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 (Ca2+F) 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 Ca2+F transients: 1.81 +/- 0.17 ms and 16.14 +/- 2.60 ms, respectively). After correction, the [Ca2+] transients (Ca2+C) measured in both the adult mouse fibres and the myotubes rose more rapidly (mean rise time of Ca2+C 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 Ca2+F transient. In the adult mouse fibres, correction increased the mean peak [Ca2+] of the Ca2+F transients by a factor of 7 from 0.53 +/- 0.08 microM to 3.76 +/- 0.71 microM. 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 microM to 0.97 +/- 0.15 microM. During the decay phase of the Ca2+ transients elicited in the adult mouse fibres and the myotubes, the corrected Ca2+C signal largely followed the unmodified Ca2+F 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.
Bakker
A J
AJ
School of Physiology and Pharmacology, University of New South Wales, Sydney, Australia. A_Bakker@unsw.edu.au
Head
S I
SI
Stephenson
D G
DG
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000378:metabolism*; D016257:Fura-2 / Q000031:analogs & derivatives / Q000378:metabolism*; D051379:Mice; D018656:Muscle Fibers, Fast-Twitch / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D051381:Rats; D013997:Time Factors
D005456:Fluorescent Dyes; C094537:mag-Fura 5; D002118:Calcium; D016257:Fura-2