Prostaglandins & other Lipid Mediators 80 (2006) 110–119

An unexpected negative inotropic effect of prostaglandinF2� in the rat heart

Nenad Jovanovic a, Mirjana Pavlovic b, Vladimir Mircevski c,Qingyou Du d, Aleksandar Jovanovic d,∗

a Division of Experimental Pharmacology, Institute of Oncology and Radiology of Serbia, Belgrade, Serbiab Department of Physiology, School of Medicine, University of Belgrade, Serbia

c Department of Neurosurgery, University of Skopje, Skopje, Macedoniad Maternal and Child Health Sciences, Ninewells Hospital & Medical School,

University of Dundee, Dundee DD1 9SY, Scotland, UK

Received 14 May 2006; received in revised form 19 May 2006; accepted 22 May 2006Available online 30 June 2006

Abstract

Prostaglandin F2� (PGF2�) is produced during myocardial inflammation and many of the insults that trigger contractile dysfunctionalso activate prostaglandin synthesis and production. However, although PGF2� plays a significant role in the cardiac response toinflammation, the effect of this particular compound on the heart was largely studied at the cellular level and probably no dueattention was paid to the effect of PGF2� on the whole heart contractility. Therefore, in the present study we have investigated theeffect of PGF2� on isolated right ventricle of the rat heart. PGF2� (1 nM–1 �M) induced concentration-dependent decrease of theamplitude of contractions of the ventricular muscle. Real time RT-PCR has revealed that prostaglandin FP receptors are expressedin the rat myocardium and the level of expression was similar to those of creatine kinase and adenylate kinase, which are proteinsabundantly present in the heart. An antagonist of FP receptors, PGF2� dimetilamide (10 nM), abolished negative inotropic effectinduced by PGF2�. To examine the possibility that PGF2� could activate non-FP prostaglandin receptor, we have measured the levelof expression of all known prostaglandin receptors in the rat heart. These experiments have shown that the order of expression ofprostaglandin receptors in the rat heart is FP � EP1 = TP > EP4 > EP3 > DP = IP. Based on the obtained results we conclude thatPGF2� induces negative inotropic effect on rat heart by activating FP prostaglandin receptors. This effect of PGF2� could contributeto cardiac dysfunction in conditions of systemic and myocardial inflammation.© 2006 Elsevier Inc. All rights reserved.

Keywords: Prostaglandin PGF2�; Heart; FP receptors

1. Introduction

Myocardial contractile dysfunction accompanies both systemic and cardiac insults. It is well established that inflam-mation of the heart can precipitate transient or permanent impairments of contractility. Prostaglandin F2�(PGF2�) isproduced in aboundant amounts during myocardial inflammation and many of the insults that trigger contractile dys-function also activate prostaglandin synthesis and production [1,2]. However, although PGF2� plays a significant role

∗ Corresponding author. Tel.: +44 1382 496 269; fax: +44 1382 632 597.E-mail address: a.jovanovic@dundee.ac.uk (A. Jovanovic).

1098-8823/$ – see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.prostaglandins.2006.05.014

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in cardiac response to inflammation, the effect of this particular compound on the heart was largely studied at thecellular level [3] and probably no due attention was paid to the effect of PGF2� on the whole heart contractility.

Therefore, the main purpose of this study was to determine the effect of PGF2� on the cardiac contractility. Wereport that PGF2� decreases contractility of right ventricle of the rat heart by activating prostaglandin FP receptors.This particular effect of PGF2� could contribute to development of heart failure observed in inflammatory diseases ofthe myocardium.

2. Materials and methods

2.1. Animals

We used female and male Wistar rats weighing 200 ± 10 g. Care and use of animals was in accordance with therecommendations of the Guide for the Care and Use of Laboratory Animals, published by the US National Institutesof Health (NIH publication No. 85-23, revised 1985).

2.2. Isolated cardiac muscle function studies

Experiments on isolated tat right ventricular muscle have been performed as we described previously [4]. In brief,animals were sacrificed by cervical dislocation and the hearts were rapidly removed. Right ventricle was dissectedfrom the cardiac tissue and placed in 25 ml muscle bath (at 37 ◦C) containing buffer of the following composition (inmM): NaCl 136.9, KCl 2.69, CaCl2 1.8, MgCl2 1.05, NaHCO3 11.9, NaH2PO4 0.42 and glucose 5.55. The buffer wascontinuously bubbled with a mixture of 95% O2 and 5% CO2 and the pH was maintained at 7.4. Each muscle wasaffixed to a isometric force transducer set up at 0.05 g/cm sensitivity. Ventricular muscle was stimulated electrically tocontract isometrically. Electrical impulses were square wave forms (5 ms duration) set at 100% higher than thresholdvoltage with a stimulus rate of 60 contractions per min. Isometric tension was recorded on a Ugo Basile model 7050recorder. The preparations were allowed to equilibrate for 60 min and during that time contractions were continuouslymonitored. Only preparations exhibiting stable and equal (variations in amplitude less than 5%) were used for furtherexperimentation. To determine whether contractions remain stable after the incubation period, in a separate seriesof experiments we have monitored muscle action for additional 70 min. Concentration–response curve for PGF2�

was made by adding increasing concentrations of this compound; the effect of each concentration was monitored for10 min. To determine the involvement of prostaglandin FP receptors, we have compared the effect of a single 100 nMconcentration of PGF2� in the absence and presence of PGF2� dimetilamide. In the absence of PGF2� dimetilamide, theeffect of 100 nM of PGF2� was monitored after 60 min-long stabilisation period for 10 min. When PGF2� dimetilamidewas used, it was applied alone for 10 min after the incubation period and then PGF2� was added and monitored for thenext 10 min. PGF2� and PGF2� dimetilamide were purchased from Sigma (UK) and Cayman Chemical, respectively.

2.3. Real time RT-PCR

Real time RT-PCR has been performed as we previously described [5]. Total RNA was extracted from rat heartusing TRIZOL reagent (Invitrogen, UK), and extracted RNA was further purified by RNeasy Mini Kit (Qiagen, UK)according to the manufacturer’s instruction. The specific primers for all known types of prostaglandin receptors (FP, DP,IP, TP, EP1, EP2, EP3 and EP4) as well as adenylate kinase (AK) and creatine kinase (CK) were designed as depictedin Table 1. The specificity of primers was tested by melting curve analysis and for their ability to produce no signal innegative controls by dimer formation. The reverse transcription (RT) reaction was carried out with ImProm-II ReverseTranscriptase (Promega, USA). A final volume of 20 �l of RT reaction containing 4 �l of 5× buffer, 3 mM MgCl2,20 U of RNasin® Ribonuclease inhibitor, 1 U of ImProm-II reverse transcriptase, 0.5 mM each of dATP, dCTP, dGTP,and dTTP, 0.5 �g of oligo(dT), and 1 �g of RNA was incubated at 42 ◦C for 1 h and then inactivated at 70 ◦C for 15 min.The produced cDNA were used as template for the quantitative real-time PCR. A SYBR Green I system was utilized inthe reaction. The 25 �l reaction mixture contained: 12.5 �l iQTM SYBR® Green Supermix (2×), 7.5 nM each primers,9 �l of ddH2O, and 2 �l of cDNA. The thermal cycling conditions were as follows: an initial denaturation at 95 ◦C for3 min, followed by 38 cycles of 10 s of denaturing at 95 ◦C, 15 s of annealing at 56 ◦C, and 50 s of extension at 72 ◦C.The real-time PCR was performed in the same wells of a 96-well plate in the iCycler iQTM Multicolor Real-Time

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Table 1Specific primers designed for different types of prostaglandin receptors (FP, DP, IP, TP, EP1, EP2, EP3, and EP4) as well as for adenylate kinase (AK) and creatine kinase (CK)

Genes Primers Size of PCR product (bp)

