INTRODUCTION

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DURING STRENUOUS ACTIVITY, the intracellular pH (pH_i) of skeletal muscle almost always decreases as lactic acid accumulates. The importance of this fall in pH_i to the development of impaired force production is debated (for a recent review, see Ref. 9). Three observations have been interpreted as demonstrating that reduced pH_i is an important factor in the development of muscle fatigue. First, low pH inhibits activity of enzymes such as phosphofructokinase (28), which are important for maintenance of the muscle energy supply. Second, studies that used either skinned or intact muscle fibers have shown clearly that low pH has an inhibitory effect on muscle performance, reducing both force output and shortening velocity (17, 31). Finally, investigations on exercising muscle that used ³¹P-nuclear magnetic resonance have shown that there is a good correlation between the decline in pH_i and the fall in force as fatigue develops (e.g., Ref. 19).

However, other studies suggest that the importance of these three factors may be questioned. First, other metabolites and ions may counteract the inhibitory effects of acidosis on enzyme activity (3, 26). Second, inhibition of force generation in muscle by low pH_i has been demonstrated to be temperature dependent (e.g., Ref. 22). Recently, it has been established that the inhibitory effects of hydrogen ions on force production (35) and shortening velocity (20, 32) in isolated mammalian muscle are inversely related to temperature and that in the physiological range of temperatures inhibition of muscle function is slight. Third, Sahlin and Ren (24) have demonstrated in humans that, although there might be a correlation between reduced pH_i and force during the development of fatigue, this correlation disappeared during recovery, during which force increased much faster than pH. Also, in subjects with myophosphorylase deficiency, force declines during the development of fatigue despite little change in pH_i (e.g., Ref. 7).

Surprisingly little work has been done to determine whether reduced pH has an adverse effect on the ability of skeletal muscle to maintain force above a given level during high-intensity exercise or repeated tetani at short intervals. Although many studies have concluded that acidosis is not the primary cause of fatigue (e.g., Refs. 2, 3), other investigators who used rats (25) and humans (12, 24) concluded that acidosis reduces the time to fatigue or exhaustion. However, the reduced endurance might reflect other changes in the intracellular or extracellular milieu rather than being a direct effect of hydrogen ions. Use of isolated single fibers allows one to control the surrounding environment and produce an isolated acidosis without other metabolic changes. In the present study, we have measured the number of tetani needed to reduce force to 40% of the original in single skeletal muscle cells subjected to repeated tetani at short intervals (i.e., a type of high-intensity activity during which pH would be expected to have a large effect). Fatigue was induced under normal and hypercapnic conditions at near-physiological temperatures, and we found the number of tetani needed to produce fatigue to be unaffected by low pH_i.

	METHODS

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Adult male mice were killed by rapid neck disarticulation. Single muscle fibers were isolated from the surface of the flexor brevis muscles as described previously (14). Platinum clips were attached to the tendons, and the fibers were then mounted horizontally in a muscle chamber, with one clip attached to an adjustable hook and the other to an Akers AE 801 force transducer. Force was recorded on a strip-chart recorder and stored on a personal computer. The fiber length was adjusted to the length that gave maximum tetanic force. Electrical stimulation of the fiber was achieved via platinum strips running parallel to the long axis of the fiber. Biphasic current pulses with an amplitude of 120-150% of the contraction threshold were used to stimulate the fiber. The stimulation frequency and duration used to obtain tetani were 80 Hz and 400 ms, respectively. Isometric tetanic force was measured as the maximum force during a tetanus. The resistance to fatigue was defined as the number of tetani required to reduce force to 40% of the initial value. The rate of relaxation was defined as the inverse of the time from the last stimulus of a tetanus until force had declined to 70% of that at the end of the tetanus.

Solutions. Muscle fibers were superfused with a Tyrode solution containing (in mM) 121 NaCl, 5 KCl, 0.5 MgCl₂, 0.4 NaH₂PO₄, 1.8 CaCl₂, 1 EDTA, 25 NaHCO₃, and 5.5 glucose (bubbled with 95% O₂-5% CO₂, pH 7.4), which will be referred to as normal Tyrode. Muscle fibers were made acidotic by bubbling the Tyrode solution with 70% O₂-30% CO₂ (pH 6.6), and this solution will be referred to as 30% CO₂ Tyrode. Fetal calf serum (0.2%) was added to all solutions. The temperature of the perfusing solution was monitored close to the fiber and was kept constant by means of a Peltier heat exchanger mounted at the entrance to the experimental chamber.

