Department of Zoology, La Trobe University, Melbourne, Victoria, Australia
Submitted 10 June 2005 ; accepted in final form 5 August 2005
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ABSTRACT |
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muscle fatigue; excitation-contraction coupling
Inhibitory effects of Pi within the cytoplasm on maximum Ca2+-activated force and Ca2+ sensitivity of the contractile apparatus have been well characterized (4, 26) and may play a role in muscle fatigue (38), but other effects of Pi are less clear. Fryer et al. (16) first suggested that cytoplasmic Pi could enter the SR and precipitate Ca2+ (CaPi), thus reducing the amount of rapidly releasable Ca2+ and contributing to the later stages of metabolic muscle fatigue (1), when force declines steeply because of reduced Ca2+ release (2). Evidently Pi can enter the SR passively (30), possibly via small-conductance Cl channels that conduct Pi (23).
A possible criticism of the earlier studies on the effect of Pi on SR Ca2+ handling, in which Pi exposure reduced caffeine-induced force responses (16, 30), is that CrP was deliberately omitted to mimic in vivo conditions during fatigue. By omitting CrP, ADP may rise considerably in local regions, possibly causing substantial Ca2+ leak through the SR Ca2+-ATPase (SERCA) (8, 9, 25). Thus it was not fully clear whether the reduction in caffeine-induced responses reported by Fryer et al. (16) was due solely to CaPi precipitation within the SR or also to loss of SR Ca2+. Furthermore, cytoplasmic Pi has been shown to increase the open probability of a single RyR incorporated into lipid bilayers (3, 14), thus suggesting that Pi exposure may also cause SR Ca2+ leak through RyRs in addition to any leak occurring through the SERCA. However, more recently, it has been shown that the Pi-induced SR Ca2+ leak through RyRs is seemingly absent in the presence of physiological free Mg2+ concentration ([Mg2+]) levels or above (i.e., 11.5 mM) (10).
The effect of cytoplasmic Pi has also been investigated in intact fibers. A study using microinjection of Pi into unfatigued, intact murine fibers (221 mM estimated to have been added to the cytoplasm) found that the intracellular Ca2+ concentration ([Ca2+]i) at rest and during tetanic stimulation was reduced after Pi injections (37). However, in those experiments, it was unclear whether other changes had occurred upon or after Pi injection, because the expected effect of intracellular Pi on maximum force production (4, 26) did not occur (see DISCUSSION). Other intact fiber studies in which Pi accumulation in the cytoplasm was eliminated by inhibiting CK activity pharmacologically (5) or by using CK-knockout (CK/) mice (6) have indeed indicated that increased cytoplasmic [Pi] is inversely correlated with reduced tetanic Ca2+ release. This suggests that cytoplasmic Pi inhibits normal SR Ca2+ handling in some way but does not definitively show whether this is due to inhibition of Ca2+ release, to net loss of SR Ca2+, or to CaPi precipitation within the SR.
Herein we report the results of our investigation into whether exposure to elevated cytoplasmic [Pi] leads to CaPi precipitation within the SR and whether this affects normal AP-mediated E-C coupling. It is currently unknown whether or to what extent tetanic force responses are affected by a reduction in total SR Ca2+, let alone by the formation of any CaPi precipitation within the SR. It is possible that tetanic force might be unaffected by even a substantial reduction in available SR Ca2+, because the endogenous SR Ca2+ content (1.1 mmol/l fiber vol) (15, 28) may well be far more than necessary to fully activate the contractile apparatus. On the other hand, the presence of any CaPi within the SR could perhaps cause a marked reduction in AP-induced Ca2+ release, possibly even more than that accounted for by any decrease in available SR Ca2+. Mechanically skinned fibers were used because they retain the normal E-C coupling mechanism and the endogenous level of SR Ca2+. Furthermore, Pi could be added and removed rapidly and precisely from the cytoplasmic space. To obviate possible effects of ADP on the SR pump and Pi on the RyRs, and thereby to examine the effect of Pi exposure independent of possible Ca2+ leakage, the experiments were performed with 10 mM CrP and 1 mM free [Mg2+] present in the cytoplasm. In addition, by placing the skinned fiber in paraffin oil during Pi exposure, it was possible to avoid any substantial net Ca2+ leak or loading by the SR. Pi exposure caused a reduction in SR Ca2+ release to AP stimulation and also to direct stimulation with caffeine, but SR lysis showed that the total amount of Ca2+ remaining in the SR was unchanged by the Pi exposure. These observations provide strong evidence that Pi enters the SR and precipitates with Ca2+, reducing the amount of Ca2+ available for rapid release in response to AP-mediated stimulation and hence possibly contributing to muscle fatigue in certain situations.
