Ca2+ Spark Activity in Intact Dystrophin-Deficient mdx Muscle during Osmotic Challenge is Triggered by Mechanosensitive Pathways

Abstract

Inhibitory DHPR control on RyR1 in intact dystrophic mdx skeletal muscle fibres was suggested to be disrupted, resulting in ‘uncontrolled’ Ca2+ spark frequencies (CSF) during osmotic challenge (Wang et al., 2005, Nat. Cell. Biol.). However, some of their conditions must be considered completely unphysiologic (i.e. 50 mM external Ca2+). We recorded Ca2+ sparks in single intact wt, mdx and transgenic mini-dystrophin (MinD) expressing muscle fibres during hypo-/hypertonic challenge using confocal microscopy. CSF were low in wt and MinD, but twofold increased in mdx fibres under isotonic resting conditions. CSF increased faster during hypertonic than hypotonic challenge and peak CSF were about three times larger in mdx vs. wt and MinD fibres. CSF decayed exponentially (τdec) with ongoing challenge and were significantly faster in mdx fibres, thus questioning ‘uncontrolled‘ spark activity. In hypertonic solution, CSF τdec was three times larger when external Ca2+ was 50 mM compared to 2 mM. Pretreatment with streptomycine or Gd3+ to block mechanosensitive channels (MsC), completely abolished the osmotic CSF increase mdx fibres. Resting membrane potentials in mdx muscle were ∼-61 mV and ∼-73 mV in wt fibres under hypertonic conditions (2 mM Ca2+); well below thresholds for physiological RyR1 activation. Contributions of MsC to the depolarisation were minor as judged from only slightly more negative resting potentials in the presence of GsMTx4 (-64 mV). We suggest that DHPR inhibition on RyR1 is modulated by MsC in mammalian skeletal muscle and is partly relieved in mdx muscle probably either due to some Ca2+ influx through aberrant MsC or direct interactions with the DHPR. A direct DHPR-RyR activation by depolarised membrane potentials in mdx fibers is more unlikely to be a consequence of osmotic membrane stress.