Pierre Athias

Effect of change in growth environment on cultured myocardial cells investigated in a standardized medium

SummaryNeonatal rat heart cells cultivated in either of two different media which varied only in their serum supplements were transferred to chemically defined medium (Hams F10) for 24 h before measuring a variety of parameters. The 24-h period of exposure to chemically defined medium was not sufficient to reverse the effects imposed on the cells by the serum used in the first phase of growth. The cells differed in rate and duration of action potentials and contractions. The initial serum composition affected the response of the cells to calcium deficiency. Studies involving the effects of pharmaceutical reagents such as isoproterenol were also influenced by the serum. In attempting to determine the cause and possible mechanism, it was found that mitochondrial membrane permeability for nitroblue tetrazolium (NBT) was unchanged. Although the serum supplements differed in fatty acid composition, the fatty acid profiles of the cell phospholipids were relatively constant. We conclude that (a) the function of the cells is affected by the growth environment, particularly serum; (b) that a short exposure to a uniform chemically defined medium is not sufficient to reverse these effects; and (c) that the differences in effects are not the result of changes in the fatty acid composition of the whole cell phospholipids nor in mitochondrial membrane permeability as measured by NBT.

Trimetazidine: In vitro influence on heart mitochondrial function

The mechanism of action of the antianginal trimetazidine (TMZ) remains largely unknown. In cultured rat ventricular myocytes in physiologic conditions, TMZ (5 x 10(-4) M) reduced the plateau potential level, the upstroke velocity, and the spontaneous action potential rate. When the cardiomyocytes were submitted to hypoxia (150 or 240 minutes) in a glucose-free medium, treatment with TMZ largely prevented the hypoxia-induced electromechanical alterations, i.e., the decrease in plateau amplitude, in resting membrane potential, in action potential duration, in rate, and in contractility. No hypoxia-induced arrhythmia was observed in the TMZ-treated cells. Moreover, the lactate dehydrogenase leakage was significantly reduced in the TMZ-treated cardiomyocytes (-58% and -36%, after 150 and 240 minutes of hypoxia, respectively). The drug was not efficient in reducing the hypoxia-induced decrease in adenosine triphosphate (ATP) content. The cellular ATP content was slightly lower in the TMZ-treated cells in normoxic conditions and in hypoxic conditions, but only in the glucose-free medium. To investigate further the relation between TMZ and energy metabolism, the respiration parameters were measured in heart mitochondria isolated from control and TMZ-treated rats (6 mg/kg/day, 7 days) with different substrates. This treatment resulted in a slight alteration of pyruvate oxidation, which was observed in the absence and in the presence of TMZ (10(-4) M) in the respiration medium. Conversely, a potent inhibition of palmitoylcarnitine oxidation was measured when TMZ was added to the respiration medium. Neither pretreatment of the rats, nor addition of TMZ to the medium affected the oxidation of glutamate or citrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of the n-6/n-3 fatty acid ratio in the phospholipids of rat ventricular myocytes in culture by the use of synthetic media: Functional and biochemical consequences in normoxic and hypoxic conditions

The respective roles of exogenous polyunsaturated fatty acids on the lipid composition, physiological properties and enzyme release was investigated on isolated cardiac muscle cells in normoxia and hypoxia. Rat neonatal ventricular myocytes were grown for 5 days in conventional serum-supplemented medium. Cells were then incubated for 24 h in fully chemically-defined media featuring a balanced fatty acid composition containing either linoleic acid (18:2 n-6) or linolenic acid (18:3 n-3) as sole polyunsaturated fatty acid source. Transmembrane potentials were monitored with microelectrodes and contractions with a photoelectric device. The radio of n-6 to n-3 phospholipid fatty acids increased from 6.3 in control cells to 20.2 in cells exposed to n-6 fatty acids (SM6) and decreased to 1.4 in those exposed to n-3 fatty acids (SM3). These modifications had no influence on the electrical and mechanical activities and on automaticity in normoxic conditions. The action potential depression under hypoxia was less severe in SM6 cells, whereas there was a better electrophysiological recovery upon reoxygenation in SM3 cells. However, the loss of lactate dehydrogenase during sustained hypoxic treatment was not affected by changes in phospholipid fatty acid pattern. These results suggest that the effect of the polyunsaturated fatty acid balance depends on the cellular function under study and on the environmental conditions.

