muscle contraction requires the propagation of action potentials (AP) along the sarcolemma and down the transverse-tubules where they activate voltage sensors and enable Ca²⁺ release from the sarcoplasmic reticulum. Each AP comprises Na⁺ influx during the depolarization phase and K⁺ efflux for the repolarization phase. Cl⁻ also diffuses into the sarcoplasm contributing to the repolarization phase because Cl⁻ channels remain open during depolarization (9) and the Cl⁻ equilibrium potential (E_Cl) is near the resting membrane potential (E_m) (23, 40). Thus it is unremarkable that intense muscle contractions also induce pronounced perturbations in muscle Na⁺, K⁺, and Cl⁻ ions. The important question is whether these changes are linked to fatigue.

Muscle fatigue can be defined as a transient and recoverable decline in muscle force and/or power with repeated or continuous muscle contractions. Although the mechanisms of muscle fatigue have been studied extensively, considerable debate and controversy still exist. This is likely in part because fatigue appears to be a multifactorial mechanism, evidenced by other mini-reviews in this series. This review focuses on muscle K⁺, Na⁺, and Cl⁻ disturbances, on Na⁺-K⁺-ATPase (NKA; Na⁺-K⁺ pump) activity, and on membrane inexcitability as critical components in muscle fatigue. We first identify the muscle intracellular and extracellular K⁺ ([K⁺]), Na⁺ ([Na⁺]), and Cl⁻ ([Cl⁻]) concentration changes during contractions; we also examine their impact on membrane potential and excitability and thus their implications for muscle fatigue. We then review the impact of NKA on regulating muscle K⁺, Na⁺ homeostasis, the exercise-induced inactivation of NKA, and their implications for fatigue.

MUSCLE IONIC CONCENTRATION CHANGES WITH CONTRACTIONS

To understand the potential roles of K⁺, Na⁺, and Cl⁻ on muscle excitability and fatigue, it is essential to first identify their concentration changes in muscle interstitial and intracellular spaces during contractions. Changes in plasma ion concentrations, water shifts, and muscle ion contents with exercise have been reviewed elsewhere (20, 61, 85) and are beyond the scope of this review.

Muscle Extra- and Intracellular [K⁺]

Extracellular.

Studies using a microdialysis technique to collect interstitial fluid directly from contracting human muscles, reveal that muscle interstitial [K⁺] ([K⁺]_int) greatly exceeds the corresponding venous effluent [K⁺], by as much as 4–8 mM (36, 68, 88). Thus venous [K⁺] cannot be used to predict [K⁺]_int. The [K⁺]_int rises with increasing exercise intensity during submaximal and intense one-legged knee-extension exercise, reaching mean values as high as 11–13 mM during intense exhaustive exercise and with some individual values exceeding 15 mM (Fig. 1) (35, 36, 47, 68, 88).

Intracellular.

As expected, intracellular [K⁺] ([K⁺]_i) decreases substantially during muscular activity. In human muscle, [K⁺]_i declined from ∼165 mM at rest to ∼130 mM during intense exercise (86). Reductions in [K⁺]_i have been more extensively documented in animal models. Fast-twitch muscles, like extensor digitorum longus (EDL), have greater resting [K⁺]_i than slow-twitch muscles, like soleus (46, 66, 94). Moreover, continuous 20-Hz stimulation induced a greater decrease in [K⁺]_i in rat EDL (from 150 to 90 mM, a 1.7-fold decrease) than in soleus (from 130 to 100 mM, a 1.3-fold decrease) (66). The decline in [K⁺]_i with intermittent contractions is in the range of 1.2- to 1.4-fold (46). Similar (1.3- to 1.7-fold) decreases in [K⁺]_i were reported in stimulated amphibian muscles (7, 30).

Muscle Extra- and Intracellular [Na⁺]

Extracellular.

Small elevations in plasma [Na⁺] occur with exercise, which obscure an actual decline in plasma Na⁺ content, because the latter is less than the corresponding decline in plasma volume (61). A tendency to lowered interstitial [Na⁺] ([Na⁺]_int) from ∼140 to ∼130 mM has been reported during exhaustive incremental exercise, whereas venous [Na⁺] increased slightly (88). Thus, as observed for K⁺, venous [Na⁺] cannot be used as an estimate of [Na⁺]_int.

Intracellular.

