Near-membrane cytosolic [Ca²⁺] levels and Ca²⁺ transients measured in myotubes grown from normal and dystrophic (mdx) mice using the Ca²⁺ indicator FFP-18.

A.J. Bakker¹, R. Han¹ and M.D. Grounds², ¹School of Biomedical & Chemical Sciences and ²School of Anatomy & Human Biology, The University of Western Australia, Crawley, WA 6009, Australia.

Abnormal extracellular Ca²⁺ influx has been suggested to be involved in the process of muscle wasting in Duchenne muscular dystrophy. However, studies comparing the resting intracellular Ca²⁺ levels in normal and dystrophic muscle cells from patients with Duchenne muscular dystrophy and mdx mice have yielded contradictory findings (Gillis, 1996). Ca²⁺ indicators targeted to the inner sarcolemmal membrane have recently been reported to be more sensitive to sarcolemmal Ca²⁺ influx than standard cytosolic Ca²⁺ indicators such as fura-2 (Bruton et al., 1999). In this study, we measured the resting Ca²⁺ levels and Ca²⁺ transients in myotubes grown from mdx and normal mice using the near-membrane Ca²⁺ indicator FFP-18.

Skeletal muscle satellite cells were isolated from the hind limbs of neonatal normal and mdx mice that had been killed by decapitation. Myotubes were grown on glass coverslips coated with collagen. The myotubes were loaded with the Ca²⁺ indicator by exposure to FFP-18-AM (3 μM) and 0.0125% Pluronic F-127 for 45 min at room temperature (22-23°C). Ca²⁺ measurements were made with a Cairn spectrophotometer attached to a Nikon inverted microscope equipped for epifluorescence. The myotubes were stimulated by electrical field stimulation (EFS) via two small platinum wires (single 0.2 ms pulse).

Graph

Resting near membrane [Ca²⁺] ([Ca²⁺]_mem) levels increased significantly during development in the mdx myotubes, (slope; 8.19 ± 1.47, p<0.0001). However, no change in [Ca²⁺]_mem was found in normal myotubes during development (slope; -0.40 ± 1.14, p=0.73). From the fitted lines, the [Ca²⁺]_mem in 12 days old mdx and normal myotubes was estimated at 93 and 36 nM respectively (Figure). Increasing the driving force for Ca²⁺ influx by raising extracellular Ca²⁺ to 18 mM, increased the steady state [Ca²⁺]_mem by 156.1 ± 14.2 % (to ~ 208 nM) (n=14) in mdx myotubes, while in normal myotubes, the [Ca²⁺]_mem increased by only 28.8 ± 7.6 % (to ~ 49 nM) (n=6), (p=0.007, unpaired Student's t-test). The half-relaxation time of EFS-induced Ca²⁺ transients was significantly increased in mdx (314.5 ± 36.9 ms, n=8) compared to normal myotubes (163.3 ± 28.4 ms, n=6) (p=0.01, unpaired t-test), which is consistent with previous studies using standard Ca²⁺ indicators.

The results of this study further support the hypothesis that increased Ca²⁺ influx results in raised intracellular levels in dystrophin-deficent skeletal muscle cells. The use of FFP-18 to measure steady state cytosolic Ca²⁺ in normal and mdx myotubes in the presence of raised extracellular Ca²⁺ could provide a more reliable method for detecting the altered Ca²⁺ homeostasis in dystrophic muscle cells.

Bruton J.D., Katz A. & Westerblad H. (1999) Proceedings of the National Academy of Sciences USA, 96, 3281-3286.

Gillis J.M. (1996) Acta Physiologica Scandinavica, 156, 397-406.

Near-membrane cytosolic [Ca2+] levels and Ca2+ transients measured in myotubes grown from normal and dystrophic (mdx) mice using the Ca2+ indicator FFP-18.

Near-membrane cytosolic [Ca²⁺] levels and Ca²⁺ transients measured in myotubes grown from normal and dystrophic (mdx) mice using the Ca²⁺ indicator FFP-18.