Journal of Applied Physiology
Vol. 83, No. 2, pp. 444-450, August 1997
EXERCISE AND MUSCLE

Contractile apparatus and sarcoplasmic reticulum function: effects of fatigue, recovery, and elevated Ca²⁺

Jay H. Williams

Department of Human Nutrition, Foods, and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0430

ABSTRACT

INTRODUCTION

METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

FOOTNOTES

REFERENCES

ABSTRACT

Williams, Jay H. Contractile apparatus and sarcoplasmic reticulum function: effects of fatigue, recovery, and elevated Ca²⁺. J. Appl. Physiol. 83(2): 444-450, 1997.This investigation tested the notion that fatiguing stimulation induces intrinsic changes in the contractile apparatus and sarcoplasmic reticulum (SR) and that these changes are initiated by elevated intracellular Ca²⁺ concentration ([Ca²⁺]_i). Immediately after stimulation of frog semitendinosus muscle, contractile apparatus and SR function were measured. Despite a large decline in tetanic force (P_o), maximal Ca²⁺-activated force (F_max) of the contractile apparatus was not significantly altered. However, Ca²⁺ sensitivity was increased. In conjunction, the rate constant of Ca²⁺ uptake by the SR was diminished, and the caffeine sensitivity of Ca²⁺ release was decreased. During recovery, P_o, contractile apparatus, and SR function each returned to near-initial levels. Exposure of skinned fibers to 0.5 µM free Ca²⁺ for 5 min depressed both F_max and Ca²⁺ sensitivity of the contractile apparatus. In addition, caffeine sensitivity of Ca²⁺ release was diminished. Results suggest that fatigue induces intrinsic alterations in contractile apparatus and SR function. Changes in contractile apparatus function do not appear to be mediated by increased [Ca²⁺]_i. However, a portion of the change in SR Ca²⁺ release seems to be due to elevated [Ca²⁺]_i.

skeletal muscle; skinned fibers; calcium

INTRODUCTION

SUSTAINED MUSCULAR ACTIVITY often leads to a temporary reduction in the capacity of skeletal muscle to generate force. This transient loss of maximal force is defined as muscle fatigue. The degree of fatigue (i.e., the extent of force decline) varies considerably and is a function of fiber type, activation pattern, duration of activity, and numerous environmental factors. Despite nearly a century of effort, the precise mechanisms that mediate skeletal muscle fatigue have yet to be identified. This is unfortunate in light of the fact that prevention and alleviation of fatigue are critical for maintenance of normal muscular function.

Without doubt, there are numerous factors involved in the fatigue process. Nevertheless, insight into the mechanisms of fatigue can be gained by examining the sigmoidal relationship between force and free calcium concentration ([Ca²⁺]). On the basis of this relationship, Westerblad and colleagues (24) suggest three potential mechanisms of fatigue: reduced maximal Ca²⁺-activated force (F_max), decreased Ca²⁺ sensitivity {i.e., increased [Ca²⁺] needed to elicit 50% of F_max ([Ca²⁺]₅₀)}, and/or diminished myofilament activation {i.e., free myoplasmic Ca²⁺ concentration ([Ca²⁺]_i)}. Measurements of intracellular free Ca²⁺ transients during fatiguing contractions clearly show that all of these mechanisms may be operative. For example, Westerblad and Allen (21, 22) show that repetitive stimulation of single mouse muscle fibers leads to a decline in tetanic force (P_o) to ~40% of initial value. Using simultaneous measurements of force and [Ca²⁺]_i, they report that the loss of force is accompanied by a 20% reduction in F_max, a doubling of [Ca²⁺]₅₀, and a 50% decline in peak [Ca²⁺]_i. Because alterations in intracellular H⁺ and P_i concentrations are known to alter force production by the contractile apparatus (4, 11) and Ca²⁺ uptake and release by the sarcoplasmic reticulum (SR) (9, 20, 26), it is possible that the above changes simply reflect an altered intracellular milieu.

It is also possible these changes are due to intrinsic alterations in the functional properties of the contractile apparatus and SR. Williams et al. (28) have previously shown that both contractile apparatus and SR function of skinned fibers were altered after fatigue. Specifically, the Ca²⁺ sensitivity of force production was increased, and the ability of the SR to sequester and release Ca²⁺ was depressed. In addition, the rates of Ca²⁺ uptake and release are depressed in SR vesicles isolated from muscles after prolonged activity (for review, see Ref. 25). Interestingly, most studies of changes in SR function during fatigue compare rested and "fatigued" conditions. Few data are available to indicate the amount of activity needed to induce alterations. Williams et al. suggest that both contractile apparatus and SR function are only altered when force is <40-50%. However, this conclusion was on the basis of limited observations. It seems reasonable to suggest that if changes in force during fatigue are because of changes in contractile apparatus or SR function, then alterations in these parameters would parallel one another.

