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Abstract Defining the substrate that influences the most favourably the myocardial post-ischemic recovery is subject of debates, due to dissociation between functional and biochemical benefits. Hence, we studied the effects of either glucose or different fatty acids on the functional and metabolic recovery of post-ischemic cardiomyocytes in a substrate-free hypoxia model of simulated ischemia-reperfusion. Rat cardiomyocytes were submitted to a 2.5 h simulated ischemia followed by a 2 h reoxygenation without substrate (control), or with either glucose, octanoic acid, oleic acid, or elaidic acid. During simulated ischemia, electromechanical function gradually disappeared while the cellular viability and mitochondrial function declined. During control simulated reperfusion, cardiomyocytes recovered near normal function but a significant reduction in the action potential amplitude and rate persisted. The addition of glucose or oleic acid during simulated reperfusion promoted a faster, better and sustain functional recovery. Amongst the fatty acids, the functional recovery was slower with elaidic and octanoic acids as compared with oleic acid. The mitochondrial function was better improved during simulated reperfusion with glucose than with the tested fatty acids, among which elaidic acid was the less unfavourable. Paradoxically, the addition of whichever substrate during simulated reperfusion tended to worsen the cellular viability. Thus, cardiomyocytes recovery strongly relies on the characteristics of the substrate supplied at the onset of simulated reperfusion: glucidic or lipidic nature, chain-length, insaturation degree. Moreover, these data suggest that defining the appropriateness of a given substrate for the post-ischemic cardiomyocyte recovery is closely related to the functional and the biological endpoints in consideration.

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 vitrosubstrate-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.

The influence of deep hypothermia (4°C) during a substrate-free, hypoxia–reoxygenation treatment was investigated on cardiomyocytes (CM) prepared from newborn rat heart in culture in an in vitro, substrate-free model of ischemia–reperfusion. The transmembranous potentials were recorded with standard microelectrodes. The contractions were monitored photometrically. The RNA messenger (mRNA) and protein expression for protein (HSP70) were analysed by RT-PCR (reverse transcriptase-polymerase chain reaction) and Western blotting, respectively. Simultated ischemia (SI) caused a gradual decrease and then a cessation of the spontaneous electromechanical activity. During the reoxygenation, the CM recovered normal function, provided that SI did not exceed 2.5 h. When SI duration was increased up to 4 h, reoxygenation failed to restore the spontaneous electromechanical activity. Conversely, the exposure of the CM to SI together with deep hypothermia decreased the functional alterations observed, and provided a complete electromechanical recovery after 2.5 h as well as after 4 h of SI. Deep hypothermia alone failed to induce HSP70 mRNA and protein production. On the contrary, HSP70 mRNA production increased after 2.5 and 4 h of deep hypothermia followed by 1 h of rewarming, proportionally to the duration of the cooling period. This augmentation in mRNA was associated with a rise in HSP70 protein content. In summary, it appeared that deep hypothermia exerts a strong cytoprotectrive action during SI only, whereas cooling CM before SI has no beneficial effect on subsequent SI. Moreover, these results suggested the persistence of a signaling system and/or transduction in deeply cooled, functionally depressed cells. Finally, CM in culture appeared to be a model of interest for studying heart graft protection against ischemia-reperfusion and contributed to clarifying the molecular and cellular mechanisms of deep hypothermia on myocardium.

The objective was to relate the response of the HSP70 and P53 genes to the cessation and the recovery of cardiac muscle cell functions when submitted to ischemia-reperfusion. We have measured the electromechanical activity, the released enzymes and HSP70 RNA and protein levels in cultured neonatal rat cardiomyocytes (CM) in a substrate-free, hypoxia-reoxygenation model of ischemia-reperfusion. In parallel the expression of the two genes P53 (the key apoptosis regulator gene) and P21/Waf1 (the P53 target gene) has been evaluated. The functional recovery during post-'ischemic' reoxygenation was associated with an overexpression of HSP70 and P53 lasting until the functional parameters reverted back to the normal, prehypoxic values. In contrast, extending the substrate-free hypoxic treatment worsens the dysfunction of the cardiac muscle cell and, in these conditions, reoxygenation failed to restore cell functions and to activate HSP70. Finally, in the conditions of reversible 'ischemic' cell injury, an early and transitory activation of P53 was associated with the functional recovering process of the CM submitted to simulated ischemia. These observations are suggestive of a contributive role of both HSP70 and P53 to a cytoprotective program activated by reoxygenation in post-'ischemic' CM.

