Cardiac myosin binding protein C (MYBPC3) is the most commonly mutated gene associated with hypertrophic cardiomyopathy (HCM). Haploinsufficiency of full-length MYBPC3 and disruption of proteostasis have both been proposed as central to HCM disease pathogenesis. Discriminating the relative contributions of these 2 mechanisms requires fundamental knowledge of how turnover of WT and mutant MYBPC3 proteins is regulated. We expressed several disease-causing mutations in MYBPC3 in primary neonatal rat ventricular cardiomyocytes. In contrast to WT MYBPC3, mutant proteins showed reduced expression and failed to localize to the sarcomere. In an unbiased coimmunoprecipitation/mass spectrometry screen, we identified HSP70-family chaperones as interactors of both WT and mutant MYBPC3. Heat shock cognate 70 kDa (HSC70) was the most abundant chaperone interactor. Knockdown of HSC70 significantly slowed degradation of both WT and mutant MYBPC3, while pharmacologic activation of HSC70 and HSP70 accelerated degradation. HSC70 was expressed in discrete striations in the sarcomere. Expression of mutant MYBPC3 did not affect HSC70 localization, nor did it induce a protein folding stress response or ubiquitin proteasome dysfunction. Together these data suggest that WT and mutant MYBPC3 proteins are clients for HSC70, and that the HSC70 chaperone system plays a major role in regulating MYBPC3 protein turnover.
Amelia A. Glazier, Neha Hafeez, Dattatreya Mellacheruvu, Venkatesha Basrur, Alexey I. Nesvizhskii, Lap Man Lee, Hao Shao, Vi Tang, Jaime M. Yob, Jason E. Gestwicki, Adam S. Helms, Sharlene M. Day
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