Temperature. All the experiments examining the resistance to fatigue of muscle fibers were undertaken at 28°C. Our choice of 28°C was dictated by several factors. First, it is close to the subcutaneous temperature in the vicinity of the flexor brevis muscles, which, when measured in mice immediately after neck disarticulation, yielded a value of 30.5 ± 0.5°C (n = 6). Second, 28°C is in the range of temperatures at which both tetanic force and endurance are near maximal in mammalian muscle (4, 21, 23). Finally, our fibers tended to lose their ability to maintain force production when subjected to repeated fatigue and recovery protocols at higher temperatures; the causes of this were not investigated further. After production of fatigue in a muscle fiber at 28°C, the temperature of the bathing solution was reduced immediately to 22°C, and the fiber was rested for at least 90 min and up to 150 min before a further fatigue run was undertaken again at 28°C.

Induction of fatigue. Eight minutes before the induction of fatigue, the bath temperature was raised to 28°C. In the final 5 min before the start of fatiguing stimulation, fibers were exposed to either Tyrode solution bubbled with 5% CO₂ (normal Tyrode) or to 30% CO₂ (30% CO₂ Tyrode). Fibers were repeatedly stimulated with trains of tetani at intervals of 4 s for 2 min, and then the interval was reduced to 3 s for 2 min and then to 2.5 s for 2 min until force had declined to 40% of control. Fatigue was induced in a total of 24 fibers. The first 15 of these fibers were subjected to a single fatigue run. The number of tetani needed to reduce force to 40% differed considerably in different muscle fibers, as had been noted previously (15). To overcome the problem of variation in the number of tetani need to fatigue different fibers, nine fibers were subjected to three fatigue runs so that each fiber might act as its own control. In these latter nine fibers, fatigue was produced in either 5% CO₂ Tyrode or 30% CO₂ Tyrode in the first and third fatigue runs. The number of tetani required to reduce force to 40% in the first and third fatigue runs was then averaged and compared with the number of tetani needed to achieve the same reduction in force in the second fatigue run. In addition, the order of exposure to 5% CO₂ and 30% CO₂ Tyrode was randomized so that five fibers were exposed first to 5% CO₂ and four to 30% CO₂ Tyrode.

pH_i measurements. In some experiments, pH_i was measured during the induction of fatigue at 28°C by using the fluorescent probe carboxy-SNARF 1 (Molecular Probes Europe, Leiden, The Netherlands). The methods have been described in detail previously (32). Briefly, fibers were incubated in 10 µM of the membrane-permeable acetoxymethyl ester form of the dye for 10 min. Fibers were washed in normal Tyrode for 20 min before pH measurements began. A total of six fibers were used to obtain the pH measurements, and, of these fibers, four were fatigued once (3 fibers in normal Tyrode and 1 in 30% CO₂ Tyrode) and the remaining two were fatigued twice (the first run performed in 30% CO₂ Tyrode and the second in normal Tyrode) with a 90-min recovery period between the two fatigue runs. During experiments, the dye was excited with light passing through a 540 ± 10-nm interference filter. The emitted light was measured at 580 ± 5 and 640 ± 5 nm with two photomultiplier tubes. The pH_i-dependent signal was expressed as the ratio (R) of the signal at 580 to that at 640 nm. This ratio was converted into pH_i values by using the equation

(1)

Values for negative logarithm of apparent dissociation constant (pK_app; 7.6), ratio at high pH (R_min; 1.43), and ratio at low pH (R_max; 3.68) were derived from our recent study of pH_i in single mouse muscle fibers by using linear interpolation between 22 and 32°C (32).

Statistics. Values are shown as means ± SE. Paired or unpaired Student's t-tests were used as necessary to verify statistical significance, and the significance level was set to 0.05.

