Integration of Pre-Mitochondrial Death Signals by ARC

描述(由申请人提供)：在缺血-再灌注和心力衰竭期间抑制心肌细胞凋亡可以减少心肌损伤、左心室重构、收缩功能障碍，在某些情况下还可以减少死亡率。然而，对特定于心肌细胞的细胞凋亡的分子调控知之甚少。中央死亡机制从蠕虫到人类都是高度保守的，在不同类型的细胞之间几乎没有区别。尽管如此，细胞凋亡通常是以一种特定的细胞类型和刺激方式进行调控，其基础还知之甚少。ARC是一种内源性的细胞凋亡抑制因子，主要表达于心肌和骨骼肌。我们的初步研究表明，ARC通过新的蛋白质-蛋白质相互作用抑制死亡受体和线粒体途径。在死亡受体途径中，ARC直接与Fas和FADD结合，抑制死亡受体信号转导所需的死亡诱导信号复合体的形成。在线粒体途径中，ARC直接与Bax结合，以抑制Bax的激活和移位到线粒体，以响应细胞凋亡的刺激。ARC同时拮抗两条中枢死亡途径的能力是独一无二的，这表明该抑制物可能作为心肌细胞凋亡的主抑制因子发挥作用。这一应用验证了一个中心假设，即ARC通过Bax、Bak和线粒体依赖机制作为心肌细胞凋亡的关键调节因子。目的是确定(A)ARC抑制线粒体前和线粒体前凋亡事件的机制；以及(B)这种抑制在病理情况下对心肌细胞存活的生物学意义。目的1研究ARC抑制Bax易位、Bak激活和线粒体凋亡事件的机制。目的2利用BAX和BAK基因敲除小鼠来确定这些下游ARC效应分子在体内急性缺血再灌注损伤和脑梗塞后重构中的作用。目的3利用ARC基因敲除小鼠来确定ARC基因缺失是否会导致基线心肌病和/或加重体内急性缺血再灌注损伤和梗死后重构。此外，这个目的是测试是否可以通过同时缺失Bax或Bak来挽救这些表型。这些目标构成了一个高度集中和综合的计划，检查ARC的关键方面，从单个分子到它在完整动物中的作用。由此得到的信息将促进我们对心肌细胞凋亡的理解，并可能为心肌梗死和心力衰竭的心脏特异性治疗提供新的基础。

DESCRIPTION (provided by applicant): Inhibition of cardiac myocyte apoptosis during ischemia-reperfusion and heart failure decreases myocardial damage, left ventricular remodeling, contractile dysfunction and, in some cases, mortality. Little is known, however, about the molecular regulation of apoptosis specifically in cardiac myocytes. The central death machinery has been highly conserved from worm to human and differs little among various cell types. Despite this, apoptosis is often regulated in a cell type- and stimulus-specific manner, the basis of which is poorly understood. ARC (Apoptosis Represser with a CARD (caspase recruitment domain)) is an endogenous inhibitor of apoptosis that is expressed primarily in cardiac and skeletal muscle. Our preliminary studies demonstrate that ARC suppresses both the death receptor and mitochondrial pathways through novel protein-protein interactions. In the death receptor pathway, ARC binds directly to Fas and FADD to inhibit the formation of the Death Inducing Signaling Complex which is required for death receptor signaling. In the mitochondrial pathway, ARC binds directly to Bax to inhibit Bax activation and translocation to the mitochondria in response to apoptotic stimuli. The ability of ARC to simultaneously antagonize both central death pathways is unique and suggests that this inhibitor may function as a master-repressor of apoptosis in cardiac myocytes. This application tests the central hypothesis that ARC serves as a critical regulator of cardiac myocyte apoptosis through Bax, Bak, and mitochondrial-dependent mechanisms. The objectives are to determine (a) the mechanisms by which ARC inhibits apoptotic pre-mitochondrial and mitochondrial events; and (b) the biological significance of this inhibition for cardiac myocyte survival in pathological contexts. Aim 1 seeks to delineate mechanisms by which ARC inhibits Bax translocation, Bak activation, and apoptotic mitochondrial events. Aim 2 uses Bax and Bak knockout mice to define the role of these downstream ARC effectors in acute ischemia-reperfusion injury and post-infarct remodeling in vivo. Aim 3 uses ARC knockout mice to determine if the absence of ARC causes a baseline cardiomyopathy and/or exacerbates acute ischemia reperfusion injury and post-infarct remodeling in vivo. In addition, this aim tests whether these phenotypes can be rescued by simultaneous deletion of Bax or Bak. These aims constitute a highly focused and integrated program that examines critical aspects of ARC from the single molecule to its role in the intact animal. The resulting information will advance our understanding of apoptosis in the myocardium and may provide the basis for novel cardiac-specific therapies for myocardial infarction and heart failure.