Challenging the Role of pH in Skeletal Muscle Fatigue

Muscle fatigue is frequently defined as a temporary loss in force- or torque-generating ability because of recent, repetitive muscle contraction.1 The development of this temporary loss of force is a complex process and results from the failure of a number of processes, including motor unit recruitment and firing rate, chemical transmission across the neuromuscular junction, propagation of the action potential along the muscle membrane and T tubules, Ca2+ release from the sarcoplasmic reticulum (SR), Ca2+ binding to troponin C, and cross-bridge cycling (for detailed reviews, see Bigland-Ritchie and Woods,1 McLester,2 and Favero3). Muscle fatigue may limit the time a person can stand, the distance a person can ambulate, or the number of stairs a person can ascend or descend. In practical terms, however, we cannot know what actually leads to a decline in function for a given patient. For a phenomenon that may have profound clinical implications, muscle fatigue often receives inadequate attention in physiology textbooks, many of which contain a page or less of information on the entire topic.4–8 In addition, many textbooks report that muscle fatigue is mainly the result of a decrease in pH within the muscle cell due to a rise in hydrogen ion concentration (H+) resulting from anaerobic metabolism and the accumulation of lactic acid.6–8 Recent literature, however, contradicts this assertion.9–19 The purpose of this update, therefore, is to provide a brief review of the role of pH in the development of muscle fatigue. The energy source necessary for muscle contraction, adenosine triphosphate (ATP), originates from 2 main metabolic processes: glycolysis and the tricarboxylic acid (TCA) cycle. Glycolysis converts glucose into pyruvate and, in the process, yields a small amount of ATP.8 If oxygen is present, pyruvate can then be completely oxidized by the TCA cycle to produce large amounts of ATP. Excess protons (H+), formed as a by-product of glycolysis, have been implicated in the development of one form of muscle fatigue.20–23 If the rate of pyruvate production (from glycolysis) exceeds the rate of its oxidation through the TCA cycle, the excess pyruvate is converted into lactic acid, which dissociates into lactate and H+ at physiological pH. The build-up of H+ within the muscle lowers the pH and may reduce muscle force by (1) decreasing Ca2+ release from the SR, (2) decreasing the sensitivity of troponin C to Ca2+, and (3) interfering with cross-bridge cycling (Fig. 1).20,24 Potential mechanisms through which decreasing pH and elevated inorganic phosphate (Pi) could cause fatigue. ATP=adenosine triphosphate, SR=sarcoplasmic reticulum. Question marks indicate mechanisms that have been challenged by the results of recent studies. Potential mechanisms through which decreasing pH and elevated inorganic phosphate (Pi) could cause fatigue. ATP=adenosine triphosphate, SR=sarcoplasmic reticulum. Question marks indicate mechanisms that have been challenged by the results of recent studies. Muscle fatigue may also occur because of the inhibition of the release of Ca2+ from the SR. Westerblad and Allen25 found a reduction in Ca2+ release from the SR during the production of fatigue in single muscle fibers of mice. They also found that the reduction in Ca2+ release from the SR during muscle fatigue and the depression of force were lessened by the addition of caffeine, which activates Ca2+ release channels in the SR. The action of caffeine suggests that the SR Ca2+ release channel is the site responsible for the reduction in Ca2+ release seen with fatigue. Because there is a temporal correlation between the changes in muscle pH and the decline in force during fatigue (r=.76 for linear fit, r=.85 for second-order polynomial fit),16 the effect of pH on the function of the SR Ca2+ release channels has been investigated. The results from single-channel experiments support the idea that a decrease in muscle pH reduces the opening probability of the SR Ca2+ release channels.26,27 Further investigation, however, has demonstrated that, in intact single muscle fibers, a reduction in intracellular free Ca2+ occurs in the absence of changes in pH and that a reduction in pH causes an elevation in intracellular free Ca2+.9–12 Therefore, it appears that a decrease in pH does not lead to a reduction in force by the direct inhibition of the SR Ca2+ release channels. Another pH-dependent fatigue mechanism is an impairment of the Ca2+ sensitivity of troponin C. During activation of skeletal muscle, Ca2+ released from the SR binds to troponin C. Once Ca2+ binds, troponin C is thought to undergo a conformational change that exposes the myosin-binding sites on the actin filaments to allow cross-bridge formation and cycling.24 The amount of Ca2+ that is released from the SR will dictate how much force a given muscle fiber will produce. As the concentration of Ca2+ increases, the amount of troponin C that binds Ca2+ increases and more cross-bridges are formed, thus increasing force.12 A change in the ability of troponin C to bind Ca2+ (a change in its sensitivity), therefore, could reduce force generation. Chin and Allen12 observed that more Ca2+ would be needed during fatigue to produce forces equivalent to the forces produced in the nonfatigued state (Fig. 2). The mechanism behind this decreased sensitivity is not known, but evidence suggests that a low pH (≈6.8) may cause inhibition of Ca2+ binding to troponin C because of competition between H+ and Ca2+.28 