ROLE OF AUTOPHAGIC FLUX IN CARDIAC MYOCYTE VIABILITY

描述(由申请人提供)： 项目概述心肌自噬在降解细胞器和蛋白质以确保内环境稳定方面具有结构性的活性。应激诱导的自噬信号上调通过促进营养供应和清除受损的细胞器和蛋白质来提高心肌细胞的存活率，但矛盾的是，它与增加缺血再灌注损伤的心肌梗死面积有关。结构性自噬功能障碍是达能病发病机制的核心，其特征是肥厚性心肌病的发展和年轻人的暴发性心力衰竭，导致早期死亡。它是由溶酶体相关膜蛋白(LAMP2)的功能缺失突变引起的，LAMP2的两个亚型被认为在巨型自噬(2B)和伴侣介导的自噬(2A)中的自噬-溶酶体融合中发挥关键作用，确保足够的通量通过自噬途径。达农病中肥厚型心肌病发生的具体机制尚不清楚。我们观察到在体心肌和体外缺氧-复氧心肌细胞中LAMP2的丰度随着缺血-再灌注损伤的反应而迅速下降。我们推测，在没有LAMP2的情况下，自噬通量的损害会导致自噬小体积累，从而触发细胞程序性死亡。在本提案中，我们将检验LAMP2介导的自噬通量是非应激心脏和3种特定靶点(SA)下的缺血再灌注损伤的心肌细胞活性的关键决定因素的假设。SA1将决定LAMP2的缺失，在体外通过siRNA介导的敲除，在体内通过基因去除，在非应激状态下对心肌细胞存活和自噬的诱导，以及对心肌细胞肥大的影响。SA2将通过条件性转基因表达来确定恢复心肌细胞LAMP2A和LAMP2B水平对心肌缺血再灌注损伤中细胞死亡和梗塞范围的影响。SA3将确定LAMP2缺失时细胞死亡和心肌肥大增加的机制，重点是激活引起细胞程序性死亡和心肌细胞肥大的信号通路。使用一种新的双荧光标记的LC3结构来评估巨型自噬通量将被用于量化自噬小体和自噬溶酶体的丰度，作为通过巨型自噬途径的动态通量的测量。伴侣介导的自噬将用传统的放射性标记底物分解进行评估，以确定其在心脏LAMP2A信号转导中的作用。促进自噬通量的策略，例如恢复溶酶体中LAMP2A和B的水平，可以治疗达农病，并通过应激诱导自噬信号的激活来促进生存，转化为心肌梗死的肌肉挽救和预防心力衰竭，这是NIH的一项关键任务。这些研究还将提供概念框架和工具，以审问细胞死亡的新范式。 公共卫生相关性： 项目简介心力衰竭是美国最常见的死亡原因，大多数病例是由心肌梗死和冠状动脉疾病引发的。达农病是一种罕见的遗传原因，由LAMP2蛋白缺失导致心力衰竭，它可能为揭示心肌梗死时迄今尚未确定的心肌细胞死亡原因提供线索。这项建议严格审查了LAMP2在确保在缺血性损伤的背景下清除受损的细胞内物质以确保心肌细胞存活的作用，并制定了挽救心肌和预防/治疗心力衰竭的策略。

DESCRIPTION (provided by applicant): Project Summary Myocardial autophagy is constitutively active in degrading organelles and proteins to ensure homeostasis. Stress-induced upregulation of autophagic signaling enhances cardiac myocyte survival by facilitating nutrient supply and removing damaged organelles and proteins, but is paradoxically implicated in increasing infarct size with ischemia-reperfusion injury. Impairment of constitutive autophagy is central to the pathogenesis of Danon disease, characterized by development of hypertrophic cardiomyopathy and fulminant heart failure in young adults, leading to early death. It is caused by loss-of-function mutations in lysosome associated membrane protein (LAMP2), two isoforms of which are postulated to play a critical role in autophagosome-lysosome fusion in macroautophagy (2B) and chaperone mediated autophagy (2A), ensuring adequate flux through the autophagic pathway. The specific mechanisms for development of hypertrophic cardiomyopathy in Danon disease are not known. We have observed a rapid decline in LAMP2 abundance in the myocardium in response to ischemia-reperfusion injury, in vivo and in cardiomyocytes subjected to hypoxia-reoxygenation, in vitro. We posit that impairment in autophagic flux in the absence of LAMP2 causes autophagosome accumulation which triggers programmed cell death. In this proposal, we will test the hypothesis that LAMP2-mediated autophagic flux is a critical determinant of cardiac myocyte viability in the unstressed heart and in response to ischemia-reperfusion injury under 3 specific aims (SA). SA1 will determine the consequences of loss of LAMP2, in vitro with siRNA mediated knockdown and in vivo with gene ablation, on cardiac myocyte survival in the unstressed state and with induction of autophagy; and on cardiac myocyte hypertrophy. SA2 will determine the effects of restoration of cardiac myocyte LAMP2A and LAMP2B levels using conditional transgenic expression, on cell death and infarct size in myocardial ischemia-reperfusion injury. SA3 will determine the mechanism of increased cell death and myocardial hypertrophy observed with loss of LAMP2, focusing upon activation of signaling pathways provoking programmed cell death and cardiac myocyte hypertrophy. Assessment of macro-autophagic flux using a novel dual fluorescence tagged LC3 construct will be employed to quantify autophagosome and autophagolysosome abundance, as a measure of dynamic flux through the macroautophagic pathway. Chaperone mediated autophagy will be assessed with traditional radiolabelled substrate breakdown, to determine its role in LAMP2A signaling in the heart. Strategies to facilitate autophagic flux, such as restoration of LAMP2A and B levels in lysosomes, could treat Danon disease and enable pro-survival outcomes with stress-induced activation of autophagic signaling, translating into muscle salvage in myocardial infarction and prevention of heart failure, a key mission of the NIH. These studies will also provide the conceptual framework and the tools to interrogate a novel paradigm for cell death. PUBLIC HEALTH RELEVANCE: Project Narrative Heart failure, the most common cause of death in the United States, is triggered by myocardial infarction and coronary artery disease in the majority of cases. Danon disease, a rare genetic cause of heart failure caused by loss of a protein LAMP2, may provide a clue into a hitherto uncharacterized cause of heart muscle cell death, operative in myocardial infarction. This proposal critically examines the role of LAMP2 in ensuring removal of damaged intracellular material in the setting of ischemic injury to ensure heart muscle cell survival, and develop strategies to salvage cardiac muscle and prevent/treat heart failure.