Measurement of sub-membrane [Ca2+] in adult myofibers and cytosolic [Ca2+] in myotubes from normal and mdx mice using the Ca2+ indicator FFP-18

The hypothesis that intracellular Ca(2+) is elevated in dystrophic (mdx) skeletal muscle due to increased Ca(2+) influx is controversial. As the sub-sarcolemmal Ca(2+) (Ca(2+)](mem)) should be even higher than the global cytosolic Ca(2+) in the presence of increased Ca(2+) influx, we investigated Ca(2+)](mem) levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice with the near-membrane Ca(2+) indicator FFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant difference in Ca(2+)](mem) was found in FDB myofibres of normal (77.3+/-3.8 nM, n=68) and mdx (79.3+/-5.6 nM, n=21, p=0.89) mice using FFP-18. Increasing external Ca(2+) to 18 mM did not significantly affect Ca(2+)](mem) in either the normal or mdx myofibres. In the myotubes, the FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived Ca(2+)] values were similar to values obtained with Fura-2. Nevertheless, in the mdx myotubes, the Ca(2+)] measured with FFP-18 increased linearly to a level approximately 2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (>or=8 days in culture), 18 mM extracellular Ca(2+) increased the steady state cytosolic Ca(2+)] to approximately 22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca(2+) homeostasis is well maintained in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca(2+) handling is compromised in mdx myotubes.