Role of TRP channels in normal skeletal muscle physiology and in the pathophysiology of muscle dystrophy

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease characterized by progressive and continue muscle degeneration followed by inadequate muscle regeneration. DMD is due to the lack of expression of a cytoskeletal protein named dystrophin which binds to cellular actin and to extracellular matrix, therefore insuring the mechanical link between the cytoskeleton and the extracellular matrix. The absence of dystrophin induces membrane fragility. In a murine model of the disease (mdx), this membrane fragility seems responsible of the abnormal sensitivity of EDL muscle to eccentric contractions (force drop when muscle is stretched during isometric contractions). Several studies have also reported an important intracellular Ca2+ overload in dystrophic muscle fibers due to a deregulation of several cations channels at the plasma membrane. Previous studies in our lab have investigated the properties of the channels involved in DMD: there are store-operated and stretch-activated and their occurrence increases in the presence of IGF. Interestingly, two members of the transient receptor potential (TRP) channels present these characteristics: TRPC1 and TRPV2. The general aim of this thesis was to investigate the role of TRPC1 and TRPV2 in the pathophysiology of DMD. We planed to repress these two TRP isoforms in normal and dystrophic skeletal muscle and compare the rate of muscle disorders. Surprisingly, using si and shRNA strategy to repress TRPC1 in skeletal muscle cell significantly altered myogenesis. Myoblasts did not align correctly and this induced a delay in their fusion into myotubes. We indeed measured a reduction in migration and differentiation rates in TRPC1 knocked-down myoblasts compared to control myoblasts. Interestingly, control myoblasts presented at day 1 of differentiation concomitant peaks of Ca2+ influx and calpain activity which were both lost in TRPC1 knocked-down myoblasts. Moreover, TRPC1 knocked-down myoblasts accumulated MARCKS, an actin binding protein well known as calpains substrate. We then concluded that Ca2+ entry through TRPC1 channel activated calpain which in its turn cleaved MARCKS to allow myoblast migration and differentiation. We then investigated myogenesis in vivo and compared the regeneration consecutive to cardiotoxin-induced muscle degeneration in TRPC1+/+ and TRPC1-/- mice. We observed that TRPC1-/- mice showed a delay in muscle regeneration characterized by smaller fiber size, lower tension and increased immature fibers. The expression and the activity of MRFs were decreased and the PI3K/Akt/mTOR/p70S6K pathway was down regulated in these muscles. Moreover, myoblasts primary culture from TRPC1+/+ mice showed an increase in Ca2+ influx that was lost in TRPC1-/- mice. Interestingly, chelating intracellular or extracellular Ca2+ at the beginning of differentiation induced a significant decrease in PI3K/Akt activation and a subsequent decrease in the rate of myoblasts differentiation. Our results showed, for the first time, a Ca2+-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during muscle regeneration. Finally, we investigated the role of TRPC1 in adult skeletal muscle. We observed that TRPC1-/- adult mice develop less force and were more susceptible to fatigue than their controls. Their muscles were smaller, contained less myofibrillar proteins, developed less force, showed lower Ca2+ transients and accentuated loss of force during fatigue protocol. However, our investigations did not indicate that TRPC1 played any role in mdx pathophysiology. We then studied the possible involvement of TRPV2 in the disease. To this aim, we used mdx mice expressing a dominant-negative mutant of TRPV2 channel (mdx/TRPV2-DN). We observed that muscle force drop after eccentric contractions was significantly reduced in mdx/TRPV2-DN compared to control mdx. Interestingly, C57 normal muscles were not more sensitive to eccentric contractions than C57/TRPV2-DN indicating a specific deregulation of TRPV2 channel in mdx muscles. Moreover, assessment of membrane permeability after eccentric contractions indicated that the abnormal entry of Ca2+ through TRPV2 preceded and was implicated in mdx muscles sarcolemmal damage during lengthening contractions.(SBIM 3) -- UCL, 201