Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

Abstract

Background: Protein quality control (PQC) is critical for maintaining sarcomere structure and function in cardiomyocytes. Mutations in PQC pathway proteins, namely CRYAB-R120G (arginine to glycine at position 120) and BAG3-P209L (proline to lysine at position 209) induce protein aggregates and cardiomyopathy in humans. Novel observations in yeast demonstrate mitochondrial uptake of cytosolic protein aggregates. We hypothesized that mitochondrial uptake of cytosolic protein aggregates and their removal by mitophagy, a lysosomal degradative pathway, facilitates cytosolic PQC in cardiomyocytes. Methods: Mice with inducible cardiomyocyte-specific ablation of TRAF2 (TRAF2icKO), which impairs mitophagy, were assessed for protein aggregates with biochemical fractionation and super-resolution imaging. Human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with TRAF2 ablation or R120G knock-in to the CRYAB locus were assessed for protein aggregates and effects of mitophagy stimulation. Transgenic mice expressing R120G-CRYAB protein (R120G-TG) were subjected to AAV9-cardiac Troponin T promoter-driven TRAF2 or PARKIN gain-of-function and TRAF2 loss-of-function in cardiomyocytes to determine the effect of mitophagy modulation on cardiac structure, function, and protein aggregate pathology. Results: TRAF2icKO mice demonstrate accumulation of mitochondrial and cytosolic protein aggregates and DESMIN mis-localization to protein aggregates. TRAF2 null hiPSC-CMs demonstrate impaired mitophagy with accumulation of polyubiquitinated proteins and disrupted sarcomeres, which are rescued by both TRAF2 and PARKIN transduction. Isolated mitochondria take up cardiomyopathy-associated aggregate-prone cytosolic proteins, namely R120G-CRYAB and P209L-BAG3. R120G-CRYAB mutant protein increasingly localizes to mitochondria in human and mouse cardiomyocytes. R120G-TG mice demonstrate upregulation of myocardial TRAF2 with increased mitophagy. Adult-onset inducible haplo-insufficiency of Traf2 resulted in accelerated mortality, left ventricular (LV) systolic dysfunction and increased protein aggregates in R120G-TG mice. Conversely, AAV9-TRAF2 transduction in R120G-TG mice stimulated mitophagy, reduced mortality, attenuated LV systolic dysfunction, reduced cytosolic protein aggregates and restored DESMIN localization. Conclusions: Stimulation of mitophagy in cardiomyocytes facilitates removal of cytosolic protein aggregates as a mechanism to ameliorate proteotoxic cardiomyopathy.