Effect of cardiac myosin-binding protein C on stability of the thick filament.

In contrast to skeletal muscle isoforms of myosin-binding protein C (MyBP-C), the cardiac isoform has 11 rather than 10 modules (labeled C0-C10, N-C terminus), three phosphorylation sites between C1 and C2, and 28 additional amino acids in C5. Within the C5-C10 region of cardiac MyBP-C (cMyBP-C) there are interactions between C5 and C8 as well as C7 and C10. Isolated skinned cardiac trabeculae were incubated with one of three recombinant fragments of cMyBP-C to interfere with interactions of endogenous C5. 2-10 microM C5 or C5-containing peptide fragments of cMyBP-C reversibly reduced Ca sensitivity without extracting myofibrillar protein. C2-C4 fragments had no effect. This result indicated that the region of cMyBP-C that contains C5 maintains a specific structural arrangement of myosin that helps set its contractile properties. Greater than 10 microM C5 caused skinned trabeculae to lose a substantial amount of cMyBP-C and some myosin heavy chain, resulting in irreversible decline in maximum Ca-activated force. MyBP-C appears to stabilize the structure of the thick filament and modulate the way in which myosin heads extend to the thin filament.

COOH-terminal truncated human cardiac MyBP-C alters myosin filament organization.

Myosin-binding protein C (MyBP-C) is thought to play structural and/or regulatory role in striated muscles. The cardiac isoform of MyBP-C is one of the disease genes associated with familial hypertrophic cardiomyopathy and most of the mutations produce COOH truncated proteins. In order to determine the consequences of these mutations on myosin filament organization, we have characterized the effect of a 52-kDa NH2-terminal peptide of human cardiac MyBP-C on the alpha-myosin heavy chain (alpha-MyHC) filament organization. This peptide lacks the COOH-terminal MyHC-binding site and retains the two MyHC-binding domains located in the N-terminal part of MyBP-C. For this characterization, cDNA constructs (rat alpha-MyHC, full-length and truncated human cardiac MyBP-C) were transiently expressed singly or in pairwise combination in COS cells. In conformity with previous works performed on the skeletal isoform of MyBP-C, we observed that full-length cardiac MyBP-C organizes the MyHC into dense structures of uniform width. While the truncated protein is stable and can interact with MyHC in COS cells, it does not result in the same organization of sarcomeric MyHC that is seen with the full-length MyBP-C. These results suggest that the presence of truncated cardiac MyBP-C could, at least partly, disorganize the sarcomeric structure in patients with familial hypertrophic cardiomyopathy.

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.

Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy.

Five disease genes encoding sarcomeric proteins and associated with familial and classical forms of hypertrophic cardiomyopathy have been determined since 1989. In 1996 two other genes encoding ventricular regulatory and essential myosin light chains were shown to be associated with a particular phenotype of the disease characterized by mid left ventricular obstruction. The aim of the present study was to search for mutations in the ventricular regulatory myosin light chain gene (MYL2), located on chromosome 12q23q24.3, in a panel of 42 probands presenting a classical phenotype of familial hypertrophic cardiomyopathy. Single-strand conformation polymorphism analysis was used to search for mutations in the coding segments of the MYL2 gene, and the abnormal products were sequenced. Two novel missense mutations, Phe18Leu in exon 2 and Arg58Gln in exon 4 were identified in three unrelated families. None of the affected patients had hypertrophy localized only at the level of the papillary muscle with mid left ventricular obstruction. By analysis of genetic recombinations, one of these mutations identified in a large family allowed us to refine the localization of the MYL2 gene on the genetic map, in an interval of 6 cM containing six informative microsatellite markers. In conclusion, we show that mutations in the MYL2 gene may be involved in familial and classical forms of hypertrophic cardiomyopathy, and we provide new tools for the genetic analysis of patients with familial hypertrophic cardiomyopathy.