Limb Girdle Muscular Dystrophies are rare genetic diseases, characterized by weakness and progressive muscular atrophy. A subfamily of LGMD2 regroups sarcoglycanopathies caused by mutations in genes coding for α/β/γ or δ sarcoglycans (SG). These transmembrane proteins are part of the dystrophin complex which is an important link between the actin cytoskeleton, the sarcolemma and the extracellular matrix that protects muscle fibers against mechanical stress due to contraction. There is no treatment available for these diseases.

More than 65% of mutations in SG genes are missense. One of the most frequent mutations is R77C in α-SG gene. This mutation accounts for up to one-third of all mutations in European populations except in Finland where it is found in every LGMD2D patients often in both alleles.

Previous studies demonstrated that SG missense mutants are not present at the muscle fiber membrane because they are retained in the endoplasmic reticulum by the quality control (ERQC) and they are prematurely degraded by the proteasome.

In order to understand the mechanisms implicated in sarcoglycanopathies and to identify new therapeutic targets, we decided (i) to study, at the molecular level, the ERCQ pathways responsible for sarcoglycan disposal and susceptible of pharmacological intervention and (ii) to test small molecules modulating ERQC pathways and promoting rescue of disease related mutations. We first generated in vitro and in vivo models to investigate the SGs cellular trafficking mechanisms. Cell lines expressing one α-SG mutant were used to test approved drugs selected towards their known action of the ERQC. We identified promising pharmacological compounds promoting α-SG mutants membrane localization. We are now developing a cytometry protocol using Imagestream technology in order to quantify the effect of the molecules on the α-SG mutants retargeting.

In parallel, we will take advantage of these in vitro and in vivo models to screen small molecules libraries for compounds being able to rescue the targeting or the folding of the mutated proteins.

Diverse forms of cardiomyopathies, result in cardiac compensatory remodelling that may progress to ventricular dilation and heart failure. This remodeling process is characterised by an increase in the size of cardiomyocytes associated with elevated rates of RNA synthesis, the activation of a foetal cardiac gene program and concomitant repression of corresponding adult cardiac genes that regulate cardiac contractility. It has been shown that Bcl11b, a C2H2 zinc finger protein, interacts with the RNApolymeraseII regulatory complex pTefb and exert an inhibitory action on it. Microarray data comparisons showed that Bcl11b could modulate the expression of genes during cardiac hypertrophy. Furthermore, a genetic study revealed that common genetic variations in a locus harboring this gene is associated with increased risk for cardiovascular disease making it relevant to study the role of Bcl11b in the heart under normal and pathological conditions. Our aim is to determine the role of bcl11b in regulating transcription during cardiac hypertrophy. Through the use of various techniques (Western Blot, immunoprecipitation), we were first able to characterize a post-translational modification (SUMOylation) of Bcl11b in the context of cardiac hypertrophy. This SUMOylation was specific to the hypertrophic state of the heart both under physiological and pathological conditions. In addition, using the chromatin immunoprecipitation (ChIP) technique on cardiac tissue, the binding of Bcl11b on the promoters of genes involved in cardiac hypertrophy was studied (βMHC, skeletal and cardiac actin, ANF). An enrichment of Bcl11b recruitment at the level of βMHC, skeletal and cardiac actin promoters was observed in mice with induced hypertrophy (phenylephrin osmotic mini-pump treatment) as compared to control. This enrichment was correlated with an increase in the expression of the βMHC and skeletal actin genes and anti-correlated with the expression of the cardiac actin gene suggesting a dual role for Bcl11b (activator versus inhibitor). Based on these preliminary data and on the literature, we speculate that the status of Bcl11b (sumoylated versus unsumoylated state) could be implicated in this dual response. Our results will allow gaining further insights into the molecular mechanisms that govern the onset of pathological cardiac hypertrophy and more specifically the role of Bcl11b and its SUMOylation in the regulation of gene expression during cardiac hypertrophy. 

Excitation-contraction coupling relies on spatial organization of triad membranes and on the precise localization of proteins of the Calcium Release Complex (CRC) in these membranes. Sarcoplasmic Reticulum (SR) proteins of the CRC are exclusively found in terminal cisternae (TC) of the reticulum, but the mechanisms responsible for their traffic and retention in this compartment are poorly defined. Among CRC proteins, triadin was proposed to act as an anchor for the other reticulum proteins at triads. To investigate the mechanisms leading to protein targeting to triads and to the organization of triad membranes in muscle cells, we have expressed fluorescent chimeras of triadin in differencing primary myotubes from triadin KO mice.

The mobility of GFP-triadin was recorded during cell differentiation. Although fixed at late differentiation stages, SR membranes labeled with GFP-Triadin can undergo short and long distance movements at earlier stages. These movements of SR membranes require intact microtubule cytoskeleton, and may be necessary for triad organization at the A-I junctions. A photoactivatable version of Triadin (PAGFP-Triadin) was used to study the dynamics of limited pools of activated molecules. At both early and late myotube differentiation stages a continuous diffusion of triadin molecules in the SR was revealed. Triadin accumulation in the TC of SR was shown to depend on its C-terminal luminal domain. Overall, our experiments reveal that clusters of triadin move along microtubules during early differentiation stage. These movements could reflect TC formation and organization along the sarcomeres during myotube differentiation. During this process, and also at later differentiation stages when TC have a fixed localization, a continuous diffusion of triadin allows its traffic to the TC. We show that the exclusive localization of Triadin in TC rely,at least in part, on a retention-based mechanism mediated by its C-terminal part.