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Enhanced Cardiac Function in Transgenic Mice Expressinga Ca21-Stimulated Adenylyl Cyclase
Larissa Lipskaia, Nicole Defer, Giovanni Esposito, Iman Hajar, Marie-Claude Garel,Howard A. Rockman, Jacques Hanoune
Abstract—The predominant functional adenylyl cyclases normally expressed in cardiac tissue and coupled tob-adrenergicreceptors are inhibited by micromolar Ca21 concentration. To modify the overall balance of activities, we have generatedtransgenic mice expressing the Ca21-stimulatable adenylyl cyclase type 8 (AC8) specifically in the heart. AC activityis increased by at least 7-fold in heart membranes from transgenic animals and is stimulated by Ca21 in the same rangeof concentration that inhibits the endogenous activity. Moreover, the in vivo basal protein kinase A activity wasaugmented 4-fold. Overexpression of AC8 in the heart has no detrimental consequences on global cardiac function.Basal heart rate and contractile function, measured by noninvasive echocardiography, were unchanged. In contrast, onrelease of parasympathetic tone, the intrinsic contractility is heightened and unresponsive to furtherb-adrenergicreceptor stimulation. AC8 transgenic mice thus represent an original model to investigate the relative influence of Ca21
and cAMP on cardiac function within a phenotype of enhanced cardiac contractility and relaxation.(Circ Res.2000;86:795-801.)
Key Words: adenylyl cyclasen transgenesisn cardiac function
I n the heart, the force of contraction is dependent on theinflux of Ca21 ions through voltage-dependent channels.1,2
b-Adrenoceptor stimulation augments the amplitude of theL-type Ca21 current and the force of contraction3 throughbinding to b-adrenergic receptors (b-ARs), stimulation ofadenylyl cyclase (AC), and increase in the concentration ofcAMP. To understand the physiological and pathologicalconsequences of this cascade, murine models have beencreated with an enhanced efficacy of theb-AR–Gs-ACsignaling pathway.4–6 Overexpression of theb2-AR resultedin a maximal activation of theb-AR signaling pathway, evenin the absence of the agonist.4,7 Gsa overexpression resultedin cardiomyopathy and substantial cardiac histologicalabnormalities.8
Another approach to enhanceb-AR–Gs-AC signalingwould be to bypass the potential deleterious consequences ofthe receptor or G protein and directly to increase the expres-sion of the effector, AC. At least 9 isoforms of AC areknown.9 There is a significant heterogeneity in the distribu-tion and biochemical properties of the different isoforms, andeach tissue or cell type possesses a unique combination ofthese isoforms. In the heart, the Ca21-inhibitable isoformsAC5 and AC6 are the most abundant.10,11 Elevation of Ca21
concentration might inhibit cAMP synthesis and therebyprovide a sensitive negative feedback.12 In contrast, AC1 andAC8, which are essentially expressed in the central nervous
system, are activated by Ca21 through the Ca21/calmodulincomplex.13–15
In this study, we describe transgenic mice overexpressingthe Ca21/calmodulin-activatable isoform AC8 specificallytargeted to cardiomyocytes. Surprisingly, we observed thatAC8 overexpression has no effect on the viability of theanimals but leads to a higher basal intrinsic contractility thatis unresponsive to furtherb-AR stimulation.
Materials and MethodsGeneration of Transgenic MiceFor the construction of transgenic mice, the murinea-myosin heavychain (MHC) promoter16 was ligated to the cDNA coding for humanAC8.15 Mice were screened for the presence of the transgene bySouthern blot performed on tail genomic DNA. Two founders wereidentified and propagated by crossbreeding with C57BL/6 wild-typemice. Number of transgene copies was determined by slot-blotanalysis. The care and use of animals were in accordance withinstitutional guidelines.
