ROLE OF AUTOPHAGIC FLUX IN CARDIAC MYOCYTE VIABILITY

自噬流在心肌细胞活力中的作用

基本信息

批准号：
8084569
负责人：
Abhinav Diwan
金额：
$ 38万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
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/8084569
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

项目摘要

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.

描述（由申请人提供）：项目摘要心肌自噬是在降解细胞器和蛋白质以确保稳态的组成性活动中。压力诱导的自噬信号传导上调通过促进营养供应并去除受损的细胞器和蛋白质，从而增强了心肌细胞的存活，但在矛盾的是与缺血 - 重新灌注损伤的梗塞大小相关。构型自噬的损害是达诺疾病发病机理的核心，其特征是年轻人的肥厚性心肌病和暴发性心力衰竭的发展，导致早期死亡。它是由溶酶体相关膜蛋白（LAMP2）的功能丧失突变引起的，其中两种同工型假定在大量自源噬菌体（2B）和伴侣介导的自噬（2A）中在自噬体 - 溶酶体融合中起关键作用，从而确保通过自动path的速度进行适当的速度。尚不清楚达诺疾病中肥厚性心肌病的特定机制。我们已经观察到，在体内和患有低氧抗氧化合物的心肌细胞和心肌细胞中，心肌的LAMP2丰度迅速下降。我们认为，在没有LAMP2的情况下，自噬通量的损害会导致自噬体积累，从而触发编程的细胞死亡。在此提案中，我们将检验以下假设：LAMP2介导的自噬通量是无数心脏中心肌细胞生存能力的关键决定因素，并且在3个特定目标（SA）下响应缺血 - 重新灌注损伤。 SA1将在体外，在体外与siRNA介导的敲低和基因消融的体外，对心肌细胞的生存以及自动噬的诱导，并在体内确定损失的后果；以及心肌肥大。 SA2将确定使用条件转基因表达，心肌死亡和梗死大小在心肌缺血 - 重新灌注损伤中恢复心肌细胞LAMP2A和LAMP2B水平的影响。 SA3将确定因LAMP2丢失而观察到的细胞死亡和心肌肥大增加的机制，重点是激活信号通路引发程序性细胞死亡和心肌肌细胞肥大。使用新型的双荧光标记的LC3构建体对宏观 - 自助通量进行评估，以量化自噬体和自噬物质体丰度，以衡量通过大型嗜血杆菌通路的动态通量。伴侣介导的自噬将通过传统的放射性标记的底物分解来评估，以确定其在LAMP2A信号中的作用。促进自噬通量的策略，例如溶酶体中LAMP2A和B水平的恢复，可以治疗Danon疾病，并通过压力诱导的自噬信号激活自动噬菌学的激活，使心肌梗死和预防心力衰竭的心力衰竭，是NIH的关键任务。这些研究还将提供概念框架和质疑细胞死亡范式的工具。公共卫生相关性：在大多数病例中，心肌梗塞和冠状动脉疾病引发了项目叙事心力衰竭，这是美国最常见的死亡原因。 Danon病是由蛋白质LAMP2丧失引起的心力衰竭的罕见遗传原因，它可能为心肌梗死的手术造成心脏肌肉细胞死亡的迄今为止未表征的原因提供了线索。该提案批判性地研究了LAMP2在缺血性损伤中确保损坏的细胞内材料中的作用，以确保心肌细胞的存活，并制定策略来挽救心脏肌肉并预防/治疗心力衰竭。