Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle

T. L. Dutka, L. Cole, and G. D. Lamb

Department of Zoology, La Trobe University, Melbourne, Victoria, Australia

Submitted 10 June 2005 ; accepted in final form 5 August 2005


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During vigorous exercise, Pi concentration levels within the cytoplasm of fast-twitch muscle fibers may reach ≥30 mM. Cytoplasmic Pi may enter the sarcoplasmic reticulum (SR) and bind to Ca2+ to form a precipitate (CaPi), thus reducing the amount of releasable Ca2+. Using mechanically skinned rat fast-twitch muscle fibers, which retain the normal action potential-mediated Ca2+ release mechanism, we investigated the consequences of Pi exposure on normal excitation-contraction coupling. The total amount of Ca2+ released from the SR by a combined caffeine/low-Mg2+ concentration stimulus was reduced by ~20%, and the initial rate of force development slowed after 2-min exposure to 30 mM Pi (with or without the presence creatine phosphate). Peak (50 Hz) tetanic force was also reduced (by ~25% and ~45% after 10 and 30 mM Pi exposure, respectively). Tetanic force responses produced after 30 mM Pi exposure were nearly identical to those observed in the same fiber after depletion of total SR Ca2+ by ~35%. Ca2+ content assays revealed that the total amount of Ca2+ in the SR was not detectably changed by exposure to 30 mM Pi, indicating that Ca2+ had not leaked from the SR but instead formed a precipitate with the Pi, reducing the amount of available Ca2+ for rapid release. These results suggest that CaPi precipitation that occurs within the SR could contribute to the failure of Ca2+ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca2+ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses.

muscle fatigue; excitation-contraction coupling


CONTRACTION IN VERTEBRATE skeletal muscle is initiated by a burst of action potentials (APs) in a sequence of events known as excitation-contraction (E-C) coupling. Briefly, APs propagate into the transverse tubular (T) system, causing rapid depolarization and activating dihydropyridine receptors (DHPRs), which in turn trigger Ca2+ release through Ca2+ release channels [also known as ryanodine receptors (RyRs)] located in the terminal cisterna of the sarcoplasmic reticulum (SR). Energy for the many processes involved in E-C coupling is derived from the hydrolysis of ATP, thus making ATP a crucial cytoplasmic factor that must be well buffered at high levels (~7–8 mM at rest and expressed per liter of cytoplasmic water). Rapid buffering of ATP during either aerobic or anaerobic activity is achieved predominantly by the enzyme creatine kinase (CK) and the substrate creatine phosphate (CrP), which is present in high concentrations (~40 mM) in muscle (2, 13). As ATP is hydrolyzed to ADP and Pi, CrP donates its phosphate to the ADP to resynthesize ATP and the Pi concentration ([Pi]) within the cytoplasm may reach high levels (≥30 mM).

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., 1–1.5 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 (2–21 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.


    METHODS
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 ABSTRACT
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Preparations. Male Long-Evans hooded rats (~6 mo old) were anesthetized with Fluothane (2% vol/vol) in a glass chamber and killed by asphyxiation in accordance with the guidelines of the La Trobe University Animal Ethics Committee and with the approval of that committee. Both extensor digitorum longus (EDL) muscles were swiftly excised and immediately pinned at their resting length under paraffin oil (Ajax Chemicals, Sydney, Australia) in a petri dish lined with Sylgard 184 (Dow Corning, Midland, MI). When the muscles were not in use, they were kept cool (~10°C) on an ice pack. Individual muscle fibers were mechanically skinned with fine forceps, and a suitable segment (~3-mm length, 30- to 50-µm diameter) was attached to a force transducer (AME801, resonance frequency >2 kHz; SensoNor, Horten, Norway) and then stretched to 120% of the resting length. The mounted skinned fiber segment was then transferred to a Perspex well containing 2 ml of the standard K+-hexamethylenediamine-N,N,N',N'-tetraacetate (K+-HDTA) solution (see below) with 75 µM total EGTA for 2 min to replace all of the in vivo diffusible cytoplasmic constituents with the experimental solutions (18, 19). All experiments were performed at ~24°C, data are expressed as mean ± SE, and statistical significance was set at P < 0.05 using Student's t-test (paired or unpaired as appropriate).

