Cellular mechanisms of fatigue in skeletal muscle.

Cellular

mechanisms

of fatigue in skeletal muscle

HAKAN WESTERBLAD, JOHN A. LEE, JAN LANNERGREN, AND DAVID G. ALLEN Department of Physiology, University of Sydney, Sydney, New South Wales 2006, Australia; Division of Pathology, University of Newcastle upon Tyne, Royal Victoria Znfirmary, Newcastle upon Tyne NE1 4LP, United Kingdom; and Department of Physiology, Karolinska Znstitutet, S-l 04 01 Stockholm, Sweden

WESTERBLAD, HAKAN, JOHN A. LEE, JAN LANNERGREN, AND DAVID G. ALLEN. Cellular mechanisms of fatigue in skeletal muscle. Am. J. Physiol. 261 (Cell Physiol. 30): Cl95C209, 1991.-Prolonged activation of skeletal muscle leads to a decline of force production known as fatigue. In this review we outline the ionic and metabolic changes that occur in muscle during prolonged activity and focus on how these changes might lead to reduced force. We discuss two distinct types of fatigue: fatigue due to continuous high-frequency stimulation and fatigue due to repeated tetanic stimulation. The causes of force decline are considered under three categories: 1) reduced Ca” release from the sarcoplasmic reticulum, 2) reduced myofibrillar Ca”’ sensitivity, and 3) reduced maximum Ca”+-activated tension. Reduced Ca”’ release can be due to impaired action potential propagation in the T tubules, and this is a principal cause of the tension decline with continuous tetanic stimulation. Another type of failing Ca’)+ release, which is homogeneous across the fibers, is prominent with repeated tetanic stimulation; the underlying mechanisms of this reduction are not fully understood, although several possibilities emerge. Changes in intracellular metabolites, particularly increased concentration of Pi and reduced pH, lead to reduced Ca”’ sensitivity and reduced maximum tension, which make an important contribution to the force decline, especially with repeated tetanic stimulation. excitation-contraction coupling; intracellular pH; myoplasmic concentration; sarcoplasmic reticulum; T tubules

MUSCLES ARE ADMIRABLE designs by nature, “motors” which are able to convert chemical energy into mechanical work with a reasonable degree of efficiency and with minimal pollution. Their activity can be precisely controlled by the nervous system, thus forming the basis for animal movement; in addition, they can adapt to altering demands by changing their size and to some extent their functional properties. Yet, as motors, skeletal muscles have a serious weakness: their performance deteriorates with prolonged activity both in terms of force production and speed, resulting in a decreased power output. This reduced performance, muscle fatigue in a general sense, has been measured and defined in various ways during the long history of its study. Exercise physiologists often favor a definition such as “failure to maintain the required or expected force” (36), whereas others tend to designate any decline in force from a rested state as fatigue (e.g., Refs. 14, 125). Because the “required force” often varies and also because we will

SKELETAL

0363-6143/91

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free calcium

mainly deal with experiments on isolated muscle preparations, we shall use the latter definition here. POSSIBLE

SITES

OF

FATIGUE

In principle, fatigue might be due to a failure anywhere along the path illustrated in Fig. 1. This path starts with the central command (i) which will lead to activation of cu-motoneurones and conduction of action potentials (ii) down to the neuromuscular junction. At the neuromusculular junction (iii) an action potential will cause release of acetylcholine from the nerve ending, and this causes a local depolarization in the end-plate area of the surface membrane of the muscle cell. The depolarization initiates an action potential by activating voltage-gated sodium and potassium channels (iv). Action potentials then propagate along the surface membrane and also into the transverse tubular system (v), which consists of narrow invaginations of the surface membrane forming a system

G> 1991 the American

Physiological

Society

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of branched tubules which are open to the exterior and therefore contain extracellular fluid. T tubule action potentials cause release of Ca2’ stored in the sarcoplasmic reticulum (SR) into the myoplasm (vi). The intracellular free calcium concentration ( [ Ca2+] i) then increases and Ca”+ binds to troponin C, resulting in the removal of tropomyosin from the active sites of actin (vii). Interaction between myosin and actin is then facilitated, cross-bridge cycling starts (viii), and the cell contracts. An ATP-dependent pump continuously transports Ca2’ back into the SR (ix). When activation ceases and the Ca” release from SR stops, [ Ca2+]i is rapidly reduced mainly due to the active SR uptake. This results in Ca2+ removal from troponin C, cessation of crossbridge cycling, and relaxation. An important landmark in studies of fatigue is the classic study by Merton (91) on the human adductor pollicis. Merton showed that the reduced force production in a prolonged voluntary contraction could not be improved by direct motor nerve stimulation and concluded that reduced central drive and impaired neuromuscular transmission were unimportant. Although this conclusion has been challenged (e.g., Ref. 119), it is now considered that in well-motivated subjects “central fatigue” is not an important cause of reduced force production (10, 14, 126). Furthermore, there appears to be a sufficient safety factor for neuromuscular transm isson to prevent blockade at this point during maximal efforts in normal subjects (13, 14, 92). Thus in most cases the principal locus of fatigue is the muscle itself. SCOPE

OF

THE

REVIEW

In this review we will deal with mechanisms of fatigue within the muscle (Fig. 1, iv to ix), and we will focus on studies at the single fiber level for the following reasons. First, the interpretation of results from studies on whole muscles or multicellular preparations is difficult due to the fact that muscles generally consist of several fiber types with different fatigue properties. Second, with single fiber preparations it is possible to directly compare recordings of tension with measurements of other cell properties, e.g., membrane potential or intracellular ion activity. Third, in single fiber experiments the extracel-

, ,(i)

I FIG.

denote

1. Schematic drawing of events in muscle different steps that are discussed in text.

(viii)

activation.

Numbers

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lular milieu can be controlled, whereas in studies of multifiber preparations the composition of the extracellular fluid will change during fatiguing stimulation. Although changes of the extracellular fluid may be of importance for development of fatigue in vivo, the elucidation of basic cellular mechanisms is simplified when such changes are minimized. In the first part of this review we will briefly describe changes in metabolite concentrations and electrolyte composition that occur with fatiguing stimulation. We will then focus on factors that may cause the tension decline during fatiguing stimulation. These factors fall into three categories: 1) reduced Ca2’ release from the SR, 2) reduced Ca2’ sensitivity of the myofilaments, and 3) reduced maximum Ca2+-activated tension. The relative importance of each of these factors in various forms of fatigue will be discussed as well as mechanisms underlying them. A detailed account of the slowing of relaxation which often accompanies fatigue will not be given in this review. Moreover, force production and metabolite concentrations during recovery from fatigue will not be considered in detail. However, we stress that it is essential in studies of fatigue that recovery, i.e., a return to control force production, can be demonstrated. In the absence of recovery there will be uncertainty about whether one is studying genuine fatigue or merely an irreversible deterioration of the preparation. TYPES

OF

FATIGUE

The time course of fatigue, and possibly also the underlying mechanisms, depends to a large extent on the experimental protocol. Unfortunately, nearly every group that studies fatigue uses its own stimulation scheme, which makes comparisons difficult. Two general types of regimes are, however, commonly used: continuous highfrequency stimulation and repeated tetani. In this review we will mainly deal with these two models. Fatigue resulting from continuous high-frequency stimulation (high-frequency fatigue) develops rapidly, and tension declines with a half-time of 5-30 s. Recovery after high-frequency fatigue is also rapid and has an initial component requiring only a few seconds to be completed (12,59,60,78,97,131). Tension declines more slowly during a sustained maximum voluntary contraction than it does during continuous high-frequency stimulation (12, 60). This is due to the fact that during a prolonged maximum voluntary contraction there is a gradual reduction of the a-motoneuron firing frequency coincident with a progressive slowing of relaxation, i.e., late during a prolonged contraction adequate mechanical summation can occur at a reduced stimulation frequency (9, 11, 12, 60). Thus continuous high-frequency stimulation does not occur during normal activity, but the accompanying type of fatigue, high-frequency fatigue, is useful as a model for a type of fatigue which depends mainly on ionic changes (see below). Fatigue resulting from repeated tetanic stimulation is in general much slower. Exact figures for time to 50% force decline are difficult to give because they depend on the stimulation scheme and the motor unit or fiber type


