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Biomedical subjects

D Charlemagne

Publications and source records attributed to D Charlemagne.

At least 37 records · Page 2Linked to original sources
PubMed1993

Arrhythmogenicity of the hypertrophied and senescent heart and relationship to membrane proteins involved in the altered calcium handling.

The high incidence of arrhythmias in human left ventricular hypertrophy has been well established but the mechanisms of arrhythmias are not well defined. In attempt to clarify these mechanisms, we tried to determine if a relationship might exist in the hypertrophied or senescent hearts between the incidence of arrhythmias and alterations in the gene expression of the main membrane proteins involved in the regulation of calcium movements. Holter monitoring was used in young and senescent rats where hypertrophy had been induced by aortic stenosis and hyperthyroidism (young rats) or by DOCA-salt treatment (senescent rats). Different types of spontaneous arrhythmias were detected. In the aortic stenosis group, the heart rate and the number of supraventricular premature beats were increased significantly, whereas the number of ventricular premature beats was increased in some animals but not in all. In senescent rats, the numbers of ventricular and supraventricular premature beats and the incidence of atrioventricular block were very high. At the cellular level, the density of calcium channels from the sarcolemma and of the alpha 1 subunit of the Na+/K(+)-ATPase were unchanged in the hypertrophied and senescent hearts but most of the proteins involved in the regulation of calcium movements (calcium release channel and Ca(2+)-ATPase from the sarcoplasmic reticulum, Na+/Ca2+ exchange, and beta adrenergic and muscarinic receptors from the sarcolemma) have a decreased density or activity. These changes might account for the slowing of the maximum shortening velocity and the impaired contractility of the hypertrophied and senescent hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1993

Molecular and cellular level of action of digitalis.

The pharmacological receptor of cardiac glycosides is the Na+/K(+)-ATPase which consists of a catalytic alpha (M(r) = 112,000) and glycosylated beta (M(r) = 35,000) subunit. The enzyme is responsible for the vectorial transport across the sarcolemma of three Na+ ions outward and two K+ ions inward against their electrochemical gradient. Specific inhibition of the Na+ pump by digitalis induces a positive inotropic effect by increasing the intracellular Na+ concentration which in turn induces an increase in the intracellular Ca2+ concentration by the Na+/Ca2+ exchange and an increase in the Ca2+ pool of the sarcoplasmic reticulum; toxic effects are observed at higher doses of cardiac glycosides leading to spontaneous calcium release from the sarcoplasmic reticulum. Three isoforms of the alpha catalytic subunit have been identified by molecular cloning. They share a high homology in the deduced amino acid sequence with eight transmembrane domains. The ouabain binding domain is located on the extracellular side and ouabain sensitivity depends mainly on the two residues at the border of the first extracellular domain. The isoforms differed by their ouabain sensitivity, are expressed in a tissue-specific and hormonally-regulated manner. Moreover, expression of the isoforms and their ouabain sensitivity vary from species to species with an alpha 1 isoform of very low affinity being the major isoform (80%) in the adult rat heart and an alpha 1 isoform of high affinity representing 50% of total alpha mRNA abundance in the human heart. Therefore the effect of digitalis on the heart depends mainly on the isoform which is expressed and on the regulation of their expression according to age, hormonal influence and pathology.

Animals↗
PubMed1993

Cardiac hypertrophy and failure--a disease of adaptation. Modifications in membrane proteins provide a molecular basis for arrhythmogenicity.

Cardiac hypertrophy is the physiological adaptation of the heart to chronic mechanical overload. Cardiac failure indicates the limits of the process. Cardiac hypertrophy is only one example of biological adaptation and results from the induction of several changes in gene expression, mostly of the fetal type, including those coding for the myosin heavy chain or the alpha-subunit of the Na+,K(+)-ATPase. From a thermodynamic point of view, the decrease in Vmax allows the heart to produce a normal tension at a lower cost. This process results from changes both in the sarcomere and in the expression of certain membrane proteins. The decrease in calcium transient is determined by several changes in membrane proteins that result in a rather fragile equilibrium in terms of calcium homeostasis. Any abnormal input in calcium will have exaggerated detrimental consequences on a hypertrophied myocyte and may cause automaticity and arrhythmias or an exaggerated response to anoxia in terms of compliance.