Oxidative injury of isolated cardiomyocytes: dependence on free radical species

The contribution of lipid peroxidation to myocardial injury by free radicals (FR) is still unclear. Consequently, we examined the functional damages inflicted on cultured rat cardiomyocytes (CM) during FR stress provoked by the xanthine/xanthine oxidase system (X/XO) or by a hydroperoxidized fatty acid ((9 Z, 11 E, 13 (S), 15 Z)-13-hydroperoxyocta-decatrienoic acid; 13-HpOTrE), in order to simulate in vitro the initial phase and the propagation phase of the FR attack, respectively. Transmembrane potentials were recorded with glass microelectrodes and contractions were monitored photometrically. The EPR spectroscopy showed that X/XO produced superoxide and hydroxyl radicals during 10 min. The X/XO system altered sharply and irreversibly the spontaneous electrical and mechanical activities of the CM. However, the gas chromatographic analysis showed that these drastic functional damages were associated with comparatively moderate membrane PUFA degradation. Moreover, the EPR analysis did not reveal the production of lipid-derived FR. 13-HpOTrE induced a moderate and reversible decrease in electrical parameters, with no change in CM contractions. These results indicate that the functional consequences of FR attack are dependent on the radical species present and do not support the idea that the membrane lipid breakdown is a major factor of myocardial oxidant dysfunction.

Protective effects of trimetazidine on hypoxic cardiac myocytes from the rat

Summary— The electrophysiological effects of the antianginal drug trimetazidine (TMZ) were investigated in cultured rat ventricular myocytes using a substrate‐free hypoxia model of ischemia. The transmembrane potentials were recorded with glass microelectrodes and the contractions were simultaneously monitored with a video motion detector. The cardiomyocytes were treated with TMZ (1–5.10−4 M final concentration) in the bath. The untreated and the drug‐treated cells were submitted either to 150 min normoxia or to 150 min hypoxia followed by 90 min reoxygenation in the absence of oxidizable substrate. In normoxic conditions, TMZ did not affect the maximal diastolic potential (MDP) but significantly lowered the plateau potential level (OS) and decreased the upstroke velocity (Vmax) and the spontaneous action potential rate (APR). Conversely, TMZ significantly increased action potential duration at 80% repolarization (APD80). Under substrate‐free hypoxia, the untreated cells displayed a progressive contractile failure and an important decrease in OS and APD. In parallel, early postdepolarizations triggering high rate spikes were observed. Prolonging oxygen depletion led to the cessation of the spontaneous electrical activity and thereafter to a gradual decrease in MDP. Near normal rhythmic action potentials and contractions resumed after reoxygenation. Comparatively, the treatment by 5.10−4 M TMZ almost completely prevented the decrease in plateau amplitude, resting membrane potential, Vmax, APD80, and rate caused by substrate‐free hypoxia. Moreover, the hypoxia‐induced arrhythmias and the cessation of spontaneous electromechanical activities did not occur in the presence of TMZ (5.40−4 M). After reoxygenation, the TMZ‐treated cells exhibited a higher action potential amplitude than that of the untreated cells, although the TMZ‐induced depressive effects on the spontaneous frequency and the Vmax persisted. In conclusion, this study shows that TMZ (5.10−4 M) is efficient in protecting the isolated cardiac myocytes against the functional alterations induced by substrate‐free hypoxia and led thus to a better recovery upon reoxygenation. This cytoprotective action may be linked, at least in part, to apparent ion channel blocking effects of the drug, which appeared in basal conditions at concentrations used in this study.

Influence of phospholipid polyunsatured fatty acid composition on some metabolic disorders induced in rat cardiomyocytes by hypoxia and reoxygenation

The influence of membrane polyunsaturated fatty acid (PUFA) composition on lactate production, energy status, enzyme leakage and cell defences against oxygen free radical production was studied in cultured rat ventricular myocytes during hypoxia and reoxygenation. After 4 days in a conventional serum-supplemented medium, the cardiomyocytes were incubated for 24 h in synthetic media containing either linoleate and arachidonate (SM6 Medium) or linolenate and eicosapentaenoate (SM3 Medium) as unique source of PUFA. The fatty acid n-6/n-3 ratio of phospholipid was 13.1 in SM6 cells and 0.9 in SM3 cells. Hypoxia induced an increase in lactate production, severe decreases in ATP and ADP, leakage of cellular lactate dehydrogenase and reduction of superoxide dismutase and glutathione peroxidase activities. Reoxygenation of hypoxic cells reduced lactate production to normal aerobic values and allowed slight resynthesis of ATP from AMP. However, lactate dehydrogenase release was not stopped by reoxygenation, and decreases in superoxide dismutase and glutathione peroxidase activities were not avoided. The majority of the biochemical parameters measured during normoxia, hypoxia and reoxygenation were not significantly affected by changes in the fatty acid composition of membrane phospholipids, except for reduced superoxide dismutase activity which appeared earlier in SM3 cells during hypoxia. We conclude that the sarcolemmal PUFA composition of cultured rat ventricular myocytes does not significantly influence altered cell metabolism elicited by hypoxia and reoxygenation.