The polyunsaturated fatty acids (PUFAs) of theω3 series are known to modulate adrenergic functions in ventricular myocytes. This study evaluated the influence of hypoxia duration and PUFA composition on the ability of cultured rat cardiomyocytes in producingα- andβ-adrenergic messengers (IPs and cAMP). After hypoxia (1.5, 2.5 or 3.5 h) followed by reoxygenation (1h), IP and cAMP production was induced by phenylephrine or isoproterenol stimulation, respectively. Hypoxia did not affect the basal level of messenger production in unstimulated cells, but decreased the cAMP production elicited by isoproterenol stimulation (up to 50%). The decrease in IP production after phenylephrine stimulation was observed only after long-term hypoxia duration close to irreversible cellular damages. The use of modified culture media supplemented with either arachidonic acid (AA) or docosahexaenoic acid (DHA) induced cardiomyocytes displaying either an arachidonic acid membrane profile (35% AA and 2% DHA in the phospholipids) or a docosahexaenoic acid membrane profile (15% AA and 20% DHA). These modifications did not alter the basal level of either messenger production in unstimulated cells nor the IP released afterα-adrenergic stimulation. Conversely, the decrease in cAMP production was significantly more pronounced in docosahexaenoic acid-enriched cells than in arachidonic acid-enriched cells. This study suggests that hypoxia alters theβ-adrenergic messenger production, and that theα-system may balance the depression of theβ-system. The depression of theβ-adrenergic function induced by the incorporation of docosahexaenoic acid in membrane phospholipids may contribute to the beneficial effect of this fatty acid in the reperfused heart.

The specific effect of docosahexaenoic (DHA; C22:6 n-3), as compared to eicosapentaenoic acid (EPA; C20:5 n-3), on adrenoceptor function was investigated in cultured rat myocardial cells. The cardiomyocytes were grown for 24 h in a conventional seric medium, and then incubated for 96 h in a medium enriched with either DHA or EPA. After this treatment, the phospholipids of the DHA-treated cells contained approximately 20% of the total fatty acids as C22:6 n-3, and those of EPA-treated cells displayed a high content in C20:5 n-3 and its elongation product C22:5 n-3 (30% of total fatty acids). Additionally, the n-3/n-6 polyunsaturated fatty acid ratio was the same in the two groups of cells. These modifications were roughly similar in all the phospholipid classes. The contractions were monitored photometrically and no significant difference in basal frequency and contraction parameters could be detected. The stimulation of theβ-adrenergic receptors (isoproterenol 10−7m) resulted in a positive chronotropic effect, which was significantly higher in the DHA-rich cells. Conversely, the higher DHA content in the phospholipids appeared to induce a decrease in the affinity of theβ-receptors for the ligand (dihydroalprenolol) without alteration of the number ofβ-receptor binding sites and provoked a significant decrease in isoproterenol-stimulated cAMP production (−19%). To investigate further these controversial data, the cardiomyocytes were treated with dibutyryl-cAMP, which elicited a positive chronotropic response significantly higher in the DHA-rich cells. Theα-adrenergic stimulation by phenylephrine (3×10−6m) increased the spontaneous rate, but in a similar manner in the DHA- and EPA-enriched cells. Similarly, neither theα-adrenergic receptor binding characteristics nor the production of phosphoinositides was modulated by the membrane DHA content, although the phosphatidylinositol PUFAs were significantly altered. In conclusion, increasing the DHA content in membrane phospholipids did not affect theα-adrenergic system, but exerted a specific positive influence on theβ-adrenergic transduction mechanism, essentially through an increase of cAMP efficiency.