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investigated. For example, in a classical study of motor unit properties in the cat gastrocnemius muscle, Burke et al. (20) used a stimulation scheme in which a 330-ms tetanus was given every 1 s and found 50% decay times ranging from -1 min to >I h depending on the type of unit. Recovery after fatigue produced by repeated tetani is often a slow process and may involve several different phases. An example of this is the very long-lasting force depression at low stimulation frequencies which is frequently observed after strenuous contractions (low-frequency fatigue; e.g., Refs. 37, 61). Another and more dramatic example of postexercise weakness is the transient, almost complete loss of tension frequently observed in frog fibers (postcontractile depression; e.g., Ref. 127). Thus recovery does not necessarily represent a straightforward reversal of the fatigue process, and for this reason comparisons of force production and metabolite concentration during fatigue and recovery may be misleading. METABOLIC

FACTORS

IN

FATIGUE

When a muscle is brought from rest to full activity its metabolic rate increases considerably. This increase varies between different animals and fiber types; values of the ratio of maximum to basal metabolic rate have been reported to range from -20 in slow mammalian muscle to -500 in fast frog muscle (42a). As a consequence of the increased metabolic rate, the concentration of various metabolites starts to change immediately after the onset of contraction (for a comprehensive account of muscle metabolism, see Ref. 132). The energy for cross-bridge cycling and active transport is derived from the cleavage of the terminal bond of ATP, but because any ATP used is immediately resynthesized by the creatine phosphokinase reaction (Lohmann reaction), it is actually phosphocreatine (PCr) that is consumed with a concomitant formation of creatine and Pi. After some short time, depending on temperature and fiber type, glycogenolysis and glycolysis are accelerated, leading, via various intermediates, to the formation of pyruvate. The fate of pyruvate depends on the availability of oxygen; in its absence it is converted to lactic acid, in its presence some lactic acid is produced but pyruvate is mainly metabolized in the mitochondria to form CO2 and water. Oxidation of glycogen produces 12 times more ATP per glycosyl unit than does anaerobic glycogenolysis but is slower in onset and can only sustain a lower rate of ATP formation. Apart from glycogen (or glucose), fat can often be used as an energy source under aerobic conditions. Thus energy for ATP regeneration comes from three major sources: PCr splitting, anaerobic glycogenolysis, and oxidative phosphorylation. The contribution of each of these sources will depend on the intensity of work, the time after the onset of activity, and the specific metabolic profile of the muscle or muscle fiber. Fiber Types It is well known for mammalian muscle that the oxidative capacity, which is related to the number of mitochondria, as well as the glycolytic capacity vary over a

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substantial range. This, together with the contractile speed, can be used for classification of mammalian fibers into three major types: fast glycolytic (FG, type IIb), fast oxidative glycolytic (FOG, type IIa), and slow oxidative (SO, type I) (for overview, see Ref. 113). There are also different fiber types in amphibian muscle (76, 77, ill), but these do not correspond directly to those in mammalian muscle and the nomenclature is different. The inverse relation between glycolytic and oxidative capacity generally seen in mammalian muscle fibers does not exist in frog fibers. Type 1 fibers, which have the highest ATPase activity and therefore are fastest, have the lowest glycolytic as well as oxidative capacity; type 2 fibers are intermediate in all respects; and type 3 fibers are slow and have the highest glycolytic and oxidative capacity. For mammalian muscle it is well known that there is a close correlation between oxidative capacity and fatigue resistance (e.g., Refs. 20, 71), and such a correlation has also been observed in frog fibers (e.g., Ref. 77). In Xenopus it is possible to calculate the highest duty cycle (= tetanus duration/tetanus interval) which can be sustained by a particular fiber type without the development of a marked oxygen dept and major metabolic changes such as lactic acid accumulation. Such calculations of the maximum duty cycle, which take into account both the rate of ATP consumption and the maximum rate of oxidative ATP production, show a good agreement with the duty cycle single fibers can sustain without a marked tension decline (124). The duty cycle of individual fibers cannot be established during human exercise but ought to be related to the work load. A number of studies have shown that there is little lactate accumulation in muscle until subjects exercise at levels of -70% of the maximum oxygen uptake (e.g., Refs. 58,63). Thus at moderate work loads fatigue develops more slowly, and factors such as the glycogen content in the muscle cells may eventually limit performance (e.g., Refs. 7, 47, 51); this type of fatigue will not be considered in this review. Metabolite

Concentrations

in Fatigue

Because most muscles are mixtures of different fiber types, it is difficult to make meaningful comparisons between overall metabolite change and the tension decline from a whole muscle, since these will reflect the contributions of different fiber types in a complex way. For this reason, although there is a wealth of data from biopsy and nuclear magnetic resonance measurements on human muscle, we will focus on studies on isolated muscle or single fibers. The levels of metabolites in resting muscle have been studied by many investigators and are reviewed in Dawson and Wilkie (29) and Godt and Maughan (45). Some representative values for frog muscle are the following: ATP, -6 mM; PCr, -35 mM; Pi, -3 mM; pH -7.0. Similar values have been obtained with mammalian fast muscle, but it appears that the Pi concentration is higher and the PCr concentration is lower in mammalian slow muscle (94). Although the time course of changes in metabolites occurring during intense activity varies considerably be-


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tween different fiber types, the general pattern of changes is relatively stereotyped and can in fact be predicted by calculation assuming that the creatine kinase and myokinase reactions remain at equilibrium (3, 22). Thus, in the early stages of prolonged activity, PCr declines and Pi and creatine show equivalent increases. ATP shows no significant change over this period, and it will not display a substantial decline until a late stage when PCr has fallen to close to zero. To give an indication of the metabolite changes that occur in fatigue, we have taken data from studies on isolated frog muscles and single fibers in which metabolite levels were measured both in the rested state and when tension was reduced to -50% of the original tetanic tension (P,,) by intermittent tetani (Refs. 27, 98; A. S. Nagesser, W. J. van der Laarse, and G. Elzinga, personal communication). The average change in metabolites from control to fatigue has then been applied to the resting values listed above. This approach indicates that ATP falls from 6 to 4.6 mM, and PCr falls from 35 to 2.4 mM. Pi was only measured in one of these studies (27) and increased from 5 to 25 mM; a calculation from the given changes in PCr and ATP suggests a larger change from 3 to -38 mM. If the creatine phosphokinase reaction is assumed to be at equilibrium, a calculation of free ADP reveals an increase from -30 to 200 mM (27). The changes of intracellular pH (pHi) that occur in fatigue produced by repeated tetani have been measured in a number of studies, and a typical change is from 7.0 to 6.5 (27, 65, 103, 104, 129). It should be noted that the pHi change will depend on the glycolytic vs. oxidative capacity, the buffer capacity, and the rate at which lactate and proton equivalents are removed. Thus it is likely that the pHi reduction will vary between fibers and will be dependent on fiber type. This is clearly illustrated in a study in which isolated Xenopus fibers were used (129), and the pHi in equally fatigued fibers ranged from 6.15 in an easily fatigued fiber to 6.85 in a fatigueresistant fiber. Relation Between Metabolic Changes and Fatigue As discussed above, during most forms of fatiguing activity substantial changes occur in the intracellular concentration of a number of metabolites. Correlations between metabolic changes and force reduction could come about either through 1) a decrease in high-energy compounds (including reduced phosphorylation potential) leading to reduced function of some critical energydependent process or 2) an accumulation of breakdown products with deleterious effects. There are, however, also some forms of fatigue in which metabolic changes probably play little role. One example is high-frequency fatigue in which force falls quickly, but characteristically also recovers very rapidly, either after the end of stimulation or even during stimulation if the frequency is lowered (9, 12, 59, 60, 78, 131). In this case recovery appears to be too rapid for restitution of metabolic changes to occur, and there is now direct experimental evidence that this form of fatigue is associated with T tubule conduction failure (see below). There are also cases of dissociation of fatigue from metabolite changes

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in which high-frequency fatigue plays little role. Thus in both human studies (126) and animal experiments (84) repeated contractions may cause large changes in force with little change in metabolite levels. In the majority of cases,however, one finds that fatigue is accompanied by marked metabolite changes and the resistance to fatigue (endurance) is closely related to the metabolic capacity of the preparation whether it is a whole muscle, a single motor unit, or an isolated fiber. ELECTROLYTE

CHANGES

DURING

FATIGUE

Repetitive activation of excitable cells, including skeletal muscle cells, leads to ionic shifts across the cell membrane. From measurements in frog muscle fibers it has been calculated that for each action potential there would be an increase in the intracellular Na’ concentration ([Na+]i) of -7.7 mM and a decrease of the intracellular K+ concentration ([ K’]i) of -4.7 mM (52). During intense activation these small changes in [Na’]; and [K’]i will, although counteracted by the Na’-K’ pumps, add up and, consequently, substantial changes of the intracellular ionic composition may occur. In addition, fatigue is generally associated with an increased cell volume (48, 75, 112, 115), and this will amplify a reduction of [K+]i, while an increase in [Na+]i will be diminished. A decline of [K’]; of -lo-20% and an -1.5fold increase of [Na’]; have been found in fatigue in a variety of experimental systems (85, 112, 115); representative values taken from a study in which ion-sensitive microelectrodes were used and mouse soleus muscles were fatigued by intermittent tetani until tension was down to -0.4 P, are as follows: [Na’]; increased from 11 to 15 mM and [K+]i decreased from 174 to 145 mM (66). It is also worth noting that the change of these ions can be fiber type dependent so that the changes are bigger in fast than in slow muscle (64). The transmembrane fluxes of K+ and Na’ during intense activation may also result in marked changes of the extracellular ionic composition, especially in the narrow lumen of the T tubules, where an increased [K+] and a reduced [Na+] are very likely to occur (see Ref. 1). Changes in the relation between the intracellular and the extracellular ionic content will then affect the resting membrane potential as well as the action potentials, and the possible contribution of these factors to fatigue will be discussed in a later section. Changes of the myoplasmic concentration of other ions, such as Ca”‘, Mgg+, H+, and lactate ions, will also occur during fatiguing stimulation. In these cases the changes are not so much due to transmembrane fluxes as to metabolic processes and ionic movements between different compartments within the cell. These ions will be considered in greater detail in following sections. CALCIUM

RELEASE

FROM

THE

SR

In a classical paper Eberstein and Sandow (33) proposed that a failure of Ca”+ release may contribute to fatigue. The proposition was based on the fact that the force produced by fatigued muscle showed substantial


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recovery when the muscle was rapidly depolarized with high K+ or when caffeine was applied, maneuvers which are now known to increase the SR Ca*+ release. These results have been confirmed many times (2, 21, 49, 62, 67, 81,82,97, 126a, 128), but identification of the mechanism(s) by which Ca*+ handling is affected during fatigue is still at an early stage. In this section we consider the evidence for changes in Ca”’ release and discuss possible mechanisms involved.

1) Myoplasmic Ca”+ buffers might become loaded with Ca”‘. The gradual rise in resting [Ca*+]i observed during fatiguing stimulation (see Fig. 2 and Refs. 2, 83, 126a) is likely to mean that Ca*+ binding sites such as troponin and parvalbumin are more loaded with Ca*’ at the start of each tetanus. For this reason a given release of Ca”+ from SR during the tetanus will lead to a larger increase of the myoplasmic Ca”’ concentration. While this mechanism can explain the increase in tetanic [Ca*+];, it cannot explain the concomitant decrease in tension. 2) Fatigue is generally accompanied by acidosis, which Measurements of Myopkzsmic Ca”’ Concentration reduces the binding of Ca”+ to troponin C (15) and A direct way to determine changes in Ca*’ release is therefore also the Ca”’ buffering of troponin. This posto measure [Ca*+]; during fatigue. This has now been sible mechanism is supported by the finding that intracellular acidosis produced by CO2 also increases tetanic achieved using aequorin in Xenopus fibers and using fura- in Xenopus and mouse fibers (2, 4, 83, 126a, 131). [Ca*+]i (2). While this mechanism may explain the early The results of all studies in which repeated tetanic stim- increase in tetanic [Ca’+]; in most preparations, it cannot ulation was used showed the same major features (illus- be the explanation in single mouse fibers where there was no concomitant reduction in pHi (5). trated in Fig. 2) and will be considered together. 3) The concentration of Pi is known to increase during After -10-20 tetani, there is a moderate increase in peak tetanic [Ca’+]; and at this time tension has fallen fatiguing stimulation, and Pi reduces the apparent Ca”’ to 0.8-0.9 P,. Thus the initial tension decline appears sensitivity of the myofilaments (17, 46, 68) without affecting the Ca”+ binding of isolated troponin C (69). not to be caused by reduced Ca”’ release; it is more likely to be due to reduced maximum Ca*+-activated tension, Because Pi also reduces maximum Cap+-activated tenwhich will be discussed in a later section. The increase sion, the reduced Ca*+ sensitivity could arise because of in tetanic [Ca”‘]i could occur either because the amount an interaction between attached cross bridges and the Ca”’ binding of troponin as first suggested by Bremel of Ca2+ released has increased or because the Ca”+ buffering of the myoplasm has decreased. While there is no and Weber (18). On this kind of model, attachment of direct evidence to separate these possibilities at present, cross bridges leads to an increase in Ca2’ binding by it seems more likely that Ca*’ buffering has decreased. troponin; consequently, interventions which reduce force and the number of cross bridges in force-generating Possible causes of reduced Ca’+ buffering are the followstates, such as an increase in Pi, could lead to a reduction ing. in Ca”’ binding (19, 50). A a b The early increase in tetanic [Ca’+]; eventually re400 verses and is followed by a decline to values lower than Tension the original. Thus, when tension is reduced to 0.3-0.5 P,, Wa) tetanic [Ca’+]; has fallen to -50% of control levels. The 0 I reduction of tetanic [Ca”+]; could arise either because of a reduced Ca*’ release or because of an increased Ca*+ 1.0n b buffering. We have already argued that the Ca2+ bufferB 2a ing is reduced in early fatigue, and the buffering is likely to decrease further as stimulation continues. If this is Lca2+li 1 _ h _ fi C the case, the moderate decline in tetanic [Ca’+]; toward b-d * the end of fatiguing stimulation could represent a much -A--m ---------larger decline in Ca*’ release. 0 The tension decline in high-frequency fatigue is also 400 accompanied by reduced tetanic [Ca”+] i (131). In contrast Tension to fatigue produced by repeated short tetani, there is in ~kpa~o~:; jy \ this case both a general reduction and an uneven distribution of [Ca*+]; across a fiber; the relevance of the latter finding will be discussed in the following section. There are many possible causes for reduced Ca”’ release in fatigue. For the purpose of discussion, we will FIG. 2. Original records of tension and intracellular free calcium divide them into three categories: failure of action potenconcentration ([ Ca’+]i) obtained from a single mouse muscle fiber tial propagation either in the surface or T tubular memduring a fatigue run (modified from Ref. 126a). A: continuous tension brane, impaired coupling of T tubular depolarization to record in which each vertical line represents a tetanus. B: [Ca’+]i SR Ca”+ release, and reduced Ca” content in the SR. (measured with fura-2) and tension records obtained from the individual tetani indicated above record in A. Three major features are illustrated: I) the initial tension decline is accompanied by an increase in tetanic [ Ca’+],, 2) in late fatigue the tetanic [ Ca”]; is reduced, and 3) the resting calcium increases during fatiguing stimulation (dashed line indicates resting [Ca”], in control). Stimulation periods are shown below tension records in B.