Adaptation, Physiological↗
PubMed1992

The membrane proteins of the overloaded and senescent heart.

Cardiac hypertrophy which occurs during chronic mechanical overload is one of the numerous examples of biological adaptation to environmental requirements. As such, it is obtained at random by trial and error, and adaptation represents the sum of various modifications in gene expression, including the shift in isoform of myosin or in iso Na+, K+ ATPase, the decrease in beta-adrenergic and muscarinic receptors, ryanodine channels or SR Ca2+ ATPase densities and the unchanged density in Ca2+ current. Some of these changes are beneficial at the cellular level, but are finally detrimental for the organism as a whole, as is the slowing of Vmax. It was suggested that the calcium homeostasis of the hypertrophied cardiocyte was fragile and that this modified cell was less able to buffer the changes in the intracellular calcium, thus providing a biological basis for the arrhythmogenicity of the hypertrophied heart. These various modifications may provide a new key for future pharmaceutical research.

Adaptation, Physiological↗
PubMed1992

Cell and membrane lipid analysis by proton magnetic resonance spectroscopy in five breast cancer cell lines.

The lipid composition of five human breast cancer cell lines (MCF-7, T47D, ZR-75-1, SKBR3 and MDA-MB231) was assessed by proton magnetic resonance spectroscopy (MRS) in whole cells and membrane-enriched fractions. The proportions of the three main lipid resonances in 1D spectra were different for each cell line. These resonances included mobile methyl and methylene functions from fatty acids of triglycerides and phospholipids and N-trimethyl from choline of phospholipids. T47D and ZR-75-1 cells presented a high methylene/methyl ratio (6.02 +/- 0.35 and 6.28 +/- 0.90). This ratio was significantly lower for SKBR3, MCF-7 and MDA-MB231 cells (2.76 +/- 0.22, 2.27 +/- 0.57 and 1.39 +/- 0.39). The N-trimethyl/methyl ratio was high for MDA-MB231 and SKBR3 cells (1.38 +/- 0.54 and 0.86 +/- 0.32), but lower for MCF-7, T47D and ZR-75-1 cells (0.49 +/- 0.11, 0.16 +/- 0.07 and 0.07 +/- 0.03). 2D COSY spectra confirmed these different proportions in mobile lipids. From 1D spectra obtained on membrane preparations, T47D and ZR-75-1 were the only cell lines to retain a signal from mobile methylene functions. These differences might be related to the heterogeneity found for several parameters of these cells (tumorigenicity, growth rate, hormone receptors); an extended number of cases from fresh samples might enable clinical correlations.

Breast Neoplasms↗
PubMed1992

Remodeling of the heart (membrane proteins and collagen) in hypertensive cardiopathy.

The basis for impaired left ventricular function of hearts in moderate to severe stages of hypertrophy and congestive heart failure remains uncertain. At the cellular level, the mechanisms governing the movements of calcium in the myocardium are actually depressed and might at least in part account for the slowing of the maximum shortening velocity and the impaired relaxation. These alterations of membrane proteins seem particularly important in species where the slowing of Vmax cannot be a consequence of the myosin heavy chain shift. They lead to an unstable equilibrium of calcium homeostasis and to calcium overload in heart failure. On the other hand, the enhanced density and remodeling of collagen in the hypertrophied heart, which would depend on elevation in circulating aldosterone, impair myocardial stiffness with diastolic dysfunction and lead to altered pumping capacity of the heart. Disturbances of calcium metabolism and matrix collagen remodeling enhance early afterdepolarizations and arrhythmias.

Animals↗
PubMed1991

The density of ryanodine receptors decreases with pressure overload-induced rat cardiac hypertrophy.

We investigate the possibility that alterations in the calcium movements of the hypertrophied rat heart might involve sarcoplasmic reticulum (SR) ryanodine receptors. A decreased receptor density was observed with severe hypertrophy (0.26 +/- 0.05 and 0.35 +/- 0.06 pmol/mg protein and 170 and 366 receptors/micron2 of SR in 50-80% hypertrophy and control, respectively); however, the total number of receptors per left ventricle was unchanged. The dissociation constant (0.7 nM) was similar in both hypertrophied and control left ventricles. Thus the decreased density of the ryanodine receptors may participate in altered calcium movements in hypertrophied rat heart.