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METHODS |
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Solutions. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. The standard K+-HDTA solution (control solution) contained (in mM) 50 HDTA2 (Fluka, Buchs, Switzerland), 8 total ATP, 10 CrP, 37 Na+, 126 K+, 8.53 total Mg2+ (giving 1 mM free [Mg2+]), 0.075 total EGTA, and 90 HEPES, pH 7.1, along with log10 [Ca2+] (pCa) of 6.9 except where stated. A similar solution made by replacing all K+ with Na+ (Na+-HDTA solution) was used to depolarize the T-system by ionic substitution (22) (see Fig. 4). All solutions had an osmolality of 295 ± 5 mosmol/kgH2O and a free [Mg2+] of 1 mM on the basis of apparent Mg2+ affinity constants of 6.9 x 103 M1 for ATP, 8 M1 for HDTA, and 15 M1 for CrP (12, 35). The pCa of solutions (for pCa <7.2) was measured with a Ca2+-sensitive electrode (Orion Research, Cambridge, MA).
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Pi (purchased from Ajax Chemicals, Sydney, Australia) was used to make a 30 mM Pi solution, similar to the standard K+-HDTA solution in terms of, for example, osmolality, ionic strength, and EGTA concentration, with the only differences being that the total HDTA concentration was decreased from 50 to 27 mM and the total [Mg2+] was raised from 8.53 to 9.33 mM (producing 1 mM free [Mg2+]) to take into account the amount of Mg2+ bound to the 30 mM Pi (16). In experiments requiring 10 mM Pi, the 30 mM Pi solution was mixed at a 1:2 ratio with the standard K+-HDTA solution. An oxalate stock solution was made with 30 mM oxalate replacing 30 mM HDTA, with the total Mg2+ increased by 10.5 mM to keep the free [Mg2+] at 1 mM on the basis of an apparent Mg2+ affinity for oxalate of 5.75 x 102 M1 (33). This stock solution was mixed with the standard K+-HDTA solution to produce a final concentration of 5 or 10 mM oxalate.
Pi exposure.
Mathematical modeling has shown that molecules can rapidly equilibrate within a skinned EDL fiber upon a change in the bathing solution (e.g., caffeine reaching 60% of the added level within 0.9 s in a fiber of
30-µm radius) (36). Therefore, it was assumed that 10 s was ample time for Pi to be added to or washed out of the cytoplasm of the skinned fibers, such that it reached close to equilibrium with the bathing solution. Because it has been suggested that the presence of cytoplasmic Pi induces Ca2+ leak from the SR (3, 9, 10), the fiber was bathed in 10 or 30 mM Pi (or 0 mM Pi control) solution for only 10 s and then transferred to paraffin oil (1 min), making the fiber a closed system and hence allowing enough time for Pi to enter the SR and precipitate with Ca2+ while ensuring that most of any Ca2+ that may have leaked into the cytoplasm (in which there was only low Ca2+ buffering) would be recovered by the SR via the SERCA. This 10-s Pi exposure and 1-min oil incubation procedure was repeated as many times as necessary (twice unless stipulated otherwise), and then the fiber was washed for 30 s in control solution before being stimulated.
Caffeine/low-[Mg2+]-induced SR Ca2+ release experiments. The total amount of releasable Ca2+ released into the SR was assayed by directly activating the RyRs with a nonphysiological stimulus {30 mM caffeine, 0.05 mM free [Mg2+] (total adjusted from 8.53 to 2.15 mM) and 0.5 mM EGTA (pCa 8.0) to chelate released Ca2+} (15, 20). The endogenous level of releasable SR Ca2+ was determined initially by exposing the freshly skinned fiber to the caffeine/low [Mg2+] solution (termed "full release") and then reloading the SR with Ca2+ (standard K+-HDTA solution, pCa 6.7, 1 mM total EGTA) for a period that produced a force response similar to that observed when the SR initially contained its normal endogenous level. The time integral (i.e., area) of the force response was indicative of the relative amount of Ca2+ loaded into the SR (21). The entire load (30 s), release (1 min), and wash (1 min) cycle was performed before and after Pi exposure (Fig. 1). This type of procedure has been detailed extensively elsewhere (20, 21). With each fiber, the SR was reproducibly reloaded to the same set level on each cycle, with this level being approximately the same or slightly above the normal endogenous level.
