Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers

Abstract

1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability.

Figure 1. Intracellular free Ca²⁺ concentrations in dystrophin-deficient and control muscle fibres

Ca²⁺ concentrations in resting mouse interosseus muscle fibres from control (con), dystrophic (MDX) and myotonic-dystrophic (ADR-MDX) animals. Number of analysed fibres indicated for each genotype above bar (means ±s.d.).

Figure 2. Sarcoplasmic Ca²⁺ transients of an MDX muscle fibre

A, ratio of emission intensities (340 nm/380 nm, sampled every 5 ms) vs. recording time. Stimulus frequency, 1 Hz. B, single transient plotted on expanded time scale. Ascribed decay time constant (τ) obtained by fitting single exponential to falling phase (continuous line).

Figure 3. Decay time constants of Ca²⁺ transients of electrically stimulated muscle fibres

Data determined from 49 MDX and 36 control fibres grouped into 10 ms intervals. Percentages of fibres are plotted for each interval for MDX (▪) and control fibres (□).

Figure 4. Sarcoplasmic Ca²⁺ transients in control muscle

A, ratio of emission intensities (sampled every 5 ms) vs. recording time. Stimulus frequency: 1 Hz (3 pulses), 10 Hz (10 pulses) and 30 Hz (30 pulses). B, Ca²⁺ deregulation after 30 Hz stimulation.

Figure 5. Quench of fura-2 fluorescence generated by entry of Mn²⁺ into MDX muscle fibres

Sudden drop of fluorescence intensity upon application of 10 μM ACh in the presence of 500 μM Mn²⁺ (•). Removal of Mn²⁺ and ACh does not restore original fluorescence intensity. After preincubation with α-bungarotoxin (0.1 μM, 15 min; ^) quench rate is as small (4.8 % min⁻¹) as in the absence of ACh (for control see Fig. 7).

Figure 7. Quench rates obtained from different muscle fibres

A, determination of quench rate (5 % min⁻¹) in an MDX fibre using linear regression analysis. Calibration of both axes different from those in Fig. 5. The decline of the fura-2 fluorescence is expressed as percentage per minute (initial fluorescence intensity = 100 %). Bar indicates presence of 500 μM Mn²⁺. B, overall results from control (□), MDX (▪) and ADR-MDX () fibres.

Figure 6. Absence of Mn²⁺ entry during short-term muscle activity

Application of 500 μM Mn²⁺ to an MDX fibre during electrically induced contractions (10 Hz, 10 pulses, lower trace) does not reduce fluorescence intensity (upper trace). Ca²⁺-independent fluorescence intensity calculated as described in Methods.

Figure 8. Block of Mn²⁺ entry into an MDX fibre by Gd³⁺

Application of 50 μM Gd³⁺ immediately prevents entry of Mn²⁺. Presence of Mn²⁺ and Gd³⁺ indicated by bars.

Figure 9. Block of Mn²⁺ entry into MDX, ADR-MDX and control fibres by various ion channel blockers

Quench rates determined in the presence of 500 μM Mn²⁺ and the agent indicated (□, before; ▪, after application of channel blocker). A, Gd³⁺; B, La³⁺; C, Ni²⁺; D, amiloride.