Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca²⁺ overload during simulated ischemia and reperfusion: Possible mechanism of cardioprotection

Mitochondrial ATP-sensitive potassium (mitoK_ATP) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca²⁺ concentration ([Ca²⁺]m), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoK_ATP channel opener diazoxide attenuated the accumulation of [Ca²⁺]_m during simulated I/R (EC₅₀=18 μmol/L). These effects of diazoxide were blocked by the mitoK_ATP channel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca²⁺]m accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, ΔΨ_m, in permeabilized myocytes revealed that diazoxide depolarized ΔΨ_m (by 12% at 10 μmol/L, P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca²⁺ overload, as a consequence of partial mitochondrial membrane depolarization by mitoK_ATP channels, underlies cardioprotection. Furthermore, mitoK_ATP channels and the MPT differentially affect mitochondrial calcium homeostasis: mitoK_ATP channels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion.