Muscle fatigue is frequently defined as a temporary loss in force- or

Update 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, Ca 2 release from the sarcoplasmic reticulum (SR), Ca 2 binding to troponin C, and cross-bridge cycling (for detailed reviews, see Bigland-Ritchie and Woods, 1 McLester, 2 and Favero 3 ). 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. [Stackhouse SK, Reisman DS, Binder-Macleod SA. Challenging the role of ph in skeletal muscle fatigue. Phys Ther. 2001;81:1897 1903.] Key Words: Fatigue, Inorganic phosphate, Lactate, ph, Skeletal muscle. Scott K Stackhouse, Darcy S Reisman, Stuart A Binder-Macleod Therapy. Volume. Number 12. December 2001 1897

Increased ph and Skeletal 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 Ca 2 release from the SR, (2) decreasing the sensitivity of troponin C to Ca 2, and (3) interfering with cross-bridge cycling (Fig. 1). 20,24 ph and SR Ca 2 Release Muscle fatigue may also occur because of the inhibition of the release of Ca 2 from the SR. Westerblad and Allen 25 found a reduction in Ca 2 release from the SR during the production of fatigue in single muscle fibers of mice. They also found that the reduction in Ca 2 release from the SR during muscle fatigue and the depression of force were lessened by the addition of caffeine, which activates Ca 2 release channels in the SR. The action of caffeine suggests that the SR Ca 2 release channel is the site responsible for the reduction in Ca 2 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 Ca 2 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 Ca 2 concentrations of inorganic phosphate, not hydrogen ions, are likely a major causative factor in skeletal muscle fatigue at the level of the cross-bridge. release channels. 26,27 Further investigation, however, has demonstrated that, in intact single muscle fibers, a reduction in intracellular free Ca 2 occurs in the absence of changes in ph and that a reduction in ph causes an elevation in intracellular free Ca 2. 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 Ca 2 release channels. ph and Troponin C Sensitivity Another ph-dependent fatigue mechanism is an impairment of the Ca 2 sensitivity of troponin C. During activation of skeletal muscle, Ca 2 released from the SR binds to troponin C. Once Ca 2 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 Ca 2 that is released from the SR will dictate how much force a given muscle fiber will produce. As the concentration of Ca 2 increases, the amount of troponin C that binds Ca 2 increases and more cross-bridges are formed, thus increasing force. 12 SK Stackhouse, PT, MS, is a doctoral student in the Interdisciplinary Graduate Program in Biomechanics and Movement Science, University of Delaware. DS Reisman, PT, MA, is a doctoral student in the Interdisciplinary Graduate Program in Biomechanics and Movement Science, University of Delaware. SA Binder-Macleod, PT, PhD, is Chair and Professor, Department of Physical Therapy, University of Delaware, Newark, DE 19716 (USA) (sbinder@udel.edu). Address all correspondence to Dr Binder-Macleod. All authors provided concept/idea and writing. Michael Higgins, Michael Lewek, Ryan Mizner, David Russ, Wayne Scott, Jennifer Stevens, and Glenn Williams provided critical review of this manuscript. Dr Binder-Macleod was supported by a grant from the National Institutes of Health (HD36787). Ms Reisman was supported by grants from the Foundation for Physical Therapy (Mary McMillan Doctoral Scholarship) and from the National Institutes of Health (HD35857 02) to John P Scholz. Mr Stackhouse was supported by a grant from the Foundation for Physical Therapy (PODSI). 1898. Stackhouse et al Physical Therapy. Volume 81. Number 12. December 2001 Downloaded from https://academic.oup.com/ptj/article-abstract/81/12/1897/2857605

Figure 1. Potential mechanisms through which decreasing ph and elevated inorganic phosphate (P i ) could cause fatigue. ATP adenosine triphosphate, SR sarcoplasmic reticulum. Question marks indicate mechanisms that have been challenged by the results of recent studies. A change in the ability of troponin C to bind Ca 2 (a change in its sensitivity), therefore, could reduce force generation. Chin and Allen 12 observed that more Ca 2 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 Ca 2 binding to troponin C because of competition between H and Ca 2. 28 ph and Cross-bridge Formation 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 [P i ], 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 Ca 2 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 approx- Therapy. Volume. Number 12. December 2001 Stackhouse et al. 1899

muscles at a more physiologically realistic temperature (37 C), and Lännergren and Westerblad 31 studied the mouse muscles at 25 C. These temperatures are much closer to physiologic temperatures for these animals ( 39 C). Figure 2. An example of the force-[ca 2 ] relationship in single muscle fibers under nonfatigued and fatigued conditions. Adapted with permission of The Physiological Society from Chin and Allen. 12 imately 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 colleagues 21 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 Effects of Temperature In contrast to the studies using skinned muscle fibers, Adams and colleagues 30 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 Westerblad 31 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 colleagues 30 tested the cat 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. Lack of Temporal Association 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 forcegenerating 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 colleagues 16 and Saugen and colleagues 17 used phosphorus nuclear magnetic resonance ( 31 P-NMR) spectroscopy to evaluate the effects of fatiguing exercise on force production and metabolite levels. 31 P-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 1900. Stackhouse et al Physical Therapy. Volume 81. Number 12. December 2001 Downloaded from https://academic.oup.com/ptj/article-abstract/81/12/1897/2857605

Figure 3. 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 P i binding states are possible. The isomerization of actomyosin ADP P i shifts the cross-bridge into a higher force-generating state. This strongly bound force-generating state is followed closely by the release of P i and a large change in free energy that stabilizes the force-generating cross-bridges. An increase in [P i ] 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, P i inorganic phosphate, [P i ] phosphate concentration. Information synthesized from McLester 2 and Gordon et al. 37 minutes 16 and intermittent isometric voluntary contractions of the knee extensors 17 ), 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 authors 16 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 colleagues 17 and Vøllestad and colleagues 19 (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 Therapy. Volume. Number 12. December 2001 Stackhouse et al. 1901

