Résumé :
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Calcium dysbalance is expected to be one of the triggering events causing muscular degeneration in Duchenne muscular dystrophy (DMD). It has been proposed that the increased Ca2+ influx could result from transient membrane lesions (Menke et al., 1995; Petrof et al., 1993) or from influx through plasma membrane Ca2+ channels. Recent results indicate that the increased permeability of the sarcolemma of dystrophic fibres may be due to increased activity of cationic channels belonging to the TRP family which could result in an increase of Ca2+ concentration. Both, stretch-activated channels (SAC) and store-operated channels (SOC) have been proposed to be involved in the enhanced Ca2+ entry occurring in dystrophic skeletal muscle cell. In our study we focused mainly on TRPC family. We and others have shown that TRPC1 as well as TRPC 2,3,4 and 6 are expressed in skeletal muscle and were proposed to be candidates for these Ca2+ channels (Vandebrouck et al., 2002). In a series of experiments we have shown by quantitative RT-PCR that TRPC3 mRNA is overexpressed in mdx muscles compared to wt. This prompted us to consider TRPC3 as a potential candidate for the increase of Ca2+ influx described in mdx muscle. TRPC1 has been described as important and necessary for (SOC) and (SAC). Therefore we have cloned a series of shRNAs directed against TRPC1 an TRPC3 into the adeno-associated viral vector type 2 and 6 (pAAV) together with the Pol III promoter H1. We have validated the efficiency of our constructions in heterologous systems and we have obtained an inhibition of TRPC3 and TRPC1 mRNAs from 50% up to 90%. We are currently producing high titer viruses in order to transduce our myotubes from EDL-mdx cell line as well as mdx FDB fibres and we will analyse the effect of their inhibition on calcium
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