Cardiac myosin binding protein C mutants interact with Hsp70‐family chaperones and disrupt protein quality control
Authors: Glazier Amelia Ann , Helms Adam , Kotlo Srisha , Hafeez Neha , Yob Jaime , Tang Vi , Mellacheruvu Dattatreya , Nesvizhskii Alexey , Day Sharlene 
Published: November 2023

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease with mutations in cardiac myosin binding protein C (MYBPC3) being most prevalent. Mechanisms underlying pathogenesis of MYBPC3‐linked HCM remain unclear. >90% of MYBPC3 mutations are nonsense and whether these mutations lead to loss‐ or gain‐of‐function is unresolved. Expression of mutant MYBPC3 in neonatal rat ventricular cardiomyocytes (NRVMs) is sufficient to induce UPS dysfunction, suggesting a gain‐of‐function mechanism affecting proteostasis is present. Myocardium from patients with MYBPC3 mutations displays impaired ubiquitin proteasome system (UPS) activity. In preliminary studies, transcripts of stress‐responsive chaperones Hsp70 and BiP were significantly elevated. The objective of this study was to identify whether MYBPC3 is a client of Hsp70‐family chaperones and to evaluate if this interaction contributes to proteostatic burden in cardiomyocytes.We generated constructs of WT and mutant MYBPC3 expressed via adenovirus in NRVMs. Expression of mutant MYBPC3 induced accumulation of the UPS reporter GFPu compared to WT and untreated cells, indicating impaired UPS function. To investigate whether MYBPC3 and Hsp70 chaperones interact, we performed affinity purification‐mass spectrometry of MYBPC3. Several major Hsp70‐family proteins were identified, and Hsc70 in particular had enriched interaction with some MYBPC3 mutants. Immunofluorescence showed Hsc70 localized to the sarcomere in both NRVMs and human myocardium, placing it in close proximity to MYBPC3. To determine if Hsc70 functionally interacts with MYBPC3, we measured degradation rates of MYBPC3 by cycloheximide chase assay. siRNA knockdown of Hsc70 resulted in a significantly longer t½ for both WT and mutant MYBPC3. Conversely, enhancement of Hsc70 activity using the small molecule activator YM‐1 accelerated MYBPC3 degradation. Interestingly, YM‐1 dramatically reduced the t½ of W1098* MYBPC3 mutant compared to WT. In order to examine the effect of mutant MYBPC3 expression on Hsc70, we measured uptake of transferrin by clathrin‐mediated endocytosis, a critical Hsc70‐dependent function. Fluorescently‐labeled transferrin uptake was significantly decreased in NRVMs expressing mutant MYBPC3 compared to WT and untreated cells, suggesting an adverse effect on Hsc70 activity.We conclude that mutant MYBPC3 significantly impacts proteostatic systems including the UPS and Hsc70 function. Evidence of physical and functional interactions indicate MYBPC3 is a client for Hsp70 chaperones and that Hsc70 plays a role in its degradation. Additionally, mutant MYBPC3 may be selectively targeted by Hsp70 chaperones, as YM‐1 demonstrated a greater effect on the t½ of W1098* MYBPC3. Furthermore, expression of mutant MYBPC3 interferes with normal Hsc70 activity. Overall, these results support the hypothesis that proteotoxic gain‐of‐function effect of mutant MYBPC3 may potentially be relevant to HCM pathogenesis.Support or Funding InformationAHA 15PRE25090023NHLBI F31 HL131327‐01NIH T32 GM008322