Murata M, Akao M, O'Rourke B, Marbán E
Institute of Molecular Cardiobiology, The Johns Hopkins University, Baltimore, MD, USA.
Circ Res. 2001 Nov 9;89(10):891-8. doi: 10.1161/hh2201.100205.
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 Ca(2+) concentration (Ca(2+)), 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 Ca(2+) during simulated I/R (EC(50)=18 micromol/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 Ca(2+) accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, DeltaPsi(m), in permeabilized myocytes revealed that diazoxide depolarized DeltaPsi(m) (by 12% at 10 micromol/L, P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca(2+) 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.
线粒体ATP敏感性钾通道（mitoK(ATP)）在心脏缺血预处理中起关键作用。然而，心脏保护机制仍存在争议。我们使用延时共聚焦显微镜测量成年兔心室肌细胞中罗丹明2荧光，作为线粒体基质Ca(2+)浓度（Ca(2+)）的指标。为模拟缺血和再灌注（I/R），将细胞暴露于代谢抑制（50分钟），随后用对照溶液冲洗。在模拟缺血期间，罗丹明2荧光逐渐增加，再灌注时进一步升高。mitoK(ATP)通道开放剂二氮嗪减弱了模拟I/R期间Ca(2+)的积累（半数有效浓度[EC(50)] = 18 μmol/L）。二氮嗪的这些作用被mitoK(ATP)通道拮抗剂5-羟基癸酸（5HD）阻断。相反，线粒体通透性转换（MPT）抑制剂环孢素A和硼酸霉素在缺血期间不改变Ca(2+)的积累，但显著抑制再灌注期间罗丹明2荧光的激增。对透化心肌细胞中线粒体膜电位DeltaPsi(m)的测量表明，二氮嗪以5HD可抑制的方式使DeltaPsi(m)去极化（在10 μmol/L时降低12%，P<0.01）。我们的数据支持这样的假说，即mitoK(ATP)通道引起的部分线粒体膜去极化导致线粒体Ca(2+)超载的减轻，是心脏保护的基础。此外，mitoK(ATP)通道和MPT对线粒体钙稳态有不同影响：mitoK(ATP)通道在I/R期间抑制钙积累，而MPT仅在再灌注时起作用。