FP Sense 5′-ACAGGCAAGGCAGGTCTC-3′, antisense 5′-AGCGTCGTCTCACAGGTC-3 151AK Sense 5′-ATGAATTCGTGGTGGGCGGACCTGGCTC-3′, antisense 5′-GCCTCGAGTTAAATGCCACGCTTGTCATAGA-3′ 471CK Sense 5′-ATGAATTCGAGTACCCAGACCTCAGCAA-3′, antisense 5′-GCCTCGAGCTACTTCTGCGCGGGGATCA-3′ 1089DP Sense 5′-CGCCTTCCTGATGTCCTTC-3′, antisense 5′-AGCCTATTACCGAGAATGAGC-3′ 272IP Sense 5′-CGGGCACGAGAGGATGAAG-3′, antisense 5′-ACACAGACAACACAACCAGAAC-3′ 246TP Sense 5′-GTGAGGTGGAGATGATGGTTC-3′, antisense 5′-CGTAGGTAGATGAGCAGTTGG-3′ 169EP1 Sense 5′-TCTCTGCCGCTGCTCAAC-3′, antisense 5′- GTAGGAGGCGAAGAAGTTGG-3′ 162EP2 Sense 5′-CGGGCACGAGAGGATGAAG-3′, antisense 5′-CTCCTCCGCCATAGAAGTCC-3′ 270EP3 Sense 5′-CACCACGGAGACGGCTATC-3′, antisense 5′-AGGCGAACGGCGATTAGG-3′ 174EP4 Sense 5′-CGGAGACCAGGCATTGTG-3′, antisense 5′-AAGAGAAGGCGGCGTAGG-3′ 299

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Detection System (Bio-Rad, Hercules, USA). The data were collected following each cycle and displayed graphically(iCycler iQTM Real-time Detection System Software, version 3.0 A, BioRad, Hercules, USA). Threshold cycle (CT)values were determined automatically by software. The melting curve data were collected to check the PCR specificity.The cDNA sample was run in duplicate and the corresponding no-RT mRNA sample was included as a negative control.

2.4. Statistical analysis

Data are presented as mean ± S.E.M., with n representing the number of examined hearts. The difference betweenmeans where assessed using t-test. P < 0.05 was considered statistically significant.

3. Results

3.1. Contractions of right ventricles are stable for 70 min

Following 60 min-long incubation period contractions of right ventricles were continuously monitored for the next70 min. As depicted in Fig. 1, no changes in amplitude of cardiac contractions were observed during this period (n = 5,P > 0.2).

Fig. 1. Right ventricular contractions are stable over the course of 70 min. (A) Time course of the amplitude of right ventricular contractions. Eachpoint represent mean ± S.E.M. (n = 5 for each point). (B) Bar graph shows the amplitude of right ventricular contractions at 60 and 130 min timepoints. Each bar represents mean ± S.E.M. (n = 5 for each bar).

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Fig. 2. Prostaglandin F2� (PGF2�) induces negative inotropic effect on cardiac muscle. (A) Concentration-dependent curve for PGF2� action onright ventricular cardiac muscle. Each point represent mean ± S.E.M. (n = 3 for each point). (B) Time course of the amplitude of right ventricularcontractions. Each point represent mean ± S.E.M. (n = 7 for each point). (C) Bar graph shows the amplitude of right ventricular contractions in theabsence and presence of PGF2� (100 nM). Each bar represents mean ± S.E.M. (n = 7 for each bar);*P < 0.05.

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3.2. PGF2α induces concentration-dependent decrease in amplitude of ventricular contractions

When cumulatively applied, PGF2� (1 nM–1 �M) induced concentration-dependent decrease in amplitude of cardiaccontractions (Fig. 2A). The estimated EC50 was ∼100 nM. When 100 nM of PGF2� was applied on beating rightventricle, negative inotropic effect was clearly observed and the difference between contractions in the absence andpresence of PGF2� was statistically significant (Fig. 2B and C, P = 0.009, n = 7).