EchocardiographyEchocardiography was performed in anesthetized mice (Avertin[tribromoethanol] 2.5%, 14mL/g IP) using an ATL HDI 5000 (ATLUltrasound, Bothell, Wash) echocardiograph as previously de-scribed.17 The following parameters were measured: left ventricular(LV) end-diastolic dimension (LVEDD), LV end-systolic dimension(LVESD), posterior and septal wall thickness, heart rate, percentageof fractional shortening (%FS) (calculated as [LVEDD–LVESD]/
Received July 13, 1999; accepted January 11, 2000.Unite de Recherches, INSERM U-99 (L.L., N.D., J.H.) and INSERM U-474 (I.H., M.-C.G.), Hopital Henri Mondor, Creteil, France, and University
of North Carolina at Chapel Hill (G.E., H.A.R.), Chapel Hill, NC. Present affiliation ofG.E. and H.A.R. is Duke University Medical Center, Durham, NC.Correspondence to Jacques Hanoune, Unite de Recherches, INSERM U-99, Hopital Henri Mondor, F-94010 Creteil, France. E-mail
LVEDD3100), and mean velocity of circumferential fiber shorten-ing (mean Vcfc).
Hemodynamic EvaluationMice were anesthetized with a mixture of ketamine (100 mg/kg) andxylazine (2.5 mg/kg) and analyzed as previously described.6,18
Briefly, after endotracheal intubation, mice were connected to arodent ventilator. After bilateral vagotomy, a 1.4F high-fidelitymicromanometer catheter (Millar Instruments) was inserted into theright carotid artery and retrograde across the aortic valve into the LV.Hemodynamic measurements were recorded at baseline and 45 to 60seconds after injection of incremental doses of isoproterenol (ISO).Doses of ISO were specifically chosen to maximize the contractileresponse but limit the increase in heart rate. Ten sequential beatswere averaged for each measurement.
RNA Preparations and Northern BlottingTotal RNA was extracted,19 and Northern blots were carried out asdescribed.20 RNAs were hybridized with [a-32P]dCTP-labeled AC8,AC5, or AC6 cDNA probes.20 A rat GAPDH cDNA was used tocontrol the equal RNA loading.
AC AssaysAC activity was measured as described20 on purified cardiac mem-branes. Hearts were homogenized in 10 volumes of ice-cold lysisbuffer (in mmol/L, Tris-HCl [pH 7.6] 10, EDTA 0.1, DTT 0.5, andPMSF 0.5) and centrifuged at 500gfor 5 minutes at 4°C. Thesupernatant was centrifuged at 15 000gfor 30 minutes and the pelletwashed 3 times in the same buffer. For analysis of Ca21 effect, themembranes were previously washed twice with 1 mmol/L EGTA.Free Ca21 concentrations were calculated as described.21
Protein Kinase A (PKA) AssayPKA activity was measured on crude myocardial extracts using theSigna TECT PKA Assay System (Promega). Assays were performedwith or without exogenous cAMP (5mmol/L). Addition of the PKApeptide inhibitor completely abolished the enzyme activity.
b-AR Bindingb-ARs were estimated by saturation binding of [125I]iodocyanopin-dolol (125I-CYP) as described.6,22
Western BlottingCrude cardiac homogenates were prepared as described (UpstateBiotechnology). Proteins (25 to 100mg/lane) were separated on 12%SDS-PAGE and transferred to a nitrocellulose membrane. Themembrane was incubated with anti-calmodulin antibody as recom-mended by the manufacturer, and antigen was visualized using the
enhanced chemiluminescence system from Amersham PharmaciaBiotech.
Statistical AnalysisAll results are expressed as mean6SEM of at least 3 determinations.To examine the effect of ISO on changes in hemodynamic parame-ters between control and transgenic animals, a repeated-measuresANOVA was used. For echocardiographic data, a 1-factor ANOVAwas used. Post hoc analysis with regard to differences in mean valuesbetween groups was conducted with a Scheffe test.P,0.05 wasconsidered significant.
An expanded Materials and Methods section is available online athttp://www.circresaha.org.
ResultsGeneration and Genetic Characterization of theTransgenic MiceThe a-MHC-AC8-SV40 intron/pA transgene (Figure 1A)was microinjected into the pronuclei of fertilized mouse eggs.Two founders were obtained (L7 and L8). The transmissionof the transgene was demonstrated by Southern blotting of theoffspring (Figure 1B). Slot-blot analyses of genomic DNAfrom tail biopsies showed 7 to 8 copies of the transgene pergenome (not shown). Both strains expressed AC8 mRNA andactivity at a high level in the heart. For further studies, onlythe offspring of the L7 founder were used.
