Peroxynitrite in Cardiac Ischemia/Reperfusion

描述（由申请人提供）：NO在细胞凋亡信号传导中的作用是近年来研究最深入的课题之一。然而，已发表的研究结果令人困惑且充满争议。我们最近证明NO本身具有显著的抗凋亡作用，而其二次反应产物（如过氧亚硝酸盐）则具有促凋亡作用。越来越多的证据表明，蛋白质硝化是一个关键的翻译后修饰，心肌缺血/再灌注（MI/R）引起显著的蛋白质硝化。此外，我们的初步实验首次证明，体外培养的心肌细胞暴露于病理相关浓度的过氧亚硝酸盐或体内MI/R会导致硫氧还蛋白（一种新型抗氧化和抗凋亡蛋白）以及硫氧还蛋白还原酶（负责硫氧还蛋白还原和再激活的唯一酶）的硝化失活。我们的长期目标是揭示心肌梗死/再灌注后心肌凋亡的机制，并寻找减少心肌再灌注损伤的最佳治疗策略。本研究的总体假设是，硫氧还蛋白/硫氧还蛋白还原酶硝化作用在心肌凋亡信号传导中起着致病作用，抑制硫氧还蛋白/硫氧还蛋白还原酶硝化作用可能是一种减少心肌梗死/心肌梗死损伤的新治疗策略。为了验证这一假设，我们将使用体外和体内实验模型来解决以下具体目标：探讨过氧亚硝酸盐处理成人心肌细胞中硫氧还蛋白/硫氧还蛋白还原酶硝化作用与细胞凋亡之间的因果关系；具体目标2。确定硫氧还蛋白/硫氧还蛋白还原酶硝化导致心肌细胞凋亡的下游信号机制；具体目标3。鉴定体内MI/R后负责硫氧还蛋白/硫氧还蛋白还原酶硝化的分子/细胞来源；4.具体目标验证抗硝化干预可阻断硫氧还蛋白/硫氧还蛋白还原酶硝化作用，从而减少心肌梗死，改善心肌功能恢复。缺血性心脏病仍然是美国的头号杀手。阐明Ml后细胞死亡的机制和确定新的治疗策略将有助于减少我国缺血性疾病相关的死亡。

DESCRIPTION (provided by applicant): The role of NO in apoptosis signaling has been one of the most intensely studied topics in the past few years. However, published results are confusing and controversial. We have recently demonstrated that NO by itself exerts significant anti-apoptotic effect, whereas its secondary reaction products (e.g., peroxynitrite) are pro-apoptotic. Accumulating evidence suggests that protein nitration is a critical post-translational modification and myocardial ischemia/reperfusion (MI/R) causes significant protein nitration. Moreover, our preliminary experiments demonstrated for the first time that in vitro exposure of cultured cardiomyocytes to a pathologically relevant concentration of peroxynitrite or in vivo MI/R results in nitrative inactivation of thioredoxin, a novel anti-oxidant and anti-apoptotic protein, as well as thioredoxin reductase, the exclusive enzyme responsible for thioredoxin reduction and reactivation. Our long-term goals are to uncover the mechanisms responsible for myocardial apoptosis after MI/R, and to search for the optimal therapeutic strategies that will reduce myocardial reperfusion injury. The overall hypothesis to be tested in the present grant application is that thioredoxin/thioredoxin reductase nitration plays a causative role in myocardial apoptosis signaling, and that inhibiting thioredoxin/thioredoxin reductase nitration may be a novel therapeutic strategy to reduce MI/R injury. To test this hypothesis, we will address the following specific aims using both in vitro and in vivo experimental models: Specific Aim 1. To establish a causative link between thioredoxin/thioredoxin reductase nitration and apoptosis in cultured adult cardiomyocytes treated with peroxynitrite; Specific Aim 2. To determine the downstream signaling mechanisms by which nitration of thioredoxin/thioredoxin reductase leads to apoptotic cardiomyocyte death; Specific Aim 3. To identify molecular/cellular sources that are responsible for thioredoxin/thioredoxin reductase nitration after MI/R in vivo; and Specific Aim 4. To test the hypothesis that anti-nitration interventions may block thioredoxin/ thioredoxin reductase nitration and thus reduce myocardial infarction and improve myocardial functional recovery after MI/R. Ischemic heart disease remains to be the number 1 killer in the USA. Clarifying the mechanisms responsible for cell death after Ml and identifying novel therapeutic strategies will help to reduce ischemic disease related death in this country.