Muscle intracellular [Na⁺] ([Na⁺]_i) increases markedly during contractions. In humans, [Na⁺]_i rose from 6 mM at rest to 24 mM at the end of intense exercise, calculated from muscle Na⁺ content and inulin space determinations (86). Contrary to the situation observed with [K⁺]_i, fast-twitch muscles have lower [Na⁺]_i (10–15 mM) than slow-twitch muscles (14–25 mM) (25, 46, 66, 94). Electrical stimulation studies in animal muscles also revealed large increases in [Na⁺]_i. Fatiguing mouse soleus with tetanic contractions at a rate of 1 contraction per second for 5 min raised [Na⁺]_i from 14 mM to ∼35 mM, representing a 1.5-fold increase above rest (25). Similarly, one tetanic contraction per s for 1 min raised [Na⁺]_i from 12 to 24 mM in mouse soleus muscle (46). Increases in [Na⁺]_i by as much as 2.5-fold have also been reported in rat EDL and frog sartorius muscles (30, 66).

Muscle Extra- and Intracellular [Cl⁻]

Extracellular.

Plasma [Cl⁻] changes little during exercise in humans because of a concomitant loss of Cl⁻ and water (11, 49, 55, 57). We are unaware of any measures of muscle interstitial [Cl⁻] ([Cl⁻]_int) with exercise, but a decline can be anticipated, as observed for [Na⁺]_int.

Intracellular.

Whereas muscle Cl⁻ content increases during fatiguing exercise (30, 58), increases in [Cl⁻]_i are less clear, in part due to a gain in intracellular water. After intense cycling exercise, [Cl⁻]_i was unchanged in human muscles (49). In a perfused rat hindlimb, stimulation to fatigue causing 40% force reduction, [Cl⁻]_i was unchanged in soleus and gastrocnemius but almost doubled from 12 to 23 mM in plantaris (56). Muscle [Cl⁻]_i was increased after electrical stimulation in rat red gastrocnemius and plantaris muscles but not in soleus or white gastrocnemius; exhaustive swimming did not increase [Cl⁻]_i in any of these muscles (58). Apparently, changes in [Cl⁻]_i and water uptake may vary significantly between muscles and activation patterns. Further studies investigating the effects of exercise on muscle [Cl⁻]_i in humans are required.

DEPRESSING EFFECTS OF NA⁺, K⁺, AND CL⁻

K⁺

The resting E_m in unfatigued muscles ranges between −75 and −85 mV and decreases by ∼10–15 mV when fatigue is elicited by intermittent contractions (7, 8, 21, 48, 54, 78, 79, 81, 92). Greater depolarizations of 25–30 mV occurred with prolonged and continuous stimulation of Xenopus toe muscle (51, 52). Two studies have reported E_m hyperpolarization with fatigue (39, 60); in both studies, the fatigue-resistant soleus muscle was fatigued at 37°C. The fatigue-induced depolarization is most likely related to the increased extracellular [K⁺] ([K⁺]_e) and decreased [K⁺]_i, which are known to cause membrane depolarization in unfatigued muscle (1, 15, 16, 23, 40, 80, 93). However, the depolarization observed during fatigue is considerably less than what is expected from the changes in [K⁺]_e and [K⁺]_i. For example, doubling [K⁺]_int from 4 to 8 mM with a concomitant decrease in [K⁺]_i from 150 to 90 mM reduces the K⁺ equilibrium potential (E_K) by 33 mV, and it reduces it by 48 mV when [K⁺]_int reaches 14 mM as during intense contractions. Thus, whereas the dramatic changes in [K⁺]_int and [K⁺]_i that occur during fatigue most likely contribute to E_m depolarization, other ions, e.g., Cl⁻ (23), and the electrogenic effects of NKA activity (20) reduce the extent of K⁺-induced depolarization during fatigue. The main effect of membrane depolarization is the inactivation of Na⁺ channels (2, 41, 44, 45, 82), which lowers AP amplitude (80, 93, 94).

