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: 8459893
• 负责人: Abhinav Diwan
• 金额: $ 36.18万
• 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/8459893
• 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）： 项目摘要 心肌自噬在降解细胞器和蛋白质以确保体内平衡方面具有持续活性。应激诱导的自噬信号上调通过促进营养供应和去除受损的细胞器和蛋白质来增强心肌细胞的存活，但矛盾的是，它与缺血再灌注损伤导致的梗塞面积增加有关。组成性自噬受损是 Danon 病发病机制的核心，其特点是在年轻人中发展为肥厚性心肌病和暴发性心力衰竭，导致过早死亡。它是由溶酶体相关膜蛋白 (LAMP2) 的功能丧失突变引起的，推测其两种亚型在巨自噬 (2B) 和分子伴侣介导的自噬 (2A) 中的自噬体-溶酶体融合中发挥关键作用，确保通过自噬途径的足够流量。 Danon 病发展为肥厚型心肌病的具体机制尚不清楚。我们观察到，体内缺血再灌注损伤引起的心肌中 LAMP2 丰度迅速下降，体外缺氧复氧的心肌细胞中 LAMP2 丰度迅速下降。我们假设，在 LAMP2 缺失的情况下，自噬流受损会导致自噬体积累，从而引发程序性细胞死亡。在本提案中，我们将测试以下假设：LAMP2 介导的自噬流是无应激心脏中心肌细胞活力的关键决定因素，并在 3 个特定目标 (SA) 下响应缺血再灌注损伤。 SA1 将确定 LAMP2 丢失的后果，在体外通过 siRNA 介导的敲低，在体内通过基因消融，对无应激状态下心肌细胞的存活和诱导自噬产生影响；以及心肌细胞肥大。 SA2 将确定使用条件转基因表达恢复心肌细胞 LAMP2A 和 LAMP2B 水平对心肌缺血再灌注损伤中细胞死亡和梗塞面积的影响。 SA3 将确定 LAMP2 缺失时观察到的细胞死亡增加和心肌肥大的机制，重点关注引发程序性细胞死亡和心肌细胞肥大的信号通路的激活。使用新型双荧光标记的 LC3 构建体评估宏观自噬通量将用于量化自噬体和自噬溶酶体丰度，作为通过宏观自噬途径的动态通量的测量。将使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 LAMP2A 信号传导中的作用。促进自噬流的策略，例如恢复溶酶体中的 LAMP2A 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的结果，转化为心肌梗死的肌肉挽救和预防心力衰竭，这是 NIH 的一项关键使命。这些研究还将提供概念框架和工具来质疑细胞死亡的新范式。