The main factors of myocardial ischemia are hypoxia, substrate deprivation, acidosis, and high extracellular potassium concentration ([K+]e), but the influence of each of these factors has not yet been evaluated in a cardiomyocyte (CM) culture system. Electromechanical responses to the individual and combined components of ischemia were studied in CM cultured from newborn rat ventricles. Action potentials (APs) were recorded using glass microelectrodes and contractions were monitored photometrically. Glucose-free hypoxia initially reduced AP duration, amplitude, and rate and altered excitation-contraction coupling, but AP upstroke velocity (Vmax) remained unaffected. Early afterdepolarizations appeared, leading to bursts of high-rate triggered impulses before the complete arrest of electromechanical activity after 120 min. Acidosis reduced Vmax whereas AP amplitude and rate were moderately decreased. Combining acidosis and substrate-free hypoxia also decreased Vmax but attenuated the effects of substrate-free hypoxia on APs and delayed the cessation of the electrical activity (180 min). Raising [K+]e reduced the maximal diastolic potential and Vmax. Total ischemia (substrate deletion, hypoxia, acidosis, and high [K+]e) decreased AP amplitude and Vmax without changing AP duration. Moreover, delayed afterdepolarizations appeared, initiating triggered activity. Ultimately, 120 min of total ischemia blocked APs and contractions. To conclude, glucose-free hypoxia caused severe functional defects, acidosis delayed the changes induced by substrate-free hypoxia, and total ischemia induced specific dysfunctions differing from those caused by the former conditions. Heart-cell cultures thus represent a valuable tool to scrutinize the individual and combined components of ischemia on CMs.

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.

Mycophenolate mofetil (MMF) is a new immunosuppressive drug used to reduce acute rejection after heart transplantation. As with other immunosuppressive drugs, MMF therapy is associated with several adverse effects. However, the direct effects of MMF on myocardial tissue has not been yet evaluated. The aim of the work was thus to evaluate the effects of MMF on isolated cardiomyocytes (CM) in normal conditions and in an in vitro model of simulated ischemia (SI; substrate-free hypoxia) and reperfusion (R; reoxygenation). Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded using conventional microelectrodes and the cell contractions were monitored with a photoelectric device. In basal conditions, MMF (10(-6) and 10(-5) M) exerted no significant effects on the survival and on the electrical and contractile activities of CM in culture, even during long-term exposure (up to 48 h). SI per se led to a gradual decrease and then an abortion of the spontaneous automaticity and electromechanical activity of CM. Pretreating CM with either 10(-6) or 10(-5) M MMF was able to reduce the SI-induced cell dysfunctions. The presence of MMF at these concentrations did not hamper the post-SI functional recovery of CM during reoxygenation. At 10(-5) M, MMF applied during reoxygenation only permitted a better recovery of CM. However, the mitochondrial function after reoxygenation, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl-tetrazolium bromide (MTT) test, was not significantly influenced by the addition of MMF before as well as after ischemia. Conversely, MMF was able to reduce in this model the postischemic rise in xanthine and hypoxanthine. These data from CM-enriched model show that MMF: (i) had no cytotoxic effect, (ii) displayed a cytoprotective effect during SI, and (iii) exerted its beneficial effect at least partly through the decrease in the xanthine oxidase-dependent free radical production.

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