Role of Protein Kinase C in Mitochondrial K ATP Channel–Mediated Protection Against Ca 2+ Overload Injury in Rat Myocardium

Abstract

Abstract —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 Ca 2+ 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 Ca 2+ preconditioning; and whether diazoxide reduces Ca 2+ paradox (PD) injury via protein kinase C (PKC) signaling pathways. Langendorff-perfused rat hearts were subjected to the Ca 2+ PD (10 minutes of Ca 2+ depletion followed by 10 minutes of Ca 2+ repletion). The effects of the MitoK ATP channel and other interventions on functional, biochemical, and pathological changes in hearts subjected to Ca 2+ PD were assessed. In hearts treated with 80 μmol/L diazoxide, left ventricular end-diastolic pressure and coronary flow were significantly preserved after Ca 2+ 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 Ca 2+ PD hearts. The salutary effects of diazoxide on the Ca 2+ PD injury were similar to those in hearts that underwent Ca 2+ preconditioning or pretreatment with phorbol 12-myristate 13-acetate before Ca 2+ 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 Ca 2+ PD. Blockade of Ca 2+ entry during diazoxide treatment by inhibiting L-type Ca 2+ channel with verapamil or nifedipine also completely reversed the beneficial effects of diazoxide on the Ca 2+ 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.