Northern blot analysis revealed a high, cardiac-specificexpression of the AC8 transgene in these animals (Figure1C), which is consistent with the previously documentedpattern of expression achieved with the murinea-MHCpromoter.4,6,23,24 In contrast, no variation in the mRNAexpression of the 2 major cardiac cyclases, AC5 and AC6,was detected.
Anatomical examination of 2-month-old animals showedno fibrosis or any obvious differences between hearts of AC8transgenic mice (AC8TM; n56) and control littermates(n56) with respect to gross morphology or myocyte appear-ance (not shown). Body weight (control, 3460.4 g; trans-genic, 3360.7 g), heart weight (control, 153.962.8 mg;transgenic, 156.967.8 mg), tibia length (control, 1960.1 g;transgenic, 18.660.1 g), and LV wet weight (control,113.462.16 g; transgenic, 117.866.6 g) were unchanged bytransgene expression (control mice [CM], n514; transgenic
Figure 1. Genetic characterization ofthe transgenic mice. A, DNA constructused for generation of transgenic miceoverexpressing human AC8 in the heart.B, Southern blot analysis of HpaI-digested genomic DNA from tail biop-sies. Blots were hybridized with thea-32P–labeled cDNA probe for AC8,washed (0.53 SSC, 0.1% SDS, at 65°Cfor 15 minutes), and exposed for 24hours. Lanes 1 through 8 representDNA from 13 littermates after the cross-ing (AC8TM3C57BL/6). N indicatesC57BL/6 control animal; P-AC8TM,transgenic parent (L7 founder). C,Cardiac-specific mRNA expression ofthe AC8 transgene. Shown are totalRNAs (30 mg) from heart (H), kidney (K),
brain (B), and skeletal muscle (M) of 1 AC8TM or of 1 control mouse. Autoradiograms were obtained after 2 hours (GAPDH) or 12hours (AC8, AC5, and AC6) of exposure.
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mice, n512). No differences in behavior or exterior aspectwere observed. Neonatal mortality was not different betweentransgenic and nontransgenic animals.
AC Activity in Cardiac Membranes From AC8TMAC activity was assayed in cardiac membranes prepared frompools of 10 transgenic or 10 CM. Basal AC activity wasincreased at least 7-fold in AC8TM as compared with theirlittermates (156.2665.5 pmol cAMP/min mg–1 proteins ver-sus 21.063.4 for control hearts [n55;P50.001]; Figures 2Aand 2B). In the presence of NaF (10 mmol/L), AC activitywas increased by 3-fold in AC8TM and by 8-fold in CM(470618.59 versus 178.4366.75 pmol cAMP/min mg–1 pro-teins in cardiac membranes from AC8TM and CM,respectively).
To document the AC activity in cardiac membranes fromAC8TM heart, the enzyme activity was assayed in thepresence of increasing concentrations of Ca21. As expected,micromolar concentrations of Ca21 inhibited the FSK-stimulated AC activity in normal nontransgenic animals by'25% (Figure 2C). In membranes from AC8TM, forskolin(FSK)-stimulated AC activity was higher by 3- to 4-fold thanin CM hearts; it was only slightly inhibited by Ca21. Additionof calmodulin (1mmol/L) had no effect on control mem-branes (Figure 2A) but evoked a 3-fold stimulation of ACactivity in transgenic membranes (Figure 2B). This stimula-tion was completely abolished by the addition of the calmod-ulin inhibitor, W7 (100mmol/L). Because the basal activityincreased from 21.063.4 to 156.2665.5 pmol cAMP/minmg–1 proteins in membranes from control and transgenichearts, respectively, and to 450.47639.0 pmol cAMP/minmg–1 proteins in transgenic heart membranes under calmod-ulin stimulation, these results demonstrate that, in hearts fromtransgenic mice, AC8 represents the major part of the ACactivity.
In mammalian cardiomyocytes, calmodulin plays an im-portant role as a regulator of cell proliferation and func-tion.25–28 Although its concentration decreases after birth inthe heart, it remains high in the adult.28,29We did not find anymodification in the calmodulin expression in the heart ofAC8TM as compared with their littermates (Figure 3).
