Role of phosphate and calcium stores in muscle fatigue

Abstract

Intensive activity of muscles causes a decline in performance, known as fatigue, that is thought to be caused by the effects of metabolic changes on either the contractile machinery or the activation processes. The concentration of inorganic phosphate (P_i) in the myoplasm ([P_i]_myo) increases substantially during fatigue and affects both the myofibrillar proteins and the activation processes. It is known that a failure of sarcoplasmic reticulum (SR) Ca²⁺ release contributes to fatigue and in this review we consider how raised [P_i]_myo contributes to this process. Initial evidence came from the observation that increasing [P_i]_myo causes reduced SR Ca²⁺ release in both skinned and intact fibres. In fatigued muscles the store of releasable Ca²⁺ in the SR declines mirroring the decline in SR Ca²⁺ release. In muscle fibres with inoperative creatine kinase the rise of [P_i]_myo is absent during fatigue and the failure of SR Ca²⁺ release is delayed. These results can all be explained if inorganic phosphate can move from the myoplasm into the SR during fatigue and cause precipitation of CaP_i within the SR. The relevance of this mechanism in different types of fatigue in humans is considered.

There is a decline in the performance of muscles when they are used at near their maximum capacity. The changes in performance include reduced force production, decreased velocity of shortening, and slowed relaxation; the combination of these factors can lead to profound reductions in performance particularly for rapidly repeated movements. This decline of performance, or fatigue, is particularly obvious and well recognized in athletes who use their muscles very close to their maximum capacity. What is less appreciated is that many patients suffer serious consequences from muscle fatigue and could benefit from strategies to reduce fatigue. Patients with a variety of diseases that affect muscle directly (e.g. muscular dystrophy) or indirectly (e.g. strokes, multiple sclerosis) have reduced muscle function. If the muscles involved are used for vital functions such as breathing or walking, then the remaining functional muscles are used much nearer to their maximal capacity. Consequently essential muscles fatigue rapidly with potentially serious outcomes. Elderly humans suffer a gradual loss of muscle mass and consequently muscles may fatigue during everyday activities causing loss of mobility and independence. Heart failure patients suffer from breathlessness on exertion but also experience excessive muscle fatigue and both of these factors contribute to their reduced exercise capacity (Wilson, 1996; Lunde et al. 2001b).

In the present article we explain the reasons for thinking that increased concentration of inorganic phosphate in the myoplasm ([P_i]_myo) has a central role in muscle fatigue including causing activation failure. We propose that inorganic phosphate (P_i), which increases substantially during fatigue, may enter the sarcoplasmic reticulum (SR), combine with Ca²⁺ and form an insoluble precipitate of calcium phosphate (CaP_i), leading to reduced SR Ca²⁺ release and a consequent decline in muscle performance as originally suggested by Fryer et al. (1995). This mechanism provides a simple explanation for the failure of SR Ca²⁺ release in fatigue and may lend itself to therapeutic interventions in the future.

Mechanisms of fatigue

In muscles contracting at high work loads, stores of energy within the muscle are consumed and products of these reactions accumulate. Thus muscles become acid because glycogen is broken down anaerobically to lactic acid; P_i accumulates because phosphocreatine (PCr) is broken down to creatine (Cr) and P_i. In addition there are, of course, numerous other metabolic changes in fatigue (for review see Fitts, 1994).

In principle, the cellular mechanisms which control force include: (i) the Ca²⁺ concentration surrounding the myofilaments, (ii) the sensitivity of the myofilaments to Ca²⁺, and (iii) the force produced by the crossbridges characterized by the maximum Ca²⁺-activated force. Thus, at a cellular level, fatigue is generally thought to be caused by the cumulative effects of the various metabolite changes acting through these three mechanisms (for review see Westerblad et al. 1991; Allen et al. 1995). Historically by far the most popular hypothesis has been that intracellular acidosis associated with lactic acid accumulation is the major cause of fatigue acting through mechanisms (ii) and (iii). We have recently reviewed the evidence for this theory and outline why this theory is losing support, at least in mammals (Westerblad et al. 2001). As an alternative P_i has also been intensively investigated. Studies in intact muscles show that the resting [P_i]_myo is 1–5 mm (Kushmerick et al. 1992) while during intense contraction it can rise to 30–40 mm (Cady et al. 1989) and the increased [P_i]_myo may cause fatigue by acting through all three of the above mechanisms.

