SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    Intracellular calcium ([Ca2+]i) and tension were measured from single muscle fibres dissected from the cane toad (Bufo marinus). The amount of Ca2+ which could be released from the sarcoplasmic reticulum (SR) was estimated by brief (≈20 s) exposures to 4-chloro-m-cresol (4-CmC) or caffeine.
  • 2
    Muscle fatigue was produced by repeated tetani at 4 s or shorter intervals and continued until tension had fallen to 50 % of the control. The intracellular free calcium concentration during a tetanus (tetanic [Ca2+]i) first increased and then steadily declined to 43 ± 2 % of control by the time tension had fallen to 50 %. Over the period of fatigue the rapidly releasable Ca2+ from the SR fell to 46 ± 6 % of control. Tension and tetanic [Ca2+]i recovered to 93 ± 3 % and 100 ± 4 % of the control values after 20 min of rest. Over the same period rapidly releasable SR Ca2+ recovered to 98 ± 12 %.
  • 3
    When a similar number of tetani (200) were repeated at longer intervals (10 s), fibres showed only a small reduction in tension (to 85 ± 1 %) and tetanic [Ca2+]i did not change significantly. Under these conditions the rapidly releasable SR Ca2+ did not change significantly.
  • 4
    The recovery of rapidly releasable SR Ca2+ after fatigue was unaffected by removal of extracellular calcium but did not occur when oxidative phosphorylation was inhibited with cyanide.
  • 5
    These results suggest that an important cause of the decline of tetanic [Ca2+]i during fatigue is an equivalent decline in the amount of rapidly releasable SR Ca2+. The results show that the decline of rapidly releasable SR Ca2+ is related to a metabolic consequence of fatigue and are consistent with the hypothesis that Ca2+ precipitates with phosphate in the SR during fatigue.

Muscles which are used repeatedly at near their maximum tension output show a gradual reduction in tension production, in shortening velocity and a slowing of relaxation. These changes are collectively known as muscle fatigue and they represent an important limitation on exercise performance in athletes and on respiration and other activities in patients with muscle-wasting diseases. Current ideas on the mechanism of muscle fatigue are that it has several contributions (for review see Fitts, 1994; Allen et al. 1995). Intracellular accumulation of products of metabolism, such as inorganic phosphate (Pi), causes (i) reduced force production by the crossbridges and (ii) reduced Ca2+ sensitivity of the myofibrillar proteins (Dawson et al. 1978; Godt & Nosek, 1989; Millar & Homsher, 1990). In addition (iii) there is a reduction of sarcoplasmic reticulum (SR) Ca2+ release whose mechanism is unknown (Allen et al. 1989; Westerblad & Allen, 1991).

In principle the reduction of Ca2+ release during fatigue could involve any of the components of excitation-contraction coupling such as (i) the action potential, (ii) the voltage sensor, (iii) the Ca2+ release channels in the SR, (iv) the store of Ca2+ available for release in the SR, (v) the SR Ca2+ pump. Of these factors, the SR Ca2+ store has received relatively little attention mainly because there was no simple and repeatable way to measure it. The total SR Ca2+ can be measured by electron probe microanalysis and Gonzalez-Serratos et al. (1978) showed that in a fatigued muscle the total Ca2+ in the store was substantially increased. This combination of increased store Ca2+ combined with reduced SR Ca2+ release led to the hypothesis that the SR release channels were failing to open normally during fatigue (Allen et al. 1995).

Although Ca2+ stores have not been studied in detail in fatigued muscles, there are a number of suggestions that reduced Ca2+ release during fatigue might be a consequence of changes in the fraction of SR Ca2+ available for release. Fryer et al. (1995) demonstrated that in a skinned muscle fibre preparation with intact SR, a high myoplasmic Pi caused a reduced Ca2+ release. They suggested that this was because Ca2+ and Pi precipitated within the SR and that this precipitate (CaPi) redissolved relatively slowly so that the precipitated Ca2+ was not available for immediate release. This idea was supported when injection of Pi into intact fibres caused a marked reduction in Ca2+ release which reversed over 1 h (Westerblad & Allen, 1996).

