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An efficient gene delivery system is a prerequisite for myocardial gene therapy. Among the various procedures studied so far, catheter-based percutaneous gene delivery to the myocardium through the coronary vessels seems the most relevant to routine clinical practice; however, the optimal conditions remain to be determined. We selectively infused adenoviral vectors encoding luciferase (1 x 10(9) PFU) or beta-galactosidase (1 x 10(10) PFU) into coronary arteries of adult rabbits in various experimental conditions. Coronary artery occlusion for 30 sec, during and after adenovirus delivery, was required to observe luciferase activity in the target area of the circumflex artery (4.0 +/- 1.0 x 10(5) vs. 1.1 +/- 0.2 x 10(4) RLU/mg with and without coronary occlusion, respectively, p < 0.01, and 1.0 +/- 0.1 x 10(3) RLU/mg using nonselective infusion). When adenoviruses were delivered using high-pressure infusion (82 +/- 12 vs. 415 +/- 25 mmHg before and during infusion, respectively, p < 0.01), luciferase activity increased to 8.5 +/- 2.5 x 10(5) RLU/mg (p < 0.05 vs coronary occlusion alone). Coronary venous sinus occlusion with saline buffer retroinfusion starting before and during anterograde adenovirus delivery resulted in a further 4.7-fold increase in luciferase activity (4.4 +/- 0.8 x 10(6) RLU/mg, p < 0.01) with 5-25% blue-stained myocytes in the target area, compared with 0-5% with the other procedures. Histamine or VEGF-A(165) pretreatment, used to increase vascular permeability, slightly increased gene transfer efficiency (8.5 +/- 2.0 x 10(5) and 9.0 +/- 2.5 x 10(5) RLU/mg respectively, p < 0.05 vs. coronary occlusion alone). We conclude that catheter-mediated adenoviral gene transfer to cardiac myocytes through coronary vessels can be a very efficient procedure for myocardial gene therapy, particularly when the vector residence time and perfusion pressure in the vessels are increased.

Objective: Cardiomyocytes can be transplanted successfully into skeletal and cardiac muscle. Our goal was to determine the feasibility of grafting cardiomyocytes onto various synthetic supports to create an excitable and viable tissue for implantation., Methods: Adult rat cardiomyocytes were cultured over an 8-week period onto different substitutes, including human glutaraldehyde-treated pericardium (n = 3), equine glutaraldehyde-treated pericardium (n = 3), polytetrafluoroethylene (n = 8), Dacron polyester (n = 16), and Vicryl polyglactin (n = 8)., Results: Only the cells seeded on the Dacron survived, with the synthetic fibers colonized at 8 weeks. On the other supports, the number of myocytes progressively decreased from the first week, with their density (number of cells per square millimeter) being, after 20 days, 17 +/- 2 on the polytetrafluoroethylene and 5 +/- 1 on the human or equine pericardium compared with 45 +/- 3 on the Dacron. After 8 weeks of culture on Dacron, the sarcomeric protein (sarcomeric alpha-actinin) was detected in all cells. In addition, the staining was regularly arranged and well aligned in a striated pattern. Spontaneous beating activity was obtained. Moreover, electrical stimulation of the cell preparation resulted in the generation of calcium transients, the frequency of which followed the frequency of the electrical stimulation., Conclusions: These results suggest that adult cardiac myocytes remain viable and excitable during long-term culture on a 3-dimensional Dacron support, which might constitute a new synthetic cardiac tissue.

Catecholamines have complex effects on cardiac myocyte growth and survival, including the triggering of apoptosis at high concentration. Here, we examined whether at a lower concentration, catecholamine protected adult rat ventricular myocytes from apoptosis in vitro. Myocytes were exposed to staurosporine (ST, 10 microM) for 18 h, with or without epinephrine (0.1 or 10 microM) or fetal calf serum (10%). Apoptosis was assessed after 48 h of culture in terms of DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method, DNA gel electrophoresis). Epinephrine (0.1 microM) and serum reduced ST-induced myocyte apoptosis by approximately 50% (n = 12 cultures, P <.001), whereas epinephrine and serum alone did not influence the low apoptotic rate in control cultures. In contrast, 10 microM epinephrine induced marked apoptosis in ST-free conditions. The protective effects of 0.1 microM epinephrine and serum were blunted by the tyrosine kinase inhibitor genistein (n = 12 cultures, P <. 001). Extracellular signal-regulated kinase (ERK) activity was stimulated by 0.1 microM epinephrine but not by 10 microM epinephrine. Furthermore, the protective effect of epinephrine was mimicked by isoproterenol (1 microM) and forskolin (1 microM) but not by phenylephrine (10 microM) and was blunted by propranolol (10 microM) but not by prazozin (10 microM). Finally, isoproterenol and forskolin activated ERK, an effect that was blunted by propranolol. In conclusion, low epinephrine concentrations attenuate ST-induced apoptosis of adult cardiac myocytes in vitro, an effect mediated by coupling between the cAMP pathway and ERK activation. This suggests that a minimal adrenergic tone is essential for myocyte survival in conditions of unusual stress.

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.

