Nonsense Mutations In Atrial Myofilament Regulatory Protein MyBP-HL Are Subject To Proteolytic Degradation And Loss Of Functional Protein

Background: Myosin binding protein-H like (MyBP-HL) is a recently identified atrial myofilament protein encoded by the gene MYBPHL. The Arg255Stop mutant has been linked to atrial enlargement, atrial fibrillation, and dilated cardiomyopathy. Deletion of MYBPHL in mice recapitulates this human phenotype. MyBP-HL is composed of two Immunoglobulin domains (Ig) and one Fibronectin domain (FnIII), like the structure and sequence of the last three C-terminal domains (C8-C9-C10) of cardiac myosin binding protein-C (cMyBP-C). cMyBP-C nonsense mutations fail in myofilament incorporation and result in a rapid degradation of the truncated protein and are a major cause of hypertrophic cardiomyopathy. There are 9 premature stop mutations in MYBPHL identified in humans (Gln29, Trp54, Arg113, Tyr123, Trp158, Trp192, Lys250, Arg255, and Tyr307). Hypothesis: We hypothesize that MYBPHL nonsense mutations will share a common mechanism that leads to MyBP-HL failing to integrate into the sarcomere, resulting in truncated MyBP-HL degradation and loss myofilament incorporation. Results: We demonstrate heterologous expression of MyBP-HL in neonatal rat cardiomyocytes can incorporate in a similar sarcomere pattern as cMyBP-C. Expression of the nine nonsense mutations do not localize into the sarcomere. Neonatal rat cardiomyocytes transfected with MyBP-HL constructs were treated with proteasome and/or calpain inhibitors. We observed that wild-type MyBP-HL and MyBP-HL truncating mutations are degraded mainly by the proteasome, although we also identified a significant calpain involvement in MyBP-HL degradation. Shorter MyBP-HL truncated peptides are degraded as a poisoning peptides, but longer peptides are not degraded. Conclusion: Based on these data, we demonstrated that MYBPHL nonsense mutations prevent MyBP-HL incorporation into the thick filament as are subject to degradation. The differential expression of short vs. long truncating MyBP-HL mutants suggest a differential pathogenic mechanism for subsets of these mutations.