Recently we showed that brief application of either caffeine or 4-chloro-m-cresol (4-CmC), agents which open SR Ca2+ release channels (Rousseau et al. 1988; Herrmann-Frank et al. 1996; Westerblad et al. 1998), produced a large rise in [Ca2+]i (Kabbara & Allen, 1999). The amplitude of this rise in [Ca2+]i can be used to estimate the amount of Ca2+ released from the SR over 10–20 s. In the present study we apply this approach to muscle fatigue. The rapidly releasable SR Ca2+ declines during fatigue and returns to control during recovery in a manner which closely parallels the changes in the tetanic [Ca2+]i. We show that the decline and recovery of the rapidly releasable SR Ca2+ is dependent on a metabolic change associated with repeated activity. These experiments support the hypothesis that the precipitation of CaPi within the SR, in a form which dissociates slowly, is one cause of the decline of Ca2+ release in fatigue (Fryer et al. 1995; Westerblad & Allen, 1996; Posterino & Fryer, 1998).

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Single fibre dissection and mounting

Adult cane toads (Bufo marinus) were anaesthetized by immersion in 0.5 % tricaine methanosulphonate in water and then double pithed. These experiments were approved by the Animal Ethical Committee of the University of Sydney. Single muscle fibres were dissected from any of the lumbrical muscles (III-V). Fibres were 30–50 μm in diameter and transparent in appearance. The fibres were mounted in a chamber (volume 0.2 ml) which allowed simultaneous measurements of tension and fluorescence (Westerblad & Allen, 1991). Fibres were superfused under normal conditions at a flow rate of 1 ml min−1 with a Ringer solution containing (mM): NaCl, 115; KCl, 2.5; CaCl2, 1.8; sodium phosphate buffer, 3; pH 7.3 (22°C). Before application of 4-CmC or caffeine the pump rate was increased to 4 ml min−1. The change between solutions was made with solenoid valves close to the chamber (0.1 ml dead space, 1.5 s delay). In order to minimize the exposure to 4-CmC and the resulting high [Ca2+]i, we used the following procedure. 4-CmC was applied while observing the indo-1 ratio. As soon as the peak had been reached, the washout of 4-CmC was started. At least 10 min was allowed after application of 4-CmC for recovery of the fibre (Kabbara & Allen, 1999). We measured the amplitude (peak - resting) of the [Ca2+]i rise and refer to this as the rapidly releasable SR Ca2+.

Tetani were produced by 0.5 ms stimuli (1.2 × threshold) at 100 Hz and lasted 500 ms. Fatigue was produced by repeated tetani with a gradually reducing interval so that all fibres fatigued in less than 10 min. The protocol was 4 s intervals for 2 min, 3 s intervals for 2 min, 2.5 s intervals for 2 min, 2 s intervals for 2 min, 1.5 s intervals for 2 min (see Fig. 1). Stimulation was stopped when tension was less than 50 % of control. Recovery was determined after a 20 min rest.

image

Figure 1. Isometric force and [Ca2+]i during repeated tetanic stimulation and recovery

Force (upper panel) and [Ca2+]i (lower panel) from a single cane toad muscle fibre during repeated tetanic stimulation. For stimulation protocol see Methods. Stimulation was stopped when force had declined to 50 % and a 20 min rest was allowed before a recovery tetanus (g) was produced. Force record is continuous (except for 20 min rest) while [Ca2+]i records are shown for selected tetani marked by the letters a-g on the upper panel. Time scale: 120 s for force and 5 s for [Ca2+]i. Dotted line in lower panel indicates initial resting [Ca2+]i; resting [Ca2+]i increased from 120 to 200 nM in this experiment.

Download figure to PowerPoint

Measurement of [Ca2+]i

[Ca2+]i was measured with the fluorescent indicator indo-1. The indicator was either pressure injected into the fibre (Westerblad & Allen, 1992) or the fibre was loaded with 10–20 μM indo-1 AM for 30–60 min (Kabbara & Allen, 1999). No obvious differences were apparent between the two methods of loading and the majority of experiments utilized AM loading. Excitation was at 360 nm; emitted fluorescent light was measured simultaneously at 400 and 500 nm with two photomultiplier tubes. The signals from the photomultiplier tubes had the background subtracted electronically and were then passed to an analog divide circuit which provided a continuous (400 nm/500 nm) ratio signal.

Photobleaching was reduced by illuminating the fibres for as short a period as possible and by reducing the intensity of the illuminating light by a factor of 30 with a neutral density filter. For this reason we normally only recorded selected [Ca2+]i signals during fatigue.

Calibration of indo-1

The [Ca2+]i was obtained from the ratio signal using the following equation (Grynkiewicz et al. 1985):

  • image

where KD is the dissociation constant of indo-1 for Ca2+, β is the ratio of the 500 nm fluorescence under Ca2+-free and saturating [Ca2+] conditions, R is the ratio (400 nm/500 nm), Rmin is the ratio under Ca2+-free conditions, and Rmax is the ratio at saturating [Ca2+]. KD (283 ± 26 nM) was taken from in vivo measurements (Andrade et al. 1998), β was measured by the method of Bakker et al. (1993), Rmin and Rmax were measured by the methods described in Kabbara & Allen (1999).