The effects of tyrosine protein kinases (TK) on the L-type Ca(2+) current (I(Ca)) were examined in whole cell patch-clamped human atrial myocytes. The TK inhibitors genistein (50 microM), lavendustin A (50 microM), and tyrphostin 23 (50 microM) stimulated I(Ca) by 132 +/- 18% (P < 0.001), 116 +/- 18% (P < 0.05), and 60 +/- 6% (P < 0.001), respectively. After I(Ca) stimulation by genistein, external application of isoproterenol (1 microM) caused an additional increase in I(Ca). Dialyzing the cells with a protein kinase A inhibitor suppressed the effect of isoproterenol on I(Ca) but not that of genistein. Inhibition of protein kinase C (PKC) by pretreatment of cells with 100 nM staurosporine or 100 nM calphostin C prevented the effects of genistein on I(Ca). The PKC activator phorbol 12-myristate 13-acetate (PMA), after an initial stimulation (75 +/- 17%, P < 0.05), decreased I(Ca) (-36 +/- 5%, P < 0.001). Once the inhibitory effect of PMA on I(Ca) had stabilized, genistein strongly stimulated the current (323 +/- 25%, P < 0.05). Pretreating myocytes with genistein reduced the inhibitory effect of PMA on I(Ca). We conclude that, in human atrial myocytes, TK inhibit I(Ca) via a mechanism that involves PKC.

Objectives: The aim of the present study was to determine if myocytes can die by apoptosis in fibrillating and dilated human atria., Background: The cellular remodeling that occurs during atrial fibrillation (AF) may reflect a degree of dedifferentiation of the atrial myocardium, a process that may be reversible., Methods: We examined human right atrial myocardium specimens (n = 50) for the presence of apoptotic myocytes. We used immunohistochemical and Western blotting analysis to examine the expression of a final effector of programmed cell death, caspase-3 (CASP-3) and of regulatory proteins from the BCL-2 family., Results: Sections from atria in AF contained a high percentage of large myocytes with a disrupted sarcomeric apparatus replaced by glycogen granules (64.4 +/- 6.3% vs. 12.2 +/- 5.8%). These abnormal myocytes, which also predominated in atria from hearts with decreased left ventricular ejection fraction (42.3 +/- 10.1%), contained large nuclei, most of which were TUNEL positive, indicating a degree of DNA breakage. None of these abnormal myocytes expressed the proliferative antigen Ki-67. A small percentage of the enlarged nuclei (4.2 +/- 0.8%) contained condensed chromatin and were strongly TUNEL positive. Both the pro- and activated forms of CASP-3 were detected in diseased myocardial samples, which also showed stronger CASP-3 expression than controls. Expression of the antiapoptotic BCL-2 protein was decreased in diseased atria, whereas that of the proapoptotic BAX protein remained unchanged., Conclusions: In fibrillating and dilated atria, apoptotic death of myocytes with myolysis contributes to cellular remodeling, which may not be entirely reversible.

Besides the well-documented effect of the chemotherapeutic drug doxorubicin on free radical generation, the exact signaling mechanisms by which it causes cardiac damage remain largely unknown and are of fundamental importance in understanding anthracycline cardiotoxicity. In this study, we describe that a 1 h treatment of isolated adult rat cardiac myocytes with doxorubicin (0.5 microM) induced DNA fragmentation associated with the classical morphological features of apoptosis observed after 7 days of culture. The doxorubicin toxicity was preceded by an increase in intracellular ceramide levels with a concurrent decrease in sphingomyelin. Anthracycline-induced ceramide accumulation resulted from the activation of a sphingomyelinase assayed under acidic conditions, an effect related to an increase in V(max). Pretreatment of cardiac myocytes with L-carnitine (200 microgram/ml), a compound known for its protective effect on cardiac metabolic injuries, was found to dose-dependently inhibit the doxorubicin-induced sphingomyelin hydrolysis and ceramide generation as well as subsequent cell death. However, L-carnitine did not protect cardiac myocytes from apoptosis induced by exogenous cell-permeant ceramide. L-carnitine pretreatment did not affect the sphingomyelinase basal activity but abolished the doxorubicin-induced increase in V(max). Moreover, in vitro studies conducted on cell extracts or with purified acid sphingomyelinase demonstrated that L-carnitine exerted a dose-dependent, sphingomyelinase inhibitory effect (through V(max) reduction). Taken together, these findings show that by inhibiting a (perhaps novel) drug-activated acid sphingomyelinase and ceramide generation, L-carnitine can prevent doxorubicin-induced apoptosis of cardiac myocytes.

