Cardiac myocyte protein quality control is critical for maintenance of the contractile apparatus and cardiac function. Mutations in chaperone proteins that affect protein quality control induce proteotoxicity, sarcomere dysfunction and cardiac myocyte cell death. One such point mutation in the CRYAB gene in humans, results in an arginine to glycine change at position 120 in the heat-shock protein beta-5 (R120G HSPB5) protein, and provokes autosomal dominant cardiomyopathy resulting in heart failure, need for cardiac transplantation and premature mortality. Strategies to prevent proteotoxicity have focused on stimulating the autophagy-lysosome pathway to remove protein aggregates; and need to be refined for maximizing therapeutic benefit. Our studies during the current grant-funding period have uncovered an essential role for TRAF2, an innate immunity protein, in mediating basal mitophagy in cardiac myocytes. Our preliminary data show that loss of mitophagy with TRAF2 ablation in cardiac myocytes induces marked protein aggregation in cardiac myocytes, despite a lack of effect on general macro-autophagy. In a mouse model of cardiac myocyte specific human HSPB5 R120G mutant protein expression, we have uncovered evidence for mitochondrial accumulation of HSPB5 protein, along with polyubiquitinated proteins and p62 (an adaptor protein essential for aggregate formation). This is accompanied with markedly increased TRAF2 expression in the mitochondria and reduced mitochondrial protein content, pointing to a role for mitophagy in handling protein aggregate pathology. Given recent observations in yeast and mammalian cell lines indicating that mitochondria may facilitate uptake and degradation of cytosolic protein aggregates, we hypothesize that mitophagy facilitates removal of cytosolic protein aggregates of HSPB5 R120G mutant protein in cardiac myocytes to prevent cardiac myocyte death and cardiomyopathy. In this renewal application, we have generated reagents and developed collaborations to test this hypothesis via the following aims. In aim 1, we will examine if mitophagy is required for removal of protein aggregates in mice. Studies will also be performed in human induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs) with CRISPR targeted ablation of TRAF2, and in those with CRISPR-knock-in of R120G mutation. In aim 2, we will examine if TRAF2-induced mitophagy is sufficient to facilitate removal of protein aggregates in mice and in human iPSC-CMs. Aim 3 will focus on mechanistic studies to understand the steps involved in mitochondrial uptake of HSPB5 R120G mutant protein uptake in mitochondria. Studies in mice will focus on impairing p62-mediated protein aggregation to examine the role of p62 in mitochondrial protein aggregate uptake. Experiments in yeast and mouse model systems will examine if hsp104, a yeast disaggregase protein shown to be functional in mammalian cells, is required and sufficient for mitochondrial protein aggregate upt...