	RESULTS

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Figure 1 shows the typical changes in pH_i of two different fibers during the induction of fatigue (shown in bottom traces in A and B) in normal (A) and 30% CO₂ Tyrode (B). It can be seen that, in the fiber fatigued in normal Tyrode (Fig. 1A), pH_i fell during the period of stimulation by ~0.15 pH unit. In five fibers fatigued in normal Tyrode, the mean pH_i value of 6.91 ± 0.09 at the end of stimulation was significantly less (P = 0.004) than the initial starting value of 7.18 ± 0.07. The changes in pH_i in a different fiber when the solution was changed to 30% CO₂ Tyrode and the subsequent induction of fatigue are shown in Fig. 1B. In this fiber, on exposure to 30% CO₂ Tyrode, pH_i decreased by ~0.3 pH unit and was reduced by a further 0.3 pH unit by the end of the period of stimulation. In the three fibers in which pH_i was monitored during the induction of fatigue in 30% CO₂ Tyrode, the mean pH_i was 7.17 ± 0.06 in normal Tyrode. Exposure to 30% CO₂ reduced pH_i to 6.77 ± 0.13. During fatigue in 30% CO₂ Tyrode, pH_i showed an additional decrease and had decreased significantly (P = 0.008) to 6.46 ± 0.12 by the time force was reduced to 40% of its initial value. Thus, in both normal and 30% CO₂ Tyrode, pH_i was significantly reduced by ~0.3 pH unit during the development of fatigue.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Intracellular pH (pHi) was measured with carboxy-SNARF 1 in 2 different fibers during induction of fatigue in normal (A) and 30% CO2 Tyrode (B). A: top trace shows that pHi fell slightly during course of repetitive tetanic contractions. B: change from 5 to 30% CO2 (indicated by solid line) caused a fall in pHi (top trace), and there was a further decrease in pHi during the course of repetitive stimulation. Dotted line indicates a gap of 150 s. Note that there was no reduction of tetanic force (bottom traces) with switch from 5 to 30% CO2 despite a large fall in pHi.

At 28°C, tetanic force was little affected by acidosis, being 354.9 ± 11.3 kPa (n = 14) in fibers before the first fatigue run in normal Tyrode and 347.1 ± 13.2 kPa (n = 10) before the first fatigue run in 30% CO₂ Tyrode. The mean number of tetani needed to reduce force to 40% of the initial value for fibers fatigued in normal Tyrode (63 ± 10 tetani; n = 14) was not significantly different (P = 0.08) from that needed in 30% CO₂ Tyrode (98 ± 18 tetani, n = 10). However, the number of tetani required to fatigue different muscle fibers varied considerably, and, to overcome this problem, we subjected nine fibers to three fatigue runs so that each fiber might act as its own control. Figure 2A shows the tetanic force generated during three fatigue runs carried out on the same fiber. The first and last records were obtained in normal Tyrode, and the middle fatigue run was obtained in 30% CO₂ Tyrode. In this fiber, the number of tetani needed to reduce force to 40% of the initial value was clearly greater in 30% CO₂ Tyrode than in normal Tyrode. A total of nine fibers were subjected to triple fatigue runs. The number of tetani required to reduce force to 40% of the initial value for each of the individual fibers in both normal and 30% CO₂ Tyrode solution is plotted in Fig. 2B. Although there was a large spread in the number of tetani needed to fatigue individual fibers, the number of tetani needed under control and acidic conditions for a given fiber was similar. The slope of the relationship was 0.98 ± 0.09, which was not significantly different from the slope of the line of identity (dotted line in Fig. 2B, slope = 1). Figure 2C shows the mean number of tetani needed to reduce force to 40% of the starting value in the nine fibers subjected to triple fatigue runs. The data show clearly that there was no significant difference (P = 0.7) in the number of tetani needed to reduce force to 40% of the initial value in control (94 ± 14) and acidic (98 ± 12) conditions.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Acidification did not reduce number of tetani needed to induce fatigue in mouse single muscle fibers. A: typical example of 3 fatigue runs in the same fiber in which the fiber was stimulated until force had fallen to 40% of initial value. Top and bottom traces show fatigue in normal Tyrode (bubbled with 5% CO2), while middle trace shows fatigue in Tyrode bubbled with 30% CO2. Time and force calibration bars are common to all 3 records. B: plot of number of tetani needed to induce fatigue in each of the 9 fibers subjected to 3 fatigue runs. , Fibers exposed to 5% CO2 in first and last fatigue runs; , fibers exposed to 30% CO2 in first and last fatigue runs. Dashed line shown for reference is line of identity. C: values (means ± SE) are no. of tetani needed to reduce force to 40% of control in 9 single muscle fibers subjected to 3 fatigue runs. Values are not significantly different from each other.