Objectives: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubicin induces cardiac myocyte apoptosis through ceramide generation., Methods: Adult rat ventricular myocytes were grown in the presence of 10% fetal calf serum, and exposed to 0.5 microM doxorubicin (Dox) for 1 h on the day of cell isolation (day 0). We used the membrane-permeant ceramide analog C2-ceramide (C2-cer) to mimic the effects of endogenous ceramide and PDMP to induce endogenous ceramide accumulation. Apoptosis was assessed upon morphological criteria and DNA fragmentation by the TUNEL method and agarose gel electrophoresis. Ceramide concentration was assessed using the DAG kinase assay., Results: Myocyte exposure to Dox was associated with cellular and nuclear alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treated cultures compared to control (Cl) cultures (82 +/- 3 vs. 12 +/- 1%, n = 7; p < 0.001); internucleosomal DNA fragmentation was confirmed by the observation of DNA ladders. These alterations were associated with an increase in the intracellular ceramide concentration (1715 +/- 243 vs. 785 +/- 99 pmol/mg prot, n = 5; p < 0.01), a phenomenon also detected on day 3 (731 +/- 59 vs. 259 +/- 37 pmol/mg prot, n = 5; p < 0.001). Incubation of myocytes at day 0 with 50 microM C2-cer induced rapid cell shrinkage and DNA fragmentation (45 +/- 3 vs. 10 +/- 1% TUNEL-positive myocytes on day 1 in C2-cer-treated and Cl cultures, respectively; n = 6, p < 0.001). Myocyte exposure to 10 microM PDMP for 7 days (n = 5), caused ceramide accumulation (1.7-fold increase vs. Cl, p < 0.01), and a marked increase in the percentage of TUNEL-positive myocytes (62 +/- 6 vs. 11 +/- 3% in Cl cultures, p < 0.001). Ventricles of rats injected intraperitoneally with a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed an increased ceramide concentration 2 weeks later (11.01 +/- 0.64 vs. 5.24 +/- 0.88 pmol/mg prot, n = 6; p < 0.001)., Conclusion: Our study confirms the existence of a functional ceramide pathway related to apoptosis in cardiac myocytes, and points to its possible involvement in doxorubicin-induced cardiomyopathy.

Objective: To address the effect of longstanding left ventricular (LV) hypertrophy and failure on LV adenylyl cyclase (AC) gene expression, mRNA concentrations of the main cardiac AC isoforms were measured in the non-infarcted area of LV from rats with myocardial infarction (MI), without (H) or with (F) LV failure, and in control (C) rats. Basal, GTP- and forskolin-stimulated Mg(2+)- and Mn(2+)-dependent AC activities were also measured in F and C rats., Methods: Two- and six months after MI, steady-state AC mRNA concentrations were assessed by Northern blot analysis and RNase protection assay with isoform-specific cDNA and cRNA probes, respectively. AC activities were assessed on LV microsomal fractions using standard procedures., Results: Types V and VI, and types IV and VII were the major and minor AC mRNA isoforms in both the LVs of F and C rats. Two months after MI, no difference in LV type V or VI mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios was observed in rats with H or F compared to C. Six months after MI, no difference in LV type V mRNA concentration was observed between the three rat groups, whether this level was normalized to GAPDH, poly-(A+) or 18S RNAs. In contrast, a 35% decrease in the type VI mRNA to poly-(A+) RNA ratio and a 29% decrease in the type VI mRNA to 18S RNA ratio was observed only in rats with F compared to C (p < 0.05 vs. C for the two comparisons). Two- and six months after MI, basal and forskolin-stimulated Mg(2+)-dependent AC activities were decreased by 30-35% in F rats compared to C (p < 0.05), whereas Mn(2+)-dependent activities were unchanged., Conclusion: Longstanding LV hypertrophy and failure resulting from MI in rats is not associated with altered expression of the most abundant, type V, AC mRNA isoform, whereas that of type VI is decreased. The lack of change in Mn(2+)-dependent AC activities in the LV of F rats suggests that this decrease has no functional consequence on overall AC activity and that decreased Mg(2+)-dependent activities are related to alterations occurring upstream.

Several studies have shown that the Na-Ca exchange is electrogenic in cardiac tissues and that the current carried by the exchange, iNa-Ca, participates in the development of the cardiac action potential plateau. The aim of the present study was to assess the contribution of iNa-Ca to the development of the plateau in different preparations, i.e. normal and hypertrophied (DOCA-salt) perfused rat hearts, normal and dilated (MS 200 strain) perfused hamster hearts and normal human atrial myocytes and to examine the repercussion of iNa-Ca suppression on the global electrogram of the perfused guinea-pig heart. iNa-Ca was suppressed by briefly substituting lithium for sodium (Li test). In the normal rat heart, the Li test resulted in a depression of the late component of the plateau. In the severely hypertrophied rat heart, the action potential was lengthened, the two components of the plateau barely distinguishable. The part of the plateau suppressed during Li perfusion was not larger in amplitude than in the normal rat heart, but was of much longer duration. In the dilated heart of hamsters older than 60 days of age the shortening effect of the Li test was much larger than in the normal hamster heart indicating a strongly increased contribution of iNa-Ca to the plateau development during dilatation. The contribution of iNa-Ca to the action potential plateau of human atrial myocytes was as large as that found in animal ventricles and varied with the type of cell studied. In the perfused guinea-pig heart the Li test resulted in a sizeable shortening of QT duration and a marked decrease in T wave amplitude suggesting that iNa-Ca may be a major source of current in the building up of the ventricular complex of the electrocardiogram in normal and pathological conditions.

