Abstract 18826: Cardiac Myosin Binding Protein C Mutants Interact With and Cause Mislocalization of the Hsp70 Family Chaperones

Abstract

Introduction: Myosin binding protein C (MYBPC3) is the most frequently mutated gene in hypertrophic cardiomyopathy (HCM) and >90% of MYBPC3 mutations produce truncated proteins. Ubiquitin proteasome dysfunction has been observed in human HCM hearts with MYBPC3 mutations and in experimental models, but the mechanisms responsible are unknown. We hypothesize that interactions between truncated MYBPC3 and molecular chaperones could underlie proteasome dysfunction by exhausting a critical chaperone pool required to sustain normal cellular protein quality control. Methods and Results: FLAG-tagged wild-type and two mutant truncated MYBPC3 proteins of different lengths were expressed in neonatal rat cardiomyocytes via adenovirus. Spontaneous contractions per minute were markedly reduced in myocytes expressing mutant compared to wild-type MYBPC3 (non-transduced 49±8; WT 57±7; W1098* 25±3; I154Lfs*5 4±1; n=6, p<0.05). Mutant MYBPC3 expression also caused proteasome dysfunction as evidenced by increased expression of the GFPμ degron reporter (% non-transduced myocytes: WT 55.3±17.6%; W1098* 324.4±117.5%; I154Lfs*5 307.4±52.8%; n=3, p<0.05). Affinity purification mass spectroscopy identified heat shock protein 70kDa (Hsp70) and heat shock cognate 70kDa (Hsc70) as highly abundant interacting proteins with wild-type and W1098* MYBPC3 but not I154Lfs*5. Interaction confidence scores as determined by SAINT analysis were ~3 fold higher for W1098* than for wild-type MYBPC3 (23.5 vs 8.0 for Hsc70 and 77.7 vs. 12.3 for Hsp70). These interactions were confirmed by Western blot and co-localization experiments. In non-transduced myocytes, and in myocytes expressing wild type MYBPC3, Hsc70 showed distinct M-line and Z-disc banding, while in myocytes expressing either mutant MYBPC3, Hsc70 was relocalized from the sarcomere to cytosolic, nuclear, and perinuclear aggregates, similar to non-transduced myocytes that were subjected to heat shock. Conclusions: These results suggest MYBPC3 is a client of Hsp70 chaperones and that mutant proteins may interfere with regular functions and localization of Hsp70 and Hsc70. Further experiments will determine if these interactions are important for maintaining cellular proteostasis in MYBPC3-mutant HCM.