Biomedical subjects

J J Mercadier

Publications and source records attributed to J J Mercadier.

At least 37 records · Page 2Linked to original sources

PubMed1998

Myosin heavy chain gene expression changes in the diaphragm of patients with chronic lung hyperinflation.

In striated muscle, chronic increases in workload result in changes in myosin phenotype. The aim of this study was to determine whether such changes occur in the diaphragm of patients with severe chronic obstructive pulmonary disease, a situation characterized by a chronic increase in respiratory load and lung volume. Diaphragm biopsies were obtained from 22 patients who underwent thoracic surgery. Myosin was characterized with electrophoresis in nondenaturing conditions, SDS-glycerol PAGE, and Western blotting with monoclonal antibodies specific for slow and fast myosin heavy chain (MHC) isoforms. Flow volume curves, total lung capacity, and functional residual capacity were measured before surgery in 20 patients. We found that the human diaphragm is composed of at least four myosin isoforms, one slow and three fast, resulting from the combination of three MHC species. Chronic overload was associated with an increase in the slow beta-MHC species at the expense of the fast species (beta-MHC, 78.2 +/- 4.6 and 50.0 +/- 6.5% in emphysematous and control patients, respectively; P < 0.005). Linear correlations were found between beta-MHC percentage and forced expiratory volume in 1 s (r = -0.52; P < 0.02), total lung capacity (r = 0.44; P < 0.05), and functional residual capacity (r = 0.65; P < 0.003). The human adult diaphragm is composed of a balanced proportion of slow and fast myosin isoforms. In patients with chronic obstructive pulmonary disease, the proportion of fast myosins decreases, whereas that of slow myosin increases. This increase appears to be closely related to lung hyperinflation and may reflect an adaptation of the diaphragm to the new functional requirements.

Adult↗

PubMed1998

[Transient outward potassium current and repolarization of cardiac cells].

The transient 4-aminopyridine-sensitive outward potassium current, Ito, is one of the ionic membrane currents involved in the repolarization of cardiac action potentials. It is present in several species (rat, dog, human) but not in guinea pig ventricle. It induces both a marked lowering of the ventricular action potential plateau level and an early repolarization wave in the ventricular ECG complex of hypothermic rats. In dog ventricle where Ito is much shorter than the action potential plateau it can induce only a transient initial repolarization (notch). The distribution of Ito is heterogeneous across the dog left ventricular free wall, the current being of sizeable amplitude in epicardial and midmyocardial layers but absent in the endocardial layer. As a result, ventricular action potentials exhibit a notch only in epicardial and mid layers. Although the physiological role of Ito remains unclear, we suggest that it can participate in the control of calcium current intensity by influencing the level of the initial part of the plateau. In pathophysiological conditions, Ito may exert unfavourable effects, specially during simulated ischemia when the notch reaches the cellular repolarization threshold, thus inducing premature termination of the action potential, an obvious cause of drastic electrical heterogeneity and resulting severe arrhythmias. The current Ito is reduced in moderate cardiac hypertrophy and dilatation and almost entirely suppressed in severe hypertrophy. Ito is of larger amplitude in human atrial than in ventricular myocytes. The heterogeneous distribution of Ito described in the dog has also been found in human ventricles. Because Ito is markedly prolonged at low temperatures it is suggested that it can be responsible for the early repolarization wave (J wave) observed in the ECG of subjects submitted to hypothermia.

