Myocardial Injury Associated with Mitochondria-derived Oxygen Free Radical(s)

描述(申请人提供)：线粒体氧化应激与缺血-再灌注(I/R)损伤有关。在缺血后心脏的心肌水平上，能量代谢缺陷与氧自由基的显著过量产生有关(S)。现有证据表明，氧自由基和呼吸链蛋白之间的特异性相互作用在I/R病理生理学中起关键作用，氧自由基诱导的氧化应激可作为氧化还原信号改变线粒体的硫醇氧化还原状态！蛋白质，随后触发细胞事件，如细胞凋亡、增殖和衰老。两种含有关键氧化还原硫醇的线粒体蛋白质，NADH脱氢酶(NDH)和琥珀酸泛醌还原酶(SQR)，也是线粒体产生超氧化物的催化成分。该项目的长期目标是阐明线粒体中氧自由基的产生如何导致心肌损伤的分子机制。利用分离的酶复合体和缺血后的心脏，通过追求以下特定目标，将检验超氧化物歧化和蛋白质S-谷胱甘肽氧化的关键假说。[1]确定NDH产生超氧化物的机理。[2]确定SQR产生超氧化物的机理。[3]阐明了氧自由基产生的主要途径导致蛋白质S-谷胱甘肽化的分子机制。[4]评价心肌缺血后蛋白质S-谷胱甘肽和非脱氢酶/SQR衍生的S-谷胱甘肽。酶介导的超氧化物生产将通过EPR自旋捕获和固定化酶EPR(IE-EPR)进行测量。酶介导的NO消耗速率将通过使用NO电极的电化学检测来测量。用免疫印迹法检测蛋白质衍生的S-谷胱甘肽和蛋白质自由基的形成。蛋白质自由基(S)、反应性硫醇(S)和谷胱甘肽结合(GS)结构域(S)将通过质谱学和肽图谱进行鉴定。每个识别区域的功能作用将通过使用针对构象表位多肽的特定抗体的免疫化学来确定。在缺血后的心脏系统中，将评估S-谷胱甘肽作为氧化应激引起的氧化还原改变的生物标志物的应用。这项研究的结果将提供有关氧自由基和蛋白质氧化还原硫醇(S)在线粒体功能障碍中的病理生理作用的基本信息，并有可能改进心脏疾病的临床诊断、治疗和预防。

DESCRIPTION (provided by applicant): Mitochondrial oxidative stress is associated with ischemia-reperfusion (I/R) injury. At the myocardial level of the post-ischemic heart, defective energy metabolism has been linked to marked overproduction of oxygen free radical(s). Available evidence indicates that [a] specific interactions between oxygen free radicals and respiratory chain proteins are critical in the I/R pathophysiology, and [b] oxygen free radical- induced oxidative stress can act as a redox signal to change the thiol redox state of mitochondria! proteins, subsequently triggering cellular events such as apoptosis, proliferation and senescence. Two mitochondrial proteins containing critical redox thiols, NADH dehydrogenase (NDH) and succinate ubiquinone reductase (SQR), are also catalytic components responsible for superoxide production in mitochondria. The broad long-term goal of this project is to elucidate the molecular mechanism of how oxygen free radical production in mitochondria contributes to the myocardial injury. The key hypotheses of superoxide generation and protein S-glutathiolation will be tested by pursuing the following specific aims using the isolated enzyme complex and post-ischemic hearts. [1] Determine the mechanism of superoxide production by NDH. [2] Determine the mechanism of superoxide production by SQR. [3] Elucidate the molecular mechanism of protein S-glutathiolation resulting from the major pathway of oxygen free radical production. [4] Evaluate protein S-glutathiolation and NDH/SQR-derived S- glutathiolation in post-ischemic hearts. Enzyme-mediated superoxide production will be measured by EPR spin trapping and immobilized enzyme EPR (IE-EPR). Enzyme-mediated NO consumption rates will be measured by electrochemical detection using a NO electrode. Protein-derived S-glutathiolation and protein radical formation will be detected by immunoblotting. Protein radical(s), reactive thiol(s), and glutathione-binding (GS-binding) domain(s) will be identified by mass spectrometry and peptide mapping. The functional role of each identified domain will be defined by immunochemistry using specific antibodies against conformational epitope peptides. The utilization of protein S-glutathiolation as a biomarker of redox change resulting from oxidative stress will be evaluated in the systems of post- ischemic heart. Results from this proposal will provide fundamental information concerning the pathophysiological role of oxygen free radicals and protein redox thiol(s) in mitochondrial dysfunction, and potentially improve clinical diagnosis, therapeutics, and prevention of heart diseases.