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: 8506698
• 负责人: YEONG-RENN CHEN
• 金额: $ 37.42万
• 依托单位: NORTHEAST OHIO MEDICAL 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 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8506698
• 关键词: Address; Affect; Animal Model; Antibodies; Aorta; Area; Atherosclerosis; Bioenergetics; Blood Vessels; Cardiac; Cardiomyopathies; Cardiovascular Diseases; Carmustine; Cell physiology; Cells; Complex; Cysteine; Defect; Disease; Disease Marker; Electron Transport; Endothelium; Energy Metabolism; Energy-Generating Resources; Enzymes; Etiology; Event; Generations; Genotype; Glutathione Disulfide; Glutathione Reductase; Grant; Heart; Heart Diseases; Heart Mitochondria; Heart failure; Homeostasis; Hypertension; Immunochemistry; In Vitro; Injury; Knowledge; Light; Link; Mass Spectrum Analysis; Measures; Mediating; Mediation; Metabolic stress; Metabolism; Mitochondria; Mitochondrial Proteins; Modeling; Modification; Molecular; Molecular Biology; Mus; Myocardial; Myocardial Contraction; Myocardial Infarction; Myocardial Ischemia; Myocardial Reperfusion Injury; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Pathway interactions; Phase; Play; Post-Translational Protein Processing; Predisposition; Production; Protein S; Proteins; Proteomics; Regulation; Relaxation; Reperfusion Injury; Reperfusion Therapy; Reporting; Research; Role; Route; SOD2 gene; Signal Pathway; Signal Transduction; Source; Spectrometry; Spin Trapping; Sulfhydryl Compounds; Techniques; Testing; Transgenic Mice; Tyrosine; Up-Regulation; design; free radical oxygen; in vivo; in vivo Model; insight; mitochondrial dysfunction; mouse model; nitration; novel; oxidation; patient population; protein function; public health relevance; therapy design

DESCRIPTION (provided by applicant): Mitochondria as the major source of energy generation are essential for proper cellular function. There is considerable evidence supporting the key role of mitochondrial dysfunction in heart disease such as myocardial infarction and heart failure. At the myocardial level of the post-ischemic heart, a defect in energy metabolism associated with overproducing oxygen free radicals and NO in mitochondria was marked. Alterations of protein S-glutathionylation (PrSSG) and protein nitration have been detected in the mitochondrial complex I (NQR), complex II (SQR), and other ETC proteins during myocardial ischemia and reperfusion injury. Alterations of NQR/SQR-derived oxidative modifications are closely linked to oxygen free radical production, NO metabolism, and homeostasis of redox thiols in mitochondria. S-glutathionylation of the reactive and labile cysteine residues in the NQR or SQR is a reversible modification, whereas S-sulfonation of reactive cysteine residues is an irreversible modification. Our central hypotheses are that both cysteinyl modifications are highly regulated by the redox status in the mitochondria of the post-ischemic heart, and the mitochondrial redox status is controlled by ROS production, NO metabolism, and the homeostasis of the GSH pool. The long term objectives of this research are to elucidate the molecular mechanism of mitochondrial redox signals in the mediation of myocardial injury, to understand the pathogenesis, and to develop a treatment for cardiovascular diseases. The key hypotheses of the major signal pathway leading to protein S-glutathionylation/sulfonation in mitochondria will be tested by pursuing the following specific aims using novel animal models, EPR spectrometry, and mass spectrometry. Specific aim 1 will determine whether irreversible protein S- sulfonation of NQR and SQR is induced in the mitochondria of the post-ischemic myocardium. The protein sulfonation marked in the NQR and SQR after myocardial infarction will be characterized by mass spectrometry. A sequence-specific antibody for sulfonation will be generated to detect this event in vitro and in vivo. EPR spectrometry with a spin probe and a spin trap will be used to measure the redox status and "O2- generation activity of mitochondria isolated from the post-ischemic heart. Specific aim 2 will determine the role of glutathione reductase (GR) in the mechanism of glutathionylation of NQR/SQR and regulation of overall mitochondrial function in the post-ischemic heart. We will use a pharmacologic approach and mice deficient in GR (gsr-/-) to determine whether (i) enhancing GSSG in vivo will increase glutathionylation of NQR and SQR, and (ii) whether or not increasing NQR/SQR glutathionylation in vivo will be protective and reduce the susceptibility of mitochondria to post-ischemic injury. Specific aim 3 will ascertain the role of eNOS in the mechanism of NQR/SQR glutathionylation and regulation of overall mitochondrial function in the post-ischemic heart. Mice with an eNOS-/- and a cardiac-specific eNOS-/- genotype will serve as an excellent in vivo model for studying the regulation of NQR/SQR glutathionylation, mitochondrial redox status, and its "O2- generation activity via NO metabolism. We will also create a novel mouse model by crossing cardiac-specific SOD2 transgenic mice with eNOS-/- mice in order to determine whether increased SOD2 signaling in mitochondria is sufficient to correct oxidative injury resulting from eNOS deficiency and post-ischemic injury.