Animals↗
PubMed1991

Two functional Na+/K(+)-ATPase isoforms in the left ventricle of guinea pig heart.

Guinea pig left ventricular muscle contains two distinct molecular forms of the Na+/K(+)-ATPase catalytic alpha subunit. Sarcolemmal vesicles highly enriched in Na+/K(+)-ATPase were isolated by a new procedure that yielded specific activities of 60-100 mumol Pi.h-1.mg-1. SDS/PAGE of isolated sarcolemma after reduction and alkylation of the sulfhydryl groups and identification on immunoblots with specific anti-(alpha subunit) antibodies indicated the presence of two major polypeptides of 100 kDa and 103 kDa, respectively. The two alpha subunits were functional: the dose/response curves of Na+/K(+)-ATPase activity with ouabain, dihydroouabain and digitoxigenin were biphasic, revealing the presence of high-affinity [concentration of drug causing 50% inhibition (IC50) = 10 nM] and low-affinity (IC50 = 2 microM) forms with proportional contributions of 55% and 45%, respectively. The involvement of the high-affinity form in the positive inotropic effect of digitalis and of the low-affinity sites in both inotropy and toxicity are consistent with the literature data on rodents.

Animals↗
PubMed1991

Effect of pressure overload on cardiac Ca2+ antagonist binding sites of guinea pig. Comparison with the adaptational response of the hypertrophied rat heart.

STUDY OBJECTIVE: The aim was to determine if the adaptational process of the cardiac calcium channel to pressure overload observed in rat heart also occurs in species characterised by a higher sensitivity to external calcium than in the rat. This adaptation occurs via a maintained density of dihydropyridine receptors and calcium current in hypertrophied rat heart. DESIGN: The guinea pig was chosen and the dissociation constant (Kd), association and dissociation rate constants (k+1,k-1), and maximal number (Bmax) of the dihydropyridine receptors were measured through binding of [3H]PN 200-110 to crude sarcolemma fractions from control and hypertrophied guinea pig left ventricle. EXPERIMENTAL MATERIAL: Hypertrophy of the left ventricle was obtained by stenosis of the abdominal aorta in guinea pigs. MEASUREMENTS AND MAIN RESULTS: Hypertrophy reached at least 50% in 15% of the surviving animals. No significant differences in the binding of [3H]PN 200-110 to the dihydropyridine receptor were observed between control and hypertrophied left ventricle microsomal preparations: Kd = 1.59(SEM 0.22) and 1.17(0.36) nM; Bmax = 225(18) and 213(4) fmol.mg-1 of protein; k-1 = 2.30(0.26) and 2.00(0.13) min-1 x 10(-2); k+1 = 3.8(0.7) and 3.5(0.3) nM-1.min-1 x 10(-2) respectively. CONCLUSIONS: In guinea pig as in rat, the total number of dihydropyridine receptors per left ventricle increased proportionately to the hypertrophy. This is consistent with an unchanged density of the cardiac Ca2+ channels in the hypertrophied guinea pig heart as previously shown in hypertrophied rat heart.

Adaptation, Physiological↗
PubMed1991

Membrane proteins of the myocytes in cardiac overload.

Hypertrophy of the cardiac myocytes resulting from a mechanical overload may be responsible for major membraneous modifications, either at a sarcolemmal or at a sarcoplasmic level. In the present report, evaluation of several sarcolemmal markers such as beta-receptors, muscarinic receptors or (Na+,K+)-ATPase has been realized on an experimental model of cardiac hypertrophy performed on adult rats. Special attention has been taken to compare results expressed in densities or expressed in number of receptors present at the cellular level. For example, the beta-receptors density was 30% decreased whereas calculations of number of receptors present in the myocytes revealed an unchanged number (around 20,000 in both groups). The unmodified number could suggest a non regulation for the genes coding for the receptors.

Animals↗
PubMed1990

Membrane proteins of the myocytes in cardiac overload.

