Cardiac Myocyte Apoptosis: Mechanism and Significance

描述（由申请人提供）：有或没有再灌注的长时间缺血会引发心肌细胞的凋亡和非凋亡性死亡。这些细胞的死亡是心肌梗死发病机制的一个重要组成部分。细胞凋亡和一些非凋亡形式的细胞死亡是积极介导的过程，表明它们可以被抑制以治疗心脏病。我们和其他人已经证明，在缺血再灌注期间抑制心肌细胞死亡可以限制梗死面积并保持心功能。细胞凋亡是由两条中枢通路介导的，这两条通路在缺血再灌注心肌细胞死亡中都起重要作用。ARC是一种内源性细胞凋亡抑制剂，在心肌细胞中含量丰富。ARC在细胞凋亡抑制剂中是独特的，因为它通过我们已经阐明的机制拮抗两个中心细胞凋亡途径。ARC还通过未知的途径抑制坏死细胞的死亡。考虑到ARC的丰度和效力，为什么大量心肌细胞在缺血-再灌注期间死亡？在缺血-再灌注过程中，ARC蛋白水平的急剧下降提示了一种可能性。我们已经证明，这些减少是导致细胞死亡的触发因素，而不是结果，因为即使维持在低于基线水平的ARC也会抑制细胞死亡。我们已经确定，ARC蛋白水平在缺血-再灌注期间下降，主要是因为ARC蛋白降解增加，并且ARC蛋白通过泛素-蛋白酶体途径和自噬在溶酶体中降解。该项目的目的是确定体内缺血-再灌注期间介导ARC降解的分子机制，并确定每种机制对ARC蛋白丢失和导致心肌细胞死亡的贡献。目的1研究这一过程中的泛素-蛋白酶体途径。特别是，我们将描述E3连接酶MDM2作用于ARC的机制，以及我们已经确定的一种新型磷酸化子在ARC中的潜在作用。目的2侧重于溶酶体途径，并试图确定自噬的形式，介导ARC降解。值得注意的是，我们在ARC中发现了一个新的基序，可能针对ARC进行伴侣介导的自噬。目的3在基因工程的小鼠中测试了早期目的的机制发现，这些发现在关键蛋白质降解机制中存在缺陷。这些机制在体内ARC降解和心肌梗死发生中的作用将被描述。这些研究将为心肌梗死期间触发心肌细胞死亡的事件提供新的见解。此外，它们可能为确定小分子治疗的靶点提供概念基础，以维持心肌梗死期间的ARC水平，从而(a)限制梗死面积，(b)扩大初级血管成形术再灌注治疗的时间窗口。公共卫生相关性：ARC是一种保护心肌细胞免于死亡的蛋白质，但在心脏病发作初期，ARC被破坏，导致心肌细胞死亡。这项研究的目的是确定ARC被摧毁的原因和方式。由此产生的知识可能有助于设计一种药物来防止ARC破坏和减轻心脏病发作造成的损害。

DESCRIPTION (provided by applicant): Prolonged ischemia with or without reperfusion triggers the apoptotic and non-apoptotic death of cardiac myocytes. The death of these cells is a major component in the pathogenesis of myocardial infarction. Apoptosis and some non-apoptotic forms of cell death are actively mediated processes, suggesting that they can be inhibited to therapeutic advantage in heart disease. We and others have shown that inhibition of cardiac myocyte death during ischemia-reperfusion in the intact mouse limits infarct size and preserves cardiac function. Apoptosis is mediated by two central pathways, both of which play important roles in cardiac myocyte death during ischemia-reperfusion. ARC is an endogenous inhibitor of apoptosis that is abundant in cardiac myocytes. ARC is unique among apoptosis inhibitors in that it antagonizes both central apoptosis pathways through mechanisms that we have elucidated. ARC also inhibits necrotic cell death through unknown means. Given the abundance and potency of ARC, why do large numbers of cardiac myocytes die during ischemia- reperfusion? A possibility is suggested by the dramatic decreases in ARC protein levels during ischemia- reperfusion. We have shown that these decreases are the trigger - not the consequence - of the resulting cell death because maintenance of ARC even at sub-baseline levels inhibits cell death. We have determined that ARC protein levels decrease during ischemia-reperfusion primarily because of increases in ARC protein degradation, and that ARC protein is degraded by both the ubiquitin-proteasomal pathway and in the lysosome via autophagy. The objective of this project is to define the molecular mechanisms that mediate ARC degradation during ischemia-reperfusion in vivo and to determine the contribution of each to the loss of ARC protein and resulting cardiac myocyte death. Aim 1 investigates the ubiquitin-proteasomal pathway in this process. In particular, the mechanisms by which the E3 ligase MDM2 act on ARC will be delineated, as will the potential role of a novel phosphodegron in ARC that we have identified. Aim 2 focuses on the lysosomal pathway and seeks to identify the form(s) of autophagy that mediate ARC degradation. Of note, we have identified a novel motif in ARC that may target ARC for chaperone-mediated autophagy. Aim 3 tests the mechanistic findings from the earlier aims in mice genetically engineered to have defects in key protein degradation mechanisms. The roles of these mechanisms in ARC degradation and the genesis of myocardial infarction in vivo will be delineated. These studies will provide novel insights into the events that trigger cardiac myocyte death during myocardial infarction. In addition, they may provide the conceptual basis for the identification of targets for small molecule therapies to maintain ARC levels during myocardial infarction so as to (a) limit infarct size and (b) expand the time window for reperfusion therapy with primary angioplasty. PUBLIC HEALTH RELEVANCE: ARC is a protein that protects heart muscle cells from dying, but at the beginning of a heart attack ARC is destroyed allowing heart muscle cells to die. The purpose of this research is to determine why and how ARC gets destroyed. The resulting knowledge may be useful in devising a medication to prevent ARC destruction and lessen the damage from heart attacks.