We examined changes in the structural and physiological characteristics of human atrial myocytes during primary culture in the presence of serum. Action potentials and ionic currents were recorded in freshly dissociated (FM) and cultured (CM) whole-cell patch-clamped myocytes, alpha-smooth muscle actin, sarcomeric alpha-actinin and beta-myosin heavy chains (beta-MHC) were stained with monoclonal antibodies. From day 5 to day 21, myocytes lost their rod shape, spread and exhibited reorganized sarcomeres. These morphological changes were associated with a marked increase in membrane capacitance (+266%). Both beta-MHC and alpha-smooth muscle actin were expressed in CM but not in FM, indicating a dedifferentiation process. CM were characterized by a lower resting potential (-30 +/- 2 v -60 +/- 4 mV, P < 0.05) and, when repolarized, by a shorter action potential duration (APD) than FM (APD-60: 126.9 v 159.6 ms, P < 0.05). The inward rectifier K+ current was absent in CM, thus explaining the low resting potential. The density of the transient component of the voltage-activated K+ current Ito1 was not modified during culture, while that of the sustained component Isus was increased fourfold. The amplitude of ICa was increased, but its density was unchanged, indicating that CM maintained a normal density of functional calcium channels. Neither the voltage dependence nor the inactivation of ICa was modified in CM. The time constants of inactivation of ICa were unchanged, although the amplitude of the rapidly inactivating component of ICa was increased in CM compared to FM. Moreover, ICa was increased by the beta-adrenergic agonist isoproterenol (1 microM) throughout the culture period. Our results demonstrate that in long-term serum-supplemented culture, adaptation of human atrial myocytes to their new environment is associated with differential alterations of the main ionic currents and phenotypic changes characteristic of immature myocardium.

Ventricular hypertrophy is associated with an increase in action potential (AP) plateau amplitude and duration. In a model of cardiac hypertrophy by DOCA-pellet implantation in uninephrectomized saline-drinking rats, we have previously demonstrated that the influence of hypertrophy was to reduce Ito1 density, the process being fully reversible after elimination of DOCA pellets. In that study the decrease of Ito1 density appeared to vary from moderate reduction to complete suppression which could explain, at least in part, the AP lengthening. In the present study the effect of the degree of hypertrophy (moderate and severe hypertrophy) was investigated on rat ventricular action potential plateau amplitude and duration, high threshold calcium current, Ica-L, Na-Ca exchange current, INa-Ca, transient outward potassium current, Ito1, and sustained outward potassium current Isus. Ventricular action potentials of isolated perfused hearts were recorded by means of standard floating microelectrodes and ionic currents of single ventricular myocytes were measured using the whole-cell recording patch-clamp technique. We show that: (1) AP plateau amplitude and duration increase more markedly in severe than in moderate hypertrophy; (2) the decrease in Ito1 density is much larger in severe than in moderate hypertrophy whereas ICa-L, INa-Ca and i(sus) densities remain unaltered in either state of hypertrophy. After suppression of Ito1 by 3 mM 4-aminopyridine, action potential plateau amplitude and duration remain increased in severely hypertrophied rat hearts compared to sham rats. Therefore, although these results designate Ito1 reduction as the main cause of hypertrophy-induced AP changes, those occurring in severe hypertrophy cannot be uniquely explained by a quasi-complete extinction of Ito1.

Objective: The aim of our study was to determine the main electrophysiological alterations associated with cardiac dilation in MS200 strain Syrian hamsters, a model of genetically determined cardiomyopathy., Methods: Ventricular action potentials (APs) were recorded with standard microelectrodes in isolated hearts from 120-day-old cardiomyopathic (strain MS200) and age-matched control (strain CHF148) Syrian hamsters. Ionic currents were recorded from single ventricular myocytes using the whole-cell patch-clamp technique., Results: In MS200, AP was prolonged and the plateau phase was markedly increased as compared to CHF148. Differences in both AP duration and 4-aminopyridine-induced AP lengthening between epicardial and endocardial tissues were less marked in MS200 than in CHF148 ventricles. Cell size and membrane capacitance were not higher in MS200 than in CHF148 myocytes, indicating the absence of cell hypertrophy in myopathic ventricles. The L-type calcium current (ICa,L) density was significantly reduced in MS200 and the voltage-dependence of both steady-state activation and inactivation was altered. The voltage-dependent outward current was composed of both transient (Ito1) and sustained (Iss) components, respectively sensitive and insensitive to 4-aminopyridine. Ito1 density was strongly depressed in MS200 compared to CHF148, whereas Iss density was only slightly reduced. The conductance-voltage and steady-state inactivation relationships for Ito1 were shifted to more positive potentials in MS200. The Ito1 recovery process was markedly slower in MS200 than in CHF148. The steady-state current-voltage relationships, in the physiological voltage range, were superimposable in MS200 and CHF148., Conclusions: In ventricular myocytes from dilated heart of MS200 Syrian hamsters, Ito1 is more drastically depressed than ICa,L. Such an observation might partially explain dilation-induced AP lengthening.