Drugs

Caffeine was obtained from Sigma and was prepared in aqueous solutions. 4-CmC (Aldrich Chemicals) and indo-1 AM (Teflabs, Austin, TX, USA) were prepared as stock solutions in dimethyl sulfoxide (DMSO). The DMSO concentration applied to the muscle fibre did not exceed 0.2 %. Cyanide was freshly prepared and added to the Ringer solution to give a final concentration of 2 mM NaCN.

Analysis of data

All data are presented as means ±s.e.m. Student's paired and unpaired t tests were used to assess the statistical significance. P values < 0.05 were accepted as statistically significant. Note that the errors in the rapidly releasable SR Ca2+ are relatively large because the ratio approaches Rmax during this measurement leading to larger changes in [Ca2+]i per unit change in ratio (Kabbara & Allen, 1999).

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Effects of fatiguing stimulation on [Ca2+]i and tension in the cane toad muscle fibre

Figure 1 shows isometric tension and [Ca2+]i from a toad fibre during repeated tetanic stimulation and recovery. Note that the tension declines monotonically unlike the three phases which are often prominent in mouse fibres (Lännergren & Westerblad, 1991). In 10 fibres 204 ± 10 tetani were required to reduce tension to 50 % of control. Selected tetanic [Ca2+]i signals are shown in the lower panel and show a small initial rise followed by a monotonic fall. In 10 fibres the peak tetanic [Ca2+]i when tension had fallen to 50 % was 43 ± 2 % of control. Note that there is no sign of the more pronounced fall in tetanic [Ca2+]i in the final phase of fatigue which is seen in mouse (Westerblad & Allen, 1991). Towards the end of fatigue there was a small increase in resting [Ca2+]i (increase of 63 ± 9 nM; n= 5) and a slowing of the rate of decline of [Ca2+]i as described in other muscle types (Allen et al. 1989; Westerblad & Allen, 1991).

After fatiguing stimulation, muscles were rested for 20 min and then a test tetanus given. In six experiments tension recovered to 93 ± 3 % and tetanic [Ca2+]i recovered to 100 ± 4 %. Note that the Ringer solution contains no glucose so this recovery in the absence of glucose is quite different from that seen in mouse fibres (Chin & Allen, 1997). We did not observe the failure of early recovery (post-contractile depression) described in Xenopus fibres (Westerblad & Lännergren, 1986).

Rapidly releasable SR Ca2+ during fatigue

Changes in SR Ca2+ stores could be one of the factors contributing to the decline in tetanic tension and [Ca2+]i in late fatigue (see Introduction). In a recent study we showed that brief application of 2 mM 4-CmC or 30 mM caffeine allowed repeated measurements of the rapidly releasable SR Ca2+ without affecting muscle cell function (Kabbara & Allen, 1999). In 10 experiments under control conditions the peak tetanic [Ca2+]i was 1130 ± 70 nM and the peak 4-CmC- or caffeine-induced [Ca2+]i was 2460 ± 180 nM. In the remainder of the results we express the tetanic and 4-CmC/caffeine [Ca2+]i as a percentage of the control values for that experiment.

The aim of the present experiments was to measure the rapidly releasable SR Ca2+ during fatigue. Figure 2 shows [Ca2+]i records before, during and after fatigue. On the left are a single tetanus followed by a 4-CmC (5 mM) exposure. A 10 min rest was then allowed and muscle fatigue was produced by our standard protocol which in this case required 180 tetani (7.8 min; note breaks in the record). A few seconds after the end of fatigue 4-CmC was reapplied. Note that the amplitude of tetanic [Ca2+]i was reduced to about one-half during fatigue while the amplitude of the 4-CmC-induced [Ca2+]i was also reduced to about one-half of the control. Then a 20 min recovery period was allowed and the right-hand panel shows the recovery tetanic [Ca2+]i and 4-CmC-induced [Ca2+]i which in this experiment were slightly bigger than control.

image

Figure 2. Rapidly releasable SR Ca2+ during fatigue

Record of [Ca2+]i illustrating procedure used to estimate rapidly releasable SR Ca2+ changes during fatigue and recovery. Rapidly releasable SR Ca2+ was estimated by rapid application of 5 mM 4-CmC 10 min before fatigue, at the end of fatigue, and after 20 min rest. Note that each 4-CmC application is preceded by a test tetanus. Note breaks in the record.