Investigations on mechanism (i) started with the classic work of Eberstein & Sandow (1963). They fatigued intact muscles with repeated tetani until force was greatly reduced and then increased the level of activation by increasing extracellular K⁺ or application of caffeine. Both these manoeuvres increased force substantially in the fatigued muscle suggesting that a reversible failure of activation was an important contributor to fatigue. A recent example of this approach is shown in Fig. 1 A which illustrates how caffeine can reverse much of the decline of force in a fatigued muscle. The rise in tetanic [Ca²⁺]_myo, which activates the contractile proteins (Fig. 1 B (i)), initially increases (Fig. 1 B (ii)), but then declines during fatigue (Fig. 1 B (iii)). Agents such as caffeine, which increase the opening of the SR Ca²⁺ release channels (ryanodine receptors), can increase the amplitude of tetanic [Ca²⁺]_myo (Fig. 1 B (iv)) and thus overcome much of fatigue. Thus the partial failure of SR Ca²⁺ release is accepted to be one of the causes of muscle fatigue (Allen et al. 1995; Williams & Klug, 1995; Favero, 1999).

Figure 1. Muscle fatigue is partly caused by failure of SR Ca²⁺ release.

A, force production from a mouse single fibre stimulated to give repeated brief tetani at gradually reducing intervals until force had declined to ∼40 % of control. At that time caffeine (10 mm) was applied, which reversed much of the decline of force. B, [Ca²⁺]_myo records of selected tetani from experiments similar to A; (i) is the first tetanus, (ii) is at the end of the early decline of force, (iii) is a fatigued tetanus just before the addition of caffeine, and (iv) is in the presence of caffeine. A is taken from Lännergren & Westerblad (1991). B is modified from data in Westerblad & Allen (1991).

The main focus of the present review is the role of P_i in the failure of SR Ca²⁺ release during fatigue. It is already established that increasing [P_i]_myo reduces crossbridge force and Ca²⁺ sensitivity of the myofilaments (Cooke & Pate, 1985; Millar & Homsher, 1990) and probably contributes to the early fall in force (within 1 min) shown in Fig. 1 A. Inorganic phosphate also increases the open probability of the SR Ca²⁺ release channels (Fruen et al. 1994) and is a probable cause of the early increase in tetanic [Ca²⁺]_myo illustrated in Fig. 1 B (ii). Inorganic phosphate also contributes to a slowing of the SR Ca²⁺ pump (Dawson et al. 1980a; Duke & Steele, 2000). These roles of increasing [P_i]_myo in early fatigue are each supported by the finding that when creatine kinase is inoperative (see later section) the early fall of force, the early rise of tetanic [Ca²⁺]_myo and the early slowing of the decline of [Ca²⁺]_myo during relaxation are all abolished (Dahlstedt et al. 2001).

Dawson et al. (1978) initially suggested that slowing of the SR Ca²⁺ pump could explain the reduced force in fatigue. This could come about if the Ca²⁺, which is not returned to the SR, were sequestered in some organelle, e.g. mitochondria, or removed from the cell. However, the amount of Ca²⁺ sequestered by mitochondria during fatigue seems to be relatively small (Gonzalez-Serratos et al. 1978; Lännergren et al. 2001) and the rate of removal of Ca²⁺ to the extracellular space is also very slow (Balnave & Allen, 1998). Furthermore, studies in which the SR pump is slowed by pharmacological means show that the amplitude of the tetanic [Ca²⁺]_myo becomes larger during pump inhibition presumably because the activity of the pump usually opposes the rise in tetanic [Ca²⁺]_myo caused by SR Ca²⁺ release (Westerblad & Allen, 1994). Thus current evidence shows that slowing of the SR Ca²⁺ pump, while important to relaxation, is not the primary cause of reduced SR Ca²⁺ release.

