INTRODUCTION

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MUSCULAR ACTIVITIES requiring either short-term, high-intensity, or prolonged, low-intensity efforts typically result in progressive declines in the muscle's ability to generate force. This process, defined as fatigue, is thought to be a key factor limiting the individual's exercise and/or work capacity. Despite years of investigation, the precise mechanisms that mediate the fatigue process are not fully understood. Numerous investigators have provided a wealth of experimental evidence in an attempt to explain all or part of the fatigue process. In general, hypotheses concerning the mechanisms of fatigue fall into three broad categories: 1) depletion hypotheses that suggest that reduced availability of energy results in depressed force output, 2) accumulation hypotheses that propose that the build-up of metabolic by-products impairs the contractile process, and 3) calcium (Ca²⁺) exchange hypotheses that state that intrinsic alterations in the sarcoplasmic reticulum (SR) Ca²⁺ uptake and/or release properties limit activation of the contractile apparatus and reduce force output. Although proposed early by Eberstein and Sandow (8), this last hypothesis has recently gained increasing support. Much of this support comes from experiments that show that intracellular Ca²⁺ transients are depressed during the development of fatigue (1, 15, 19) and that the rates of SR Ca²⁺ release and uptake are reduced in skinned fibers and SR vesicles obtained from fatigued muscles (6, 9, 16, 17, 20, 21).

Without doubt, the fatigue process is complex, involving multiple factors and several mechanisms. The importance of each of these mechanisms likely depends on the type of muscle being studied, the type of activity employed, as well as model being used. Interestingly, a recent survey of pertinent literature indicates that in several different models using varied activity patterns, the rates of SR Ca²⁺ release and uptake are consistently depressed by 40-60% (21). For example, Williams et al. (22) found in frog muscle stimulated to fatigue within 3 min that Ca²⁺ uptake and release in skinned fibers were depressed, whereas Luckin et al. (16) found qualitatively similar results using rats and treadmill exercise lasting nearly 90 min. Studies such as these suggest that depressions in SR function are closely linked to the development of fatigue in a wide range of muscle activity patterns. That is, regardless of the type of activity used to induce fatigue, depressions in SR Ca²⁺ uptake and release occur and may contribute to the fatigue process.

Unfortunately, the disparate experimental approaches used and the differing degrees of fatigue elicited make it difficult to directly compare changes in SR Ca²⁺ uptake and release kinetics between varied activity patterns. Thus firm conclusions regarding the role of SR dysfunction in the fatigue process are somewhat tenuous. We suggest that if changes in SR function truly contribute to the fatigue process, then alterations should be apparent regardless of the mode of activity employed. With this in mind, the purpose of this investigation was to examine changes in SR function in muscles subjected to different stimulation patterns. In the first set of experiments, we used varying durations of stimulation, which resulted in similar degrees of fatigue. In the second set, we applied the stimulation protocols for similar durations, which resulted in varied degrees of fatigue. We then compared changes in SR Ca²⁺ uptake and release rates between protocols.

	METHODS

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In vitro muscle preparation. For all experiments, male grass frogs (Rana pipiens) were used and all procedures were approved by the Virginia Tech Animal Care and Use Committee. Animals were first placed in a cold-induced torpor (crushed ice-water slurry, 10 min). They were then rapidly decapitated, and both sartorius muscles were removed and placed in ice-cold Ringer solution.

The Ringer incubation solution contained (in mM) 115 NaCl, 2.5 KCl, 1.8 CaCl₂, 2.15 Na₂HPO₄, and 0.85 NaH₂PO₄ and was continually bubbled with room air (pH 7.2). Whole muscles were mounted in a temperature-controlled chamber (21°C), with one end of the muscle attached to a fixed post and the other to an isometric force transducer (Grass FT-03, 7P122 low-level direct current amplifier). Tetanic contractions were electrically evoked by 100-ms trains of pulses (0.2 ms, 100 Hz; Grass S-88 stimulator, SIU-5 stimulus isolation unit) delivered across platinum wire electrodes, which were situated at either end of the muscle. Muscles were allowed to equilibrate for 15-20 min, during which time optimal length was determined and tetanic force stabilized. All contractions were displayed in an oscilloscope (Techtronix 2201) and then digitized on a chart recorder [1 kHz, 12-bit analog/digital (A/D), Keithley-MetraByte DAS1608] and stored on disk via microcomputer. Contractions were analyzed off-line for peak tetanic force (P_o). In the rested state, P_o ranged from 20 to 25 N/cm² cross-sectional area (CSA), where CSA was computed as muscle wet mass divided by the product of muscle length and density (1.06 g/ml).

