title: Alterations in triad ultrastructure following repetitive stimulation and intracellular changes associated with exercise in amphibian skeletal muscle.
abstract: This study used Rana temporaria sartorius muscles to examine the effect of fatiguing electrical stimulation on the gap between the T-tubular and sarcoplasmic reticular membranes (T-SR distance) and the T-tubule diameter and compared this with corresponding effects on resting fibres exposed to a range of extracellular conditions that each replicate one of the major changes associated with muscular activity: membrane depolarisation, isotonic volume increase, acidification and intracellular lactate accumulation. Following each treatment, muscles were immersed in isotonic fixative solution and processed for transmission electron microscopy (TEM). Mean T-SR distances were estimated from orthogonal intercepts to provide estimates of diffusion distances between T and SR membranes and T-tubule diameter was estimated by measuring its shortest axis in the sampled J-SR complexes. Measurements from muscles fatigued by low frequency intermittent stimulation showed significant (P << 0.05) reversible increases in both T-SR distance and T-tubule diameter from 15.97 +/- 0.37 nm to 20.15 +/- 0.56 nm and from 15.44 +/- 0.60 nm to 22.26 +/- 0.84 nm (n = 40, 30) respectively. Exposure to increasing concentrations of extracellular [K(+)] in the absence of Cl(-) to produce membrane depolarisation without accompanying cell swelling reduced T-SR distance and increased T-tubule diameter, whilst comparable increases in [K(+)](e) in the presence of Cl(-) suggested that isotonic cell swelling has the opposite effect. Acidification alone, produced by NH(4)Cl addition and withdrawal, also decreased T-SR distance and T-tubule diameter. A similar reduction in T-SR distance occurred following exposure to extracellular Na-lactate where such acidification was accompanied by elevations of intracellular lactate, but these conditions produced a significant swelling of T-tubules attributable to movement of lactate from the cell into the T-tubules. This study thus confirms previous reports of significant increases in T-SR distance and T-tubule diameter following stimulation. However, of membrane depolarisation, isotonic cell swelling, intracellular acidification and lactate accumulation, only isotonic cell swelling increases T-SR distance whilst membrane depolarisation and intracellular lactate likely contribute to the observed increases in T-tubule diameter.
authors.affiliation: Physiological Laboratory, Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3EG, UK. jau20@cam.ac.uk
authors.forename: Juliet A
authors.initials: JA
authors.lastname: Usher-Smith
authors.forename: James A
authors.initials: JA
authors.lastname: Fraser
authors.forename: Christopher L-H
authors.initials: CL
authors.lastname: Huang
authors.forename: Jeremy N
authors.initials: JN
authors.lastname: Skepper
mesh_terms: D000818:Animals; D004558:Electric Stimulation; D006863:Hydrogen-Ion Concentration; D007773:Lactates / Q000378:metabolism*; D008564:Membrane Potentials; D032342:Muscle Cells / Q000502:physiology* / Q000648:ultrastructure; D009119:Muscle Contraction / Q000502:physiology*; D018763:Muscle Fatigue / Q000502:physiology*; D018482:Muscle, Skeletal / Q000502:physiology* / Q000648:ultrastructure; D005082:Physical Exertion; D011896:Rana temporaria; D012519:Sarcoplasmic Reticulum / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D007773:Lactates