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Am J Physiol Cell Physiol 294: C97-C105, 2008. First published October 24, 2007; doi:10.1152/ajpcell.00155.2007
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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Comparison of the effects of inorganic phosphate on caffeine-induced Ca2+ release in fast- and slow-twitch mammalian skeletal muscle

Giuseppe S. Posterino and Stacey L. Dunn

Discipline of Physiology, School of Molecular and Biomedical Sciences, The University of Adelaide, Adelaide, South Australia, Australia

Submitted 12 April 2007 ; accepted in final form 22 October 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We compared the effects of 50 mM Pi on caffeine-induced Ca2+ release in mechanically skinned fast-twitch (FT) and slow-twitch (ST) skeletal muscle fibers of the rat. The time integral (area) of the caffeine response was reduced by ~57% (FT) and ~27% (ST) after 30 s of exposure to 50 mM Pi in either the presence or absence of creatine phosphate (to buffer ADP). Differences in the sarcoplasmic reticulum (SR) Ca2+ content between FT and ST fibers [~40% vs. 100% SR Ca2+ content (pCa 6.7), respectively] did not contribute to the different effects of Pi observed; underloading the SR of ST fibers so that the SR Ca2+ content approximated that of FT fibers resulted in an even smaller (~21%), but not significant, reduction in caffeine-induced Ca2+ release by Pi. These observed differences between FT and ST fibers could arise from fiber-type differences in the ability of the SR to accumulate Ca2+-Pi precipitate. To test this, fibers were Ca2+ loaded in the presence of 50 mM Pi. In FT fibers, the maximum SR Ca2+ content (pCa 6.7) was subsequently increased by up to 13 times of that achieved when loading for 2 min in the absence of Pi. In ST fibers, the SR Ca2+ content was only doubled. These data show that Ca2+ release in ST fibers was less affected by Pi than FT fibers, and this may be due to a reduced capacity of ST SR to accumulate Ca2+-Pi precipitate. This may account, in part, for the fatigue-resistant nature of ST fibers.

fatigue; Ca2+ precipitation; excitation-contraction coupling

IN VERTEBRATE SKELETAL MUSCLE, the generation of an action potential at the neuromuscular junction ultimately leads to muscle contraction. There are several important intermediary steps linking these two events, which together describe a process that is broadly termed excitation-contraction coupling. More specifically, the action potential rapidly spreads across the surface membrane of a muscle fiber and into the transverse tubular system to elicit a depolarization that is subsequently sensed by specialized voltage-dependent Ca2+ channels termed the dihydropyridine receptors. The dihydropyridine receptors, in turn, activate closely associated Ca2+ channels (termed the ryanodine receptors) located in the terminal cisternae of the sarcoplasmic reticulum (SR). Calcium is subsequently released into the cytoplasm, where it then binds to the Ca2+ regulatory system and initiates contraction (28).

Muscle contraction is an energy-intensive process. The basic energy currency of a muscle cell is ATP, which is formed intracellularly from the degradation of several fuels (e.g., glucose) (19). The importance of ATP to the many steps in excitation-contraction coupling ensures that the cytoplasmic ATP concentration ([ATP]) remains relatively well buffered at high concentrations (~7–8 mM per liter of cytoplasmic water) (1). This is accomplished by the generation of ATP through both aerobic and anaerobic processes and by the rapid hydrolysis of creatine phosphate (CP) (19). However, with repeated and prolonged activity, skeletal muscle force output declines with time. This phenomenon is termed fatigue (12).

Fatigue is more profound in the fast-twitch (FT) fibers than in the slow-twitch (ST) fibers, which leads to the latter fiber type being often referred to as fatigue resistant. The difference in the fatigability of these two fiber types seemingly arises from the different capacity of these fiber types to maintain the ATP pool. In FT fibers, during prolonged high-intensity activity, ATP synthesis can be limited. Consequently, because ATP catabolism exceeds synthesis, a rapid and large increase in the concentrations of ATP metabolites, such as ADP (~1–2 mM) and Pi (30–50 mM), occurs. The increase in Pi has been strongly implicated in metabolic fatigue in FT fibers (15).

