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 kD 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.