Propagation of Action Potentials in the Surface and T Tubular Membrane Activation of a skeletal muscle fiber involves conduction of an action potential along the surface membrane


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and into the T tubular network. In this section we consider changes in these processes that occur during fatigue. Propagation of action potentials in the surface membrane. The way muscles are stimulated is of great importance for the tension decline in any form of fatigue produced by tetanic stimulation. When punctate electrodes are used for stimulation so that the action potential must conduct along the length of fibers, action potentials may start to fail after a few seconds (conduction block) and this then leads to an unfused tetanus or a sudden decline of force (e.g., Ref. 87). Similarly, prolonged stimulation with nonphysiologically high frequencies in humans may eventually lead to conduction block (e.g., Ref. 12). As already commented, such block is unlikely to develop during maximal voluntary contractions because of the gradual decline in motoneuron impulse frequency. To safeguard against conduction block in experiments on isolated preparations, massive stimulation is usually employed so that an action potential is simultaneously initiated all along the preparation. Continuous high-frequency stimulation. With massive stimulation, force generally declines smoothly over 20 s or so during prolonged high-frequency stimulation (e.g., Ref. 78). Under these conditions the action potential declines in amplitude, slows in time course, and the membrane before each new action potential depolarizes to around -50 mV (78, 79; see also Ref. 87). In these studies conventional microelectrode technique was used, which means that activity both in the surface and T tubular membrane contributes to the recorded action potential (for details see Ref. 1). This implies that if changes in action potential configuration occur, they may reflect changes both at the surface. membrane and in the T tubular system. Thus a similar change in action potential appearance may be caused in different ways and hence there is no clear correlation between the action potential configuration and the accompanying tension response. For instance, in Xenopus fibers virtually identical action potentials were obtained in high-frequency fatigue and after exposure to a solution with increased [K’], but the accompanying tension was markedly higher in the latter case (78). Alternatively, the same twitch tension can be obtained despite marked differences in action potential configuration; decreasing the extracellular [Na’] markedly reduced the amplitude of the action potential, but the accompanying twitch was unaltered

(49) During continuous high-frequency stimulation there is likely to be accumulation of K+ and/or depletion of Na’ in the T tubules, and this may contribute to high-frequency fatigue. This can be illustrated by calculated concentration changes associated with an action potential which are in the millimolar range in the T tubules (1) but -100 times smaller in the myoplasm (52). Because of diffusion, the accumulation or depletion of ions in the T tubules will always be greater near the center of the fiber than close to the edge. Furthermore, such concentration changes are transient, disappearing over a few seconds after the end of a tetanus. A characteristic feature of high-frequency fatigue is that a substantial recovery of force takes place after a

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rest of only l- ,2 s. Because the diffusion of ions out of the T tubules also has a time course of a few secon.ds (53, 70, 96), this supports the idea that changes in ions in the T tubules could contribute to mechanical failure. There are two obvious mechanisms by which ion changes in the T tubules could be responsible for the decline of force in high-frequency fatigue. First, K+ accumulation will lead to depolarization and consequently inactivation of Na+ channels; Juel(66) showed that quite small increments in K+ lead to a reduction of force in subsequent tetani. Second, Na’ depletion will reduce the peak of the action potential which, if the reduction is sufficiently great, will reduce the Ca”’ release from SR. Bezanilla et al. (8) showed that when external Na + was reduced, tetani showed a rapid decline of force that was associated with wavy (i.e., inactivated) myofibrils in the center of the fiber. Furthermore, Westerblad et al. (131) recently showed that during continuous high-frequency stimulation, the distribution of [Ca”‘]i across the fiber displayed a radial gradient with low [Ca”‘]i in the center, which suggests failure of Ca”+ release in the center compared with the edge of the fiber. Also, this gradient disappeared and the tension increased within a few seconds when stimulus frequency was lowered. Thus failing propagation of action potentials into the T tubular system appears to be of major importance for high-frequency fatigue. Intermittent tetanic stimulation. The membrane potential has been monitored in single Xenopus fibers fatigued by repeated short tetani (127), and the changes in membrane potential and action potential configuration can be compared with those that occurred with continuous high-frequency stimulation in the same preparation (78, 79). When tension was reduced to -0.4 P, by intermittent tetani, the membrane potential was reduced from about -90 to -70 mV, and the action potentials were reduced in amplitude and broadened. Although these changes are substantial, they are much smaller than in high-frequency fatigue, and the following findings suggest that the tension decline with repeated tetani is not caused by impaired propagation of action potentials. First, the development of fatigue was similar with repeated 40- and 80-Hz tetani, although ionic changes in the T tubules, if present, would be much greater in the latter case (80). Second, tension is generally similar at the end of one tetanus and at the beginning of the next tetanus (e.g., Ref. 127); thus there is no marked rapid recovery as is the case in high-frequency fatigue. Third, with repeated tetani the distribution of [Ca”]; was found to be homogeneous across a fiber, implying that the T tubule conduction has not been compromised (131). The above results suggest that failing membrane excitation does not contribute to fatigue produced by repeated tetanic stimulation in single Xenopus fibers. The situation is, however, less clear in some other preparations. For instance, in single mouse fibers during intermittent tetani we have occasionally observed a small tension increase when the stimulation frequency was reduced from 100 to 70 Hz (unpublished observations). In isolated mouse soleus muscle, a marked depolarization has been observed during intermittent tetani and depolarization to a similar level induced by increasing the