In FT fibers, Pi has been shown to directly inhibit maximum Ca2+-activated force and the Ca2+ sensitivity of the contractile apparatus (6, 29). Pi has also been implicated in the sudden rapid decline in Ca2+ release from the SR seen in the later stages of metabolic fatigue. Fryer et al. (15) first suggested that Pi enters the SR, whereby it precipitates with Ca2+ to limit the free Ca2+ available for release. Posterino and Fryer (33) later showed that the entry of Pi into the SR is seemingly passive (ATP independent), and it is now thought that Pi enters the SR through a small conductance chloride channel (23). Other studies using single intact fibers further confirmed the importance of Pi in the failure of Ca2+ release and fatigue in FT fibers (21, 39). However, previous studies by Duke and Steele (7, 8) suggested that a failure in ADP buffering may play an important role in the apparent Pi-induced failure of Ca2+ release seen in past skinned fiber studies. Many previous skinned fiber studies examined Pi in the absence of CP and thus seemingly failed to adequately buffer ADP, which could potentially reverse the flow of Ca2+ through the SR Ca2+-ATPase. Recently, however, Dutka et al. (11) showed that Pi could reduce the available Ca2+ in the SR for release in FT even when ADP was well buffered with CP.

Although ST fibers have a higher aerobic capacity than FT fibers, during repeated and prolonged activity, the cytoplasmic Pi concentration ([Pi]) can rise to similar levels as that seen in FT fibers (20–30 mM) (22). However, there has been no previous examination of the effects of Pi on Ca2+ release from the SR in ST fibers, and, therefore, its role in fatigue in these fiber types is not known. A recent study by Macdonald and Stephenson (27) showed that Ca2+ handling in ST fibers is significantly less sensitive to rises in cytoplasmic ADP than FT fibers, which suggests that the underlying fatigue resistance of these fiber types may be a reduced sensitivity to ADP, among other metabolites thought to be involved in fatigue.

Therefore, the aim of the present study was to compare the effects of Pi in both FT and ST skeletal muscle fibers to better understand the mechanisms that underlie the differences in fatigability of these two fiber types. We used mechanically skinned fibers in the present study because this preparation allows both the SR Ca2+ content and the cytoplasmic [Pi] to be tightly controlled. We examined the effects of Pi in both fiber types, in the presence and absence of CP, to identify any possible effects of ADP on Ca2+ leak from the SR due to possible pump reversal. Furthermore, because skinned fibers are effectively an open system (due to the removal of the surface membrane), the SR Ca2+ content may fluctuate because of Ca2+ movements into and out of the fiber that are independent of any effect of Pi. Consequently, this may influence observed results. Therefore, we also examined the effects of Pi in a closed system, using paraffin oil as a barrier to limit exogenous Ca2+ movements and to ensure that the SR Ca2+ content remained constant, with the exception of any effect of Pi. We show for the first time that even at elevated Pi levels that approximate the most severe state found in FT fibers, SR Ca2+ release following Pi exposure was reduced more substantially (by ~2-fold) in FT than ST fibers, which indicates that ST fibers are more resistant to elevated Pi levels. Furthermore, this difference in the effects of Pi on Ca2+ release appears to be related to a difference in the ability to accumulate Ca2+-Pi precipitate in these fiber types. Preliminary data have been previously reported in abstract form (32).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Single Fiber Isolation The use of animals and all procedures in the present study were approved by the University of Adelaide Animal Ethics Committee and conformed to the Guidelines for the Care and Use of Experimental Animals as described by the National Health and Medical Research Council of Australia. Male Hooded-Wistar rats (4–6 mo of age) were killed by asphyxiation with CO2. Whole extensor digitorum longus and soleus muscles were then dissected from the hind limb and pinned under paraffin oil at resting length. With the use of a dissecting microscope (Leica), single muscle fibers were isolated and then mechanically skinned with a pair of fine jewelers forceps (Dumont no. 5). One end of the fiber was subsequently tied with fine suture silk (Deknatel 10/0) to a force transducer (AE-800, Memscap; resonance frequency with stainless steel pin attached of >1 kHz), and the other end of the fiber was clamped with a fixed pair of forceps. The resting length of the fiber was then measured before the fiber was stretched a further 20% [to bring the sarcomere length within the optimal force-producing range (~3.1–3.2 µM)]. The diameter of the fiber was then measured. The skinned fiber was subsequently immersed for 2 min in potassium hexamethylene-diamine-tetraacetic acid (K-HDTA) solution, which mimics the normal cytoplasmic milieu (see below).

