Draft · 2018. 8. 3. · Draft CJPP-2018-0115_R1 - 2 ABSTRACT The hyperpolarization-activated...
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HCN channels: pathophysiological, developmental and
pharmacological insights into their function in cellular excitability
Journal: Canadian Journal of Physiology and Pharmacology
Manuscript ID cjpp-2018-0115.R1
Manuscript Type: Review
Date Submitted by the Author: 02-May-2018
Complete List of Authors: Spinelli, Valentina; Universita degli Studi di Firenze Scuola di Scienze della
Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Sartiani, Laura; Universita degli Studi di Firenze Scuola di Scienze della Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Mugelli, Alessandro; Universita degli Studi di Firenze Scuola di Scienze della Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Romanelli, Maria Novella; Universita degli Studi di Firenze Scuola di Scienze della Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health Cerbai, Elisabetta; Universita degli Studi di Firenze Scuola di Scienze della
Salute Umana, Department of Neurosciences, Psychology, Drug Research and Child Health
Is the invited manuscript for consideration in a Special
Issue: IACS EU Section
Keyword: HCN channels, arrhythmias, cardiomyopathies, HCN blockers, β<sub>3</sub>-adrenergic receptors
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TITLE
HCN channels: pathophysiological, developmental and pharmacological insights into their
function in cellular excitability.
Valentina Spinelli1, Laura Sartiani
1, Alessandro Mugelli
1, Maria Novella Romanelli
1 and Elisabetta
Cerbai**1
1Department of Neurosciences, Psychology, Drug Research and Child Health (NeuroFarBa),
University of Florence, Florence, Italy;
** Corresponding author: Elisabetta Cerbai, PhD - Department of Neurosciences, Psychology,
Drug Research and Child Health (NeuroFarBa), University of Florence, Viale G. Pieraccini 6,
50139 Florence, Italy, phone: +390552758207 email: [email protected]
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ABSTRACT
The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion
channels, conducting both Na+ and K
+, blocked by millimolar concentrations of extracellular Cs
+
and modulated by cyclic nucleotides (mainly cAMP) that contribute crucially to the pacemaker
activity in cardiac nodal cells and subsidiary pacemakers. Over the last decades, much attention has
focused on HCN current, If, in non-pacemaker cardiac cells and its potential role in triggering
arrhythmias. In fact, in addition to pacemakers, HCN current is constitutively present in the human
atria and has since long been proposed to sustain atrial arrhythmias associated to different cardiac
pathologies or triggered by various modulatory signals (catecholamines, serotonin, natriuretic
peptides). An atypical If occurs in diseased ventricular cardiomyocytes, its amplitude being linearly
relate to the severity of cardiac hypertrophy. The properties of atrial and ventricular If and its
modulation by pharmacological interventions has been object of intense study, including the
synthesis and characterization of new compounds able to block preferentially HCN1, HCN2 or
HCN4 isoforms. Altogether, clues emerge for opportunities of future pharmacological strategies
exploiting the unique properties of this channel family: the prevalence of different HCN subtypes in
organs and tissues, the possibility to target HCN gain- or loss-of-function associated with disease,
the feasibility of novel isoform-selective drugs as well as the discovery of HCN-mediated effects
for old medicines.
Key words: Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channels; arrhythmias;
cardiomyopathies; HCN blockers; β3-adrenoceptors.