After the equilibration period, one muscle of each pair was subjected to a fatigue protocol in which tetanic contractions were evoked at 2.0, 0.5, or 0.2 trains per second (TPS). In the first set of experiments, stimulation was continued until force was reduced to ~20% of initial (constant fatigue, n = 8). In the second set, stimulation was continued for 1 min (constant duration, n = 6). In both experiments, contralateral control muscles were treated in a similar manner except that the fatigue protocol was not used.

Measurement of SR Ca²⁺ transport. The homogenizing buffer contained 250 mM sucrose, 20 mM HEPES (pH 7.5), 2% sodium azide (NaN₃), and 0.2 mM phenylmethylsulfonyl fluoride. Immediately after stimulation, muscles were placed in 8 volumes of ice-cold buffer and minced with scissors. They were homogenized, on ice, with a Pro 200 homogenizer and 5-mm probe using three 15-s bursts at ~12,000 rpm. The crude homogenates were then centrifuged at 1,600 g for 10 min (2°C), and the supernatant was removed and stored at 80°C. Total protein concentrations were determined with the Bradford protocol as modified by Bio-Rad.

The assay buffer consisted of 100 mM KCl, 20 mM HEPES, 7.5 mM pyrophosphate, 1 µM indo 1, 0.5 mM Mg²⁺, and 2 µM free Ca²⁺ (pH 7.0). Ca²⁺ uptake and release were measured by adding 200 µg of homogenate fraction protein to 1 ml of buffer. Uptake was initiated by adding 1 mM MgATP (10 µl of 100 mM stock) and allowed to continue until little or no change in extravesicular free Ca²⁺ concentration ([Ca²⁺]) was observed (Fig. 1). Ca²⁺ release was then initiated by the addition of 25 µM AgNO₃ (10 µl of 2,500 µM stock) or by the addition of 5 mM 4-chloro-m-cresol (4CMC) (10 µl 500 µM stock). During this procedure, the buffer solution was continually stirred and temperature was maintained at 37°C.

This level of free Ca²⁺ (2 µM) was found to be optimal for the Ca²⁺ uptake experiments. Free Ca²⁺ concentrations greater than 2 µM tended to result in slower uptake rates, possibly because of activation of Ca²⁺-induced Ca²⁺ release. We also found that 37°C was near optimal for assessing SR function in the frog homogenate fraction. At 21°C, the temperature used for the whole muscle experiments, Ca²⁺ uptake was slower than at 37°C. In addition, the indo 1 signal-to-noise ratio was increased, and the coefficient of variation within an individual muscle sample was sometimes in excess of 20%. Despite this, we found that in muscles stimulated at 2.0 TPS until force reached 20% of initial, the relative changes in Ca²⁺ uptake and release measured at 21°C were qualitatively similar to those observed at 37°C. However, because of the lower coefficient of variation obtained at 37°C (see RESULTS), this temperature was used for all experiments.

Extravesicular free [Ca²⁺] was monitored fluorometrically using a Jasco CAF-110 Intracellular Ion Analyzer and indo 1 as the extravesicular Ca²⁺ indicator. Excitation light came from a high-pressure xenon lamp equipped with a monochromator and filtered at 349 nm. Emission fluorescence was determined by a pair of photomultipliers using 410 nm (F₄₁₀) and 500 nm (F₅₀₀) filters. Fluorescence ratios (R = F₄₁₀/F₅₀₀) were sampled at 2 Hz (Keithley-MetraByte DAS1608, 12-bit A/D) and stored on disk for later analysis. Free [Ca²⁺] was computed using the ratiometric method of Grynkiewicz et al. (13) using the following equation: [Ca²⁺]_free = K_d ×  × (R  R_min)/(R  R_max), where the indo 1-Ca²⁺ dissociation constant (K_d) was assumed to be 250 nM (13), R_min and R_max are the R values measured in the uptake buffer with 10 mM EGTA added and with 1 mM Ca²⁺ added, respectively, and  is the ratio of F₅₀₀ recorded in EGTA- and Ca²⁺-supplemented buffers. The rates of Ca²⁺ uptake and release were computed from the steepest negative and positive slopes of the extravesicular free [Ca²⁺] versus time curve and normalized by the protein concentration. The initial fast phase of uptake, observed in Fig. 1 and reported by Gilchrist et al. (11), tended to be somewhat variable and was not considered in the calculation of uptake rate.