Solutions All chemicals were purchased from Sigma. The composition of skinned fiber solutions used is shown in Table 1. The K-HDTA solution was used to generate several solution types listed in Table 1. To maintain close to normal ionic strength and osmolality in solutions containing phosphate, HDTA was replaced with Pi at a ratio of 1:1.3. Osmolality was measured with a Roebling Automatic osmometer kindly lent to us by Professor Richard Ivell. The osmolality of the standard K-HDTA solution was 295 ± 5 mosmol/kgH2O, whereas 50 mM Pi solutions varied by no more than ±10 mosmol/kgH2O.


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  		Table 1. Composition of skinned fiber solutions

 
Experimental Protocols Estimation of releasable SR Ca2+. A qualitative assessment of the amount of releasable Ca2+ from the SR of skinned fibers was determined by examining changes in the time integral (area) of the caffeine-induced force response elicited by exposure to the caffeine solution (see Table 1). The area of the caffeine-induced force transient has been previously shown to be directly proportional to the SR Ca2+ content over a range of SR Ca2+ concentration ([Ca2+]) (33, 37). Figure 1 shows the relationship between the area of the caffeine-induced force response versus the length of time fibers were loaded with Ca2+, in both FT and ST fibers. We used a correction method of Macdonald and Stephenson (26) to determine the force transient that would otherwise normally fall below a detectable range due to the presence of 1 mM EGTA in the caffeine solution to ascertain the 0 time point. In this instance, no correction was needed for ST fibers, whereas all responses in FT fibers were corrected by adding 10% to the measured force transient consistent with previous work (37). It can be seen that there is a relatively linear relationship between the area versus the load time for both FT and ST fibers over a range of load times. In both fiber types, the area was saturated by 2 min of continuous loading at pCa 6.7, with longer load times (data not shown) having no additional effect on the area in either fiber type.


Figure 1
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  		Fig. 1. Relationship between load time and the area of the caffeine-induced force response in fast-twitch (FT) and slow-twitch (ST) fibers. Both FT (n = 3) and ST (n = 4) fibers were loaded with Ca2+ (load solution, pCa 6.7; see METHODS) for differing times until the sarcoplasmic reticulum (SR) was saturated with Ca2+. The area of the caffeine-induced force response after each load period was normalized to the maximum load time (120 s). Areas were corrected by using methods described by Macdonald and Stephenson (26). Curves were fitted to the data by using a single exponential (parameters: maximum = 100 and minimum = 0).

 
Initially, freshly skinned fibers were equilibrated in a K-HDTA solution (0.025 mM EGTA) for 2 min and were then briefly equilibrated in the wash solution (1 mM EGTA, 6 s) before the SR was emptied of Ca2+ by using the caffeine solution (30 mM caffeine, 0.02 mM free Mg2+, 1 mM EGTA, 1 min; see Table 1). The caffeine solution has been previously shown to rapidly empty the SR of all stored Ca2+ (16). The force response recorded initially (after the 2-min equilibration described above) was indicative of the endogenous SR Ca2+ content of the fiber. However, in many instances, neither FT nor ST muscle fibers provided a measurable endogenous force response, and possible reasons for this have been detailed by Trinh and Lamb (37). We know that the relative endogenous SR Ca2+ content of FT and ST fibers equates to an SR that is approximately one-third full and maximally loaded, respectively (16). Consequently, with the use of previously determined maximum Ca2+ content for each fiber type (see Fig. 1), fibers were washed for 1 min in the wash solution (see Table 1) and then reloaded with Ca2+ by using the load solution (pCa 6.7, 1 mM total EGTA; see Table 1) for 30 s in FT fibers and for 2 min in ST fibers to mimic the different endogenous SR capacities; for ST fibers, the SR was essentially fully loaded after ~ 30–40 s (see Fig. 1), but we extended this to 2 min to ensure uniform loads between all fibers. Fibers were then washed briefly in the wash solution (10 s) before the amount of Ca2+ loaded into the SR was redetermined by exposure to the caffeine solution. The area of the force response elicited was a measure of the total loaded Ca2+ after the 30-s and 2-min loads indicated above. The peak force response to caffeine as a percentage of the maximum Ca2+ activated force was 53 ± 6% (n = 19) and 64 ± 5% (n = 29) for FT and ST fibers, respectively. This indicated that the force responses to the caffeine solution were submaximal.

