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). 5. 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. 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.

Figure 1. Spontaneous variations of membrane potential in co-cultured normal or DMD muscle cells

A, pacemaker-like activity, aborted APs (arrows) and after-hyperpolarization phase (arrow head) in DMD (a,c) and normal (b,d) co-cultured human myotubes. B and C, global membrane potential variations recorded with a slower time base in normal (B) and DMD (C) cells.

Figure 2. Spontaneous calcium transients in normal and DMD cells

Examples of spontaneous calcium transients in DMD (A–C) and normal (D and E) myotubes. The dotted lines correspond to the basal level of intracellular free calcium concentration in each cell. F, the area under each spontaneous calcium transient recording (as shown by the hatched area in E) allows quantification of these calcium variations in normal and DMD myotubes. n corresponds to the number of tested cells.

Figure 3. Calcium current recordings and I–V curves

A, examples of calcium currents recorded for a depolarization from −90 to −30 mV (a) and to +10 mV (b) in normal (left) and DMD (right) myotubes. B, I–V curves obtained in normal (^) and DMD (•) cells. I is expressed as the peak current density (pA pF⁻¹). n corresponds to the number of tested cells. Values are given as means ±s.e.m.

Figure 4. Separation of two types of calcium current in normal and DMD cells

A, examples of calcium currents recorded in control medium (left) and in the presence of 5 μm nifedipine (right) for two depolarizations from a holding potential of −90 mV to −30 and +10 mV. Normal myotubes. B, I–V curves obtained in the presence of 5 μm nifedipine in normal (^) and DMD myotubes (•). Holding potential, −90 mV. I is expressed as the peak current density. Values are given as means ±s.e.m. n corresponds to the number of tested cells. C, I–V curves obtained as in B but in control medium and with a holding potential of −50 mV.

Figure 5. Voltage dependence of the availability of I_Ca,T and I_Ca,L in normal and DMD co-cultured cells

Steady-state inactivation (availability) curves for I_Ca,T (a) and I_Ca,L (B) in normal and DMD myotubes. The potential pulse protocols are shown as diagrams at the top of each panel. The diagrams are not scaled as indicated by interruptions of the lines. The continuous curves were calculated from the Boltzmann function.

Figure 6. Electron microscopy

A, control. Part of a myotube showing early myofibril development (asterisks). At this stage most of the myofilaments are of actin and there are very few myosin filaments. Microtubules (Mt), rough endoplasmic reticulum (RER) and ribosomes are common. Scale bar = 1.0 μm. B, DMD. A myotube showing both early myofibril development (asterisks) and a more advanced myofibril with recognizable sarcomeres containing longitudinally aligned myosin filaments in the A-bands (A). In the same field there is a clear Golgi apparatus (Go) and vesicles (arrowheads) that may be precursors of the sarcotubular system. I, I-band; Z, Z-line. Scale bar = 1.0 μm. C, DMD. The edge of a myotube rich in caveolae (arrowheads). Some of these are fusing (or budding) and may be T-tubule precursors. The edge of the adjacent fibre contains coated vesicles (cv). Scale bar = 1.0 μm. D, DMD. A myofibril showing an example of early triad formation, although the terminal cisternae of the sarcoplasmic reticulum are not yet well defined. SR, sarcoplasmic reticulum; T, T-tubule; Mt, microtubules; My, myofilaments. Scale bar = 1.0 μm.