Download figure to PowerPoint

We performed 10 experiments of this type; three using 2 mM 4-CmC, three using 5 mM 4-CmC and four using 30 mM caffeine; recovery was followed in six experiments. The rapidly releasable SR Ca2+ in fatigue was reduced to 53 ± 2, 45 ± 5 and 45 ± 8 %, respectively. There were no significant differences between them so we present the global average here. The amplitude of rapidly releasable SR Ca2+ declined to 46 ± 6 % after fatigue and recovered to 98 ± 12 %.

Interpretation of these results depends on comparisons of the amplitude of 4-CmC or caffeine exposures before and after fatigue and before and after recovery. We therefore performed control experiments to test whether a 4-CmC response changed after 20 min of rest. In three fibres examined the mean peak 4-CmC-induced [Ca2+]i after 20 min rest was 107 ± 10 % of control. Thus there was no significant change in the rapidly releasable SR Ca2+ over a 20 min rest period.

Effects of long unfatiguing stimulation on rapidly releasably SR Ca2+ and tetanic [Ca2+]i

The above results show that 150–250 tetani which cause fatigue lead to a reduction of rapidly releasable SR Ca2+. This could either be a consequence of the 150–250 tetani or a consequence of the metabolic changes associated with fatigue. To distinguish between these possibilities we stimulated the muscle with 200 tetani but at 10 s intervals (34 min). Figure 3 shows a representative example; tetanic tension shows only a small decline, tetanic [Ca2+]i is unchanged and the amplitude of the 4-CmC-induced [Ca2+]i declined slightly. In three experiments tetanic tension was reduced to 85 ± 1 % of control; no significant change was seen in either the amplitude of the tetanic [Ca2+]i (98 ± 17 %) or the amplitude of the 4-CmC-induced [Ca2+]i (105 ± 17 %). These results suggest that the reduction in rapidly releasable SR Ca2+ observed in fatigue is related to the metabolic changes occurring in fatigue rather than simply the number of tetani involved.

image

Figure 3. Effects of non-fatiguing stimulation on rapidly releasable SR Ca2+ and tetanic [Ca2+]i and force

Upper panel, [Ca2+]i (note breaks in the record); lower panel, first and last tetani. Rapidly releasable SR Ca2+ was estimated with 2 mM 4-CmC 10 min before and after 200 tetani at 10 s intervals. Note that tetanic [Ca2+]i, force and rapidly releasable SR Ca2+ were little affected by this stimulation protocol. Time scale: 30 s for [Ca2+]i and 1 s for force.

Download figure to PowerPoint

Recovery from fatigue does not require Ca2+ entry

The reduced rapidly releasable SR Ca2+ observed in fatigue could occur because Ca2+ is pumped out of the fibre because of the increase in mean [Ca2+]i during repeated tetani. If this is the case, recovery may require Ca2+ entry from the extracellular space (Balnave & Allen, 1998). We tested this hypothesis by measuring the rapidly releasable SR Ca2+ after recovery in the absence of extracellular Ca2+ (replaced by Mg2+). Figure 4 shows a representative experiment; note the usual reduction in tetanic [Ca2+]i during fatigue and the greatly reduced 4-CmC-induced [Ca2+]i after fatigue. However, the 4-CmC-induced [Ca2+]i after recovery in the absence of extracellular Ca2+ has a similar amplitude to the control. In four fibres tested the mean amplitude of the 4-CmC-induced [Ca2+]i measured after recovery was 112 ± 20 % of the initial control. Thus recovery was normal in the absence of extracellular Ca2+.

image

Figure 4. Recovery from fatigue in the absence of extracellular Ca2+

Rapidly releasable SR Ca2+ and tetanic [Ca2+]i during fatigue and recovery. Rapidly releasable SR Ca2+ was estimated in the usual way (see legend to Fig. 2). However, during 20 min recovery the perfusing Ringer solution contained no added Ca2+, which was replaced by equimolar Mg2+ (0 Ca). Note that the rapidly releasable SR Ca2+ returned to the control level after the rest period in the absence of extracellular Ca2+.

Download figure to PowerPoint

To test whether a period of Ca2+-free perfusion had any effect under control conditions we performed 4-CmC exposures before and after a 20 min period of Ca2+-free perfusion (not shown). In three experiments the rapidly releasable SR Ca2+ after this period was 95 ± 14 % of control. Thus a 20 min period of Ca2+-free perfusion had no detectable effect on rapidly releasable SR Ca2+.

