate the slides for 1 hWash with tap waterCounterstain as desiredEmbed in karyon, cover with coverslip, dry and seal with acryl varnish

It should be noted that there is not the ‘one’ protocol for immunohistology, but manyvariations and the investigator has to find the most suitable for his purposes by varying theseveral steps. However, some problems often encountered with immunohistology will bediscussed briefly. The antibodies may bind to unspecific protein-binding sites which can beavoided with blocking agents such as BSA and gelatin. Some antibodies may react withcharged surfaces or charged proteins. This can be reduced with surfactant reagents as Tween20 and NaCl. In the tissue there can be Fc fragment-binding sites which can be blocked byincubation with serum. Alternatively the F(ab�)2 fraction of antibody may be used. It maybe possible that the antibody used can react with the same or a similar epitope in otherproteins of the host tissue (especially with interstitial areas). Cross-reactivity from the dilutedsecond antibody may be absorbed by adding to it 1–2% of serum of the host species.Antibodies and streptavidin may be bound by basic amino acids of the tissue. This can beinhibited by adding 2 mg/ml of the basic peptide poly-L-lysine (MW 3,000–6,000) to thediluted antibody. Disturbing autofluorescence may be reduced by staining the tissue withpontamine sky blue (0.05% in PBS with 1% dimethylsulfoxide) for 30 min before applyingthe first antibody.

Controls have to be carried out: incubation (and development without incubation withthe primary antibody, i.e. only with secondary antibody) to control for nonspecific reactionsof the second antibody. In addition, if immunofluorescence is used, unstained slides have tobe investigated also as controls in order to detect nonspecific background fluorescence (forexample of connective tissue), which may in some cases be enhanced by some drugs whichwere administered prior to taking the sample.

Using immunohistology, it is possible to determine the distribution of a specific connexinwithin the tissue or within cells. It can also be used for semiquantitative evaluations by


counting, for example, the percentage of positively stained cells among all cells in a viewfield, or measuring the positively stained membrane length in comparison with the totalmembrane length. This has to be repeated with many sections and many view fields, andwill then give reliable results. A quantification of the protein content by measuring theintensity of staining (e.g. of immunoflourescence) is only possible if the primary antibodyis labeled, as it cannot be assessed how many molecules of the labeled secondary antibodywill bind to the primary. However, such quantitative immunofluorescence is extremely errorprone and difficult, and great caution has to be taken in order to garantee that there is nodifference in bleeching between the samples, the optical system is unchanged, the backgroundis corrected exactly in the same way and so on. Thus, if quantification of the connexincontent is desired, other methods are recommended, as the direct isolation of connexin (seenext section).

8.5 Isolation of Cx43

Often the question arises, whether the total amount of connexin is altered. Becauseimmunohistology is mostly a semiquantitative method, the following biochemical methodfor isolation of gap junctions may be a suitable alternative. After isolation of the gap junctionpellet SDS-PAGE has to be carried out and the gels have to be stained. For control animmunoblot is recommended.

Protocol for isolation of gap junctions (as used in the author’s laboratory, adapted fromManjunath et al. [1982]). The quantities refer to the initial tissue amount of a rabbit heart.All steps have to be carried at 0–4 ºC if not stated otherwise.1 Transfer tissue (heart) to cold 1 mmol/l NaHCO3 (pH 8.2)2 Remove superficial fat and vessels3 Cut the tissue into small pieces and add these to 50 ml 1 mmol/l NaHCO3 (pH 8.2)4 Add phenylmethylsulfonyl fluoride (PMSF) to a final PMSF concentration of 1 mmol/l5 Stir 15 min at 4 ºC6 Homogenize tissue: 60 s, Vmax (Virtis homogenizer)7 Further homogenization using tissue mizer, SDT 100 EN8 Dilute the homogenate to 300 ml with 1 mmol/l NaHCO3 (pH 8.2), and filter through

6–8 sheets of medical gauze9 Centrifuge the filtrate: 15 min, 33,000 g

10 Remove the supernatant and disolve the resulting pellet in 300 ml 1 mmol/l NaHCO3

(pH 8.2)11 Centrifuge: 15 min, 33,000 g12 Remove supernatant and resuspend pellet in 100 ml 0.6 mol/l KI, 6 mmol/l Na2S2O3 in

1 mmol/l NaHCO3 (pH 8.2)13 Add PMSF (as step 4)14 Stir overnight at 4 ºC15 Filter through 6 sheets medical gauze16 Centrifuge: 30 min, 27,000 g17 Remove supernatant, resuspend the pellet in 100 ml 0.6 mol/l KI, 6 mmol/l Na2S2O3 in

1 mmol/l NaHCO3 (pH 8.2), homogenize in homogenizer (3 strokes only)


18 Centrifuge: 30 min, 27,000 g19 Remove supernatant, wash pellet in 70 ml (final volume) 0.6 mol/l KI, 6 mmol/l Na2S2O3

in 1 mmol/l NaHCO3 (pH 8.2)20 Centrifuge: 15 min, 27,000 g21 Remove supernatant, wash pellet in 30 ml 5 mmol/l Tris (pH 10) at room temperature22 Homogenize the solution in homogenizer (3 strokes only)23 While gently stirring add 30 ml 0.6% N-lauroylsarcosine in 5 mmol/l Tris (pH 10)24 Stir at room temperature for 10 min25 Prepare a density gradient in a centrifuge tube and add the solution, from top to bottom:

20 ml sample volume; 8 ml 35% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10),and 5 ml 44.5% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10)

26 Allow to stand for 20 min at room temperature27 Centrifuge: 60 min, 65,000 g, 15 ºC28 Take the band at the 35–44.5% interface and dissolve in 45 ml 0.3% deoxycholate in

5 mmol/l Tris (pH 10)29 Prepare a density gradient and add the solution, from top to bottom: 15 ml sample

volume; 9 ml 35% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10), and 9 ml 44.5%sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10)

30 Centrifuge: 60 min, 65,000 g, 15 ºC31 Take the band at the 35–44.5% interface and dissolve in 45 ml 0.3% deoxycholate in

5 mmol/l Tris (pH 10)32 Repeat step 29 with 15 ml of the solution33 Take the band at the 35–44.5% interface and dissolve 1:1 in 0.3% deoxycholate in

5 mmol/l Tris (pH 10)34 Centrifuge: 30 min, 106,000 g, 15 ºC35 Wash the pellet in 5 mmol/l Tris (pH 10)36 The pellet contains the gap junctional pellet contaminated with nonjunctional

membranes37 Prepare a density gradient and add the solution, from top to bottom: sample; 9 ml

31.5% sucrose, 0.3% deoxycholate in 5 mmol/l Tris (pH 10), and 9 ml 35% sucrose, 0.3%deoxycholate in 5 mmol/l Tris (pH 10)

38 Centrifuge: 90 min, 65,000 g, 15 ºC39 Take the material at the 31.5–35% interface40 Centrifuge: 30 min, 106,000 g, 15 ºC41 Wash as step 3542 Pellet: gap junction pellet

Following this isolation of the gap junction pellet the connexin has to be isolated usingdiscontinuous gel electrophoresis (SDS-PAGE). The stacking gel and separating gel can beprepared according to the following recipies (as used in the author’s laboratory for Maxigel,Biometra, Germany):

Stacking gel 5% (volume 6 ml)Acrylamide: 30% (w/v), 0.8% (w/v) bisacrylamide in water: 1 mlTris/HCl: 0.625 mol/l (pH 6.8): 1.2 mlSodium dodecyl sulfate (SDS): 0.5% (w/v) in water: 1.2 mlH2O: 2.6 ml


Ammonium persulfate: 10% (w/v) in water: 30 llN,N,N�,N�-tetramethylethylenediamine (TEMED): 6 ll

Separating gel 12.5% (volume 30 ml)Acrylamide: 30% (w/v), 0.8% (w/v) bisacrylamide in water: 12.5 mlTris/HCl: 1.88 mol/l (pH 8.8): 6 mlSDS: 0.5% (w/v) in water: 6 mlH2O: 5.5 mlAmmonium persulfate: 10% (w/v) in water: 150 llTEMED: 30 ll

Sample buffer0.5 mol/l Tris (pH 6.8) 1.2 ml20% SDS in H2O 1 mlGlycerin 1 ml5% b-Mercaptoethanol0.1% (v/v) bromphenol blue in ethanol 0.5 mlThe sample buffer is distributed in Eppendorf tubes in amounts of 500 ll. Finally, 25 ll b-mercaptoethanol is added prior to use.

Running buffer0.18 mol/l Tris (12.1 g/l)0.1 mol/l glycin (7.5 g/l)3 mmol/l SDS (1 g/l)

Staining solution500 ml methanol500 ml concentrated acetic acid100 ml H2O2.5 g Coomassie brillant blue R-250

Destaining wash solution50 ml methanol75 ml concentrated acetic acid

Prepare a 1-mm gel from these gel solutions with 10 indentations for 50-ll samples.Fill the indentations with running buffer and add 40 ll of sample (the sample is dissolved1:1 in sample buffer). It is necessary to reserve one lane for a molecular weight marker. Torun the stacking gel 25 mA is used, for the running gel 60 mA (this refers to Maxigel,Biometra, Germany). After running, the gel is bathed in the staining solution for 30 min.Thereafter, it is washed in the destaining wash solution for 24 h. Finally the gel is dryed at60 ºC for 90 min.

8.6 Double-Cell Voltage-Clamp

Very often it is necessary to measure the conductance of gap junctional channels. Inprinciple, there are two possibilities: one can measure the single-channel conductance, orthe total conductance between two cells. It is important to make this choice before setting


up the experiment, because for these two kinds of investigation, different amplifiers may beused. As pointed out in a previous chapter the serial resistance of the pipettes may disturbaccurate measurement of the total resistance between two cells. The problem arises from thechange in potential before and after the pipette (which serves as a resistor) during currentinjection via this serial resistance. According to a paper by Wilders and Jongsma [1992] theseries resistance resulting from the pipette and from the cytoplasm can make it difficult oreven impossible to accurately measure the voltage dependence of gap junctions.

This series resistance problem can be avoided by the use of discontinuous single-electrode voltage-clamp (dSEVC) amplifiers or switch-clamp amplifiers. These amplifiersswitch between voltage measurement and current injection thereby avoiding artefacts by theresistance of the pipettes. The use of switch-clamp amplifiers is, from a present point of view,recommended for the measurement of the total conductance between two cells. However, itshould be noted that the use of these amplifiers is a bit more complicated than that ofnormal patch-clamp or voltage-clamp amplifiers, since the switching frequency, gain, dutycycle and capacity compensation have to be adjusted very accurately. Since voltage shouldbe measured after the injection of current, it is necessary to adjust the system in a way thatthe voltage developed on the microelectrode resistance and capacitance by the injected currentis decayed prior to voltage sampling. With accurate capacity compensation it is possible tomeasure the membrane potential at a time when no current passes the recording electrode.However, switch-clamp amplifiers are often problematic if single-channel conductance is tobe measured. Some can be used for this purpose with specially designed head stages. Mostinvestigators use classic patch-clamp amplifiers to measure single-channel events.

In the author’s laboratory gap junction conductance between two cells is measuredusing the double-cell voltage-clamp method with two switch-clamp amplifiers (SEC-05, NPIElectronic, Germany) in a double whole-cell patch. The procedure is described below.

The double-cell voltage-clamp setup (as used in the author’s laboratory) consists of thefollowing components:2 SEVC amplifiers1 inverted microscope (objectives 10¶, 20¶, 40¶, 100¶, all long-working distance withcorrection for thickness of the glass ground of the bath; eyepiece: 10¶; phase contrast)2 micromanipulators with electronic remote command1 break-out box (for connection of the amplifiers to the computer)1 PC system equiped with an A/D converterSoftware for controlling the SEVC amplifiers (make sure that your software can control twoamplifiers; it is preferable if the software can record 2 voltage and 2 current traces)Bath with inlet and outlet (it is recommended to use a bath with temperature control)Roller pumpTubingsGroundFaraday cagePipette pullerMicroforge

It is absolutely necessary to provide proper grounding of all components in the setup.The microscope, the manipulators and the bath have to be shielded by a Faraday cage. Theamplifiers, the PC system, oscilloscopes and roller pump are located outside the cage. Allcomponents of the setup have to be connected to a star point. This is often represented by


a solid copper bar with banana jacks. No component should be coupled (directly or indirectly)more than once to the star point. Otherwise, so-called grounding loops will be created whichcause noise. The star point itself is connected to the ground of the building. Problems oftenarise from saline-filled tubings. Ingoing and outgoing tubings should be interrupted by somesort of a dropper as used with common infusion tubings.

Isolate or cultivate cells according to the protocols given before. Pull pipettes of 2–3 MXresistance, fire-polish in a microforge or treat with sylgard. Prepare the following solutions.

Extracellular solutionNaCl 135 mmol/lKCl 4 mmol/lCaCl2 2 mmol/lMgCl2 1 mmol/lNaH2PO4 0.33 mmol/lHEPES 10 mmol/lGlucose 10 mmol/lpH 7.4

Pipette solution (‘intracellular solution’)CsCl 125 mmol/lNaCl 8 mmol/lCaCl2 1 mmol/lEGTA 10 mmol/lNa2ATP 2 mmol/lMgATP 3 mmol/lNa2GTP 0.1 mmol/lHEPES 10 mmol/lpH 7.2 (with CsOH)This solution should be filtered through a sterile filter before use.

Transfer an aliquot of cell-containing solution to the bath which is positioned on thestage of an inverted microscope or use coverslips with cells grown on top as bath ground.Perfuse the bath at a constant rate of 1 ml/min at either room temperature (21–24 ºC) or at37 ºC as desired. Adjust the amplifiers and compensate for series resistance or capacity. IfSEVC amplifiers are used adjust the switching frequency to values of about 25–30 kHz.Make sure that the command potential is reached within 3–5 ms with an accuracy of =5 mV,even when large currents are recorded. Find a cell pair (use a 40¶ objective with 10¶eyepieces and phase contrast) and position electrodes in the direct vicinity of the cells.Establish a gigaseal (seal resistance should exceed 5 GX) for one cell, perform the breakinand achieve the whole cell configuration by applying a sharp suction pulse. Thereafter tryto establish a gigaseal and afterwards a whole-cell configuration in the other cell. 3–5 minafter establishing the whole-cell configuration the experiment is started. Sampling frequencyis adjusted to 10 kHz and the obtained signal is low-pass filtered at 1 kHz. During therecordings 1 mmol/l BaCl2 is added to the external solution.

One can use either a symmetrical or an asymmetrical protocol. First both cells areclamped to Ö40 mV holding potential in order to inactivate the sodium current. Thereafter,one cell is clamped to potential ranging from Ö90 to +10 mV for 200 ms (pulse duration;asymmetrical protocol). Thereby, a transcellular voltage difference of ×50 mV can be applied


Table 4. Dyes used for investigation of dye coupling

Dye Excitation Emission Molecularwavelength weight(nm)

Lucifer yellow 430 535 457Procion yellow 488 530 6972,7-Dichlorofluorescein 513 532 691

and the current measured in the nonpulsed cell can be taken as the gap junctional current[Spray et al., 1981; Weingart, 1986]. Gap junction conductance can be calculated as the slopeof the current-voltage relationship by linear regression analysis, if the relationship is linear.By addition of the currents flowing in the resting and the pulsed cell, the sarcolemmal currentin the pulsed cell can be calculated as a current-voltage relationship [Weingart, 1986] andthe input resistance can be estimated from the chord conductance between Ö80 and Ö40 mV.Measurements should be done in both cells alternatively.

