The main results for our team in 2004 are as follows:

 

1) Identification of a homozygous mutation (R403W) in the gene encoding the β myosin heavy chain in FHC, and in vitro functional analysis of the mutated myosin (in collaboration with C. Coirault, INSERM, Unit 572, Paris and T. Eschenhagen, Hamburg University Hospital): highlighting an increase of the mechanical and enzymatic properties of myosin in comparison with normal values (Keller, 2004).

 

2) The team has also highlighted the characterisation of cMyBP-C deficient mice (in collaboration with R. Knöll and K. Chien, UCSD, La Jolla, CA, USA). This is a demonstration of an asymmetric septal hypertrophy in heterozygous mice, and thus constitutes the first murine model presenting the principal characteristic of human familial hypertrophic cardiomyopathy (FHC) (Carrier, 2004).
Our results showed that inactivation of one or both alleles of cMyBP-C lead to different cardiac phenotypes. Heterozygous mice, possessing only a single functional allele, show a slight but significant decrease (-25%, P<0.01) of cMyBP-C level in the heart (see results in the figures below). At 10-11 months, these mice present minor cardiac hypertrophy and moderate ventricular hypertrophy. The development of asymmetric septal hypertrophy (echocardiography and histology) is associated with a considerable increase in interstitial fibrosis.

3) The development and characterisation of transgenic drosophila over-expressing different mutants of human cMyBP-C (in collaboration with L. Röder and M. Sémériva, CNRS, UMR6545, Marseille). The results show that more than 50% of transgenic flies lose their capacity to fly in less than 10 days, and that this effect is retarded when these mutants are crossed with flies with calmodulin deficiency (Vu Manh, 2005)
The incorporation of human cMyBP-C into the indirect flight muscles (IFM) of the flies caused structural anomalies and a phenotype characterised by the absence of flight. This phenotype is worsened with age and gene dosage. Transcriptional analysis of the IFM shows considerable molecular remodelling and especially an over-expression of the gene encoding calmodulin, which plays a central role in the regulation of calcium homeostasis. The crossing of flies over-expressing cMyBP-C with calmodulin-deficient mutants slows the development of the phenotype. This suggested that calmoduline could be a modifier gene in human FHC.

 

4) Molecular and cell analysis of cMyBP-C mutants in cardiac myocytes (in collaboration with T. Eschenhagen, Hamburg University Hospital, Germany, and O. Zolk, University of Erlangen, Germany): Demonstration of the inhibition of the ubiquitin-proteasome system by truncated cMyBP-Cs resulting from FHC MYBPC3 mutations (Sarikas, 2005):
Most MYBPC3 mutations shift the reading frame and produce truncated proteins. However, these were not detected in the cardiac tissue of 4 patients presenting these types of mutations. In this project, we examined if the truncated proteins are degraded by lysosome and/or the ubiquitin-proteasome system. Using adenoviruses, we analysed the expression and localisation of two truncated cMyBP-Cs (M6t and M7t, both identified in FHC patients) in comparison with the normal cMyBP-C found in the cardiomyocytes of new-born rats. The expression of truncated proteins M6t and M7t is significantly decreased compared to normal (see below, left-hand panel). After incubation of the cells with a proteasome inhibitor, expression of the truncated proteins returns to normal level (see below, left-hand panel). This indicates that the mutants are massively and rapidly degraded by the proteasome. In addition, the M7t mutant forms ubiquitin-positive aggregates in the myocytes, suggesting that the proteasome could also be inhibited.. Using an adenovirus reporting proteasome dysfunction (UbiG76V-DsRed, if the cells are red, the proteasome is blocked), we demonstrated that the M7t mutant inhibits the ubiquitin-proteasome system (see below, right-hand panel):