To determine whether the increase in the AC activityobserved in vitro in the heart membranes of transgenic micecorresponds to an increase in the AC activity in vivo, wemeasured the cAMP-dependent PKA activity on crude heartextracts from AC8TM and CM. In transgenic mice, PKAactivity was found to be higher by 4-fold than that of controlanimals (2.1460.06 [n53] versus 0.5960.04 pmol ATP/minmg–1 protein [n53]; P50.0004), whereas the total PKAactivity measured in the presence of an excess of cAMP wasunchanged (transgenic, 7.5260.49 pmol ATP/minmg–1 pro-tein, n53; control, 7.7660.43 pmol ATP/minmg–1 protein,n53; P5NS). This indicates that cAMP level in hearts ofAC8TM was considerably increased as compared with CM,suggesting that AC8 was functionally active in vivo.
Echocardiography and In Vivo Assessment ofCardiac Function in AC8-Overexpressing MiceTo determine whether the marked overexpression of AC8would affect the physiological phenotype, transthoracic echo-
Figure 2. Effects of Ca21 on basal and on FSK- and calmodulin-stimulated AC activities in cardiac membranes from AC8TM andCM. Membranes (25 mg of proteins) prepared from hearts of CM(A) or transgenic AC8 littermates (B) were incubated withincreasing amounts of Ca21 in the presence (‚ and Œ) or in theabsence (M and f ) of 1 mmol/L calmodulin. F indicates W7(100 mmol/L) added to the incubation medium. C, Effects ofCa21 on FSK-stimulated AC activity. AC activity measured in thepresence of 10 mmol/L FSK and 0.08 mmol/L free Ca21 wastaken as 100% (CM, 589.92611.99 pmol cAMP/min mg–1 pro-tein; AC8TM, 2054.746186.43 pmol cAMP/min mg–1 protein).
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cardiography was performed. Despite the increase in cardiacAC expression, basal heart rate and contractile functions wereunchanged (Table); LV end-diastolic and end-systolic dimen-sions, heart rate, %FS, and mean Vcfc were similar betweenthe 2 groups.
We further assessed the in vivo cardiac function by cardiaccatheterization in intact anesthetized control and transgenicmice after bilateral vagotomy. The following parameters wererecorded: heart rate, LV systolic pressure, and the 2 deriva-tives of LV systolic pressure (LV dP/dtmax and LV dP/dtmin)(Figure 4). Under basal conditions, LV dP/dtmax in AC8TMwas twice that of wild-type littermates (15 65161805 versus802361705 mm Hg/s for AC8TM [n514] and CM [n512],respectively; P,0.00001) and unresponsive to furtherb-adrenergic stimulation (16 73662390 mm Hg/s;P5NS).Heart rate was significantly increased in AC8TM as com-pared with wild type (485620 versus 377616 bpm forAC8TM [n514] and CM [n512], respectively;P,0.0005).These results indicate that with overexpression of AC8,cardiac contractility is markedly increased and unresponsiveto furtherb-adrenergic stimulation.
Characterization of the AC Signaling inTransgenic HeartTo document whether cardiac myocytes overexpressing AC8were responsive tob-adrenergic stimulation in vitro, AC assayswere performed in the presence of ISO and in the presence orabsence of calmodulin (Figure 5). In control heart,b-agoniststimulation increased AC activity by 2-fold (Figure 5A). Addi-tion of calmodulin (1 mmol/L) had no effect on the ISO-stimulated AC activity. In membranes from AC8TM, the ACactivity was only poorly stimulated by 10 mmol/L ISO, from219.662.99 to 262.2565.94 pmol cAMP/min mg–1 protein inthe absence of calmodulin, and increased from 600.0611.35 to685.0614.58 pmol cAMP/min mg–1 protein in the presence of1 mmol/L calmodulin (Figure 5B). The apparent affinity towardISO was not different in control and transgenic mice. Thus, ISOstimulation does not appear to affect AC8 activity directly, andit is very likely that in AC8TM, the ISO-stimulated AC activityessentially corresponds to the effect of ISO on the endogenousisoforms. Furthermore, radioligand binding assays indicated asimilar b-AR number in control (57.9563.10 fmol 125I-CYPbound/mg protein) and transgenic (59.3961.96 fmol 125I-CYPbound/mg protein) animals, with no difference in the apparentaffinity.