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with repeated short tetani (2,81) and continuous tetanic stimulation (21). This marked tension recovery shows that the Ca2+content in the SR is not reduced to a degree in which high tensions can no longer be produced and that the T tubular voltage sensors can still be activated. In high-frequency fatigue the failing activation of the voltage sensorscan be explained by impaired propagation of action potentials into the T tubules, but in fibers fatigued by repeated tetani the reduction of tetanic [Ca2+]i was homogeneous (131) and the reduced Ca2+ release must be due to some other mechanism(s). Coupling Between T Tubular Depolarization Application of caffeine in severe fatigue results in a and SR Ca2+ Release marked tension recovery, which again illustrates that To consider this issue in more detail we first briefly with an effective stimulus Ca2’ can be released from the review current ideas about the coupling of excitation to SR. In recent studies, however, it has been shown that contraction (for a more extensive review see Refs. 41, in fatigue produced by repeated tetanic stimulation, ten106) sion in the presence of caffeine increases only to -0.8 P, In’ the last few years there has been rapid progress in (34, 82) despite a substantial increase of tetanic [Ca2+]; (unpublished observations). Thus these experiments understanding of the processes that couple the T tubule show that in addition to reduced Ca2’ release, a reduction action potential to the release of Ca2’ into the myoplasm. A version of the hypothesis of Schneider and Chandler of the maximum Ca2+-activated tension may contribute (114), in which depolarization of the T tubule affects to the tension decline in fatigue (discussed in a following section). sensors located in the T tubule membrane which then open a Ca2+ channel in the SR, is now widely accepted. Possible causesof impaired coupling between T tubular Schneider and Chandler (114) based their hypothesis on depolarization and SR Ca2+release. As noted above, with the detection of charge movement whose time course and repeated tetanic stimulation the changes in tetanic voltage sensitivity seem appropriate to control a putative [Ca2+]i can be uniform across the fiber, and, therefore, Ca”’ channel in the SR. Recent studies suggest that a the T tubular action potential conduction is probably dihydropyridine-binding protein, thought to be a modiadequate. The reduced Ca2’ release could then be exfied Ca2’ channel, acts as the voltage sensor (103, 123). plained in the following ways: 1) the voltage sensorshave become less sensitive to voltage changes, 2) the SR Ca2+ This molecule is coupled to a ryanodine-binding protein channels have become less sensitive to stimulus from the which is now known to be both the foot protein observed in the electron microscope and the Ca2’ channel found voltage sensors, and 3) the opening probability of the Ca2+ channels has become reduced. As far as we are in the sarcoplasmic reticulum membrane (55-57, 72). The SR Ca2’ channel was first identified by Smith et al. aware, there is no evidence at present to determine the (116) and has now been extensively studied. The channel, relative importance of these possibilities. One valuable approach to obtain further knowledge which has a high conductance to Ca2+, can be opened by micromolar Ca”+, millimolar ATP, and caffeine and it is would be to measure charge movements in fatigued fibers inhibited by Mg2+ (e.g., Refs. 109, 117). The channel has or perhaps in cut-end fibers in which the intracellular some voltage sensitivity, but it is widely accepted that it solution has been modified in ways that occur in fatigue. Another very promising approach is the use of skinned is not directly activated by T tubular depolarization. Under physiological resting conditions (ATP, -6 mM; fibers with intact T tubules (e.g., Ref. 73). In this prepCa 2+ -50 nM; Mg”+, -1 mM) the channel would be aration, change in perfusate ion concentration can cause T tubular depolarization and activate the normal voltageexpected to be closed and the hypothesis is that charge movement within the voltage sensor in some way triggers gated mechanism of SR Ca”+ release. Of course such a preparation cannot be fatigued in the usual sense, but channel opening. Application of high K+ and caffeine in fatigue. Rapid the effect of metabolite and ion changes on the coupling application of solutions containing either high [K’] or between T tubule depolarization and Ca2’ release can be caffeine has frequently been used to study the role of examined. In this regard Lamb and Stephenson (74) have shown that a rise in [Mg”+] inhibits Ca2+ release from factors preceding cross-bridge activity in fatigue. Both the SR. This may be of importance in fatigue because a these maneuvers act on the coupling between T tubular depolarization and SR Ca2+release, but the site of action breakdown of MgATP would be expected to lead to a rise in [Mg2+]i; this would also provide a link between reduced is different. High K+ causes a continuous depolarization of the T tubular membrane, which is a more effective Ca2+ release and metabolic factors. As noted above, the reduced Ca2+ release in fatigue stimulus for the voltage sensors than a series of brief action potentials, as evidenced by a much increased must in some way be related to the metabolic changes. [Ca2+]i (2, 16); caffeine has several modes of action (42) To our knowledge, there is no experimental data sugof which the most important in the present context is its gesting that the function of the T tubular voltage sensors ability to facilitate opening of or to directly activate the is affected by any intracellular metabolites. The SR Ca2’ SR Ca2+channels (109). channels, on the other hand, are sensitive to various Rapid application of a solution with high [K’] causes metabolites that change during fatigue, e.g., pH and a substantial tension increase in muscles fatigued both ATP. The isolated channels show a maximal open prob-

extracellular [K’] caused a tension decline (66). Moreover, when the Na+-K+ pump activity was increased by &adrenoceptor stimulation, the ionic changes in the muscle fibers were reduced, the depolarization was smaller, and the muscle became slightly more fatigue resistant. Because the function of the Na+-K+ pump is energy dependent, this finding might provide one link between metabolic capacity and fatigue resistance, on the one hand, and failing Ca”’ release, on the other.


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ability near pH 7.4 which drops to -0 at pH 6.5 (88, 110). However, this was observed under nonphysiological conditions (low or 0 ATP, 0 Mg’+), so it is not clear that this kind of channel activity is the same as that which occurs after activation by the voltage sensor in vivo. In support of this latter idea, Lamb et al. (personal communication) have found that in their skinned fiber preparation (see Ref. 73) changes in cytoplasmic pH from 7.6 to 6.0 have little effect on SR Ca2+ release induced by strong T tubular depolarization. The rate of Ca”+ release from SR is slowed at low ATP concentrations [apparent Michaelis constant (K,) -1 mM; Ref. 901, and this might suggest that a reduction in ATP concentration in fatigue could inhibit SR Ca2’ release. However, a reduction of MgATP from 7 to 2 mM had little effect on Ca2’ release in skinned fibers (74). Thus the change in ATP concentration in fatigue (-6.0 to 4.6 mM) appears to be too small to affect SR Ca2’ channel opening. Furthermore, Ca2’ release from the SR can be activated both by AMP (95) and by the nonhydrolyzable ATP analogue &y-methyleneadenosine 5’triphosphate (AMP-PCP) (90, 116). Thus any adenine 2+ nucleotide appears to be effective in activating SR Ca release and hence the release would not be inhibited until the total concentration of adenine nucleotides is markedly reduced. Inositol triphosphate (IP3) is known to be involved in the coupling between membrane excitation and contraction in smooth muscle (118). Many studies have failed to show a similar role of IP3 in skeletal muscle, but there is also some experimental data suggesting that IP3 does have a role, possibly by acting as a messenger between the voltage sensors and the SR Ca”’ channels (for discussion see Ref. 108). If IP:j is involved in some way in the signal transmission between T tubules and SR, this could provide a link to energy metabolism since IPR turnover requires ATP (discussed in Ref. 31). Ca”

Content of the SR

A possible explanation for the decline in SR Ca2’ release in fatigue is that the SR has become depleted of Ca.?. An early attempt to measure the SR Ca2+ content in fatigue was the electron probe study of GonzalezSerratos et al. (48). They found a small increase in total Ca”’ in the terminal SR of frog fibers fatigued by intermittent tetani until developed tension was almost abolished. In separate experiments these authors (48) showed that high K+ or caffeine could increase the force in fatigued muscles, and they concluded that there was a reduced Ca” release in fatigue despite a normal SR Ca”’ content. However, several concerns about these experiments should lead to caution. First, little detail is given on the nature of fatigue development; the sole tension record shows a very rapidly fatigued fiber. Second, despite the rapid tension decline, pHi fell from 7.3 to 6.3, which is a surprisingly large change especially as the measurements were apparently made 8-20 min after the end of stimulation when a major recovery of pH would be expected (e.g., Refs. 65, 129). Third, the samples used for the ion probe were not taken until 60-150 s after the end of fatiguing stimulation, and it is very likely that

REVIEW

changes in the ionic composition of va rious compartments would have occurred during this time. Thus we believe that these experiments are of such importance in the analysis of Ca2’ handling during fatigue that they deserve repetition under a wider range of well-defined conditions. Fatigue i s generally act ompanied by a slowing of relaxation which could, in addition to slowed cross-bridge cycling (e.g., Ref. 35), be due to the ATP-dependent SR Ca”+ pump working m.ore slowly or being incapable of achieving a sufficient gra.dient oIf Ca2’. For instance, Dawson et al. (28) showed a close correlation between slowing of relaxation and the free energy of ATP hydrolysis (AGATP) during the development of fatigue. This led to the suggestion that the decline in AGATp was reducing the effectiveness of the Ca”’ pump, thus leading to slower relaxation. The rate of decline of tetanic [ Ca2+]i also slows in fatigue (2,83), and it is possible that after a period of repeated tetani more Ca2+ remains in the myoplasm where it will bin d to buffers, such as parvalbumin (reviewed in Ref. 43), and possibly also lead to mitochondrial loading. Such a rise in resting [C a2+]i in fatigue has been observed with both aequorin and fura- and in both Xenopus and mouse fibers (2, 83, 126a). Furthermore, if a severely fatigued mouse fiber is allowed to rest for - 10 s, this results in 1) an approximate halving of the increase in resting [Ca2+]i, 2) a substantial tension increase accompanied by increased tetanic [Ca”‘]i, and 3) a marked recovery of the slowing of relaxation (126a, 130). The latter three findings can all be explained by improved function of the SR Ca”+ pumps and hence increased SR Ca2+ loading. Because even small changes in AGATP can have large effects on active transport systems (28), one possible way to explain the improved pumping is that AGATP has become reduced during fatiguing stimulation and shows some recovery during the 10-s pause. Thus it is likely that a critical factor in fatigue is a metabolite-induced reduction in SR Ca2+pump effectiveness, which leads to reduced SR Ca2’ content. Two more findings might be taken to support a reduced Ca2’ content in the SR in fatigue. First, in Xenopus fibers the [Ca”‘] i signal associated with rapid application of high K+ was substantially smaller in fatigue than under control conditions (2). Second, in fatigued mouse fibers the tetanic [Ca’+]; in the presence of caffeine was markedly smaller than in controls (126a). Although there may be alternative interpretations of these two findings and many of the above results, most are compatible with a reduced SR Ca2’ content in fatigue. CALCIUM