Precipitation of calcium phosphate

None of the established effects of P_i described above appear capable of explaining the substantial changes in activation observed during fatigue. The first suggestion that increased [P_i]_myo might have novel effects through precipitation of CaP_i came from an important study by Fryer et al. (1995). They used a skinned fibre preparation with intact SR and tested the effects of increased [P_i]_myo on the SR Ca²⁺ content, which was estimated from the force produced by caffeine. As background information it has long been known that the Ca²⁺ uptake of isolated SR vesicles can be greatly increased by agents such as oxalate or P_i (Hasselbach, 1964). These substances enter the SR, bind to Ca²⁺, and eventually precipitate with Ca²⁺. Consequently such substances buffer the Ca²⁺ within the SR and greatly increase the Ca²⁺ uptake capacity of the SR. Fryer et al. confirmed such effects of elevated [P_i]_myo when the solution around the preparation was large. More importantly, they also showed that when the volume of solution around the fibre was restricted, elevation of [P_i]_myo reduced the subsequent caffeine-induced contraction (Fig. 2 A). They proposed that P_i is capable of entering and leaving the SR leading to the events shown schematically in Fig. 2 B. It is known that the Ca²⁺ concentration in the SR ([Ca²⁺]_SR) is of the order of 1 mm (Somlyo et al. 1981) with the total Ca²⁺ being much higher because much of the Ca²⁺ is bound to calsequestrin (CS), a Ca²⁺-binding protein with high capacity and low affinity (MacLennan & Holland, 1975). Ca²⁺ is thought to bind to, and dissociate from, calsequestrin relatively quickly so that it acts as a rapid buffer for Ca²⁺. The solubility product of CaP_i is 6 mm² (Fryer et al. 1995) so that with a [Ca²⁺]_SR of 1 mm, if [P_i]_SR exceeds 6 mm then CaP_i will start to precipitate. The net effect is that [Ca²⁺]_SR declines as an increasing fraction of the total SR Ca²⁺ becomes precipitated as CaP_i. This mechanism can explain the reduced contraction caused by elevated [P_i]_myo (Fryer et al. 1995).

Figure 2. Increased [P_i]_myo reduces SR Ca²⁺ release.

A, force records from skinned fibres with intact SR. Caffeine was used to release SR Ca²⁺ producing the contractures shown; thus the size of the contracture is an indication of the Ca²⁺ available for release in the SR. In the middle record, the muscle was exposed to 50 mm P_i for 20 s, the P_i was then washed off and caffeine applied. Adapted from Fryer et al. (1995) with permission. B, schematic diagram of Ca²⁺ and P_i movements across the SR membrane and binding sites within the SR. Under control conditions [P_i]_myo =[P_i]_SR = 3 mm and [Ca²⁺]_SR = 1 mm. Thus [Ca²⁺]_SR×[P_i]_SR = 3 mm² and because this is below the solubility product of CaP_i (which is 6 mm²) none of this product is present. Ca²⁺ in the SR, however, binds rapidly and reversibly to calsequestrin (CS) so that there is a large pool of CaCS which buffers [Ca²⁺]_SR. When the SR Ca²⁺ release channel opens, a large flux of Ca²⁺ into the myoplasm occurs because [Ca²⁺]_SR is high and [Ca²⁺]_SR is maintained high by the buffering of CaCS. In fatigue [P_i]_myo is 30 mm and P_i enters the SR via anion channels. Once [P_i]_SR exceeds 6 mm, the product of [Ca²⁺]_SR×[P_i]_SR exceeds the solubility product of CaP_i and precipitation of CaP_i starts to occur slowly in the SR. As a consequence [Ca²⁺]_SR and CaCS fall and when the SR Ca²⁺ release channels are open the flux is smaller both because [Ca²⁺]_SR is lower and the buffering of [Ca²⁺]_SR by CaCS is reduced. Dissociation of CaP_i is assumed to be too slow to contribute to Ca²⁺ release. Heavy arrows indicate changes of key concentrations during fatigue.