As stated above, there was no significant effect of intracellular acidification on tetanic force production or the number of tetani required to induce fatigue. However, there was a very clear difference in the relaxation rate in the two experimental conditions, especially by the end of the repeated tetanic stimulation used to induce fatigue (Fig. 3A). Mean values of the relaxation rate are shown in Fig. 3B. In control Tyrode, the relaxation rate had been significantly reduced (P = 0.04) from its starting value by the end of the period of stimulation, being 26.3 ± 2.1 and 19.8 ± 2.2 s¹ for the first and last tetani, respectively (n = 8). Compared with normal Tyrode, the relaxation rate in 30% CO₂ Tyrode was already significantly decreased (P = 0.006) at the start of stimulation and was further reduced when electrical stimulation was ended. In normal Tyrode before exposure to 30% CO₂ Tyrode the relaxation rate was 26.1 ± 1.8 s¹, and in 30% CO₂ Tyrode the relaxation rate for the first tetanus was 20.5 ± 1.4 s¹ and that for the last tetanus of the fatigue run was significantly reduced (P < 0.001) to 10.6 ± 2 s¹ (n = 8). Thus acidification of the muscle fibers caused a marked reduction in the rate of relaxation.

View larger version (11K): [in this window] [in a new window]   Fig. 3.   Acidification induced with 30% CO2 reduced rate of relaxation in fatigue. Records in A and B are from the same fiber, which was fatigued first in normal Tyrode and then, after it recovered for 120 min, was fatigued a second time in 30% CO2 Tyrode. A: first tetanus of fatigue run in either normal (solid lines) or 30% CO2 (broken lines) Tyrode is shown at left, and last tetanus in fatigue runs is shown at right. Solid lines under force records indicate period of 80-Hz electrical stimulation at right. Calibration bars are common to both records in A. B: mean rate of relaxation of tetanic force at start and end of fatigue in fibers exposed either to normal Tyrode only () or to normal Tyrode and then to 30% CO2 Tyrode 5 min before start of the fatigue run (). First, relaxation rate of first tetanus in fatigue run; Last, relaxation rate of last tetanus in fatigue run; Pre 30%, relaxation rate in normal Tyrode before induction of fatigue in 30% CO2 Tyrode. Values are means ± SE from 8 fibers.

	DISCUSSION

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The main finding of this study is that at near-physiological temperatures the number of tetani required to induce muscle fatigue in single mouse muscle fibers was little affected by acidification. However, acidification caused a marked reduction in the rate of relaxation of force, which became especially pronounced as fatigue developed. The stimulation protocol used to induce fatigue in the mouse muscle fibers was intense, and presumably usage of anaerobic energy sources would dominate. Obviously, extrapolation of the present results to animal or human models of fatigue must be done with caution. Nonetheless, in situations such as all-out sprinting, in which maximal effort is exerted for a short period, muscle acidification and the accompanying slowing of relaxation would impair performance. The slowed relaxation rate means that maintenance of coordinated alternating movements would be possible only if the frequency or magnitude of these movements is reduced.

Repetitively stimulating fibers in normal Tyrode until force had fallen to 40% of control caused a significant decrease in pH_i of ~0.3 pH unit. The decrease in pH is less than that reported by other groups that have examined the relationship between pH and fatigue and found that pH_i falls from ~7.0 at rest to 6.3-6.4 when fatigue was established (16, 19, 26). This probably reflects the different preparations and fatigue protocols used. In an earlier study that used a similar preparation and fatigue protocol, Westerblad and Allen (30) found a far smaller decline (0.063 pH unit) in pH_i compared with that observed here. The explanation for the discrepancy might lie in the lower temperature that they employed (22°C compared with 28°C here). It is unlikely to be due to the different indicators used because they observed pH_i changes similar to those reported here when CO₂ was increased from 5 to 30%.