SENSITIVITY

OF

THE

MYOFILAMENTS

Metabolic changes that occur in fatigue are known to have marked effects on the Ca2’ sensitivity of the myofibrils; thus they will affect the tension produced at a given [Ca”+]i. The Ca2’ sensitivity is defined by the position of the force-calcium relation along the [Ca”‘] axis and it is frequently characterized by the [Ca”‘] needed to produce half-maximal tension (Ca& (see Fig. 3, inset). Changes in Ca2’ sensitivity can occur for the following two reasons: 1) ions, such as H’, compete for


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’ max

8

7

1

I

6

5

PCs 3. Values of force vs. [Ca”]i obtained in control and during late fatigue in a single mouse muscle fiber (data replotted from Ref. 126a). Fatigue was produced by repeated 350-ms lOO-Hz tetani (PO refers to tension of 1st tetanus of fatiguing stimulation). Open circles show force-calcium relation of unfatigued tetani whose force was varied by stimulating at different frequencies (25-200 Hz); leftmost point was obtained at rest. Open triangles represent unfatigued tetani (70-150 Hz) in the presence of 10 mM caffeine. Filled circles were obtained during the final part of fatiguing stimulation. Inset: schematic relation between force (ordinate) and [Ca’+]; (abscissa). [ Ca”+]i which causes half-maximum force is indicated (CaSo) and is a measure of the Ca” sensitivity. Downward pointing arrow indicates the effect of factors which reduce maximum Ca”+-activated force; horizontal arrow indicates the effect of factors which reduce myofibrillar Ca”’ sensitivity. Thus the main plot shows an example in which both the maximum tension and the Ca” sensitivity are reduced. Curves were drawn according to a Hill equation of the form: relative tension = P,,, x [Ca’+]“/{(Caso)” + [Ca”‘]“], where N is a constant related to the steepness of the relationship and P,,, is the maximum tension as a fraction of P, obtained either under control conditions or in fatigue ( Pmax,fat). Control values: P,,, = 1.09, N = 2.40, CasO = 316 nM. Fatigue values: Pmax,fat = contribution of reducing 0.85, N = 2.30, CaSo = 602 nM. The relative tetanic [Ca”li, Gag’ sensitivity, and maximum Ca’+-activated force on overall tension production was assessed as follows. These 3 factors were considered to act independently so that the product of the relative tension due to each factor individually equals the relative tension in The contribution of reduced fatigue ( Pfat = 0.35 in this experiment). tetanic [ Ca’+]; was determined by noting the relative force (PA) under control conditions which corresponds to the [Ca’+]; at Pfat (PA = 0.82). The reduction in maximum tension was taken as the ratio Pmax,fat/Pmax (0.78). The remaining reduction in force is then due to reduced sensitivity [0.35/(0.82 X 0.78) = 0.551. FIG.

the same troponin C binding site as Ca2+ (15), and, consequently, reduced pH leads to a fall in Ca2+ sensitivity; and 2) other interventions, such as an increased Pi concentration, lead to a reduction in Ca2+ sensitivity without affecting Ca2+ binding to isolated troponin C (69). Purely changing the Ca2’ sensitivity has the effect of shifting the entire force-calcium relation parallel to the Ca2’ axis; thus reduced Ca2+ sensitivity increases the contraction threshold and saturating [C a 2+ *]i but does not affect the shape of the curve in between or the maximum force which can be generated. The extent to which changes in Ca2’ sensitivity are important depends on the [Ca2+]i in fatigue. We have shown in a previous section that [ Ca2+]i falls during fatigue, and later in this section we show that [Ca”‘]i in fatigue is on the steep part of the force-calcium relation. Thus changed Ca” sensitivity will affect the tension in fatigue. The contribution of changes in Ca2’ sensitivity also depends on the steepness of the force-calcium rela-

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tion. A very steep force-calcium relation means that a small change in Ca2+ sensitivity can have a dramatic effect on force over a small range of tetanic [ Ca2+]i, whereas a shallow force-calcium relation would mean a smaller effect. There are notable differences in the steepness of the force-calcium relation between different fiber types (e.g., Ref. 121) and also in their responses to the metabolic changes of fatigue (32, 93). These differences are expected on the grounds of the presence of different isoforms of troponin (122). It should be noted that most changes in fatigue that affect myofibrillar Ca2+ sensitivity also affect the maximum Ca2+-activated force. An illustration of this is given in Fig. 3, which shows a plot of force-[Ca2+]i values obtained from a single mouse fiber under control conditions and during the final part of a fatiguing stimulation. Although changes in Ca2’ sensitivity and maximum Ca2+-activated force often occur together, it is important to separate them conceptually to understand the mechanisms involved in fatigue. In the rest of this section the possible role of changes in Ca2+ sensitivity is considered, while the following section deals with changes in maximum Ca2+-activated force. Ca2’ Sensitivity

Studied

in Skinned

Fibers

The Ca2+ sensitivity has mostly been investigated in skinned muscle fibers, that is, fibers in which the cell membrane is removed, which allows access of the bathing solution to the myofilaments. The skinned fiber approach has the advantage that the effects of various factors on Ca2+ sensitivity can be studied on intact myofilaments. On the other hand, it has the disadvantage that the results obtained may depend critically on the bathing solutions chosen. The solutions are designed to represent the cytosol in most respects and a great deal is now known about the cytoplasmic composition of rested fibers (45). One remaining uncertainty concerns the possible presence of low-molecular-weight organic compounds which may affect the Ca2’ sensitivity. For example, several recently discovered nitrogen-containing compounds present in the myoplasm may have marked effects on the Ca2+ sensitivity of myofilaments (e.g., Ref. 101) but have generally not been included in bathing solutions. There are also other factors that affect Ca2’ sensitivity and hence need to be considered, e.g., temperature (44), sarcomere length (120), and ionic strength (40) In spite of the various uncertainties about exactly where the baseline for Ca2+ sensitivity in skinned fibers should be drawn, it is now clear that several myoplasmic factors which change during fatigue have important effects on Ca2+ sensitivity. Quantitatively the most important modulators of Ca2+ sensitivity appear to be hydrogen and Pi ions. Decreasing pH by -0.5 units from 7.0 causes an increase in the CasO by a factor of -2 (31, 38, 46, 93). A similar reduction of the Ca2+ sensitivity has been obtained in skinned rabbit psoas muscle when Pi was raised from 0 to 15 mM (17, 46). Several other myoplasmic factors also change in fatigue. Godt and Nosek (46) made a systematic attempt to determine the relevance of these factors by investigat-