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Verification of Pi washout. Cytoplasmic Pi is known to reduce maximum force output and the Ca2+ sensitivity of the contractile apparatus (4, 26). Thus it was important that all cytoplasmic Pi be washed out of the fiber before assaying the effect of the Pi exposure on SR Ca2+ handling. This effect was verified in the following way. A rapid step rise in [Ca2+] to a level producing submaximal force was applied to a fiber to detect whether the force response of the contractile apparatus was affected by any Pi remaining within the fiber. The rapid rise in [Ca2+] within the skinned fiber was produced by transferring the fiber from a solution in which [Ca2+] was only weakly buffered (e.g., 75 µM EGTA) at a low level (pCa 6.9) to a solution in which [Ca2+] was heavily buffered (50 mM Ca2+-EGTA-EGTA, pCa 5.8) at a higher submaximal level (27). This rapidly produced a submaximal force response that should have been sensitive to even subtle changes in Ca2+ sensitivity or maximum force production. All solutions also contained 50 µM SERCA-specific inhibitor 2,5-di-(tert-butyl)-1,4-hydroquinone to prevent the SR from taking up any Ca2+ and interfering with the level set by Ca2+ buffering (31). Any decrease in the rate of force production or the size of the force response would be indicative that Pi was still affecting the contractile apparatus. It was found that after Pi exposure, a 30-s washout period (in control solution) was sufficient to fully eliminate any effect of the Pi exposure on the rate of force development and the level of force attained after 8 s (data not shown), thus indicating that effectively all of the Pi had been removed from the cytoplasm.
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RESULTS |
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Effect of Pi exposure on AP-mediated Ca2+ release.
We next examined the effect of Pi exposure on force responses elicited via the normal AP-mediated E-C coupling mechanism (Fig. 2). Tetanic (50-Hz) stimulation produced maximum AP-mediated force (see Table 2), because the cytoplasmic Ca2+ buffer parvalbumin is removed from mechanically skinned fibers, thus causing summation at a comparatively low frequency. The oil exposure sequence itself (control treatment) had no significant effect on peak tetanic force, whereas exposure to 10 and 30 mM Pi caused a concentration-dependent decrease in the amplitude of the tetanic responses (to 79% and
55% of the control level with 10 and 30 mM Pi, respectively) (Figs. 2 and 3; mean data are shown in Table 2). In three fibers, the twitch response after 30 mM Pi exposure also was examined, and the reduction in twitch peak amplitude was proportionally larger than that observed in the tetani (see Fig. 2B and Table 2). T-system depolarization-induced force elicited by ionic (Na+) substitution was also reduced after 2-min exposure to 30 mM Pi (see Fig. 4 and Table 2), thus ruling out the failure of AP propagation as the cause of the reduction in force. In addition to the reduction in peak force, the rate of force development of the twitch, tetanic, and Na+ depolarization-induced force responses after Pi exposure was slowed compared with the pretreatment level (Table 2).
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Comparison of tetani after SR depletion and Pi exposure.
The effect on tetanic force of Pi exposure and of depleting the SR Ca2+ to a certain level were compared in the same fiber to ascertain whether they affected the responses in a similar manner. To deplete the SR of some of its releasable Ca2+, the fiber was tetanically stimulated (50 Hz for 0.4 s) in the presence of 2 mM free [BAPTA], which rapidly chelated the released Ca2+, preventing it from being resequestered by the SR. The BAPTA was then washed away and the SR was allowed to recover Ca2+ slowly from the weakly Ca2+-buffered control solution (pCa 6.9, 75 µM EGTA) to eventually produce a tetanus of approximately similar amplitude (e.g., 5060% maximum force) as that elicited after Pi exposure. In the five fibers examined in this way, the tetanic force responses after partial SR depletion and Pi exposure were clearly similar in terms of peak force, rate of rise from 10 to 90% peak force (RR1090), and in fall rate from 90 to 10% peak force (FR9010) (see Table 2 for mean data).
