Calcium currents and transients in co‐cultured contracting normal and Duchenne muscular dystrophy human myotubes

Abstract

1. 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.2. 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-1.3. The time integral under the spontaneous Ca 2+ transients was significantly greater in DMD myotubes (97 ± 8 nM s) than in normal myotubes (67 ± 13 nM s).4. 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 potentialat 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.6. 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.7. 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.

Mini‐dystrophin restores L‐type calcium currents in skeletal muscle of transgenic mdx mice

“…Furthermore, no changes of C m were reported in juvenile single fibres of 3‐ to 8‐week‐old wt and mdx mice (Collet et al 2003). Therefore, our results support findings reported for DMD myotubes where reduced i Ca amplitudes were suggested to be due to a reduction in the density of functional Ca 2+ channels (Imbert et al 2001).…”

“…So far, no single channel data on L‐type Ca 2+ channels in adult mdx mice are available. It is interesting to note, that in a recent study using whole‐cell patch clamp in co‐cultured human normal and DMD myotubes, L‐type and T‐type Ca 2+ current densities were also found to be significantly smaller in DMD myotubes compared to normal myotubes (Imbert et al 2001). Concerning i Ca kinetics in our preparation, TTP was significantly slowed in young mdx fibres (2–3 months) but similar to wt fibres in old mdx fibres (12–18 months).…”

“…Much circumstantial evidence suggests that intracellular Ca 2+ ions are involved in the degenerative process but a clear view of the cause‐effect mechanism remains to be obtained. Several reports have shown that the activities of various types of Ca 2+ channels such as mechano‐sensitive (Franco & Lansman, 1990), Ca 2+ ‐leak (Franco & Lansman, 1994) and voltage‐dependent T‐ and L‐types (Imbert et al 2001) are affected in dystrophin‐lacking myotubes and juvenile skeletal muscle fibres (Collet et al 2003). However, the impact of these changes on the intracellular calcium homeostasis of adult and fully differentiated fibres is still a matter of debate, as changes in cytosolic Ca 2+ concentration observed in myotubes were no longer present in adult fibres (e.g.…”

Duchenne muscular dystrophy

“…[41,54,55] In addition, altered membrane permeability and abnormal calcium homeostasis are thought to play a role, with increased cytosolic calcium concentration leading to activation of proteases such as calpains. [55][56][57][58] The absence of nitric oxide synthase, delocalized from the subsarcolemmal membrane, may contribute to damage, but is not thought to directly cause dystrophic features. [59][60][61] Various animal models of DMD exist; the most Yiu, et al: Duchenne muscular dystrophy commonly studied and used is the mdx mouse.…”

Regulation of TRPC1 and TRPC4 Cation Channels Requires an α1-Syntrophin-dependent Complex in Skeletal Mouse Myotubes

“…Moreover, we recorded the resting membrane potential after the depletion protocol in minidysϩ SolD6 myotubes and obtained a mean value of Ϫ61 mV. The depletion protocol leads to low depolarization (around 6 mV depolarization for minidysϩ SolD6 and around 4 mV depolarization for dysϪSolC1), but the membrane potential remains underneath the activation threshold of L-type calcium channels, which is normally around Ϫ30 mV in human (34,35) and rat (36) myotubes and in wild type fibers (37). Nifedipine was used in minidysϩ SolD6 transfected with ␣1-syntrophin siRNA (supplemental Fig.…”