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 M–1 for ATP, 8 M–1 for HDTA, and 15 M–1 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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Fig. 4. Effect of 30 mM Pi exposure on transverse tubular (T) system depolarization-induced force responses of a skinned EDL fiber. Depolarization (Depol)-induced force responses were elicited by exchanging the standard K+-hexamethylenediamine-N,N,N',N'-tetraacetate (K+-HDTA) bathing (control) solution with Na+-HDTA, and the fiber was repolarized by being returned to the control solution for 60 s. The peak amplitude of the first response after the 30 mM Pi exposure was substantially reduced (see Table 2 for mean data), and subsequent responses progressively increased, consistent with a modest rate of SR Ca2+ loading occurring when the fiber was left in the control solution. The numbers above the force responses indicate the number of responses after 30 mM Pi exposure.

 
Maximum Ca2+-activated force was elicited in fibers when exposed to a solution that was similar to the K+-HDTA solution, but with all 50 mM HDTA replaced with 50 mM Ca2+-EGTA (pCa 4.5), and the total Mg2+ was adjusted to produce 1 mM free [Mg2+] (35). Force returned to the baseline level when the fiber was exposed to a similar solution with 50 mM free EGTA (pCa >10). Another maximum Ca2+-activated force solution was used in the Triton X-100 oil experiments (see below) to ensure the closest comparison with the intracellular solution present in the fiber during SR lysis (28). This maximum Ca2+-activating solution contained 45 mM HDTA and a total Ca2+-EGTA concentration of only 5 mM (~pCa 4.5). There was no significant difference in the level of force produced by either maximum force-activating solution (data not shown).

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 M–1 (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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Fig. 1. Exposure to 30 mM Pi [with 10 mM creatine phosphate (CrP)] for 2 min under paraffin oil reduces the amount of Ca2+ rapidly released from the sarcoplasmic reticulum (SR) by the caffeine/low Mg2+ concentration ([Mg2+]) solution in a mechanically skinned extensor digitorum longus (EDL) fiber. The force response after Pi exposure displayed 1) an appreciable delay in initial force development, 2) a reduced rate of force production, and 3) a smaller area (see Table 1 for mean data). Maximum Ca2+-activated force {–log10 Ca2+ concentration ([Ca2+]) (pCa) of 4.5} was ~0.7 mN (data not shown). Note that Pi was washed out of the fiber for 30 s before the response was tested. In the bracketing control responses, the fiber was treated identically, except that Pi was absent. The SR was reloaded with Ca2+ for 30 s before each response and was fully depleted by exposure to the caffeine/low-[Mg2+] solution for 1 min.

 
T-system depolarization-induced force responses. DHPR activation was achieved by using transverse electrical field stimulation (11, 29, 31, 32) to elicit APs or by ionic (Na+) substitution (see 18, 19, 22) to depolarize the T-system directly. For electrical field stimulation, the fiber was placed midway between two platinum wire electrodes in a small stimulating bath (~130 µl) containing the K+-HDTA standard solution (control). Application of a single 75 V·cm–1, 2-ms duration pulse (delivered by an in-house stimulator) elicited a single AP within the sealed T-system, which then activated the DHPRs and triggered SR Ca2+ release through the RyRs, producing twitch responses (Fig. 2B). Tetanic force responses were generated by applying a train of 20 identical pulses (as described above) at 50 Hz. Na+ depolarization-induced, twitch, and tetanic force responses were elicited before and after Pi exposure, and these responses were then amplified and recorded simultaneously on both a chart recorder (Linear) and a computer with PowerLab series 4/20 software (Chart 5).