The dose-response curve for the K⁺ effect on maximal tetanic force reveals a range of K⁺]_e causing little or no force depression despite the decrease in AP amplitude, followed by a precipitous decrease in force at higher [K⁺]_e (Fig. 2). Regardless of the species and experimental temperature, 90% of the decreases in twitch and tetanic force occur within a range of 2–3 mM [K⁺]_e (12, 15, 16, 22, 74, 80, 93). The [K⁺]_e at which force abruptly declines, here referred as the critical [K⁺]_e, is 9–10 mM in frog sartorius muscle (12, 80), whereas in Xenopus toe muscle, 14 mM [K⁺]_e still does not suppress twitch force (51). In mammalian muscles, the critical [K⁺]_e is 8–9 mM at 25–30°C (15, 16), but it reaches 10–12 mM at 35–37°C (22, 74, 93). The abrupt K⁺-depressor effect is even more drastic if force is plotted against E_m, as the depression is completed within 5 mV, starting at −65 mV in frog sartorius (81) and at −60 mV in mouse soleus and EDL muscles (15, 16).

Na⁺

Investigation of Na⁺ effects on muscle contractility is more complex than K⁺, because the substantial [Na⁺]_i changes during fatigue are difficult to induce in unfatigued muscle without potentially complicating pharmacological interventions. However, if Na⁺ effects occur via an effect on E_m, these can be mimicked by reducing [Na⁺]_e to replicate the fall in [Na⁺]_int/[Na⁺]_i gradient observed during fatigue (13). For example, the decline in the [Na⁺] gradient due to two- and threefold increases in [Na⁺]_i would be mimicked by experimentally reducing [Na⁺]_e from 150 mM to 75 mM and to 50 mM, respectively.

Contrary to K⁺, Na⁺ has little effects on resting E_m (1, 13, 40, 93). Instead, lower [Na⁺]_int/[Na⁺]_i gradients reduce Na⁺ influx during APs, resulting in lower amplitudes (10, 13, 42). Decreases in extracellular [Na⁺] ([Na⁺]_e) or increases in [Na⁺]_i in unfatigued muscle reduce both twitch and tetanic force (10, 12, 13, 17, 59, 67). However, large force depressions only occurred when the [Na⁺] gradient is substantially reduced. For example, in mouse soleus muscle, at least 2.5-fold decrease in [Na⁺]_int/[Na⁺]_i gradient was required to cause a 50% reduction in force (13). Furthermore, the decreases are observed within a very narrow change in [Na⁺]_e, i.e., between 40 and 30 mM (13).

Cl⁻

Cl⁻ significantly contributes to the resting E_m (23, 40). In amphibian muscles, E_Cl is very close to the resting E_m, and unlike changes in [K⁺]_e, a change in extracellular ([Cl⁻]_e) only causes transient changes in resting E_m (40). In mammalian muscles at 37°C, E_Cl is closed to resting E_m in some muscles, such as rat diaphragm, whereas it is less negative in others, such as for rat red sternomastoid and for mouse EDL and soleus muscle (23). During fatigue, any increase in [Cl⁻]_i is expected to lower E_Cl and to thus contribute to E_m depolarization. There are, however, very few studies on the effect of Cl⁻ on muscle contractility. One of these studies has demonstrated that lowering [Cl⁻]_e increased the rate of fatigue during continuous and intermittent contractions in rat and mouse soleus (14, 18).

PARADOXICAL EFFECTS OF K⁺, NA⁺, AND H⁺ ON CONTRACTILITY

Recent studies have demonstrated that changes in Na⁺, K⁺, and H⁺ not only have depressing effects but they can also enhance muscle contractility.

K⁺ Elevation Can Potentiate Muscle Force

Although elevated [K⁺]_e that remains below the critical [K⁺]_e has no effect on the maximum force generated during a completely fused tetanus, it actually enhances forces generated with submaximal tetanic stimulation. In frog sartorius muscle, twitch potentiation reached a maximum of 60% at 8–9 mM (12, 80), whereas in Xenopus toe muscle, peak twitch force was twofold higher at 14 mM compared with 2.5 mM K⁺ (51). In mammalian muscles, twitch potentiation reaches a maximum at 9–10 mM [K⁺]_e, being 15–20% at 20–25°C (16) and 80–100% at 37°C (43, 93). Similar twitch potentiation was observed for rat gastrocnemius muscle in situ at a plasma [K⁺] of 14–15 mM (91) and in rabbit hindlimb extensor muscles at 10 mM (37). The K⁺-induced force potentiation is also not limited to twitch contraction. Raising [K⁺]_e from 2 to 8 mM at 37°C, increases subtetanic peak force by 60% in soleus stimulated at 12 Hz and by twofold in EDL muscles stimulated at 40 Hz (43). In rabbit hindlimb extensor muscles, 10 mM [K⁺] potentiated subtetanic peak force up to 80 Hz, and it reversed fatigue elicited with 0.25 Hz (37). Finally, force potentiation is 60–100% when the stimulation frequency is ≤110 Hz in mouse EDL, and it reached two- to threefold in soleus at stimulation frequencies up to 40 Hz (Z. He and J. M. Renaud, unpublished results). Interestingly, the typical in vivo motor unit discharge rates for rat soleus muscles are ∼10–20 Hz and are 90–100 Hz for EDL muscles (38). Thus the K⁺-induced submaximal tetanic force potentiation is within a physiological range of stimulation frequencies for each of soleus and EDL. The mechanism of the K⁺-induced force potentiation is still not understood, but at least in mouse EDL, it is not due to a prolongation of the AP allowing for longer Ca²⁺ release (93).