The results of Williams et al. also indicate that once the contractile apparatus and SR are removed from the fatigued intracellular environment, changes in function persisted. Unfortunately, few factors that might "trigger" such changes in contractile apparatus and SR function have been identified. During repetitive stimulation, resting intracellular Ca²⁺ concentration (i.e., [Ca²⁺]_i) increases 10-fold from ~0.05 to 0.5 µM (22). In both intact and skinned muscle fibers, elevations in intracellular Ca²⁺ initiate myofibrillar degradation leading to muscle damage (5, 6). In addition, exposure to physiological levels of free Ca²⁺ causes force deterioration and SR dysfunction in skinned fibers (14, 15). These changes are often observed after prolonged exposure to Ca²⁺. However, some myofibrillar and force degradation occur in as little as 90 s (5). Chin and Allen (3) have shown that elevated resting [Ca²⁺]_i might initiate transient structural alterations that lead to excitation-contraction coupling (ECC) failure and diminished force output. Thus it is possible that changes in contractile apparatus and SR dysfunction reported by Williams et al. (28) are initiated by elevated [Ca²⁺]_i.

The purpose of this investigation was to determine the extent of force depression during activity needed to evoke changes in contractile apparatus and SR function. This was accomplished by examining the time courses of changes in force as well as in contractile apparatus and SR function during fatigue and recovery. In addition, the notion that elevated intracellular Ca²⁺ mediates these effects was evaluated.

METHODS

RESULTS

The functional properties of the contractile apparatus were assessed by F_max and [Ca²⁺]₅₀. Values recorded in rested fibers were 223.9 ± 13.5 kN · m² and 1.80 ± 0.06 µM, respectively. Alterations in these parameters as well as changes in P_o during fatigue and recovery are shown in Fig. 1. As can be seen, P_o rapidly declined during the first minutes of stimulation to ~25% of initial value. Thereafter, P_o steadily declined to ~3%. Despite the large decline in P_o, F_max was not significantly affected. On the other hand, [Ca²⁺]₅₀ was significantly decreased but only at the 3.75- and 5-min measurements, when P_o was reduced to <10%. This latter result indicates that an increase in Ca²⁺ sensitivity occurs during fatigue but only when P_o is markedly reduced. The recovery of P_o was somewhat slow. It rapidly increased to ~30% during the first 20 min and then slowly returned to near initial levels (i.e., 95%) within 180 min. During this time, F_max remained constant. The reduction in [Ca²⁺]₅₀ rapidly returned to initial levels within 20 min. N was not significantly altered by either fatigue or recovery. In addition, similar effects of fatigue on F_max and [Ca²⁺]₅₀ were found by using Triton X-100 skinned fibers.

The functional properties of the SR were determined by k_Ca and CT. In rested fibers, values were 4.88 ± 0.10 min and 3.0 ± 0.3 mM, respectively. Changes in SR function during fatigue and recovery are indicated in Fig. 2. During the initial minute of stimulation, k_Ca declined by ~50%. Thereafter, it remained depressed to the same extent despite further reductions in P_o. On the other hand, CT steadily increased throughout the fatigue protocol, eventually doubling its initial value. The initial recovery of k_Ca was somewhat delayed, remaining <70% of initial value during the first 40 min of recovery. Thereafter, k_Ca steadily recovered, with full restoration accomplished by 60 min. CT rapidly declined during the first 40 min of recovery, reaching initial levels by 80 min.

To determine whether changes in [Ca²⁺]₅₀, k_Ca, or CT were transient, skinned fibers taken from muscles subjected to the 5-min fatigue protocol were allowed to incubate in relaxing solution for 0-30 min before measurements were performed. As can be seen in Fig. 3, reductions in [Ca²⁺]₅₀ and k_Ca and increases in CT were similar at each measurement interval. F_max remained constant throughout the 30-min incubation period. Thus it appears that fatigue-induced changes in the contractile apparatus and SR persist once the sarcolemma is disrupted.

After exposure to 0.5 µM free Ca²⁺, F_max was reduced by 17% from 214.1 ± 13.2 to 177.4 ± 22.9 kN · m² (P < 0.05) (Fig. 4). In addition, [Ca²⁺]₅₀ was increased from 1.73 ± 0.16 to 2.29 ± 0.19 µM (P < 0.05) without change in N.