For a symmetrical protocol both cells are clamped to the common holding potentialof Ö40 mV. Thereafter, both cells are pulsed equally but with opposite polarity. Thus, cell1 is clamped to Ö50 mV and cell 2 to Ö30 mV, then to Ö60 and Ö20 mV, respectively,and so forth.

If voltage dependence is to be investigated the pulse duration must be enhanced tovalues of 1–2 s or even more, and transjunctional voltages of up to 100 mV have to beapplied.

A common problem with gap junction measurements is a rundown of gj in thesepreparations, for example in neonatal rat heart cells Schmilinsky-Fluri et al. [1990] found adecrease in gj of 16.4% in 6 min which could be antagonized by addition of a phospholipaseinhibitor, 20 lmol/l bromophenacyl bromide, to 1.8% within 6 min. They suggested thatendogenous arachidonic acid is involved in spontaneous uncoupling. Others favored a wash-out of ATP and cyclic nucleotides as a possible cause and prevented their preparations fromspontaneous uncoupling by addition of ATP, GTP or cAMP to the pipette solution [Mulleret al., 1997a, b].

8.7 Dye-Coupling Studies

A technique often used for the investigation of gap junctions is the injection of luciferyellow (LY). However, it should be noted that dye coupling and electrical coupling can differfrom each other in certain situations. For dye-coupling studies, LY is dissolved in water atconcentrations of 3–5%. The cell is penetrated with a LY-filled pipette and stained byapplication of constant hyperpolarizing current pulses of 4–10 nA (1 s pulse duration, 0.5Hz frequency) for 1–2 min. LY-filled pipettes exhibit higher resistance than 3 mol/l KClelectrodes and give unstable recordings. Alternatively, LY can be dissolved in 3 mol/l LiCl.If LY is used with the patch-clamp technique, LY is dissolved in the standard pipette solutionat a concentration of about 0.1–0.5%. After establishing the whole cell configuration, the


cell is clamped to negative potential for 1–5 min in order to increase the intracellular dyeconcentration.

Staining of the injected cell and the adjacent cells can be observed directly using anepifluorescence microscope (table 4). The excitation light is emitted from a mercury high-pressure lamp and filtered by a band-pass excitation filter (BP 450–490). The light thenpasses a dichroic mirror (FT 510) which serves as a beam splitter. Light with wavelengthsof =510 nm is selectively reflected by the mirror, whereas longer wavelengths pass it. Therebyexcitation light of 450–510 nm reaches the specimen and excites the dye. The light emittedby the dye passes the beam splitter and is filtered by a long-pass emission filter (LP 520)which enables wavelengths of ?520 nm to pass. It is necessary to use objectives with themicroscope which are designed for high UV light transmission (e.g. Neofluar, Zeiss).

Acknowledgements

I greatly thank Dr. Aida Salameh for the superior help with the figures, Mrs. MichaelaGottwald for intensive corrections and help with the search for literature, Mrs. Kathi Kruse-mann for taking the histological photographs, and Mr. Rajiv Grover for language revision.


...........................References

Allessie MA, Bonke FIM, Schopman FJG: Circus movement in rabbit atrial muscle as a mechanism oftachycardia. Circ Res 1973;32:54–62.

Allessie MA, Bonke FIM, Schopman FJG: Circus movement in rabbit atrial muscle as a mechanism oftachycardia. III. The ‘leading circle’ concept: A new model of circus movement in cardiac tisuewithout the involvement of an anatomical obstacle. Circ Res 1977;41:9–18.

Aonuma S, Kohama Y, Akai K, Iwasaki S: Studies on heart. XX. Further effect of bovine ventricleprotein (BVP) and antiarrhythmic peptide (AAP) on myocardial cells in culture. Chem Pharm Bull(Tokyo) 1980a;28:3340–3346.

Aonuma S, Kohama Y, Akai K, Komiyama Y, Nakajima S, Wakabayashi M, Makino T: Studies onheart. XIX. Isolation of an atrial peptide that improves the rhythmicity of cultured myocardial cellclusters. Chem Pharm Bull (Tokyo) 1980;28:3332–3339.

Aonuma S, Kohama Y, Makino T, Hattori K: Studies on heart. XXII. Inhibitory effect of an atrialpeptide on several drug-induced arrhythmias in vivo. Yakugaku Zasshi 1983;103:662–666.

Aonuma S, Kohama Y, Makino T, Fujisawa Y: Studies on heart XXI. Amino acid sequence of antiarrhyth-mic peptide isolated from bovine atria. J Pharmacobiol Dyn 1982;5:40–48.

Anumonwo JMB, Wang HZ, Trabka-Janik E, Dunham B, Veenstra RD, Delmar M, Jalife J: Gap junctionalchannels in adult mammalian sinus nodal cells. Immunolocalization and electrophysiology. CircRes 1992;71:229–239.

Argentieri T, Cantor E, Wiggins JR: Antiarrhythmic peptide has no direct cardiac actions. Experientia1989;45:737–738.

Arluk DJ, Rhodin AG: The ultrastructure of calf heart conducting fibers with special reference to nexusesand their distribution. J Ultrastruct Res 1974;49:11–23.

Arnsdorff MF, Sawick GJ: The effects of lysophosphatidylcholine: A toxic metabolite of ischemia on thecomponents of cardiac excitability in sheep Purkinje fibers. Circ Res 1981;49:16–30.

Ashraf M, Halverson C: Ultrastructural modifications of nexuses (gap junctions) during early myocardialischemia. J Mol Cell Cardiol 1978;10:263–269.

Auerbach AA, Bennett MVL: A rectifying electrotonic synapse in the central nervous system of a vertebrate.J Gen Physiol 1969;53:211–237.

Azarnia R, Dahl G, Loewenstein WR: Cell junction and cyclic AMP. III. Promotion of junctionalmembrane permeability and junctional membrane particles in a junction-deficient cell type. J MembrBiol 1981;63:133–146.

Azarnia R, Loewenstein WR: Intercellular communication and the control of growth. X. Alteration ofjunctional permeability by the src gene. A study with temperature-sensitive mutant Rous sarcomavirus. J Membr Biol 1984;82:191–205.

Banach K, Weingart R: Connexin 43 gap junctions exhibit asymmetrical gating properties. Pfluger’s Arch1996;431:775–785.

Bastiaanse EM, Jongsma H, van der Laarse A, Takens-Kwak BR: Heptanol-induced decrease in cardiacgap jnctional conductance is mediated by a decrease in the fluidity of membranous cholesterol-richdomains. J Membr Biol 1993;136:135–145.

Bastide B, Herve JC, Cronier L, Deleze J: Rapid onset and calcium independence of the gap junctionuncoupling induced by heptanol in cultured heart cells. Pfluger’s Arch 1995;429:386–393.

Bastide B, Neyses L, Ganten D, Paul M, Willecke K, Traub O: Gap junction protein connexin40 ispreferentially expressed in vascular endothelium and conductive bundles of rat myocardium and isincreased under hypertensive conditions. Circ Res 1993;73:1138–1149.

Bayer BL, Forster W: Actions of prostaglandin precursors and other unsaturated fatty acids on conductiontime and refractory period in the cat heart. Br J Pharmacol 1979;66:191–195.

121

Beblo DA, Wang HZ, Beyer EC, Westphale EM, Veenstra RD: Unique conductance, gating, and selectivepermeability properties of gap jnction channels formed by connexin40. Circ Res 1995;77:813–822.

Becker DL, Davies CS: Role of gap junctions in the development of the preimplantation mouse embryo.Microsc Res Tech 1995;31:364–374.

Bennett MVL, Barrio LC, Bargiello TA, Spray DC, Herzberg E, Saez JC: Gap junctions: new tools, newanswers, new questions. Neuron 1991;6:305–320.

Bennett MVL, Zheng X, Sogin ML: The connexin family tree; in Kanno Y, Kataoka K, Shiba Y, ShibataY, Shimazu T (eds): Intercellular Communication Through Gap Junctions. Science, Amsterdam,Elsevier, 1995, pp 3–8.

Beny JL, Connat JL: An electron-microscopic study of smooth muscle cell dye coupling in the pig coronaryarteries. Circ Res 1992;70:49–55.

Beny JL, Pacicca C: Bidirectional electrical communication between smooth muscle and endothelial cellsin the pig coronary artery. Am J Physiol 1994;266:H1465–H1472.

Bergoffen J, Scherer SS, Wang S, Scott MO, Bone LJ, Paul DL, Chen K, Lensch MW, Chance PF,Fischbeck KH: Connexin mutations in X-linked Charcot-Marie-Tooth disease. Science 1993;262:2039–2042.

Berridge MJ: Inositol triphosphate and diacylglycerol as second messengers. Biochem J 1984;220:345–360.Berthoud VM, Ledbetter MLS, Hertzberg EL, Saez JC: Regulation of gap junctions by cell contact and

phosphorylation in MDCK cells; in Hall JE, Zampighi GA, Davis RM (eds): Gap Junctions.Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 269–274.

Beyer EC: Molecular cloning and developmental expression of two chick embryo gap-junction proteins.J Biol Chem 1990;265:14439–14443.

Beyer EC, Goodenough DA, Paul DL: The connexins, a family of related gap junction proteins; inHertzberg EL, Johnson RG (eds): Gap Junctions. New York, Liss, 1988, pp 167–175.

Beyer EC, Kistler J, Paul DL, Goodenough DA: Antisera directed against connexin 43 peptides reactwith a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol 1989;108:595–605.

Beyer EC, Paul DL, Goodenough DA: Connexin 43: A protein from rat heart homologous to a gapjunctional protein from liver. J Cell Biol 1987;105:2621–2629.

Beyer EC, Veenstra RD, Kanter HL, Saffitz JE: Molecular structure and patterns of expression of cardiacgap junction proteins. In: Zipes D, Jalife J (eds): Cardiac electrophysiology. From cell to bedside.2nd ed. WB Saunders, Philadelphia, 1995, pp 31–38.

Blackburn JP, Peters NS, Yeh HI, Rothery S, Severs NJ: Upregulation of connexin 43 gap junctionsduring early stages of human coronary atherosclerosis. Arterioscler Thromb Vasc Biol 1995;15:1219–1228.

Blackshear PJ, Nairn AC, Kuo JF: Protein kinases 1988: A current perspective. FASEB J 1988;2:2957–2969.Bredikis J, Bukauskas F, Veteikis R: Decreased intercellular coupling after prolonged rapid stimulation

in rabbit atrial muscle. Circ Res 1981;49:815–820.Brink PR: Patch-clamp studies of gap junctions; in Peracchia C (ed): Biophysics of Gap Junction Channels.

Boca Raton, CRC Press, 1991, pp 29–42.Britz-Cunningham SH, Shah MM, Zuppan CW, Fletcher WH: Mutations of the connexin 43 gap junction

gene in patients with heart malformations and defects of laterality. N Engl J Med 1995;332:1323–1329.

Brugada P, Wellens HJJ: Arrhythmogenesis of antiarrhythmic drugs. Am J Cardiol 1988;61:1108–1111.Bruzzone R, Haefliger JA, Gimlich RL, Paul DL: Connexin 40, a component of gap junctions in vascular

endothelium is restricted in its ability to interact with other connexins. Mol Biol Cell 1993;4:7–20.

Bukauskas FF, Elfgang C, Willecke K, Weingart R: Biophysical properties of gap junction channelsformed by mouse connexin40 in induced pairs of transfected human HeLa cells. Biophys J 1995;68:2289–2298.

Bukauskas FF, Weingart R: Temperature dependence of gap junction properties in neonatal rat heartcells. Pflugers Arch 1993;423:133–139.

Burt JM: Block of intercellular communication: Interaction of intracellular H+ and Ca2+. Am J Physiol1987;253:C607.

122References

Burt JM, Massey KD, Minnich BN: Uncoupling of cardiac cells by fatty acids: Structure-activity relation-ships. Am J Physiol 1991;260:C439–C448.

Burt JM, Minnich BN, Massey KD, Ovadia M, Moore LK, Hirschi KK: Influence of lipophilic compoundson gap junction channels; in Hall JE, Zampighi GA, Davies RM (eds): Gap Junctions. Progress inCell Research, vol 3. Amsterdam, Elsevier, 1993, pp 113–120.

Burt JM, Spray DC: Inotropic agents modulate gap junctional conductance between cardiac myocytes.Am J Physiol 1988;254:H1206–H1210.

Burt JM, Spray DC: Single channel events and gating behaviour of the cardiac gap junctional channel.Proc Natl Acad Sci USA 1988b;85:3431–3434.

Burt JM, Spray DC: Volatile anaesthetics block intercellular communication between neonatal rat myocar-dial cells. Circ Res 1989;65:829–837.

Campos de Carvalho AC, Masuda MO, Tanowitz HB, Wittner M, Goldenberg RC, Spray DC: Conductiondefects and arrhythmias in Chagas’ disease: Possible role of gap junctions and humoral mechanisms.J Cardiovasc Electrophysiol 1994;5:686–698.

Campos de Carvalho AC, Moreno AP, Christ GJ, Melman A, Roy C, Hertzberg EL, Spray DC: Gapjunctions formed of connexin43 interconnect smooth muscle cells of the human corpus cavernosum.J Urol 1993;149:1568–1575.

Campos de Carvalho AC, Tanowitz HB, Wittner M, Dermietzel R, Roy C, Hertzberg EL, Spray DC:Gap junction distribution is altered between cardiac myocytes infected with Trypanosoma cruzi.Circ Res 1992;70:733–742.

Carlsson L, Almgren O, Duker G: QTU-prolongation and torsade de pointes induced by putative classIII antiarrhythmic agents in the rabbit: Etiology and interventions. J Cardiovasc Pharmacol 1990;16:276–285.

Caterall WA: Structure and function of voltage sensitive channels. Science 1988;242:50–61.Chanson M, Meda P: Rat pancreatic acinar cell coupling: Comparison of extent and modulation in vitro

and in vivo; in Hall JE, Zampighi GA, Davies RM (eds): Gap Junctions. Progress in Cell Research,vol 3. Amsteradam, Elsevier, 1993, pp 199–205.

Chapman RA: Excitation-contraction coupling in cardiac muscle. Prog Biophys Mol Biol 1979;35:1–52.Chapman RA, Fry CH: An analysis of the cable properties of frog ventricular myocardium. J Physiol

(Lond) 1978;283:263–282.Chen YH, DeHaan RL: Multiple channel conductance states in gap junctions; in Hall JE, Zampighi

GA, Davies RM (eds): Gap Junctions. Progress in Cell Research, vol 3. Amsteradam, Elsevier,1993, pp 97–103.

Chen L, Goings GE, Upshaw-Earley J, Page E: Cardiac gap junctions and gap junction-associated vesicles:Ultrastructural comparison of in-situ negative staining with conventional positive staining. CircRes 1989;64:501–514.