Eicosapentaenoic and Docosahexaenoic Acids in Cultured Rat Ventricular Myocytes and Hypoxia-induced Alterations of Phospholipase-A Activity

Hypoxia was reported to induce a decrease in phosphatidylcholine-hydrolyzing phospholipase activity (PC-PLA) in cultured rat cardiomyocytes. This work was intended to compare the influence of the presence of either eicosapentae noic acid (EPA) or docosahexaenoic acid (DHA) in the phospholipids on the PC-PLA activity in normoxic and hypoxic conditions. The enrichment of the medium with EPA or DHA resulted in cell phospholipids containing about 2% or 22% DHA, respectively. These cells were then submitted for 3.5 h to either normoxia or hypoxia and the PC-PLA activities were assayed using [1-14C] dioleoyl-PC (pH 8.4 for PC-PLA2 and 4.9 for PC-PLAT). The results show that both enzymic activities are significantly higher in DHA-rich cardiomyocytes. Hypoxia induced a significant decrease in PC-PLA2 (about 25%) which was not statistically different between the two groups of cells. The hypoxia-induced decrease in PC-PLA1 was not found significant. In conclusion, the nature of the long chain n-3 polyunsaturated fatty acids in the phospholipids appears to contribute to the regulation of PC-PLA activity but not to influence its decrease during hypoxia. (Mol Cell Biochem116: 75–78, 1992)

Microtubule alteration is an early cellular reaction to the metabolic challenge in ischemic cardiomyocytes.

Cytoskeleton damage, particularly microtubule (MT) alterations, may play an important role in the pathogenesis of ischemia-induced myocardial injury. However, this disorganization has been scarcely confirmed in the cellular context. We evaluated MT network disassembly in myoblast cell line H9c2 and in neonatal rat cardiomyocytes in an in vitro substrate-free hypoxia model of simulated ischemia (SI). After different duration of SI from 30 up to 180 min, the cells were fixed and the microtubule network was revealed by immunocytochemistry. The microtubule alterations were quantified using a house-developed image analysis program. Additionally, the tubulin fraction were extracted and quantified by Western blotting. The cell respiration, the release of cellular LDH and the cell viability were evaluated at the same periods. An early MT disassembly was observed after 60 min of SI. The decrease in MT fluorescence intensity at 60 and 90 min was correlated with a microtubule disassembly. Conversely, SI-induced significant LDH release (35%) and decrease in cell viability (34%) occurred after 120 min only. These results suggest that the simulated ischemia-induced changes in MT network should not be considered as an ultrastructural hallmark of the cell injury and could rather be an early ultrastructural correlate of the cellular reaction to the metabolic challenge.

A simple gas-flow chamber for cultured cell electrophysiology in a controlled atmosphere

A simple and inexpensive device is described to control the gaseous environment while recording membrane potentials and contractile motion from single cultured cells. This equipement was used to study the electrophysiological and mechanical responses to hypoxia of cultured rat heart cells, but should also be suitable for a wide range of applications with several cell types.

Membrane Docosahexaenoic Acid vs. Eicosapentaenoic Acid and the Beating Function of the Cardiomyocyte and Its Regulation Through the Adrenergic Receptors

The β-adrenergic system in cardiac muscle cells is influenced by the n-3 polyunsaturated fatty acid (PUFA) content in cell membranes. This study deals with the specific effect of docosahexaenoic acid (DHA) as compared to other n-3 PUFA, without modification of the arachidonic acid content. Increasing the DHA content in the phospholipids of isolated cardiomyoxytes did not provoke electrical or contraction modifications, except for a slightly lower plateau phase (−2 mV). Conversely, the β-adrenergic function was affected at several levels: (i) the receptor affinity for dihydroalprenolol tended to decrease (Kd) without alteration of the number of β-binding sites (βmax); (ii) the isoproterenol-induced (10−7 M and 10−6M) cAMP production was significantly decreased (−20%); and (iii) the positive chronotropic response to β-adrenergic stimulation (isoproterenol, 10−7 M) was significantly enhanced (+80%). In order to further investigate the relationship between the decreased cAMP and the increased chronotropic response, the cells were treated with dibutyryl-cAMP, a perment analogue of cAMP, which elicited a significantly higher chronotropic response in DHA-enriched cells than in EPA-enriched cells (+50%). The increase in DHA content in cardiac cell membranes phospholipids may thus affect the β-adrenergic system through an increase of cAMP efficiency. Although the membrane phosphatidylinositols were largely involved in the PUFA alterations, none of the parameters related to α-adrenergic system (chronotropic response, receptor density, affinity for prasozin, and inositide phosphate production) were influenced by the membrane DHA content.