Exposure to elevated Ca²⁺ increased the CT from 3.0 ± 0.5 to 4.6 ± 0.4 mM. Peak contracture forces in response to increasing caffeine concentrations are shown in Fig. 5A. For comparison, responses of rested fibers, those exposed to elevated Ca²⁺, and fatigued fibers are shown. As can be seen, both elevated Ca²⁺ and fatigue shifted this relationship to the right. The caffeine concentrations needed to evoke 50% of F_max were 4.5 ± 0.8, 6.2 ± 0.7, and 7.2 ± 1.1 mM for rested, elevated Ca²⁺, and fatigued fibers, respectively (P < 0.05). Unfortunately, the alterations in the force-free Ca²⁺ relationship due to elevated Ca²⁺ and fatigue make interpretation of the contracture force data difficult. This is particularly evident in the elevated Ca²⁺ fibers, in which F_max and [Ca²⁺]₅₀ were substantially affected. However, it is possible to gain some insight into the amount of Ca²⁺ released in response to caffeine by comparing contracture forces to the force-free Ca²⁺ relationships determined for each condition. Figure 5B shows the Ca²⁺ concentration corresponding to the caffeine contracture force. For each measurement of caffeine-induced force, free Ca²⁺ was estimated by using the mean values of [Ca²⁺]₅₀ and N determined for each condition in parallel experiments. It is important to point out that, although these data do not provide quantitative evidence of altered SR Ca²⁺ release, they do suggest that qualitative changes occur with elevated Ca²⁺ and fatigue. As can be seen, less Ca²⁺ is released by a given amount of caffeine after exposure to Ca²⁺ of the development of fatigue. In addition, the depression in release by elevated Ca²⁺ appears to be somewhat less than that induced by fatigue. The only exception to this was at 10 mM caffeine, where F_max was reached in all three conditions.

Exposure to elevated Ca²⁺ also reduced the contracture force evoked after timed Ca²⁺ loading (Fig. 6A) and reduced k_Ca from 4.93 ± 0.19 to 3.62 ± 0.25 min (P < 0.05). Figure 6B shows the estimated amount of Ca²⁺ released after loading for varying durations. As described in the previous paragraph, these values were estimated from the contracture forces shown in Fig. 6A and the mean [Ca²⁺]₅₀ and N values determined for each condition in parallel experiments. This amount is thought to reflect the amount sequestered during the loading period (7). When adjustment is made for the effects on the contractile apparatus, it appears as though elevated Ca²⁺ did not markedly affect Ca²⁺ sequestration. Fatigue, however, clearly depressed the amount of Ca²⁺ sequestered during loading for 5-60 s. Loading for 120 s was not affected by elevated Ca²⁺ or fatigue.

DISCUSSION

It is well documented that many conditions that mimic a fatigued intracellular milieu (e.g., low pH, elevated P_i) affect [Ca²⁺]₅₀ and F_max of the contractile apparatus and alter Ca²⁺ uptake and release by the SR (4, 9, 11, 20, 26). Westerblad et al. (24) argue that these changes account for, in part, changes in [Ca²⁺]_i and the force-free Ca²⁺ relationship measured in intact, fatigued muscle fibers. It should be noted, however, that such direct effects of an altered intracellular environment could not account for the changes in contractile apparatus and SR function presented here. In the present experiments, the contractile apparatus and SR were removed from the fatigued intracellular milieu of the fiber and examined in one that more closely simulated conditions at rest. Therefore, the results of these experiments indicate that repetitive stimulation leading to fatigue also induces intrinsic alterations in the functional properties of the SR and contractile apparatus. These changes include increased Ca²⁺ sensitivity of the contractile apparatus and reductions in Ca²⁺ uptake and caffeine sensitivity of the SR. In addition, the present results indicate that alterations in k_Ca, CT, and [Ca²⁺]₅₀ are completely reversible if the intact cell is allowed to recover (see Fig. 2). It is important to point out that because changes in these parameters are reversible in muscles allowed to recover, they represent transient alterations rather than permanent damage or injury. On the other hand, once the sarcolemma is disrupted, alterations in these parameters persist. Apparently some mechanism or agent responsible for the recovery of contractile apparatus and SR function is lost or inactivated once the sarcolemma is removed. Thus changes in [Ca²⁺]₅₀, k_Ca, and CT most likely reflect transient, fatigue-induced intrinsic alterations in SR and contractile apparatus function.

ACKNOWLEDGEMENTS

The author thanks Dr. Gary Klug and Christopher Ward for timely discussions of this work.

FOOTNOTES

Address for reprint requests: J. H. Williams, Dept. of Department of Human Nutrition, Foods, and Exercise, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0430 (E-mail: JHWMS{at}VT.EDU).

Received 23 December 1996; accepted in final form 7 April 1997.

REFERENCES