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INTRODUCTION
The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion
channels, conducting both Na+ and K
+ ions, blocked by millimolar concentrations of extracellular
Cs+ and modulated by cyclic nucleotides (mainly cAMP) (see (Sartiani et al. 2017) for a
comprehensive review). HCN-current was first described in cardiac nodal cells and subsidiary
pacemakers and termed If (funny current) by Dario Di Francesco and coworkers (Brown et al. 1979;
DiFrancesco 1981a, b). Similar recordings were obtained in neurons possessing rhythmic activity
(Yanagihara and Irisawa 1980; Yanagihara et al. 1980), and altogether these findings led to the
classification of If as a pacemaker current playing a relevant yet limited role in cells able to set the
pace. However, during the last decades and mainly after two groups succeeded sequencing the
genes coding for the four HCN isoforms (Ludwig et al. 1998; Santoro et al. 1998), it became more
and more evident that these channels are expressed almost ubiquitously. The expression of the four
different isoforms and the resulting HCN-current varies depending on tissue and cell subtype, age
and developmental stage, disease and genotype. Besides the canonical expression in cardiac
sinoatrial and atrioventricular nodes and in the conducting system, HCN current is constitutively
present in the human atria and has since long been proposed to sustain atrial arrhythmias associated
to different cardiac pathologies or triggered by various modulatory signals (catecholamines,
serotonin, natriuretic peptides) (Sartiani et al. 2017). An atypical If occurs in diseased human and
rodent ventricular cardiomyocytes, its amplitude being linearly related to the severity of cardiac
hypertrophy. This overexpression has been interpreted as the consequence of remodeling toward a
fetal phenotype, hence attention has been devoted to the fate of HCN channels during cardiac
differentiation of embryonic stem cells and in fetal cardiomyocytes. This review will recapitulate
available information concerning the pathophysiological and developmental features of HCN
channel expression in cardiomyocytes, with recent original data from our lab on modulation by β3-
adrenergic stimulation. Finally, we will remark on the progress toward HCN-isoform selective
blockers, and the opportunities for future pharmacological strategies.
MATERIAL AND METHODS
Methods described thereafter refers to original data described in the following sections;
experimental conditions and appropriate ethical statements related to previously published results
have been extensively described in corresponding literature.
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Culture of mouse embryonic stem cells
Mouse embryonic stem cells (mESC) of the line CGR8 commercially available, were used
(Nichols et al. 1990). Cells were propagated at 37 °C and 5 % CO2 in gelatin coated dishes, using
Glasgow Minimum Essential Medium BHK-21 (G-MEM BHK-21), supplemented with 10% fetal
bovine serum (FBS) (GIBCO Invitrogen, Milan, Italy), 2 mM glutamine, 0.1 mM 2-
mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids, 1%
penicillin/streptomycin (Sigma Aldrich, Milan, Italy) and 1000 units/ml of Leukemia Inhibiting
Factor (LIF, 10 µg/ml – MILLIPORE, Milan, Italy). All other products for cell cultures were
purchased from GIBCO Invitrogen. Prior to splitting, cultures were treated with trypsin/EDTA
(Sigma Aldrich) at room temperature for 30-60 sec in order to eliminate differentiated cells. These
culture conditions allowed CGR8 mESCs to maintain an undifferentiated phenotype characterized
by round-growing, tightly-packed, refrangent colonies.
Cardiac differentiation of CGR8 embryonic stem cells
CGR8 mESC were differentiated using the hanging drop method (Sartiani et al. 2007). Drops of
differentiating medium containing 600 cells were seeded on the lid of bacteriological dishes. When
the dish was closed, this allowed cells to aggregate and grow in suspension (for 2 days) and
eventually form mouse embryoid bodies (mEBs). Differentiation medium, in control condition,
consisted of medium G-MEM BHK21 supplemented with 10% FBS, 2 mM glutamine, 0.1 mM 2-
mercaptoethanol, 1 mM sodium pyruvate, 1% non-essential amino acids, 1%
penicillin/streptomycin. Thereafter, mEBs were re-suspended in differentiation medium and grown
in suspension in bacteriological dishes for 4 days. On the 6th day, mEBs were plated on gelatin-
coated dishes, where they attached. After 24-36 h from plating, cardiac differentiation was
recognizable from the occurrence of spontaneous contracting areas. mEBs were monitored daily by
inverted microscopy, the beating mEBs were counted from day 7th to 21th (D7-D21). mEBs
maturation was followed for no longer than 21 days according to experimental protocols.
To evaluate the modulating effect of β3-adrenergic receptor (β3-AR) signaling during cardiac
maturation, CGR8 mESCs were differentiated in the presence of the selective β3-AR agonist
BRL37344 (BRL, 7 µM) (Sigma Aldrich) and antagonist SR59230A (SR, 10µM) (Sigma Aldrich).
Drugs were administered from day zero (hanging drop formation) to the end of experiments,
renewing media every 2 days.