An example of the force-Ca2+ relationship in single muscle fibers under nonfatigued and fatigued conditions. Adapted with permission of The Physiological Society from Chin and Allen.12 An example of the force-Ca2+ relationship in single muscle fibers under nonfatigued and fatigued conditions. Adapted with permission of The Physiological Society from Chin and Allen.12 Single muscle fiber analysis has played a major role in the investigation of metabolic factors associated with muscle fatigue. These preparations allow for systematic manipulation of the concentration of different metabolic components (eg, adenosine diphosphate ADP, inorganic phosphate Pi, and hydrogen ions H+) to determine their role in muscle fatigue. Furthermore, single muscle fibers that have had their muscle membranes removed (ie, skinned fibers) allow investigators to directly manipulate intracellular calcium concentrations idependent of Ca2+ release from the SR. This allows the investigation of fatigue that results directly from problems in cross-bridge cycling. Before the early 1990s, skinned muscle preparations could not be kept stable above a temperature of approximately 15°C; therefore, all experiments using this type of preparation were tested at or below 15°C.21–23 Using this type of skinned muscle preparation, Cooke and colleagues21 showed that a drop in pH from 7.0 to 6.5 reduced isometric force by about 35%. These results were replicated several times and in other laboratories.21–23 Thus, there was strong support for the idea that an increase in H+ directly inhibited force production at the cross-bridge level. Although little evidence exists to explain why a drop in pH would reduce force, one hypothesis suggests that a decrease in pH would reverse the equilibrium of the ATP-hydrolysis step, thereby limiting the binding of actin and myosin.29 In the cross-bridge cycle (Fig. 3), the hydrolysis of ATP is required to provide the free energy necessary for the power stroke of the myosin head, and a reversal of this step would interfere with normal cross-bridge cycling.2 A reduction in the amount of hydrolyzed ATP would reduce the number of myosin heads undergoing a power stroke and, therefore, produce a lower amount of force.2,13 The validity of extrapolating the findings from these earlier studies that used nonphysiological temperatures has recently been challenged.13 Cross-bridge kinetics. On the right-hand side of the cycle, the ATP-hydrolysis step provides the necessary change in free-energy for the power stroke of the myosin head to occur. A reversal in the equilibrium of this step was hypothesized to explain how pH could reduce force.29 Also note that on the left-hand side of the cycle, 2 actomyosin·ADP·Pi binding states are possible. The isomerization of actomyosin·ADP·Pi shifts the cross-bridge into a higher force-generating state. This strongly bound force-generating state is followed closely by the release of Pi and a large change in free energy that stabilizes the force-generating cross-bridges. An increase in Pi has been hypothesized to reduce isometric force by shifting the equilibrium to the weakly bound, low force-generating state. ATP=adenosine triphosphate, ADP=adenosine diphosphate, Pi=inorganic phosphate, Pi=phosphate concentration. Information synthesized from McLester2 and Gordon et al.37 Cross-bridge kinetics. On the right-hand side of the cycle, the ATP-hydrolysis step provides the necessary change in free-energy for the power stroke of the myosin head to occur. A reversal in the equilibrium of this step was hypothesized to explain how pH could reduce force.29 Also note that on the left-hand side of the cycle, 2 actomyosin·ADP·Pi binding states are possible. The isomerization of actomyosin·ADP·Pi shifts the cross-bridge into a higher force-generating state. This strongly bound force-generating state is followed closely by the release of Pi and a large change in free energy that stabilizes the force-generating cross-bridges. An increase in Pi has been hypothesized to reduce isometric force by shifting the equilibrium to the weakly bound, low force-generating state. ATP=adenosine triphosphate, ADP=adenosine diphosphate, Pi=inorganic phosphate, Pi=phosphate concentration. Information synthesized from McLester2 and Gordon et al.37 In contrast to the studies using skinned muscle fibers, Adams and colleagues30 and Lännergren and Westerblad,31 using intact (nonskinned) cat and mouse skeletal muscle fibers, respectively, did not find a dramatic effect of reduced pH on maximum isometric tension or shortening speed. Lännergren and Westerblad31 attributed these disparate findings to differences in intact versus skinned fibers. Another major difference between the studies using skinned and nonskinned muscle fibers was the temperature at which the studies were conducted. In contrast to the typical 15°C temperature used in skinned preparations, Adams and colleagues30 tested the cat muscles at a more physiologically realistic temperature (37°C), and Lännergren and Westerblad31 studied the mouse muscles at 25°C. These temperatures are much closer to physiologic temperatures for these animals (≈39°C). In 1995, Pate and colleagues,13 using “temperature jump” techniques that allow testing of skinned fibers at temperatures above 15°C, found that, with increasing temperatures, the effect of pH on maximum isometric tension and shortening speed was dramatically reduced in rabbit psoas muscle. For example, at 10°C, maximum isometric tension dropped 53% with a drop in pH from 7.0 to 6.2, whereas, at 