Cheung WY: Calmodulin: An overview. Fed Proc 1982;41:2253–2257.Chien KR, Han A, Sen A, Buja LM, Willerson JT: Accumulation of unesterified arachidonic acid in

ischemic canine myocardium. Circ Res 1984;54:313–322.Christ GJ, Spray DC, El-Sabban M, Moore LK, Brink PR: Gap junctions in vascular tissues. Evaluating

the role of intercellular communication in the modulation of vascular tone. Circ Res 1996;79:631–646.Christ GJ, Zao W, Moss J, Gondre CM, Roy C, Brink PR, Spray DC: Gap junctions modulate tissue

contractility and a1-adrenergic agonist efficacy in isolated rat aorta. J Pharmacol Exp Ther 1993;266:1054–1065.

Colatsky TJ, Tsien RW: Electrical properties associated with wide intercellular clefts in rabbit Purkinjefibers. J Physiol (Lond) 1979;290:227–252.

Cole WC, Garfield RE: Evidence for physiological regulation of myometrial gap junction permeability.Am J Physiol 1986;251:C411–C420.

Cole WC, Picone JB, Sperelakis N: Gap junction uncoupling and discontinuous propagation in the heart.A comparison of experimental data with computer simulations. Biophys J 1988;53:809–818.

Corr PB, Gross RW, Sobel BE: Amphipathic metabolites and membrane dysfunction in ischemic myocar-dium. Circ Res 1984;55:136–142.

Corr PB, Creer MH, Yamada KA, Saffitz JE, Sobel BE: Prophylaxis of early ventricular fibrillation byinhibition of acylcarnitine accumulation. J Clin Invest 1989;83:927–936.

123References

Coronel R: Distribution of Extracellular Potassium during Acute Myocardial Ischemia; thesis, Amsterdam,1988.

Crow DS, Beyer EC, Paul DL, Kobe SS, Lau AF: Phosphorylation of connexin43 gap junction proteinin uninfected and Rous sarcoma virus-transformed mamalian fibroblasts. Mol Cell Biol 1990;10:1754–1763.

Darrow BJ, Fast VG, Kleber AG, Beyer EC, Saffitz JE: Functional and structural assessment of intercellularcommunication. Increased conduction velocity and enhanced connexin expression in dibutyrylcAMP-treated cultured cardiac myocytes. Circ Res 1996;79:174–183.

Darrow BJ, Laing JG, Lampe PD, Saffitz JE, Beyer EC: Expression of multiple connexins in culturedneonatal rat ventricular myocytes. Circ Res 1995;76:381–387.

DaTorre SD, Creer MH, Pogwizd SM, Corr PB: Amphipathic lipid metabolites and their relation toarrhythmogenesis in the ischemic heart. J Mol Cell Cardiol 1991;23(suppl 1):11–22.

Daut J: The passive electrical properties of guinea pig ventricular muscle as examined with a voltageclamp technique. J Physiol (Lond) 1982;330:221–242.

Davidenko JM, Kent PF, Chialvo DR, Michales DC, Jalife J: Sustained vortex like waves in normalisolated ventricular muscle. Proc Natl Acad Sci USA 1990;87:8785–8789.

Davis LM, Kanter HL, Beyer EC, Saffitz JE: Distinct gap junction protein phenotypes in cardiac tissueswith disparate conduction properties. J Am Coll Cardiol 1994;24:1124–1132.

Davis LM, Rodefeld ME, Green K, Beyer EC, Saffitz JE: Gap junction protein phenotypes of the humanheart and conduction system. J Cardiovasc Electrophysiol 1995;6:813–822.

Dekker LRC, Fiolet JWT, VanBavel E, Coronel R, Opthof T, Spaan JAE, Janse MJ: Intracellular Ca2+,intercellular electrical coupling and mechanical activity in ischemic rabbit papillary muscle. Effectsof preconditioning and metabolic blockade. Circ Res 1996;79:237–246.

De Leon JR, Buttrick PM, Fishman GI: Functional analysis of the connexin 43 gene promoter in vivoand in vitro. J Mol Cell Cardiol 1994;26:379–389.

Delmar M, Michaels DC, Johnson T, Jalife J: Effects of increasing intercellular resistance on transverseand longitudinal propagation in sheep epicardial muscle. Circ Res 1987;60:780–785.

Delmar M, Liu S, Morley GE, Ek JF, Pertsova RN, Anumonwo JMB, Taffet SM: Toward a molecularmodel for the pH regulation of intercellular communication in the heart. In: Zipes D, Jalife J(eds): Cardiac electrophysiology. From Cell to bedside. 2nd ed. WB Saunders, Philadelphia, 1995,pp 135–143.

De Maziere AMGL, Scheuermann DW: Structural changes in cardiac gap junctions after hypoxia andreoxygenation: A quantitative freeze fracture study. Cell Tissue Res 1990;261:183–194.

De Mello WC: Effects of intracellular injection of calcium and strontium on cell communication in heart.J Physiol 1975;250:231–245.

De Mello WC: Influence of the sodium pump on intercellular communication in heart fibers: Effect ofintracellular injection of sodium ion on electrical coupling. J Physiol (Lond) 1976;263:171–197.

De Mello WC: Effect of intracellular injection of cAMP on the electrical coupling of mammalian cardiaccells. Biochem Biophys Res Commun 1984;119:1001–1007.

De Mello WC: Interaction of cyclic AMP and Ca in the control of electrical coupling in heart fibers.Biochim Biophys Acta 1986a;888:71.

De Mello WC: Increased spread of electrotonic potentials during diastolic depolarization in cardiacmuscle. J Mol Cell Cardiol 1986b;18:23–29.

De Mello WC: Cyclic nucleotides and junctional permeability; in Hertzberg EL, Johnson RG (eds): GapJunctions. New York, Liss, 1988, pp 245–254.

De Mello WC: Effect of isoproterenol and 3-isobutyl-1-methylxanthine on the junctional conductance inheart cell pairs. Biochim Biophys Acta 1989;1012:291–298.

De Mello WC: Cyclic AMP and junctional communication viewed through a multi-biophysical approach;in Peracchia C (ed): Biophysics of Gap Junction Channels. Boca Raton, CRC Press, 1991, pp 229–239.

De Mello WC: The role of the renin angiotensin system in the control of cell communication in the heart:Effects of enalapril and angiotensin II. J Cardiovasc Pharmacol 1992;20:643–651.

De Mello WC: Is an intracellular renin-angiotensin system involved in control of cell communication inheart? J Cardiovasc Pharmacol 1994;23:640–646.

124References

De Mello WC: Renin-angiotensin system and cell communication in the failing heart. Hypertension 1996;27:1267–1272.

Dermietzel R, Leibstein A, Frixen U, Janssen-Timmen U, Traub O, Willecke K: Gap junctions in severaltissues share antigenic determinants with liver gap junctions. EMBO J 1984;3:2261–2270.

Dermietzel R, Traub O, Hwang TK, Beyer EC, Bennet MVL, Spray DC, Willecke K: Differential expressionof three gap junction proteins in developing and mature brain tissues. Proc Natl Acad Sci USA1989;86:10148–10152.

De Vries SH, Schwartz EA: Modulation of an electrical synapse between solitary pairs of catfish horizontalcells by dopamine and second messengers. J Physiol 1989;414:351–375.

Dhein S, Gerwin R, Ziskoven U, Schott M, Rump A, Zhao Y, Salameh A, Klaus W: Propranolol unmasksclass III like electrophysiological properties of norepinephrine. Naunyn Schmiedebergs Arch Phar-macol 1993a;348:643–649.

Dhein S, Manicone N, Muller A, Gerwin R, Ziskoven U, Irankhahi A, Minke C, Klaus W: A newsynthetic antiarrhythmic peptide reduces dispersion of epicardial activation recovery interval anddiminishes alterations of epicardial activation patterns induced by regional ischemia. A mappingstudy. Naunyn Schmiedebergs Arch Pharmacol 1994;350:174–184.

Dhein S, Muller A, Gerwin R, Klaus W: Comparative study on the proarrhythmic effects of someantiarrhythmic agents. Circulation 1993b;87:617–630.

Dhein S, Muller A, Klaus W: Prearrhythmia: changes preceeding arrhythmia, new aspects by epicardialmapping. Basic Res Cardiol 1990;85:285–296.

Dhein S, Poeppel P, Krusemann K, Gottwald M: Changes in connexin 43 distribution after 24 hours ofatrial fibrillation. Proc Intern Gap Junction Conference, Key Largo, 1997a; pp 51.

Dhein S, Gottwald M, Schafer T, Muller A, Gover R, Tudyka T: Improvement of intercellular coup-ling by an antiarrhythmic peptide during ischemia and hypoxia. Eur Heart J 1997;18(suppl):568.

Dhein S, Schott M, Gottwald E, Muller A, Klaus W: The contribution of neutrophils to reperfsuionarrhythmias and a possible role for antiadhesive pharmacological substances. Cardiovasc Res 1995a;30:881–888.

Dhein S, Schott M, Gottwald E, Tudyka T, Rutten P: Antiarrhythmic effects of the antiarrhythmic peptideAAP10 in regional ischemia: preservation of longitudinal propagation of activation. Circulation1996;94(suppl I):715.

Dhein S, Schott M, Tudyka T, Piecha D, Gottwald E, Klaus W: Antiarrhythmic peptides: A new antiar-rhythmic principle. Exp Clin Cardiol 1997c;2:51–58.

Dhein S, Tudyka T: Therapeutic potential of antiarrhythmic peptides. Cellular coupling as a new antiar-rhythmic target. Drugs 1995;49:851–855.

Dhein S, Tudyka T, Schott M, Gottwald E, Muller A, Klaus W: A new antiarrhythmic peptide improvescellular coupling: A possible new antiarrhythmic mechanism. Circulation 1995b;92(suppl I):641.

Dillon SM, Allessie MA, Ursell PC, Wit AL: Influences of anisotropic tissue structure on reentrantcircuits in the epicardial border zone of subacute canine infarcts. Circ Res 1988;63:182–206.

Doble BW, Chen Y, Bosc DG, Litchfield DW, Kardami E: Fibroblast growth factor-2 decreases metaboliccoupling and stimulates phosphorylation as well as masking of connexin43 epitopes in cardiacmyocytes. Circ Res 1996;79:647–658.

Doble BW, Kardami E: Basic fibroblast growth factor and gap junction mediated intercellular communica-tion of cardiac fibroblasts. Mol Cell Biochem 1995;143:81–87.

DoeringAE,EisnerDA,LedererWJ:CardiacNa-CaexchangeandpH.AnnNYAcadSci1996;779:182–198.Dostal DE, Rothblum KN, Chermin MJ, Cooper GR, Baker MK: Intracellular detection of angio-

tensinogen and renin: A localized renin-angiotensin system in neonatal rat heart. Am J Physiol1992;263:C838–C850.

Dupont E, Aoumari A, Briand JP, Fromaget C, Gros D: Cross linking of cardiac gap junction connexonsby thiol/disulfide exchanges. J Membr Biol 1989;108:247–252.

Eberth CJ: Die Elemente der gestreiften Muskeln. Virchows Arch Pathol Anat Physiol 1866;37:100.Ebihara L: Expression of dog connexin 40 and 45 in paired Xenopus oocytes; in Kanno Y, Kataoka K,

Shiba Y, Shibata Y, Shimazu T (eds): Intercellular Communication through Gap Junctions. Progressin Cell Research, vol 4. Amsterdam, Elsevier, 1995, pp 395–398.

125References

Ebihara L, Beyer EC, Swenson KI, Paul DL, Goodenough DA: Cloning and expression of a Xenopusembryonic gap junction. Science 1989;243:1194–1195.

Echt DS, Liebson PR, Mitchell LB et al: Mortality and morbidity in patients receiving encainide, flecainideor placebo. The cardiac arrhythmia suppression trial. New Engl J Med 1991;324:781–788.

Ek JF, Delmar M, Perzova R, Taffet SM: Role of histidine 95 on pH gating of the cardiac gap junctionprotein connexin 43. Circ Res 1994;74:1058–1064.

Enomoto T, Martel N, Kanno Y, Yanmasaki H: Inhibition of cell communication between Balb/c3T3cells by tumor promotors and protection by cAMP. J Cell Physiol 1984;121:323–333.

Epstein ML, Sheridan JD, Johnson RG: Formation of low resistance junctions in vitro in the absence ofprotein synthesis and ATP production. Exp Cell Res 1977;104:25–30.

Factor SM, Sonnenblick EH, Kirk ES: The histologic border zone of acute myocardial infarction: Islandsor peninsulas? Am J Pathol 1978;92:111–124.

Falk MM, Kumar NM, Gilula NB: Biosynthetic membrane integration of connexin proteins; in KannoY, Kataoka K, Shiba Y, Shibata Y, Shimazu T (eds): Intercellular Communication through GapJunctions. Progress in Cell Research, vol 4. Amsterdam, Elsevier, 1995, pp 319–322.

Fast VG, Darrow BJ, Saffitz JE, Kleber AG: Anisotropic activation spread in heart cell monolayersassessed by high resolution optical mapping. Circ Res 1996;79:115–127.

Fast VG, Kleber AG: Microscopic conduction in cultured strands of neonatal rat heart cells measuredwith voltage sensitive dyes. Circ Res 1993;73:914–925.

Fast VG, Kleber AG: Two-dimensional high-resolution optical mapping of anisotropic impulse conductionin neonatal rat heart cell monolayers (abstracts). Circulation 1994;90(suppl I):411.

Firek L, Weingart R: Modification of gap junction conductance by divalent cations and protons inneonatal rat heart cells. J Mol Cell Cardiol 1995;27:1633–1643.

Fischbach PS, Corr PB, Yamada KA: Long-chain acylcarnitine increases intracellular calcium and inducesafterdepolarizations in adult ventricular myocytes (abstracts). Circulation 1992;86(suppl I):748.

Fishman GI, Eddy RE, Shows TB, Rosenthal L, Leinwand LA: The human connexin gene family ofgap junction proteins: Distinct chromosomal locations but similar structure. Genomics 1991a;10:250–256.

Fishman GI, Hertzberg EL, Spray DC, Leinwand LA: Expression of connexin 43 in the developing ratheart. Circ Res 1991b;68:782–787.

Fishman GI, Moreno AP, Spray DC, Leinwand LA: Functional analysis of human cardiac gap junctionchannel mutants. Proc Natl Acad Sci USA 1991c;88:3525–3529.

Fishman GI, Spray DC, Leinwand LA: Molecular characterization and functional expression of thehuman cardiac gap junction channel. J Cell Biol 1990;111:589–598.

Fitzgerald DJ, Knowles SE, Ballard FJ, Murray AW: Rapid and reversible inhibition of junctional commu-nication by tumor promotors in a mouse cell line. Cancer Res 1983;43:3614–3618.

Frank JS, Beydler S, Mottino G: Membrane structure in ultrarapidly frozen, unpretreated, freeze fracturedmyocardium. Circ Res 1987;61:141–147.

Furshpan EJ, Potter DD: Transmission at the giant motor synapses of the crayfish. J Physiol 1959;145:289–325.

Furukawa T, Kimura S, Furukawa N, Bassett AL, Myerburg RJ: Role of cardiac ATP-regulated potassiumchannels in differential responses of endocardial and epicardial cells to ischemia. Circ Res 1991;68:1693–1702.

Gainer HStC, Murray AW: Diacylglycerol inhibits p junctional communication in culured epidermal cells:Evidence for a role of protein kinase C. Biochem Biophys Res Commun 1985;126:1109–1113.