1. Hypertrophy of the cardiac myocytes resulting from a mechanical overload may be responsible for major membraneous modifications, either at the sarcolemmal or at the sarcoplasmic level. In this study several sarcolemmal markers such as beta-adrenoceptors, muscarinic receptors or (Na+, K+)-ATPase were investigated in an experimental model of cardiac hypertrophy, the chronic aortic stenosis in adult rats. 2. Left ventricular beta-adrenoceptor density (expressed in fmol mg-1 protein) was decreased in the aortic stenosis group by about 30%; however, when expressed in number of receptors per cardiac cell beta-adrenoceptor number in the hypertrophied myocytes was unchanged. 3. Similarly, the number of muscarinic receptors in the hypertrophied cells, expressed as number of receptors per cardiac cell, was unchanged. 4. The number of (Na+, K+)-ATPase molecules with high affinity for ouabain was markedly increased in the hypertrophied myocytes, while those with low affinity for ouabain were not. 5. These results indicate the necessity in chronic hypertrophy to calculate receptors not only in density (fmol mg-1 protein) but also in number per cardiac cell. The unchanged number of beta-adrenergic and muscarinic receptors present on the hypertrophied myocytes suggests a non-regulation for the genes coding for these receptors.

Animals↗
PubMed1990

Normal responsiveness to external Ca and to Ca-channel modifying agents in hypertrophied rat heart.

In rat heart, the maximum velocity of shortening is decreased in response to chronic pressure overload. This is in part explained by an isomyosin shift, but several arguments suggest changes in membrane proteins. Inotropic response to calcium channel modifiers and to external calcium were simultaneously determined to explore this possibility. Left ventricular hypertrophy was induced by abdominal aortic stenosis and after 4-5 wk the left ventricular-to-body weight ratio increased by 61%. The effects of BAY K 8644 (10(-9) to 10(-6) M), a calcium channel activator, nifedipine (10(-9) to 10(-7) M), and external calcium (0.25-2.50 mM) were studied on isolated hearts at a coronary flow of 20 ml.min-1.g of left ventricle-1. The inotropic response (in percent changes in developed pressure and in dP/dtmax) was unmodified in the hypertrophied hearts. This work is in agreement with previous findings that both the total number of dihydropyridine binding sites and the peak magnitude of calcium current increase in proportion to the degree of hypertrophy. It suggests that the slowing of velocity could not be explained by a decreased number of Ca2+ channels but may more likely reflect modifications of the sarcomeres or sarcoplasmic reticulum.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1990

Calcium current in single cells isolated from normal and hypertrophied rat heart. Effects of beta-adrenergic stimulation.

The L-type calcium current was investigated in normal and hypertrophied rat ventricular myocytes as a possible cause of the action potential lengthening that has been reported during hypertrophy. Regulation of the calcium current (ICa) by a beta-adrenergic agonist (isoproterenol) was also analyzed since beta-agonist-induced positive inotropy is less marked in hypertrophied heart. Left ventricular hypertrophy was induced by stenosis of the abdominal aorta. For recording ICa, the whole-cell patch-clamp technique was used. Potassium currents were suppressed by replacing K+ ions with Cs+ ions in both the extracellular and intracellular media, and sodium current was blocked by 50 microM tetrodotoxin. The Ca2+ current was larger in hypertrophied cells (2.2 +/- 0.6 nA [n= 31]) than in normal cells (1.2 +/- 0.5 nA [n = 33]). However, if one relates ICa amplitude to the cell membrane area, as estimated by membrane capacitance measurement, no significant difference was observed in current density (8.5 +/- 2.5 pA/pF [n = 31] and 8.3 +/- 2.1 pA/pF [n = 33] in hypertrophied and in normal cells, respectively). In both cell types, ICa displayed the same voltage and time dependence. When expressed as a percentage, the maximal increase in ICa amplitude that was obtained with 100 nM isoproterenol was less in hypertrophied cells (+78%) than in normal cells (+120%). The sensitivity of ICa to beta-adrenergic stimulation was not modified: EC50 was 3.8 nM for hypertrophied cells and 4.8 nM for normal cells. Forskolin and cyclic AMP were as effective in both cell types. Stimulation of ICa by beta-adrenergic agonist was decreased in agreement with a reduced number of binding sites of beta-agonists and/or an altered coupling of the G-proteins.

Action Potentials↗
PubMed1989

Signal and adaptational changes in gene expression during cardiac overload.