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ing the effects on Ca2+ sensitivity of changes similar to those in fatigue: no effect on the Ca”+ sensitivity was produced by reduction of MgATP, increase of ADP, or increase of AMP; a small increase in Ca”’ sensitivity was produced by either reducing PCr or increasing creatine. During stimulation the myosin P light chain becomes phosphorylated (6, 89), and this may (102) or may not (46) increase the Ca2’ sensitivity. From the results quoted above it is apparent that overall, the myoplasmic changes occurring during fatigue would be expected to cause a net desensitization of the myofilaments to Ca”+. This expectation was tested and confirmed by Godt and Nosek (46), who found that a solution mimicking all the metabolite changes in fatigue caused CasO to increase by a factor of -1.7. Ca”’ Sensitivity Studied in Intact Fibers In an intact muscle fiber, the only way to conclusively determine the importance of sensitivity changes in fatigue is to simultaneously measure [ Ca2+]i and force and to construct force-calcium curves both under control conditions and in fatigue. At present this has only been performed in isolated Xenopus and mouse fibers. Xenopus fibers. The method used to produce a control force-calcium relation depends on the type of fiber studied. For Xenopus fibers, which have a high twitch-totetanus ratio, partial activation under control conditions has been achieved by submaximal potassium contractures (2). An alternative method has been to use the tetanic [Ca2+]i during recovery to construct the control force-calcium curve (83). This approach can be used in fibers in which the tetanic [ Ca2+]i recovers much more slowly than metabolic factors associated with fatigue and has been validated by comparing tetanic [Ca2+]; during recovery with tetanic [Ca2+]; produced by potassium contractures in the control state (2). In our initial study using aequorin (2), the [Ca2+]i required to produce a particular tension was found to be similar in fatigue produced by repeated tetani and under control conditions, and thus the Ca2’ sensitivity appeared to be unchanged in fatigue. However, the results from a subsequent study using fura- (83) indicated that a reduction of the Ca2+ sensitivity does occur in fatigue. One possibility to account for this difference is that the [ Mg”+]i may increase during fatigue, and this would tend to disguise a reduction in myofibrillar Ca2+ sensitivity when aequorin is used to measure [Ca2+]i, because of the effect of Mg”+ on aequorin light emission. It thus seems likely that a reduced Ca2’ sensitivity contributes to the force reduction in fatigued Xenopus muscle fibers. Mouse fibers. In mouse fibers in which the twitch-totetanus ratio is low, a control force-calcium curve can be produced by using tetani at various submaximal frequencies (Fig. 3; Refs. 4, 126a). In these fibers the [Ca”‘]; for a given tension was markedly higher during a series of fatiguing tetani than in control tetani. The CasO was -1.6-fold higher in late fatigue than in control, which is similar to the increase obtained in skinned fibers bathed in a solution mimicking the milieu in fatigue (W-fold increase; Ref. 46). Thus in mouse fibers a reduced Ca2’ sensitivity appears to be of considerable importance for

REVIEW

the tension

decline in fatigue.

MAXIMUM

CALCIUM-ACTIVATED

Skinned

TENSION

Fibers

By elevating [Ca2+]i above saturation for troponin C, the properties of maximally activated myofilaments can be studied. In skinned fiber experiments this can easily be achieved just by increasing the Ca”+ concentration of the bath solution. Using skinned fibers it has been shown that among the changes occurring in fatigue, increased Pi and reduced pH cause a marked reduction of the maximum tension-generating capacity. In skinned skeletal muscle fibers an increase of Pi from -1 to 15-30 mM results in a tension reduction to -0.7 P, (23-25, 46, 99, loo), and a reduction of pH from -7.0 to 6.5 causes a tension depression of approximately the same size (2325, 32, 38, 46, 93, 100, 107); it should be noted that the tension depression due to acidosis is fiber type dependent so that fast fibers are more sensitive to pH changes than slow fibers (23, 32, 93, 100). The combined effect of the changes of Pi and pH in fatigue have experimentally been found to reduce tension to about half the control (24, 25, 99). However, there are also changes in fatigue which can potentiate tension production: low levels of ATP (24, 25, 39) and of PCr (46). Thus when fibers were exposed to a solution mimicking all changes thought to occur in fatigue, the maximum tension only declined to -0.7 P, (46) . Intact Fibers In intact muscle fibers it is more difficult to assess the maximum Ca2+ -activated tension. Caffeine has been extensively used for this purpose and among its modes of action (for review see Ref. 42) the most important in this context is its ability to open or facilitate opening of Ca” channels in the SR (109). Caffeine may act in two different ways: 1) high doses cause a large Ca2’ release from the SR which results in a contracture and 2) low doses do not result in a Ca2’ release sufficient to produce any clear contracture tension, but it facilitates the Ca” release associated with stimulation. There is a considerable difference in the sensitivity to caffeine between frog and mammalian muscle: 8 mM caffeine induces a maximum contracture in frog fibers (67, 81), whereas doses as high as 25 mM may be used without any contracture in mammalian fibers (82). To be sure that the maximum tension is produced, it should be possible to raise [Ca’+]; without any increase of tension. This has recently been shown to be the case when 10 mM caffeine is applied to isolated mouse fibers. In these fibers 10 mM caffeine does not produce any contracture, but the tetanic [ Ca”+] i becomes substantially higher, whereas tetanic tension remains almost unchanged (triangles in Fig. 3; 126a). Based on this result, we will assume that the tension in the presence of an appropriate concentration of caffeine is maximum, or very close to maximum. Application of high doses of caffeine to Xenopus fibers fatigued by repeated tetani (tetanic tension reduced to -0.4 P,) produced a contracture tension similar to that


INVITED

obtained under control conditions (81, 128). However, even in control conditions the tension produced in caffeine contractures was only -0.8 P, and, therefore, a small reduction of the maximum tension-generating capacity cannot be excluded. Application of a low dose of caffeine (0.5 mM) did not result in any contracture or increase of tetanic tension in frog fibers fatigued by repeated tetani at low temperature (l-3”C), but the twitches were greatly potentiated (34). The tetanic tension in these fibers was reduced to -0.75 P,, and this tension reduction would then solely be due to depressed maximum tension-generating capacity. However, if a more severe fatigue is produced in this preparation, application of caffeine results in an increase of tetanic tension (86). A recent study of fatigue in isolated mouse fibers (82) has shown that the tetanic tension in the presence of 15-25 mM caffeine is significantly depressed already after 20 fatiguing tetani. Because tetanic [ Ca’+]; is elevated at this stage (126a), reduced tension-generating capacity appears to be the dominant cause of the force reduction. Thereafter a small additional depression occurs, and at the end of fatiguing stimulation (tetanic tension reduced to -0.3 P,), the maximum tension-generating capacity is reduced to -0.8 P,. Mechanisms

Underlying

Reduction

of Maximum Ca2’-Activated Tension

The above results indicate that a reduction of the maximum Ca2+-activated tension is involved in the force decline in fatigue produced by repeated tetani. It appears to be of greatest importance at moderate degrees of fatigue and may cause a decline to -0.75 P,, which is similar to the tension decline observed in skinned fibers exposed to a fatigue solution (46). What mechanisms underlie the reduction of maximum tension-generating capacity? In principle, the reduction can be due to reduced force output of each attached cross bridge or to a declining number of attached cross bridges. The relative importance of these two factors can be evaluated by measuring the stiffness during contraction, which is considered to be proportional to the number of attached cross bridges (54). Edman and Lou (34) have shown that the reduction in stiffness is much smaller than the depression of maximum tension and consequently most of the depression would be attributable to reduced force production per attached cross bridge. Altered cross-bridge function in fatigue is strongly indicated by the reduction in shortening speed that is also observed (e.g., Refs. 35, 81). Among the metabolic changes occurring in fatigue, the reduction of pHi appears to be of greatest importance for the reduced shortening speed (24, 35); the exact mechanism by which hydrogen ions impair cross-bridge function is at present not completely understood, and it probably involves changes at multiple sites on the contractile proteins (see Ref. 24). In addition to the reduction in pHi, increased concentration of Pi is of major importance for the reduction of maximum Ca2+-activated tension in fatigue and consequently Pi must affect cross-bridge function. In contrast to hydrogen ions, Pi does not reduce the shortening

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velocity and its force-depressing effect is proposed to be caused by a reduced proportion of cross bridges in a strongly bound, force-producing state due to a shift of bridges to a weakly attached, low-force state (19, 24). The nature of interaction between the effects of pH and Pi on maximum force production is currently controversial. In mammalian slow muscle they appear to act independently (loo), i.e., if increased Pi reduces tension to 0.7 P, and acidosis reduces tension to 0.7 PO,then the combined effect will be to reduce tension to 0.7 x 0.7 = 0.49 PO. In mammalian fast muscle, on the other hand, some studies suggest that pH and Pi act independently (e.g., Ref. 23), while others suggest a synergistic interaction (99, 100). The latter authors (99, 100) found a good correlation between the concentration of diprotonated Pi and tension reduction and concluded that it is this species of Pi which causes the tension decline in fast muscle. Despite the mechanistic importance of this topic, the quantitative consequence for tension reduction in fatigue is rather similar with the two models. CONCLUDING