Action Potentials↗

PubMed1997

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

EHNA (Erythro-9-[2-hydroxy-3-nonyl]adenine) is a wellknown inhibitor of adenosine deaminase. Recently, EHNA was shown to block the activity of purified soluble cGMPstimulated phosphodiesterase (PDE2) from frog, human, and porcine heart with an apparent Ki value of approximately 1 microM and with negligible effects on Ca2+/calmodulin PDE (PDE1), cGMP-inhibited PDE (PDE3), and low Km cAMP-specific PDE (PDE4) (Méry, P.F., C. Pavoine, F. Pecker, and R. Fischmeister. 1995. Mol. Pharmacol. 48:121-130; Podzuweit, T., P. Nennstiel, and A. Muller. 1995. Cell. Signalling. 7:733- 738). To investigate the role of PDE2 in the regulation of cardiac L-type Ca2+ current (ICa), we have examined the effect of EHNA on ICa in freshly isolated human atrial myocytes. Extracellular application of 0.1-10 microM EHNA induced an increase in the amplitude of basal ICa ( approximately 80% at 1 microM) without modification of the current-voltage or inactivation curves. The maximal stimulatory effect of EHNA on ICa was comparable in amplitude with the maximal effect of isoprenaline (1 microM), and the two effects were not additive. The effect of EHNA was not a result of adenosine deaminase inhibition, since 2'-deoxycoformycin (1-30 microM), another adenosine deaminase inhibitor with no effect on PDE2, or adenosine (1-10 microM) did not increase ICa. In the absence of intracellular GTP, the substrate of guanylyl cyclase, EHNA did not increase ICa. However, under similar conditions, intracellular perfusion with 0.5 microM cGMP produced an 80% increase in ICa. As opposed to human cardiomyocytes, EHNA (1-10 microM) did not modify ICa in isolated rat ventricular and atrial myocytes. We conclude that basal ICa is controlled by PDE2 activity in human atrial myocytes. Both PDE2 and PDE3 may contribute to keep the cyclic nucleotides concentrations at minimum in the absence of adenylyl and/or guanylyl cyclase stimulation.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Primary culture of human atrial myocytes is associated with the appearance of structural and functional characteristics of immature myocardium.

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.

Action Potentials↗

PubMed1997

Troponin T mRNA and protein isoforms in the human left ventricle: pattern of expression in failing and control hearts.

We and others have previously cloned several cDNAs of human cardiac troponin T (cTnT), demonstrating the multiplicity of cTnT isoforms in the human heart. Four of them named cTnT1, 2, 3 and 4 result from a combinatorial alternative inclusion of 30- and 15-nucleotides in the 5' coding region of the cDNAs. In failing human ventricles, increased expression of cTnT4 has been reported at the protein level. More recent RT-PCR experiments showed increased expression of fetal-type splicing products in the 5' region, one of them corresponding to cTnT1. To clarify this issue, we examined the accumulation of the 4 cTnT mRNA and protein species in left ventricular specimens at the time of heart transplantation, and in control left ventricular samples using RNase protection and Western blotting. In all samples, cTnT3 was the major mRNA isoform, cTnT4 a minor isoform while cTnT1 and cTnT2 mRNAs were present but barely detectable. At the protein level, cTnT3, 4 and 1 were detected with the same relative abundance as that seen at the mRNA level. In addition, we detected a fourth TnT species of very low abundance corresponding either to a skeletal or to a "short" cardiac TNT isoform. Compared to controls, increased levels of cTnT4 mRNA and protein were detected in only half the failing ventricles independently of the cause of failure, suggesting that this increase may not be a general characteristic of left ventricular failure but instead could be related to stress. Unexpectedly, we found a decrease in cTnT1 protein expression in all failing ventricular samples studied, compared to controls.

Adult↗

PubMed1997

The antiarrhythmic agent bertosamil induces inactivation of the sustained outward K+ current in human atrial myocytes.

1. In whole-cell patch-clamped human atrial myocytes, the antiarrhythmic agent bertosamil (10 microM) inhibited the sustained component, Isus (38.6 +/- 3.1%), and enhanced the inactivating component, I(t) (9.1 +/- 6.1%), of the outward K+ current elicited by 750 ms test pulses from -60 mV to +50 mV. Higher concentrations of bertosamil (> 10 microM) inhibited both I(t) and Isus. 2. Suppression of Isus and stimulation of I(t) by 10 microM bertosamil was observed on renewed stimulation following a 2 min rest period during which the drug was applied and persisted after washout, indicating a rest-dependent effect of bertosamil on the outward K+ current. 3. Cell dialysis with an internal solution containing 10 microM bertosamil increased both I(t) (78.0 +/- 14.7%) and Itotal (26.7 +/- 8.4%) and inhibited Isus (28.9 +/- 6.3%, n = 6). In the presence of intracellular bertosamil, external application of the drug inhibited I(t) and Isus in a concentration-dependent and use-dependent manner. 4. Following the suppression of Isus by 200 microM 4-aminopyridine (4-AP), bertosamil (10 microM) inhibited I(t). Washout of 4-AP was associated with a larger I(t) amplitude than that observed in control conditions. In myocytes characterized by a prominent Isus and lack of I(t), bertosamil (10 microM) induced a rapid and partial inactivation of the current, together with inward rectification of the current measured at the end of the test pulse. 5. In the presence of bertosamil the activation/voltage relationships, steady-state inactivation and recovery from inactivation of I(t) were markedly modified, pointing to changes in the conductance underlying I(t). 6. We conclude that bertosamil induces rapid inactivation of sustained outward current which leads to an apparent increase in I(t) and decrease in Isus. This effect, which was distinct from the use-dependent inhibition of the outward K+ current, could represent a new antiarrhythmic mechanism.