The GTPgS dose-response curve is shown in Figure 5C. Theactivity assayed on membranes from CM increased from13.7563.25 pmol cAMP/min mg–1 protein in the absence ofGTPgS to 126.6763.48 in the presence of 10mmol/L GTPgS.Addition of calmodulin does not affect this activity. In cardiacmembranes from transgenic animals, the activity increased from150.3364.63 (GTPgS50) to 320.67641.09 (GTPgS510mmol/L) pmol cAMP/min mg–1 protein in the absence ofcalmodulin and from 313.067.21 (GTPgS50) to 641.0638.8(GTPgS510 mmol/L) pmol cAMP/min mg–1 protein in thepresence of 1mmol/L calmodulin. Thus, the increased ACactivity in transgenic mice did not affect either the number ofb-ARs or the GTPgS responsiveness of AC activity.
Figure 6 shows the effect of increasing Ca21 concentration onISO-stimulated AC activity. As expected, Ca21 inhibited theISO-stimulated endogenous activity by'30% in heart mem-branes from CM and to a lesser extent in heart membranes fromAC8TM. Whereas calmodulin had no effect on inhibition ofISO-stimulated AC activity in CM, in the AC8TM, Ca21 andcalmodulin increased the ISO-stimulated activity from265.065.34 to 463.0632.25 pmol cAMP/min mg–1 protein.
DiscussionIn the heart, the inotropic effect ofb-adrenergic agonists ismediated by the stimulation of AC activity and the subsequentphosphorylation of specific proteins by cAMP-dependent pro-tein kinase. The prevalence of AC5 and AC6 in the cardiomyo-cytes hints at a crucial role for the susceptibility of these ACs toCa21 inhibition in the regulation of cardiac function. It has beenproposed that elevated [Ca21]i could inhibit cAMP synthesis byAC5 and AC6 and thereby provide sensitive negative feed-back.12,30–32We have developed an in vivo model of transgenicmice overexpressing the Ca21-stimulatable isoform AC8 specif-ically in cardiomyocytes. Because of the presence of calmodulinin the heart,28,29 the activity of this isoform should be activated
Figure 3. Western blot analysis of calmodulin (CaM) expression.A, Expression in mouse brain and heart (50 mg protein/lane).Various concentrations of pure calmodulin were used as controlas well as a positive control proposed by the manufacturer. B,Expression in mouse heart from 3 control and transgenic ani-mals (50 mg/lane). C, Increasing amounts of cardiac extract fromcontrol and transgenic mouse hearts.
Echocardiography Parameters in Control and AC8Transgenic Mice
Control (n510) Transgenic (n59)
Heart rate, bpm 493613 491613
Velocity of circumferential fibershortening, circ/s
7.7660.54 8.0560.34
Posterior wall thickness, mm 0.6160.03 0.5760.07
Septal wall thickness, mm 0.7760.04 0.8260.04
Left ventricular diastolicdiameter, mm
4.0060.09 3.8860.09
Left ventricular systolicdiameter, mm
2.5060.09 2.3760.10
Fractional shortening, % 37.461.94 39.261.42
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by Ca21 when the activity of the endogenous isoforms, AC5 andAC6, are inhibited.
Two transgenic lines have been obtained, both expressingAC8 at high levels in cardiomyocytes. For both, AC activitywas increased'7-fold and was strongly activated by Ca21/calmodulin, with AC8 representing at least 80% of the totalactivity in the cardiomyocyte membranes. ISO does notstimulate directly AC8 activity in heart membranes fromtransgenic mice. The inability of AC8 to respond to ISO byincreased cAMP accumulation has already been described inAC8-transfected HEK293 cells.14 Furthermore, Fagan et al33
demonstrated that HEK 293 cells possess the capability tolocalize transfected AC appropriately, suggesting that thetargeting information is encoded within the protein sequence.In this context, AC8 would appear to function as a “pure Ca21
” detector.34,35 Baker et al36 have demonstrated that theGs-coupled receptor, 5-HT7 receptor, stimulates AC8 activityin vivo, by increasing [Ca21]i concentration. We cannotexclude a similar mode of action for theb-AR to account forsome effect on AC8 activity in vivo.