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Gene expression of pluripotency and cardiac development markers
Samples of mEBs were collected at different time points of differentiation for molecular assessment
by RNA extraction, reverse transcription and gene expression analysis using quantitative Real-Time
PCR (Q-PCR). Total RNA was isolated from mEBs using NucleoSpin® RNA kit (Macherey-Nagel,
Düren, Germany). Subsequently, complementary DNA (cDNA) was synthesized from 1 �g total
RNA using iScript ™ cDNA Synthesis kit (Bio-Rad, Milan, Italy). All these steps were performed
according to the manufacturer’s instructions. Real-time quantitative PCR (Q-PCR) was performed
using the default thermocycler program for all genes: 1 minutes of pre-incubation at 95°C followed
by 40 cycles for 15 seconds at 95°C, 1minute at 60°C and 15 seconds at 95°C, 1minute at 60°C and
1minute 95 °C. Individual real-time PCR reactions were carried out in 10 µl volumes in a 96-well
plate (Applied Biosystems™, London, UK) containing 2 µl RNAse free water, 1 µl of sense and
antisense primers (Bio-Rad) and 5 µl iTaq™ SYBR® Green Universal Supermix (Bio-Rad) plus 2
µl of cDNA sample (5 ng/µl). Each experiment was repeated in triplicate, and quantitative PCR
analysis was performed in triplicate and analyzed with Delta Ct-method. Mouse Glyceraldehyde-3-
Phosphate Dehydrogenase (mGapdh) was used for internal normalization. Results were analyzed by
assuming as 100% the maximal value of gene expression in each experiment.
RESULTS AND DISCUSSION
If current in ventricular cardiomyocytes: a hallmark of cardiac maladaption
The occurrence of a non-canonical pacemaker current in ventricular cardiomyocytes was first
inferred by Yu et al (Yu et al. 1993), based on original and somehow surprising observation of a
cesium-sensitive inward current, activated upon hyperpolarization, in guinea-pig cells. At that time,
some of us were trying to clarify an unexpected finding in papillary muscles from hypertrophied
(but not in normal) rat hearts. Ventricular samples from spontaneously hypertensive rats (SHR)
showed a sort of diastolic depolarization whose steepness was increased by isoproterenol,
eventually giving rise to spontaneous activity (Barbieri et al. 1994). In the following years, we
proved that the mechanism underlying abnormal depolarization and spontaneous activity was the
occurrence of an If -like current (Cerbai et al. 1994), whose amplitude correlated with the severity
of hypertrophy and was therefore very large in SHR with signs of heart failure (Cerbai et al. 1996).
The translational impact of this observation grew up with the demonstration the same held true for
human cardiomyocytes from failing explanted hearts (Cerbai et al. 1997).
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In line with other changes occurring during the progression to cardiac hypertrophy and failure
(Swynghedauw 1999), we inferred that If overexpression was a hallmark of a “regression” to a fetal
phenotype. Supporting this interpretation, If with similar properties (density, occurrence) was
present in immature rat cardiomyocytes from 2-day-old rats and declined thereafter during
maturation (Cerbai et al. 1999).
At that time, two groups succeeded to identify genes coding for the f-channel (HCN1-3) (Ludwig et
al. 1998; Santoro et al. 1998); soon after, a fourth isoform (HCN4) was detected (Ludwig et al.
1999). This advancement made it possible to widen our knowledge concerning gene and protein
expression in different species and tissues and, at a cardiac level, in different sites and experimental
or clinical settings. In parallel, the 2000’s were characterized by an exponential growth of studies
aimed at clarify the ontogenetic and phylogenetic features of this unique family of channels
(Christoffels et al. 2010; Jackson et al. 2007; Mommersteeg et al. 2007).
In our lab, we focused first to changes in cardiac HCN expression occurring in response to different
pathophysiological settings. The left panels of Figure 1 show typical recordings of action potentials
stimulated at 0.2 Hz pacing rate, and If obtained in patch-clamped left ventricular cardiomyocytes
from control and failing human hearts. Current density measured at -120 mV resulted significantly
higher in ischemic cardiomyopathy (2.0±0.2 pA/pF) than in dilated cardiomyopathy (1.2±0.1
pA/pF) or control (1.0±0.1 pA/pF). In the same panels of Figure 1, normalization to maximal
current at -120 mV allows comparing activation kinetics and voltage dependence: If activates at less
negative potentials in cardiomyocytes from failing hearts, the voltage of half maximal activation
being shifted by about 10 mV rightwards (Cerbai et al. 2001; Stillitano et al. 2008). Together with If
gain-of-function, the expression of HCN isoforms, remarkably HCN2 and HCN4 but not HCN1,
increased by 7 to 9-fold (Stillitano et al. 2008).