ph as a major causative factor in fatigue has been questioned. 16,17 Roles of Lactate and Inorganic Phosphate While evidence that challenges the role of ph as a major causative factor in fatigue has accumulated, other metabolites such as lactate and P i have been investigated. The effects of elevated lactate concentration on Ca 2 release from the SR and cross-bridge formation have been studied in muscle fibers from toads, rats, and rabbits. 32,33 Dutka and Lamb 32 reported that the presence of lactate comparable to what is seen during moderate aerobic exercise ( 15 mm) caused no reduction in depolarization-induced Ca 2 release from the SR, whereas lactate levels comparable to those seen during strenuous anaerobic exercise ( 30 mm) reduced Ca 2 release by 10%. At the cross-bridge level, 15 and 30 mm of lactate only decreased the maximum Ca 2 - activated force by approximately 2% to 8%. 32,33 It would appear, therefore, that lactate plays a small role in the production of fatigue. Inorganic phosphate, however, has a strong relationship to fatigue and has been implicated in the decreased force production observed with fatigue presumably through its effect on cross-bridge cycling. 2,34,35 An increased P i concentration ([P i ]), which occurs with fatigue, can lead to a greater number of cross-bridges in the weakly bound actomyosin ADP P i state and thus, lower force production 2 (Figs. 1 and 3). An increased [P i ] has also been demonstrated to decrease Ca 2 release from the SR. 36 This may occur through the formation of a P i -Ca 2 precipitate in the SR. 37 Formation of this precipitate would decrease the amount of free Ca 2 available for release from the SR. 2,36,37 Although the mechanism is still hypothetical, an increased [P i ] can lead to decreased force production by decreasing Ca 2 release from the SR. Thus, in theory, an increase in [P i ] can cause fatigue through 2 of the 3 mechanisms by which ph was once believed to do so, decreasing maximum Ca 2 -activated force (through increasing the number of actomyosin cross-bridges in the low force state) and decreasing Ca 2 release from the SR. Consequently, it is likely that increased concentrations of inorganic phosphate, not hydrogen ions, are a major causative factor in skeletal muscle fatigue at the level of the cross-bridge. Conclusion The evidence regarding the effect of a declining ph, as observed with fatigue, on skeletal muscle function suggests that, although it may play a role in fatigue through indirect mechanisms, it is not a major causative factor in fatigue at the cross-bridge level. The previously hypothesized mechanisms through which ph was believed to cause fatigue have not been substantiated by recent work. In addition, there has not been evidence to suggest that this type of fatigue is what is occurring in patients with functional limitations and disability. Evidence from studies on nonhuman mammals suggests that the effect of ph on maximal isometric tetanic force and shortening speed is small at near physiologic temperatures ( 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. References 1 Bigland-Ritchie B, Woods JJ. Changes in muscle contractile properties and neural control during human muscular fatigue. Muscle Nerve. 1984;7:691 699. 2 McLester JR Jr. Muscle contraction and fatigue: the role of adenosine 5 -diphosphate and inorganic phosphate. Sports Med. 1997;23:287 305. 3 Favero TG. Sarcoplasmic reticulum Ca 2 release and muscle fatigue. J Appl Physiol. 1999;87:471 483. 4 Guyton AC, Hall JE. Textbook of Medical Physiology. 10th ed. Philadelphia, Pa: WB Saunders Co; 2000. 5 Berne RM, Levey MN. Physiology. 4th ed. St Louis, Mo: Mosby Year-Book; 1998. 6 Hole JW. Human Anatomy and Physiology. 6th ed. Dubuque, Iowa: WC Brown Publishers; 1993. 7 Marieb EN. Human Anatomy and Physiology. 2nd ed. Redwood City, Calif: The Benjamin/Cummings Publishing Co Inc; 1991. 8 McArdle WD, Katch FI, Katch VL. 2nd ed. Essential of Exercise Physiology. Philadelphia, Pa: Lea & Febiger; 2000. 9 Westerblad H, Allen DG. Myoplasmic free Mg 2 concentration during repetitive stimulation of single fibres from mouse skeletal muscle. J Physiol. 1992;453:413 434. 10 Westerblad H, Allen DG. The contribution of [Ca 2 ] i to the slowing of relaxation in fatigued single fibres from mouse skeletal muscle. J Physiol. 1993;468:729 740. 11 Lamb GD, Recupero E, Stephenson DG. Effect of myoplasmic ph on excitation-contraction coupling in skeletal muscle fibres of the toad. J Physiol. 1992;448:211 224. 12 Chin ER, Allen DG. The contribution of ph-dependent mechanisms to fatigue at different intensities in mammalian single muscle fibers. J Physiol. 1998;512:831 840. 13 Pate E, Bhimani M, Franks-Skiba K, Cooke R. Reduced effect of ph on skinned rabbit psoas muscle mechanics at high temperatures: implications for fatigue. J Physiol. 1995;486:689 694. 14 Westerblad H, Bruton JD, Lännergren J. The effect of intracellular ph on contractile function of intact, single fibres of mouse muscle declines with increasing temperature. J Physiol. 1997;500:193 204. 1902. Stackhouse et al Physical Therapy. Volume 81. Number 12. December 2001 Downloaded from https://academic.oup.com/ptj/article-abstract/81/12/1897/2857605

