Christian Cognard

The goal of the present study was to investigate differences in calcium movements between normal and Duchenne muscular dystrophy (DMD) human contracting myotubes co‐cultured with explants of rat spinal cord with attached dorsal root ganglia. Membrane potential, variations of intracellular calcium concentration and T‐ and L‐type calcium currents were recorded. Further, a descriptive and quantitative study by electron microscopy of the ultrastructure of the co‐cultures was carried out. The resting membrane potential was slightly less negative in DMD (‐61.4 ± 1.1 mV) than in normal myotubes (‐65.5 ± 0.9 mV). Both types of myotube displayed spontaneous action potentials (mean firing frequency, 0.42 and 0.16 Hz, respectively), which triggered spontaneous calcium transients measured with Indo‐1. The time integral under the spontaneous Ca²⁺ transients was significantly greater in DMD myotubes (97 ± 8 nm s) than in normal myotubes (67 ± 13 nm s). The L‐ and T‐type current densities estimated from patch‐clamp recordings were smaller in DMD cells (2.0 ± 0.5 and 0.90 ± 0.19 pA pF⁻¹, respectively) than in normal cells (3.9 ± 0.7 and 1.39 ± 0.30 pA pF⁻¹, respectively). The voltage‐dependent inactivation relationships revealed a shift in the conditioning potential at which inactivation is half‐maximal (V_h,0.5) of the T‐ and L‐type currents towards less negative potentials, from ‐72.1 ± 0.7 and ‐53.7 ± 1.5 mV in normal cells to ‐61.9 ± 1.4 and ‐29.2 ± 1.4 mV in DMD cells, respectively. Both descriptive and quantitative studies by electron microscopy suggested a more advanced development of DMD myotubes as compared to normal ones. This conclusion was supported by the significantly larger capacitance of the DMD myotubes (408 ± 45 pF) than of the normal myotubes (299 ± 34 pF) of the same apparent size. Taken together, these results show that differences in T‐ and L‐type calcium currents between normal and DMD myotubes cannot simply explain all observed alterations in calcium homeostasis in DMD myotubes, thus suggesting that other transmembrane calcium transport mechanisms must also be altered in DMD myotubes compared with normal myotubes.

The effect of stimulation rate and of external ionic composition on the repriming period of contractures induced by 6 mM caffeine was tested on isolated skeletal muscle fibres of the frog (Rana ridibunda). The repriming period, which was 11.2 ± 0.1 min (mean ± SEM, n = 9) on quiescent fibres, was shortened in fibres stimulated at a frequency ranging from 3 to 12 min⁻¹ (optimal rate, 8 min⁻¹; full repriming 5.7 ± 0.2 min; n = 10). A 10-fold increase in the extracellular calcium concentration shortened the repriming period on both stimulated and quiescent fibres, whereas decreasing external calcium (1/10) delayed it. In a Na⁺-free solution (Li⁺ substituted) the repriming period of stimulated fibres was markedly delayed (14 min), whereas quiescent fibres never recover more than 10% of their ability to develop subsequent caffeine contractures. In contrast, with a 35% Na solution, the repriming period was greatly shortened (stimulated, 5.4 ± 0.2 min, n = 7; quiescent, 6.2 ± 0.5 min, n = 8). It is concluded that repriming depends on three mechanisms that seem to refill a calcium store and trigger recovery: the slow inward calcium current, a Na⁺–Ca²⁺ exchange, and perhaps a passive Ca² influx.