Mitochondrial K_ATP channel activation reduces anoxic injury by restoring mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨ_m) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive K⁺ (mitoK_ATP) channel activation prevents the collapse of ΔΨ_m in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mite Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed ΔΨ_m, as monitored by 5,5′,6,6′-tetrachloro-1,1′,3,3′- tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome c immunostaining. Activation of mitoK_ATP channel with diazoxide (100 μmol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized ΔΨ_m. This protection was blocked by 5-hydroxydecanoate (500 μmol/l), a mitoK_ATP channel-selective inhibitor, but not by HMR-1098 (30 μmol/l), a putative sarcolemmal K_ATP channel-selective inhibiter. Dissipation of ΔΨ_m also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts ΔΨ_m and induces apoptosis, which are prevented by the activation of the mitoK_ATP channel. This further emphasizes the therapeutic significance of mitoK_ATP channel agonists in the prevention of ischemia-reperfusion cell injury.