Role of protein kinase C in mitochondrial K(ATP) channel-mediated protection against CA²⁺ overload injury in rat myocardium

Growing evidence exists that ATP-sensitive mitochondrial potassium channels (MitoK(ATP) channel) are a major contributor to the cardiac protection against ischemia. Given the importance of mitochondria in the cardiac cell, we tested whether the potent and specific opener of the MitoK(ATP) channel diazoxide attenuates the lethal injury associated with Ca²⁺ overload. The specific aims of this study were to test whether protection by diazoxide is mediated by MitoK(ATP) channels; whether diazoxide mimics the effects of Ca²⁺ preconditioning; and whether diazoxide reduces Ca²⁺ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca²⁺ PD (10 minutes of Ca²⁺ depletion followed by 10 minutes of Ca²⁺ repletion). The effects of the MitoK(ATP) channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca²⁺ PD were assessed. In hearts treated with 80 μmol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca²⁺ PD; peak lactate dehydrogenase release was also significantly decreased, although ATP content was less depleted. The cellular structures were well preserved, including mitochondria and intercalated disks in diazoxide-treated hearts compared with nontreated Ca²⁺ PD hearts. The salutary effects of diazoxide on the Ca²⁺ PD injury were similar to those in hearts that underwent Ca²⁺ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca²⁺ PD. The addition of sodium 5-hydroxydecanoate, a specific MitOK(ATP) channel inhibitor, or chelerythrine chloride, a PKC inhibitor, during diazoxide pretreatment completely abolished the beneficial effects of diazoxide on the Ca²⁺ PD. Blockade of Ca²⁺ entry during diazoxide treatment by inhibiting L-type Ca²⁺ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca²⁺ PD. PKC-δ was translocated to the mitochondria, intercalated disks, and nuclei of myocytes in diazoxide-pretreated hearts, and PKC-α and PKC-ε were translocated to sarcolemma and intercalated disks, respectively. This study suggests that the effect of the MitoK(ATP) channel is mediated by PKC- mediated signaling pathway.