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

• Published in: Journal of molecular biology 1999-11, Vol.294 (2), p.443-456
• Main Authors: Flavigny, Jeanne, Souchet, Michel, Sébillon, Pascale, Berrebi-Bertrand, Isabelle, Hainque, Bernard, Mallet, Alain, Bril, Antoine, Schwartz, Ketty, Carrier, Lucie
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Animals; Binding Sites; cardiac myosin-binding protein C; cardiomyocytes; Cardiomyopathy, Hypertrophic - genetics; Cardiomyopathy, Hypertrophic - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cells, Cultured; cMyBP-C gene; Connectin; COS Cells - metabolism; familial hypertrophic cardiomyopathy; Gene Expression; Heart - embryology; Humans; Molecular Sequence Data; Muscle Proteins - metabolism; Mutation; mutations; Myocardium - cytology; Myocardium - metabolism; myomesin; myosin-binding protein; Myosins - metabolism; protein sequence analysis; Rats; Rats, Wistar; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sarcomeres - metabolism; Sequence Analysis, Protein
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1999.3276

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 NH 2-terminal C0 domain of cMyBP-C exhibits a consistent homology (sequence similarity score of 42 %) with a segment of the NH 2-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.