Measurement of sub-membrane [Ca 2+] in adult myofibers and cytosolic [Ca 2+] in myotubes from normal and mdx mice using the Ca 2+ indicator FFP-18

Abstract

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 ∼2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (≥8 days in culture), 18 mM extracellular Ca 2+ increased the steady state cytosolic [Ca 2+] to ∼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.