Attenuation of the K⁺-Induced Force Depression by Na⁺, H⁺, and Lactic Acid

In electrically stimulated, mechanically skinned single fibers, the depressive effects of substantially lowering [K⁺]_i (from 120 to 20 mM) were reduced by elevated [Na⁺]_i (70). This was suggested to reflect effects of increased NKA activity on the t-tubular membrane (70). Thus, whereas a reduced [Na⁺]_int/[Na⁺]_i gradient by reducing [Na⁺]_e causes force depression, elevated [Na⁺]_i can also attenuate this effect.

Lactic acid, at 20 mM, increases the critical [K⁺]_e at which force depression occurs, by almost 2 mM [K⁺] (22, 74) (Fig. 2), likely due to an increase in the concentration of H⁺ ions and not lactic acid per se (22). The higher force was associated with a recovery of the M-wave area, suggesting that the effect of the concentration of H⁺ ions involves a recovery of membrane excitability. In a subsequent study, it was shown that an acidic pH_i lowers Cl⁻ conductance, allowing for greater AP amplitude at 11 mM [K⁺] and thus greater force development (76). An improved force development due to lower Cl⁻ conductance at 11 mM [K⁺]_e was further supported by findings that inhibiting Cl⁻ channels with 9-anthracene-carboxylic acid increased force at 11 mM [K⁺] (75). Finally, the force of mechanically skinned fibers, elicited by electrical stimulation of T tubules, was less depressed by lowering [K⁺]_i in acidic than normal pH, an effect not observed when Cl⁻ was removed (76). Together, these studies demonstrated that when the AP is largely depressed at high [K⁺]_e, a reduction in Cl⁻ conductance lowers the Cl⁻ influx during the AP allowing for greater Na⁺-induced depolarization. As the amplitude of the AP increases, the amount of Ca²⁺ release and thus force increases.

NA⁺, K⁺, AND CL⁻ EFFECTS: IMPLICATIONS FOR FATIGUE

For years, increases in lactic acid, increases in [H⁺]_i and changes in [Na⁺]_i, [K⁺]_i, [K⁺]_e, and [Cl⁻]_i have been proposed as being factors contributing to the decrease in force during fatigue. Recent studies using unfatigued muscles have demonstrated that some ions can alleviate fatigue effects or can even potentiate submaximal tetanic force. Thus the effects of Na⁺, K⁺, and Cl⁻ on contractility are more complex than originally thought.

The effects observed in unfatigued muscles may reflect events occurring early in exercise. For example, during a 30-min knee-extensor exercise bout at 30 W, [K⁺]_int increases to 10 mM within 5 min and remains above 8 mM for the remaining exercise period (68). Any depressive effect of 10 mM [K⁺] may have been minimized by increased catecholamines and [H⁺]_i (via reducing Cl⁻ conductance) and by NKA activation (via increased [Na⁺]_i). The increased [K⁺]_int may also have enhanced muscle performance via potentiation of submaximal force and vasodilatation of blood vessels (3). Thus the effects of K⁺, Na⁺, H⁺, and Cl⁻ in unfatigued muscle may represent a mechanism that maximizes muscle performance early in exercise.

