Abstract
BackgroundIn 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 ResultsWe 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 (imax) 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. imax amplitudes were fully restored in MinD fibres.ConclusionsWe 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.
Highlights
Duchenne muscular dystrophy (DMD) is a common Xchromosomal hereditary disease that involves progressive muscle wasting and eventually results in immobility and death from respiratory and cardiac failure early in adulthood [1,2]
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
C57/SV129 wild-type mice served as a control. mdx mice represent an animal model for DMD [46], and MinD mice are a transgenic strain with the mdx background (CVBA3’, [42]) expressing the Dexon17–48 mouse mini-dystrophin construct with a molecular weight of 228 kDa [42,47]
Summary
Duchenne muscular dystrophy (DMD) is a common Xchromosomal hereditary disease that involves progressive muscle wasting and eventually results in immobility and death from respiratory and cardiac failure early in adulthood [1,2]. [12,13,14,15,16]), mitochondrial function [17], motor protein interaction [18,19] or gene transcription [20,21] From these studies, dystrophin has been implicated in the regulation of cellular signalling cascades either directly by regulating membrane-associated proteins, including ion channels [13], or indirectly via second messenger cascades [22,23]. 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
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