Mitochondrial Ca-sensitive K channel-induced superoxide and cardiac protection

描述(由申请人提供)：药物预适应(PPC)是一种限制缺血再灌注(IR)损伤的方法，它不需要事先短暂的缺血或一旦发生缺血就需要药物的存在。KATP通道激动剂本身通过记忆通路诱导PPC，通过更好的组织灌流、改善代谢和机械功能、减少心律失常和缩小梗塞范围来评估对心脏的保护。我们对PPC的特征和机制的了解有限，阻碍了对这一有效现象的最佳利用，以保护心脏免受IR损伤。尤其缺乏对线粒体在PPC中的作用的了解。大(B)电导钙敏感性钾通道(BKCa)也存在于心肌细胞内线粒体膜(IMM)中，可能参与心肌保护作用。我们有证据表明，IMM中的小电导(S)KCA通道也有保护作用。我们认为，药物诱导的K+进入线粒体(M)基质改变了生物能量学，增加了电子泄漏，诱导了触发下游保护作用所需的活性氧物种(ROS)的增加。初步结果表明，mKCa通道开放的保护作用可被超氧阴离子自由基的歧化所阻断。对PPC启动机制的一个统一假设可能是基质K+内流诱导的ROS的产生。我们将a)在豚鼠分离的心肌线粒体中检测由基质K+内流引发的导致ROS产生的生物能量机制，以及b)确定在豚鼠分离的心脏中负责触发PPC的特定ROS。此外，我们还将通过蛋白质印迹、2D凝胶电泳法、MALDI-TOF和LIT SNCE质谱仪鉴定这些通道，并在人工脂质双层中对这些通道进行表征。我们将使用最好的技术、方法(线粒体呼吸、胞浆和mCa2+、NADH、MFAD、MPH、IMM电位和几种ROS)和可用的药物来寻找启动PPC的因素、事件顺序和特定的ROS。公共卫生相关性：这些研究将使人们更好地了解钙、钾和ROS对线粒体生物能量功能的调节，并选择线粒体作为药物操作的目标。这项研究应该会导致线粒体靶向药物的实际应用，以使用新的方法预防性治疗冠心病患者。

DESCRIPTION (provided by applicant): Pharmacologic preconditioning (PPC) is an approach to limit ischemia reperfusion (IR) injury that does not require prior brief ischemia or the presence of the drug once ischemia occurs. KATP channel agonists, per se, induce PPC via memory pathways that lead to cardioprotection assessed by better tissue perfusion, improved metabolic and mechanical function, fewer dysrhythmias and reduced infarct size. Limits to our understanding of features and mechanisms of PPC have impeded optimal utilization of this potent phenomenon to protect against cardiac IR injury. Particularly lacking is an understanding of the role of the mitochondrion in PPC. Big (B) conductance Ca2+ -sensitive K+ channels (BKCa) are also present in cardiac cell inner mitochondrial membrane (IMM) and appear to mediate cardioprotection. We have evidence of protection also by small conductance (S) KCa channels in the IMM. We propose that drug -induced K+ entry into the mitochondrial (m) matrix alters bioenergetics in a way that increases electron leak to induce an increase in reactive oxygen species (ROS) required to trigger downstream protective effects. Preliminary results indicate that protective effects of mKCa channel opening are blocked by dismutation of the superoxide radical. A unifying hypothesis for the initiating mechanism of PPC may be a matrix K+ influx -induced ROS generation. We will a) examine in guinea pig isolated cardiac mitochondria the bioenergetic mechanisms initiated by matrix K+ influx that lead to ROS generation and b) determine the specific ROS responsible for triggering PPC in guinea pig isolated hearts. In addition we will c) identify these channels by Western blots, 2D gel electrophoresis and MALDI- TOF and LIT SNCE mass spectrometry and characterize these channels in artificial lipid bilayers. We will use the best techniques, measures (mitochondrial respiration, cytosolic and mCa2+, NADH, mFAD, mpH, IMM potential, and several ROS) and drugs available to search for the factors, the sequence of events, and the specific ROS that initiate PPC. PUBLIC HEALTH RELEVANCE: These studies will result in a better understanding of the regulation of mitochondrial bioenergetic function by Ca2+, K+, and ROS, and selection of the mitochondrion as a target for pharmacologic manipulation. This research should lead to the practical application of mitochondrial-targeted drugs to prophylactically treat patients with coronary artery disease using novel approaches.