HCN expression in cardiac remodeling and differentiation: a mirror-image process?
Both transcriptional and post-translational processes contribute to gain- or loss-of-function of If in
adult cardiac cells. The over-expression of If, detected in ventricular myocytes from hypertrophied
or failing rat hearts, was counteracted by drugs interfering with the renin angiotensin system, such
as losartan or irbesartan (Cerbai et al. 2000; Cerbai et al. 2003), as well as by the bradycardic agent
ivabradine (Suffredini et al. 2012). Electrophysiological reverse remodeling was associated with
downregulation of mRNA and protein level for the two major ventricular isoforms, HCN2 and
HCN4. The promoter regions of the two isoforms appear to be trans-activated by the DNA-binding
protein Sp1, belonging to the family of zinc-finger transcription factors: hindering Sp1
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overexpression during stimulation with hypertrophic factors (e.g., angiotensin II) prevents
HCN2/HCN4 re-expression in adult ventricular cardiomyocytes (Lin et al. 2009).
Recent evidence highlighted the role of microRNAs, miR-1 and miR-423-5p, targeting HCN4 in the
SAN of trained rats as well as athletes (D’Souza et al. 2014; D’Souza et al. 2017); of note,
endurance athletes often suffer from bradycardia and supraventricular arrhythmias at old age.
Oppositely, decreased miR-1 level in samples from patients with atrial fibrillation is associated to If
gain-of-function in cardiomyocytes (Li et al. 2015; Stillitano et al. 2013).
As mentioned above, upregulation of HCN protein and, correspondingly, f-current likely represents
a marker of re-expression of fetal genes. Indeed, HCN4 expression occurs ever since the appearance
of the pacemaker activity in the precardiac mesoderm and plays a fundamental role thereafter,
(between E9.5 and E11.5 in mice) (Stieber et al. 2003). Developmental changes of HCN expression
during cardiogenesis in mice has been deeply investigated (Barbuti and Robinson 2015; Christoffels
et al. 2010). Similar studies are obviously lacking for human hearts; however, developmental
changes of cardiomyocytes from embryonic or induced Pluripotent Stem (iPS) cells can provide
interesting insights. The right panels of Figure 1 exemplify this concept. Using human embryonic
stem cells (hESC) committed and differentiated toward the cardiac phenotype, we observed that If
evolves from “less mature” to “more mature” biophysical features (smaller amplitude, more
negative threshold of activation), in parallel with changes in action potential profile, during in vitro
maturation from early to late stages (Sartiani et al. 2007). Again, these changes were accompanied
by parallel decline of expression of all HCN isoforms (Bosman et al. 2013; Sartiani et al. 2007), i.e.,
in the reverse direction tracked by maladaption remodeling. This process goes hand-in-hand with
flattening of diastolic depolarization and decrease of spontaneous rhythmicity.
As recently reviewed by Barbuti and Robinson (2015), several groups have been trying to
counteract the fading of spontaneous activity, that is, to enrich the percentage of pacemaker-like
myocytes. Cell-engineering, pharmacological tools and cell selection processes have been applied,
and invariably the maintenance (or increase) of HCN4 expression has been considered a reliable
marker (Garcia-Frigola et al. 2003; Morikawa et al. 2010; Scavone et al. 2013). Among
pharmacological approaches, the most successful were endothelin-1 (Gassanov et al. 2004), the K-
channel modulator EBIO (1-ethyl-2-benzimidazolinone) (Kleger et al. 2010) and suramin (Wiese et
al. 2011). Interestingly enough, in most cases the enrichment in SAN-like cardiomyocytes was
accompanied by upregulation of transcription factors involved in specification of the conducting
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system such as Isl-1, Tbx3 and Shox2 (Kleger et al. 2010). Although very promising, all these tools
were far from the physiological setting.