Phosphate experiments. After the SR of single fibers was reloaded with Ca2+ to reestablish the near endogenous Ca2+ content (see above), fibers were then exposed to a wash solution (1 total EGTA) for 10 s (to stop further Ca2+ uptake) and then a further 16–36 s in the same solution before the SR was depleted of Ca2+ by using the caffeine solution. The caffeine-induced force response subsequently elicited was indicative of the Ca2+ content remaining in the fiber after equilibration for various times in the wash solution that contained 1 mM EGTA. Under these conditions, the SR is expected to leak (lose) Ca2+. After a 30-s equilibration (maximum equilibration period), we estimated this to be ~30% of the initial loaded Ca2+ content in FT fibers and 40% of the loaded Ca2+ in ST fibers. This is similar to previously reported estimates by others (2, 34). This response formed the control response to which subsequent caffeine-induced force responses following Pi exposure were compared. Control responses were repeated twice to ensure reproducibility. Subsequently, fibers were again reloaded with Ca2+ in the load solution, immersed briefly (6 s) in the wash solution (to stop Ca2+ uptake), and then exposed for 10–30 s in a solution containing either 50 mM Pi and 0 mM CP (50Pi/0CP solution) or 50 mM Pi and 10 CP (50Pi/10CP solution) (see Table 1). Fibers were then washed in a wash solution for 10 s to completely remove Pi from the fiber before being exposed to the caffeine solution to ascertain the amount of releasable Ca2+ present within the SR. Fibers were then washed for 1 min (wash solution), and the load-deplete protocol was repeated again in the absence of Pi exposure. The area of the caffeine-induced force response elicited after Pi treatment was compared with the average control responses obtained before and after Pi treatment. Providing that the response to caffeine for bracketed controls remained relatively reproducible, the effect 50 mM Pi exposure in both the presence and absence of CP was examined in the same fiber.

Because the SR loses Ca2+ when allowed to leak in the wash solution for various times, the endogenous content may not be well represented (fibers end up underloaded). Nevertheless, it was still expected that the SR contained a proportionately larger Ca2+ store in ST fibers than FT fibers after the load leak protocol described above, consistent with the differences in SR contents between the fiber types. Therefore, in some experiments, to ensure that the SR contents remained close to their original endogenous levels, both FT and ST fibers were equilibrated in paraffin oil (20 s) following a 10 s exposure to the wash solution (control conditions) or equivalent 50Pi/0CP solution, to limit the amount of Ca2+ leaked from the SR over this time (30 s). Under control conditions, there was <10% reduction in the SR Ca2+ content, which indicates that the paraffin oil acted as an effective barrier to the movements of Ca2+ into and out of the fiber.

In other experiments (see RESULTS), we were also interested in examining the effect of approximately matching the SR Ca2+ contents of FT and ST fibers on the effect of a 30-s exposure to Pi on Ca2+ release. In this instance, ST fibers were Ca2+ loaded for only 20 s and 30 s to bring the proportional SR Ca2+ content closer to that of FT fibers. From Fig. 1, it can be seen that even after a 20-s load, the SR Ca2+ content of ST fibers was still large (~50% of its maximum Ca2+ content). Loading for shorter periods was deemed too inconsistent and was subsequently not done. Nevertheless, this load period reduced the total SR Ca2+ content and brought fibers closer to the range typically seen in FT fibers. Experiments were otherwise conducted as described above.