The effects observed in unfatigued muscles do not, however, eliminate perturbations in these ions as potential causative factors in the etiology of muscle fatigue. Numerous observations make it attractive to hypothesize that high [K⁺]_int per se contributes to fatigue development in human muscle. First, the high [K⁺]_int (12–14 mM) reported in human muscle with fatigue is within the range of critical [K⁺]_e, identified by in vitro studies, that caused marked force depression. Secondly, the decline in human vastus lateralis E_m from ∼90 to ∼75 mV during exhaustive exercise (86) might be an underestimate. When average values of [K⁺]_int and [Na⁺]_int at exhaustion are used rather than venous concentrations, E_m at exhaustion would be as low as ∼60 mV, at which muscle fiber contractility is significantly impaired. This calculation does not however, take into account the effects of changes in K⁺ and Cl⁻ conductance, but these are unknown in exercising human muscles. Third, when knee-extensor exercise is preceded by arm exercise, muscle [K⁺]_int increases more rapidly and fatigue occurs earlier compared with a control exercise without any arm exercise (71). Fourth, training increases fatigue resistance while reducing the rate of increase in [K⁺]_int (68).

Some observations do not fully support a role for elevated [K⁺]_int per se in fatigue. A marked range of [K⁺]_int has been reported at exhaustion, suggesting that increases in [K⁺]_int per se do not always depress muscle function and exercise performance (71). When intense exercise is repeated, exhaustion occurs within 5.5 min at [K⁺]_int of 11.5 mM during the first bout of exercise, but it occurs within 3.5 min during the third bout at a lower [K⁺]_int of 9 mM (Fig. 1) (64). Considering the force-[K⁺]_e relationship, [K⁺]_int reaches the critical [K⁺]_e near the end of fatigue, especially in the presence of lactic acid and catecholamines, suggesting that most of the decrease in force may not be related to changes in [K⁺]_int and [K⁺]_i.

Although there are factors that increase the critical [K⁺]_e for force depression, there are also factors that reduce the critical [K⁺]_e, including Na⁺ and Cl⁻ (Fig. 2). In rat soleus muscle (73), a 1.3-fold increase in [K⁺]_e, or a 1.7-fold decrease in [Na⁺]_e from 147 to 85 mM reduces peak tetanic force by 10%. When [K⁺]_e and [Na⁺]_e are changed simultaneously, peak tetanic force decreased by 50%, suggesting a synergistic rather than an additive effect for Na⁺ and K⁺. Furthermore, all reductions in peak tetanic force correlated well with reductions in M-wave area, suggesting that the Na⁺ and K⁺ depressor effects involved a decrease in membrane excitability. A similar Na⁺ and K⁺ synergistic effect was reported in frog sartorius muscle (12). Interestingly, both the 1.3-fold increase in [K⁺]_e and 1.7-fold reduction in [Na⁺] gradient are smaller than the changes observed during fatigue. Similarly, whereas lowering [Cl⁻]_e causes a transient decrease in force and 9 mM [K⁺]_e almost none in unfatigued muscle, concomitantly lowering [Cl⁻]_e and increasing [K⁺]_e result in large and synergistic decrease in force (18). It appears that [K⁺]_int together with changes in [K⁺]_i, [Na⁺]_i, and [Cl⁻]_i must be considered not separately, but in combination, to better understand their role in the etiology of muscle fatigue. Furthermore, NKA activity modulates each of [K⁺]_i, [K⁺]_int, [Na⁺]_i, and E_m and thus must also impact on muscle force and fatigability.

NKA AND MUSCLE EXCITABILITY

Muscle Contraction Elicits a Rapid Increase in NKA Activity

Muscle excitation results in a dramatic increase in the NKA activity, as measured in vitro by ²²Na extrusion or ⁸⁶Rb uptake (20). Even brief contractions of 2 s are sufficient to markedly stimulate the NKA in isolated muscles, indicating that elevated NKA activity must occur at the commencement of all muscular exercise. In isolated rat soleus muscles subjected to high frequency electrical stimulation, NKA activity may reach the maximal theoretical activity of the pump, indicating that all available pumps are fully activated (20, 24, 69). It is likely however, that the extent of NKA activation is considerably less under physiological conditions. In contracting muscles in humans, evidence for in vivo rates of NKA activation has been based on venous [K⁺] changes using an indwelling K⁺-sensitive microelectrode; these studies suggest that NKA activity increases to only 25–50% of the maximal theoretical activity (85). These calculations will, however, likely be underestimates due to the very large (4–8 mM) interstitial-venous [K⁺] gradient that develops during exercise (36, 68, 88). Regardless, a vigorous activation of NKA clearly occurs with exercise, evidenced by the rapid and pronounced decline in venous [K⁺] postexercise (90). Most studies indicate increased [Na⁺]_i with exercise, which is a potent NKA stimulator; this suggests ongoing NKA activity regardless of exercise duration. Furthermore, NKA activity remains elevated even with declining intracellular [Na⁺] (69), with NKA activation related to events associated with membrane excitation. Thus elevated NKA activity must be an important feature of all muscular contraction, which has important implications both for constraining fatigue through regulation of transsarcolemmal K⁺ and Na⁺ gradients and E_m, as well as exacerbating fatigue when at least some pumps become inactivated.

