Peroxynitrite in Cardiac Ischemia/Reperfusion

描述(申请人提供)：在过去的几年中，NO在细胞凋亡信号中的作用一直是最热门的研究课题之一。然而，公布的结果令人困惑，也有争议。我们最近已经证明，NO本身具有显著的抗凋亡作用，而其次级反应产物(如过氧亚硝酸盐)则是促凋亡的。越来越多的证据表明，蛋白质硝化是一种关键的翻译后修饰，而心肌缺血/再灌注(MI/R)导致了显着的蛋白质硝化。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会导致新型抗氧化剂和抗凋亡蛋白硫氧还蛋白和硫氧还蛋白还原酶亚硝化失活，硫氧还蛋白还原酶是负责硫氧还蛋白还原和重新激活的唯一酶。我们的长期目标是揭示MI/R后心肌细胞凋亡的机制，并寻找减少心肌再灌注损伤的最佳治疗策略。本研究拟验证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转导中起重要作用，抑制硫氧还蛋白/硫氧还蛋白还原酶硝化可能是减轻心肌缺血/再灌注损伤的一种新的治疗策略。为了验证这一假说，我们将利用体外和体内实验模型来解决以下特定目的：特定目的1.建立过氧亚硝酸盐处理的培养的成年心肌细胞中硫氧还蛋白/硫氧还蛋白还原酶硝化与细胞凋亡之间的联系；特定目的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性死亡的下游信号机制；特定目的3.确定体内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；4.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，从而减少MI/R后心肌梗死，促进心肌功能恢复的假说。在美国，缺血性心脏病仍是头号杀手。弄清ML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与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.