Impairment of sarcoplasmic reticulum functions, metabolic perturbations during muscle fatigue

Abstract

The purpose of these three studies was to examine the role of altered metabolic perturbations as a mechanism modulating sarcoplasmic reticulum (SR) function during the development of muscle fatigue. The first study examined the role of SR CA^2+ release during and following fatigue and attempted to define the role of specific intracellular metabolites in Ca^2+ release and recovery of mechanical unction. Following a 3 min stimulation protocol (15 Hz; 250 ms; 50% duty cycle) designed to produce fatigue and metabolic perturbations (decreases in ATP and PCr and increases in Pi and H+), in vitro rat diaphragms were subjected to one of the following three conditions: (1) two min of additional stimulation (2MS), (2) five min of passive recovery (5MR) and (3) twenty min of anoxic recovery (20MR). Similarly, a second fatigued diaphragm was exposed to one of these three conditions but in the presence of caffeine.

In the 2 MS group, fatigue at low frequencies (LFF) was only partially reversed by caffeine (P < 0.05), suggesting that part of the reduction in tension was due to an impaired cross-bridge activation. Caffeine exaggerated the reduction in tension (P < 0.05), with high-frequency fatigue (HFF), compared to the no caffeine group suggesting that the [Ca^2+]i was reduced beyond the level induced by the stimulation protocol only. For the 5MR, which was designed to allow for the normalization of ATP and PCr, the recovery period was not sufficient to reverse the effect of LFF in the no-caffeine diaphragm. However, caffeine mediated recovery produced increases in tension that were higher than the non-stimulated control (P < 0.05). Such an effect was not found for HFF. This finding suggested that with LFF, there was no inhibition at the cross-bridges, that caffeine was capable of neutralizing the inhibition placed on the Ca^2+ release channels by H^+ and that differences in the sensitivity of the myofibrillar apparatus to Ca^2+ occurred. The lack of tension recovery for the 20MR condition, which would have allowed sufficient time for the dissipation of H^+, suggests that a suppressed phosphorylation potential and increased metabolic by-products are the main mechanism responsible for the reduced tension.

In the second study, the sensitivity Ca^2+-MG^2+-ATPase activity to Ca^2+ ,H^+ and ATP was examined. This was done using homogenate preparations of muscles (white gastrocnemius medialis, WGM; red gastrocnemius medialis, RGM; soleus, S) containing different proportions of slow- and fast-twitch fibres known to reflect differences in SERCA 2a and 1 isoform content (Lytton et al., 1992). It was hypothesised that differences in sensitivity of Ca^2+-Mg^2+-ATPase activity in the homogenate to the above metabolic factors would be dependent on the fibre type composition, reflecting differences in SERCA 2a and 1 isoform content (Lytton et al., 1992). It was hypothesised that differences in sensitivity of Ca^2+-Mg^2+-ATPase activity in the homogenate to the above metabolic factors would be dependent on the fibre type composition, reflecting different contents of the two isoforms. Although the results showed that the dependency to Ca^2+ and the ATP by the three muscle fibre types were similar, the dependency to H^+ between homogenates of WGM and RGM compared to S was different (P < 0.05). These results suggested that the sensitivity of SERCA 1a isoform to pH was greater than the sensitivity to pH of the SERCA 2a isoform.

The final study was designed to determine if the selectivity of the isolation protocol could be a contributing factor in explaining the discrepancy in results that exist between laboratories (Byrd et al., 1989a; Chin & Green, 1996) concerning the effect of prolonged exhaustive exercise on Ca^2+-Mg^2+-ATPase activity. The general procedures of Byrd et al. (1989a) were carefully reproduced to minimise the experimental variability between the two studies. Rats were run to exhaustion on a slope treadmill. The red muscles (gastrocnemius and vastus) of two animals were pooled together for the control and run groups. Ca^2+-Mg^2+-ATPase activity and activities of marker enzyme's for mitochondrial and sarcolemmal contamination were measured in homogenate, various sub-fractions and final vesicle fractions. The results showed no differences in total, basal and maximal Ca^2+-stimulated activity between the control and run animals for the homogenate, sub-fractions and vesicle fractions. The results also confirmed previous published results (Dossett-Mercer et al., 1994; Chin & Green, 1996) from this laboratory that found no depression in Ca^2+-Mg^2+-ATPase activity following running exercise. The discrepancy that exists regarding the on the effect of prolonged exercise on Ca^2+-Mg^2+-ATPase activity cannot be explained on the basis of differences in species of rats, exercise protocol or isolation procedure, since these variables were similar in the two studies.