Recovery from fatigue requires oxidative phosphorylation

An alternative explanation for the decline and recovery of rapidly releasable SR Ca2+ is that it is related in some way to the metabolic consequences of fatigue. This interpretation is supported by the earlier finding that the reduction of SR Ca2+ release does not occur if substantial fatigue does not develop. To test whether the recovery of rapidly releasable SR Ca2+ required metabolic recovery we inhibited the metabolic recovery by preventing oxidative phosphorylation. Figure 5 shows that the rapidly releasable SR Ca2+ did not recover when 2 mM cyanide was present during the recovery period. In three fibres fatigue reduced rapidly releasable SR Ca2+ to 49 ± 2 %. After recovery in the presence of cyanide, rapidly releasable SR Ca2+ was 51 ± 4 %. Tetanic [Ca2+]i after fatigue was 46 ± 3 % and after recovery in cyanide it was 31 ± 5 %; similarly tetanic tension was 50 % after fatigue and 33 ± 7 % after recovery in cyanide. These results indicate that recovery of tension, tetanic [Ca2+]i and rapidly releasable SR Ca2+ from fatigue all require oxidative phosphorylation and raise the possibility of a common metabolic component to all three.

image

Figure 5. Effect of inhibition of oxidative phosphorylation on recovery from fatigue

Rapidly releasable SR Ca2+ and tetanic [Ca2+]i during fatigue and recovery measured using standard protocol. During the 20 min recovery period Ringer solution contained 2 mM sodium cyanide (CN), an inhibitor of oxidative phosphorylation. Both rapidly releasable SR Ca2+ and tetanic [Ca2+]i remained small after 20 min recovery in the presence of cyanide. During a second recovery in the absence of cyanide, tetanic [Ca2+]i partially recovered while the rapidly releasable SR Ca2+ fully recovered.

Download figure to PowerPoint

In the experiment shown in Fig. 5, after a further 20 min recovery in the absence of cyanide, the rapidly releasable SR Ca2+ was restored to control level. In the two other experiments the fibre did not recover after fatigue and 20 min in cyanide.

To test whether cyanide had any effect on rapidly releasable SR Ca2+ under control conditions, in three experiments cyanide was applied to resting (unfatigued) fibres for 20 min (not shown). The rapidly releasable SR Ca2+ at the end of this exposure was unaffected (107 ± 10 %).

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Measurement of Ca2+ stores during fatigue

Kabbara & Allen (1999) showed that under control conditions brief applications of 4-CmC or caffeine caused a large rise in [Ca2+]i which did not appear to damage the muscle and was repeatable. They argued that the pool from which this Ca2+ was being released was completely depleted on the grounds that application of a SR Ca2+ pump blocker did not increase the magnitude of the myoplasmic [Ca2+]i. However, calculation suggested that this pool was less than the total SR Ca2+. It is generally accepted that most of the Ca2+ within the SR (total Ca2+ 30–50 mM) is bound to calsequestrin, with only ∼1 mM remaining free (Somlyo et al. 1981). Since the Ca2+ released from the SR by 4-CmC approximates to half the Ca2+ within the SR (Somlyo et al. 1981; Kabbara & Allen, 1998), this implies that the Ca2+ bound to calsequestrin must be capable of dissociating relatively quickly. Note that the opposite is also true; if Ca2+ bound to some species within the SR from which it did not dissociate during the time course of a tetanus or a 4-CmC exposure, then this component of Ca2+ would not be measured by this test. For these reasons we refer to the 4-CmC-induced Ca2+ rise as measuring the rapidly releasable SR Ca2+.

A preliminary question is whether 4-CmC is still capable of opening the SR Ca2+ channels sufficiently during fatigue to enable the rapidly releasable SR Ca2+ to be measured reliably. An important observation by Kanaya et al. (1983) was that the sensitivity of contracture tension to caffeine was reduced considerably in fatigued muscles (the half-maximal sensitivity increased from ∼3 mM to ∼7 mM). This could arise either because the SR Ca2+ release channels in fatigued muscles had become less sensitive to caffeine or because the contractile proteins had a reduced Ca2+ sensitivity so that a greater Ca2+ release was required to produce the same tension. We previously showed that under control conditions 2 mM 4-CmC gave a just-maximal Ca2+ release and that 5 mM 4-CmC gave the same maximal release (Kabbara & Allen, 1999). In the present study we have shown that in fatigued conditions the rapidly releasable SR Ca2+ as a fraction of the control value was 53 ± 2 % for 2 mM 4-CmC and 45 ± 5 % for 5-CmC. Since these values are not significantly different it follows that the sensitivity of the SR release channels to 4-CmC is not reduced in fatigue. Furthermore it is known that the SR Ca2+ pump is inhibited during fatigue (Byrd, 1992; Westerblad & Allen, 1993) so that for a given increase in SR Ca2+ channel permeability the fraction of Ca2+ in the myoplasm compared with the SR will be greater. We conclude that rapid application of 4-CmC can effectively measure the rapidly releasable SR Ca2+ during fatigue as well as under control conditions.

