Programme Contents

The role of ryanodine receptor Ca²⁺ leak in t-system Ca²⁺ handling in skeletal muscle fibres

The tubular (t-) system of skeletal muscle forms a junction with the sarcoplasmic reticulum (SR), with some 12nm between the membranes. In the resting muscle, [Ca²⁺] within the small volume bound by the junctional membranes should be determined by the leak of Ca ²⁺ through the SR ryanodine receptors (RyRs), the Ca²⁺ handling ability of the t-system and diffusion of Ca²⁺ from the junctional space (js). The [Ca²⁺]_js is expected to be higher than [Ca²⁺ ]_bulk with a standing gradient set between the RyRs and SR Ca²⁺-pumps. We aimed to detect the effects of RyR Ca²⁺ leak into the junctional space by harnessing the ability of the t-system to detect changes in this domain by monitoring a Ca-sensitive dye inside the t-system.

All experimental procedures were approved by The Animal Ethics Committee of The University of Queensland. Wistar rats were euthanized by CO₂ asphyxiation and the extensor digitorum longus and soleus muscle rapidly excised. Muscles were pinned down in a Petri dish above a layer of Sylgard under a layer of Paraffin oil. Bundles of fibres were isolated from the muscle and exposed to a physiological solution containing 2.5mM rhod-5N. After 10 mins a fibre was isolated and mechanically skinned, trapping the rhod-5N in the t-system as it sealed.

To detect the RyR Ca²⁺ leak and its effects on t-system handling we exploited the fact that t-system Ca²⁺ uptake activity will be set by [Ca²⁺]_js. T-system Ca²⁺-uptake activity was tracked with rhod-5N trapped in the t-system of mechanically skinned fibres of rat slow- and fast-twitch muscles on a confocal microscope. Chronic depletion of [Ca²⁺]_SR with caffeine reduced [Ca²⁺]_t-systo 0.1 mM via chronic activation of store-operated Ca²⁺ entry. We then exposed Ca²⁺-depleted preparations to 28nM-1.3μM [Ca²⁺]_cyto in 50mM EGTA to allow observation of t-system Ca²⁺ uptake rates at known [Ca²⁺]_bulk. Experiments were repeated in the presence of 1mM tetracaine or 10mM Mg²⁺ 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. [Ca²⁺]_bulk and peak t-system Ca²⁺ fluxes were fitted by Hill curves. Vmax was significantly depressed in slow- compared to fast-twitch fibres. The k_D for both fibre types was right-shifted by tetracaine. It followed that at 67nM [Ca²⁺]_bulk, [Ca²⁺]_js was 165 and 220nM in slow and fast-twitch fibres, respectively. By lowering [Mg²⁺]_cyto to 0.13mM to increase RyR leak further than physiological levels in the presence of a broad range of [Ca²⁺]_cyto, t-system Ca²⁺ uptake rate and steady state [Ca²⁺]_t-sys was reduced to below resting conditions but above that where SOCE was activated in the absence of Ca²⁺. The addition of 25mM BAPTA with varied [Ca²⁺]_cyto also produced a decreased steady state [Ca²⁺]_t-sys and a reduced Ca²⁺ uptake rate.

We conclude that increasing RyR Ca²⁺ leak activates SOCE, allowing SOCE to simultaneously act as a counter-current against Ca²⁺ being extruded from the fibre. Thus SOCE holds Ca²⁺ within the fibre rather than sequentially recovering Ca²⁺ previously lost from the fibre, as commonly presumed. These results also highlight that t-system Ca²⁺ fluxes can be used as a nanodomain sensor of RyR Ca²⁺ leak, an important factor in many muscle diseases.