Measurement of NKA Activity in Human Skeletal Muscle

NKA activity in isolated muscles can be assessed by ouabain-inhibitable ²²Na transport or ⁸⁶Rb uptake, but such approaches are impossible in exercising humans in vivo. Thus in vitro approaches have been applied to muscle obtained via biopsy to determine NKA activity. Because of limited tissue yield, the traditional measures of NKA activity, P_i liberation or ATP hydrolysis have not been employed; rather an activity measure utilising the properties of a partial reaction of the NKA enzyme cycle have been utilised. This assay measures the phosphatase activity that is stimulated by K⁺ and inhibitable by ouabain, and it is modified for human skeletal muscle (28). The phosphatase cleaves the artificial substrate 3-O-methyl fluorescein phosphate, yielding the fluorescent compound 3-O-methyl fluorescein and is referred to as the 3-O-methyl fluorescein phosphatase (3-O-MFPase) activity (28).

Exercise Inhibits Maximal NKA Activity in Human Muscle

Work from two laboratories in recent years has investigated the effects of fatiguing exercise on the maximal NKA (in vitro 3-O-MFPase) activity in human muscle. Findings have consistently revealed reduced maximal NKA activity after exhaustive exercise (Fig. 3), including after sustained submaximal isometric contractions (26); dynamic sprinting or quadriceps contractions lasting 1–6 min in duration (4, 27, 77); repeated high-intensity bouts (6); incremental exercise (5, 83); and in submaximal cycling ranging from 70 to 90% peak oxygen uptake (V̇o_2peak), leading to fatigue in excess of 1 h (53, 65). Furthermore, this depression has been demonstrated in untrained and highly trained athletes, and is repeatable in the same athlete, indicating a persistent effect (4, 27). There are two interesting exceptions. A recent report indicated elevated in vitro NKA activity during exercise at 57% V̇o_2peak when participants undertook glucose feeding; this was attributed to an increased appearance of pumps due to translocation, evidenced by an accompanying increase in ouabain binding (34). Furthermore, no decline in maximal in vitro NKA activity was observed during the placebo exercise trial (34). This seems unlikely to reflect a minimum level of exercise and accompanying intramuscular disturbance required for this effect to be manifest, because the same group reported a decline in activity at a similar relative work rate (83). Our laboratory was recently unable to detect any decline in NKA activity in electrically stimulated, isolated EDL muscle in the rat, despite a marked loss of force (31). The latter may reflect increased resistance to exercise-induced inactivation in the rodent, consistent with small inhibitory effects observed in running rats only after very prolonged exercise (26). Indeed an increased maximal NKA activity has even been reported in rats during prolonged running exercise (84).

The mechanisms underlying these inhibitory exercise effects on muscle NKA activity have not yet been extensively studied. Several studies have demonstrated that the total number of pumps in skeletal muscle, as fully quantified in human muscle by ouabain binding, is not reduced with exercise (4, 53, 65). Thus the decline in NKA activity is referred to as NKA inactivation rather than a loss of existing pumps. A recent study has implicated reactive oxygen species (ROS) in NKA inactivation with exercise (62). During prolonged submaximal exercise comprising 45 min at 70% V̇o_2peak followed by exercise to exhaustion at 70%-90% V̇o_2peak, maximal NKA activity underwent a substantial decline. Infusion of the nonspecific antioxidant N-acetylcysteine (NAC) prolonged the time to fatigue and almost halved the relative decline in NKA activity (62). Thus redox disturbances may be responsible for at least an important fraction of NKA inactivation. This observation is also consistent with the important role for ROS in muscle fatigue (24a). Another potential mechanism is related to the pulsatile increases in [Ca²⁺]_i during muscle contraction. Incubation of diaphragm in high [Ca²⁺]_e, which increased [Ca²⁺]_i, reduced NKA activity (90). Whether such effects occur in a physiological preparation or in vivo remain to be seen.