SR total Ca2+ content determined by lysing the SR. A method of assaying the total amount of Ca2+ within the SR of a fiber (see Refs. 15, 28) was used to determine whether the reduction in the amount of releasable SR Ca2+ after Pi exposure was caused by a loss of Ca2+ from the SR or by CaPi formation within the SR. With the use of this method, any CaPi present in the permeabilized SR dissolves (i.e., forming Ca2+ and Pi) as the BAPTA binds available Ca2+ and keeps the free [Ca2+] within the fiber space low. By preequilibrating the fiber for 20 s in a particular [BAPTA] and then permeabilizing all membranes with the TX-oil emulsion (see Fig. 5), we ascertained the total amount of Ca2+ remaining in the SR after 1) a control sequence without Pi exposure, 2) 30 mM Pi exposure, and 3) depletion of SR Ca2+ to produce approximately the same mean peak force as occurred with 30 mM Pi exposure (Fig. 6). If a detectable but nonmaximal level of force was produced upon lysis, the total amount of Ca2+ within the fiber could be calculated on the basis of the [BAPTA] preequilibrated within the fiber and the level of force produced (see METHODS). If zero force or maximum force was elicited upon lysis, then only the upper and lower [Ca2+] estimates, respectively, could be obtained (see means ± SE with arrows in Fig. 7). These latter estimates were not used to calculate the mean total amount of Ca2+ remaining in the SR after each treatment but were nevertheless included in Fig. 7 to help demarcate the ranges in the different cases. The mean total amounts of Ca2+ remaining in the SR (expressed relative to intact fiber volume) immediately after each treatment were 1.16 ± 0.04 mM (n = 9), 0.74 ± 0.03 mM (n = 5), and 1.16 ± 0.07 mM (n = 3) for control, depleted, and 30 mM Pi exposure fibers, respectively. The mean total amount of Ca2+ remaining in the SR after depletion was significantly lower (P < 0.05; unpaired Student's t-test) than both the control and Pi-treated cases, while there was no significant difference between control and Pi treatments (P > 0.05; unpaired Student's t-test). Thus the reduction in releasable Ca2+ caused by Pi exposure was not due to SR Ca2+ loss by a leakage pathway, because the total amount of Ca2+ was unchanged (i.e., still in the SR). This finding is consistent with Pi precipitating with Ca2+ (CaPi).
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DISCUSSION |
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Creating a closed system in a skinned fiber.
It has been reported that cytoplasmic Pi can cause SR Ca2+ leak either through the RyRs and/or through the SERCA (3, 8, 9, 34). For this reason, we created a closed system around the skinned fiber by immersing it in paraffin oil for two 1-min periods, which was enough time for Pi to enter the SR and form CaPi but prevented any substantial net loss or uptake of Ca2+ by the SR. While under oil, the great majority of any Ca2+ leaking out of the SR would have been recovered by the SERCA, because the Ca2+ could not diffuse away and was only very weakly buffered in the cytoplasm (75 µM total EGTA). Hence, only a small amount of SR Ca2+ would be required to increase the free cytoplasmic [Ca2+], which would then both increase active uptake and decrease passive efflux through the pump (25) until it stopped net Ca2+ loss from the SR. Consequently, under these conditions, the observed reduction in the amount of releasable Ca2+ cannot be attributed to net loss of Ca2+ from the SR. This was also confirmed directly by the SR permeabilization experiments (see below). In addition, having the skinned fiber under oil also prevented the fiber from loading any appreciable amount of exogenous Ca2+, which otherwise occurs (albeit slowly) if Ca2+ is able to diffuse continuously into the fiber from the bathing solution. While the fiber was under oil, the total amount of Ca2+ available in the enclosed solution was only 30 µmol/l cytoplasmic water, with most of this being bound to the 75 µM EGTA at pCa 6.9, equivalent to
23 µM when expressed per liter of total fiber volume. Thus, even if the SR were able to load all of this Ca2+ during each of the two equilibration oil periods, the total SR Ca2+ would be increased by only
46 µM, or
4% of that already present endogenously (
1.16 mM). The weak Ca2+ buffering of the control solution also meant that there was only a very limited amount of total Ca2+ available to diffuse into the fiber space and hence that little net Ca2+ was taken up into the fiber during the 30-s washout period. This limitation on uptake can be observed in the slow recovery of tetanic force after depletion of SR Ca2+ (mean increase, 9 ± 1% of maximum force/min; n = 4) (see, e.g., Fig. 6), which indicated that net Ca2+ uptake from the open bathing solution occurred at
90 µM/min over this SR load range.