Transduction of the beta-adrenergic signal plays an important role in the regulation of cardiac contractility. It is mediated by three sarcolemmal proteins: the beta-adrenergic receptor, G proteins and adenylyl cyclase which is the catalytic unit of the system which generates cAMP, the second messenger of the system. Each protein comprises a number of isoforms which yields a wide range of potential regulations, many of which are not yet elucidated. Among the three proteins, the adenylyl cyclase is the one which has been less studied. However, the recent cloning of many of its isoforms allows now investigations of their expression in many tissues and cell types. We have shown in rats that among the five isoforms detected in the myocardium, type V and VI adenylyl cyclase mRNAs are the most abundant ones. Type V and VI adenylyl cyclase mRNA abundance is similar in late fetal hearts. Type V mRNA accumulates in the heart during postnatal development whereas type VI mRNA concentration remains unchanged. Consequently, type V mRNA becomes highly predominant compared to type VI mRNA in the adult rat ventricle (type V/type VI adenylyl cyclase mRNAs approximately 10). Whatever the developmental stage, cardiac adenylyl cyclase activity is inhibited by submicromolar calcium concentrations. In adult ventricles, adenylyl cyclase activity in the presence of 1 mM ATP is at least three times higher than that observed in fetal and new born rat hearts. Since this increase parallels the accumulation of type V adenylyl cyclase mRNA, one can hypothesize that the former is due to the latter. In contrast, our preliminary results seem to indicate that during heart failure in rats, decreased adenylyl cyclase activity is not associated with decreased cardiac concentrations of type V and VI adenylyl cyclase mRNAs. Isoform specific antibodies are now required to understand the reasons for such discrepancy.

Type V and VI adenylyl cyclase mRNAs are the two main cyclase isoforms expressed in the mammalian heart. A recent report has shown that their expression is differentially regulated during ontogenic development, but the accumulation of the two mRNA species and their concentration ratio have not been determined. We thus determined the accumulation and the relative amounts of type V and VI adenylyl cyclase mRNA in fetal, neonatal and adult rat hearts, using a sensitive ribonuclease protection assay. In 18-day-old fetuses, the two adenylyl cyclase mRNA isoforms were weakly expressed in approximately equal amounts (type V mRNA/type VI mRNA = 0.93 +/- 0.09). Further development was characterized by a sharp increase in type V adenylyl cyclase mRNA (x 1.9 in neonates v fetuses, P < 0.01; x 2.4 and x 4.5 in adults v neonates and fetuses, respectively, P < 0.01 for both comparisons) and a slight, non-significant fall in type VI mRNA (P = 0.16). As a result, the type V mRNA/type VI mRNA ratio was 2.86 +/- 0.57 and 9.09 +/- 1.21 in neonatal hearts and adult ventricles, respectively (P < 0.01 v ratio in fetal hearts for both comparisons; P < 0.01 for ratio in adult ventricles v ratio in neonatal hearts), and the overall amount of the two mRNA isoforms was 2.3 times greater in adult than in fetal hearts (P < 0.01). This increase was paralleled by an increase in basal and isoproterenol- and forskolin-stimulated adenylyl cyclase activities in adult hearts compared to fetal and neonatal hearts (P < 0.01 for the three comparisons). Our results demonstrate that type V adenylyl cyclase mRNA accumulates in the rat heart after birth to become the highly predominant isoform in the adult heart. They further suggest that the increase in cardiac adenylyl cyclase activity observed during rat development is due to this accumulation.

Troponin T, like many myofibrillar proteins, exists as multiple isoforms encoded by distinct genes or generated by splicing of the same primary RNA transcript. We have previously cloned the first human cardiac troponin T (cTnT) cDNA and showed the differential expression of cTnT in cardiac and skeletal muscle during ontogenic development. In this work we located the human cTnT gene by means of fluorescent in situ hybridization to 1q32 and, by sequencing thirteen cDNAs isolated from a human fetal heart cDNA library, identified three new isoforms resulting from specific combinations of three variable regions in human cTnT cDNA. The first variable region is a 30-bp box located at the 5' end of the cDNA, which can be excised either totally or only from the first 3 bp onwards; the second is a codon which can be completely excised; and the third is a 9-bp box in the 3' half of the cDNA, which can also be excised either totally or only from the first 3 bp. The existence of the corresponding RNAs in fetal and adult ventricles was confirmed by RNase protection studies. No accumulation of the fetal isoforms was found in failing ventricles compared with controls.

The characteristics of cultured myocardial cells isolated from small mammals are well documented, but there is a dearth of data on cultured human cardiocytes. The aim of this study was to determine the main features of myocytes isolated from human atria and maintained in culture in the presence of 10% fetal calf serum (FCS), according to the age of the donor. The following characteristics were analysed: (1) yield and viability; (2) adhesive properties; and (3) changes in cell morphology. Myocytes preferentially adhered to laminin-coated dishes and could be maintained in culture for at least 2 weeks, whatever the age of the donor (which was from 6 days to 85 yr). Maintenance in culture induced morphologic changes characterized by myocyte spreading and changes in myofibrillar organization. Interestingly, the time of onset of these changes depended on the age of the donor: they occurred earlier in young atrial myocytes (< 1 yr) than in older cells (> 13 yr).

We have isolated a full-size cDNA coding for cardiac troponin T (cTnT) from a human adult heart library, using a slow skeletal TnT probe. This cDNA detected a 1.2 kb mRNA in fetal and post-natal human heart, the amount of which increased during ontogenic development. Interestingly, a similar transcript was coexpressed in fetal skeletal muscle, together with the 0.9 kb slow skeletal muscle mRNA, and its expression was down-regulated during further development.

