Résumé :
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Muscular dystrophies are characterized by the primary wasting of skeletal muscle, which compromises patient mobility and can lead to a complete paralysis and premature death. These diseases are among the most difficult to treat, since current pharmacological approaches are implicated in modifying pathways such as calcium fluxes and protease activities that result in systemic toxicity. Several studies performed on mouse models suggest that impairment of calcium transport is one contributing factor to the clinical phenotype of LGMD2A and Duchenne muscular dystrophies, although there are no studies performed in human patients that verify this.We have analyzed the effect of the ryanodine receptor stabilizer S107 on human myotubes derived from skin fibroblasts, and innervated by rat spinal cords. Treatment of mdx mice with S107 has been shown to protect against muscle damage and improve function, however, it has not been tested on human samples yet.We have obtained skin fibroblasts from dystrophic patients and controls, and forced expression of MyoD to derive them into myoblasts. Rat spinal cord explants were added to the cultures after several days in a differentiation media, when myoblasts started fusing to form myotubes. Soon after, neurite outgrowth from explants was observed and 10 days after explant addition we could see contracting myotubes with a well-developed cross-striated pattern. We used the ratiometric fluorochrome Fura-2-AM to measure the levels of free intracellular calcium, and found that contracting myotubes displayed spontaneous intracellular calcium sparks that preceded contractions. Addition of S107 to this system resulted in significant decreases in the frequency and amplitude of calcium sparks. Normal frequency was immediately restored after washing S107 away, however, calcium spark amplitude remained diminished long after the drug was withdrawn.The in vitro coculture system developed here will be an important muscular Muscular dystrophies are characterized by the primary wasting of skeletal muscle, which compromises patient mobility and can lead to a complete paralysis andpremature death. These diseases are among the most difficult to treat, since current pharmacological approaches are implicated in modifying pathways such as calciumfluxes and protease activities that result in systemic toxicity. Several studies performed on mouse models suggest that impairment of calcium transport is one contributingfactor to the clinical phenotype of LGMD2A and Duchenne muscular dystrophies, although there are no studies performed in human patients that verify this.We haveanalyzed the effect of the ryanodine receptor stabilizer S107 on human myotubes derived from skin fibroblasts, and innervated by rat spinal cords. Treatment of mdxmice with S107 has been shown to protect against muscle damage and improve function, however, it has not been tested on human samples yet.We have obtained skinfibroblasts from dystrophic patients and controls, and forced expression of MyoD to derive them into myoblasts. Rat spinal cord explants were added to the culturesafter several days in a differentiation media, when myoblasts started fusing to form myotubes. Soon after, neurite outgrowth from explants was observed and 10 daysafter explant addition we could see contracting myotubes with a well-developed cross-striated pattern. We used the ratiometric fluorochrome Fura-2-AM to measure thelevels of free intracellular calcium, and found that contracting myotubes displayed spontaneous intracellular calcium sparks that preceded contractions. Addition of S107to this system resulted in significant decreases in the frequency and amplitude of calcium sparks. Normal frequency was immediately restored after washing S107 away,however, calcium spark amplitude remained diminished long after the drug was withdrawn.The in vitro coculture system developed here will be an important musculardystrophy model system for drug screening and therapy design.
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