The cause of the reduction of rapidly releasable SR Ca2+ during fatigue

It is clear that the rise of [Ca2+]i caused by 4-CmC declines during fatigue and returns to control levels during the period when the tetanic [Ca2+]i and tension recover. We argue above that this is not because the Ca2+ release channels become less sensitive to 4-CmC during fatigue and therefore this observation can be interpreted either as a reduction of the total amount of Ca2+ stored in the SR or, alternatively, Ca2+ binds to some substance within the SR to form a product from which Ca2+ cannot be released during a short 4-CmC exposure or a tetanus.

To test the possibility that Ca2+ stored in the SR is reduced we made use of an observation by Balnave & Allen (1998). They showed that during a prolonged period (20–30 min) of elevated [Ca2+]i, Ca2+ was removed from the cell on the Na+-Ca2+ exchanger. At the end of this period, the SR Ca2+ store was depleted and could be replenished by a 30 min period in normal extracellular [Ca2+]. However, if extracellular [Ca2+] was removed during the 30 min recovery period then the store was not replenished. The present results show that the reduced 4-CmC-releasable Ca2+ observed in fatigue recovers even in the absence of extracellular calcium. This result suggests that the SR Ca2+ store is not depleted, but instead that the Ca2+ is present but not able to be released and that this process can reverse during recovery of the muscle.

Our experiments provide strong evidence that the reduction in releasable Ca2+ is in some way related to the metabolic changes associated with fatigue. First, when the same number of tetani was performed with longer intervals between tetani so that no fatigue occurs, then no such reduction in releasable Ca2+ was observed. Second, the releasable Ca2+ recovered during a 20 min rest period, which is sufficient for many of the metabolic changes associated with fatigue to be reversed (Kushmerick & Meyer, 1985; Cady et al. 1989). Third, when oxidative phosphorylation was blocked during the recovery period, no such recovery of releasable Ca2+ occurred. It is known that preventing oxidative phosphorylation greatly reduces the degree of metabolic recovery in a resting muscle (Dawson et al. 1980). In particular the myoplasmic Pi remains elevated when the muscles are kept anaerobic during recovery.

The CaPi hypothesis

The present experiments provide no direct evidence as to the nature of the metabolic change which leads to a reduction of rapidly releasable SR Ca2+. However, our data are consistent with the hypothesis that Ca2+ precipitates with Pi within the SR, forming a species of CaPi which redissolves slowly.

This hypothesis was first proposed by Fryer et al. (1995) based on studies of Ca2+ release in skinned rat and cane toad fibres with intact SR (Owen et al. 1992). They showed that, in the presence of high Pi concentrations and a finite pool of Ca2+, Ca2+ release from the SR was reduced in a manner consistent with precipitation of CaPi in the SR. In subsequent studies Fryer et al. (1997) investigated the mechanism of Pi uptake by the SR and showed a delay of several minutes between the rise of Pi and the development of CaPi in the SR. Posterino & Fryer (1998) measured the time course of recovery of SR Ca2+ release when [Pi] was reduced from a high to a low level. This process had a half-time of ∼35 s which they attributed to Pi removal from the SR. All the above studies were in skinned fibres but further support for the development of CaPi in the SR came from experiments in which NaPi was injected into intact fibres. This produced a substantial reduction in Ca2+ release and tension production consistent with the above hypothesis (Westerblad & Allen, 1996). The above studies strongly suggest that precipitation of CaPi in the SR can occur under some conditions but they do not show whether this mechanism operates during fatigue.

The present experiments make the case that this mechanism operates during fatigue much stronger. The key items of evidence, discussed above, are as follows. (i) Releasable SR Ca2+ is reduced in fatigue and returns to control values during recovery. (ii) This phenomenon is closely associated with the metabolic change occurring during fatigue.

Interestingly, the early electron probe studies by Gonzalez-Serratos et al. (1978) showed not only a rise in the SR total Ca2+ but also a small rise in the phosphorus content which would be consistent with increased CaPi. However, it is important to note that there is, as yet, no direct evidence of precipitation of CaPi in the SR of fatigued muscles to our knowledge.