Gettes LS: What are the effects of potassium on the electrophysiology of acute ischemia?; in Hearse DJ,Manning AS, Janse MJ (eds): Life Threatening Arrhythmias during Ischemia and Infarction. NewYork, Raven Press, 1987, pp 77–90.

Giaume C: Application of the patch clamp technique to the study of junctional conductance; in PeracchiaC (ed): Biophysics of Gap Junction Channels. Boca Raton, CRC Press, 1991, pp 175–190.

Giaume C, Kado RT, Korn H: Voltage clamp analysis of a crayfish rectifying synapse. J Physiol (Lond)1987;386:91–112.

Giaume C, Randriamampita C, Trautmann A: Arachidonic acid closes gap junction channels in ratlacrimal glands. Pflugers Arch 1989;413:273–279.

126References

Gilman AG, Goodman LS, Rall TW, Murad F: The pharmacological basis of therapeutics. 7th ed.Macmillan Publishing Company, New York, 1985.

Gimlich RL, Kumar NM, Gilula NB: Sequence and developmental of mRNA coding for a gap junctionprotein in xenopus embryos. J Cell Biol 1988;107:1065–1073.

Gimlich RL, Kumar NM, Gilula NB: Differential regulation of the levels of three gap junction mRNAsin xenopus embryos. J Cell Biol 1990;110:597–605.

Girsch SJ, Peracchia C: Calmodulin binding sites in connexins (abstract). Biophys J 1992;61:A506.Gogol E, Unwin N: Organization of connexons on isolated rat liver gap junctions. Biophys J 1988;54:

105–112.Goldstein DB: The effects of drugs on membrane fluidity. Annu Rev Pharmacol Toxicol 1984;24:43–64.Goshima K: Formation of nexuses and electrotonic transmission between myocardial and FL cells in

monolayer culture. Exp Cell Res 1970;63:124–130.Gottwald M, Dhein S: Prevention from cellular uncoupling by Hoe 642 in the guinea pig papillary muscle

after application of sodiumpropionate. Eur Heart J 1997;18:pp 270.Gottwald M, Gottwald E, Dhein S: Increased dispersion of epicardial potential duration caused by

PMN’s is decreased by diminishing cellular uncoupling: A study with antiarrhythmic peptide andnordihydroguartic acid (abstract). Pflugers Arch 1997;433(suppl):135.

Gourdie RG, Green CR, Severs NJ, Thompson RP: Immunolabelling patterns of gap junction connexinsin the developing and mature rat heart. Anat Embryol (Berl) 1992;185:363–378.

Gourdie RG, Harfst E, Severs NJ, Green CR: Cardiac gap junctions in rat ventricle: Localization usingsite-directed antibodies and laser scanning confocal microscopy. Cardioscience 1990;1:75–82.

Grassi F, Monaco L, Fratamico G, Dolci S, Iannini E, Conti M, Eusebi F, Stefanini M: Putative secondmessengers affect cell coupling in the seminiferous tubules. Cell Biol Int Rep 1986;10:631–639.

Green CR, Severs J: Robert Feulgen Prize Lecture. Distribution and role of gap junctions in normalmyocardium and human ischaemic heart disease. Histochemistry 1993;99:105–120.

Gros D, Jary-Guichard T, ten Velde I, de Maziere A, van Kempen MJA, Davoust J, Briand JP, MormanAFM, Jongsma HJ: Restricted distribution of connexin 40, a gap junctional protein, in mammalianheart. Circ Res 1994;74:839–851.

Gruber HJ, Low PS: Interaction of amphiphiles with integral membrane proteins. I. Structural destabiliza-tion of the anion transport protein of the erythrocyte membrane by fatty acids, fatty alcohols andfatty amines. Biochim Biophys Acta 1988;944:414–424.

Haas HG, Meyer R, Einwachter HM, Stockem W: Intercellular coupling in frog heart muscle. Electro-physiological and morphological aspects. Pflugers Arch 1983;399:321–335.

Haefliger JA, Bruzzone R, Jenkins NA, Gilbert DJ, Copeland NG, Paul DL: Four novel members of theconnexin family of gap junction proteins: Molecular cloning, expresion and chromosome mapping.J Biol Chem 1990;116:187–194.

Han J, Moe GK: Nonuniform recovery of excitability in ventricular muscle. Circ Res 1964;16:46–60.Hartl FU: Molecular chaperones in cellular protein folding. Nature 1996;381:571–580.Haworth RA, Goknur AB, Berkoff HA: Inhibition of Na-Ca exchange by general anaesthetics. Circ Res

1989;65:1021–1028.Haydon DA, Elliot JR, Hendry BM: Effects of anesthetics on the squid giant axon; in Kleinzeller A (ed):

Current Topics in Membranes and Transports, vol 22. New York, Academic Press, 1984, pp 445–482.

Hennemann H, Schwarz J, Willecke K: Characterization of gap junction genes expressed in F9 embryoniccarcinoma cells: Molecular cloning of mouse connexin31 and -45 cDNAs. Eur J Cell Biol 1992a;57:51–58.

Hennemann H, Suchyna T, Lichtenberg-Frate H, Jungbluth S, Dahl E, Schwarz J, Nicholson BJ, WilleckeK: Molecular cloning and functional expression of mouse connexin40, a second gap junction genepreferentially expressed in lung. J Cell Biol 1992b;117:1299–1310.

Hermans MMP, Kortekaas P, Jongsma HJ, Rook MB: pH sensitivity of the cardiac gap junction proteins,connexin 45 and 43. Pflugers Arch 1995;431:138–140.

Hermans MMP, Kortekaas P, Jongsma HJ, Rook MB: The role of histidine residues on pH sensitivityof the gap junction protein connexin 43 (abstract 0047). Circulation 1996;94(suppl I):8.

Hille B: Ionic Channels of Excitable Membranes, ed 2. Sunderland, Sinauer, 1992.

127References

Hirche HJ, Franz C, Bos L, Bissig R, Lang R, Schramm M: Myocardial extracellular K+ and H+ increaseand noradrenaline release as possible cause of early arrhythmias following acute coronary occlusionin pigs. J Mol Cell Cardiol 1980;12:579–593.

Hirschi KK, Minnich BN, Moore LK, Burt JM: Oleic acid differentially affects gap junction-mediatedcommunication in heart and vascular smooth muscle cells. Am J Physiol 1993;265:C1517–C1526.

Hodgkin AL, Rushton WAH: The electrical constants of a crustacean nerve fibre. Proc R Soc Lond [B]1946;133:444–479.

Hoffman BF, Bigger JT: Digitalis and allied cardiac glycosides; in Gilman AG, Goodman LS, Rall TW,Murad F (eds): The Pharmacological Basis of Therapeutics, ed 7. New York, Macmillan, 1985,pp 716–747.

Hoyt RH, Cohen ML, Corr PB, Saffitz JE: Alterations of intercellular junctions induced by hypoxia incanine myocardium. Am J Physiol 1990;258:H1439–H1448.

Hoyt RH, Cohen ML, Saffitz JE: Distribution and three-dimensional structure of intercellular junctionsin canine myocardium. Circ Res 1989;64:563–574.

Imanaga I: Cell-to-cell diffusion of procion yellow in sheep and calf Purkinje fibers. J Membr Biol 1974;16:381–388.

Imanaga I: Cell-to-cell coupling studied by diffusional methods in cardiac cells. Experientia 1987;43:1080–1083.

Imanaga I, Kameyama M, Irisawa H: Cell-to-cell diffusion of fluorescent dyes in ventricular paired cellsisolated from guinea pig heart. Am J Physiol 1987;252:H223–H232.

In’t Veld P: Gap junctions between pancreatic B cells are modulated by cyclic AMP. Eur J Cell Biol 1985;36:269–276.

Iwatsuki N, Petersen OH: Pancreatic acinar cells: Acetylcholine-evoked electrical uncoupling and its ionicdependency. J Physiol 1978;274:81–96.

Jackson P, Blythe D: Immunolabelling techniques for light microscopy; in Beesley JE (ed): Immunocyto-chemistry. A Practical Approach. Oxford, Oxford University Press, 1993, pp 15–41.

Janse MJ, van Capelle FJL: Electrotonic interactions across an inexcitable region as a cause of ectopicactivity in acute regional myocardial ischemia. Circ Res 1982;50:527–537.

Janse MJ, van Capelle FJL, Morsink H, Kleber AG, Wilms-Schopman F, Cardinal R, Naumann d’Alnon-court C, Durrer D: Flow of ‘injury current’ and patterns of excitation during early ventriculararrhythmias in acute myocardial ischemia in isolated porcine and canine hearts. Circ Res 1980;47:151–165.

Janse MJ, Wit AL: Electrophysiological mechanisms of ventricular arrhythmias resulting from myocardialischemia and infarction. Physiol Rev 1989;69:1049–1169.

John SA, Revel JP: Connexon integrity is maintained by non-covalent bonds: Intramolecular disulfide bondslink theextracellular domains inrat connexin 43.Biochem Biophys ResCommun 1991;178:1312–1318.

Jongsma HJ, Wilders R, Takens-Kwak BR, Rook MB: Are cardiac gap junctions voltage sensitive?; inHall JE, Zampighi GA, Davies RM (eds): Gap Junctions, Progress in Cell Research, vol 3. Amster-dam, Elsevier, 1993, pp 187–192.

Joyner RW: Effects of the discrete pattern of electrical coupling on propagation through an electricalsyncytium. Circ Res 1982;50:192–200.

Kameyama M: Electrical coupling between ventricular paired cells isolated from guinea pig heart. J Physiol(Lond) 1983;336:345–357.

Kanno Y, Enomoto T, Shiba Y, Yamasaki H: Protective effect of cAMP on tumor-promotor mediatedinhibition of cell-cell communication. Exp Cell Res 1984;152:31–37.

Kanno Y, Sasaki Y, Hirono C, Shiba Y: Delayed change in gap junctional cell communication in theacinus of the rat submandibular gland after secretion of saliva; in Hall JE, Zampighi GA, DaviesRM (eds): Gap Junctions. Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 207–209.

Kanter HL, Davis LM, Green KG, Beyer EC, Saffitz JE: Unique connexin phenotype in the caninesinoatrial node (abstract). Circulation 1993a;88(suppl I):175.

Kanter HL, Laing JG, Beau SL, Beyer EC, Saffitz JE: Distinct patterns of connexin expression in caninePurkinje fibers and ventricular muscle. Circ Res 1993b;72:1124–1131.

Kanter HL, Laing JG, Beyer EC, Green KG, Saffitz JE: Multiple connexins colocalize in canine ventricularmyocyte gap junctions. Circ Res 1993c;73:344–350.

128References

Kanter HL, Saffitz JE, Beyer EC: Cardiac myocytes express multiple gap junction proteins. Circ Res1992;70:438–444.

Kanter HL, Saffitz JE, Beyer EC: Molecular cloning of two human cardiac gap junction proteins, connexin40 and connexin 45. J Mol Cell Cardiol 1994;26:861–868.

Kardami E: Stimulation and inibition of cardiac myocate proliferation in vitro. Mol Cell Biochem 1990;92:129–135.

Kardami E, Stoski RM, Doble BW, Yamamoto T, Hertzberg EL, Nagy JI: Biochemical and ultrastructuralevidence for the association of basic fibroblast growth factor with cardiac gap junctions. J BiolChem 1991;266:19551–19557.

Kass S, McRae C, Graber HL, Sparks EA, McNamara D, Boudoulas H, Basson CT, Baker PB 3rd,Cody RJ, Fishman MC, Cox N, Kong A, Wooley CF, Seidman JG, Seidman CE: A gene defectthat causes conduction system disease and dilated cardiomyopathy maps to chromosome 1p1–1q1.Nat Genet 1994;7:546–551.

Katz AM: Physiology of the Heart. New York, Raven Press, 1992.Kessler JA, Spray DC, Saez JC, Bennett MVL: Development and regulation of electrotonic coupling

between cultured sympathetic neurons; in Bennett MVL, Spray DC (eds): Gap Junctions. ColdSpring Harbour, Cold Spring Harbour Laboratory, 1985, pp 231–240.

Kieval RS, Spear JF, Moore EN: Gap junctional conductance in ventricular myocyte pairs isolated frompostischemic rabbit myocardium. Circ Res 1992;71:127–136.

Kimura H, Oyamada Y, Ohshika H, Mri M, Oyamada M: Reversible inhibition of gap junctionalcommunication, synchronous contraction and synchronism of intracellular Ca2+ fluctuation incultured neonatal rat cardiac myocytes by heptanol. Exp Cell Res 1995;220:348–356.

Klausner RD, Kleinfeld AM, Hoover RL, Karnovsky MJ: Lipid domains in membranes: evidence derivedfrom structural perturbations induced by free fatty acids and lifetime heterogeneity analysis. J BiolChem 1980;255:1286–1295.

Kleber AG, Janse MJ, van Capelle FJL, Durrer D: Mechanism and time course of ST and TQ segmentchanges during acute regional myocardial ischemia in the pig heart determined by extracellular andintracellular recordings. Circ Res 1978;42:603–613.

Kleber AG, Janse MJ, Wilms-Schopman FJG, Wilde AAM, Coronel R: Changes in conduction velocityduring acute ischemia in ventricular myocardium in the isolated procine heart. Circulation 1986;73:189–198.

Kleber AG, Riegger CB, Janse MJ: Electrical uncoupling and increase of extracellular resistance afterinduction of ischemia in isolated, arterially perfused rabbit papillary muscle. Circ Res 1987;61:271–279.

Kohama Y, Iwabuchi K, Shibahara T, Okabe M, Mimura T: Response of immunoreactive antiarrhythmicpeptide (IR-AAP) level associated with experimental arrhythmia in rats. J Pharmacobiodyn 1986;9:806–810.

Kohama Y, Kawahara Y, Kabe M, Mimura T, Aonuma S: Determnation of immunoreactive antiarrhythmicpeptide (AAP) in rats. J Pharmacobiodyn 1985;8:1024–1031.

Kohama Y, Okimoto N, Mimura T, Fukaya C, Watanabe M, Yokoyama K: A new antiarrhythmic peptide,N-3-(4-hydroxyphenyl)-propionyl-Pro-Hyp-Gyl-Ala-Gly. Chem Pharm Bull 1987;35:3928–3930.

Kolb HA, Somogyi R: Biochemical and biophysical analysis of cell-to-cell channels and regulation ofgap junctional permeability. Rev Physiol Biochem Pharmacol 1991;118:1–47.

Kramer JB, Corr PB: Mechanisms contributing to arrhythmias during ischemia and infarction. Eur HeartJ 1984;5(suppl B):11–18.

Krinsky VI: Spread of excitation in an inhomogeneous medium (state similar to cardiac fibrillation).Biofizika 1966;11:676–683.

Krinsky VI: Mathematical models of cardiac arrhythmias (spiral waves); in Szekeres L (ed): Pharmacologyof Antiarrhythmic Agents. Oxford, Pergamon Press, 1981, pp 105–124.

Kumar NL, Gilula NB: Cloning and characterization of human and rat liver cDNA’s coding for a gapjunction protein. J Cell Biol 1986;103:767–776.

Kuo CS, Munakata K, Reddy CP, Surawicz B: Characteristics and possible mechanism of ventriculararrhythmia dependent on the dispersion of action potential durations. Circulation 1983;67:1356–1367.

