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

与线粒体衍生的氧自由基相关的心肌损伤

• 批准号: 7474076
• 负责人: YEONG-RENN CHEN
• 金额: $ 36.41万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7474076
• 关键词: Antibodies; Apoptosis; Binding; Biological Markers; Cell Death; Complex; Condition; Consumption; Cysteine; Cytochrome c Reductase; Defect; Detection; Electrodes; Electron Transport; Energy Metabolism; Enzymes; Epitopes; Evaluation; Event; Flavins; Flavoproteins; Functional disorder; Generations; Glutathione; Glutathione Disulfide; Goals; Heart; Immobilized Enzymes; Immunoblotting; Immunochemistry; In Vitro; Injury; Ischemia; Link; Mass Spectrum Analysis; Measures; Mediating; Mediation; Mitochondria; Mitochondrial Proteins; Molecular; Multienzyme Complexes; Myocardial; Myocardial Ischemia; Oxidation-Reduction; Oxidative Stress; Pathway interactions; Peptide Mapping; Peptides; Physiological; Physiological reperfusion; Production; Proteins; Rate; Rattus; Reactive Oxygen Species; Reperfusion Injury; Reperfusion Therapy; Research Personnel; Research Project Grants; Research Proposals; Respiratory Chain; Role; Signal Transduction; Spin Trapping; Succinate dehydrogenase (ubiquinone); Sulfhydryl Compounds; Superoxides; System; Testing; Therapeutic; Ubiquinone; clinical Diagnosis; free radical oxygen; heart disease prevention; improved; mitochondrial dysfunction; programs; response; senescence

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.

描述（由申请人提供）：线粒体氧化应激与缺血-再灌注（I/R）损伤相关。在缺血后心脏的心肌水平，能量代谢缺陷与氧自由基的显著过量产生有关。现有证据表明，[a]氧自由基和呼吸链蛋白之间的特异性相互作用在I/R病理生理学中至关重要，[B]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巯基氧化还原状态！蛋白质，随后触发细胞事件，如凋亡，增殖和衰老。含有关键氧化还原硫醇的两种线粒体蛋白质，NADH脱氢酶（NDH）和琥珀酸泛醌还原酶（SQR），也是负责线粒体中超氧化物产生的催化组分。本项目的长期目标是阐明氧自由基在线粒体中的产生如何导致心肌损伤的分子机制。超氧化物生成和蛋白S-谷胱甘肽化的关键假设将通过使用分离的酶复合物和缺血后心脏追求以下特定目标来测试。[1]确定NDH产生超氧化物的机制。[2]通过SQR确定超氧化物的产生机制。[3]阐明了氧自由基产生的主要途径导致蛋白S-谷胱甘肽化的分子机制。[4]评价缺血后心脏中蛋白S-谷胱甘肽化和NDH/SQR衍生的S-谷胱甘肽化。酶介导的超氧化物产生将通过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。将通过免疫印迹法检测蛋白源性S-谷胱甘肽化和蛋白自由基形成。将通过质谱法和肽图谱鉴定蛋白质自由基、反应性巯基和谷胱甘肽结合（GS结合）结构域。每个鉴定的结构域的功能作用将通过免疫化学使用针对构象表位肽的特异性抗体来定义。将在缺血后心脏系统中评价蛋白S-谷胱甘肽化作为氧化应激引起的氧化还原变化的生物标志物的利用。该提案的结果将提供有关氧自由基和蛋白质氧化还原硫醇在线粒体功能障碍中的病理生理作用的基本信息，并可能改善心脏病的临床诊断，治疗和预防。