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Fig. 2. Effect of 10 mM and 30 mM Pi exposure on tetanic force responses in two typical EDL fibers. A: total of 2 min under paraffin oil in control solution (control treatment) had no effect on peak force (third response) compared with the pretreatment level (first two tetani), whereas 2-min exposure to 10 mM Pi under oil reduced peak tetanic force by ~15% (fourth response). The tetanic force response recovered when the fiber was left in the control solution for several minutes (stimulation every 1 min). B: twitch and tetanic force responses were unchanged before and after exposure to the control solution under oil but were markedly reduced after exposure to 30 mM Pi under oil. Note that after 30 mM Pi exposure, tetani did not fully recover to the levels observed before Pi exposure. Maximum Ca2+-activated force was determined by exposure to 50 mM Ca2+-EGTA (pCa 4.5) shown at slow time scale.

 
Measurement of total SR Ca2+ content. After first investigating the effects of SR Ca2+ depletion or Pi exposure on tetanic force, the amount of Ca2+ remaining in the SR after the given treatment was ascertained. This measurement was performed by preequilibrating the skinned fiber for 20 s in a known BAPTA concentration ([BAPTA], a fast Ca2+ buffer with well-known binding properties) added to the control solution and then lysing all membranous compartments within the fiber by exposing the fiber to an emulsion of Triton X-100 (10% vol/vol) in paraffin oil (TX oil) (see Fig. 6). This procedure has been described comprehensively elsewhere (15, 28). Briefly, the Ca2+ released from compartments upon lysing rapidly binds to the known amount of BAPTA within the fiber and to other sites, predominantly troponin C (TnC). If force is produced but is not maximal (i.e., saturated), a relatively accurate estimate of the total Ca2+ liberated can be determined in absolute terms on the basis of the known Ca2+ binding sites and affinities (see Ref. 28 for details). In these experiments, maximum force was ascertained first with the fiber in open solution (maximum Ca2+-activated force) and then when moved back into the TX oil emulsion (e.g., closed system). This step was necessary to check whether the maximum force produced by the fiber was the same in the TX oil environment. As found previously (28), maximum Ca2+-activated force typically decreased during prolonged activation, particularly when the fiber was moved to the TX oil emulsion, although in some fibers it increased to a greater level in TX oil (see, e.g., Fig. 6B).



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Fig. 6. Tetanic force responses in the same fiber before and after depleting SR Ca2+ by stimulating in 2 mM free [BAPTA] and 30 mM Pi exposure. A: after depletion in BAPTA, the SR was able to slowly reload Ca2+ from the control solution (pCa 6.9, 75 µM EGTA) between tetani (1-min intervals), and additional brief (5-s) loading (pCa 6.7, 1 mM total EGTA) was used to restore SR Ca2+ rapidly to the initial level. The fiber was exposed to 30 mM Pi (2 min) and then returned to the control solution in which all force responses were elicited. B: tetanic force responses expanded from A under the following conditions: control (1), SR depleted (2), and after 30 mM Pi exposure (3). Responses after depletion and after 30 mM Pi exposure display similar peaks, rates of force development, and force fading during stimulation.

 
Amounts of Ca2+ were expressed millimolar concentrations per liter of total fiber volume (rather than cytoplasmic water), in keeping with previous studies (15, 16, 28). The total amount of Ca2+ remaining in the SR after each treatment was calculated (see Ref. 28) using the following steps: 1) the free [BAPTA] within the fiber was taken as 1.06x [BAPTA] in solution to account for the initial fiber swelling when placed in the bathing solution; 2) this number was then multiplied by the fractional amount of BAPTA occupied by Ca2+ as determined from the amount of force (relative to maximum force) achieved upon lysis (i.e., 50% force corresponded to 88% BAPTA with Ca2+ bound; see Ref. 28); 3) 0.03 mM was deducted to take into account the contaminating Ca2+ already bound to BAPTA in the bathing solution; 4) the amount of Ca2+ bound to TnC as indicated by the force response was added (see Ref. 31 for estimates); 5) the amount of Ca2+ occupying binding sites other than BAPTA or TnC (~0.3 mM) (see Ref. 15) was added; and 6) the estimated Ca2+ present in the T-system (~0.23 mM) (28) was deducted.

