The most commonly mutated gene in hypertrophic cardiomyopathy (HCM) is cardiac myosin binding protein C (MYBPC3). Over 90% of MYBPC3 mutations are nonsense, but whether these mutations manifest in loss- or gain-of-function is unresolved. Evidence suggests MYBPC3 mutants impact protein quality control mechanisms. The objective of this study was to evaluate interactions of MYBPC3 with proteostatic systems and test the hypothesis that these interactions affect protein homeostasis in cardiomyocytes.
WT and mutant MYBPC3 constructs were expressed in neonatal rat ventricular myocytes (NRVMs) via adenovirus. Mutant MYBPC3 induced ubiquitin proteasome system reporter GFPu accumulation (fold increase in GFPu-positive cells vs control: WT 138±14.0%, mutant 198±27.2%, mean±SEM, p<0.05 vs control and WT), indicating proteasome dysfunction. Affinity purification/mass spectrometry identified molecular chaperones Hsp70 and Hsc70 as prominent interactors with MYBPC3. We observed MYBPC3 degradation by cycloheximide chase in response to Hsc70 siRNA knockdown or pharmacological treatment with Hsp70 activator YM-1. Hsc70 knockdown slowed degradation of WT and mutant MYBPC3 (WT control t
½
=5.47±0.70 hr, WT Hsc70 knockdown t
½
=13.5±1.62 hr; mutant control t
½
=3.42±0.61 hr, mutant Hsc70 knockdown t
½
=9.87±0.95 hr), while YM-1 treatment accelerated degradation (WT DMSO t
½
=10.2±3.28 hr, WT YM-1 t
½
=3.16±0.61 hr; mutant DMSO t
½
=11.7±2.67 hr, mutant YM-1 t
½
=1.37±0.16 hr). We then evaluated whether transferrin uptake via clathrin mediated endocytosis, a critical Hsc70-dependent activity, was affected by mutant MYBPC3. Transferrin uptake was significantly decreased in NRVMs expressing mutant MYBPC3 compared to WT and untreated controls (transferrin-positive cells: control 22.93±3.34%, WT 17.47±0.70%, mutant 9.30±1.63%, mean±SEM, p<0.05 vs control and WT).
In conclusion, we have demonstrated that Hsp70 chaperones interact with MYBPC3 in cardiomyocytes and affect MYBPC3 degradation, suggesting MYBPC3 is a client of Hsp70 and Hsc70. Additionally, expression of mutant MYBPC3 causes ubiquitin proteasome impairment and interferes with normal Hsc70 function. These results support our hypothesis that mutant MYBPC3 affects protein homeostasis in HCM.
Ubiquitin-Proteasome System Impairment Caused by a Missense Cardiac Myosin-binding Protein C Mutation and Associated with Cardiac Dysfunction in Hypertrophic Cardiomyopathy
