Abnormal calcium homeostasis in Duchenne muscular dystrophy myotubes contracting in vitro

Resting intracellular calcium activity was recorded in three kinds of human muscle cells in culture: normal (control) and dystrophic (DMD and FSH), by means of a ratiometric fluorescence method using the calcium probe Indo-1 under laser illumination. DMD cells are characterized by a lack of dystrophin whereas FSH cells express normal dystrophin. The aim of this study was to determine whether, in dystrophin-deficient muscle cells (DMD), contraction destabilized internal calcium homeostasis. Muscle cells were cocultured with rat spinal cord explants to improve the maturation of human myotubes up to the stage where contraction appears. The resting intracellular calcium level was significantly higher in contracting DMD cells (107 +/- 8 nM; n = 44) compared to control cells (66 +/- 6 nM; n = 43) or in FSH cells (56 +/- 6 nM; n = 35). DMD myotubes cocultured in the presence of TTX which inhibited contractile activity, did not develop an increase in free cytosolic Ca2+ concentration. The amplitudes of calcium transients elicited by exposure to acetylcholine (ACh) or high K+ medium (100K) were significantly higher in contracting DMD myotubes than in control ones. The extra-responses were not observed in DMD myotubes cocultured with TTX. This study strongly suggest that: (i) contraction is a dominant factor contributing to Ca2+ abnormalities in DMD cells; and (ii) contracting dystrophin-deficient cells have defective calcium handling mechanisms during electrical events which involve sarcolemma.