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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Effect of Pi exposure on SR Ca2+ handling. In the first set of experiments, the normal AP-mediated Ca2+ release mechanism was bypassed by using a caffeine/low-[Mg2+] stimulus to open the RyRs directly, triggering Ca2+ release from the SR and force development. The time integral of the force response elicited by the full-release solution is indicative of the amount of Ca2+ released from the SR (21). Exposure to 30 mM Pi for 2 min (followed by 30-s washout) reduced the time integral of the force response produced by the full-release solution to ~80%, and the rate of force development to ~45%, of the bracketing control level (see Fig. 1 and Table 1). Neither the reduction in the time integral nor the slowing in force development caused by 30 mM Pi exposure was significantly different with or without CrP present (Table 1).


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Table 1. Summarized data of the effect 30 mM Pi exposure on caffeine/low-[Mg2+]-induced force responses

 
The duration of the 30 mM Pi exposure was varied to ascertain whether the reduction in the amount of releasable Ca2+ elicited by the full-release solution had reached a steady state. The degree of reduction in the time integral and the rate of force development caused by 30 mM Pi was not significantly different after either a 2-min or longer (4- to 5-min) exposure (Table 1), indicating that the reduction in releasable SR Ca2+ had reached an approximately steady state within 2 min of exposure.

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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Table 2. Summarized data of the effect of 2-min Pi exposure and partial SR Ca2+ depletion on AP-induced and depolarization-induced force responses

 


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Fig. 3. Summary of the effect of control oil treatment or either 10 or 30 mM Pi treatment for 2 min on peak tetanic force (as described in Fig. 2). The mean ± SE peak force of each treatment was expressed relative to that before treatment. Results were 97.5 ± 3.2% (control oil), 78.6 ± 2.4% (10 mM Pi), and 54.9 ± 4.0% (30 mM Pi). Both 10 and 30 mM Pi exposures significantly reduced peak tetanic force compared with the pretreatment control level. *P < 0.05; Student's paired t-test. Furthermore, exposure to 30 mM Pi reduced peak tetanic force significantly more than did 10 mM Pi exposure. #P < 0.05; Student's unpaired t-test. Control treatment was performed in a subset (n = 7) of the 18 fibers used for 10 or 30 mM Pi treatment.

 
Fibers exposed to only 10 mM Pi reloaded enough Ca2+ slowly from the control solution (pCa 6.9, 75 µM EGTA) to return eventually (typically ~3 min) to the pretreatment force level; however, after 30 mM Pi exposure, tetanic force usually did not return fully to the pretreatment level (see Fig. 2B). In these cases, to facilitate the return of force to the pretreatment level, the SR of the fiber was loaded more rapidly with a load solution (1 mM total EGTA, pCa 6.7, for 5 s). However, even with this loading regime, force never returned to the pretreatment level in some instances, possibly because not all of the CaPi in the SR had redissolved (see DISCUSSION).

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., ~50–60% 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 (RR10–90), and in fall rate from 90 to 10% peak force (FR90–10) (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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Fig. 5. Typical examples of fiber lysis in Triton X-100 paraffin oil emulsion TX oil after various treatments. Each skinned fiber was preequilibrated in a particular BAPTA concentration ([BAPTA]) (1.2 mM in A and C, 0.7 mM in B) and then placed into TX oil emulsion to liberate all Ca2+ stored within the skinned fiber. Control tetani (A), SR depletion (B), or 30 mM Pi exposure (C) was examined in different fibers. Upon removing the fiber from the TX oil emulsion, force increased when the fiber was in the air being transferred between solutions. Maximum force was ascertained first with the fiber in open solution (maximum Ca2+-activated force) and then when moved back to the TX oil emulsion (see METHODS). Time scale, 1 s for tetani and 20 s during lysing and maximum Ca2+-activated force.