3.3. PGF2α induces concentration-dependent decrease in amplitude of ventricular contractions by activating FPreceptors

Some functional studies on isolated cardiomyocytes have suggested that PGF2� may act on FP receptors on car-diomyocytes [3]. However, no direct evidence has been provided so far to suggest that these receptors can be found in theheart. Therefore, we have applied real time RT-PCR methodology to test whether FP is expressed in the myocardium.In these regards, we have designed FP-specific primers that have produced a single product as indicated by the shapeof melting curves (Fig. 3). Real time RT-PCR has shown that right ventricular tissue contains FP receptors (cycle

Fig. 3. FP receptors are highly expressed in cardiac ventricular tissue. Melting curve analysis of FP, adenylate kinase (AK) and creatine kinase (CK)amplicons (shown are plots of fluorescence (−dF1/dT) vs. temperature; the presence of a single peak is consistent with the formation of a singleamplicon and indicates the lack of primer-dimer formation) and representative progress curves done in duplicate for the real-time PCR amplificationof FP, AK and CK cDNA as depicted on the figure itself.

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Fig. 4. Negative inotropic effect of PGF2� is inhibited by PGF2� dimetilamide. Time course of the amplitude of right ventricular contractions in thepresence of PGF2� dimetilamide (10 nM) and PGF2� (100 nM) plus PGF2� dimetilamide (10 nM). Each point represent mean ± S.E.M. (n = 7 foreach point).

threshold was 23.8, Fig. 3). To put the level of this expression into prospective, we have compared FP expression withthe expression of two abundant myocardial proteins, creatine kinase (CK) and adenylate kinase (AK). Cycle thresholdwas 19.9 for CK and 26.6 for AK (Fig. 3). These results show that the level of expression of FP is high and comparablewith the expression of highly expressed myocardial proteins. To prove that the activation of FP receptors mediatethe negative inotropic action of PGF2� we have tested the effect of PGF2� dimetilamide, a compund know to be apartial agonist of FP receptors [6]. When applied on its own PGF2� dimetilamide (10 nM) has induced a decrease inthe amplitude of ventricular contractions, which is in agreement with the partial agonistic nature of this compound(Fig. 4). However, when applied in the presence of PGF2� dimetilamide, PGF2�-induced negative inotropic effect wasabolished and even slightly positive inotropic effect was observed (Fig. 4). To examine the possibility that PGF2� hasactivated non-FP prostaglandin receptor we have examined the level of expression of all known prostaglandin receptorsin the rat heart. These experiments have shown that cycle threshold was 27.8, 30.1, 30.3, 28.5, 28.0, 31.6 and 31.7 forEP1, EP2, EP3, EP4, TP, DP and IP receptors, respectively (Fig. 5). Thus, the order of expression of prostaglandinreceptors in the rat heart was FP � EP1 = TP > EP4 > EP3 > DP = IP.

4. Discussion

In previous studies, cardiac effects of PGF2� has been examined primarily on isolated cardiomyocytes. It has beenshown that PGF2� activates inositol triphosphate (IP3) signalling pathway, a pathway leading to an increase of intra-cellular Ca2+ and positive inotropic effect [7–9]. It has been reported that PGF2� stimulates contractility in isolatedcardiomyocytes by increasing intracellular Ca2+ levels [3]. However, the results obtained on the whole heart, were nottotally in agreement with the findings reported on isolated cells and the findings published so far seem to be contro-versial. To this end, it has been suggested that PGF2� induces positive inotropic effect by activating phospholipase C(PLC)/IP3 signalling pathway/increasing cardiac Ca2+ levels [10] or that stimulates cardiac contractility independentlyof intracellular Ca2+ [11] or even that PGF2� has no cardiac effects whatsoever [12]. Our present finding that PGF2�

induces negative inotropic effect is, at first sight, in contradiction with previous reports. The concentration-dependentnature of negative inotropic effect would suggest an involvement of specific receptors in this particular PGF2� action.In support of our results, some studies have suggested that PLC/IP3/Ca2+ signalling pathway could induce negativeinotropic effect [13]. As an example, it has been shown that that PLC bound � adrenoceptors could mediate negative,

