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

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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 μm). Bath application of 50 μm 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.