Cardiac Myocyte Apoptosis: Mechanism and Significance

描述(由申请人提供)：长时间的缺血，无论有没有再灌注，都会引发心肌细胞的凋亡性和非凋亡性死亡。这些细胞的死亡是心肌梗死发病机制的主要组成部分。细胞凋亡和一些非凋亡形式的细胞死亡是主动介导的过程，这表明它们可以被抑制，从而在心脏病中具有治疗优势。我们和其他人已经证明，在完整的小鼠中，抑制心肌细胞在缺血-再灌注期间的死亡限制了梗死范围，并保留了心功能。细胞凋亡是由两条中枢通路介导的，这两条通路在心肌细胞缺血再灌注过程中的死亡中起重要作用。ARC是一种内源性的细胞凋亡抑制因子，广泛存在于心肌细胞中。ARC是唯一的凋亡抑制剂，因为它通过我们已经阐明的机制来拮抗这两条中枢凋亡途径。ARC还通过未知的方式抑制坏死性细胞死亡。鉴于ARC的丰度和效力，为什么大量的心肌细胞在缺血再灌注期间死亡？在缺血再灌流过程中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ARC即使维持在低于基线水平也会抑制细胞死亡。我们已经确定，在缺血-再灌注过程中，ARC蛋白水平的下降主要是由于ARC蛋白降解的增加，并且ARC蛋白通过泛素-蛋白酶体途径和自噬在溶酶体中被降解。本项目的目的是明确在活体缺血-再灌流过程中介导ARC降解的分子机制，并确定每种机制在ARC蛋白丢失和导致的心肌细胞死亡中的作用。目的1研究这一过程中的泛素-蛋白酶体途径。特别是，E3连接酶MDM2作用于ARC的机制将被描述，以及我们已经确定的一种新的磷酸降解子在ARC中的潜在作用。目的2重点研究溶酶体途径，并试图确定介导ARC降解的自噬形式(S)。值得注意的是，我们已经在ARC中确定了一个新的基序，该基序可能针对ARC进行伴侣介导的自噬。AIM 3在基因工程小鼠身上测试了早期AIMS的机制发现，这些小鼠在关键的蛋白质降解机制上存在缺陷。这些机制在体内ARC降解和心肌梗死发生中的作用将被勾画出来。这些研究将为在心肌梗死期间引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靶点以在心肌梗死期间维持ARC水平，从而(A)限制梗塞范围和(B)扩大直接血管成形术再灌注治疗的时间窗口提供概念性基础。与公共健康相关：ARC是一种保护心肌细胞免于死亡的蛋白质，但在心脏病发作开始时，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。由此得到的知识可能有助于设计一种药物来防止ARC的破坏，并减少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.