Chronic growth hormone (GH) hypersecretion in rats leads to increased isometric force without affecting the unloaded shortening velocity of isolated cardiac papillary muscles, despite a marked isomyosin shift toward V3. To determine if alterations occurred at the level of the contractile proteins in rats bearing a GH-secreting tumor (GH rats), we examined the mechanical properties of skinned fibers to eliminate the early steps of the excitation-contraction coupling mechanism. We found that maximal active tension and stiffness at saturating calcium concentrations (pCa 4.5) were markedly higher in GH rats than in control rats (tension, 52.9 +/- 5.2 versus 38.1 +/- 4.6 mN.mm-2, p < 0.05; stiffness, 1,105 +/- 120 versus 685 +/- 88 mN.mm-2.microns-1, p < 0.01), whereas values at low calcium concentrations (pCa 9) were unchanged. In addition, the calcium sensitivity of the contractile proteins was slightly but significantly higher in GH rats than in control rats (delta pCa 0.04, p < 0.001). The crossbridge cycling rate, reflected by the response to quick length changes, was lower in GH rats than in control rats (62.0 +/- 2.6 versus 77.4 +/- 6.6 sec-1, p < 0.05), in good agreement with a decrease in the proportion of alpha-myosin heavy chains in the corresponding papillary muscles (45.5 +/- 2.0% versus 94.6 +/- 2.4%, p < 0.001). The changes in myosin heavy chain protein phenotype were paralleled by similar changes of the corresponding mRNAs, indicating that the latter occurred mainly at a pretranslational level. These results demonstrate that during chronic GH hypersecretion in rats, alterations at the myofibrillar level contribute to the increase in myocardial contractility observed in intact muscle.

Expression of the two sarcomeric actins, alpha-skeletal and alpha-cardiac, is regulated in the rodent heart in response to developmental, hormonal, and hemodynamic stimuli. Little is known in man, except that both isogenes were found to be coexpressed in three adult ventricles. In this report, we investigated the isoactin mRNA composition in ventricles from 21 control patients (4 fetal, 5 juvenile, 12 adult) and from 15 patients undergoing cardiac transplantation (5 idiopathic dilated cardiomyopathies, 5 ischemic myopathies with myocardial infarcts, 5 diverse etiologies) by two different and complementary techniques: RNA dot blot analysis with specific cDNA probes, and primer extensions with an oligonucleotide common to alpha-cardiac and alpha-skeletal actins. In the case of dot blot analysis, quantification of each isoform was performed by using as standards RNA transcripts obtained from cloned human alpha-actin sequences, and the total amount of sarcomeric actin mRNA was evaluated as a function of total poly(A+)RNA. We found that both isogenes are always coexpressed, and that the isoactin pattern changes during development. In utero and in neonatal hearts, alpha-skeletal actin mRNA represents less than or equal to 20% of sarcomeric actins, it increases to 48 +/- 6% during the first decade after birth and becomes the predominant isoform of adult hearts (60.4 +/- 8.5%). The 15 adult failing hearts exhibited the same isoactin pattern as the control ones (62.84 +/- 11.06%), and there was no difference in expression between patients with dilated cardiomyopathy or ischemic heart disease. These observations demonstrate that cardiac development in man, in contrast to rodent heart, is characterized by an up-regulation of the skeletal actin gene, the expression of which does not change in hypertrophied and failing hearts, and suggest that the actin and myosin heavy chain families are independently regulated in human heart.

A decrease in the myocardial level of the mRNA encoding the Ca2(+)-ATPase of the sarcoplasmic reticulum (SR) has been recently reported during experimental cardiac hypertrophy and failure. To determine if such a deficit occurs in human end-stage heart failure, we compared the SR Ca2(+)-ATPase mRNA levels in left (LV) and right ventricular (RV) specimens from 13 patients undergoing cardiac transplantation (6 idiopathic dilated cardiomyopathies; 4 coronary artery diseases with myocardial infarctions; 3 diverse etiologies) with control heart samples using a rat cardiac SR Ca2(+)-ATPase cDNA probe. We observed a marked decrease in the mRNA for the Ca2(+)-ATPase relative to both the 18S ribosomal RNA and the myosin heavy chain mRNA in LV specimens of patients with heart failure compared to controls (-48%, P less than 0.01 and -47%, P less than 0.05, respectively). The LV ratio of Ca2(+)-ATPase mRNA to 18S RNA positively correlated with cardiac index (P less than 0.02). The RV ratio correlated negatively with systolic, diastolic and mean pulmonary arterial pressures (P less than 0.02, P less than 0.02, and P less than 0.01, respectively). We suggest that a decrease of the SR Ca2(+)-ATPase mRNA in the myocardium plays an important role in alterations of Ca2+ movements and myocardial relaxation reported during human end-stage heart failure.

