The Changes in Ca²⁺ Sparks Associated with Measured Modifications of Intra-store Ca²⁺ Concentration in Skeletal Muscle

In cardiac muscle and amphibian skeletal muscle, the intracellular Ca²⁺ release that signals contractile activation proceeds by discrete local packets, which result in Ca²⁺ sparks. The remarkably stereotyped duration of these release events requires a robustly timed termination mechanism. In cardiac muscle the mechanism of spark termination appears to crucially involve depletion of Ca²⁺ in the lumen of the sarcoplasmic reticulum (SR), but in skeletal muscle, the mechanism is unknown. We used SEER (shifted excitation and emission ratioing of fluorescence) of SR-trapped mag-indo-1 and confocal imaging of fluorescence of cytosolic rhod-2 to image Ca²⁺ sparks while reversibly changing and measuring [Ca²⁺] in the SR ([Ca²⁺]_SR) of membrane-permeabilized frog skeletal muscle cells. Sparks were collected in cells immersed in a solution promoting production of events at moderate frequency. Just after permeabilization, event frequency was zero, and in 10 minutes it reached close to a steady value. Controlled interventions modified [Ca²⁺]_SR reversibly between a low value (299 μM on average in 10 experiments) and a high value (433 μM, a 45% average increase). This change increased sparks frequency by 93%, spatial width by 7%, rise time by 10%, and peak amplitude by 38% (provided that it was calculated in absolute terms, rather than normalized by resting fluorescence). The changes in event frequency and amplitude were statistically significant. The “strength” of the effect of [Ca²⁺]_SR on frequency, quantified by decomposition of variance, was <6%. While the average change in [Ca²⁺]_SR was limited, it reached up to 200% in individual fibers, without causing massive Ca²⁺ release or an increase of >3.5-fold in event frequency. Taken together with existing evidence that depletion is modest during Ca²⁺ sparks or release elicited by an action potential, the mild effects of [Ca²⁺]_SR reported here do not support a major role of depletion in either the termination of sparks or the strong inactivation that terminates Ca²⁺ release at the global level in frog skeletal muscle.