A second indication that P_i has effects other than directly on the myofilaments came from a study in which P_i was directly injected into muscle cells (Westerblad & Allen, 1996). We were expecting to see reduced force and Ca²⁺ sensitivity due to the direct effects of P_i on the myofilaments but, to our surprise, these effects were hardly apparent and instead there was a drastic reduction in SR Ca²⁺ release which caused a fall in force. Since the expected effects of P_i on myofilaments were largely absent, we reasoned that most of the injected P_i had entered the SR, precipitated as CaP_i, and consequently reduced SR Ca²⁺ release.

Role of calcium stores in fatigue

The studies cited above show that precipitation of CaP_i can occur but it still has to be established that this process takes place in the more complex intracellular environment of fatigue. An important contribution to this debate arises from a landmark study by Gonzalez-Serratos et al. (1978). They fatigued single frog muscle fibres with repeated tetani and measured ionic concentrations by electron probe microanalysis. The electron microprobe could be focused down onto individual terminal cisternae of the SR and the total Ca²⁺ and phosphorus were measured. The important finding was that both total Ca²⁺ and phosphorus (which would be largely P_i) increased significantly in the fatigued muscle. The authors concluded that the Ca²⁺ store was not depleted during fatigue and hence the decline in tetanic [Ca²⁺]_myo which occurs during fatigue has usually been interpreted as a failure of opening of the SR Ca²⁺ release channels.

Another approach to this problem is to try to measure the size of the SR Ca²⁺ store in intact muscle. This has frequently been done in cardiac myocytes in which caffeine causes rapid SR Ca²⁺ release. We have recently applied this approach to single cane toad fibres, as illustrated in Fig. 3 (Kabbara & Allen, 1999). Note that application of a drug which increases the opening probability of SR Ca²⁺ release channels, causes a large rise in [Ca²⁺]_myo which is typically twice the size of a maximally activated tetanus. We call this the rapidly releasable SR Ca²⁺ content to indicate that Ca²⁺ bound to slowly releasable sites within the SR would not be detected by this approach. During fatigue it is clear from Fig. 3 that the rapidly releasable SR Ca²⁺ falls substantially; it also recovers after a rest period as does the tetanic [Ca²⁺]_myo. In order to try to identify the whereabouts of the missing SR Ca²⁺ two types of experiments were performed. First, recovery from fatigue was performed in the absence of extracellular Ca²⁺; if the missing Ca²⁺ had been pumped out of the cell this would presumably have prevented recovery of SR Ca²⁺ release (Balnave & Allen, 1998). However, SR Ca²⁺ stores recovered completely in the absence of extracellular Ca²⁺ suggesting that the missing Ca²⁺ remained within the fibre. Second, recovery was performed in the presence of cyanide to delay the recovery of metabolic changes associated with fatigue (Fig. 3). It is known, for instance, that the elevated [P_i]_myo recovers very slowly in the absence of oxidative metabolism (Dawson et al. 1980b). Cyanide prevented the recovery of the SR Ca²⁺ release, showing that oxidative metabolism is required for store recovery. These experiments show that the rapidly releasable SR Ca²⁺ declines during fatigue and are compatible with the hypothesis that the missing Ca²⁺ is within the SR as a precipitate of CaP_i. Note that there is no conflict between these results, which show reduced rapidly releasable SR Ca²⁺ ([Ca²⁺]_SR+[CaCS], see Fig. 2B), and the earlier results of Gonzalez-Serratos et al. (1978) which showed increased total SR Ca²⁺, which would include [Ca²⁺]_SR+[CaP_i]+[CaCS].

Figure 3. SR Ca²⁺ stores decline during fatigue and recovery is prevented by inhibition of oxidative metabolism.

