Disturbances of the sarcoplasmic reticulum and transverse tubular system in 24-h electrostimulated fast-twitch skeletal muscle

Abstract Chronic low-frequency stimulation of rabbit tibialis anterior muscle over a 24-h period induces a conspicuous loss of isometric tension that is unrelated to muscle energy metabolism (J.A. Cadefau, J. Parra, R. Cusso, G. Heine, D. Pette, Responses of fatigable and fatigue-resistant fibres of rabbit muscle to low-frequency stimulation, Pflugers Arch. 424 (1993) 529-537). To assess the involvement of sarcoplasmic reticulum and transverse tubular system in this force impairment, we isolated microsomal fractions from stimulated and control (contralateral, unstimulated) muscles on discontinuous sucrose gradients (27–32–34–38–45%, wt/wt). All the fractions were characterized in terms of calcium content, Ca 2+ /Mg 2+ -ATPase activity, and radioligand binding of [ 3 H]-PN 200-110 and [ 3 H]ryanodine, specific to dihydropyridine-sensitive calcium channels and ryanodine receptors, respectively. Gradient fractions of muscles stimulated for 24 h underwent acute changes in the pattern of protein bands. First, light fractions from longitudinal sarcoplasmic reticulum, enriched in Ca 2+ -ATPase activity, R 1 and R 2 , were greatly reduced (67% and 51%, respectively); this reduction was reflected in protein yield of crude microsomal fractions prior to gradient loading (25%). Second, heavy fractions from the sarcoplasmic reticulum were modified, and part (52%) of the R 3 fraction was shifted to the R 4 fraction, which appeared as a thick, clotted band. Quantification of [ 3 H]-PN 200-110 and [ 3 H]-ryanodine binding revealed co-migration of terminal cisternae and t-tubules from R 3 to R 4 , indicating the presence of triads. This density change may be associated with calcium overload of the sarcoplasmic reticulum, since total calcium rose three- to fourfold in stimulated muscle homogenates. These changes correlate well with ultrastructural damage to longitudinal sarcoplasmic reticulum and swelling of t-tubules revealed by electron microscopy. The ultrastructural changes observed here reflect exercise-induced damage of membrane systems that might severely compromise muscle function. Since this process is reversible, we suggest that it may be part of a physiological response to fatigue.