It should also be noted that placing the fiber under paraffin oil had no adverse effects at all on the tetanic responses, with this being part of the normal skinning procedure for all fibers and also used in all of the control sequences without Pi exposure. Two 1-min oil sequences were used instead of a single 2-min period, because replenishing the solution trapped within the fiber between oil immersions should have prevented any appreciable buildup of metabolites (e.g., Pi, ADP, Mg2+) or depletion of substrates (e.g., ATP, CrP). Furthermore, when the fiber was under oil and Pi moved into the SR to form CaPi, the amount of Pi present in the cytoplasmic space should not have decreased substantially, because the total amount of Ca2+ in the SR is equivalent to only 1.16 mM and hence the amount of Pi lost from the cytoplasm must have been less than that concentration. Interestingly, for this reason, it remains unclear what actually happened in the experiments (37) when up to 20 mM Pi was injected into intact fibers (see the introduction), in which the Pi seemingly had largely disappeared from the cytoplasm within 5 min because it had no evident effect on maximum force production.
Full release of SR Ca2+ by caffeine/low [Mg2+].
The total amount of available Ca2+ rapidly released from the SR by the caffeine/low-[Mg2+] solution was substantially reduced (to 80%) after 30 mM Pi exposure for 2 min (Table 1). Furthermore, the rate of force development of these responses was slowed and also delayed (by
1 s) after Pi exposure (see Fig. 1). It appeared that Pi had reached some form of equilibrium within the 2-min period, because longer exposure times did not appreciably alter the level of inhibition caused by Pi exposure (Table 1). The amount and rate of Ca2+ release and consequent force development of caffeine-induced responses is highly dependent on the amount of releasable Ca2+ in the SR (20). The slower rate of force production observed in the present study after Pi exposure is readily explained by a reduction in the free [Ca2+] within the SR, which not only decreases the driving force for Ca2+ efflux from the SR but also results in less stimulation of the release channels exerted via the stimulatory Ca2+ binding sites located within the SR lumen (24).
The estimate of the amount of Ca2+ made unavailable for release by Pi exposure (i.e., 20% of the total), presumably because it formed some type of CaPi complex, is likely an underestimate of the overall effect. Comparable in vitro chemical assays have shown that the CaPi precipitate dissolves with a half-time of
10 s if the free [Ca2+] is greatly reduced (16). Thus, because the caffeine/low-[Mg2+] exposure induced most of the Ca2+ efflux for a total of
10 s (Fig. 1), it seems probable that there was appreciable dissolution of CaPi within the SR during this period, increasing the total proportion of Ca2+ released during stimulation.
The reduction in the amount of Ca2+ available for release after Pi exposure was approximately the same, regardless of whether 10 mM CrP was present in the Pi solution. Thus it appears that, in agreement with the conclusions of previous studies (16, 30), CaPi precipitated in the SR in both cases. In another skinned fiber study, Duke and Steele (9) concluded that CaPi formation within the SR may not occur when CrP is absent from the cytoplasmic solution. Their conclusion was based on their finding of net efflux of Ca2+ from the SR when Pi was added to the cytoplasm in the absence of CrP. Thus, under the conditions used in that study, the elevated levels of ADP and Pi evidently caused a relatively high rate of Ca2+ efflux from the SR that may simply have outweighed any increase in Ca2+ uptake that occurred upon formation of CaPi within the SR. As mentioned above, in the fibers used under oil in the present study, any such net efflux would cease, owing to the ability of Ca2+ to build up in the cytoplasm. Thus the present results suggest that CaPi precipitation would likely occur in the SR of intact fibers even if most or all of the cytoplasmic CrP had been used.
SR Ca2+ content assay.