129References

Kwak BR, Hermanns MMP, De Jonge HR, Lohmann SM, Jongsma HJ, Chanson M: Differential regula-tion of distinct types of gap junction channels by similar phosphorylating conditions. Mol BiolCell 1995a;6:1707–1719.

Kwak BR, Jongsma HJ: Regulation of cardiac gap junction channel permeability and conductance byseveral phosphorylating conditions. Mol Cell Biochem 1996;157:93–99.

Kwak BR, Van Veen TAB, Analbers LJS, Jongsma HJ: TPA increases conductance but decreases permeabil-ity in neonatal rat cardiomyocyte gap junction channels. Exp Cell Res 1995b;220:456–463.

Kyte J, Doolittle RF: A simple method for displaying the hydropathic character of a protein. J Mol Biol1982;157:105–132.

Laing JG, Westphale EM, Engelmann GL, Beyer EC: Characterization of the gap junction protein,connexin 45. J Membr Biol 1994;139:31–40.

Laird DW, Puranam KL, Revel JP: Turnover and phosphorylation dynamics of connexin43 gap junctionprotein in cultured cardiac myocytes. Biochem J 1991;273:67–72.

Larsen WJ: Biological implications of gap junction structure, distribution and composition: A review.Tissue Cell 1983;15:645–671.

Larson DM, Haudenschild CC, Beyer EC: Gap junction messenger RNA expression by vascular wallcells. Circ Res 1990;66:1074–1080.

Lau AF, Kurata WE, Kanemitsu MY, Loo LMW, Warn-Cramer BJ, Eckhart W, Lampe PD: Regulationof connexin 43 function by activated tyrosine protein kinases. J Bioenerg Biomembr 1996;28:359–368.

Lazzara R, Scherlag BJ: Current concepts of the genesis of ischemic arrhythmias and sudden coronarydeath; in Lucchesi R, Dingell JV, Schwarz RP (eds): Clinical Pharmacology of AntiarrhythmicTherapy. New York, Raven Press, 1984, pp 9–23.

Lesh MD, Pring M, Spear JF: Cellular uncoupling can unmask dispersion of action potential durationin ventricular myocardium. Circ Res 1989;65:1426–1440.

Lindl T, Bauer J: Zell- und Gewebekultur, 3. Aufl. Stuttgart, Fischer, 1994.Lindner E: Die submikroskopische Morphologie des Herzmuskels. Z Zellforsch 1957;45:702–746.Little TL, Xia J, Duling BR: Dye tracers define differential endothelial and smooth muscle coupling

patterns within the arteriolar wall. Circ Res 1995;76:49–504.Liu S, Tafet S, Stoner L, Delmar M, Vallano ML, Jalife J: A structural basis for the unequal sensitivity

of the major cardiac and liver gap junctions to intracellular acidification: The carboxy tail length.Biophys J 1993;64:1422–1433.

Loewenstein WR: Some reflections on growth and differentiation. Perspect Biol Med 1968;11:260–272.

Loewenstein WR: Junctional intercellular communicaiton: The cell-to-cell membrane channel. PhysiolRev 1981;61:829–913.

Loewenstein WR, Kanno Y, Socolar SJ: Quantum jumps of conductance during formation of conductancechannels at cell–cell junction. Nature 1978;274:133–136.

Luke RA, Saffitz JE: Remodeling of ventricular conduction pathways in healed canine infarct borderzones. J Clin Invest 1991;87:1594–1602.

Luque EA, Veenstra RD, Beyer EC, Lemanski LF: Localization and distribution of gap junctions innormal and cardiomyopathic hamster heart. J Morphol 1994;222:203–213.

Macdonald RL, Hsu D, Mann JE, Sperelakis N: An analysis of the problem of K+ accumulation in theintercalated disk clefts of cardiac muscle. J Theor Biol 1975;52:455–474.

Makowski L: Structural domains in gap junctions: Implications for the control of intercellular communica-tion; in Bennett MVL, Spray DC (eds): Gap Junctions. Cold Spring Harbour, Cold Spring HarbourLaboratory, 1985, pp 5–12.

Makowsky L: X-ray diffraction studies of gap junction structure. Adv Cell Biol 1988;2:119–158.Makowsky L, Caspar DLD, Phillips WC, Goodenough DA: Gap junction structures. II. Analysis of the

X-ray diffraction data. J Cell Biol 1977;74:629–645.Makowsky L, Caspar DLD, Phillips WC, Goodenough DA: Gap junction structures. V. Structural chem-

istry inferred from X-ray diffraction measurements on sucrose accessibility and trypsin susceptibility.J Mol Biol 1984;174:449–481.

Maldonado PE, Rose B, Loewenstein WR: Growth factors modulate junctional cell-to-cell communication.J Membr Biol 1988;106:203–210.

130References

Manjunath CK, Goings GE, Page E: Isolation and protein composition of gap junctions from rabbithearts. Biochem J 1982;205:189–195.

Manjunath CK, Nicholson BJ, Teplow D, Hood L, Page E, Revel JP: The cardiac gap junction protein(Mr 47,000) has a tissue-specific cytoplasmic domain of Mr 17,000 at its carboxy terminus. BiochemBiophys Res Commun 1987;142:228–234.

Manjunath CK, Page E: Cell biology and protein composition of cardiac gap junctions. Am J Physiol1985;248:H783–H791.

Manjunath CK, Page E: Rat heart gap junctions as disulfide-bonded connexon multimers: Their depoly-merization and solubilization in desoxycholate. J Membr Biol 1986;90:43–57.

Massey KD, Minnich BN, Burt JM: Arachidonic acid and lipoxygenase metabolites uncouple neonatalrat cardiac myocyte pairs. Am J Physiol 1992;263:C494–C501.

Maurer P, Weingart R: Cell pairs isolated from guiea pig and rat hearts: Effects of [Ca2+]i on nexalmembrane resistance. Pflugers Arch 1987;409:394–402.

Mazet F, Wittenberg BA, Spray DC: Fate of intercellular junctions in isolated adult rat cardiac cells. CircRes 1985;56:195–204.

McCallister LP, Trapukdis S, Neely JR: Morphometric observations of the effects of ischemia in theisolated perfused rat heart. J Mol Cell Cardiol 1979;11:619–630.

McMahon DG, Kanpp AG, Dowling JE: Horizontal cells gap junctions: Single channel conductance andmodulation by dopamine. Proc Natl Acad Sci USA 1989;86:7639–7643.

Mege RM, Matsuzaki F, Gallin WF, Golber JI, Cunningham BA, Edelman GM: Construction of epi-thelioid sheets by transfection of mouse sarcoma cells with cDNAs for chicken cell adhesionmolecules. Proc Natl Acad Sci USA 1988;85:7274–7278.

Meszaros J, Pappano AJ: Electrophysiological effects of L-palitoylcarnitine in single ventricular myocytes.Am J Physiol 1990;258:H931–H938.

Milks LC, Kumar NM, Houghton R, Unwin N, Gilula NB: Topology of the 32-kd liver gap junctiondetermined by site directed antibody localisations. EMBO J 1988;7:2967–2975.

Miller T, Dahl G, Werner R: Structure of a gap junction gene: Rat connexin 32. Biosci Rep 1988;8:455–464.

Miner P, Lampe P, Atkinson M, Johnson R: Extracellular calcium and cadherins regulate the process ofgap junction assembly between cells in culture; in Kanno Y, Kataoka K, Shiba Y, Shibata Y, ShimazuT (eds): Intercellular Communication through Gap Junctions. Progress in Cell Research, vol 4.Amsterdam, Elsevier, 1995, pp 331–334.

Moe GK, Rheinboldt WC, Abildskov JA: A computer model of atrial fibrillation. Am Heart J 1964;67:200–220.

Moore DH, Ruska H: Electron microscope study of mammalian cardiac muscle cells. J Biophys BiochemCytol 1957;3:261–267.

Moreno AP, Fishman GI, Spray DC: Phosphorylation shifts unitary conductance and modifies voltagedependent kinetics of human connexin43 gap junction channels. Biophys J 1992;62:51–53.

Moreno AP, Laing JG, Beyer EC, Spray DC: Properties of gap junction channels formed of connexin45 endogenously expressed in human hepatoma (SKHep1) cells. Am J Physiol 1995;268:C356–C365.

Moreno AP, Rook MB, Fishman GI, Spray DC: Gap junction channels: Distinct voltage-sensitive andinsensitive conductance states. Biophys J 1994a;67:113–119.

Moreno AP, Saez JC, Fishman GI, Spray DC: Human connexin 43 gap junction channels. Regulationof unitary conductances by phosporylation. Circ Res 1994b;74:1050–1057.

Morley GE, Anumonwo JMB, Delmar M: Effects of 2,4-dinitrophenol or low [ATP]i on cell excitabi-lity and action potential propagation in guinea pig ventricular myocytes. Circ Res 1992;71:821–830.

Morley GE, Taffet SM, Delmar M: Intramolecular interactions mediate pH regulation of connexin 43channels. Biophys J 1996;70:1294–1302.

Muller A, Dhein S: Sodium channel blockade enhances dispersion of the cardiac action potential duration.A computer simulation study. Basic Res Cardiol 1993;88:11–15.

Muller A, Gottwald M, Tudyka T, Linke W, Klaus W, Dhein S: Increase in gap junction conduction byan antiarrhythmic peptide. Eur J Pharmacol 1997a;327:65–72.

131References

Muller A, Schaefer T, Gottwald M, Tudyka T, Linke W, Klaus W, Dhein S: Effect of the antiarrhythmicpeptide AAP10 on cellular coupling. Naunyn Schmiedebergs Arch Pharmacol 1997b;356:76–82.

Munster PN, Weingart R: Effects of phorbol ester on gap junctions of neonatal rat heart cells. PflugersArch 1993;423:181–188.

Murphy E, Freudenreich CC, Levy LA, London RE, Liebermann M: Monitoring cytosolic free magnesiumin cultured chicken heart cells by use of the fluorescent indicator furaptra. Proc Natl Acad Sci USA1989;86:2981–2984.

Murray AW, Fitzgerald DJ: Tumor promotors inhibit metabolic cooperation in cocultures of epidermaland 3T3 cells. Biochem Biophys Res Commun 1979;91:395–401.

Musil LS, Beyer EC, Goodenough DA: Expression of the gap junction protein connexin43 in embryonicchick lens: Molecular cloning, ultrastructural localization, and posttranslational phosporylation.J Membr Biol 1990a;116:163–175.

Musil LS, Cunningham BA, Edelman GM, Goodenough DA: Differential phosphorylation of the gapjunction protein connexin 43 in junctional communication competent and deficient cell lines. J CellBiol 1990b;111:2077–2088.

Musil LS, Goodenough DA: Multisubunit assembly of an integral plasma membrane channel protein,gap junction connexin43, occurs after exit from the ER. Cell 1993;74:1065–1077.

Musil LS, Goodenough DA: Biochemical analysis of connexon assembly; in Kanno Y, Kataoka K, ShibaY, Shibata Y, Shimazu T (eds): Intercellular communication through gap junctions. Progress in CellResearch, vol 4. Amsterdam, Elsevier, 1995, pp 327–330.

Nakamura TY, Yamamoto I, Kanno Y, Shiba Y, Goshima K: Intercellular metabolic coupling of gluta-thione between mouse and quail cardiac myocytes and its protective role against oxidative stress;in Kanno Y, Kataoka K, Shiba Y, Shibata Y, Shimazu T (eds): Intercellular Communication throughGap Junctions. Progress in Cell Research, vol 4. Amsterdam, Elsevier, 1995, pp 167–170.

Nedergaard M, Cooper AJL, Goldman SA: Gap junctions are required for the propagation of spreadingdepression. J Neurobiol 1995;28:433–444.

Nedergaard M, Goldman S, Desai S, Pulsinelli W: Acid induced death in neurons and glia. J Neurosci1991;11:2489–2497.

Nelson WL, Makielski JC: Block of cardiac sodium current by heptanol and octanol (abstract). BiophysJ 1990;57:299.

Neyton J, Trautmann A: Single channel curents of an intercellular junction. Nature 1985;317:331–335.Neyton J, Trautmann A: Acetylcholine modulation of the conductance of intercellular junctions between

rat lacrimal cells. J Physiol 1986;377:283–295.Nicholson BJ, Gros DB, Kent SBH, Hood LE, Revel JP: The Mr 28,000 gap junction proteins from rat

heart and liver are different but related. J Biol Chem 1985;260:6514–6517.Nicholson BJ, Gros DB, Kent SBH, Hood L, Revel JP: The Mr 28,000 gap junction proteins from rat

heart and liver are different but related. J Biol Chem 1990;260:6514–6517.Nicholson BJ, Suchyna T, Xu LX, Hammernick P, Cao FL, Fourtner C, Barrio L, Bennett MVL: Divergent

properties of different connexins expressed in xenopus oocytes; in Hall JE, Zampighi GA, DaviesRM (eds): Gap Junctions. Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 3–13.

Niggli E, Rusisuli A, Maurer P, Weingart R: Effects of general anaesthetics on current flow acrossjunctional and non-junctional membranes and contractility in guinea pig myocytes. Am J Physiol1989;256:C273–C281.

Nishizuka Y: The molecular heterogeneity of protein kinase C and its implications for cellular regulation.Nature 1988;334:661–665.

Noma A, Tsuboi N: Dependence of junctional conductance on proton, calcium and magnesium ions incardiac paired cells of guinea pig. J Physiol (Lond) 1987;382:193–211.

Oliveira-Castro GM, Loewenstein WR: Junctional membrane permeability: Effects of divalent cations.J Membr Biol 1971;5:51–77.

Oosthoek P, Viragh S, Lamers WH, Moorman AFM: Immunohistochemical delineation of the conductionsystem. I. The atrioventricular node and Purkinje fibers. Circ Res 1993a;73:482–491.

Oosthoek P, Viragh S, Mayen AEM, van Kempen MJA, Lamers WH, Moorman AFM: Immuno-histochemical delineation of the conduction system. I. The sinuatrial node. Circ Res 1993b;73:473–481.

132References

Ormerod MG, Imrie SF: Immunohistochemistry; in Lacey AJ (ed): Light Microscopy in Biology. Apractical approach. Oxford, Oxford University Press, 1989, pp 103–136.

Ovadia M, Burt JM: Developmental modulation of susceptibility to arhythmogenesis in myocardialischemia: Reduced sensitivity of adult versus neonatal rat heart cells to uncoupling by lipophilicsubstances (abstract). Circulation 1991;84(suppl II):324.

Padua RR, Fandrich RR, Kardami E: Increased basic fibroblast growth factor accumulation and distinctpattern of localization in isoproterenol-induced cardiomyocytes injury. Growth Factors 1993;8:291–306.

Page E: Cardiac gap junctions. In Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE (eds):The cardiovascular system. 2nd ed. Raven Press, New York, 1992, pp 1003–1047.

Paul DL: Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol 1986;103:123–134.Paul DL: New functions for gap junctions. Curr Opin Cell Biol 1995;7:665–672.Paul DL, Ebihara L, Takemoto LJ, Swenson KI, Goodenough DA: Connexin46, a novel lens gap-junction

protein, induces voltage gated currents in non-junctional plasma membrane of xenopus oocytes.J Cell Biol 1991;115:1077–1089.

Pepper MS, Meda P: Basic fibroblast growth factor increases junctional communication and connexin43 expression in microvascular endothelial cells. J Cell Physiol 1992;153:196–205.

