Time course of calcium transients derived from Fura-2 fluorescence measurements in single fast twitch fibres of adult mice and rat myotubes developing in primary culture

In this study, we applied a method to correct for the altered binding kinetics of Fura-2 for Ca2+ in vivo, on Ca2+ fluorescence transients (Ca-F(2+)) measured using Fura-2 in single adult fast twitch skeletal muscle fibres of the mouse, which exhibit very fast [Ca2+] responses, and rat myotubes developing in culture which exhibit slower [Ca2+] responses (rise time [20-80% of peak] of Ca-F(2+), transients: 1.81 +/- 0.17 ms and 16.14 +/- 2.60 ms, respectively). After correction, the [Ca2+] transients (Ca-C(2+)) measured in both the adult mouse fibres and the myotubes rose more rapidly (mean rise time of Ca-C(2+) transients: adult mouse fibres, 0.76 +/- 0.12 ms; rat myotubes, 8.25 +/- 2.83 ms) and often exhibited a Ca2+ spike which exceeded the peak of the Ca-F(2+), transient. In the adult mouse fibres, correction increased the mean peak [Ca2+] of the Ca-F(2+) transients by a factor of 7 from 0.53 +/- 0.08 mu M to 3.76 +/- 0.71 mu M. The accuracy of the time course of the corrected Ca2+ transients was confirmed by comparison to the time course of Ca2+ transients measured with Mag-Fura-5, which had a similar mean rise time (0.94 +/- 0.10 ms, t-test, P = 0.80). The more slowly rising Ca2+ transients measured in the rat myotubes were less affected by the correction process, increasing in mean peak [Ca2+] by a factor of only 1.2 from 0.82 +/- 0.17 mu M to 0.97 +/- 0.15 mu M. During the decay phase of the Ca2+ transients elicited in the adult mouse fibres and the myotubes, the corrected Ca-C(2+), signal largely followed the unmodified Ca-F(2+) transient. The correction process was found to have little effect on Ca2+ transients with rise time values greater than 10 ms, which included most of the Ca2+ transients measured in the myotubes.