Cardiovascular Research Advance Access first published online on October 22, 2009
This version [Corrected Proof] published online on November 18, 2009
Cardiovascular Research, doi:10.1093/cvr/cvp348
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Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms
1 Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany
2 Institute of Anesthesiology and Operative Intensive Medicine, University of Mannheim, Mannheim, Germany
3 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
4 Department of Biochemistry and Immunology, Faculty of Medicine Ribeir
o Preto, University of So Paulo, So Paulo, Brazil
5 Carolina Cardiovascular Biology Center, University of North Carolina, Chapell Hill, NC, USA
6 Inserm, U974, Institut de Myologie, Paris F-75013, France
7 Université Pierre et Marie Curie-Paris 6, UMR-S974, CNRS, UMR7215, Institut de Myologie, IFR14, Paris F-75013, France
* Corresponding author. Tel: +49 40 7410 57208; fax: +49 40 7410 55925. E-mail: l.carrier{at}uke.uni-hamburg.de
Aims: Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin–proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C.
Methods and results: Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels.
Conclusion: These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.
KEYWORDS E3 ubiquitin ligase; Sarcomere; Myosin-binding protein C; MuRF1; Atrogin-1
Time for primary review: 21 days