Statistical analyses. The effects of condition (rest, fatigue) and stimulation protocol on Ca²⁺ uptake and release were determined by repeated-measures analyses of variance adjusted for repeated measures made on contralateral muscles (split-plot). When needed, a Student-Newman-Keuls post hoc exam was used. Significance was set at the P < 0.05 level of confidence.

	RESULTS

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Validation of SR Ca²⁺ uptake and release measurement. A typical example of an SR Ca²⁺ uptake and release experiment is shown in Fig. 1. Our approach to measuring SR Ca²⁺ uptake and release using a homogenate fraction was found to be very reliable. Coefficients of variation for triplicate measurements made on an individual muscle were 3.56 ± 0.51 and 4.02 ± 0.49% (mean ± SE, n = 10) for uptake and release, respectively.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Typical example of the Ca2+ uptake and release measurements. In this example, uptake was initiated by the addition of 1 mM MgATP and release by 25 µM AgNO3. The initial, fast phase of uptake was not used in calculation of peak uptake rate. Brackets indicate concentration.

The technique of assessing SR Ca²⁺ uptake and release used here relies on measurements of extravesicular Ca²⁺ concentration. As opposed to measurements using isolated SR vesicles, the rates of extravesicular Ca²⁺ change presented here could be influenced by Ca²⁺ binding to other proteins and organelles present in the homogenate fraction. To ensure that our measurements accurately reflect SR Ca²⁺ uptake and release, several initial experiments were performed (Table 1). First, we found that cyclopiazonic acid (20 µM), which inhibits the SR Ca²⁺ ATPase (12), completely abolished Ca²⁺ uptake. Second, AgNO₃, which is known to evoke SR Ca²⁺ release via modification of specific sulfhydryl groups on the Ca²⁺ release channel (18), initiated release in our samples. We found that dithiothrietol, which prevents the actions of AgNO₃ (18), completely inhibited Ca²⁺ release. Finally, the inclusion of sodium azide (NaN₃) had no effect on either Ca²⁺ uptake or release. Taken together, these results suggest that the rates of Ca²⁺ uptake and release accurately reflect rates of SR Ca²⁺ uptake and release rather than some nonspecific Ca²⁺ binding and/or release.

Changes in SR Ca²⁺ uptake and release. Changes in P_o during the constant-fatigue experiments are shown in Fig. 2. There were no significant differences in the relative decline in P_o between the three protocols. When data of all protocols were pooled, the average reduction in P_o was 83% of initial. As anticipated, the time required for this reduction in force varied widely based on the protocol. The duration of activity was significantly longer (P < 0.05) at both 0.5 and 0.2 TPS than at 2.0 TPS. In addition, the number of stimuli required to reach the desired force output varied between protocols. Values at 0.2 TPS (202.8 ± 12.9) were significantly greater (P < 0.05) than those at 0.5 (130.5 ± 5.6) and 2.0 TPS (118.4 ± 5.7), which were not different from each other (P > 0.05). For the constant-duration experiments, stimulation was terminated after 1 min, which resulted in final forces of 15.3 ± 1.6, 55.3 ± 1.3, and 90.7 ± 2.3% of initial, respectively (P < 0.05 between all three conditions).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Changes in peak tetanic force during the 3 stimulation protocols. , 2.0 trains per second (TPS); , 0.5 TPS; and , 0.2 TPS. Values are means ± SE.

Changes in the rates of SR Ca²⁺ uptake following fatigue during the constant-fatigue experiments are shown in Fig. 3. As can be seen, all three protocols resulted in significant reductions in uptake rate. Qualitatively similar results for the rates of Ca²⁺ release are shown in Fig. 4. When either 25 µM AgNO₃ or 5 mM 4CMC was used, significant depressions in the Ca²⁺ release rates occurred. It is important to point out that the amounts of Ca²⁺ sequestered and released were not different between conditions (P > 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Changes in rates of Ca2+ uptake in control (open bars) and fatigued (solid bars) muscles of the constant-fatigue experiments. * P < 0.05 vs. control.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Changes in rates of Ca2+ release in control (open bars) and fatigued (solid bars) muscles of the constant-fatigue experiments. A: release initiated by 25 µM AgNO3. B: release initiated by 5 mM 4-chloro-m-cresol (4CMC). * P < 0.05 vs. control.

