title: Sarcoplasmic reticulum Ca2+ permeation explored from the lumen side in mdx muscle fibers under voltage control.
abstract: Under resting conditions, external Ca(2+) is known to enter skeletal muscle cells, whereas Ca(2+) stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the sarcolemmal Ca(2+) entry and in the SR Ca(2+) leak is still a matter of debate, but several lines of evidence suggest that these Ca(2+) fluxes are up-regulated in Duchenne muscular dystrophy. We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the mouse model of Duchenne muscular dystrophy. Using the cytosolic Ca(2+) dye Fura2, we first demonstrated that the rate of Ca(2+) increase in response to cyclopiazonic acid (CPA)-induced inhibition of SR Ca(2+)-ATPases at resting potential was significantly higher in mdx fibers, which suggests an elevated SR Ca(2+) leak. However, removal of external Ca(2+) reduced the rate of CPA-induced Ca(2+) increase in mdx and increased it in control fibers, which indicates an up-regulation of sarcolemmal Ca(2+) influx in mdx fibers. Fibers were then loaded with the low-affinity Ca(2+) dye Fluo5N-AM to measure intraluminal SR Ca(2+) changes. Trains of action potentials, chloro-m-cresol, and depolarization pulses evoked transient Fluo5N fluorescence decreases, and recovery of voltage-induced Fluo5N fluorescence changes were inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR Ca(2+) changes. Voltage dependence and magnitude of depolarization-induced SR Ca(2+) depletion were found to be unchanged in mdx fibers, but the rate of the recovery phase that followed depletion was found to be faster, indicating a higher SR Ca(2+) reuptake activity in mdx fibers. Overall, CPA-induced SR Ca(2+) leak at -80 mV was found to be significantly higher in mdx fibers and was potentiated by removal of external Ca(2+) in control fibers. The elevated passive SR Ca(2+) leak may contribute to alteration of Ca(2+) homeostasis in mdx muscle.
authors.affiliation: Université Lyon 1, Centre National de la Recherche Scientifique UMR 5534, Centre de Génétique et de Physiologie Moléculaire et Cellulaire, 69622 Villeurbanne Cedex, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Robin
authors.forename: Christine
authors.initials: C
authors.lastname: Berthier
authors.forename: Bruno
authors.initials: B
authors.lastname: Allard
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000378:metabolism; D004195:Disease Models, Animal; D055724:Electrophysiological Phenomena; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008810:Mice, Inbred C57BL; D018101:Mice, Inbred mdx; D018613:Microscopy, Confocal; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D000252:Calcium-Transporting ATPases