The tubular (t-) system of skeletal muscle forms a junction with the sarcoplasmic reticulum (SR), with some 12nm between the membranes, at every sarcomere for the main purpose of conducting excitation-contraction coupling. 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 [Ca2+]js is expected to be higher than the bulk cytoplasmic [Ca2+] ([Ca2+]js) with a standing gradient set between the RyRs and SR Ca2+-pumps in the resting muscle. The value of [Ca2+]js is unknown but has significant implications for signalling cascades initiating in this nanodomain. The value of [Ca2+]js could also be expected to change under conditions of RyR mutation, which underlie a number of myopathies. Our aim was to develop a method to determine the [Ca2+]js in fast- and slow-twitch muscle fibres. To do this we exploited the fact that t-system Ca2+ uptake activity will be set by [Ca2+]js (and the t-system Ca2+ gradient) to determine [Ca2+]js.
All experimental procedures were approved by The Animal Ethics Committee of The University of Queensland. Rats were euthanized by CO2 asphyxiation and the extensor digitorum longus and soleus muscle were 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.
Chronic depletion of [Ca2+]SR with caffeine reduced [Ca2+]t-sys to 0.1 mM via chronic activation of store-operated Ca2+ entry, providing a 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 0.1 mM 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. [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 of Hill curves fitted to data for both fibre types was right-shifted by tetracaine compared to the absence of tetracaine. It followed that at 100nM [Ca2+]bulk, [Ca2+]js was 165 and 220nM in slow and fast-twitch fibres, respectively. These results show that t-system Ca2+ fluxes can be used as a nanodomain sensor of RyR leak.