Abstract Body (Do not enter title and authors here): Hypertrophic cardiomyopathy (HCM) is one of the common genetic heart diseases and causes hypercontractility and pathological thickening of the myocardium. Cardiac myosin binding protein-C (cMyBP-C) is an important regulator of cardiac contractility and MYBPC3 mutations accounts for 40% of HCM cases, resulting in the truncation and loss of cMyBP-C. While cMyBP-C is expressed in both atria and ventricle, its recently identified homologous partner, myosin binding protein-HL (MyBP-HL) is solely expressed in the atria. Loss of function mutations in MYBPHL cause dilated cardiomyopathy (DCM) in humans and mice. cMyBP-C and MyBP-HL bind to specific positions on the thick filament within the C-zone of sarcomere. Within the atria, there is a 50:50 ratio between MyBP-HL and cMyBP-C, whereas the ventricle solely consists of cMyBP-C, specifically double the amount found within the atria, establishing a stoichiometric relationship between these two proteins in their ability to bind to the thick filament within the sarcomere.
Within the ventricle, MYBPC3 missense mutations within the myosin binding regions would not result in cardiac dysregulation unless these mutations prevent proper myosin binding and sarcomere incorporation. Since the atria expresses MyBP-HL and cMyBP-C, we hypothesize that MYBPC3 and MYBPHL missense mutations are more severe due to the competition for binding sites within the sarcomere. We have identified uncertain MYBPC3 and MYBPHL missense mutations to evaluate their pathogenicity and sarcomere incorporation via immunofluorescence microscopy within neonatal rat ventricular cardiomyocytes. To test these mutations, we created a construct, named Mini-C, consisting of the thick filament binding domains of MyBP-C. We have validated the use of our Mini-C construct via transfection and co-localization with endogenous cMyBP-C within neonatal rat ventricular cardiomyocytes (NRVMs). Our data demonstrates MYBPHL missense mutations have shown improper sarcomere incorporation while MYBPC3 missense mutations have variable changes in proper sarcomere incorporation within NRVMs. To test the effects of missense mutations on myosin binding protein stoichiometry within the sarcomere, we have created and validated a T2A/P2A construct, expressing our Mini-C construct, a fluorescent reporter, Td-Tomato, and our hMyBP-HL construct at consistent and reproducible expression levels via western blot and mass spectrometry.