[Ca²⁺]_myo recorded from a single cane toad muscle fibre. The first record shows a single short tetanus followed by ∼10 s application of 4-chloro-m-cresol (4-CmC, indicated by bar above record). This drug opens SR Ca²⁺ release channels and the large rise in [Ca²⁺]_myo represents the amount of rapidly releasable SR Ca²⁺. Similar results are obtained with caffeine. The fibre was then rested for 20 min and then fatigued with repeated brief tetani until the tetanic force (not shown) was reduced to 40 %. 4-CmC was then reapplied and the amount of rapidly releasable SR Ca²⁺ was reduced compared to control. The fibre was then allowed to rest for 20 min in the presence of 2 mm cyanide, which inhibits oxidative metabolism, and a tetanus and 4-CmC application repeated. Neither tetanic [Ca²⁺]_myo, the rapidly releasable SR Ca²⁺ store, nor the force (not shown) recovered. The fibre was then rested for 20 min in cyanide-free solution and partial recovery of all three parameters occurred. Adapted from Kabbara & Allen (1999).

One weakness of the above approach is the possibility that some change in the SR Ca²⁺ release channels or their sensitivity to caffeine (Duke & Steele, 1998) is the cause of the reduced SR Ca²⁺ release in fatigue. Furthermore, we have no information about the time course of the changes in stored Ca²⁺. Both these weaknesses can be resolved by direct measurement of [Ca²⁺]_SR. This has been achieved in a number of tissues (Golovina & Blaustein, 1997; Shmigol et al. 2001), and we have recently shown that it is possible in skeletal muscle (Kabbara & Allen, 2001). The essence of the method is to use a low affinity Ca²⁺ indicator loaded in its membrane-permeant form. Assuming there is esterase present in the SR, some indicator should be localized within the SR site and the indicator in the myoplasm will give little response to [Ca²⁺]_myo because of the low Ca²⁺ sensitivity of the indicator. These studies showed that [Ca²⁺]_SR declined throughout a period of fatiguing stimulation and recovered afterwards. These experiments do not allow unequivocal identification of the mechanism involved but they do support the previous studies in showing a reduction and recovery of [Ca²⁺]_SR which presumably contribute to the changes in SR Ca²⁺ release.

Inhibition of creatine kinase prevents the rise of P_i

A prerequisite for precipitation of CaP_i in the SR to occur is that [P_i]_myo increases. The P_i accumulating in the myoplasm during fatigue mainly stems from PCr breakdown. Creatine kinase (CK) holds the following reaction near equilibrium:

Thus the net reaction when ATP is rapidly consumed is as follows:

Consequently when the CK reaction is inhibited, fatigue occurs without any major increase in [P_i]_myo (Dahlstedt et al. 2000) and then precipitation of CaP_i within the SR should be avoided. On these grounds it might be hypothesized that tetanic [Ca²⁺]_myo will remain unchanged during fatiguing stimulation in muscle fibres with inoperative CK. This hypothesis has recently been tested both with genetically engineered mice, which have no detectable CK in their skeletal muscles (CK^−/−) (Dahlstedt et al. 2000), and with pharmacological inhibition of the CK reaction using 2,4-dinitro-1-fluorobenzene (DNFB) (Dahlstedt & Westerblad, 2001). The basic results of these two studies were similar: there was a markedly slower decline of tetanic [Ca²⁺]_myo during fatigue after CK inhibition (Fig. 4). Both these models have limitations: CK^−/− muscle displays adaptations with, for instance, an increased mitochondrial content (Steeghs et al. 1997) and DNFB has non-specific inhibitory effects on muscle function (for discussion see Dahlstedt & Westerblad, 2001). Since the limitations of the two models are quite disparate and they both cause a slower decline of tetanic [Ca²⁺]_myo during fatigue, it appears safe to conclude that the preserved tetanic [Ca²⁺]_myo in fatigue is due to inhibition of CK. Thus, the results from muscle fibres with inoperative CK strengthen the proposal that CaP_i precipitation is an important mechanism underlying the failure of SR Ca²⁺ release in fatigue.

Figure 4. The decline of tetanic [Ca²⁺]_myo is delayed in muscle fibres which fatigue without an increase in [P_i]_myo.

