👤 Bodvael Fraysse

Hypertrophic cardiomyopathy (HCM) is frequently caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). The mechanisms leading from gene mutations to the HCM phenotype remain incompletely understood, partially because current mouse models of HCM do not faithfully reflect the human situation and early hypertrophy confounds the interpretation of functional alterations. The goal of this study was to evaluate whether myofilament Ca(2+) sensitization and diastolic dysfunction are associated or precede the development of left ventricular hypertrophy (LVH) in HCM. We evaluated the function of skinned and intact cardiac myocytes, as well as the intact heart in a recently developed Mybpc3-targeted knock-in mouse model carrying a point mutation frequently associated with HCM. Compared to wild-type, 10-week old homozygous knock-in mice exhibited i) higher myofilament Ca(2+) sensitivity in skinned ventricular trabeculae, ii) lower diastolic sarcomere length, and faster Ca(2+) transient decay in intact myocytes, and iii) LVH, reduced fractional shortening, lower E/A and E'/A', and higher E/E' ratios by echocardiography and Doppler analysis, suggesting systolic and diastolic dysfunction. In contrast, heterozygous knock-in mice, which mimic the human HCM situation, did not exhibit LVH or systolic dysfunction, but exhibited higher myofilament Ca(2+) sensitivity, faster Ca(2+) transient decay, and diastolic dysfunction. These data demonstrate that myofilament Ca(2+) sensitization and diastolic dysfunction are early phenotypic consequences of Mybpc3 mutations independent of LVH. The accelerated Ca(2+) transients point to compensatory mechanisms directed towards normalization of relaxation. We propose that HCM is a model for diastolic heart failure and this mouse model could be valuable in studying mechanisms and treatment modalities. Show less

Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin-proteasome system in lowering the level of mutant proteins. Show less