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.
Han
Renzhi
R
School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia. renzhi-han@uiowa.edu
Grounds
Miranda D
MD
Bakker
Anthony J
AJ
D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism*
D005456:Fluorescent Dyes; C098105:fura-FFP18; D002118:Calcium; D016257:Fura-2