MYBPHLnonsense mutations have poor sarcomere binding, are degraded, and cause abnormal contraction

Abstract

AbstractHeart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation (AF). Recently, a novel sarcomere component, myosin binding protein-H like (MyBP-HL) was identified. MyBP-HL is mainly expressed in cardiac atria and shares homology to the last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). TheMYBPHLR255X mutation has been linked to atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. Similar nonsense mutations inMYBPC3result in no myofilament incorporation and a rapid degradation of the truncated protein and are highly associated with development of hypertrophic cardiomyopathy. However, theMYBPHLR255X mutation occurs too frequently in the human population to be highly pathogenic. We sought to determine whether allMYBPHLnonsense mutations lead to impaired MyBP-HL sarcomere integration and degradation of the mutant protein, or if theMYBPHLR255X mutation has a different consequence. We mimicked humanMYBPHLnonsense mutations in the mouseMybphlcDNA sequence and tested their sarcomere incorporation in neonatal rat cardiomyocytes. We demonstrated that wild type MyBP-HL overexpression showed the expected C-zone sarcomere incorporation, like cMyBP-C. Nonsense mutations showed defective sarcomere incorporation. We demonstrated that wild type MyBP-HL and MyBP-HL nonsense mutations were degraded by both proteasome and calpain mechanisms. Additionally, we observed changes in contraction kinetics and calcium transients in cells transfected with MyBP-HL nonsense mutations compared to MyBP-HL full length. Together, these data support the hypothesis thatMYBPHLnonsense mutations are largely similar.Short summaryPremature stop mutations in myosin binding protein H-like prevent sarcomere incorporation of the translated protein. Overexpression of truncating mutants causes contractile defects in neonatal rat cardiomyocytes. These effects occur regardless of the location of the premature stop along the protein.