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

Heart 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). The MYBPHL R255X mutation has been linked to atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. Similar nonsense mutations in MYBPC3 result in no myofilament incorporation and a rapid degradation of the truncated protein and are highly associated with development of hypertrophic cardiomyopathy. However, the MYBPHL R255X mutation occurs too frequently in the human population to be highly pathogenic. We sought to determine whether all MYBPHL nonsense mutations lead to impaired MyBP-HL sarcomere integration and degradation of the mutant protein, or if the MYBPHL R255X mutation has a different consequence. We mimicked human MYBPHL nonsense mutations in the mouse Mybphl cDNA 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 that MYBPHL nonsense mutations are largely similar.