Peroxynitrite in Cardiac Ischemia/Reperfusion

描述（由申请人提供）：NO在细胞凋亡信号转导中的作用是过去几年中研究最深入的主题之一。然而，发表的结果令人困惑和有争议。我们最近已经证明，NO本身发挥显着的抗凋亡作用，而其次级反应产物（例如，过氧亚硝酸盐）是促凋亡的。越来越多的证据表明，蛋白质硝化是一种重要的翻译后修饰，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，在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在体内MI/R的结果在硝化失活的硫氧还蛋白，一种新的抗氧化剂和抗凋亡蛋白，以及硫氧还蛋白还原酶，负责硫氧还蛋白还原和再活化的独家酶。我们的长期目标是揭示MI/R后心肌细胞凋亡的机制，并寻找最佳的治疗策略，将减少心肌再灌注损伤。在本授权申请中待测试的总体假设是硫氧还蛋白/硫氧还蛋白还原酶硝化在心肌细胞凋亡信号传导中起致病作用，并且抑制硫氧还蛋白/硫氧还蛋白还原酶硝化可能是减少MI/R损伤的新的治疗策略。为了检验这一假设，我们将使用体外和体内实验模型来解决以下具体目标：具体目标1。建立硫氧还蛋白/硫氧还蛋白还原酶硝化与过氧亚硝酸盐处理的培养成人心肌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传导机制;具体目标3。确定负责体内MI/R后硫氧还蛋白/硫氧还蛋白还原酶硝化的分子/细胞来源;具体目标4。验证抗硝化干预可能阻断硫氧还蛋白/硫氧还蛋白还原酶硝化，从而减少心肌梗死并改善MI/R后心肌功能恢复的假设。缺血性心脏病仍然是美国的头号杀手。阐明MI后细胞死亡的机制并确定新的治疗策略将有助于减少该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.