Fast calcium removal during single twitches in amphibian skeletal muscle fibres

Abstract

Fluorescence signals from the calcium sensitive dyes Fluo-3 or Rhod-2 were obtained simultaneously with isometric tension in single fibres isolated from the anterior tibialis muscle of Leptodactylus insularis (20–22°C). Fluo-3 fluorescence signals were transformed into [Ca²⁺]_i transients as previously described. Most of the decay phase of single twitch transient is well fitted by a single exponential (τ of about 10 ms), followed by a slower declining component lasting tens of milliseconds. During short periods, 10 to 20 s, of low frequency stimulation, between 0.2 and 5 Hz, the basal [Ca²⁺]_i increased slowly from 0.1 to about 0.4 μM, with only minor changes in the exponentially decaying phase. In fibres poisoned with thapsigargin or cyclopiazonic acid (1–2 μM) the τ of decay of fluorescence or Ca²⁺ transients of single twitches was very similar to that observed in non-poisoned fibres. Nevertheless, in poisoned fibres challenged with repetitive stimulation, the τ of Ca²⁺ transients decay increased from about 10 ms to > 40 ms, while the basal [Ca²⁺]_i increased from 0.1 to 2 μM. Short rest periods (about 5 min) could reverse these effects, indicating that they were not a direct consequence of SR Ca²⁺-ATPase inhibition. The correlation coefficient between τ of decay and basal [Ca²⁺]_i was > 0.8 (P < 0.0001). Qualitatively similar results were obtained measuring Rhod-2 fluorescence signals. A lumped, two-compartment model could account for these results. Loading the fibres with EGTA-AM, diminished the effects of prolonged stimulation observed in poisoned fibres. Moreover, we show that the Na⁺ – Ca²⁺ exchange mechanism does not participate appreciably in fast Ca²⁺ removal.