Isozymes of myosin have been localized with respect to individual cardiac myocytes in hearts from 3-week-old, adult controls, and adult hypophysectomized rats, and in cultured cardiac cells. For this purpose, affinity-purified antibodies reacting specifically with the heavy chains of each of the two major myosin isozymes of adult rat heart, V1 and V3, were used. The distribution of the two isomyosins was determined by double immuno-labeling of the same cell, V1 myosins being revealed by rhodamine and V3 myosins by fluorescein. A procedure is described which allows optimum immunological visualization of the myosin filaments of rod-shaped isolated myocytes. It was found that the response of the cardiac cells to the two antimyosins varied depending on the state of the animal. In 3-week-old rats, all cells were stained with the anti-V1, and almost none with the anti-V3 myosin. In the hypophysectomized animals, on the contrary, all cells were stained with the anti-V3 and none with the anti-V1. A mixed pattern of reactivity was observed in adult controls since 50% of the cells reacted with the anti-V1, 10% with the anti-V3, and 40% with both antibodies. In the latter case, the distributions of V1 and V3 reactivities were homogeneous throughout the cell, and absolutely superimposable. The same double reactivity and homogeneous repartition were observed in cultured cells. These findings indicate that myocytes from adult rat myocardium are heterogeneous in terms of their isomyosins content and show for the first time that two isomyosins can coexist and be equally distributed in one cardiac cell. These observations are relevant to the regulation of individual heart cell contractility.

Two distinct myosin heavy chain isoforms, referred to as alpha and beta, were identified in the human heart with specific antimyosin antibodies. By indirect immunofluorescence, myosin heavy chain alpha was found to be a major component of atrial myosin and a minor component of ventricular myosin, while heavy chain beta was found to be a major component of ventricular myosin and a minor component of atrial myosin. In the normal heart, there was marked individual variability in the proportion of ventricular myocytes reactive for heavy chain alpha. Atrial myocytes staining for heavy chain beta were rare in the left atrium and more numerous in the right atrium, especially in the crista terminalis and in the interatrial septum. Surgical and autoptic specimens from hypertrophied left ventricles of patients with mitral regurgitation showed a myosin immunoreactivity pattern similar to that of normal specimens. Very rare muscle cells reactive for heavy chain alpha were seen in the hypertrophied left ventricles of subjects with hypertension and in the hypertrophied right ventricles of subjects with tetralogy of Fallot. A dramatic transformation of myosin heavy chain composition was observed in hypertrophied left atria of patients with mitral stenosis, with a shift to heavy chain beta in a large proportion of atrial myocytes. The findings indicate that chronic exposure to hemodynamic overload can induce marked changes in the myosin heavy chain composition of human atria, whereas it affects only slightly that of the ventricles.

We have investigated the level of expression of the atrial natriuretic factor (ANF) gene in the human heart during ontogenic development by determining the concentrations of ANF messenger ribonucleic acid (ANF mRNA), of immunoreactive ANF (IR ANF) and of receptor reactive ANF (RR ANF), in myocardial samples of the various heart chambers. We found the level was high and almost identical in the left and right ventricles in utero. It gradually decreased during ontogenic development to reach the low adult levels, with a more rapid decrease in the right than in the left ventricle after birth. In the atria, ANF gene expression was high as early as the 13th week of gestation, was higher in the right than in the left atrium, and appeared little affected by ontogenic development.

The phenotype of myocardial proteins is not set once and for all: it changes during ontogenesis, haemodynamic overload and in many other circumstances (e.g. dysthyroidism, diabetes). These changes are attributed either to the differential expression of multigenic protein families, or to modulation of single gene expression. In haemodynamic overload, this versatility of the myocardium enables it, usually by re-expression of its foetal phenotype, to find in itself the capacity for getting adjusted to new functioning conditions. The mechanisms that link the heart work overload to these phenotypic transitions, as well as their possible physiopathology, remain to be determined.

Isolated adult myocytes incubated with [35S]methionine were used to study the expression of proteins in the rat heart during the first 2 wk after either pressure or volume overload. In both models an early (2-4 d) and transient expression of three major stress proteins (heat shock protein [HSP] HSP 70, HSP 68, and HSP 58) was observed together with an increased synthesis of putative ribosomal proteins. Only traces of 35S-labeled HSPs were detected in controls and sham-operated animals. The three stress proteins were identified by their migration in two-dimensional gels, by comigration with HSPs, which had been induced in myocytes by incubation at 41 degrees C and immunoblot analysis using antisera directed against the 70-kD protein. Immunohistochemical staining of HSP 70 in rod-shaped myocytes and detection by immunoblot showed that HSP 70 was equally present and distributed in both sham-operated and overloaded hearts, and provided no evidence for a subpopulation of myocytes acutely involved in the increased expression of HSP 70. It is suggested that the transient expression of HSPs that occurs during the early adaptation of the myocardial cells to overload could confer some degree of protection to the actively growing myocytes.