Despite the high AC and PKA activities, the basal cardiacfunction of the transgene-positive animals, as measured byechocardiography, was not affected. In contrast, when thephenotype was evaluated by invasive hemodynamics, LVdP/dtmax (an index of contractility) was increased and found tobe unresponsive to furtherb-AR stimulation. Our physiolog-ical data demonstrate that overexpression of AC8 does nothave deleterious consequences on global cardiac function,because chamber size and fractional shortening are normal.Furthermore, heart rate is not affected as long as the auto-nomic nervous system is intact. However, release of para-sympathetic tone shows that the intrinsic contractility isheightened, in part related to the higher heart rate, with lossof normalb-AR function as shown by the lack of responsive-ness to catecholamine stimulation. Because echocardiographyis most sensitive for the determination of chamber dimensionand not contractile function, it is not surprising that echoparameters of %FS and Vcfc are the same. For instance,overexpression of theb2-AR results in a marked increase indP/dt max4 but has no effect on %FS or Vcfc.37 Importantly,under certain conditions, these mice lose normal regulation ofb-AR coupling. Whether this will have an impact in theconscious animal will require further study.
Cardiac overexpression of theb-AR, Gsa, or b-AR kinaseinhibitor only slightly increases the basal AC activity and theb-ARsignaling.4–6,38 On the other hand, unlike our AC8 mice, AC6overexpression in mice resulted in a strong amplification of theb-AR signaling,39 as evidenced by cAMP accumulation in isolatedcardiomyocytes and physiological assessment of cardiac function,although the echocardiographic parameters were unchanged. Theoverall published literature points to a very complex relationship
Figure 4. In vivo assessment of LV contractile function in responseto b-agonist stimulation. Parameters measured were LV end-sys-tolic and end-diastolic pressure, LV dP/dtmax and LV dP/dtmin, andheart rate. Four measured parameters are shown at baseline andafter progressive doses of ISO in wild-type mice (E, n514) andAC8TM (F; n512). A, Heart rate. B, LV systolic pressure. C, LVdP/dtmax. D, LV dP/dtmin. Data were analyzed with a repeated-measures ANOVA. *P,0.0001, †P,0.05 vs AC8TM.
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Figure 5. Effect of ISO or GTPgS on basal or Ca21/calmodulin-stimulated AC activities. AC activity was measured in the pres-ence of indicated concentrations of ISO and 10 mmol/L GTP orin the presence of GTPgS (C). Effect of ISO and GTPgS onCa21/calmodulin-stimulated AC activity was assayed in the pres-ence of GTP (10 mmol/L), calmodulin (1 mmol/L), and 1.7 mmol/Lfree Ca21. M, Control (basal); ‚, control (1 mmol/L calmodulin);f, AC8TM (basal); Œ, AC8TM (1 mmol/L calmodulin).
Figure 6. Effect of Ca21 and calmodulin on ISO-stimulated ACactivities. AC activity was measured in the presence of ISO(5 mmol/L), GTP (10 mmol/L), and indicated concentrations offree Ca21, with or without addition of calmodulin (1 mmol/L). M,Control (basal); ‚, control (1 mmol/L calmodulin); f, AC8TM(basal); Œ, AC8TM (1 mmol/L calmodulin).
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betweenb1- and b2-ARs, Ca21, and cardiac contraction, whichcould explain the differences observed in the various models oftransgenic animals. At present, the relationship between Ca21,cAMP, and the various parameters of cardiac function still needs tobe clarified. From this point of view, AC8TM represent an originalmodel in which the AC activity is stimulatable by Ca21. These micecan be used to investigate in more detail the relative influence ofCa21 and cAMP on cardiac function within a phenotype of en-hanced contractility and relaxation.
AcknowledgmentsThis work was supported by the INSERM, the Universite Paris XII,and the NIH Grant HL 56 687 (to H.A.R.). L.L. was a recipient of afellowship from the Fondation pour la Recherche Medicale. Wethank Dr Jeffrey Robbins (Children’s Hospital Medical Center,Cincinnati, Ohio) for providing thea-myosin heavy chain promoterand Dr J.-F. Authier (Hopital Henri Mondor, Creteil, France) forhistology of the heart. We are grateful to F. Pecker, R. Fischmeister,and M. BestBelpomme for their helpful discussions.
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