In the constant-duration experiments, the rate of Ca²⁺ uptake was significantly reduced following the 2.0 and 0.5 TPS protocols only (Fig. 5). In addition, the magnitude of reduction following the 2.0 TPS protocol was greater than that which occurred following the 0.5 TPS protocol. Changes in the rates of AgNO₃- and 4CMC-induced release followed a similar pattern (Fig. 6). That is, they were reduced following 2.0 and 0.5 TPS stimulation only, with the depression being larger following the higher-frequency stimulation protocol (P < 0.05).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Changes in rates of Ca2+ uptake in control (open bars) and fatigued (solid bars) muscles of the constant-duration experiments. * P < 0.05 vs. control.

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Changes in rates of Ca2+ release in control (open bars) and fatigued (solid bars) muscles of the constant-duration experiments. A: release initiated by 25 µM AgNO3. B: release initiated by 5 mM 4CMC. * P < 0.05 vs. control.

The difference in the Ca²⁺ uptake and release rates between contralateral rested and fatigued muscles is shown in Fig. 7. As can be seen, the percent difference in rates during the constant-fatigue experiments was not significantly different between protocols. Each protocol resulted in a 40-50% reduction in Ca²⁺ uptake and a similar reduction in Ca²⁺ release induced by either AgNO₃ or 4CMC. On the other hand, the percent reductions in Ca²⁺ uptake and release during the constant-duration experiments varied among each of the three protocols. The reductions in rates following stimulation at 0.5 TPS were approximately one-half of those that occurred following stimulation at 2.0 TPS, and the reductions in rates following 0.2 TPS were not different from zero.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Percent difference in Ca2+ uptake (open bars), AgNO3-stimulated release (hatched bars), and 4CMC-stimulated release (solid bars) between control and contralateral stimulated muscles. A: constant-fatigue experiments. B: constant-duration experiments. * P < 0.05 vs. 2.0 and 0.2 TPS.  P < 0.05 vs. 2.0 and 0.5 TPS.

	DISCUSSION

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The results of the constant-fatigue experiments show that when frog sartorius muscles are fatigued to similar extents, depressions in SR Ca²⁺ exchange kinetics are independent of the pattern of muscular activity. In each of the protocols used in the constant-fatigue experiments, an 83% reduction in P_o resulted in 40-50% depressions in Ca²⁺ uptake and release rates, values that were not significantly different between protocols. These similar alterations occurred despite the fact that 1-17 min of stimulation were required to reach the desired reduction in P_o. The results of the constant-duration experiments show that when muscles are stimulated for identical intervals, depressions in SR Ca²⁺ uptake and release rates vary considerably depending on the protocol used. With more intense activity patterns, where P_o is reduced to a greater extent, larger depressions in SR function occur. With less intense activity, where little force depression is induced, SR function is not markedly altered.

It is important to point out that small reductions in force occurred without alterations in SR function. For example, stimulation at 0.2 TPS for 60 s resulted in a ~10% reduction in P_o without any reductions in Ca²⁺ uptake or release. Allen and colleagues (1, 7, 15, 19) show that during the initial minutes of stimulation, a small reduction in P_o is associated with a rise in intracellular [Ca²⁺] ([Ca²⁺]_i) during the tetanus, possibly reflecting enhanced SR Ca²⁺ release and/or depressed uptake. These authors suggest that the reductions in P_o during "early fatigue" are likely the result of an altered intracellular milieu and the direct effects of elevated metabolite concentrations on force production by the contractile apparatus (1, 15, 19).

It is also important to recognize that Fitts et al. (10) found 26 and 74% reductions in rat soleus and extensor digitorum longus P_o following 8 h of swimming. These changes in force, however, were not associated with alterations in the rates of SR Ca²⁺ uptake or in Ca²⁺ ATPase activities. On the other hand, several studies show that treadmill exercise lasting in excess of 1 h does result in depression in both Ca²⁺ uptake and release (6, 9, 16, 17). Unfortunately, muscle force output was not measured in these studies. It is possible that the mechanisms of force reduction during very prolonged activity such as that employed by Fitts et al. (10) do not involve changes in SR function. Perhaps some other mechanism such as glycogen depletion is operative.