描述(申请人提供)：线粒体作为能量产生的主要来源，对于正常的细胞功能是必不可少的。有相当多的证据支持线粒体功能障碍在心肌梗死和心力衰竭等心脏病中的关键作用。在缺血后心脏的心肌水平，能量代谢缺陷与线粒体中氧自由基和NO的过度产生有关。在心肌缺血再灌注损伤过程中，线粒体复合体I(NQR)、复合体II(SQR)等蛋白质中存在蛋白质S谷胱甘肽基化和蛋白质硝化的改变。NQR/SQR衍生的氧化修饰的改变与线粒体中氧自由基的产生、NO代谢和氧化还原硫醇的动态平衡密切相关。NQR或SQR中活性和不稳定的半胱氨酸残基的S-谷胱甘肽基化是可逆修饰，而活性半胱氨酸残基的S-磺化是不可逆修饰。我们的中心假设是，这两种半胱氨酸修饰都受到缺血后心脏线粒体氧化还原状态的高度调控，而线粒体的氧化还原状态受ROS产生、NO代谢和GSH池的动态平衡控制。本研究的长期目标是阐明线粒体氧化还原信号在介导心肌损伤中的分子机制，了解其发病机制，并开发治疗心血管疾病的方法。我们将利用新的动物模型、电子顺磁共振波谱和质谱学来验证线粒体中导致蛋白质S的主要信号通路-谷胱甘肽基化/磺化的关键假说。具体目的1将确定在缺血后心肌线粒体中是否诱导不可逆蛋白NQR和SQR的S磺化。心肌梗死后NQR和SQR中标记的蛋白质磺化将用质谱仪进行表征。将产生用于磺化的序列特异性抗体，以在体外和体内检测这一事件。带有自旋探针和自旋陷阱的EPR光谱分析将被用来测量从缺血后心脏分离的线粒体的氧化还原状态和产生O2的活性。 确定谷胱甘肽还原酶(GR)在缺血后心脏NQR/SQR谷胱甘肽基化和整体线粒体功能调节机制中的作用。我们将使用药理学方法和GR缺陷小鼠(GSR-/-)来确定(I)体内增强GSSG是否会增加NQR和SQR的谷胱甘肽基化，以及(Ii)体内增加NQR/SQR谷胱甘肽基化是否具有保护作用，并降低线粒体对缺血后损伤的易感性。具体目标3将确定eNOS在缺血后心脏NQR/SQR谷胱甘肽基化和整体线粒体功能调节机制中的作用。携带eNOS-/-和心脏特异性eNOS-/-基因的小鼠将成为研究NQR/SQR谷胱甘肽化、线粒体氧化还原状态及其通过NO代谢产生O2-活性的良好体内模型。我们还将通过将心脏特异的SOD2转基因小鼠与eNOS-/-小鼠杂交来创建一个新的小鼠模型，以确定线粒体中增加的SOD2信号是否足以纠正eNOS缺乏和缺血后损伤所导致的氧化损伤。