REMARKS

Muscle fatigue is a complex phenomenon, and in this review we have mainly discussed a relatively artificial situation in which small preparations are directly stimulated while being constantly superfused by fresh solution. Another limitation is that we have only dealt with stimulation schemes that cause major force depression over a limited time, in general ~20 min. However, such a reductionist approach is useful for analysis of basic mechanisms of fatigue. We have discussed two principal fatigue models: highfrequency fatigue, in which stimulation is continuous, and fatigue produced by repeated tetani. To divide fatigue into two distinct types is clearly an oversimplification compared with the in vivo situation, but for the sake of analysis of fatigue mechanisms we feel that the two-model approach is useful and in the rest of this section we will summarize the main limiting factors in the two situations. High-Frequency Fatigue A major factor in high-frequency fatigue is impaired impulse propagation into the T tubules. The rate at which such an impairment develops will depend on factors such as 1) the stimulation frequency, 2) the density of Na’ and K+ channels in the T tubules, 3) the density and activity of Na’-K+ pumps in the T tubules, 4) the volume of the T tubular system, and 5) the dimension of the opening of T tubules at the surface membrane. On this kind of model, the time course of tension decline in high-frequency fatigue will reflect the muscle fiber’s ability to sustain continuous stimulation without the occurrence of a substantial change of the ionic composition in the T tubules. If a fiber is able to produce high tension for a long period of high-frequency stimulation, there will be a high demand on the metabolic system due to cross-bridge cycling and ion pumping. Metabolic changes will then become gradually more important as the tetanus is pro-


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longed, and increased concentrations of H+ and Pi may, for example, depress tension by interfering with crossbridge cycling. That cross-bridge function may change in high-frequen cy fatigu .e is indic ated by the finding that the shortening velocity bet omes reduced as the tetanus duration is increased (26, 30). The measurements in single Xenopus fibers which reveal gradients of [ Ca”+]i across a fiber in high-frequency fatigue also show a general reduction of [Ca2+]i (131). Thus part of the tension reduction in high-frequency fatigue appears to be caused by a homogeneous decline of [Ca2+];; the mechanism by which this can occur is discussed in connection with fatigue produced by repeated tetani. In multifiber preparations it is likely that during continuous tetanic stimulation ionic changes, such as an accumulation of K+ and a depletion of Na’, will also take place in the restricted space between fibers. This will affect propagation of action potentials in the surface membrane; it may also reduce the rate at which ions diffuse in and out of the T tubular system and in that way it can affect impulse propagation in the T tubules. To sum up, we believe that the dominant cause of high-frequency fatigue is impaired propagation of action potentials into the T tubules resulting in declining [Ca2+]i in the center of the muscle fiber, but other factors also contribute. Intermittent

Tetanic Fatigue

The tension decline with repeated tetanic stimulation is caused by a combination of 1) reduced Ca2+ release from the SR, 2) reduced Ca”’ sensitivity of the myofibrils, and 3) reduced maximum Ca2+-activated tension. The relative importance of these factors depends on the degree of fatigue. A reduction of the maximum tension appears to be most important in moderate fatigue, and it can depress tension to -0.8 P,. In more severe fatigue, reduced Ca” release and reduced Ca2’ sensitivity become gradually more important. Force-calcium curves obtained under control conditions and late during fatiguing stimulation can be used to estimate the relative role of these factors in fatigue. Such curves are at present available for mouse fibers fatigued to -0.3 PO, and one example is shown in Fig. 3. In this preparation the decline in tetanic [Ca”‘]i reduces tension to -0.7 PO, and the decreased Ca2’ sensitivity by itself reduces tension to -0.6 P, (values obtained from Ref. 126a; for a detailed account of calculations see legend to Fig. 3). It should be noted that the reduction of tetanic [ Ca2+]i represents a combination of reduced Ca2’ release from the SR, which tends to reduce tetanic [Ca2+]i and hence tension, and reduced myoplasmic Ca2’ buffering, which tends to increase tetanic [ Ca2+]i and tension; at present we cannot separate these two factors. Force-calcium curves are also available for Xenopus fibers fatigued to -0.4 P, (2, 83), but in this preparation the results are less straightforward; the reduction in maximum tension has not been unambiguously established, and reduced Ca2’ sensitivity was found to be unimportant when aequorin was used to measure [Ca”‘];, while it was of some importance when fura- was

REVIEW

used. Thus in Xenopus fibers absolute values of the relative importance cannot be given, but it does appear that reduced Ca2+ release from the SR is more important in this preparation than in mouse fibers. The reduction in maximum Ca2+-activated tension and myofibrillar Ca2’ sensitivity is likely to be mainly caused by reduced pHi and/or increased Pi. The reduction in SR Ca2’ release is more difficult to explain. Some link must exist between metabolic factors and reduced SR Ca2+ release, since the relation between declining tetanic [ Ca2+]i and reduced tension is similar in fibers with markedly different fatigue resistance and since fatigue resistance is related to a fiber’s metabolic profile. We have discussed five possible connections between Ca2’ release and metabolic factors: 1) Na+-K+ pumps in the surface and T tubular membrane require ATP, and depressed function may lead to depolarization, reduced action potential amplitude, and hence impaired voltage sensor activation; 2) IP3 may have some role in the transmission of signals between the T tubules and the SR, and IP, turnover requires ATP; 3) the SR Ca2’ channels may be affected by pH; 4) the SR Ca2+ release is inhibited by high Mg2+, and [Mg2+]i is likely to increase during fatigue due to a breakdown of MgATP; and 5) the function of the SR Ca2+ pumps depends on the AGATP, and if this falls, reduced Ca2’ pumping will reduce the Ca2’ content in the SR and hence the Ca2’ release. Present data do not allow the relative importance of these possibilities to be distinguished. Also, because the exact mechanism by which signals are transmitted between the T tubules and the SR is still unknown, it is quite possible that some factor or some mechanism that we have not considered will turn out to be of major importance for the reduced Ca2’ release. We thank Simeon Cairns for useful discussion and Lorraine Kerr for help with preparation of the manuscript. J. Lannergren was supported by funds at the Karolinska Institutet; J. Lannergren and H. Westerblad were supported by grants from the Swedish Medical Association (SLS). Address for reprint requests: H. Westerblad, Dept. of Physiology, Fl3, Univ. of Sydney, Sydney, New South Wales 2006, Australia. REFERENCES 1. ADRIAN,

potential 1973.

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Mechanisms of protein balance in skeletal muscle.

Mechanisms modulating skeletal muscle phenotype.

Cellular players in skeletal muscle regeneration.

Fatigue of long duration in human skeletal muscle after exercise.

Altered cellular distribution of hexokinase in skeletal muscle after exercise.

Free radicals may contribute to oxidative skeletal muscle fatigue.

Electrically-induced muscle fatigue affects feedforward mechanisms of control.

Skeletal muscle cellular metabolism in older HIV-infected men.

Autophagic cellular responses to physical exercise in skeletal muscle.

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Skeletal muscle Pi transport and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles.

Oxidative Stress-Mediated Skeletal Muscle Degeneration: Molecules, Mechanisms, and Therapies.

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Direct measurement of skeletal muscle fatigue in patients with chronic heart failure.

Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro.

Force decline due to fatigue and intracellular acidification in isolated fibres from mouse skeletal muscle.

Use of a catchlike property of human skeletal muscle to reduce fatigue.

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Neurobiology of muscle fatigue.

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Neural mechanisms of mental fatigue.

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Orai1-dependent calcium entry promotes skeletal muscle growth and limits fatigue.