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

描述（由申请人提供）：药物预处理（PPC）是一种限制缺血再灌注（IR）损伤的方法，不需要预先短暂缺血或缺血发生后药物的存在。KATP通道激动剂本身通过记忆通路诱导PPC，其导致通过更好的组织灌注、改善的代谢和机械功能、更少的节律障碍和减小的梗死面积评估的心脏保护。我们对PPC特征和机制的理解有限，阻碍了对这种有效现象的最佳利用，以防止心脏IR损伤。特别缺乏的是对PPC中的P2P的作用的理解。大（B）电导Ca 2+敏感性K+通道（BKCa）也存在于心肌细胞线粒体内膜（IMM）中，并且似乎介导心脏保护。我们也有证据表明IMM中的小电导（S）KCa通道具有保护作用。我们提出药物诱导的K+进入线粒体（m）基质以增加电子泄漏的方式改变生物能量学，从而诱导触发下游保护作用所需的活性氧（ROS）增加。初步结果表明，mKCa通道开放的保护作用被超氧自由基的歧化阻断。PPC的启动机制的统一假设可能是基质K+内流诱导的ROS产生。我们将a）在豚鼠离体心脏线粒体中检查由基质K+内流引发的导致ROS产生的生物能量机制，和B）确定负责触发豚鼠离体心脏中PPC的特定ROS。此外，我们将c）通过蛋白质印迹、2D凝胶电泳和MALDI-TOF和LIT SNCE质谱鉴定这些通道，并表征人工脂质双层中的这些通道。我们将使用最好的技术，措施（线粒体呼吸，胞质和mCa 2+，NADH，mFAD，mPH，IMM潜力，和几个ROS）和药物，可用于搜索的因素，事件的顺序，和特定的ROS启动PPC。公共卫生相关性：这些研究将导致更好地了解线粒体生物能量功能的调节Ca 2+，K+，和ROS，并选择作为药理学操作的靶点。这项研究将导致脑靶向药物的实际应用，使用新的方法对冠状动脉疾病患者进行非特异性治疗。

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.