A, left: tetanic [Ca²⁺]_myo records obtained in a wild-type mouse muscle fibre at the start and the end (tetanic force reduced to 30 %; not shown) of fatigue caused by repeated tetani. This fibre was fatigued after 88 tetani and at that time tetanic [Ca²⁺]_myo was markedly reduced. A, right: records from a genetically engineered fibre lacking creatine kinase (CK^−/−). In this fibre 100 tetani had no significant effect on either tetanic [Ca²⁺]_myo or force (not shown). Adapted from Dahlstedt et al. (2000). B, the first two records show tetanic [Ca²⁺]_myo at the start and end of fatigue produced in a mouse muscle fibre under control conditions. In this fibre 42 tetani were required to reduce force to 30 %. The fibre was then allowed to recover for 90 min before 10 μm 2,4-dinitro-1-fluorobenzene (DNFB), which inhibits CK, was applied and the fibre fatigued again. This procedure produced some reduction in the first tetanic [Ca²⁺]_myo (not shown; for discussion of this point see Dahlstedt & Westerblad, 2001). Note that after DNFB exposure, tetanic [Ca²⁺]_myo was not reduced by 42 tetani. Adapted from Dahlstedt & Westerblad (2001).

P_i appears to enter the SR via ATP-sensitive anion channels

The results of an early study looking at P_i transport over the SR membrane indicate that P_i enters the SR via an ATP-dependent P_i transporter (Stefanova et al. 1991; Fryer et al. 1997). However, the results of a subsequent study suggest that P_i entry into the SR is a passive process, which was inhibited in the presence of ATP (Posterino & Fryer, 1998). It has now become clear that the SR membrane contains small conductance chloride channels, which may conduct P_i (Ahern & Laver, 1998; Laver et al. 2001). Interestingly the open probability of these channels increases at low [ATP]_myo confirming the above study on intact SR (Posterino & Fryer, 1998). This dependence on [ATP]_myo can explain one apparent weakness of the hypothesis that raised [P_i]_myo causes CaP_i precipitation in the SR: [P_i]_myo increases relatively early during fatiguing stimulation while the decline of tetanic [Ca²⁺]_myo generally occurs quite late. Moreover, in mouse fast-twitch fibres the decline of tetanic [Ca²⁺]_myo temporally correlates with an increase in [Mg²⁺]_myo, which presumably stems from a net breakdown of ATP (Westerblad & Allen, 1992), and it is not obvious why CaP_i precipitation in the SR should show a temporal correlation with ATP breakdown. The ATP dependence of the presumed SR P_i channels can explain both why P_i enters the SR with a delay and why there is a temporal correlation between declining [ATP]_myo and declining tetanic [Ca²⁺]_myo.

Relevance to exercise in humans

The evidence is now reasonably strong that precipitation of CaP_i in the SR can contribute to the decline of SR Ca²⁺ release in muscles and is therefore one important cause of fatigue. In what kinds of exercise is this mechanism likely to be important? Clearly a number of processes are involved and these place some limit on the time course over which such a mechanism could develop. The first process is breakdown of PCr which is the main source of P_i. This can occur quickly (∼1 min) if the muscle is maximally and continuously activated (Kushmerick & Meyer, 1985; Cady et al. 1989). The second process is that the P_i has to move from the myoplasm to the SR presumably by the P_i permeable channel in the SR membrane noted above. To calculate the time course of entry of P_i into the SR requires knowledge of the number of channels and their P_i permeability. An upper limit can be obtained by assuming the channels exhibit their maximum permeability and that [P_i]_myo = 20 mm and these assumptions suggest an initial flux of 0.7 mm s⁻¹ (Laver et al. 2001). Assuming further that [P_i]_SR = 2 mm, it would only take 6 s for the [P_i]_SR to exceed 6 mm. Fryer et al. (1997) measured P_i flux as 0.05 mm s⁻¹ which would give a much longer equilibration time though this time would be shorter if [P_i]_SR is higher than 2 mm. Thirdly, in many systems it is common for precipitation to fail to occur immediately the solubility product threshold is exceeded; a nucleus for precipitation is required and the solubility product may be exceeded transiently before precipitation occurs. Since these processes are in series it would seem unlikely that precipitation of CaP_i would occur in fatiguing activities shorter than at 1–2 min. During recovery all these processes have to be reversed and this may be one factor why P_i recovery often precedes force recovery by some minutes (Cady et al. 1989).

