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

描述（由申请人提供）：线粒体作为能量产生的主要来源，对正常的细胞功能至关重要。有大量证据支持线粒体功能障碍在心脏病如心肌梗死和心力衰竭中的关键作用。在缺血后心脏的心肌水平，线粒体中与过量产生氧自由基和NO相关的能量代谢缺陷是显著的。在心肌缺血和再灌注损伤过程中，线粒体复合物I（NQR）、复合物II（SQR）和其他ETC蛋白质中已检测到蛋白质S-谷胱甘肽化（PrSSG）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稳态密切相关。NQR或SQR中反应性和不稳定半胱氨酸残基的S-谷胱甘肽化是可逆修饰，而反应性半胱氨酸残基的S-磺化是不可逆修饰。我们的中心假设是，这两个半胱氨酰修饰是高度调节的氧化还原状态的线粒体缺血后的心脏，和线粒体的氧化还原状态是由ROS的生产，NO代谢，和GSH池的稳态控制。本研究的长期目标是阐明线粒体氧化还原信号介导心肌损伤的分子机制，了解其发病机制，并开发心血管疾病的治疗方法。主要信号通路的关键假设，导致蛋白质S-谷胱甘肽/磺化线粒体将通过追求以下具体目标，使用新的动物模型，EPR光谱和质谱测试。具体目标1将确定在缺血后心肌的线粒体中是否诱导NQR和SQR的不可逆蛋白S-磺化。心肌梗死后NQR和SQR中标记的蛋白质磺化将通过质谱表征。将产生用于磺化的序列特异性抗体以在体外和体内检测该事件。将使用带有自旋探针和自旋捕获器的EPR光谱法来测量从缺血后心脏分离的线粒体的氧化还原状态和“O2-产生活性。具体目标2将 确定谷胱甘肽还原酶（GR）在缺血后心脏中NQR/SQR谷胱甘肽化机制和整体线粒体功能调节中的作用。我们将使用药理学方法和GR（gsr-/-）缺陷小鼠来确定（i）体内增强GSSG是否会增加NQR和SQR的谷胱甘肽化，以及（ii）体内增加NQR/SQR谷胱甘肽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在缺血后心脏中NQR/SQR谷胱甘肽化机制和整体线粒体功能调节中的作用。具有eNOS-/-和心脏特异性eNOS-/-基因型的小鼠将作为研究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活性的调节的极好体内模型。我们还将通过将心脏特异性SOD 2转基因小鼠与eNOS-/-小鼠杂交来创建一种新型小鼠模型，以确定线粒体中增加的SOD 2信号传导是否足以纠正由eNOS缺乏和缺血后损伤引起的氧化损伤。

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.