30°C, the same drop in pH led to only an 18% drop in maximum isometric tension. At 10°C, maximal shortening speed decreased by ∼30% with a drop in pH from 7.0 to 6.2, whereas, at 30°C, the same drop in pH led to a slight increase in maximal shortening velocity (∼6%). Similar results have since been found by other researchers using animal tissue.14,15 These experiments, therefore, demonstrate that, when muscle is studied at temperatures that are closer to the normal body temperatures of living organisms, the effect of a decreasing pH on maximum isometric tension and shortening speed is greatly reduced. Although there is good general agreement in the timing between changes in pH and muscle force, there is also evidence to suggest that this association is not maintained when force and pH are measured at frequent, multiple points throughout exercise and recovery.16–19 A lack of temporal association is said to occur when increases or decreases in metabolite levels do not occur at the same time as increases or decreases in force-generating capacity.17 This lack of temporal association is often demonstrated when the relationship between pH and force is studied at frequent time intervals (eg, less than 1 second between measurements).16,17 Many researchers who have investigated the temporal association between pH and voluntary force have used human subjects performing voluntary sustained or intermittent exercises. DeGroot and colleagues16 and Saugen and colleagues17 used phosphorus nuclear magnetic resonance (31P-NMR) spectroscopy to evaluate the effects of fatiguing exercise on force production and metabolite levels. 31P-NMR spectroscopy allowed for the evaluation of metabolic changes in the muscle at small time intervals (≈1 second) throughout exercise and recovery. The researchers, therefore, were able to track the temporal relationship of changes in pH and force with greater resolution than had previously been reported. Although different exercise protocols were used and different muscles were tested (maximal voluntary isometric contraction of the ankle plantar flexors sustained for 4 minutes16 and intermittent isometric voluntary contractions of the knee extensors17), the results were similar. In the first minute of exercise, when the MVC had already begun to decline, pH increased slightly. Thus, by evaluating the relationship of pH and force very early in exercise, the researchers were able to detect an early concomitant increase in pH and decline in force. Another lack of association between changes in pH and force has been found during recovery from fatigue.16,17 Several authors16–18 found that, during the initial phase of recovery from fatigue, pH either remains stable or continues to drop, whereas MVC steadily increases toward control levels. Researchers investigating fatigue during voluntary ankle plantar flexion and knee extension found that in the first 1.5 to 2 minutes after the end of exercise, pH continued to drop to a level of 6.7, whereas the MVC showed an initial rapid recovery.17,18 DeGroot and colleagues,16 using a 4-minute sustained MVC, found that in the first 20 seconds of recovery, H+ did not change, whereas force increased to 58% of the control group levels. Thus, in all of these studies, pH changes were not associated with recovery of force following fatigue. In addition to a lack of association between changes in pH and force early in exercise and recovery, no temporal association has been noted during the fatiguing exercise.17,19 Saugen and colleagues17 and Vøllestad and colleagues19 (using the same exercise protocol) found that, although pH stabilized at a steady state level during exercise, MVC continued to drop almost linearly throughout the exercise. Thus, a steady decrease in force was not associated with concomitant declines in pH. The results of these studies, which were done with human subjects, demonstrate that, in certain phases of fatiguing exercise, there is a clear lack of temporal association between changes in pH and changes in force. Because of the lack of temporal association between changes in pH and changes in force and because of the limited effect of pH when muscles are studied at temperatures similar to those in living organisms, the role of pH as a major causative factor in fatigue has been questioned.16,17 While evidence that challenges the role of pH as a major causative factor in fatigue has accumulated, other metabolites such as lactate and Pi have been investigated. The effects of elevated lactate concentration on Ca2+ release from the SR and cross-bridge formation have been studied in muscle fibers from toads, rats, and rabbits.32,33 Dutka and Lamb32 reported that the presence of lactate comparable to what is seen during moderate aerobic exercise (≈15 mM) caused no reduction in depolarization-induced Ca2+ release from the SR, where as lactate levels comparable to those seen during strenuous anaerobic exercise (≈30 mM) reduced Ca2+ release by 30°C). Furthermore, there is a lack of association between changes in pH and MVC throughout fatiguing exercise and in recovery in humans. The recent evidence regarding the role of pH in muscle fatigue may help to dispel previously held misconceptions about the development of muscle fatigue.6–8 Additional research will be needed to provide a greater understanding of the mechanisms underlying skeletal muscle fatigue and particularly as it occurs in patients. This potentially could lead to interventions that treat this phenomenon when and if it becomes a limiting factor in daily activities.