Abstract 2729: Atrogin-1 and MuRF1 Mediate Degradation of Cardiac Myosin-binding Protein C by the Ubiquitin-Proteasome System
Background. Familial hypertrophic cardiomyopathy (FHC) is a sarcomeropathy frequently associated with cardiac myosin-binding protein C (cMyBP-C) gene mutations. Most of them result in a frameshift and C-terminal truncated proteins. However, truncated mutants were not detected in myocardial tissue of FHC patients. A recent study showed that a truncated cMyBP-C (M7t) is rapidly and quantitatively degraded by the ubiquitin-proteasome system (UPS) after adenoviral gene transfer in neonatal rat cardiac myocytes (NRCM). Since the diversity and specificity of UPS regulation lies in E3 ubiquitin ligases, which specifically target proteins and direct the ubiquitination process, the present study investigated whether the sarcomere-specific E3 ligases, atrogin-1 and MuRF1, mediate degradation of wild-type (WT) and truncated M7t cMyBP-C.
Methods and Results. NRCM were co-infected with atrogin-1 and WT or M7t cMyBP-C adenoviruses. Both overexpressed WT and M7t were co-immunoprecipitated with atrogin-1 suggesting that both forms of cMyBP-C interact with atrogin-1. However, whereas the amount of overexpressed WT cMyBP-C did not change, the M7t protein level was 80% lower in NRCM infected with atrogin-1 than without. This suggests that atrogin-1 is the E3 ligase mediating proteasome degradation of truncated M7t cMyBP-C only. We then investigated whether MuRF1 could regulate the level of endogenous cMyBP-C in MuRF1-KO mice and compared to WT. The heart to body weight ratio and the chymotrypsine-like activity of the proteasome were similar in KO and WT mice. The mRNA levels of cMyBP-C, and other E3 ligases, atrogin-1 and MuRF3, were similar between KO and WT ventricles. Strikingly, the protein level of cMyBP-C was 150% and 30% higher in neonatal and 24 wks-old KO compared to age-matched WT mice.
Conclusion. These data suggest that
atrogin-1, located at the Z-band, mediates the proteasome degradation of truncated M7t cMyBP-C resulting from a FHC mutation,
the lack of MuRF1 is not compensated by other E3 ligases and is not associated with hypertrophy in KO mice, and
most importantly, MuRF1, mainly located at the M-band, specifically regulates the protein level of cMyBP-C.