The cardiac myosin phenotype, an important determinant of myocardial contractility, is modified by chronic increases in hemodynamic load. To quantify the proportion of atrial alpha-myosin heavy chain in various types of left atrial overload and to assess the possible relation between this proportion and atrial size, 34 patients were studied, 4 with Wolff-Parkinson-White syndrome, 29 with various types of mitral valve dysfunction and 1 with an atrial septal defect. Four normal autopsy hearts were also studied. The proportion of alpha-myosin heavy chain among total (alpha plus beta) myosin heavy chains was determined in each atrial sample, using an enzyme-linked immunosorbent assay. The size of the left atrium was assessed by one- and two-dimensional echocardiography. Alpha-myosin heavy chain was the main isoform present in the normal atria (85.5 +/- 9% of total myosin heavy chains). Patients with pure tight mitral stenosis (n = 9), mitral stenosis plus mild regurgitation (n = 8) and severe mitral regurgitation (n = 8), who had a higher indexed left atrial transverse diameter than those with Wolff-Parkinson-White syndrome (33 +/- 6, 39 +/- 10 and 46 +/- 5 versus 19.5 +/- 2 mm/m2, p less than 0.01, p less than 0.001 and p less than 0.001, respectively), also demonstrated a much smaller percent of alpha-myosin heavy chain content (28 +/- 20, 23.5 +/- 13 and 12 +/- 10 versus 58 +/- 18%, p less than 0.01, p less than 0.01 and p less than 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Aortic insufficiency was induced in rats. Left ventricular papillary muscle function was studied after 5, 12, and 40 wk and compared with the papillary muscles from sham-operated animals. The maximum unloaded velocity of shortening, Vmax, was decreased in the rats with aortic insufficiency relative to controls by 15, 20, and 34% at 5, 12, and 40 wk, respectively. The decrease in Vmax occurred concomitantly with a change in the myosin isoenzyme composition such that the V1 isoform content decreased and the V3 isoform increased. Relative to age-matched controls, the V3 content in the hearts with aortic insufficiency had increased by 80, 180, and 125% at 5, 12, and 40 wk, respectively. The decrease in Vmax in the aortic insufficiency group muscles correlated with the change in myosin isozyme composition and could not be explained by changes in collagen content. Thus aortic insufficiency induced changes in myosin isozyme content and Vmax similar to those previously observed with aortic stenosis, thus suggesting a common mechanism of myocardial adaptation to different types of mechanical overload.

Rats were given amiodarone (50 mg X kg-1 X day-1, orally) for 4 weeks and the distribution of ventricular isomyosins, a sensitive index of the effects of thyroid hormones on cardiac tissue, was analyzed. Amiodarone treatment induced a marked increase in both T4 and rT3 and tended to decrease T3 serum levels. At the pharmacologically active dosage we used, the drug induced a moderate redistribution of ventricular isomyosins in favour of V, at the expense of V1. Our results do not support the hypothesis that the major mechanism of action of amiodarone is mediated through hypothyroid-like effects.

We tested the hypothesis that hypertrophy of the human heart is associated with the redistribution of ventricular isomyosins. Human cardiac myosin was isolated from autopsy samples of left ventricular free wall of patients with cardiac hypertrophy and of fetal, young, and adult subjects without heart disease. The following parameters were studied: electrophoretic migration in denaturing and non-denaturing conditions; immunological cross-reactivities with three different types of antibodies; and early phosphate burst size and steady state ATPase activities stimulated by K+-EDTA, Ca++, Mg++, and actin. The antibodies were chosen for their ability to recognize selectively the rat V1 and V3 cardiac isomyosins. The first type was a monoclonal antibody, CCM-52, prepared against embryonic chick cardiac myosin, the second was an anti-beef atrial myosin, and the third was an anti-rat V1 myosin. CCM-52 reacted with a greater affinity with rat V3 than with rat V1, and was a probe of mammalian V3. Anti-beef atrial myosin and anti-rat V1 myosin both recognized specifically beef atrial and rat V1 myosins, and were thus considered as probes of mammalian V1. Under non-denaturing conditions, human myosins migrated as rat V3 isomyosin; under denaturing conditions, no difference was observed in any of the electrophoretic parameters between all samples tested, except for the fetal hearts which contained a fetal type of light chain. The immunological studies indicated that human myosins were composed mostly of a V3 type (HV3), but contained also some V1 isomyosin. A technique was developed to quantify the amount of human VI isomyosin which was found to range from almost 0 to 15% of total myosin, and to vary from one heart to the other, regardless of the origin of the heart. Enzymatic studies showed no significant difference between normal, hypertrophied, and fetal hearts in any of the activities tested. However, there was a significant correlation between Ca++-stimulated ATPase activities and HV1 amount (at 0.05 M KCl, n = 18, r2 equal 0.49, P less than 0.01; at 0.5 M KCl, n = 18, r 2 = 0.5, P less than 0.01). These data demonstrate the heterogeneity of human ventricular myosin, which appears to be composed, as in other mammalian species, of V1 and V3 isoforms of different ATPase activities (V1 greater than V3). However it seems that V1 to V3 shifts do not appear to be of physiological significance in the adaptation of human heart to chronic mechanical overloads.

The atria produce several peptides that have natriuretic and vasoactive properties, collectively called atrial natriuretic factor. All these peptides share a single messenger ribonucleic acid, the amount of which greatly increases in the rat left ventricle when the latter is submitted to chronic volume overload. Using the molecular hybridization technique and a desoxyribonucleic acid probe complementary to the atrial natriuretic factor messenger ribonucleic acid, we now report that a very important increase in the amount of this messenger ribonucleic acid is also observed in rat ventricle at at the compensatory stage of a pressure overload induced cardiac hypertrophy. This result suggests that the pressure overload hypertrophied rat ventricle also has the potential to itself regulate it's loading conditions via the regulation of extracellular fluid volume and vascular resistance.