Research in the mechanism of ischemic cell death of cardiomyocyte and the role of mitochondria

在大鼠脑缺血再灌注模型上，观察缺血预适应、线粒体K-ATP通道开放剂、尼可地尔和二氮嗪对心肌梗死面积的影响。所有干预措施均可显著缩小心肌梗死面积，提示线粒体K-ATP通道开放对减轻脑缺血损伤具有重要意义。心肌梗死后3周以左室压-容量关系评价左室重构。线粒体K-ATP通道开放使左室重构减弱，重构程度与心肌梗死面积相关。尼可地尔和二氮卓开放线粒体K-ATP通道的机制不同，这是因为尼可地尔的作用被PKC抑制剂部分抑制，而二氮卓的作用不受影响。用Western blotting检测PKC亚型从胞浆到线粒体的移位。这说明蛋白激酶Cε和δ被尼可地尔转位到线粒体部分，这一作用可被蛋白激酶C抑制剂抑制。此外，NO猝灭剂减少了这些异构体向线粒体部分的移位，表明尼可地尔的NO供体效应激活了PKCε和δ，从而与尼可地尔的直接开放效应协同激活线粒体K-ATP通道。用免疫沉淀法探索了线粒体中与PKCε和δ结合的适配蛋白，并检测到新的适配蛋白，这些适配蛋白可能在线粒体K-ATP通道的激活中起重要作用。

In the rat ischemia-reperfusion model, the effect of ischemic preconditioning, mitochondrial K-ATP channel openers, nicorandil and diazoxide, on the reduction of infarct size was tested. All these interventions reduced the infarct size significantly, indicating the importance of the opening of mitochondrial K-ATP cahnnels for the reduction of ischemic injury. The left ventricular remodeling was assessed by the LV pressure-volume relation at 3 weeks after infarction. The LV remodeling was attenuated by the opening of mitochondrial K-ATP channels and the extent of remodeling and infarct size was correlated. The mechanism of opening the mitochondrial K-ATP channels is distinct between nicorandil and diazoxide, since the effect of nicorandil was partially inhibited by PKC inhibitor, although the effect of diazoxide was not influenced. The PKC isoform translocation from cytosolic to mitochondrial fraction was assessed by Western blotting. This revealed that the PKC ε and δ was translocated to mitochondrial fraction by nicorandil, which is inhibited by PKC inhibitor. Furthermore, NO quencher reduced the translocation of these isoforms to the mitochondrial fraction indicates that the NO donor effect of nicorandil activates the PKC ε and δ, which subsequently activates the mitochondrial K-ATP cahnnels synergistically with the direct opening effect of nicorandil. The adaptor protein in mitochondria which bind to PKC ε and δ were explored by immunoprecipitation and new adaptor proteins are detected, which may play an important role for the activation of mitochondrial K-ATP channels.