The total amount of Ca2+ contained in all compartments of the skinned fiber was liberated by exposing the fiber to the TX oil emulsion. The liberated Ca2+ was then free to bind to the set amount of BAPTA preequilibrated within the fiber and to other sites to which estimates of Ca2+ binding have been ascribed (see Refs. 15, 28). Because the assumptions and method of calculating the total SR Ca2+ were the same for all treatments (i.e., control, SR depletion, and 30 mM Pi exposure), the relative estimates of Ca2+ content should be reliable, regardless of any systematic error possibly arising from inaccuracies in the estimation of the number of fixed Ca2+ binding sites within the fiber (see METHODS). Furthermore, it should be noted that the estimate of the total Ca2+ content of the fiber should be relatively accurate because it is determined predominantly by the concentration of BAPTA preequilibrated in the fiber and not by the relative size of the force response found upon fiber lysing or other assumptions made. This is because BAPTA has a comparatively high affinity for Ca2+ (affinity constant 5 x 106 M1) and was present at a relatively high concentration (0.6 to 1.3 mM). Thus, if any force at all was produced during the lysing procedure, the BAPTA contained in the fiber space had to be at least 84% occupied with Ca2+, and if the force was not maximal, BAPTA could not have been more than
93% occupied with Ca2+ (see Fig. 2 in Ref. 28).
SR Ca2+ content and Ca2+ release.
The total amount of Ca2+ in the SR of the rat EDL fibers under control conditions in the present study was estimated to be 1.16 mM (Fig. 7), which is similar to that found in previous studies (
1.01.15 mM after taking account of the Ca2+ present in the sealed T-system; Refs. 15, 28); all amounts are expressed relative to total fiber volume. Most important, the SR lysing procedure further showed that the total amount of Ca2+ remaining in the SR after the Pi exposure was also in fact
1.16 mM. Hence, the Pi exposure did not cause any appreciable net loss of total Ca2+ from within the SR under the conditions used.
It was demonstrated that the content assay did indeed detect the reduction when the SR was purposely depleted of some Ca2+ (see also Ref. 28). The total amount of Ca2+ remaining in the SR after the partial depletion protocol was 0.75 mM. This
35% reduction in total SR Ca2+ resulted in
50% reduction in tetanic peak force, a response similar to that found after 2-min exposure to 30 mM Pi. This finding is strongly suggestive that the Pi exposure reduces the amount of Ca2+ available for release during tetanus by a similar amount, that is, by
35%. That this amount is substantially larger than that observed when Ca2+ is released with caffeine/low [Mg2+] (
20% reduction; see above) might simply reflect the fact that little Ca2+ could be become available by CaPi dissolution during the course of the tetanic stimulation, which lasted only 0.4 s. It is also possible that the presence of some CaPi within the SR causes a proportionately larger reduction in AP-induced Ca2+ release than that due solely to the reduction in free [Ca2+] in the SR, such as might occur if the precipitate interfered with the rapid unloading of Ca2+ from calsequestrin or under conditions near the SR luminal end of Ca2+ release channel. In this regard, it is interesting to note that the tetanic response after exposure to 30 mM Pi commonly did not recover fully even after additional Ca2+ loading of the SR (see RESULTS).
The amount of Ca2+ released by AP stimulation nevertheless was generally in good accord with what could be expected from the reduction in amount of SR Ca2+. It was previously found that when the SR contained only 0.75 mM total releasable Ca2+, the amount released by a single AP stimulation (i.e., in a twitch) was decreased to
165 µM (Fig. 12 in Ref. 31), which was
30% less than that released (230 µM) at the normal endogenous SR Ca2+ content (
1.1 mM) (all values expressed per liter of fiber volume). When the amount of Ca2+ released by an AP is reduced by 20%, the observed twitch force decreases by
50% (11) and thus the finding reported herein that the twitch response after exposure to 30 mM Pi was decreased to
21% of the control level (see Table 2) fits well with the previous findings. When a tetanic response was triggered with a 50-Hz train of APs for 400 ms, the peak force after Pi exposure (or partial SR depletion) reached
50% of maximum force (Fig. 6 and Table 2), only slightly higher than that for a single twitch with normal SR loading conditions. This relative reduction in peak force and rate of force production of the tetanic response in these circumstances highlights how the rapid Ca2+ release induced by the first AP in the train depresses the release by the following APs (31) and also that the SR Ca2+ pump resequesters a substantial proportion of the released Ca2+ simultaneously during the release. Important, too, is that the results demonstrate that the SR of a fast-twitch fiber must be loaded at close to its normal endogenous level, which is about fourfold that needed to saturate all Ca2+ binding sites on TnC (15, 31) for the fiber to generate maximum tetanic force.
