Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246 Hamburg, Germany.
Giulia
G
Mearini
Christina
C
Gedicke
Saskia
S
Schlossarek
Christian C
CC
Witt
Elisabeth
E
Krämer
Peirang
P
Cao
Marcelo D
MD
Gomes
Stewart H
SH
Lecker
Siegfried
S
Labeit
Monte S
MS
Willis
Thomas
T
Eschenhagen
Lucie
L
Carrier
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D051379:Mice; D009124:Muscle Proteins / Q000502:physiology*; D009206:Myocardium / Q000737:chemistry*; D032383:Myocytes, Cardiac / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000502:physiology; D044843:SKP Cullin F-Box Protein Ligases / Q000502:physiology*; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D009124:Muscle Proteins; D046988:Proteasome Endopeptidase Complex; D044843:SKP Cullin F-Box Protein Ligases; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases; D000099163:Myosin Binding Protein C; C486691:Fbxo32 protein, mouse; C499084:Trim63 protein, mouse