Abstract 504: LAMP-2B Regulates Human Cardiomyocyte Function by Mediating Autophagosome-lysosome Fusion

Abstract

Autophagy plays a crucial role in cell homeostasis and function. Two types of autophagy have been well studied. Macroautophagy (referred to as autophagy hereafter) is mediated by double-membrane autophagosomes that enclose cytosolic cargoes, followed by fusion with late endosomes/lysosomes for degradation. Chaperone-mediated autophagy (CMA) is a process of chaperon-dependent selection of cytosolic proteins that are translocated into the lysosome for degradation. LAMP-2A, the A isoform of LAMP-2, is an essential receptor for CMA. However, alternative splicing of pre-LAMP-2 mRNA produces another two isoforms, LAMP-2B and LAMP-2C, with unclear functions. In non-cardiomyocytes, STX17 is required for autophagosome-lysosome fusion by interacting with SNAP29 and VAMP8. Whether cardiomyocytes (CMs) utilize the STX17-dependent mechanism to mediate autophagic fusion remains elusive. Using CMs derived from induced human pluripotent stem cells (hiPSC-CMs) and genome editing approaches, we identified previously undefined biological and molecular roles for LAMP-2B in the control of autophagosome-lysosome fusion. Remarkably, LAMP-2B functions independently of STX17. Instead, LAMP-2B interacts with ATG14 and VAMP8 through its C-terminal coiled-coil domain to promote autophagic fusion. Knockout of LAMP-2B in hiPSC-CMs decreased colocalization of ATG14 with VAMP8, and autophagosome-lysosome fusion. Forced expression of LAMP-2B in LAMP-2 deficient hiPSC-CMs restored autophagic signaling. In addition, LAMP-2B -deficient hiPSC-CMs recapitulated the metabolic and contractile defects observed in hiPSC-CMs derived from Danon patients. Danon disease represents a severe form of cardiomyopathy and has been associated with mutations in X-linked LAMP-2 gene. However, mechanisms by which LAMP-2 deficiency leads to cardiomyopathy are poorly understood. Given the similarity of phenotype between Danon and LAMP-2B knockout hiPSC-CMs, we conclude that LAMP-2B deficiency is sufficient and necessary to cause Danon cardiomyopathy. Therefore, our findings not only reveal a STX17-independent autophagic fusion mechanism mediated by LAMP-2B in human CMs, but also provide a molecular mechanism underlying Danon cardiomyopathy.