Very recently, we approached this issue starting from a different point of view consistent with our
experience with the pathophysiology of cardiac adaption. Beta3-adrenergic receptors (β3-AR),
constitutively expressed in human and rodent cardiomyocytes, undergo upregulation and gain-of-
function in heart failure (Gauthier et al. 1996; Moniotte et al. 2001; Sartiani et al. 2006). At
variance with β1- and β2-AR subtypes, β3-AR couple to nitric oxide synthase through activation of
inhibitory G-proteins (Gauthier et al. 1998). To note, this peculiar signaling pathway apparently
results in a negative shift of If activation in rat (Sartiani et al. 2006) and human (unpublished
observations) ventricular cardiomyocytes, i.e., the reverse effect caused by β1- and β2-AR
stimulation).
We investigated whether β3-AR overexpression in myocardial hypertrophy and failure also
represents a regression toward a fetal phenotype. Therefore, we analyzed changes in expression of
β3-AR in murine embryonic stem cells differentiated into cardiomyocytes (mESC-CM). Figure 2A
shows a typical blot obtained in mESC-CM at different stages. Interestingly, these receptor
subtypes were not only present, but also functional: treatment with a well-known receptor
antagonist (SR) or an agonist (BRL) results in opposite effects on growth of embryonic bodies (data
not shown) and timing/percentage of beating areas (Fig. 2B).
This acceleration/impairment of cardiac commitment and maturation with BRL or SR, respectively,
was accompanied by opposite up- and down-regulation of genetic markers of cardiac and sinoatrial
differentiation. Results summarized in Figure 2C show the effect of treatment with the β3-AR
agonist and antagonist on the relative expression of selected markers (mesoderm, pre-cardiac, early
and late cardiac markers) in a narrow, sensitive window, around 7-day post differentiation, i.e.,
when EBs start beating. However, our attention pointed to the evidence that not only the number of
beating areas, but also their firing rate at 14 days after differentiation was significantly affected by
β3-AR: increased by the agonist, decreased by the antagonist (Fig 3A). This effect was paralleled by
a remarkable upregulation of SAN markers, HCN1 and Cav1.3, in the presence of the β3-AR
agonist, BRL (Fig 3B). The expression of HCN4, in terms of mRNA level, was only slightly
changed by BRL with respect to CTR. However, in the same time window, immunohistochemistry
assay suggests that protein and especially membrane localization of HCN4 was enhanced by BRL
with respect to control and, vice versa, hindered by SR treatment (Fig. 3C); a similar tendency was
observed with α-actinin. Such a discrepancy between HCN4 mRNA and protein expression has
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been already described in atrial tissue likely due to post-transcriptional processing (Stillitano et al
2013). At variance with previous data from our lab obtained in cardiomyocytes from humans
embryonic stem cells (Bosman et al., 2013), we did not observe a membrane “spotty” staining of
HCN4. This difference might be due to the early stage of cardiac differentiation and membrane
organization (i.e., low caveolin-3 expression); however, similar appearance was reported for HCN4
staining in mESC-CM (Saito et al., 2015) and cardiomyocytes isolated from embryonic mouse
hearts and cultured thereafter (Scavone et al., 2013). Overall, although preliminary, these
observations open a new perspective on the mutual relationship between β3-AR and HCN4
(over)expression, whose mechanistic basis deserves to be further investigated also in view of its
possible role in SAN specification.
Pharmacological insights into HCN function in cellular excitability: the dark site of channels
The role of If in primary and subsidiary cardiac pacemakers has been deeply investigated (and also
debated, see for example (DiFrancesco and Noble 2012; Maltsev and Lakatta 2012); the reader can
refer to several reviews dealing with this matter (Biel et al. 2009; Sartiani et al. 2017; Wahl-Schott
et al. 2014). More uncertainty exists on rhythm disturbances due to ectopic or augmented HCN
expression in other cardiac districts. Upregulation of HCN2 and HCN4 channels in a transgenic
model characterized by severe heart failure was associated with ventricular tachycardia and sudden
cardiac death, sensitive to the If blocker ivabradine (Kuwabara et al. 2013; Yamada et al. 2014).
