title: Disruption of excitation-contraction coupling and titin by endogenous Ca2+-activated proteases in toad muscle fibres.
abstract: This study investigated the effects of elevated, physiological levels of intracellular free [Ca(2+)] on depolarization-induced force responses, and on passive and active force production by the contractile apparatus in mechanically skinned fibres of toad iliofibularis muscle. Excitation-contraction (EC) coupling was retained after skinning and force responses could be elicited by depolarization of the transverse-tubular (T-) system. Raising the cytoplasmic [Ca(2+)] to approximately 1 microm or above for 3 min caused an irreversible reduction in the depolarization-induced force response by interrupting the coupling between the voltage sensors in the T-system and the Ca(2+) release channels in the sarcoplasmic reticulum. This uncoupling showed a steep [Ca(2+)] dependency, with 50% uncoupling at approximately 1.9 microm Ca(2+). The uncoupling occurring with 2 microm Ca(2+) was largely prevented by the calpain inhibitor leupeptin (1 mm). Raising the cytoplasmic [Ca(2+)] above 1 microm also caused an irreversible decline in passive force production in stretched skinned fibres in a manner graded by [Ca(2+)], though at a much slower relative rate than loss of coupling. The progressive loss of passive force could be rapidly stopped by lowering [Ca(2+)] to 10 nm, and was almost completely inhibited by 1 mm leupeptin but not by 10 microm calpastatin. Muscle homogenates preactivated by Ca(2+) exposure also evidently contained a diffusible factor that caused damage to passive force production in a Ca(2+)-dependent manner. Western blotting showed that: (a) calpain-3 was present in the skinned fibres and was activated by the Ca(2+)exposure, and (b) the Ca(2+) exposure in stretched skinned fibres resulted in proteolysis of titin. We conclude that the disruption of EC coupling occurring at elevated levels of [Ca(2+)] is likely to be caused at least in part by Ca(2+)-activated proteases, most likely by calpain-3, though a role of calpain-1 is not excluded.
authors.affiliation: Department of Zoology, La Trobe University, Bundoora, Victoria 3086, Australia. e.verburg@latrobe.edu.au
authors.forename: Esther
authors.initials: E
authors.lastname: Verburg
authors.forename: Robyn M
authors.initials: RM
authors.lastname: Murphy
authors.forename: D George
authors.initials: DG
authors.lastname: Stephenson
authors.forename: Graham D
authors.initials: GD
authors.lastname: Lamb
mesh_terms: D000818:Animals; D002024:Bufo marinus; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002154:Calpain / Q000378:metabolism*; D002478:Cells, Cultured; D064211:Connectin; D004558:Electric Stimulation; D007527:Isoenzymes / Q000378:metabolism*; D007537:Isometric Contraction / Q000502:physiology*; D008564:Membrane Potentials / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D009124:Muscle Proteins / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013314:Stress, Mechanical
chemical_list: D064211:Connectin; D007527:Isoenzymes; D009124:Muscle Proteins; C577285:TTN protein, human; D011494:Protein Kinases; C105884:CAPN3 protein, human; D002154:Calpain; D002118:Calcium
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