Pi-assisted SR Ca2+ loading. One way to indirectly determine the total capacity of the SR to store Ca2+-Pi (and or Ca2+) in both FT and ST fibers is to examine the ability of the SR to load Ca2+ in the presence of Pi. If the SR is permeable to Pi, then Ca2+ can be continuously loaded into the SR indefinitely until either the SR structure physically impedes further loading or the SR ruptures. This is possible because Ca2+ and Pi seemingly accumulate in the SR with a 1:1 stoichiometry (8, 15). Therefore, as Pi enters the SR, the free Ca2+ in the SR lumen falls; thus the SR appears empty, and Ca2+ uptake continues. The subsequent area of the response to caffeine should then give a reasonable indication of the amount of Ca2+-Pi stored. However, one would expect this to be an underestimate given that not all of the stored Ca2+ precipitated with Pi would be liberated from the SR over the course of the caffeine stimulus, thus limiting the area of the caffeine response.

Skinned fibers from both FT and ST muscles were initially Ca2+ loaded in the load solution for 2 min to saturate the SR with Ca2+. Following a brief wash (10 s) in the wash solution, the total SR Ca2+ content was determined by subsequently emptying the SR with the caffeine solution. Fibers were then washed for 1 min in the wash solution and were then reloaded with Ca2+ for 2–5 min in a modified 50Pi/10CP solution containing 1 mM total EGTA, pCa 6.7 (similar to the load solution). The fiber was then washed for 10 s before the SR was again emptied of Ca2+ with the caffeine solution. The area of the response to caffeine was then normalized to the area of the caffeine-induced force response obtained after 2 min loading in the absence of Pi.

Muscle fiber typing. The soleus and extensor digitorum longus muscles predominately contain one major fiber type (slow and fast, respectively). However, there is a small proportion (~10%) of the opposite fiber type in each muscle group. To confirm that selected skinned fibers were either of the FT or ST type, we exposed fibers to a strontium-based EGTA solution (pSr 5.4; Table 1) at the end of each experiment. On the basis of the ~10-fold difference in the sensitivity of FT and ST muscle fibers to Sr2+ activation, we were able to confirm the fiber type of each fiber tested (31).

Statistical Analysis All data are presented as means ± SE unless otherwise indicated. Data were compared by using either a one-way or two-way ANOVA where appropriate, and data were considered significant if P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of Pi on Caffeine-Induced Ca2+ Release in FT Fibers in the Presence and Absence of CP We previously reported that Pi equilibrated in the absence of CP substantially reduced caffeine-induced Ca2+ release in FT fibers consistent with the formation of a Ca2+-Pi precipitate in the SR (15, 33). However, Duke and Steele (8) had attributed this reduction in caffeine-induced Ca2+ release to a leak of Ca2+ from the SR arising from the absence of CP rather than an effect of Pi on free SR luminal Ca2+ per se. Thus, in the first instance, we were interested in comparing the effects of Pi equilibration in the presence and absence of 10 mM CP. Figure 2 shows a typical force trace obtained in a FT muscle fiber. It can be seen that neither the presence nor absence of CP during equilibration in 50 mM Pi markedly altered the ability of Pi to reduce Ca2+ release and peak force to caffeine exposure. In both instances, the area of the caffeine-induced force response following Pi treatment was greatly reduced (by ~72% and 69% of bracketing controls, respectively).


