Sukhodub Andrey, Jovanović Sofija, Du Qingyou, Budas Grant, Clelland Allyson K, Shen Mei, Sakamoto Kei, Tian Rong, Jovanović Aleksandar
Maternal and Child Health Sciences, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK.
J Cell Physiol. 2007 Jan;210(1):224-36. doi: 10.1002/jcp.20862.
Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP-activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the alpha2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC-1, a mitochondrial membrane potential-sensitive dye, and laser confocal microscopy. In contrast, experiments with di-8-ANEPPS, a sarcolemmal-potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning-induced shortening of the action membrane potential. The preconditioning-induced activation of sarcolemmal K(ATP) channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal K(ATP) channels opening, inhibited preconditioning-induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning-induced recruitment of sarcolemmal K(ATP) channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal K(ATP) channels without being a part of signaling pathway that regulates mitochondrial membrane potential.
在持续性缺血之前出现的短暂缺血和再灌注会导致心肌梗死面积减小。这种现象被称为缺血预处理，由复杂且尚未完全明确的信号通路介导。AMP激活的蛋白激酶（AMPK）在与ATP耗竭及AMP/ATP比值增加相关的条件下在细胞中被激活。在本研究中，我们利用了一种过表达AMPKα2亚基显性负性形式的心脏表型来分析AMPK在心脏预处理中所起的作用（如果有作用的话）。我们发现心肌预处理可在野生型小鼠而非转基因小鼠中激活AMPK。与野生型细胞不同，转基因小鼠的心脏细胞无法进行预处理。如线粒体膜电位敏感染料JC-1及激光共聚焦显微镜所示，AMPK的细胞保护作用与预处理对线粒体膜电位的影响无关。相比之下，使用肌膜电位敏感染料di-8-ANEPPS进行的实验表明，完整的AMPK活性是预处理诱导动作膜电位缩短所必需的。在野生型小鼠而非转基因小鼠中观察到了预处理诱导的肌膜KATP通道激活。肌膜KATP通道开放的选择性抑制剂HMR 1098抑制了预处理诱导的动作膜电位缩短以及AMPK提供的心脏保护作用。免疫沉淀后进行的蛋白质印迹分析表明，AMPK对于预处理诱导的肌膜KATP通道募集至关重要。基于所获得的结果，我们得出结论，AMPK通过调节肌膜KATP通道的活性和募集来介导心脏细胞的预处理，而不是作为调节线粒体膜电位的信号通路的一部分。