15 Wiseman RW, Beck TW, Chase PB. Effect of intracellular ph on force development depends on temperature in intact skeletal muscle from mouse. Am J Physiol. 1996;271:C878-C886. 16 DeGroot M, Massie BM, Boska M, et al. Dissociation of [H ] from fatigue in human muscle detected by high time resolution 31 P-NMR. Muscle Nerve. 1993;16:91 98. 17 Saugen E, Vøllestad NK, Gibson H, et al. Dissociation between metabolic and contractile responses during intermittent isometric exercise in man. Exper Physiol. 1997;82:213 226. 18 Wong R, Lopaschuk G, Zhu G, et al. Skeletal muscle metabolism in the chronic fatigue syndrome: in vivo assessment by 31 P-nuclear magnetic resonance spectroscopy. Chest. 1992;102:1716 1722. 19 Vøllestad NK, Sejersted OM, Bahr R, et al. Motor drive and metabolic responses during repeated submaximal contractions in humans. J Appl Physiol. 1988;64:1421 1427. 20 Allen DG, Westerblad H, Lännergren J. The role of intracellular acidosis in muscle fatigue. Adv Exp Med Biol. 1995;384:57 68. 21 Cooke R, Franks K, Luciani GB, Pate E. The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate. J Physiol. 1988;395:77 97. 22 Pate E, Cooke R. Addition of phosphate to active muscle fibers probes actomosin states within the powerstroke. Pflugers Arch. 1989; 414:73 81. 23 Chase PB, Kushmerick MJ. Effects of ph on contraction of rabbit fast and slow skeletal muscle fibers. Biophys J. 1988;53:935 946. 24 McComas AJ. Skeletal Muscle: Form and Function. Champaign, Ill: Human Kinetics; 1996. 25 Westerblad H, Allen DG. Changes in myoplasmic calcium concentration during fatigue in single mouse muscle fibers. J Gen Physiol. 1991;98:615 635. 26 Ma J, Fill M, Knudson CM, et al. Ryanodine receptor of skeletal muscle is a gap junction-type channel. Science. 1988;242:99 102. 27 Rousseau E, Pinkos J. ph modulates conducting and gating behavior of single calcium release channels. Pflugers Arch. 1990;415:645 647. 28 Ball KL, Johnson MD, Solaro RJ. Isoform specific interactions of troponin I and troponin C determine ph sensitivity of myofibrillar Ca 2 activation. Biochemistry. 1994;33:8464 8471. 29 Taylor EW. Transient phase of adenosine triphosphate hydrolysis by myosin, heavy meromyosin, and subfragment 1. Biochemistry. 1977;16: 732 739. 30 Adams GR, Fisher MJ, Meyer RA. Hypercapnic acidosis and increased H 2 PO - 4 concentration do not decrease force in cat skeletal muscle. Am J Physiol. 1991;260:C805-C812. 31 Lännergren J, Westerblad H. Force decline due to fatigue and intracellular acidification in isolated fibres from mouse skeletal muscle. J Physiol. 1991;434:307 322. 32 Dutka TL, Lamb GD. Effect of lactate on depolarization-induced Ca 2 release in mechanically skinned skeletal muscle fibers. Am J Physiol Cell Physiol. 2000;278:C517-C525. 33 Andrews MA, Godt RE, Nosek TM. Influence of physiological L ( )-lactate concentrations on contractility of skinned striated muscle fibers of rabbit. J Appl Physiol. 1996;80:2060 2065. 34 Westerblad H, Allen DG, Bruton JD, et al. Mechanisms underlying the reduction of isometric force in skeletal muscle fatigue. Acta Physiol Scand. 1998;162:253 260. 35 Stephenson DG, Lamb GD, Stephenson GM. Events of the excitation-contraction-relaxation (ECR) cycle in fast- and slow-twitch mammalian muscle fibres relevant to muscle fatigue. Acta Physiol Scand. 1998;162:229 245. 36 Posterino GS, Fryer MW. Mechanisms underlying phosphateinduced failure of Ca 2 release in single skinned skeletal muscle fibres of the rat. J Physiol. 1998;512:97 108. 37 Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000;80:853 924. Therapy. Volume. Number 12. December 2001 Stackhouse et al. 1903