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Activation of PKC‐ε occurs upstream of mitoKATP channel activation in the mechanism of diazoxide‐induced cardioprotection

In this study, we examined which isoforms of protein kinase C (PKC) family are modulated by diazoxide (DZX) and evaluated their roles in hypoxia‐induced apoptotic cell death mediated via mitochondrial death pathway. We further investigated the interrelationship between PKC isoforms and the mitochondrial ATP‐sensitive potassium (mitoKATP) channel in the mechanism of cardioprotection by DZX in rat heart‐derived H9c2 cell lines and neonatal rat cardiomyocytes. Treatment with 100 μM DZX induced isoform‐specific translocation of PKC‐ε from the cytosolic to the mitochondrial fraction in both H9c2 and neonatal cardiomyocytes. A specific blockade of PKC‐ε translocation by εV1‐2 peptide abrogated the anti‐apoptotic effect of DZX, suggesting an essential role of PKC‐ε in cardioprotection. DZX‐induced flavoprotein oxidation was completely inhibited by 10 μM εV1‐2, and DZX‐induced translocation of PKC‐ε remained unaltered after treatment with 100 μM 5‐hydroxydecanoate (5‐HD). A direct PKC activation by phorbol ester mimicked flavoprotein‐oxidizing effect of DZX, and this effect was completely blocked by εV1‐2 or 5‐HD. During hypoxia, the treatment with 100 μM DZX prevented the increase in mitochondrial Ca2+, mitochondrial depolarization and cytochrome c release induced by hypoxia, and all these effects of DZX were almost completely blocked by εV1‐2 as well as 5‐HD. In summary, these results suggest that DZX induces translocation of PKC‐ε as an upstream signaling molecule for the mitoKATP channel, leading to cardioprotection through inhibition of mitochondrial death pathway.