Little work has been done to investigate what effect acidification has on the ability of muscle to maintain force production when subjected to repeated maximal contractions. Part of the reason for this is the difficulty in changing the pH_i in an intact animal without at the same time causing other changes. In the present study, maintaining the fibers in 30% CO₂ Tyrode caused no significant changes in the number of tetani required to induce muscle fatigue. Hogan and Welch (12) induced changes in blood lactate by first making their human subjects breathe gas containing different amounts of O₂ before undergoing exercise to exhaustion while breathing 21% O₂. They found that, after breathing 60% O₂, subjects had a lower blood lactate level and were able to exercise for longer than after breathing 16% O₂. They concluded that performance could be significantly affected by lactate. In humans, elevated muscle lactate as a result of a prior bout of exercise resulted in a reduced endurance during a second bout of exercise (3, 24). The greater apparent effect of acidosis on endurance in these studies might not be due to direct effects on muscle function. For example, elevated levels of blood lactate might result in a heightened perception of the development of fatigue. Alternatively, as previous results have indicated, it might be due to accumulation of potassium in the interstitial spaces (3). In isolated muscle fibers this latter type of complication is avoided by the constant flow of Tyrode solution, which ensures that the fiber's milieu is kept relatively constant.

The present result that acidification has no effect on the ability of mouse muscle fibers to maintain force production during repetitive stimulation apparently conflicts with the conclusions of a previous study in which the same type of single-fiber preparation and fatiguing protocol was used (30). In that study, fatigue occurred more rapidly and was accompanied by a significant fall of pH_i after application of cinnamate, which was presumed to inhibit sarcolemmal lactate transport, or when the mitochondria were inhibited by cyanide. This might indicate that acidification causes fatigue to occur more rapidly. However, the earlier onset of fatigue may not reflect a direct effect of acidosis but rather some other effect of the drugs used. Inhibition of mitochondrial function with cyanide will severely affect energy metabolism, and some factor related to changes of the concentration of high-energy phosphates may be the direct cause of the faster fatigue development. Cinnamate, which was used to inhibit lactate transport over the cell membrane, may also inhibit mitochondrial function by blocking the mitochondrial pyruvate uptake (6, 10), and, in line with this, fibers exposed to cinnamate eventually went into contracture (30). Thus, on the basis of present results, we conclude that the more rapid onset of fatigue observed with cyanide and cinnamate in the previous study of mouse muscle fibers may not be due to the acidification but rather may be a consequence of inhibited mitochondrial function.

Other studies have concluded on the basis of a correlation between force recovery and normalization of pH_i after muscle fatigue (force decline of ~80%) in frog sartorius and rat diaphragm that at least part of the cause of fatigue is due to an elevation of hydrogen ion concentration (16, 27). However, these studies have no data concerning changes in pH_i during the development of fatigue. With use of a less-exhaustive fatigue protocol, no correlation was observed between the recovery of pH_i and force in isolated frog single muscle fibers (33).

The most obvious effect of hypercapnia on muscle fibers was the reduced rate of relaxation of tetanic force, which was slight in the fiber at rest and marked by the end of the fatiguing process. Many but not all previous studies on nonfatigued acidified muscle have reported a similar slowed rate of relaxation (11, 31, 32). In fatigued frog muscle, the reduced rate of relaxation of force seems to be due predominantly to a slowed calcium removal from the myoplasm (1, 8, 34). However, in mouse skeletal muscle fibers, although there is some slowing of the sarcoplasmic reticulum pump, the major cause of the slower relaxation in acidified or fatigued muscle is slowed cross-bridge kinetics (31, 34). Our measure of the relaxation rate is dominated by the linear phase of relaxation, during which fibers have been shown to relax isometrically (13). The rate of force redevelopment after a shortening step can be used to assess the rate of cross-bridge cycling under isometric conditions (5). Metzger and Moss (18) have shown that after a shortening step the rate of force redevelopment and, hence, the rate of cross-bridge cycling is not pH sensitive when saturating levels of Ca²⁺ are used to activate skinned muscle fibers. However, when submaximal levels of Ca²⁺ are used (which is similar to the situation during relaxation of tetanic force in our fibers), this rate declines markedly as pH is reduced. Thus there is experimental evidence supporting the idea that the slowing of relaxation with acidification is due to a reduced rate of cross-bridge cycling. Interestingly, tetanic Ca²⁺ is reduced during the final phase of fatiguing stimulation (29) and, in acidified fibers, this might lead a marked fall of the rate of cross-bridge cycling during the contractions. This would then lead to a decline of the rate of ATP consumption, which minimizes the effects of any inhibition of glycolysis, and hence increase the time to fatigue.

In conclusion, our results show that at 28°C hydrogen ions per se do not impair the ability of isolated mouse muscle fibers to produce force when subjected to repetitive tetanic stimulation at short intervals.