Abstract 485: Enhanced Chaperone Mediated Autophagy in Cardiomyocytes Ameliorates Pathology Induced by Hypoxic and Proteotoxic Stresses

Abstract

Rationale: Protein degradation pathways play a critical role in maintaining cardiac homeostasis. Chaperone mediated autophagy (CMA) is a protein degradation pathway unique to mammalian cells, but little is known about CMA in cardiac disease. Preliminary studies in our lab showed increased CMA activity in mouse hearts subjected to MI (1.81 fold) and TAC surgery (3.5 fold), as well as, in hearts from mutant CryAB R120G transgenic mice (3 fold). Thus, we hypothesized that CMA plays a critical role in hypoxia- and proteotoxicity-induced cardiac stresses. CMA selectively targets substrate proteins for lysosomal degradation using CMA-specific lysosome membrane protein type 2a (LAMP2a) receptor. LAMP2a is both necessary and sufficient for CMA activity. Methods: An adenovirus and a siRNA were created to overexpress and silence LAMP2a levels to study the gain and loss of CMA in neonatal rat ventricular cardiomyocytes (NRVMs). Two models of cardiomyocyte stress were employed: 1) a hypoxic stress model that exposed NRVMs to CoCl 2 , a hypoxia mimetic agent; and 2) a proteotoxic model where mutant CryAB R120G , a missense mutation of αB-crystallin, was expressed causing the accumulation of insoluble protein aggregates. A more specific measure of CMA activity was also determined in lysosomal fractions of NRVMs. Results: The LAMP2a adenovirus and siRNA showed successful increase and knockdown of LAMP2a levels in NRVMs, respectively. CoCl 2 -induced hypoxia significantly increased CMA activity in NRVMs (26%). LAMP2a overexpression further augmented CoCl 2 -induced CMA activity (55%) and increased the percent of CMA-active lysosomes. LAMP2a siRNA blunted the CoCl 2 -induction of LAMP2a protein levels (65%) and silenced CMA activity in intact lysosomes. Further, CoCl 2 increased apoptosis which was rescued by enhanced CMA activity as shown by TUNEL staining. Thus, CMA is regulated by hypoxic stress and modulates cardiac cell survival. In a proteotoxic model, CryAB R120G and LAMP2a co-infection decreased CryAB R120G protein levels and insoluble aggregate pathology. Our data suggest that CMA may be cardioprotective against hypoxic and proteotoxic stresses. Impact: Defining the role of CMA in the heart could open new therapeutic pathways for the treatment of heart failure.