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

AimsHypertrophic 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. MethodsWe probed the mechanisms of 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, and (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. ResultsWe found that in patients with HCM who had any MYBPC3 pathogenic variant, 17.9% of them had a missense variant localized to the C3 or C6 subdomain. Unlike truncating variants, missense variants did not reduce MyBP-C content 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) altered proximity of 789 proteins (69.4% increased and 30.5% decreased relative to wild-type MyBP-C). Proteins that were increased in proximity to missense MyBP-C were enriched for proteins within thin filament. ConclusionPathogenic MYBPC3 missense variants within the C3 and C6 subdomains are present in a substantial subset of patients with HCM. Our findings implicate unique mechanisms of these variants distinct from haploinsufficiency, potentially driven in part by enhanced protein-protein interactions with the thin filament within the sarcomere. Translational perspectivePathogenic missense variants in the gene myosin binding protein C (MYBPC3) cause hypertrophic cardiomyopathy via an unknown mechanism. These variants exhibit normal cellular localization and stability. We have demonstrated that these variants are present in 17.9% of patients with HCM and a pathogenic MYBPC3 variant and do not reduce the level of total MyBP-C, the protein encoded by MYBPC3, within human left ventricular tissue. Using Flag-immunoprecipitation and proximity labeling mass spectrometry, we identified differential interacting proteins and proximity proteins, respectively, with proximity labeling mass spectrometry displaying much higher sensitivity. This was particularly true for the detection of potential gain-of-function shifts in MyBP-C environment within the sarcomere. Three of the four pathogenic missense proteins evaluated demonstrated increased proximity to thin filament proteins. This may suggest that particular missense variants within C3 and C6 internal subdomains result in a conformational change that favors thin filament binding and activation. These findings have important implications for the development of sarcomeric modulators that address the underlying mechanism HCM caused by MYBPC3 missense variants.