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

Abstract

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