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.

Fig. 1

Schematic diagram illustrating the orthogonal method of measurement of the T-SR distance. A linear test system was overlain and, where a test line intersected either the T-tubular (T) or sarcoplasmic reticular (SR) component of a junction, the length of an orthogonal intercept (↔) was measured between the T and SR

Fig. 2

Representative transmission electron microscopy images of triads (arrows) in (a) control muscles, (b) muscles exposed to intermittent low frequency electrical stimulation and (c) muscles exposed to electrical stimulation but then allowed to recover in normal Ringer solution for 60 min

Fig. 3

Measurements (means ± SEM; n = 3) of T-SR distance and T-tubule diameter from serial EM sections of control muscles (blank bars), muscles exposed to intermittent low frequency electrical stimulation (grey bars) and muscles exposed to electrical stimulation but then allowed to recover in normal Ringer solution for 60 min (black bars). *indicates a significant (P < 0.05) difference from control muscles

Fig. 4

(a) Shows the effects of 15 mM (grey bars) and 60 mM (black bars) extracellular [K⁺] in Cl⁻-free SO₄²⁻ containing Ringer solution on T-SR distance and T-tubule diameter (means ± SEM) compared to normal Cl⁻-free SO₄²⁻ Ringer solution (open bars). (b) Shows the corresponding effects of 15 mM (grey bars) and 60 mM (black bars) extracellular [K⁺] in Cl⁻ containing Ringer solution compared to normal Ringer solution (open bars). *Indicates a significant (P < 0.05) difference from control muscles and ⁺a significant difference between two conditions

Fig. 5

The effects of intracellular acidification brought about by addition and withdrawal of NH₄Cl (grey bars) and exposure to extracellular lactate (black bars) on T-SR distance and T-tubule diameter compared to normal Ringer solution (open bars). *Indicates a significant (P < 0.05) difference from control muscles and ⁺a significant difference between two conditions