Activation of mitochondrial K(ATP) (mitoK(ATP)) channel induces acute ischemic preconditioning (PC) against ischemic injury. The ability of this channel to elicit late PC remains unknown. The present study tests the hypothesis that stimulation of mitoK(ATP) channel induces late PC via the protein kinase C (PKC) signaling pathway. Rats were subjected to 30 minutes of regional ischemia and 120 minutes of reperfusion (I/R). In other groups, rats were pretreated with diazoxide, a specific opener of the mitoK(ATP) channel (7 mg/kg, IV), 12, 24, 48, and 72 hours before they were subjected to I/R. A maximum reduction in infarct size was observed after 24 hours (33.3+/-2.2% versus I/R group, 62.1 +/-2.4%). Pretreatment with diazoxide did not reduce the infarct size significantly after 12, 48, and 72 hours (50.2+/-4.3%, 50.5+/-4.6%, and 58.2+/-4.9%) compared with the I/R group. The protection was blocked with 5-hydroxydecanoic acid (5-HD, 5 mg/kg IV), a relatively selective mitoK(ATP) channel blocker (56.5+/-2.7%), and chelerythrine (5 mg/kg IV), an effective PKC inhibitor (57.1+/-3.4%) administered either on the first day before diazoxide pretreatment or 10 minutes before I/R on the second day. Cell necrosis was decreased by approximately 50% in the diazoxide preconditioned hearts compared with control I/R hearts. Cell death by apoptosis was also significantly decreased in diazoxide pretreated hearts (3.2%) as compared with I/R (11.3%). In conclusion, activation of mitoK(ATP) channel with diazoxide produces late PC against reperfusion injury. The effect of mitoK(ATP) channel appears to be dependent on the PKC-mediated signal pathway.

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Background-The mitochondrial K ATP (mitoK ATP ) channel has been shown to confer short-and long-term cardioprotection against prolonged ischemia via protein kinase C (PKC) signaling pathways. However, the exact association between PKC or its isoforms and mitoK ATP channels has not yet been clarified. The present study tested the hypothesis that the activity and translocation of PKC to the mitochondria are important for cardiac protection elicited by mitoK ATP channels. Methods and Results-PKC was downregulated by prolonged (24-hour) treatment with phorbol 12-myristate 13-acetate (4 g/kg body weight) before subsequent experiments in rats. Langendorff-perfused rat hearts were subjected to 40 minutes of ischemia followed by 30 minutes of reperfusion. Effects of PKC downregulation on the activation of mitoK ATP channels and other interventions on hemodynamic, biochemical, and pathological changes were assessed. Subcellular localization of PKC isoforms by Western blot analysis and immunocytochemistry demonstrated that PKC-␣ and PKC-␦ were translocated to the sarcolemma and that PKC-␦ was translocated to the mitochondria after diazoxide treatment. In hearts treated with diazoxide (80 mol/L), a significant improvement in cardiac function and an attenuation of cell injury were observed. In PKC-downregulated hearts, protection was abolished because mitoK ATP channels could not be activated by diazoxide. Conclusions-These data suggest that PKC activation is required for the opening of mitoK ATP channels during protection against ischemia and that this effect is linked to isoform-specific translocation of PKC-␦ to the mitochondria.