This study was undertaken to evaluate the direct cardioprotective effect of trimetazidine (TMZ), an anti-anginal drug devoid of haemodynamic action, on isolated myocytes. Cultured rat ventricular myocytes were treated with the drug 16 h and 1 h before the experiments. The drug-treated cells and control cells were placed in a substrate free medium and submitted in a specially designed device to either normoxia (N4), or hypoxia (150 min. H2.5, or 240 min, H4), or 150 min hypoxia followed by 90 min reoxygenation (HR). The treatment of the cells with TMZ (5 × 10-4M) resulted in a significant decrease of lactate dehydrogenase (LDH) leakage (-58% in H2.5, -36% in H4 and -37% in HR). The LDH release provoked by oxidizing agents, H2O2and 13-s-HpOTrE (13(S)-hydroperoxyoctadecatrienoic acid) during post-hypoxic reoxygenation was also lowered by TMZ. However, this effect reflected the beneficial action of TMZ during hypoxia since the drug was not efficient in altering the LDH leakage induced by the oxidizing agents in normal conditions. Moreover, the hypoxia-induced decreased of ATP content was not affected by TMZ, and resynthesis of ATP during substrate-free reoxygenation was similar in TMZ-treated and control cells. The respiration parameters have been studied in rat heart mitochondria isolated from control and TMZ-treated rats, in the presence or absence of TMZ in the respiration medium (10-4M). The main result was a rapid and potent inhibition of palmitoylcarnitine oxidation, when TMZ was added to the respiration medium. The chronic treatment only resulted in a slight alternation of pyruvate oxidation. In conclusion, a pre-treatment of ventricular myocytes with TMZ resulted in an increased cell resistance to hypoxic stress, as evidenced by LDH leakage. This cytoprotective effect to TMZ should not be mediated through an antioxidant activity, but could be related to a modification of lipid metabolism.

TaiCatoxin (TCX), a complex toxin isolated from Taipan snake venom, is believed to have a specific blocking activity on voltage-dependent cardiac calcium channels. The aim of this study was to investigate the effects of TCX on a broad range of heart muscle cell functions, i.e. electrophysiology, contractility, automaticity and the related biochemical modifications. Myocyte-enriched cultures were prepared from newborn rat heart ventricles. The transmembrane potentials were recorded with glass microelectrodes. The contractions were monitored photometrically. TCX decreased the action potential amplitudes, mainly by lowering the plateau. The action potential duration and the contraction parameters were decreased. Although TCX has a minor overall negative chronotropic effect, it evoked transient but severe arrhythmias and prolonged changes in the intercellular electrical coupling. Moreover, the action of TCX appeared to be dose-dependent. These effects are consistent with a specific blockade of the L-type, voltage-dependent calcium channels, but effects of other components of the toxin complex cannot be excluded. TCX also exhibits phospholipase A2 activity leading to the release of lysophospholipids and FFA (acyl CoA and acyl carnitine), which have detrimental effects on cellular integrity and function.

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)

Summary: Neonatal rat heart cells cultivated in either of two different media which varied only in their serum supplements were transferred to chemically defined medium (Ham's 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.

The mechanism of action of the antianginal trimetazidine (TMZ) remains largely unknown. In cultured rat ventricular myocytes in physiologic conditions, TMZ (5 × 10−4M) 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 hypoxiainduced 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 hypoxiainduced 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 (10−4M) in the respiration medium. Conversely, a potent inhibition of palmitoylcamitine 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. In conclusion, pretreatment of ventricular myocytes with TMZ resulted in an increased cell resistance to hypoxic stress, as was evident from electromechanical functions and lactate dehydrogenase leakage. This cytoprotective action of TMZ could be related to an effect of the drug in the fatty acid metabolism.

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.