Peracchia C: Calmodulin-like proteins and communicating junctions – electrical uncoupling of crayfishseptate axons is inhibited by the calmodulin inhibitor W7 and is not affected by cyclic nucleotides.Pflugers Arch 1987;408:379–385.

Peracchia C: The calmodulin hypothesis for gap junction regulation six years later; in Hertzberg EL,Johnson RG (eds): Gap Junctions. New York, Liss, 1988, pp 267–282.

Peracchia C: Possible involvement of caffeine and ryanodine-sensitive calcium stores in low pH-inducedregulation of gap junction channels; in Peracchia C (ed): Biophysics of Gap Junction Channels.Boca Raton, CRC Press, 1991a, pp 13–28.

Peracchia C: Effects of the anesthetics heptanol, halothane and isoflurane on gap junction conductancein crayfish septate axons: A calcium- and hydrogen-independent phenomenon potentiated by caffeineand theophylline, and inhibited by 4-aminopyridine. J Membr Biol 1991b;121:67–78.

Peracchia C, Bernardini G, Peracchia L: Is calmodulin involved in the regulation of gap junction permeabil-ity? Pflugers Arch 1983;399:152–154.

Peracchia C, Shen L: Gap junction channel reconstitution in artificial bilayers and evidence for calmodulinbinding sites in MIP26 and connexins from rat heart, liver and xenopus embryo; in Hall JE,Zampighi GA, Davis RM (eds): Gap Junctions. Progress in Cell Research, vol 3. Amsterdam,Elsevier, 1993, pp 163–170.

Perkins G, Goodenough DA, Sosinsky G: Three-dimensional structure of the gap junction connexon.Biophys J 1997;72:533–544.

Peters NS: Myocardial gap junction organization in ischemia and infarction. Microsc Res Tech 1995;31:375–386.

Peters NS: New insights into myocardial arrhythmogenesis: Distribution of gap junctional coupling innormal, ischaemic and hypertrophied hearts. Clin Sci 1996;90:447–452.

Peters NS, Green CR, Poole-Wilson PA, Severs NJ: Reduced content of connexin43 gap junctions in ven-tricular myocardium from hypertrophied and ischaemic human hearts. Circulation 1993;88:864–875.

Peters NS, Severs NJ, Rothery SM, Lincoln C, Yacoub MH, Green CR: Spatiotemporal relation betweengap junctions and fascia adherens junctions during postnatal development of human ventricularmyocardium. Circulation 1994;90:713–725.

Petersen OH, Ueda N: Pancreatic acinar cells: The role of calcium in stimulus-secretion coupling. J Physiol1976;254:583–606.

Piccolino M, Neyton J, Gerschenfeld HM: Decrease of gap junction permeability induced by dopamineand cyclic adenosine 3�,5�-monophosphate in horizontal cells of turtle retina. J Neurosci 1984;4:2477–2488.

Piper HM (ed): Cell Culture Techniques in Heart and Vessel Research. Berlin, Springer, 1990.Poche R, Lindner E: Untersuchungen zur Frage der Glanzstreifen des Herzmuskelgewebes beim Warm-

bluter und beim Kaltbluter. Z Zellforsch 1955;43:104–120.

133References

Podrid PJ: Aggravation of ventricular arrhythmia. A drug induced complication. Drugs 1985;29(suppl 4):33–44.

Podrid PJ, Lampert S, Grayboys TB, Blatt CM, Lown B: Aggravation of arrhythmia by antiarrhythmicdrugs: Incidence and predictors. Am J Cardiol 1987;59:38E–44E.

Pogwizd SM, Corr PB: Reentrant and non-reentrant mechanisms contribute to arrhythmogenesisduring early myocardial ischemia: Results using three dimensional mapping. Circ Res 1987;61:352–371.

Pogwizd SM, Corr PB: Mechanisms underlying the development of ventricular fibrillation during earlymyocardial ischemia. Circ Res 1990;66:672–695.

Pogwizd SM, Onufer JR, Kramer JB, Sobel BE, Corr PB: Induction of delayed afterdepolarisations andtriggered activity in canine Purkinje fibers by lysophosphatidylglycerides. Circ Res 1986;59:416–426.

Pringle MJ, Brown KB, Miller KW: Can the lipid theories of anaesthesia account for the cutoff inanaesthetic potency in homologous series of alcohols? Mol Pharmacol 1981;19:49–55.

Puranam KL, Laird DW, Revel JP: Trapping an intermediate form of connexin43 in the Golgi. Exp CellRes 1993;206:85–92.

Quan W, Rudy Y: Unidirectional block and reentry of cardiac excitation: A model study. Circ Res 1990;66:367–382.

Ramon F, Arellano RO, Rivera A: Regulatory mechanisms of gap junctional communication in crayfishaxons; in Peracchia C (ed): Biophysics of Gap Junction Channels. Boca Raton, CRC Press, 1991,pp 241–255.

Rash JE, Hudson CS (eds): Freeze Fracture: Methods, Artifacts and Interpretations. New York, RavenPress, 1979.

Reaume A, deSousa PA, Kulkarni S, Langille LL, Zhu D, Davies TC, Juneja SC, Kidder GM, RossantJ: Cardiac malformation in neonatal mice lacking connexin 43. Science 1995;267:1831–1834.

Reber WR, Weingart R: Ungulate cardiac Purkinje fibers: The influence of intracellular pH on the elctricalcell-to-cell coupling. J Physiol (Lond) 1982;328:87–104.

Rechsteiner M: Regulation of enzyme levels by proteolysis: The role of pest regions. Adv Enzyme Regul1988;27:135–151.

Reed KE, Westphale EM, Larson DM, Wang HZ, Veenstra RD, Beyer EC: Molecular cloning andfunctional expression of human connexin 37, an endothelial cell gap junction protein. J Clin Invest1993;91:997–1004.

Rennick RE, Connat JL, Burnstock G, Rothery S, Severs NJ, Green CR: Expression of connexin 43 gapjunctions between cultured vascular smooth muscle cells is dependent upon phenotype. Cell TissueRes 1993;271:323–332.

Reverdin EC, Weingart R: Electrical properties of the gap junctional membrane studied in rat liver cellpairs. Am J Physiol 1988;254:C226–C234.

Risek B, Guthrie S, Kumar N, Gilula NB: Modulation of gap junction transcript and protein expressionduring pregnancy in the rat. J Cell Biol 1990;110:269–282.

Rohr S: Determination of impulse conduction characteristics at a microscopic scale in patterned growthheart cell cultures using multiple site optical recording of transmembrane voltage. J CardiovascElectrophysiol 1995;6:551–568.

Rohr S, Kucera JP, Fast VG, Kleber AG: Paradoxical improvement of impulse conduction in cardiactissue by partial cellular uncoupling. Science 1997;275:841–844.

Rook MB, Jongsma HJ, de Jonge B: Single channel currents of homo- and heterologous gap junctionsbetween cardiac fibroblasts and myocytes. Pflugers Arch 1989;414:95–98.

Rook MB, Jongsma HJ, van Ginneken ACG: Properties of single gap junctional channels between isolatedneonatal rat heart cells. Am J Physiol 1988;255:H770–H782.

Rose B, Loewenstein WR: Permeability of cell junctions depends on local cytoplasmic calcium activity.Nature 1975;254:250–252.

Rose B, Loewenstein WR: Permeability of cell junctions and the local cytoplasmic free ionized calciumconcentration: A study with aequorin. J Membr Biol 1976;28:87–119.

Rosen MR, Janse MJ, Myerburg RJ: Arrhythmias induced by coronary artery occlusion: What are theelectrophysiological mechanisms?; in Hearse DJ, Manning AS, Janse MJ (eds): Life ThreateningArrhythmias during Ischemia and Infarction. New York, Raven Press, 1987, pp 11–48.

134References

Rouet-Benizeb P, Mohammadi K, Perennec J, Poyard M, Bouanani NEH, Crozatier B: Protein kinaseC isoform expression in normal and failing rabbit hearts. Circ Res 1996;79:153–161.

Rudisuli A, Weingart R: Electrical properties of gap junction channels in guinea pig ventricular cell pairsrevealed by exposure to heptanol. Pflugers Arch 1989;415:12.

Rudisuli A, Weingart R: Gap junctions in adult ventricular muscle; in Peracchia C (ed): Biophysics ofGap Junctions. Boca Raton, CRC Press, 1991, pp 44–56.

Saez JC, Gregory WA, Watanabe T, Dermietzel R, Hertzberg EL, Reid L, Bennett MVL, Spray DC:cAMP delays disappearance of gap junctions between pairs of rat hepatocytes in primary culture.Am J Physiol 1989;257:C1–C11.

Saez JC, Nairn AC, Czernick A, Spray DC, Hertzberg EL, Greengard P, Bennett MVL: Phosphorylationof connexin 32, the hepatocyte gap junction protein, by cAMP-dependent protein kinase, proteinkinase C and Ca/calmodulin-dependent protein kinase II. Eur J Biochem 1990;192:263–273.

Saez JC, Spray DC, Nairn AC, Hertzberg EL, Greengard P, Bennett MVL: cAMP increases junctionalconductance and stimulates phosphorylation of the 27 kDa principal gap junction polypeptide.Proc Natl Acad Sci USA 1986;83:2473–2477.

Saffitz JE, Davis LM, Darrow BJ, Kanter HL, Laing JG, Beyer EC: The molecular basis of anisotropy:Role of gap junctions. J Cardiovasc Electrophysiol 1995;6:498–510.

Saffitz JE, Hoyt RH, Luke RA, Kanter HL, Beyer EC: Cardiac myocyte interconnections at gap junctions:Role in normal and abnormal electrical conduction. Trends Cardiovasc Med 1992;2:56–60.

Saffitz JE, Kanter HL, Green KG, Tolley TK, Beyer EC: Tissue specific determinants of anisotropicconduction velocity in canine atrial and ventricular myocardium. Circ Res 1994;74:1065–1070.

Schmilinsky-Fluri G, Rudisuli A, Willi M, Rohr S, Weingart R: Effects of arachidonic acid on the gapjunctions of neonatal rat heart cells. Pflugers Arch 1990;417:149–156.

Schwarzmann G, Weigandt H, Rose B, Zimmermann A, Ben Haim D, Loewenstein WR: Diameter of thecell-to-cell junctional membrane channels as probed with neutral molecules. Science 1981;213:551–553.

Severs NJ: Freeze-fracture cytochemistry: Review of methods. J Electron Microsc Tech 1989;13:175–203.Severs NJ: The cardiac gap junction and intercalated disk. Int J Cardiol 1990;26:137–173.Severs NJ: Pathophysiology of gap junctions in heart disease. J Cardiovasc Electrophysiol 1994a;5:462–475.Severs NJ: Gap junction alterations in the failing heart. Eur Heart J 1994b;15(suppl D):53–57.Severs NJ, Shovel KS, Slade AM, Powell T, Twist VW, Green CR: Fate of gap junctions in isolated adult

mammalian cardiomyocytes. Circ Res 1989;65:22–42.Shaw RM, Rudy Y: The vulnerable window for unidirectional block in cardiac tissue: Characterization

and dependence on membrane excitability and intercellular coupling. J Cardiovasc Electrophysiol1995;6:115–131.

Shibata Y, Miyahara A, Okayama T, Kuraoka A, Iida H: Gap junction formation and regulation incultured adult rat and guinea pig cardiac muscle cells; in Kanno Y, Kataoka K, Shiba Y, ShibataY, Shimazu T (eds): Intercellular Communication through Gap Junctions. Progress in Cell Research,vol 4. Amsterdam, Elsevier, 1995, pp 151–154.

Sikewar S, Unwin PNT: Three-dimensional structure of gap junctions in fragmented plasma membranesform rat liver. Biophys J 1988;54:113–119.

Simpson I, Rose B, Loewenstein WR: Size limit of molecules permeating the junctional membranechannels. Science 1977;195:294–296.

Sjostrand FS, Andersson E: Electron microscopy of intercalated disks of cardiac muscle tissue. Experientia1954;9:369–371.

Smith JH, Green CR, Peters NS, Rothery S, Severs NJ: Altered patterns of gap junction distribution inischemic heart disease. An immunohistochemical study of human myocardium using laser scanningconfocal microsopy. Am J Pathol 1991;139:801–821.

Somogyi R, Kolb HA: Cell-to-cell channel conductance during loss of gap junctional coupling in pairsof pancreatic acinar and Chinese hamster ovary cells. Pflugers Arch 1988a;412:54–65.

Somogyi R, Kolb HA: Modulation of gap junctional coupling in pairs of pancreatic acinar cells by cAMP,OAG and protein kinase C. Ber Bunsenges Phys Chem 1988b;92:993–998.

Somogyi R, Kolb HA: Possible involvement of a G-protein in carbamycholine induced gap junctionclosure. J Protein Chem 1989;8:434–436.

135References

Sosinsky GE, Jesior JC, Caspar DLD, Goodenough DA: Gap junction structures. VIII. Membrane cross-sections. Biophys J 1988;53:709–722.

Spach MS, Dolber PC: Relating extracellular potentials and their derivatives to anisotropic propagationat a microscopic level in human cardiac muscle. Evidence for electrical uncoupling of side-to-sidefiber connections with increasing age. Circ Res 1986;58:356–371.

Spach MS, Dolber PC: Discontinuous anisotropic propagation; in Rosen MR, Janse MJ, Wit AL (eds):Cardiac Electrophysiology: A Textbook. Mount Kisco, Futura, 1990, pp 517–534.

Spach MS, Dolber PC, Heidlage JF: Influence of the passive anisotropic properties on directional differ-ences in propagation following modification of the sodium conductance in human atrial muscle. Amodel of reentry based on anisotropic discontinuous propagation. Circ Res 1988;62:811–832.

Spach MS, Heidlage JF: The stochastic nature of cardiac propagation at a microscopic level. Electricaldescription of myocardial architecture and its application to conduction. Circ Res 1995;76:366–380.

Spach MS, Josephson ME: Initiating reentry: The role of non-uniform anisotropy in small circuits.J Cardiovasc Electrophysiol 1994;5:182–209.

Spach MS, Miller WT III, Geselowitz DB, Barr RC, Kootsey JM, Johnson EA: The discontinuousnature of propagation in normal canine cardiac muscle: Evidence for recurrent discontinuities ofintracellular resistance that affect the membrane currents. Circ Res 1981;48:39–45.

Sperelakis N: Propagation mechanisms in the heart. Annu Rev Physiol 1979;41:441–457.Sperelakis N, Mann JE: Evaluation of electric field changes in the cleft between excitable cells. J Theor

Biol 1977;64:71–96.Spira AW: The nexuses in the intercalated disc of the canine heart: Quantitative data for an estimation

of its resistance. J Ultrastruct Res 1971;34:409–425.Spray DC, Bennett MVL, Campos de Carvalho AC, Eghbali B, Moreno AP, Verselis V: Transjunctional

voltage dependence of gap junction channels; in Peracchia C (ed): Biophysics of Gap JunctionChannels. Boca Raton, CRC Press, 1991, pp 97–116.

Spray DC, Burt JM: Structure-activity relations of the cardiac gap junction channel. Am J Physiol 1990;258:C195–C205.

Spray DC, Harris AL, Bennett MVL: Equilibrium properties of a voltage dependent junctional conduct-ance. J Gen Physiol 1981;77:77–93.

