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

描述（由申请人提供）：药理学预处理（Pharmacologic preconditioning， PPC）是一种限制缺血再灌注（IR）损伤的方法，不需要事先短暂缺血，也不需要在缺血发生后使用药物。KATP通道激动剂本身通过记忆途径诱导PPC，从而通过更好的组织灌注、改善的代谢和机械功能、减少心律失常和减少梗死面积来评估心脏保护作用。我们对PPC的特征和机制的理解的限制阻碍了对这一有效现象的最佳利用，以防止心脏IR损伤。尤其缺乏的是对线粒体在PPC中的作用的理解。大(B)电导Ca2+敏感K+通道（BKCa）也存在于心肌细胞线粒体内膜（IMM）中，并似乎介导心脏保护。我们也有证据表明，IMM中的小电导(S) KCa通道也有保护作用。我们提出，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，从而导致触发下游保护作用所需的活性氧（ROS）增加。初步结果表明，mKCa通道打开的保护作用被超氧自由基的畸变所阻断。关于PPC启动机制的统一假设可能是基质K+内流诱导的ROS生成。我们将a)在豚鼠离体心脏线粒体中检测基质K+内流引发ROS生成的生物能量机制，b)确定在豚鼠离体心脏中触发PPC的特定ROS。此外，我们将c)通过Western blots， 2D凝胶电泳和MALDI- TOF和LIT SNCE质谱鉴定这些通道，并在人工脂质双层中表征这些通道。我们将使用最好的技术、测量方法（线粒体呼吸、细胞质和mCa2+、NADH、mFAD、mpH、IMM电位和几种ROS）和可用的药物来搜索引发PPC的因素、事件序列和特定ROS。公共卫生相关性：这些研究将有助于更好地理解Ca2+、K+和ROS对线粒体生物能量功能的调节，以及线粒体作为药理学操作靶点的选择。这项研究应该导致线粒体靶向药物的实际应用，以预防冠状动脉疾病患者使用新的方法。

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.