Regulation of Mitochondrial Function in Cardiac Myocytes : Kinetics of the Opening of mPTP and it's Physiological Roles

线粒体通透性过渡孔（Mitochondrial permeability transition pore, mPTP）不仅作为缺血再灌注损伤的一种机制，而且在生理条件下对细胞功能的维持起着重要作用。本项目旨在通过共聚焦显微镜研究(1)mPTP开放的动力学及其受线粒体Ca^<2+>浓度（[Ca^<2+>]m）和线粒体膜电位（ΔΨ_m）的调控；(2)mPTP开放对[Ca^<2+>]m和细胞质Ca^<2+>浓度（[Ca^<2+>] c）的影响；(3)线粒体与肌浆网（SR）的相互作用。我们开发了一种方法来监测完整和皂素渗透心肌细胞的[Ca^<2+>]_m和mPTP的开放。通过这些方法，我们发现线粒体ATP敏感的钾通道打开剂Diazoxide打开mPTP并将Ca^<2+>从线粒体基质释放到细胞质中。[Ca^<2+>]_c的增加增加了Ca^<2+>瞬态和Ca^<2+>火花。对[Ca^<2+>]_m的测定表明，二氮氧化物还原了[Ca^<2+>]_m。最后，我们研究了[Ca^<2+>]_m与ΔΨ_m之间的关系，发现在ΔΨ_m消失的病理生理状态下，mPTP的打开和线粒体Na^+/Ca^<2+>交换对[Ca^<2+>]_m的调节是重要的。

Mitochondrial permeability transition pore (mPTP) has been considered to play important roles not only as a mechanism of ischemia reperfusion injury, but also for the maintenance of cellular function under physiological condition. In this project we aimed to study (1) the kinetics of the opening of mPTP and it's regulation by mitochondrial Ca^<2+> concentration ([Ca^<2+>]m) and mitochondrial membrane potential (ΔΨ_m), (2) the effects of the opening of mPTP on [Ca^<2+>]_m and cytosolic Ca^<2+> concentration ([Ca^<2+>]_c) and (3) the interaction between mitochondria and sarcoplasmic reticulum (SR) by using confocal microscopy.We have developed methods to monitor [Ca^<2+>]_m and the opening of mPTP in intact and saponin permeabilized cardiac myocytes. By using these methods, we have found that mitochondrial ATP sensitive potassium channel opener, Diazoxide opened mPTP and released Ca^<2+> from mitochondrial matrix to cytosol. This increase in [Ca^<2+>]_c then increased Ca^<2+> transients and Ca^<2+> sparks. Measurement of [Ca^<2+>]_m revealed that Diazoxide reduced [Ca^<2+>]_m. Finally we have investigated the relation between [Ca^<2+>]_m and ΔΨ_m and found that in the pathophysiological condition, where ΔΨ_m was dissipated, opening of mPTP and mitochondrial Na^+/Ca^<2+> exchange were important for the regulation of [Ca^<2+>]_m.