The unique aspect of this investigation is the use of a single model for comparing SR Ca²⁺ uptake and release rates following different patterns of muscular activity. Others have shown that following many different types of activity, each resulting in fatigue or exhaustion, qualitatively similar depressions in the rates of Ca²⁺ uptake and release occur. Reductions in Ca²⁺ uptake and/or Ca²⁺ ATPase activity have been observed in humans, rodents, horses, and amphibians following both whole body exercise as well as electrical stimulation of isolated muscle (for review see Ref. 21). We found that changes in SR Ca²⁺ uptake and release rates were more dependent on the degree of fatigue rather than on the pattern of activity. In each protocol used, similar degrees of fatigue elicited similar reductions in the rates of Ca²⁺ uptake and release. Likewise, changes in Ca²⁺ uptake and release were observed only when stimulation elicited a noticeable reduction in P_o (i.e., 45%). Thus it appears that changes in SR function are closely related to the reduction in tetanic force.

Similar conclusions have been reached by Allen et al. (1) and Baker et al. (2). They found a close relationship between P_o and [Ca²⁺]_i during both stimulation and recovery. Presumably, the changes in [Ca²⁺]_i reflect altered SR Ca²⁺ release. This latter notion is supported by the present results and those of Williams and colleagues (20, 22), which show that changes in SR Ca²⁺ release are correlated with the degree of force loss. The primary difference between our results and those obtained using intact fibers (1, 2, 7, 15, 19) is that the changes in SR function shown here persist even though the SR was removed from its fatigued environment and examined in a medium whose composition more closely simulates that of a rested cell. Although the skinned fiber and homogenate fraction preparations used to assess SR function cannot fully reproduce conditions of an intact cell, our data are consistent with the idea that the relationship between P_o and [Ca²⁺]_i during fatigue (1, 2) results from reductions in the rates of SR Ca²⁺ uptake and release.

When viewed as a whole, our results and the previous work of others raise the possibility that intrinsic "dysfunctions" of the SR are important mechanisms of fatigue during activities in which noticeable reductions in force production occur. It appears that in a wide range of activities, fatigue (or exhaustion) appears to be associated with reductions in the kinetics of SR Ca²⁺ uptake and release. Such changes could directly cause the depression in P_o that defines muscle fatigue. As muscular activity progresses, the diminished capability of the SR to adequately release and sequester Ca²⁺ results in decreased intracellular Ca²⁺ levels during contraction (1, 15, 19) and lowered activation of the contractile apparatus. As a consequence, P_o is decreased.

Luckin et al. (16) argue that changes in SR Ca²⁺ uptake during fatigue are due to structural alterations in the Ca²⁺ ATPase that disrupt its catalytic cycle. In terms of Ca²⁺ release, Favero et al. (9) show that in addition to depressed AgNO₃-induced release rates, ryanodine binding to its receptor is depressed following fatigue. Since ryanodine binds specifically to open Ca²⁺ channels, this suggests that fewer channels respond to activating stimuli. In this investigation we used two agents, AgNO₃ and 4CMC, to induce SR Ca²⁺ release. AgNO₃ induces release by binding to thiols on the Ca²⁺ release channel (i.e., sulfhydryl oxidation) (18) and involves two mechanisms, one that is inhibited by ruthenium red and one that is not (5). On the other hand, 4CMC elicits Ca²⁺ release via direct interaction with the release channel (14, 23). That Ca²⁺ release rates induced by these agents were depressed to similar extents by fatigue suggests that fatigue reduces the number of release channels that respond to activating stimuli (e.g., AgNO₃ and 4CMC). Without doubt, more information is needed to establish a mechanism that accounts for the depressed Ca²⁺ release observed in fatigued muscle.

In summary, we show that despite differing stimulation protocols used to elicit fatigue in isolated muscle, similar reductions in P_o are associated with comparable reductions in SR Ca²⁺ uptake and release kinetics. In addition, SR function is only altered when P_o is markedly reduced. When coupled with previous work of others, these results suggest that SR dysfunction plays an important role in the fatigue process.

Perspectives