In fatigue of a very long duration (> 1 h), muscle activation is generally quite low and the final decline of performance correlates well with glycogen depletion (Bergström et al. 1967). In this kind of exercise [P_i]_myo is only moderately raised (Vøllestad et al. 1988) and it is unclear whether CaP_i precipitation would occur. Furthermore, in muscles which have been glycogen depleted by repeated bursts of activity in the absence of glucose, fatigue occurs very rapidly but is still caused by a failure of SR Ca²⁺ release (Chin & Allen, 1997; Kabbara et al. 2000). Interestingly, such muscles do not show reduction in SR Ca²⁺ stores at the point of fatigue (Kabbara et al. 2000) possibly because the muscles fatigue so quickly that the series of events outlined above cannot occur. These results suggest that failure of SR Ca²⁺ release in glycogen-depleted muscles involves a different mechanism. For instance, studies on skinned fibres suggest that glycogen may, in addition to its energy storage role, have a structural role which is essential for normal excitation-contraction coupling (Stephenson et al. 1999).

Patients with heart failure experience rapid and disabling fatigue in their skeletal muscles and the mechanism of this fatigue has been the subject of considerable debate (Wilson, 1996; Lunde et al. 2001b). Obviously cardiac output, particularly during exercise, is reduced in heart failure patients so that blood supply to muscles will tend to be smaller. Consequently for a given level of exercise the metabolic changes are usually larger; for instance, in heart failure subjects the resting [P_i]_myo in muscles is higher in some studies (Massie et al. 1987; van der Ent et al. 1998) and the increase in [P_i]_myo on exercise is greater in most studies (Wilson et al. 1985; Arnolda et al. 1991). However, the changes are not large and it seems unlikely that the direct effects of P_i on the myofilaments are sufficient to explain the increased degree of fatigue. Therefore we suggest that raised [P_i]_myo may contribute to the more rapid fatigue by causing precipitation of CaP_i in the SR. In addition there are many changes in the protein composition of skeletal muscles in heart failure (for review see Lunde et al. 2001b) and there have been attempts to discover whether isolated skeletal muscles taken from animals with heart failure fatigue more rapidly. Perreault et al. (1993) used bundles of fast muscle and showed that those from an animal with heart failure fatigued more rapidly and had a larger decline in tetanic [Ca²⁺]_myo. This result suggested that an activation defect develops in skeletal muscle in animals with heart failure and that reduced blood flow is not the sole or major cause. However, more recent studies on single fibres from a similar model of heart failure did not show any major increase in fatiguability and only modest changes in [Ca²⁺]_myo (Lunde et al. 2001a). If changes in activation are modest in isolated single fibres this may be because the oxygen supply is more than adequate in this preparation and consequently aerobic metabolism can provide for much of the metabolic requirements. Conversely when muscles are in situ in heart failure subjects, the oxygen supply will be more limited so that anaerobic metabolic changes, such as a rise in [P_i]_myo, will occur earlier and may lead to activation changes by means of the CaP_i precipitation mechanism.

Conclusion

Failure of SR Ca²⁺ release has been shown to be a major contributor to muscle fatigue. Increasing evidence supports the hypothesis that precipitation of CaP_i in the SR contributes to the failure of SR Ca²⁺ release. We suggest that this mechanism may be important in high intensity activities which lead to fatigue in > 1–2 min but other mechanisms are probably more important in lower intensity activities which cause fatigue in > 1 h. CaP_i precipitation in the SR may contribute to the disabling fatigue common in many muscle disabilities and is especially likely to contribute in heart failure where [P_i]_myo levels are higher.