OBJECTIVE: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHC patients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin-proteasome system (UPS). METHODS AND RESULTS: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHC patients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70+/-4% and 11+/-5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. CONCLUSION: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.
OBJECTIVE: Most cardiac myosin binding protein C (cMyBP-C) gene mutations causing familial hypertrophic cardiomyopathy (FHC) result in C-terminal truncated proteins. However, truncated cMyBP-Cs were undetectable in myocardial tissue of FHCpatients. In the present study, we investigated whether truncated cMyBP-Cs are subject to accelerated degradation by the lysosome or ubiquitin-proteasome system (UPS). METHODS AND RESULTS: By using an adenovirus-based approach, we analyzed expression and localization of myc-tagged truncated proteins (M6t 3%, M7t 80% truncation, both mutations have been identified in FHCpatients) compared to wild type (WT) in neonatal rat cardiomyocytes. Despite similar mRNA levels, protein expression of M6t and M7t was markedly lower than WT (70+/-4% and 11+/-5% of WT, respectively, p<0.05). M6t exhibited weak incorporation in the sarcomere, whereas M7t was mis-incorporated at the Z-disk and formed ubiquitin-positive aggregates. The lysosome inhibitor bafilomycin only slightly raised the protein level of M7t, whereas the UPS inhibitors lactacystin or MG132 markedly raised M6t and M7t to WT level. Using an adenovirus encoding a fluorescent reporter of UPS activity, we demonstrate that mutant cMyBP-Cs impair the proteolytic capacity of the UPS. CONCLUSION: Truncated cMyBP-Cs are preferentially degraded by the UPS, which, in turn, may competitively inhibit breakdown of other UPS substrates. Since the UPS plays an important role in a variety of fundamental cellular processes, we propose impairment of this system by mutant cMyBP-Cs as a contributing factor to the pathogenesis of FHC.