Westerblad & Allen (1996) pointed out two weaknesses of the CaPi hypothesis as an explanation for the reduced Ca2+ release during fatigue. (i). The reduction of Ca2+ release occurs mainly in the last phase of fatigue and correlates better with the fall in [ATP] and the rise of [Mg2+]i which occur at about that time (Westerblad & Allen, 1992). (ii). In the final phase of fatigue there are at least three changes in Ca2+ handling; reduced tetanic [Ca2+]i, elevated resting [Ca2+]i, and slowing of the SR Ca2+ pump. The CaPi hypothesis only explains the first of these. How can these points be addressed?

The time course issue remains a difficult and complex one. First, the time course of the decline of tetanic [Ca2+]i in amphibia (present experiments; Allen et al. 1989) is somewhat different to those reported in mouse (Westerblad & Allen, 1991, 1993) in showing a more or less monotonic decline after the initial small rise, which is more simply consistent with the CaPi hypothesis. Second, a fuller understanding of the time course requires knowledge of the time course of the rise of myoplasmic Pi, the time course of the entry of Pi into the SR and the time course of precipitation of CaPi. We need more information on all these processes to decide whether the time course of decline of releasable SR Ca2+ is consistent with the theory. Third, Posterino & Fryer (1998) make the interesting proposal that there may be an interaction between elevated Pi and reduced ATP. Similar considerations apply to the time course of recovery of releasable SR Ca2+.

It remains true that the CaPi hypothesis does not explain the elevated resting Ca2+ and the slowing of the SR pump (see Westerblad & Allen, 1996, for more detailed discussion). These two effects can be explained by metabolic inhibition of the SR pump which we suggest must be an independent contributor to fatigue, though possibly also triggered by raised myoplasmic Pi.

Conclusion

These findings are all consistent with the decline of Ca2+ release during fatigue being caused by precipitation of CaPi in the SR which reverses during recovery. A major attraction of this hypothesis is that it leads to a simple explanation of the correlation between failure of activation and the metabolic changes in fatigue. It is not yet clear whether other mechanisms, such as failure of action potentials or reduced opening of SR Ca2+ channels, also contribute to the decline in Ca2+ release which occurs in fatigue. The differences in time course of fatigue and the sensitivity to glucose during recovery in mammalian muscle may indicate that additional mechanisms operate in mammalian tissues.