Adult↗

PubMed1997

Different compartments of sarcoplasmic reticulum participate in the excitation-contraction coupling process in human atrial myocytes.

The excitation-contraction coupling process of human atrial myocytes was studied in voltage-clamped myocytes isolated from right atrial appendages obtained during cardiac surgery. Intracellular Ca2+ transients (Cai transients) were monitored with 0.1 mmol/L indo 1 added to the internal dialyzing solution. Ryanodine receptors (RyRs) and sarcomeric alpha-actinin were stained with specific antibodies and visualized using plane and confocal microscopy. L-Type Ca2+ current (Ica) elicited a prolonged Cai transient, with an initial rapidly activating phase (slope 1, 23.6 +/- 1.2 s-1) followed by a slowly activating phase (slope 2, 5.8 +/- 0.4 s-1; P < .001 versus slope 1), resulting in a dome-shaped Cai transient. Ryanodine (100 mumol/L) inhibited 79 +/- 6% of the Cai transient, indicating that it was due essentially to sarcoplasmic reticulum Ca2+ release. During step depolarizations, maximal activation of the Cai transient or tail current (Itail) (in cells dialyzed with Ca2+ buffer-free internal solution) preceded that of Ica and did not follow its voltage dependence (n = 12). Test pulses lasting from 5 to 150 milliseconds elicited a similar time course of both Cai transient and Itail (n = 5). In a given cell, the two components of the Cai transient could be dissociated by altering the intracellular Ca2+ load, by increasing the stimulation rate from 0.1 to 1 Hz, or by varying the amplitude of Ica. Immunostaining of atrial sections and isolated myocytes showed that a large number of RyRs were located not only in a subsarcolemmal position but also deeper inside the cell, in a regularly spaced transverse band pattern at the level of Z lines. Together, our results indicate that, in human atrial myocytes, Ica only partially controls the activation of RyRs, with the prolonged and dome-shaped Cai transient of these cells probably reflecting the activation of RyRs not coupled to L-type Ca2+ channels.

Atrial Function↗

PubMed1997

[A study of the expression of sarcoplasmic reticulum calcium ATP-ase gene in the left ventricle of the sheep's fetus submitted to chronic increased afterload in utero].

The messenger RNA of the cardiac sarcoplasmic reticulum ATP-ase (SERCA-2a) increases in the left ventricle (LV) during ontogenic development but decreases in hypertrophy induced by increased afterload. Because of the frequency of increased left ventricular overload in congenital heart disease, the authors investigated to see if these increases were likely to interfere with the normal ontogenic program of expression of the SERCA-2a gene in the LV of sheep's foetus. A preductal coarctation of the aorta was realised by banding the transverse aorta (AoT) at 93 days' gestation in 9 foetus (CoA) matched with 9 healthy twin foetus (T). All the foetus underwent haemodynamic, anatomical, histological and molecular biological investigations 4 weeks later. The concentration of SERCA-2a mRNA in the LV was measured by hybridation of a Northern blot with a rat DNAc probe normalised with RNAr 18S. A coarctation was observed in all the CoA group and in none of the T. The ratio of LV weight/body weight was increased in 65% of CoA (p < 0.0001). The concentration of SERCA-2a mRNA in the LV was much reduced in CoA (average -28.6%) of the values observed in T (p = 0.003). Left ventricular hypertrophy of the sheep's foetus induced by pathological increases of afterload surpassed or slowed down the physiological ontogenic maturation of expression of the SERCA-2a gene in abnormalities of cardiac pump function.

Animals↗

PubMed1996

Interactions between endothelin-1 and atrial natriuretic peptide influence cultured chick cardiac myocyte contractility.