Spray DC, White RL, Mazet F, Bennett MVL: Regulation of gap junction conductance. Am J Physiol1985;248:H753–H764.

Sugiura H, Toyama J, Tsuboi N, Kamiya K, Kodama I: ATP directly affects junctional conductancebetween paired ventricular myocytes isolated from guinea pig heart. Circ Res 1990;66:1095–1102.

Swenson KI, Jordan JR, Beyer EC, Paul DL: Formation of gap junctions by expression of connexins inxenopus oocyte pairs. Cell 1989;57:145–155.

Szekeres L, Borbola J, Papp JP: Cardiac actions of arachidonic acid. Acta Biol Med Ger 1976;35:1119–1126.

Tadros PN, Laing JG, Saffitz JE, Beyer EC: The gap junction protein connexin 43 is degraded by multipleproteolytic pathways (abstract 4246). Circulation 1996;94(suppl I):726.

Takeda A, Hashimoto E, Yamamura H, Shimazu T: Phosphorylation of liver gap junction protein byprotein kinase C. FEBS Lett 1987;210:169–172.

Takeda A, Saheki S, Shimazu T, Takeuchi N: Phosphorylation of the 27-kDa gap junction protein byprotein kinase C in vitro and in rat hepatocytes. J Biochem 1989;106:723–727.

Takens-Kwak BR, Jongsma HJ: Cardiac gap junctions: Three distinct channel conductances and theirmodulation by phosphorylating treatments. Pflugers Arch 1992;422:198–200.

Takens-Kwak BR, Jongsma HJ, Rook MB, Van Ginneken AC: Mechanism of heptanol-induced uncoupl-ing of cardiac gap junctions: A perforated patch-clamp study. Am J Physiol 1992;262:C1531–C1538.

ten Velde I, de Jonge B, Verheijck EE, van Kempen MJA, Analbers L, Gros D, Jongsma HJ: Spatialdistribution of connexin43, the major cardiac gap junction protein, visualizes the cellular networkfor impulse propagation from sinoatrial node to atrium. Circ Res 1995;76:802–811.

Tibitts TT, Caspar DLD, Philipps WC, Goodenough DA: Diffraction diagnosis of protein folding in gapjunction connexons. Biophys J 1990;57:1025–1036.

136References

Trabka-Janik E, Coombs W, Lemanski LF, Delmar M, Jalife J: Immunohistochemical localization of gapjunction protein channels in hamster sinoatrial node in correlation with electrophysiologic mappingof the pacemaker region. J Cardiovasc Electrophysiol 1994;5:125–137.

Traub O, Butterweck A, Elfgang C, Hertlein B, Balzer K, Gergs U, Hafemann B, Willecke K: Immunochem-ical characterization of connexin31, -37, -40, -43, and -45 in cultured primary cells, transfected celllines and murine tissues; in Kanno Y, Kataoka K, Shiba Y, Shibata Y, Shimazu T (eds): IntercellularCommunication through Gap Junctions. Progress in Cell Research, vol 4. Amsterdam, Elsevier,1994, pp 343–347.

Traub O, Look J, Dermietzel R, Brummer F, Hulser D, Willecke K: Comparative characterization of the21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J Cell Biol 1989;108:1039–1051.

Traub O, Look J, Paul D, Willecke K: Cyclic adenosine monophosphate stimulates biosynthesis andphosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes. Eur J Cell Biol1987;43:48–54.

Tsien RW, Weingart R: Inotropic effect of cyclic AMP in calf ventricular muscle studied by a cut endmethod. J Physiol (Lond) 1976;260:117–141.

Turin L, Warner AE: Carbon dioxide reversibly abolishes ionic communication between cells of earlyamphibian embryo. Nature 1978;270:56–57.

Unwin PNT, Ennis PD: Two configurations of a channel forming protein. Nature 1984;307:609–613.Unwin PNT, Zampighi G: Structure of the junction between communicating cells. Nature 1980;283:

545–549.Ursell PC, Gardner PI, Albala A, Fenoglio JJ, Wit AL: Structural and electrophysiological changes in

the epicardial border zone of canine myocardial infarcts during infarct healing. Circ Res 1985;56:436–451.

Valiunas V, Bukauskas FF, Weingart R: Conductances and selective permeability of connexin43 gapjunction channels examined in neonatal rat heart cells. Circ Res 1997;80:708–719.

van Capelle FJL, Durrer D: Computer simulation of arrhythmias in a network of coupled excitableelements. Circ Res 1980;47:454–466.

van der Velden HMW, van Zijverden M, van Kempen MJA, Wijfels MCEF, Groenewegen WA, AllessieMA, Jongsma HJ: Abnormal expression of the gap junction protein connexin 40 during chronicatrial fibrillation in the goat (abstract). Circulation 1996;94(suppl I):593.

van Kempen MJA, Fromaget C, Gros D, Moorman AFM, Lamers WH: Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart. Circ Res 1991;68:1638–1651.

van Kempen MJA, ten Velde I, Wessels A, Oosthoek PW, Gros D, Jongsma HJ, Moorman AF, LamersWH: Differential connexin distribution accommodates cardiac function in different species. MicroscRes Tech 1995;31:420–436.

Veenstra RD: Voltage dependent gating of gap junction channels in embryonic chick ventricular cell pairs.Am J Physiol 1990;258:C662–C672.

Veenstra RD: Comparative physiology of cardiac gap junction channels; in Peracchia C (ed): Biophysicsof Gap Junction Channels. Boca Raton, CRC Press, 1991a, pp 131–144.

Veenstra RD: Physiological modulation of cardiac gap junction channels. J Cardiovasc Electrophysiol1991b;2:168–189.

Veenstra RD, Berg K, Wang HZ, Westphale EM, Beyer EC: Molecular and biophysical properties of theconnexins from developing chick heart; in Hall JE, Zampighi GA, Davis RM (eds): Gap Junctions.Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 89–95.

Veenstra RD, DeHaan RL: Cardiac gap junction channel activity in embryonic chick ventricle cells. AmJ Physiol 1988;254:H170–H180.

Veenstra RD, Wang HZ, Westphale EM, Beyer EC: Multiple connexins confer distinct regulatory andconductance properties of gap junctions in developing heart. Circ Res 1992;71:1277–1283.

Verheule S, van Kempen MJA, te Welscher PHJA, Kwak BR, Jongsma HJ: Characterization of gapjunction channels in adult rabbit atrial and ventricular myocardium. Circ Res 1997;80:673–681.

Vorperian VR, Wisialowski TA, Deegan R, Roden DM: Effect of hypercapnic acidemia on anisotropicpropagation in the canine ventricle. Circulation 1994;90:456–461.

137References

Wang HZ, Li J, Lemanski LF, Veenstra RD: Gating of mammalian cardiac gap junction channels bytransjunctional voltage. Biophys J 1992;63:139–151.

Warner A, Clements DK, Parikh S, Evans WH, DeHaan RL: Specific motifs in the external loops ofconnexin proteins can determine gap junction formation between chick heart myocytes. J Physiol(Lond) 1995;488:721–728.

Weidmann S: The electrical constants of Purkinje fibers. J Physiol (Lond) 1952;118:348–360.Weidmann S: Passive properties of cardiac fibers; in Rosen MR, Janse MJ, Wit AL (eds): Cardiac

Electrophysiology. A Textbook. Mount Kisco, Futura, 1990, pp 29–35.Weingart R: The permeability to tetraethylammonium ions of the surface membrane and the intercalated

disks of sheep and calf myocardium. J Physiol (Lond) 1974;240:741–762.Weingart R: The actions of ouabain on intercellular coupling and conduction velocity in mammalian

ventricular muscle. J Physiol (Lond) 1977;264:341–365.Weingart R: Electrical properties of the nexal membrane studied in rat ventricular cell pairs. J Physiol

(Lond) 1986;370:267–284.Weingart R, Maurer P: Cell-to-cell coupling studied in isolated ventricular cell pairs. Experientia 1987;

43:1091–1094.Weingart R, Maurer P: Action potential transfer in cell pairs isolated from adult rat and guinea pig

ventricles. Circ Res 1988;63:72–80.Werner R, Levine E, Rabadan-Diehl C, Dahl G: Formation of hybrid cell-cell channels. Proc Natl Acad

Sci USA 1989;86:5380–5384.Werner R, Rabadan-Diehl C, Levine E, Dahl G: Affinities between connexins; in Hall JE, Zampighi GA,

Davis RM (eds): Gap Junctions. Progress in Cell Research, vol 3. Amsterdam, Elsevier, 1993, pp 21–24.White RL, Doeller JE, Verselis VK, Wittenberg BA: Gap junctional conductance between pairs of

ventricular myocytes is modulated synergistically by H+ and Ca++. J Gen Physiol 1990;95:1061–1075.White RL, Spray DC, Campos de Carvalho AC, Wittenberg BA, Bennett MVL: Some electrical and

pharmacological properties of gap junctions between adult ventricular myocytes. Am J Physiol1985;249:C447–C455.

White TW, Bruzzone R, Goodenough DA, Paul DL: Mouse Cx50, a functional member of the connexinfamily of gap junction proteins, is the lens fiber protein MP70. Mol Biol Cell 1992;3:711–720.

Wiener N, Rosenblueth A: The mathematical formulation of the problem of conduction of impulses ina network of connected excitable elements, specifically in cardiac muscle. Arch Inst Cardiol Mex1946;16:205–265.

Wier WG, Cannell MB, Berlin JB, Marban E, Lederer WJ: Cellular and subcellular heterogeneity of[Ca2+]i in single heart cells revealed by Fura-2. Science 1987;235:325–328.

Wilde AAM, Escande D, Schumacher CA, Thuringer D, Mestre M, Fiolet JWT, Janse MJ: Potassiumaccumulation in the globally ischemic mammalian heart. A role for the ATP-sensitive potassiumchannel. Circ Res 1990;67:835–843.

Wilders R, Jongsma HJ: Limitations of the dual voltage clamp method in assaying conductance andkinetics of gap junction channels. Biophys J 1992;63:942–953.

Willecke K, Hennemann H, Dahl E, Jungbluth S: The mouse connexin family; in Hall JE, ZampighiGA, Davis RM (eds): Gap Junctions. Progress in Cell Research, vol.3. Amsterdam, Elsevier, 1993,pp 33–37.

Willecke K, Heynkes R, Dahl E, Stutenkemper R, Hennemann HJ, Jungbluth S, Suchyna T, NicholsonBJ: Mouse connexin37: Cloning and functional expression of a gap junction gene highly expressedin lung. J Cell Biol 1991;114:1049–1057.

Wit AL, Janse MJ: The ventricular arrhythmias of ischemia and infarction. Electrophysiological mecha-nisms. Futura Publishing Company, Mount Kisco, 1992, pp 1–160.

Wojtcak J: Contractures and increase in internal longitudinal resistance of cow ventricular muscle inducedby hypoxia. Circ Res 1979;44:88–95.

Wu J, Corr PB: Influence of long chain acylcarnitines on the voltage-dependent calcium current in adultventricular myocytes. Am J Phyisol 1992;263:H410–H417.

Wu J, McHowat J, Saffitz JE, Yamada KA, Corr PB: Inhibition of gap junctional conductance by longchain acylcarnitines and their preferential accumulation in junctional sarcolemma during hypoxia.Circ Res 1993;72:879–889.

138References

Yamada KA, McHowat J, Yan GX, Donahue K, Peirick J, Kleber AG, Corr PB: Cellular uncouplinginduced by accumulation of long-chain acylcarnitine during ischemia. Circ Res 1994;74:83–95.

Yamamoto T, Ochalski A, Hertzberg EL, Nagy JL: LM and EM immunolocalization of the gap junctionalprotein connexin 43 in rat brain. Brain Res 1990;508:313–319.

Yancey SB, Edens JE, Trosko JE, Chang C, Revel JP: Decreased incidence of gap junctions betweenChinese hamster V-79 cells upon exposure to the tumor promotor 12-O-tetradecanoyl phorbol-13-acetate. Exp Cell Res 1982;139:329–340.

Yang H, Muller-Borer BJ, Johnson TA, Sandiford DR, Lemasters JJ, Cascio WE: Na/HCO3 cotransportand Na/H exchange contribute to the rate of cell-to-cell electrical uncoupling in ischemic ventricularmyocardium (abstract 3472). Circulation 1996;94(suppl I):593.

Zampighi G: Gap junction structure; in DeMello WC (ed): Cell-to-Cell Communication. New York,Plenum Press, 1987, pp 1–28.

Zhang JT, Nicholson BJ: Sequence and tissue distribution of a second protein of hepatic gap junctions,Cx26, as deduced from its cDNA. J Cell Biol 1989;109:3391–3401.

Zimmer PB, Green RC, Evans HW, Gilula NB: Topological analysis of the major protein in isolatedintact rat liver gap junctions and gap junction derived single membrane structures. J Biol Chem1987;262:7751–7763.

139References

...........................Appendix

One-letter code of amino acids, three-letter code and codons (5�–3�) coding for theamino acid

Amino acids with apolar side chainsA Ala Alanine GCU, GCC, GCA, GCGV Val Valine GUU, GUC, GUA, GUGL Leu Leucine CUU, CUC, CUA, CUG, UUA, UUGI Ile Isoleucine AUU, AUC, AUAP Pro Proline CCU, CCC, CCA, CCGF Phe Phenylalanine UUU, UUCW Trp Tryptophane UGGM Met Methionine AUG

Amino acids with uncharged polar side chainsG Gly Glycine GGU, GGC, GGA, GGGS Ser Serine AGU, AGC, UCU, UCC, UCA, UCGT Thr Threonine ACU, ACC, ACA, ACGC Cys Cysteine UGU, UGCY Tyr Tyrosine UAU, UACN Asn Asparagine AAU, AACQ Gln Glutamine CAA, CAG

acid amino acids (negatively charged at pH 6)D Asp Aspartatic acid GAU, GACE Glu Glutamic acid GAA, GAG

basic amino acids (positively charged at pH 6)K Lys Lysine AAA, AAGR Arg Arginine CGU, CGC, CGA, CGG, AGA, AGGH His Histidine CAU, CAC

Stop codons: UAA, UAG, UGA

One-letter code for nucleic acids is: A>adenine; G>guanine; C>cytosine; U>uracil.