  • Allen, D. G., Lännergren, J. & Westerblad, H. (1995). Muscle cell function during prolonged activity: cellular mechanisms of fatigue. Experimental Physiology 80, 497527.
  • Allen, D. G., Lee, J. A. & Westerblad, H. (1989). Intracellular calcium and tension during fatigue in isolated single muscle fibers from Xenopus laevis. The Journal of Physiology 415, 433458.
  • Andrade, F. H., Reid, M. B., Allen, D. G. & Westerblad, H. (1998). Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse. The Journal of Physiology 509, 565575.
  • Bakker, A. J., Head, S. I., Williams, D. A. & Stephenson, D. G. (1993). Ca2+ levels in myotubes grown from the skeletal muscle of dystrophic (mdx) and normal mice. The Journal of Physiology 460, 113.
  • Balnave, C. D. & Allen, D. G. (1998). Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse. American Journal of Physiology 274, C940946.
  • Byrd, S. K. (1992). Alterations in the sarcoplasmic reticulum: a possible link to exercise-induced muscle damage. Medicine and Science in Sports and Exercise 24, 531536.
  • Cady, E. B., Jones, D. A., Lynn, J. & Newham, D. J. (1989). Changes in force and intracellular metabolites during fatigue of human skeletal muscle. The Journal of Physiology 418, 311325.
  • Chin, E. R. & Allen, D. G. (1997). Effects of reduced muscle glycogen concentration on force, Ca2+ release and contractile protein function in intact mouse skeletal muscle. The Journal of Physiology 498, 1729.
  • Dawson, M. J., Gadian, D. G. & Wilkie, D. R. (1978). Muscle fatigue investigated by phosphorus nuclear magnetic resonance. Nature 274, 861866.
  • Dawson, M. J., Gadian, D. G. & Wilkie, D. R. (1980). Studies of the biochemistry of contracting and relaxing muscle by the use of 31P n.m.r. in conjunction with other techniques. Philosophical Transactions of the Royal Society B 289, 445455.
  • Fitts, R. H. (1994). Cellular mechanisms of muscle fatigue. Physiological Reviews 74, 4994.
  • Fryer, M. W., Owen, V. J., Lamb, G. D. & Stephenson, D. G. (1995). Effects of creatine phosphate and Pi on Ca2+ movements and tension development in rat skinned skeletal muscle fibres. The Journal of Physiology 482, 123140.
  • Fryer, M. W., West, J. M. & Stephenson, D. G. (1997). Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle. Journal of Muscle Research and Cell Motility 18, 161167.
  • Godt, R. E. & Nosek, T. M. (1989). Changes in the intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. The Journal of Physiology 412, 155180.
  • Gonzalez-Serratos, H., Somlyo, A. V., McCellan, G., Shuman, H., Borrero, L. M. & Somlyo, A. P. (1978). Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: electron probe analysis. Proceedings of the National Academy of Sciences of the USA 75, 13291333.
  • Grynkiewicz, G., Poenie, M. & Tsien, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. Journal of Biological Chemistry 260, 34403450.
  • Herrmann-Frank, A., Richter, M., Sarkozi, S., Mohr, U. & Lehmann-Horn, F. (1996). 4-Chloro-m-cresol, a potent and specific activator of the skeletal muscle ryanodine receptor. Biochimica et Biophysica Acta 1289, 3140.
  • Kabbara, A. A. & Allen, D. G. (1999). Measurement of sarcoplasmic reticulum Ca2+ content in intact amphibian skeletal muscle fibres with 4-chloro-m-cresol. Cell Calcium 25, 227235.
  • Kanaya, H., Takauji, M. & Nagai, T. (1983). Properties of caffeine- and potassium-contractures in fatigued frog single twitch muscle fibres. Japanese The Journal of Physiology 33, 945954.
  • Kushmerick, M. J. & Meyer, R. A. (1985). Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance. American Journal of Physiology 248, C542549.
  • Lännergren, J. & Westerblad, H. (1991). Force decline due to fatigue and intracellular acidification in isolated fibres from mouse skeletal muscle. The Journal of Physiology 434, 307322.
  • Millar, N. C. & Homsher, E. (1990). The effect of phosphate and calcium on force generation in glycerinated rabbit skeletal muscle fibers; a steady-state and transient kinetic study. Journal of Biological Chemistry 265, 2023420240.
  • Owen, V. J., Lamb, G. D. & Stephenson, D. G. (1992). Effects of IMP and inorganic phosphate on depolarization-induced and caffeine-induced calcium release in skeletal muscle fibres of the toad. Proceedings of the Australian Physiological and Pharmacological Society 23, 148P (Abstract).
  • Posterino, G. S. & Fryer, M. W. (1998). Mechanisms underlying phosphate-induced failure of Ca2+ release in single skinned skeletal muscle fibres of the rat. The Journal of Physiology 512, 97108.
  • Rousseau, E., Ladine, J., Lui, Q. & Meissner, G. (1988). Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Archives of Biochemistry and Biophysics 267, 7586.
  • Somlyo, A. V., Gonzalez-Serratos, H., Shuman, H., McClellan & Somlyo, A. P. (1981). Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron probe study. Journal of Cell Biology 90, 577594.
  • Westerblad, H. & Allen, D. G. (1991). Changes of myoplasmic calcium concentration during fatigue in single mouse muscle fibers. Journal of General Physiology 98, 615635.
  • Westerblad, H. & Allen, D. G. (1992). Myoplasmic free Mg2+ concentration during repetitive stimulation of single fibres from mouse skeletal muscle. The Journal of Physiology 453, 413434.
  • Westerblad, H. & Allen, D. G. (1993). The contribution of [Ca2+]i to the slowing of relaxation in fatigued single fibres from mouse skeletal muscle. The Journal of Physiology 468, 729740.
  • Westerblad, H. & Allen, D. G. (1996). The effects of intracellular injections of phosphate on intracellular calcium and force in single fibres of mouse skeletal muscle. Pflügers Archiv 431, 964970.
  • Westerblad, H., Andrade, F. H. & Islam, M. S. (1998). Effects of ryanodine receptor agonist 4-chloro-m-cresol on myoplasmic free Ca2+ concentration and force of contraction in mouse skeletal muscle. Cell Calcium 24, 105115.
  • Westerblad, H. & Lännergren, L. (1986). Force and membrane potential during and after fatiguing, intermittent tetanic stimulation of single Xenopus fibres. Acta Physiologica Scandinavica 128, 369378.

Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

This work was supported by the National Health & Medical Research Council of Australia.