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.
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