The modest magnitude of these depressive effects might suggest that NKA inactivation has little functional adverse effect. However, these small changes are consistent with the similar small magnitude of upregulatory changes seen with training in NKA activity (6, 32), NKA content (33, 63), or NKA isoform protein abundance (32, 68). Furthermore, skeletal muscle NKA activity may be regulated independently of NKA content with training (6, 32).

IMPLICATIONS OF NKA INACTIVATION FOR FATIGUE: MUSCLE INEXCITABILITY WITH EXERCISE

The implications of NKA inactivation for fatigue are important, although not yet resolved. Declining NKA activity has the potential to impair each of the rates of Na⁺ extrusion, and K⁺ uptake, as well as the electrogenic effect on Em. These changes could exacerbate localized increases in [Na⁺]_i and in [K⁺]_int, reductions in [K⁺]_i and in E_m, culminating in a reduction in muscle force/power, via mechanisms described above. Important unresolved issues include whether the reduction in maximal NKA activity reflects a severe loss of activity in some fibers or a more generalized modest decrease in activity in many fibers. The implications would be quite different for the two scenarios: severe NKA inactivation could induce severe local cellular ionic disruptions and lead to complete loss of excitability in those fibres. A more generalized lesser decline would have only smaller effects per se on muscle ionic homeostasis and thus also on fatigue. Further work is also required to determine whether functional inactivation occurs in vivo during exercise, because present results of this phenomenon are all collected in vitro.

Evidence linking muscle NKA inactivation with membrane inexcitability in exercising humans has not yet been extensively studied. Studies from the same laboratory combining both NKA activity and M-wave characteristics produced inconsistent results: 1) both M-wave area and NKA activity were reduced after isometric contractions (26); 2) NKA activity was reduced but M-wave area unchanged after exhaustive incremental exercise (83); and 3) NKA activity was increased but M-wave area was reduced after prolonged exercise (34, 87). Further research is clearly required to resolve this important issue.

UNDERSTANDING THE ROLES OF K⁺, NA⁺ AND CL⁻: FUTURE DIRECTIONS

If the enhancing and depressing effects of K⁺, Na⁺, H⁺ and Cl⁻ are all physiologically relevant, future research needs to identify the important local conditions relevant to the opposing effects and their underlying mechanisms. Evidence exists for a mitochondrial factor that is released when ATP production is incapacitated and that reduces t-tubular membrane excitability (72). The concept that fatigue is related at least in part to a failure of membrane excitability has important implications not only for understanding factors limiting muscle force/power but also for cellular Ca²⁺ homeostasis and energy metabolism. Muscle fatigue acts to preserve cellular integrity via regulation of [Ca²⁺]i and ATP levels, and thus survival. At least three membrane components are sensitive to either ATP or Ca²⁺: 1) the ATP-sensitive K⁺ channel, 2) the CLC-1 Cl⁻ channel (9), and 3) the Ca²⁺-sensitive K⁺ channel (50). An increased activity of any of these three components undoubtedly will contribute to fatigue. This has already been shown for the Ca²⁺-sensitive K⁺ channel, which allows greater K⁺ efflux and directly lower AP amplitude (19, 29, 60). In regard to the Cl⁻ channel, an increased activity will also lower AP amplitude as Cl⁻ influx increases. It will therefore be interesting to determine whether the mitochondrial factor activates any of these channels. A final potential target is NKA because an inactivation of the pump would exacerbate increases in [K⁺]_int and [Na⁺]_i, which then would favor the depressing over enhancing effects. Further studies are required to quantify the extent of in-vivo NKA activity with exercise in human muscles and to determine the combined effects of increased activity above rest levels plus NKA inactivation on muscle excitability and fatigue effects.

CONCLUSIONS

Marked perturbations in each of [Na⁺]_i, [K⁺]_i, and [K⁺]_int occur during fatiguing exercise in humans and during electrical stimulation in in-vitro preparations, with less well established changes in [Na⁺]_int, [Cl⁻]_i, and [Cl⁻]_int. In unfatigued muscle, these ionic changes depress force, but under some conditions they can also enhance force or alleviate the fatigue effect; i.e., the effects of these ions are more complex than originally thought. It is proposed that these enhancing or alleviating effects are important early in exercise, whereas their depressing effects are dominant later and directly contribute to development of muscle fatigue. Similarly, exercise results in an early and sustained increase in NKA activity, which potentiates contractility, whereas later in exercise, or after intense contractions a decline in maximal NKA activity occurs and contributes to fatigue.