ROLE OF AUTOPHAGIC FLUX IN CARDIAC MYOCYTE VIABILITY

自噬流在心肌细胞活力中的作用

• 批准号: 8279349
• 负责人: Abhinav Diwan
• 金额: $ 38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8279349
• 关键词: Ablation; Adult; Age; Apoptosis; Autophagocytosis; Autophagolysosome; Autophagosome; Cardiac; Cardiac Myocytes; Cardiomyopathies; Caspase; Cause of Death; Cell Death; Cell Size; Cell Survival; Cells; Cessation of life; Data; Degradation Pathway; Development; Electric Capacitance; Ensure; Fibrosis; Fluorescence; Gene Expression; Genes; Glycogen Storage Disease Type IIb; Heart; Heart failure; Homeostasis; Hour; Human; Hypertrophic Cardiomyopathy; Hypertrophy; Hypoxia; Image; Impairment; In Situ Nick-End Labeling; In Vitro; Individual; Infarction; Knockout Mice; Ligation; Link; Lysosomes; Measures; Mediating; Membrane Proteins; Mission; Mitochondria; Molecular Chaperones; Mus; Muscle; Muscle Cells; Myocardial; Myocardial Infarction; Myocardial Ischemia; Myocardium; Neonatal; Nutrient; Organelles; Outcome; Pathogenesis; Pathway interactions; Permeability; Play; Prevention; Protein Isoforms; Proteins; Radiolabeled; Rattus; Recycling; Reperfusion Injury; Reperfusion Therapy; Reporter; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Stimulus; Stress; Structure; Techniques; Testing; Transgenes; Transgenic Organisms; Translating; United States; United States National Institutes of Health; Up-Regulation; Vacuole; Ventricular; Viral; Yeasts; crosslink; cytochrome c; deprivation; disease-causing mutation; gain of function; human FRAP1 protein; in vivo; loss of function; loss of function mutation; mTOR inhibition; metaperiodate; novel; overexpression; prevent; radiotracer; receptor; response; restoration; tool; yeast two hybrid system; young adult

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.

描述（由申请人提供）： 心肌自噬在降解细胞器和蛋白质以确保稳态方面具有组成性活性。应激诱导的自噬信号的上调通过促进营养供应和清除受损的细胞器和蛋白质来增强心肌细胞存活，但矛盾的是，它与缺血再灌注损伤的梗死面积增加有关。组成性自噬的损伤是Danon病发病机制的核心，其特征在于在年轻人中发展为肥厚性心肌病和暴发性心力衰竭，导致早期死亡。它是由溶酶体相关膜蛋白（LAMP 2）的功能丧失突变引起的，其中两种亚型被认为在自噬体-溶酶体融合中起关键作用，在巨自噬（2B）和分子伴侣介导的自噬（2A）中，确保通过自噬途径的足够通量。Danon病发生肥厚型心肌病的具体机制尚不清楚。我们已经观察到在心肌中响应于缺血-再灌注损伤的LAMP 2丰度的快速下降，在体内和在经受缺氧-复氧的心肌细胞中，在体外。我们认为，在缺乏LAMP 2的情况下，自噬通量的损伤导致自噬体积累，从而触发程序性细胞死亡。在这个建议中，我们将测试的假设，LAMP 2介导的自噬流量是一个关键的决定因素，心肌细胞的活力在无压力的心脏，并在3个特定的目标（SA）缺血再灌注损伤的反应。SA 1将确定LAMP 2的丧失，在体外用siRNA介导的敲低和在体内用基因消融，对无应激状态下的心肌细胞存活和诱导自噬的后果;以及对心肌细胞肥大的后果。SA 2将确定使用条件性转基因表达恢复心肌细胞LAMP 2A和LAMP 2B水平对心肌缺血-再灌注损伤中细胞死亡和梗死面积的影响。SA 3将确定在LAMP 2缺失的情况下观察到的细胞死亡和心肌肥大增加的机制，重点是引起程序性细胞死亡和心肌细胞肥大的信号传导途径的激活。使用新型双荧光标记的LC 3构建体评估宏自噬通量将用于量化自噬体和自噬溶酶体丰度，作为通过宏自噬途径的动态通量的量度。将用传统的放射性标记底物分解来评估伴侣介导的自噬，以确定其在心脏中的LAMP 2A信号传导中的作用。促进自噬流的策略，如恢复溶酶体中的LAMP 2A和B水平，可以治疗Danon病，并通过应激诱导的自噬信号激活实现促生存结局，转化为心肌梗死的肌肉挽救和心力衰竭的预防，这是NIH的关键使命。这些研究还将提供概念框架和工具，以询问细胞死亡的新范式。