Pathogenic MYBPC3 missense variants alter protein-protein interactions within the sarcomere

Aims: Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that leads to left ventricular hypertrophy, heart failure, and arrhythmias. Pathogenic missense variants in the gene myosin binding protein C (MYBPC3) cluster within its internal subdomains C3 and C6. The protein, myosin binding protein C (MyBP-C), expressing these variants, normally localizes to the myofilaments, leaving uncertainty regarding the mechanism(s) by which they cause HCM. Methods: We further characterized these variants by analyzing (1) their prevalence in an international registry of patients with HCM, (2) total MyBP-C levels and the allelic fraction of mutant MyBP-C in human left ventricular myectomy heart tissue, (3) performing flag-immunoprecipitation and proximity labeling mass spectrometry of wild-type MyBP-C and four pathogenic missense variants (Arg495Gln, Arg502Trp- C3 subdomain, Trp792Arg, Arg810His- C6 subdomain) to determine the change in MyBP-C interacting and proximity proteins induced by these variants. Results: We found that 17.9% of patients with HCM and a pathogenic MYBPC3 variant had a pathogenic missense variant within the C3 or C6 subdomain. Unlike truncating variants, these missense variants did not reduce MyBP-C relative to myosin. The mutant allelic fraction of MyBP-C varied from 10-67.0% across samples. Flag-immunoprecipitation mass spectrometry identified 252 MyBP-C interacting proteins. Pathogenic missense variants disrupted 23 MyBP-C protein interactions, including lysosomal Ragulator-Rag complex proteins (Rraga, Rragc, LAMTOR4). Proximity labeling mass spectrometry was more sensitive, identifying 3,240 MyBP-C proximity proteins. Pathogenic missense variant (s) induced altered proximity in 789 proteins (69.4% increased and 30.5% decreased). MyBP-C Arg502Trp, Trp792Arg, and Arg810His increased proximity to the troponin complex within the thin filaments. Conclusion: Pathogenic MYBPC3 missense variants within the C3 and C6 subdomains represent a significant subset of HCM with normal total MyBP-C protein levels and sarcomere localization. Our work characterizes changes in MyBP-C interacting and proximity proteins induced by these pathogenic variants.