Programme Contents

Assessment of RyR leak and t-system Ca²⁺ uptake in dystrophic skeletal muscle

The tubular (t-) system of skeletal muscle is an internalization of the plasma membrane that forms a junction with the terminal cisternae of the sarcoplasmic reticulum (SR) at every sarcomere of skeletal muscle. In the resting muscle, [Ca²⁺] within the small volume bound by the junctional membranes will be determined by the leak of Ca²⁺ through the SR ryanodine receptors (RyRs), the Ca²⁺ handling ability of the t-system and diffusion of Ca²⁺ from the junctional space (js). The value of [Ca²⁺]_js could be expected to change under conditions of RyR leak, which is believed to occur in the DMD mouse model (mdx). Our aim was to use a method recently developed in the lab to determine the [Ca²⁺]_js in dystrophic mouse muscle fibres.

All experimental procedures were approved by The Animal Ethics Committee of The University of Queensland. 4 – 6 week old Mdx and C57Bl10 mice were euthanized by CO₂ asphyxiation and the extensor digitorum longus muscle rapidly excised. Muscles were pinned down in a Petri dish above a layer of Sylgard under a layer of Paraffin oil. Fibre bundles were isolated and a Ringer solution containing rhod-5N was applied to the fibres. Fibres were isolated and mechanically skinned and placed in a custom-built experimental chamber for imaging on an Olympus FV1000 confocal microscope employing GaAsp detectors.

Chronic depletion of [Ca²⁺]_SR with caffeine reduced [Ca²⁺]_t-sys to 0.03 mM and 0.08 mM respectively in wt and mdx via chronic activation of store-operated Ca²⁺ entry, providing a consistent starting point for tracking t-system Ca²⁺ uptake. We then exposed Ca²⁺-depleted preparations to a solution containing either 50, 100, 200 or 800nM [Ca²⁺] in 50mM EGTA to allow observation of t-system Ca²⁺ uptake rates at known [Ca²⁺]_bulk. The t-system was subsequently depleted of Ca²⁺ to return [Ca²⁺]_t-sys to depletion and the cycle was repeated. Experiments were repeated in the presence of 1mM tetracaine to block RyR Ca²⁺ leak and allow [Ca²⁺]_js to equilibrate with [Ca²⁺]_bulk. Rhod-5N signals and [Ca²⁺]_t-sys were calibrated and t-system Ca²⁺ fluxes were derived. Mdx mice in this early pathological state were found to have lower t-system Ca²⁺ compared to wt, indicating a trend towards increased t-system Ca²⁺ leak. However this data is currently not statistically significant as the wt data set is underpowered. The mdx t-system Ca²⁺ could be further reduced with the application of 1mM tetracaine. Maximally activated SOCE due to caffeine application was found to be the same between wt and mdx mice when peak store dependent flux was analysed against t-system Ca²⁺. 100nM [Ca²⁺]_bulk gave a [Ca²⁺]_js of between 150 and 200nM in mdx. These results may indicate that at this early stage of the disease phenotype the Ca²⁺ handling by the t-system and sarcoplasmic reticulum of the mdx mouse is able to function similarly to healthy muscle.