L-Type Ca2+ Channel Function Is Linked to Dystrophin Expression in Mammalian Muscle

BACKGROUND: In dystrophic mdx skeletal muscle, aberrant Ca2+ homeostasis and fibre degeneration are found. The absence of dystrophin in models of Duchenne muscular dystrophy (DMD) has been connected to altered ion channel properties e.g. impaired L-type Ca2+ currents. In regenerating mdx muscle, 'revertant' fibres restore dystrophin expression. Their functionality involving DHPR-Ca2+-channels is elusive. METHODS AND RESULTS: We developed a novel 'in-situ' confocal immuno-fluorescence and imaging technique that allows, for the first time, quantitative subcellular dystrophin-DHPR colocalization in individual, non-fixed, muscle fibres. Tubular DHPR signals alternated with second harmonic generation signals originating from myosin. Dystrophin-DHPR colocalization was substantial in wt fibres, but diminished in most mdx fibres. Mini-dystrophin (MinD) expressing fibres successfully restored colocalization. Interestingly, in some aged mdx fibres, colocalization was similar to wt fibres. Most mdx fibres showed very weak membrane dystrophin staining and were classified 'mdx-like'. Some mdx fibres, however, had strong 'wt-like' dystrophin signals and were identified as 'revertants'. Split mdx fibres were mostly 'mdx-like' and are not generally 'revertants'. Correlations between membrane dystrophin and DHPR colocalization suggest a restored putative link in 'revertants'. Using the two-micro-electrode-voltage clamp technique, Ca2+-current amplitudes (i(max)) showed very similar behaviours: reduced amplitudes in most aged mdx fibres (as seen exclusively in young mdx mice) and a few mdx fibres, most likely 'revertants', with amplitudes similar to wt or MinD fibres. Ca2+ current activation curves were similar in 'wt-like' and 'mdx-like' aged mdx fibres and are not the cause for the differences in current amplitudes. i(max) amplitudes were fully restored in MinD fibres. CONCLUSIONS: We present evidence for a direct/indirect DHPR-dystrophin interaction present in wt, MinD and 'revertant' mdx fibres but absent in remaining mdx fibres. Our imaging technique reliably detects single isolated 'revertant' fibres that could be used for subsequent physiological experiments to study mechanisms and therapy concepts in DMD

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

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.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).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.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.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%.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