 


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Fig. 7. Summary of means ± SE calculated for total SR Ca2+ remaining in fiber after various treatments (as shown in Fig. 5). {bullet}, fibers in which the SR Ca2+ was ascertained accurately; {circ}, fibers in which either too much (downward arrows) or too little (upward arrows) BAPTA was present, and therefore only upper or lower estimates, respectively, of SR Ca2+ content could be rendered. The calculated amount of SR Ca2+ in control fibers was no different from those exposed to 30 mM Pi, whereas the depleted fibers had appreciably less Ca2+ remaining in the SR (see text for mean data).

 
Using the same procedures as were used for Pi, we exposed three other fibers to 5 or 10 mM oxalate under oil for a total of 2 min, and each displayed greatly reduced tetanic force responses 30 s after removal of the cytoplasmic oxalate (peak force reduced to 9% and 34% of control level after 5 mM oxalate and to 8% after 10 mM oxalate). The fibers were then kept in open control solution (i.e., not under oil, pCa 6.9, 75 µM EGTA) for between 2 and 4 min, in which circumstances the SR could slowly load more Ca2+ (and perhaps oxalate might be washed out of the SR), to follow the tetanic force response until it recovered to ~40% of the original control level. The SR was then lysed in TX oil, and the total SR Ca2+ was found to be ≥1.3 mM in each case.


    DISCUSSION
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 ABSTRACT
 METHODS
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Twitch and tetanic force responses elicited by the normal E-C coupling mechanism were reduced in a concentration-dependent manner after exposing mechanically skinned EDL fibers of the rat to high cytoplasmic [Pi] (Figs. 2 and 3 and Table 2). The decreases in peak force and in the rate of force development observed after Pi exposure were evidently due to a reduction in the amount of Ca2+ released from the SR, because all Pi had been washed out of the intracellular space before the fiber's response was tested, and thus Pi could not have had any direct inhibitory effect on the contractile apparatus. Similarly, it is evident that the reduction in Ca2+ release could not have been due to any inhibitory effect of cytoplasmic Pi directly on the Ca2+ release channels. A reduction in Ca2+ release after Pi exposure was observed when each of the three different methods of triggering Ca2+ release from the SR was used, with these involving either T-system depolarization (i.e., Na+ substitution and AP-mediated) or direct activation of the Ca2+ release channels by stimulation with caffeine/low-[Mg2+] solution (Figs. 1, 2, and 4). Thus the reduction in Ca2+ release caused by Pi exposure evidently was due primarily to a reduction in the amount of Ca2+ that could be released rapidly from the SR and not to the failure of AP propagation or depolarization of the T-system, consistent with the reduced release being the result of CaPi precipitation within the SR. Exposure to oxalate (5 or 10 mM), which can also enter the SR and precipitate with Ca2+ (9, 10), also caused a reduction in AP-induced Ca2+ release, further highlighting how Ca2+ precipitation within the SR adversely affects normal E-C coupling.

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 M–1) 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 (~1.0–1.15 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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This research was supported by National Health and Medical Research Council of Australia Grant 280623.


    ACKNOWLEDGMENTS
 
We are grateful to Prof. D. George Stephenson for helpful comments and discussions during this study and to Aida Yousef for technical assistance. We also express our gratitude to Brian Taylor for expertise in designing and building the in-house stimulator used in these experiments.


    FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Dutka, Dept. of Zoology, La Trobe Univ., Melbourne 3086, Victoria, Australia (e-mail: t.dutka{at}latrobe.edu.au)

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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