140

...........................List of Suppliers of Specialized Items

Axon Instruments1101 Chess DriveFoster City, CA 94404 (USA)Tel. +1 415 571 9400Fax +1 415 571 9500In Germany: 0130 81 0458In Switzerland: 046 05 7323(electrophysiological equipment, patch

clamp amplifiers, switch clampamplifiers)

Becton-Dickinson Labware (Falcon)1950 Willaim DriveOxnard, CA 93030 (USA)Becton Dicknson GmbHPostfach 101629D–69006 Heidelberg (Germany)Tel. +49 6221 3050Fax +49 6221 303609(cell culture equipment)

Biermann GmbH (DPC)Hohe Strasse 4-8D–61231 Bad Nauheim (Germany)Tel. +49 6032 99400Fax +49 6031 994200(antibodies)

BiologicRue de l’Europe 1F–38640 Claix (France)Tel. +33 76 986831Fax +33 76 986909(electrophysiological equipment, pullers,

patch clamp amplifiers)

BiometraRudolf-Wissell Strasse 30D–37079 Gottingen (Germany)Tel. +49 551 506860

Fax +49 551 5068666550 N. Reo Street #101Tampa, FL 33609 (USA)Tel. +1 813 287 8815Fax +1 813 282 1936(labware, SDS-PAGE equipment)

Boehringer MannheimSandhofer Strasse 116D–68305 Mannheim (Germany)Tel. +49 621 7590Fax +49 621 759 2890(biochemistry, signal transduction reagents,

immunochemistry)

Chemicon28835 Single Oak DriveTemecula, CA 92590 (USA)Tel. +1 909 676 8080Fax +1 909 676 9209(antibodies)

Clark Electromedical InstrumentsPO Box 8Pangbourne, Reading RG8 7HU (UK)Tel. +44 1734 843888Fax +44 1734 845374(electrophysiological equipment)

DianovaPostfach 301250D–20305 Hamburg (Germany)Tel. +49 40 45067 0Fax +49 40 45067 390(antibodies)

FMI Fohr Medical InstrumentsIn der Grube 13D–64342 Seeheim/Ober-Beerbach

(Germany)

141

Tel. +49 6257 962260Fax +49 6257 962262(electrophysiological equipment, pullers,


Gibco/BRLLife TechnologiesDieselstrasse 5D–76344 Eggenstein (Germany)Tel. +49 130 830902or: Gibco LtdPO Box 35Paisley PA3 4EF (UK)(cell culture media, biochemica and

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Greiner LabortechnikMaybachstrasse 2D–72636 Frickenhausen (Germany)(plastic cell culture flasks, cell culture

equipment)

HamegKelsterbacher Strasse 15–19D–60528 Frankfurt/Main (Germany)Tel. +49 69 678050Fax +49 69 6780513(oscilloscopes)

Heka ElektronikWiesenstrasse 71D–67466 Lambrecht (Germany)Tel. +49 6325 8036Fax +49 6325 8039(electrophysiological equipment, pullers,


Heraeus Instruments GmbHPostfach 1563D–63405 Hanau (Germany)Tel. +49 6181 35 413Fax +49 6181 35 739(cell culture equipment, incubators,

benches)

Hewlett-PackardRothebuhlstrasse 81D–70197 Stuttgart (Germany)Tel. +49 711 61965 0Fax +49 711 61965 50(oscilloscopes, recorders)

ImmunotechLuminy case 915F–13288 Marseille Cedex 9 (France)Postfach 101526D–20010 Hamburg (Germany)Tel. +49 40 322180Fax +49 40 323969(antibodies)

Integra BiosciencesTecnomara GmbHRuhberg 4D–35461 Fernwald (Germany)Tel. +49 6404 8090(cell culture equipment)

Leica Vertrieb GmbHLilienthalstrasse 39–45D–64606 Bensheim (Germany)Tel. +49 6251 136 0Fax +49 6251 136 155(microscopes and equipment)

List ElektronicPflungstadter Strasse 18–20D–64297 Darmstadt (Germany)Tel. +49 6151 56000Fax +49 6151 56060(electrophysiological equipment, pullers,


Luigs & NeumannBoschstrasse 19D–40880 Ratingen (Germany)Tel. +49 2102 4420 35Fax +49 2102 4420 36(micromanipulators and setups)

142List of Suppliers of Specialized Items

Marzhauser Wetzlar GmbH & Co KGAn den Fichten 35D–35579 Wetzlar (Germany)Tel. +49 6441 9116 0Fax +49 6441 9116 40(micromanipulators)

MerckFrankfurter Strasse 250D–64293 Dramstadt (Germany)Tel. +49 6151 72 0Fax +49 6151 72 2000(biochemica)

Millipore GmbHHauptstrasse 87D–65760 Eschborn (Germany)Tel. +49 6196 4940Fax +49 6196 43901or: Millipore Corporation8 Ashby RoadBedford, MA 01730 (USA)(filter technique, cell biology)

NarishigeUnit 7 Willow Business Park, Willow WayLondon SE2 64QP (UK)Tel. +44 181 699 8282Fax +44 181 699 8299(electrophysiological equipment, pullers,

micromanipulators)

NikonTiefenbroicher Weg 25D–40472 Dusseldorf (Germany)Tel. +49 211 9414 0Fax +49 211 9414 330(microscopes and equipment)

NPI electronicHaldenstrasse 62D–71732 TammTel. +49 7141 60 1534Fax +49 7141 60 1266(electrophysiological equipment, patch

clamp amplifiers, switch clampamplifiers)

Nunc GmbHHagenauer Strasse 21aD–65203 Wiesbaden-Biebrich (Germany)Tel. +49 611 67095Fax +49 611 607348or: 2000 Aurora RoadNaperville, IL 60566 (USA)(cell culture equipment)

Olympus Optical GmbH & Co.Wendenstrasse 14-19D–20097 Hamburg (Germany)Tel. +49 40 23773 0Fax +49 40 23773 647(microscopes, microforges and

equipment)

Pacer Scientific Instruments5649 Valley Oak DriveLos Angeles, CA 90068 (USA)Tel. +1 213 462 0636Fax +1 213 462 1430(recorder, micromanipulators, glass

capillaries, puller, stimulators,amplifiers)

SarstedtPostfach 1220D–51582 Numbrecht (Germany)Tel. +49 2293 3050(cell culture equipment, plastic culture

flasks)

Sartorius AGD–37070 Gottingen (Germany)Tel. +49 551 3080(filters for cell cultures, labware)

Science ProductsHofheimer Strasse 63D–65719 Hofheim (Germany)Tel. +49 6192 5046Fax +49 6192 5053(electrophysiological equipment, pullers,



Sigma ChemieGrunwalder Weg 30D–82039 Deisenhofen (Germany)Tel. +49 0130 5155Fax +49 0130 6490or: Sigma Chemical Co.PO Box 14508St. Louis, MO 63178 (USA)(chemicals, antibodies, cell culture

equipment, labware)

Sutter Instruments40 Leveroni CourtNovato, CA 94949 (USA)Tel. +1 415 883 0128Fax +1 415 883 0572(electrophysiological equipment, pullers,


TektronixColonia Allee 11D–51067 Koln (Germany)Tel. +49 221 96969 0Fax +49 221 96969 362(oscilloscopes)

Warner Instrument Corp.1125 Dixwell AvenueHamden, CT 06514 (USA)Tel. +1 203 776 0664Fax +1 203 776 1278(glass, AD/DA converter, amplifiers and

equipment)

WPI World Precision InstrumentsHarry FeinLiegnitzer Strasse 15 D–10999 Berlin

(Germany)Tel. +49 30 618 8845Fax +49 30 618 8670(microscopes, oscilloscopes and

electrophysiological equipment)

Carl Zeiss JenaTatzendpromenade 1aD–07740 Jena (Germany)Tel. +49 3641 64 2420Fax +49 3641 64 3140(microscopes, micromanipulators and

equipment)

The items which may be of interest for gap junction research as outlined in the book aregiven in brackets. This is, however, not the entire product list of the supplier. It was notpossible to incorporate all companies which supply items in the various fields of research.This is a list of suppliers mentioned somehow in this book and is not intended to representa complete list of suppliers for cell culture techniques, electrophysiology, biochemistry andlabware.


..............................Subject Index

AAP10, gap junction uncoupling 102, ATP, gap junction channel regulation44, 45104, 105

Acetic acid, gap junction uncoupling 94 Atrioventricular bundle, gap junctiondistribution 28, 30Acetylcholine, gap junction channel

regulation 46 Atrioventricular node, gap junctiondistribution 27, 29, 30Aconitine, gap junction uncoupling

95, 96 Atrioventricular reentry 9Atrium, gap junction distribution 29Action potential propagation

changes in arrhythmia 12coupling effects on transverse and Bundle branches, gap junction

distribution 30longitudinal propagation 5, 6effects of nonuniformity 4, 5

Acute cardiac disease, effects on Cable properties, muscle fibers 3, 4Caffeine, effect on uncoupled gapgap junctions 73–78

Acylcarnitines, gap junction uncoupling junctions 96Calcium, gap junction channel regulation74–76, 94

Adrenaline, gap junction channel 37, 38, 40–43cAMP, see Cyclic AMPregulation 47, 99

Aging, effects on gap junctions 88 Capacitive coupling 2, 3Carbachol, gap junction uncoupling 98, 99Angiotensin II, gap junction uncoupling

100, 101 Carbon dioxide, see pCO2

Cardiac arrhythmia suppression trialAnisotropic ratio, regional variations 6Anisotropic reentry 9, 11 (CAST), proarrhythmic risk of

antiarrhythmic drugs 1Antiarrhythmic peptides 101, 102,104, 105 Cardiac myocyte

cultureArachidonic acid, gap junction channelregulation 45, 46, 93, 94 embryonic chick cardiomyocytes

108, 109Arrhythmiaeffects on gap junctions 83, 84 neonatal rat cardiomyocytes 106–108

fibroblast-myocyte gap junctions 33types in ischemia 10, 11, 74Arrhythmogenic substrate, definition 1, 2 isolation from guinea pig 109, 110

shape effects on propagation 7, 8Assembly, gap junction channel 64–69

145

CAST, see Cardiac arrhythmia equations 51, 52voltage relationships in gapsuppression trial

Cell culture junction channels 52–54Cyclic AMP (cAMP), gap junction channelelectrophysiology, systems for study 50

embryonic chick cardiomyocytes regulation 35–38, 69, 70108, 109

neonatal rat cardiomyocytes 106–108 Decanoic acid, gap junction uncoupling 91Defective heart development, effectsChagas disease, effect on gap junctions

84–86 on gap junctions 86, 87Degradation, gap junction channel 68, 69Chronic ischemic heart disease, effects

on gap junctions 11, 79–81 Distribution, see Gap junction distributionDouble-cell voltage-clamp techniqueConnexins

assembly 66–69 50, 51, 116–119Dye-coupling studies 119, 120assembly of gap junctions 64–66, 68

Cx40 gene mutations 87Cx43 Electric field coupling 3

Electron microscopy, gap junctiongenemutation in defective heart distribution 25, 26, 34

Electrophysiology, gap junction channelsdevelopment 86, 87regulation 71, 72 cell systems for study 50

connexinsisolation 114–116physical properties 14, 15 Cx26 single-channel conductance 61

Cx37 single-channel conductanceisoform expression in gap junctions6, 13 60, 61

Cx40 single-channel conductancemultiple protein channels 34posttranslational modifications 59, 60

Cx43 single-channel conductance 5914–16, 40, 66, 69sequences 20, 21 Cx45 single-channel conductance 60

transjunctional voltage sensitivitysingle-channel conductanceCx26 channels 61 55–57

currentCx37 channels 60, 61Cx40 channels 59, 60 components 52

equations 51, 52Cx43 channels 59Cx45 channels 60 voltage relationships 52–54

double-cell voltage-clamp techniquespecies variability 23, 24staining and distribution 27–31, 110–114 50, 51, 116–119

intercellular resistance 52, 54, 58, 59structure 13, 14synthesis 64–66, 68, 69, 71, 72 ion permeability 61, 62

open probability of a single channel 57transjunctional voltage sensitivity 55–57turnover 68, 69 states of single-channel conductance 50

types of channels 50types 13, 19–23Coronary vasculature voltage sensitivity of channels 56, 57

Enalapril, effect on uncoupled gapchronic ischemic heart disease,effect on gap junctions 81 junctions 100, 101

Endothelial cells, gap junctiongap junction distribution 31Current distribution 31

Ethrane, gap junction uncoupling 90components 52

146Subject Index

Excitation, transfer between cardiac laser scanning confocal microscopy27, 34cells 2, 3

multiple connexin channels 34myocyte-fibroblast junctions 33FGF-2, see Fibroblast growth factor-2

Fibroblast growth factor-2 (FGF-2) Purkinje fibers 30sinoatrial node 29gap junction channel regulation 48, 71

release in chronic ischemic heart topology 26, 27ventricular myocardium 30, 31, 34disease 79

Gap junction channel Halothane, gap junction uncoupling 91Heart failure, effects on gap junctionsassembly 64–69

cardiac disease-induced changes 82, 83Heptanol, gap junction uncoupling 90acute cardiac disease 73–78

aging 88 Hypertension, effect on gap junctions 83arrhythmia 83, 84chronic ischemic heart disease Immunostaining

connexins 27–3179–81defective heart development 86, 87 protocol 110–114

Infective heart disease, effects onheart failure 82, 83infective heart disease 84–86 gap junctions 84–86

Ion permeability, gap junctionclosure mechanism 18clustering 17 channels 61, 62

Ischemiadegradation 68, 69developmental expression 63, 66 associated arrhythmias 10, 11

gap junction changeselectrophysiology, see Electro-physiology, gap junction channels acute cardiac disease 73–78

chronic ischemic heart diseasefunctions, overview 13, 25, 48–50new channel formation, kinetics 63, 64 11, 79–81

tissue properties in healing phase 11regulation, see specific regulatorsstructure 13, 16, 17synthesis 64–72 Laser scanning confocal microscopy,

gap junction distribution 27, 34uncouplingdrugs, see specific drugs Leading circle concept 8effects in heart disease 77, 78, 89, 90

Gap junction distribution Magnesium, gap junction channelregulation 44atrioventricular bundle 28, 30

atrioventricular node 27, 29, 30 Myocyte, see Cardiac myocyteatrium 29bundle branches 30 Noradrenaline, gap junction channel

regulation 47, 99chronic ischemic heart disease effects89, 81

connexin staining 27–31, 110–114 Octanol, gap junction uncoupling 90Oleic acid, gap junction uncoupling 92, 93coronary vasculature 31

effects on conduction 9, 10, 25 Ouabain, gap junction uncoupling 95electron microscopy 25, 26, 34endothelial cells 31 Palmitoleic acid, gap junction

uncoupling 91heart failure effects 82

147Subject Index

pCO2, gap junction channel regulation Protein kinase G (PKG), gap junctionchannel regulation 39, 4043

Permeability, gap junction channels to Purkinje fibers, gap junctiondistribution 30ions 61, 62

pH, gap junction channel regulation42, 43 Resistance, gap junction channels 52,

54, 58, 59Phospholipase C (PLC), gap junctionchannel regulation 38

Phosphorylation, gap junction channel Sinoatrial node, gap junctiondistribution 29regulation 40, 97

PKA, see Protein kinase A Sodium, gap junction channelregulation 44PKC, see Protein kinase C

PKG, see Protein kinase G Strophanthidin, gap junctionuncoupling 95PLC, see Phospholipase C

Potassium efflux, acute cardiac disease Synthesis, gap junction channel 64–7273, 74

Propagation velocity, ischemia effects Temperature, gap junction channelregulation 3575, 76, 90

Prophylaxis, proarrhythmic risk of Tyrosine kinase, gap junction channelregulation 40, 70, 71antiarrhythmic drugs 1, 2

Propionic acid, gap junction uncoupling94, 95 Uncoupling, gap junction channel

drug induction 90–105Protein kinase A (PKA), gap junctionchannel regulation 35–39 effects in heart disese 77, 78, 89, 90

Protein kinase C (PKC), gapjunction channel regulation 38, 39, 46, Ventricular myocardium, gap junction

distribution 30, 31, 3470, 97–100

148Subject Index

S. Dhein - Cardiac Gap Junctions. Physiology, Regulation, Pathophysiology and Pharmacology (1998)

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Transcript of S. Dhein - Cardiac Gap Junctions. Physiology, Regulation, Pathophysiology and Pharmacology (1998)