We have previously shown that rat atrial natriuretic peptide (ANP) reduces the contractility of cultured, spontaneously beating chick embryo ventricular cells, an effect opposite to that of endothelin-1. Endothelin-1 has been described as a secretagogue for natriuretic peptides in vitro and in vivo. Natriuretic peptides can inhibit endothelin-1 secretion from cultured endothelial cells, suggesting a negative feedback mechanism between endothelial cells and cardiomyocytes. The aim of this study was to determine whether ANP attenuated the endothelin-1-induced increase in myocyte contractility. Using a video-microscopy system we studied the contractility of isolated cultured chick ventricular myocytes in response to endothelin-1, chicken natriuretic peptide (ChNP), and both. We also used Northern blot analysis to study the time course of ChNP expression in response to endothelin-1. Endothelin-1 (10(-8) M) increased chick cardiomyocyte contractility by 20-25% between 5 and 15 min (P < 0.05). Although ChNP (3 x 10(-7) M) did not significantly change the amplitude of contraction in basal conditions, it prevented the endothelin-1-induced increase in contractility (P < 0.05) when perfused prior to endothelin-1, and reversed it when perfused 5 min after endothelin-1 exposure (P < 0.05). Endothelin-1 significantly increased the accumulation of ChNP mRNA in chick ventricular myocytes as early as the 30 min after exposure (P < 0.05), with a maximal effect after 2 h of stimulation (P < 0.01); no effect was observed after 4 h. These data support an interaction between endothelin-1 and natriuretic peptides as autocrine/paracrine factors regulating the contractile function of chick cardiac myocytes, as well as their antagonistic effects on cardiac cell contractility. The early and transient expression of ChNP mRNA in response to endothelin-1 may be involved in this interaction.

Analysis of Variance↗

PubMed1996

Depressed transient outward current density in ventricular myocytes from cardiomyopathic Syrian hamsters of different ages.

We determined whether the dilated cardiomyopathy which develops between 30 and 140 days of age in the Syrian hamster strain MS200, before the onset of cardiac hypertrophy and failure, is associated with alterations in both the action potential (AP) and the Ca(2+)-independent transient outward current, Ito1. AP was recorded in perfused hearts using microelectrodes and Ito1 was recorded in single ventricular myocytes using the whole-cell patch-clamp. The MS200 strain was compared to the control CHF148 strain at different periods of age (60, 90, 120 and 180 days). APs were markedly lengthened in MS200 compared to CHF148 hearts at all ages studied. Cell membrane capacitance increased with age in the two strains, but was not significantly higher in MS200 than in CHF148 of a given age, indicating the absence of cell hypertrophy. At 60 days of age, Ito1 density was the same in the two strains. Later on, Ito1 density increased markedly at 90-120 days then decreased at 180 days in CHF148, whereas this increase was delayed and of reduced amplitude in MS200. The sustained component of outward current, Isus, was not sizeably different in the two strains. The conductance-voltage and steady-state inactivation relationships were shifted with age towards positive potentials by 15 mV in the two strains, but earlier in MS200 (90 days) than in CHF148 (180 days). Similarly, the recovery of Ito1 from inactivation exhibited a slow component which increased with age in the two strains but was larger in MS200 than in CHF148. In conclusion, alterations of Ito1 may contribute to changes in shape of AP, but cannot entirely explain dilation-induced AP lengthening.

Action Potentials↗

PubMed1996

Action potential and plateau ionic currents in moderately and severely DOCA-salt hypertrophied rat hearts.

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.

Action Potentials↗

PubMed1996

Ionic basis of the action potential prolongation in ventricular myocytes from Syrian hamsters with dilated cardiomyopathy.

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.

Action Potentials↗

PubMed1996

Molecular and cellular biology of heart failure.

In recent years, the striking development of molecular biology and molecular genetic has brought completely new insights into the understanding of heart failure. Two aspects for which significant progress has been made in 1995 are discussed in this review: the genetic mechanisms of inherited cardiomyopathies and the molecular basis of heart failure due to chronic hemodynamic overload. In familial hypertrophic cardiomyopathy, a novel disease gene was found. It encodes myosin binding protein C, whose structure and function are poorly understood. Contractile deficits associated with the myosin mutations were demonstrated, and all this strengthened the hypothesis that hypertrophy is a compensatory mechanism that occurs in presence of a sarcomeric defect. These studies have important prognostic and clinical implications, but new and unexpected concerns have arisen, because a widespread difference in phenotype can be seen in patients harboring similar genotypes. In familial dilated cardiomyopathy, the main findings were the identification of four disease loci, but the genes are still unknown. With respect to the consequences of chronic hemodynamic overload on myocyte function and phenotype, recent data gave rise to lively discussions in the fields of reexpression of fetal troponin T isoforms and of decreased function and expression of the sarco(endo)plasmic reticulum Ca2+ ATPase in the failing human heart; at the moment it is difficult to draw definitive conclusions. Interestingly, three new concepts emerged in the understanding of the pathogenesis of heart failure: the increased contribution of the Na(+)-Ca2+ exchange, the possible recruitment of an inositol phosphate-sensitive calcium pool for myofibrillar activation, and the involvement of apoptotic myocyte and nonmyocyte cell death in myocardial remodeling.

