COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes.

:Mutations in human cardiac myosin-binding protein C (cMyBP-C) gene are associated with familial hypertrophic cardiomyopathy (FHC), and most of them are predicted to produce COOH-truncated proteins. To understand the molecular mechanism(s) by which such mutations cause FHC, we analyzed (i) the accumulation of human cMyBP-C mutants in fetal rat cardiomyocytes, and (ii) the protein sequence of the human wild-type (wt) cMyBP-C by hydrophobic cluster analysis with the aim of identifying new putative myosin-binding site(s). Accumulation and sarcomeric localization of the wt protein and of four FHC-mutant cMyBP-Cs (E542Q and three COOH-truncated proteins) were studied in cardiomyocytes by immunostaining and confocal microscopy after transfection with myc-tagged constructs. We found that: (i) 10 % of the cells expressing COOH-truncated mutants exhibit an incorporation into the A-band of the sarcomere without any alteration of the myofibrillar architecture versus 76 % of those expressing the wt or E542Q mutant cMyBP-Cs (p<0.001); (ii) 90 % of the cells expressing the truncated mutants show a diffuse localization of these proteins in the cardiomyocytes, out of which 45 % exhibit a significant alteration of the sarcomeric structure (p<0.0001 versus wt); and (iii) the two shortest mutant cMyBP-Cs accumulate at very low levels in fetal rat cardiomyocytes as compared to the wt (p<0.008). Protein sequence analysis indicated that a 45-residue sequence in the NH2-terminal C0 domain of cMyBP-C exhibits a consistent homology (sequence similarity score of 42 %) with a segment of the NH2-terminal domain of myomesin, another myosin-binding protein. This result suggests that the C0 domain of human cMyBP-C contains a novel putative myosin-binding site that could account for the A-band incorporation of the truncated mutants. In addition, the faint accumulation and the diffuse localization of truncated mutants could probably be explained by a low affinity of the C0 domain for myosin. We conclude that COOH-truncated cMyBP-Cs may act as poison polypeptides that disrupt the myofibrillar architecture and result in the defects observed in FHC.

Flavigny J ，Souchet M ，Sébillon P ，Berrebi-Bertrand I ，Hainque B ，Mallet A ，Bril A ，Schwartz K ，Carrier L ... -  《JOURNAL OF MOLECULAR BIOLOGY》

Impairment of the ubiquitin-proteasome system by truncated cardiac myosin binding protein C mutants.

Sarikas A ，Carrier L ，Schenke C ，Doll D ，Flavigny J ，Lindenberg KS ，Eschenhagen T ，Zolk O ... -  《CARDIOVASCULAR RESEARCH》

Localization of the binding site of the C-terminal domain of cardiac myosin-binding protein-C on the myosin rod.

Flashman E ，Watkins H ，Redwood C 《-》

Mutations in beta-myosin S2 that cause familial hypertrophic cardiomyopathy (FHC) abolish the interaction with the regulatory domain of myosin-binding protein-C.

:The myosin filaments of striated muscle contain a family of enigmatic myosin-binding proteins (MyBP), MyBP-C and MyBP-H. These modular proteins of the intracellular immunoglobulin superfamily contain unique domains near their N termini. The N-terminal domain of cardiac MyBP-C, the MyBP-C motif, contains additional phosphorylation sites and may regulate contraction in a phosphorylation dependent way. In contrast to the C terminus, which binds to the light meromyosin portion of the myosin rod, the interactions of this domain are unknown. We demonstrate that fragments of MyBP-C containing the MyBP-C motif localise to the sarcomeric A-band in cardiomyocytes and isolated myofibrils, without affecting sarcomere structure. The binding site for the MyBP-C motif resides in the N-terminal 126 residues of the S2 segment of the myosin rod. In this region, several mutations in beta-myosin are associated with FHC; however, their molecular implications remained unclear. We show that two representative FHC mutations in beta-myosin S2, R870H and E924K, drastically reduce MyBP-C binding (Kd approximately 60 microM for R870H compared with a Kd of approximately 5 microM for the wild-type) down to undetectable levels (E924K). These mutations do not affect the coiled-coil structure of myosin. We suggest that the regulatory function of MyBP-C is mediated by the interaction with S2, and that mutations in beta-myosin S2 may act by altering the interactions with MyBP-C.

Gruen M ，Gautel M 《JOURNAL OF MOLECULAR BIOLOGY》

Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy. Characterization Of cardiac transcript and protein.

Rottbauer W ，Gautel M ，Zehelein J ，Labeit S ，Franz WM ，Fischer C ，Vollrath B ，Mall G ，Dietz R ，Kübler W ，Katus HA ... -  《JOURNAL OF CLINICAL INVESTIGATION》