Preconditioning blocks cardiocyte apoptosis: role of KATPchannels and PKC-ε

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Abstract
The aims of this study were to determine whether preconditioning blocks cardiocyte apoptosis and to determine the role of mitochondrial ATP-sensitive K+(KATP) channels and the protein kinase C ε-isoform (PKC-ε) in this effect. Ventricular myocytes from 10-day-old chick embryos were used. In the control series, 10 h of simulated ischemia followed by 12 h of reoxygenation resulted in 42 ± 3% apoptosis ( n = 8). These results were consistent with DNA laddering and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Preconditioning, elicited with three cycles of 1 min of ischemia separated by 5 min of reoxygenation before subjection to prolonged simulated ischemia, markedly attenuated the apoptotic process (28 ± 4%, n = 8). The selective mitochondrial KATPchannel opener diazoxide (400 μmol/l), given before ischemia, mimicked preconditioning effects to prevent apoptosis (22 ± 4%, n = 6). Pretreatment with 5-hydroxydecanoate (100 μmol/l), a selective mitochondrial KATPchannel blocker, abolished preconditioning (42 ± 2%, n = 6). In addition, the effects of preconditioning and diazoxide were blocked with the specific PKC inhibitors Gö-6976 (0.1 μmol/l) or chelerythrine (4 μmol/l), given at simulated ischemia and reoxygenation. Furthermore, preconditioning and diazoxide selectively activated PKC-ε in the particulate fraction before simulated ischemia without effect on the total fraction, cytosolic fraction, and PKC δ-isoform. The specific PKC activator phorbol 12-myristate 13-acetate (0.2 μmol/l), added during simulated ischemia and reoxygenation, mimicked preconditioning to block apoptosis. Opening mitochondrial KATPchannels blocks cardiocyte apoptosis via activating PKC-ε in cultured ventricular myocytes. Through this signal transduction, preconditioning blocks apoptosis and preserves cardiac function in ischemia-reperfusion.