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Department of Zoology, La Trobe University, Bundoora, Victoria, Australia.

1. Myotubes were grown in culture from normal (C57BL/ScSn) and mdx mice and the cytosolic [Ca2+] was monitored through development (5-21 days in culture) using fura-2 loaded via ionophoresis. Simultaneous measurements of the membrane potential and cytosolic [Ca2+] were made in normal and mdx myotubes before, during and after stimulation by action potentials elicited following anode break excitation. All experiments were undertaken at 22 degrees C. All data are expressed as means +/- S.E.M. 2. A new method was developed which enabled accurate determination of the fluorescence characteristics of fura-2 in murine skeletal muscle fibres. In the under in vitro conditions by 14.60 +/- 0.05, 9.40 +/- 0.15 and 6.90 +/- 0.43% respectively. 3. The resting cytosolic [Ca2+] in the mdx myotubes was consistently higher than in the normal myotubes throughout the developmental period measured. Overall, the resting cytosolic [Ca2+] in mdx myotubes (134 +/- 9 nM, n = 22) was twofold higher than in normal myotubes (66 +/- 6 nM, n = 26). After stimulation (one to three action potentials) the cytosolic [Ca2+] of both mdx and normal myotubes remained elevated. The mdx myotubes (236 +/- 55 nM, n = 5) again had approximately double the cytosolic [Ca2+] of normal myotubes (109 +/- 19 nM, n = 9). 4. The time course and amplitude of the Ca2+ responses measured in the mdx and normal myotubes after action potential stimulation were variable. Two categories of Ca2+ response were observed in mdx and normal myotubes, the first consisted of a small, slow rise in [Ca2+] that remained elevated and the second consisted of a rapid (time to peak 7.4 +/- 1.5 ms) (n = 8) rise in [Ca2+] with amplitudes in the range 61-773 nM and a [Ca2+] decay rate constant of 4.35 +/- 1.57 s-1 (n = 8) (range 0.96-15 s-1). 5. In conclusion, the elevated cytosolic [Ca2+] reported here through development of cultured mdx myotubes suggests that this genetic disorder results in a defect which compromises the ability of the myotubes to strictly regulate cytosolic [Ca2+]. The results are consistent with the presence of functionally abnormal Ca2+ channels recently reported in mdx myotubes.

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