Chronic cardiac overload stimulates various quantitative and qualitative mechanisms of adaptation, some of them being species-specific. The signals responsible for these changes in gene expression are still speculative, nevertheless early modifications of the microtubular network have been reported. Soon after overload an increased expression of various genes coding for regulatory proteins has also been observed, this includes various oncogenes and the genes of several heat-shock proteins. Hypertrophy only, is non species-specific and is adaptational because it both multiples the number of contractile units and it lowers wall stress. The slowing of the shortening velocity allows the heart to produce normal tension, at a lower cost, and has different biological explanations depending on the species. In small rodent ventricles, the main but probably not the unique, determinant of this physiological parameter is an isomyosin shift from a high ATPase activity form V1 to a low activity form V3, discovered in our laboratory in 1979. This shift has a transcriptional origin and also occurs in atria in every mammalian including humans; nevertheless it has not been evidenced in the ventricles of humans, dog, cat or guinea-pig. In these species it is necessary to take into account other mechanisms, namely those involved intracellular calcium movements. The number of total, and possibly active, calcium channels is normal in rat overloaded heart suggesting that their synthesis is activated commensurate to the development of hypertrophy. The situation is more complex for other sarcolemma proteins such as the beta-adrenergic system and the Na+, K(+)-ATPase. For the latter there is presently some evidence that an isoenzymatic shift is likely to occur, at least in rats.

Adaptation, Physiological↗
PubMed1988

Adaptational process of the cardiac Ca2+ channels to pressure overload: biochemical and physiological properties of the dihydropyridine receptors in normal and hypertrophied rat hearts.

Inotropic responsiveness to dihydropyridines (DHP) and characterization of DHP receptors were studied during the onset of hypertrophy in rat hearts. The inotropic responsiveness of isolated hearts to external Ca2+ (0.25-2.50 mM) and nifedipine (10(-9)-10(-7) M), as expressed in percent change in dP/dtmax, was unchanged by the process of hypertrophy. Characterization of DHP receptors by Scatchard plots (Kd = 0.45 and 0.47 nM for nitrendipine), displacement curves (Kd = 0.44 and 0.42 nM for PN 200-110), and dissociation kinetics (k-1 = 4.82 X 10(-2) X min-1 and 4.85 X 10(-2) X min-1) revealed the similarity of the Ca2+ antagonist binding sites in hypertrophied and control hearts, respectively. These results on crude or purified sarcolemmal preparations from left ventricle were consistent with the presence of only one type of binding site of high affinity for DHP. The total number of Ca2+ channels was increased in hypertrophied left ventricle (LV) as compared with left ventricle from sham-operated animals (15,000 fmol/LV and 8900 fmol/LV), respectively. This increased synthesis of Ca2+ channels was observed as early as 5 days after the aortic stenosis and was related to the increase in ventricular mass. Results are in favor of an adaptational process of regulation of the total number of Ca2+ channels as an answer to pressure overload.

Adaptation, Physiological↗
PubMed1987

Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart.

The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.

Alkylation↗
PubMed1987

Two Na,K-ATPase isoenzymes in canine cardiac myocytes. Molecular basis of inotropic and toxic effects of digitalis.

Canine cardiac myocytes contain two distinct molecular forms of the Na,K-ATPase catalytic subunit. They are resolved by gel electrophoresis and identified using immunological techniques. The apparent molecular weights of the catalytic subunits are 95,000 (alpha) and 98,000 (alpha +). As judged by [3H]ouabain-binding measurements and Na,K-ATPase assays, the two forms are active and differ by a factor of 150 in their respective affinity for digitalis (ouabain and digitoxigenin). The dissociation constant of the high affinity form (alpha +) is KD, 2 nM, and that of the low affinity molecular form (alpha) is KD, 300 nM. According to both enzymatic and binding assays, up to 70% of maximum inhibition is caused by occupation of the high affinity sites (alpha +). Inasmuch as the pharmacological and toxic concentrations of digitalis in dog are 1 and 200 nM, respectively, and as maximum inhibition of Na+ pump in vivo should not exceed 80% to avoid toxicity (Akera, T. and Brody, T. (1982) Annu. Rev. Physiol. 44, 375-388), it appears that the high affinity molecular form (alpha +) is the pharmacological receptor exclusively related to positive inotropy, whereas the low affinity form (alpha) is mainly associated with toxicity.

Animals↗