Apoptosis↗

PubMed1996

Contribution of Na+/Ca2+ exchange to action potential of human atrial myocytes.

The Ca2+ dye indo 1 was used to record internal Ca2+ (Cai) transients in order to investigate the role of the Na+/Ca2+ exchange current (INa/Ca) in whole cell patch-clamped human atrial myocytes After the activation of the L-type Ca2+ current by test pulses (20 ms) at +20 mV, a tail current (I(tail)) was activated at a holding potential of -80 mV with a density of -1.29 +/- 0.06 pA/pF. The time course of I(tail) followed that of Cai transients I(tail) was suppressed by dialyzing cells with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, applying 5 mM caffeine, or substituting external Na+ with Li+, indicating that this current was mainly generated by INa/Ca. Two types of action potential were recorded: type A, which is characterized by a narrow early plateau followed by a late low plateau phase, and type B, which is characterized by a small initial peak followed by a prolonged high plateau phase. Type B action potentials were found in larger cells than type A action potentials (membrane capacitance 81.8 +/- 4.5 and 122.4 +/- 7.0 pF in types A and B, respectively, P < 0.001). Substitution of external Na+ with Li+ shortened the late plateau of the type A action potential and the prolonged plateau of the type B action potential. Suppression of Cai transients by caffeine shortens the late part of both types of action potentials, whereas its lengthening effect on the initial phase of action potentials can result from several different mechanisms. The beat-to-beat dependent relationship between Cai transients and action potentials could be mediated by Ina/Ca- Delayed afterdepolarizations were present in a significant proportion of atrial myocytes in our experimental conditions. They were reversibly suppressed by Li+ substitution for Na+, suggesting that they are generated by INa/Ca. We conclude that INa/Ca plays a major role in the development of action potentials and delayed afterdepolarizations in isolated human atrial myocytes.

Action Potentials↗

PubMed1996

Differential regulation of voltage-activated potassium currents in cultured human atrial myocytes.

To examine whether the two components of the voltage-activated outward K+ current, an initially rapidly inactivating component (Ito,1) and a slowly inactivating sustained component (Isus), in human atrial myocytes are distinct currents differentially regulated, we studied their behavior during serum-induced growth of cultured myocytes. Currents were recorded in whole cell patch clamped myocytes. After 1 wk of culture (day 8), membrane capacitance was twice the value in freshly dissociated myocytes (178.7 +/- 23 vs. 83.1 +/- 5.5 pF; P < 0.001). Ito,1 density did not differ from that in freshly dissociated myocytes (at +40 mV: 4.38 +/- 0.8 vs. 3.71 +/- 0.6 pA/pF), whereas that of Isus was markedly increased (at +40 mV: 9.76 +/- 2 vs. 2.21 +/- 0.29 pA/pF; P < 0.001). After inactivation of Ito,1 by a prepulse, sustained depolarization elicited in cultured myocytes an Isus with a density of 10.22 +/- 1.18 pA/pF and an apparent tail current reversal potential of -73.5 +/- 3.2 mV, indicating high K+ selectivity. Isus was highly sensitive to 4-aminopyridine (55.4 +/- 4.4% inhibition in 50 microM) and to D-600 (with a concentration inhibiting 50% of maximal response of 34.2 x 10(-6) M). Addition of 5-10 nM staurosporine at day 3 prevented cell growth and reduced Ito,1 density but not the increase in Isus density, which was inhibited by 10 microM staurosporine. Our results indicate that Ito,1 and Isus are regulated independently during in vitro myocyte growth in human atrial myocytes and that the increase in Isus density is not mediated by a protein kinase C-dependent pathway.

4-Aminopyridine↗

PubMed1996

[Beta adrenergic signal transduction and heart adenyl cyclase].

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.

Adenylyl Cyclases↗

PubMed1995

Type V, but not type VI, adenylyl cyclase mRNA accumulates in the rat heart during ontogenic development. Correlation with increased global adenylyl cyclase activity.

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.

Adenylyl Cyclases↗

PubMed1995

Human cardiac troponin T: cloning and expression of new isoforms in the normal and failing heart.

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.

Aged↗