An unbiased screen identified the Hsp70-BAG3 complex as a regulator of myosin binding protein C3 stability.

Abstract: Objective: We aim to identify regulators of myosin binding protein C3 (MyBP-C) protein homeostasis. Background: Variants in myosin binding protein C3 (MYBPC3) account for approximately 50% of familial hypertrophic cardiomyopathy (HCM). Most pathogenic variants in MYBPC3 are truncating variants that lead to reduced total levels of MyBP-C protein. Elucidation of the pathways that regulate MyBP-C protein homeostasis could uncover new therapeutic strategies that restore normal protein levels. Method: We developed a high-throughput screen to identify compounds that can increase or decrease steady-state levels of MyBP-C in an induced pluripotent stem cell cardiomyocyte (iPSC-CM) model derived from a patient with HCM. To normalize results, we also monitored effects on myosin heavy chain (MYH) and focused on those molecules that selectively modulated MyBP-C levels. Results: Screening a library of 2,426 known biologically active compounds, we identified compounds which either decreased (241/2426, 9.9%) or increased (29/2426, 1.2%) MyBP-C/MYH levels. After a rigorous validation process, including a counter screen for cellular toxicity, two compounds (JG98 and parthenolide) were confirmed as decreasing MyBP-C levels and no compounds were confirmed to increase MyBP-C levels. For further studies, we focused on JG98, which is an allosteric modulator of heat shock protein 70 (Hsp70), inhibiting its interaction with BAG domain co-chaperones. We found that genetic reduction of BAG3 phenocopies treatment with JG98 by reducing MyBP-C protein levels. Conclusion: An unbiased compound screen identified the Hsp70-BAG3 complex as a regulator of MyBP-C stability. Thus, approaches that stimulate function of this complex may be beneficial in the treatment of HCM.