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
J
INSERM UR523, Institut de Myologie, Paris, France.
Souchet
M
M
Sébillon
P
P
Berrebi-Bertrand
I
I
Hainque
B
B
Mallet
A
A
Bril
A
A
Schwartz
K
K
Carrier
L
L
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells / Q000378:metabolism; D002312:Cardiomyopathy, Hypertrophic / Q000235:genetics* / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D064211:Connectin; D015870:Gene Expression; D006321:Heart / Q000196:embryology; D006801:Humans; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012518:Sarcomeres / Q000378:metabolism; D020539:Sequence Analysis, Protein; D000099163:Myosin Binding Protein C
D002352:Carrier Proteins; D064211:Connectin; D009124:Muscle Proteins; D009218:Myosins; D011994:Recombinant Proteins; D000099163:Myosin Binding Protein C