Electrical instability due to HCN overexpression was also detected in a mouse model of ventricular
hypertrophy (Hofmann et al. 2012). Ivabradine was able to ameliorate cardiac function and revert
HCN overexpression in a rat model of heart failure due to myocardial infarction (Ceconi et al. 2011;
Suffredini et al. 2012). Not only gain of function, but also loss-of-function might underlie the
ventricular arrhythmic burden, as recently suggested by the identification of a novel mutation in the
HCN4 channel in a patient with Brugada syndrome (Biel et al. 2016). Seemingly, HCN channel
dysfunction in ventricular tissue, as well as in other cardiac sites, might be unveiled and emphasized
by concomitant changes of other ion currents in congenital or acquired syndromes. In long-QT
syndrome patients carrying mutations of caveolin-3 (a lipid raft component of myocyte membrane),
simultaneous alterations of HCN4 properties as described in naïve cardiomyocytes and cell lines
(Barbuti et al. 2004; Barbuti et al. 2012; Barbuti et al. 2007; Bosman et al. 2013) may contribute to
the clinical phenotype (Motloch et al. 2017). The hypothesis of “polygenic mechanisms underlying
cardiac disorders” involving HCN4 mutation has been inferred also by the association between
sinus bradycardia and left ventricular non-compaction cardiomyopathy (LVNC) in families with
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heritable HCN4 defects (Milano et al., 2014; Schweizer et al. 2014) and further confirmed by recent
investigation in an infant with LVNC (Yokoyama et al. 2018).
The predominant role of HCN4 at cardiac level prompted us to search for isoform selective blockers
(Romanelli et al. 2016). The feasibility of this approach has been demonstrated by the selectivity of
a series of compounds derived from structural modifications of the non-selective blocker
zatebradine (Del Lungo et al. 2012; Koncz et al. 2011; Melchiorre et al. 2010). This strategy opened
up a somehow unforeseen, promising field: exploitation of blockers targeting HCN1/HCN2
isoforms – instead of the “cardiac” isoform, HCN4– to treat extra-cardiac disorders without
affecting heart rhythm and conduction. In fact, HCN1 blockade by the selective inhibitor MEL57A
counteracted hyperalgesia and allodynia in chemotherapy-induced neuropathy, an ill-treated
condition in patients, at dosage devoid of bradycardic effects (Resta et al. 2018). Recent
unpublished data from our lab suggest that a similar result was achieved by simultaneous blockade
of HCN1/HCN2.
Conclusions
Almost 40 years of intensive research, since the first description of a cardiac funny current (Brown
et al., 1979), have allowed an exponential growth of our knowledge on HCN channels’ properties -
from biophysics to structure and molecular features - making it possible the pharmacological and
therapeutic exploitation of selective f-channel blockers. However, the physiological and
pathophysiological regulation of HCN expression in biologically and clinically relevant settings
unveils new and original fields of investigation. In this light, clues emerge from the observation of a
promoting effect of β3-AR stimulation of HCN functional expression during cardiac differentiation,
whose molecular mechanisms deserve further investigation.
ACKNOWLEDGEMENTS
All authors declare no conflict of interest. This work was funded by grants from the University of
Florence (ex 60%) (M.N.R.); Ente Cassa di 390 Risparmio di Firenze (2013.0683 to L.S.);
Normacor project (contract LSH 391 M/CT/2006/018676 to E.C.).
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FIGURES AND FIGURE LEGENDS
Figure 1. Representation of membrane potential or action potential, HCN-current and HCN
isoform relative expression in human adult cardiomyocytes (Panel A) and cardiomyocytes
differentiated from human embryonic stem cells (Panel B). Action potentials in normal or
failing hVCM were obtained by patch-clamp recordings at 0.2Hz pacing rate. Abbreviation: hESC,
human embryonic stem cells; hESC-CM early, late: cardiomyocytes differentiated from hESC at
early (20 days) and late (90 days) phase; hVCM Normal, Failing: cardiomyocytes dissociated the
left ventricle of control donor hearts or explanted hearts from patients with ischemic heart failure.
(Modified from Cerbai et al. 2001; Sartiani et al. 2007; Stillitano et al. 2013; Stillitano et al. 2008).