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instead of positive, inotropic effect [14]. This phenomenon is yet not entirely clear, but it could be probably explainedby the complexity of PLC signalling pathway. PLC targets phosphatydilinositol biphosphate (PIP2) to produce IP3,which, in turn, mobilises Ca2+ from sarcoplasmic reticulum leading to stimulation of cardiac contractility. However,besides IP3, diacylglycerol (DAG) is also a product of PLC catalysed reaction. DAG activates protein kinase C (PKC)an enzyme with multiple targets which could lead to a negative inotropic effect. Accordingly, it has been shown thatPKC activate Na+/K+ ATPase, troponine or ATP-sensitive K+ (KATP) channels, all of which could decrease cardiaccontractility [14–17]. It is quite possible that the outcome on cardiac contractility following activation of PLC dependson the basal state of the heart. In the experimental model we have applied here, the effect of PGF2� was examinedunder conditions that were similar to those in vivo. In most of the previous studies, the effect of PGF2� was analysedon isolated non-beating cardiomyocytes [11]. It is likely, and that was noted before, that when cycle systole–diastoleexist the negative inotropic effect of PKC activation could be predominant effect of activation PLC signalling pathway.It should be also taken into consideration that in our experimental model endocardial endothelium has remained intact[4], which might be crucial for the PGF2� negative inotropic action. In these regards, it has been shown that nitricoxide (NO) form endocardial endothelium relaxes cardiac muscle [18], which could mediate a negative inotropic effectinduced by prostaglandins [19].

In most of the tissues PGF2� induces its effect via specific prostaglandin FP receptors [20]. Some functional studieshave suggested that FP receptors are present in the heart, but no direct evidence of their existence has been provided. In

Fig. 5. The expression of non-FP receptors in cardiac ventricular tissue. Melting curve analysis of different types of prostaglandin receptor amplicons(shown are plots of fluorescence (−dF1/dT) vs. temperature; the presence of a single peak is consistent with the formation of a single ampliconand indicates the lack of primer-dimer formation) and representative progress curves done in duplicate for the real-time PCR amplification of theircDNA as depicted on the figure itself.

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Fig. 5. (Continued ).

the present study, real-time RT-PCR has revealed that FP receptors are expressed in the rat ventricular tissue. As the levelof expression was comparable to those of CK and AK it is logical to conclude that FP receptors are abundant protein inthe heart. With this level of FP expression in the heart it is logical to expect that PGF2�-induced negative inotropic effectis meditated by these receptors. To test this hypothesis, we have used PGF2� dimetilamide. This compound has beenused in the past as an antagonist of FP receptors. However, similarly to other antagonists of prostaglandin receptors,PGF2� dimetilamide has been shown to posses partial agonistic properties [21]. On its own, PGF2� dimetilamide hasexhibited a negative inotropic effect and this is a further evidence in favor of the idea that activation of FP receptorsresult in negative inotropic effect. As PGF2�-induced negative inotropic effect has been abolished in the presence ofPGF2� dimetilamide it is reasonable to conclude that this PGF2� action is mediated via FP receptors. However, there

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was a possibility that the activation of non-FP prostaglandin receptor could mediate PGF2�-induced negative inotropiceffect. Therefore, we have examined the level of expression of all known prostaglandin receptors and demonstratedthat FP receptors are expressed in the heart much more than any of the other subtypes of prostaglandin receptors.Taking into consideration that PGF2� has the highest affinity towards FP receptors [22], it is very unlikely that anyother prostaglandin receptor subtype is involved in observed PGF2� action on rat heart.

In conclusion, this study has shown that PGF2� induces negative inotropic effect on rat heart by activating FPprostaglandin receptors. This effect of PGF2� could contribute to cardiac dysfunction in conditions of systemic andmyocardial inflammation.

Acknowledgements

This research was supported by grants from BBSRC, British Heart Foundation and MRC.

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