Relevance of CaPi formation to muscle fatigue.
The presence of high [Pi] in the cytoplasm not only reduces the ability of the contractile apparatus to develop force (4, 26, but see 7) but also evidently readily leads to CaPi precipitation within the SR in skinned fibers as well as a consequent decrease in the amount of SR Ca2+ available for release during tetanus. Therefore, it seems likely that Pi accumulation within the cytoplasm in intact fibers contributes not only to the small decline in force observed during the early stages of metabolic fatigue but also eventually to the much larger decline in force that occurs later due to the failure of SR Ca2+ release (1, 2, 16, 17). The experiments described herein were performed at 24°C, and hence the findings should be referable directly to the multitude of studies that have investigated muscle fatigue in intact fibers and whole muscle preparations at this temperature. It nevertheless is possible that at normal body temperature, Pi does not enter the SR or form a CaPi precipitate as readily as occurs at room temperature; therefore, less CaPi might be produced at a given cytoplasmic [Pi] or time point in vivo than was found in the experiments in the present study. It is also possible that Pi enters the SR more rapidly in the mechanically skinned fiber used herein than occurs in an intact fiber, perhaps because some substance normally present in the cytoplasm in vivo inhibits Pi entry into the SR but is lost when the normal cytoplasm is replaced by artificial solution. However, given the evident ready access of Pi into the SR in skinned fibers and in isolated SR preparations, it seems unlikely that this phenomenon would not occur at least to some extent in intact fibers in vivo, especially given the range of different findings in intact fibers suggestive of some role of CaPi precipitation in fatigue (1, 5, 6, 17, 37, 38).
The solubility product of CaPi in solution in vitro is 6 mM2 (16). If a similar value pertained under the conditions prevailing within the SR and if the free [Ca2+] in the SR were initially
1 mM as usually assumed, the entry of only
6 mM Pi would be required to cause some CaPi to form. If Pi kept entering the SR until it reached the same free concentration as in the cytoplasm (30 mM), then the free [Ca2+] in the SR would be expected to decline to 0.2 mM, or 20% of the initial free level. Assuming that the free [Ca2+] in the SR over this range is approximately proportional to the total bound (mainly to the low-affinity, high-capacity Ca2+ binding protein calsequestrin), the Pi exposure should cause a much greater drop in the amount of releasable Ca2+ (by
80%) than that observed in the present study (by
35% or less). This disparity could result from the initial free [Ca2+] in the SR being substantially lower than presumed (1 mM), or because the conditions inside the SR were less conducive to the formation of CaPi than those in free solution in vitro, or because the free [Pi] did not reach the same level in the SR as it did in the cytoplasm.
Finally, we stress that metabolic muscle fatigue is a multifactorial phenomenon (see Refs. 2, 13, 18). Elevated free [Mg2+], which occurs concomitantly with ATP depletion, has previously been shown to inhibit the Ca2+ release channels and exacerbate the reduction in AP-mediated Ca2+ release caused by low ATP concentration (11). Furthermore, Duke and Steele (9) reported that cytoplasmic Pi has a direct inhibitory effect on the Ca2+ release channels and that this effect is augmented in the presence of elevated [Mg2+]. Such effects would be expected to compound those reported in the present study that were caused by CaPi precipitation within the SR.
Concluding remarks.
In fast-twitch fibers, high-intensity exercise brings about CrP breakdown. As a consequence, the amount of Pi present in the cytoplasm rises to high levels (possibly 30 mM). It is likely that Pi can enter the SR via anion channels (23) or by other means. The consequence of Pi entering the SR is that CaPi forms, causing a reduction in the amount of Ca2+ available for rapid release during tetanus. Thus tetanic force output is reduced as shown herein. This effect seems likely to be involved in the metabolic muscle fatigue observed in isolated preparations studied at room temperature (1) and may contribute to metabolic muscle fatigue in vivo in certain circumstances.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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