Figure 2. Molecular and functional expression of ββββ3-AR during cardiac differentiation of
mESC. Panel A: Immunoblot and relative quantification of β3-AR protein in mESC at different
stages (from day 2, D2, to day 14, D14) of cardiac differentiation. Panel B: number of beating areas
in mESC embryo bodies differentiating in control medium supplemented or not with a selective β3-
AR antagonist (SR, red) or agonist (BRL, blue). Panel C: Q-PCR analysis of mRNA levels
measured in mESC-CM cultured in the presence of SR or BRL, compared to matched controls
(CTR). Bars represent the relative expression of genes coding for markers of pluripotency (Oct4),
early mesoderm (Brachiury, Bra); pre-cardiac development (Wnt3a; Wnt11; β-catenin, Ctnnb1);
first and second heart field (Mef2c, Hand1, Hand2, Isl1, Fgf10); cardiac differentiation (Gata4,
Nkx2.5, Tbx3, Tbx18, Shox2, cTnI).
Figure 3. Modulation of mESC-CM differentiation and maturation by ββββ3-AR signaling. Panel
A: Frequency of spontaneous contraction of beating areas in embryoid bodies (EBs) differentiating
in control medium supplemented or not with a selective β3-AR antagonist (SR, red) or agonist
(BRL, blue). *** p<0.01 (BRL vs. CTR and BRL vs. SR), ** p<0.01 (SR vs. CTR). Panel B:
expression of genes coding for alpha 1D subunit of L-type calcium channels, CaV1.3, HCN4 and
HCN1 isoforms evaluated by Q-PCR analysis, expressed as ratio versus control (dashed line), in
mESC-CM cultured in the absence (CTR) or presence of SR or BRL. Panel C: Representative
results from immunohistochemistry assays showing expression and localization of HCN4 and α-
actinin in mESC-CM cultured in the absence (CTR) or presence of SR or BRL.
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Figure 1. Representation of membrane potential or action potential, HCN-current and HCN isoform relative expression in human adult cardiomyocytes (Panel A) and cardiomyocytes differentiated from human
embryonic stem cells (Panel B). Abbreviation: hESC, human embryonic stem cells; hESC-CM early, late: cardiomyocytes differentiated from hESC at early (20 days) and late (90 days) phase; hVCM Normal, Failing: cardiomyocytes dissociated the left ventricle of control donor hearts or explanted hearts from
patients with ischemic heart failure. (Modified from Cerbai et al. 2001; Sartiani et al. 2007; Stillitano et al. 2013; Stillitano et al. 2008).
209x157mm (300 x 300 DPI)
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Figure 2: Molecular and functional expression of β3-AR during cardiac differentiation of mESC. Panel A: Immunoblot and relative quantification of β3-AR protein in mESC at different stages (from day 2, D2, to day 14, D14) of cardiac differentiation. Panel B: number of beating areas in mESC embryo bodies differentiating
in control medium supplemented or not with a selective β3-AR antagonist (SR, red) or agonist (BRL, blue). Panel C: Q-PCR analysis of mRNA levels measured in mESC-CM cultured in the presence of SR or BRL,
compared to matched controls (CTR). Bars represent the relative expression of genes coding for markers of pluripotency (Oct4), early mesoderm (Brachiury, Bra); pre-cardiac development (Wnt3a; Wnt11; β-catenin,
Ctnnb1); first and second heart field (Mef2c, Hand1, Hand2, Isl1, Fgf10); cardiac differentiation (Gata4, Nkx2.5, Tbx3, Tbx18, Shox2, cTnI).
209x157mm (300 x 300 DPI)
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Figure 3. Modulation of mESC-CM differentiation and maturation by β3-AR signaling. Panel A: Frequency of spontaneous contraction of beating areas in embryoid bodies (EBs) differentiating in control medium
supplemented or not with a selective β3-AR antagonist (SR, red) or agonist (BRL, blue). *** p<0.01 (BRL vs. CTR and BRL vs. SR), ** p<0.01 (SR vs. CTR). Panel B: expression of genes coding for alpha 1D subunit of L-type calcium channels, CaV1.3, HCN4 and HCN1 isoforms evaluated by Q-PCR analysis, expressed as ratio versus control (dashed line), in mESC-CM cultured in the absence (CTR) or presence of SR or BRL.
Panel C: Representative results from immunohistochemistry assays showing expression and localization of HCN4 and α-actinin in mESC-CM cultured in the absence (CTR) or presence of SR or BRL.
209x155mm (300 x 300 DPI)
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