Figure 2
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  		Fig. 2. Effect of 50 mM Pi in a single skinned FT skeletal muscle fiber in the presence and absence of 10 mM creatine phosphate (CP). From left to right and top to bottom, the trace shows the data obtained in a single FT fiber subjected to the load-release protocol described in METHODS. Following a 30-s Ca2+ load (to approximately match the typical endogenous FT total SR Ca2+ content; see Fig. 1), the fiber was equilibrated for 30 s in either a standard potassium hexamethylene-diamine-tetraacetic acid (K-HDTA) solution, a 50 mM Pi/0 mM CP solution, or a 50 mM Pi/10 mM CP solution, each containing 1 mM total EGTA. The subsequent force responses to the caffeine solution (indicated by solid bars below force trace; slow time scale of 1 s) are indicative of the total releasable Ca2+ content after the appropriate equilibration period (e.g., control and post-Pi treatment). Each exposure to the caffeine solution fully empties the SR of Ca2+ such that subsequent Ca2+ loads result in similar total SR Ca2+ contents. Equilibration for 30 s in a Pi solution in either the absence or presence of CP resulted in a similarly large reduction in the force response to the caffeine solution (compare 2nd to 5th responses), which indicated that the loaded SR Ca2+ was not easily released, as previously described (30). Responses were normalized to the average control force response before and after Pi treatment. Maximum Ca2+-activated force in pCa 4.5 (Max) and strontium (pSr 5.4) exposure are shown at the faster time scale of 0.5 s.

 
Figure 3 shows the mean data for these experiments in FT fibers treated with 50 mM Pi for between 10 s and 30 s. It can be seen that the area of the caffeine-induced force response was greatly reduced even after a 10-s exposure to 50 mM Pi, irrespective of the presence of CP (Fig. 3, A and B). Comparison of the mean areas for FT fibers across all the time points (10 s to 30 s) in the presence and absence of CP showed no significant difference between points (P > 0.05, one-way ANOVA). Furthermore, the rapid time dependence and the extent of the reduction in force after Pi treatment were not different from that previously reported by Posterino and Fryer (33).


Figure 3
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  		Fig. 3. Effect of 50 mM Pi exposure on FT and ST fibers in the absence and presence of CP. Mean area of the caffeine-induced force responses (±SE; n in brackets) for 50 mM Pi/0 mM CP treatments (A) and 50 mM Pi/10 CP treatments (B). Mean data for each data set (A and B) were compared with a two-way ANOVA; for A and B, overall P values were <0.001 and <0.01, respectively. Significance between time points (Bonferroni posttest analysis) is shown: *P < 0.05, **P < 0.01. Y-axis, area expressed as the percentage of the bracketing control responses before and after Pi treatment (see Figs. 1 and 2); x-axis, exposure time to appropriate Pi solution.

 
Effects of Pi on Caffeine-Induced Ca2+ Release in ST Fibers in the Presence and Absence of CP There has been no previous report examining the effects of phosphate on Ca2+ handling in ST muscle. ST muscle is often referred to as fatigue resistant, although the underlying mechanisms for this resistance have not been fully examined. Figure 4 shows a typical raw data trace examining the effects of 50 mM Pi exposure on an ST fiber in the presence and absence of CP. Surprisingly, exposure to Pi for 30 s only depressed the area of the subsequent caffeine-induced force response by ~21% in this fiber (contrast with the effect of Pi observed in the FT fiber in Fig. 2). This indicated that Pi had a much smaller effect on Ca2+ release in ST fibers. Furthermore, the reduction in area was not altered by the presence of 10 mM CP in the Pi equilibration solution (P > 0.05, one-way ANOVA).


Figure 4
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  		Fig. 4. Effect of 50 mM Pi in a single skinned ST skeletal muscle fiber in the presence and absence of CP. From left to right and top to bottom, the trace shows the data obtained in a single ST fiber subjected to the load-release protocol described in METHODS. Following a 2-min Ca2+ load (to fully load the SR), the fiber was equilibrated for 30 s in either a standard K-HDTA solution, a 50 mM Pi/0 mM CP solution, or a 50 mM Pi/10 mM CP solution, each containing 1 mM total EGTA. The subsequent force responses to the caffeine solution (indicated by solid bars below force trace) are indicative of the total releasable Ca2+ content after the appropriate equilibration period (e.g., control and post-Pi treatment). Each exposure to the caffeine solution fully empties the SR of Ca2+ such that subsequent Ca2+ loads result in similar total SR Ca2+ contents. It can be seen that exposure to Pi in either the presence or absence of CP had little effect on the subsequent force response to the caffeine solution (compare 2nd and 5th responses); compare with the larger effect of Pi treatment observed in a single FT fiber in Fig. 2. Responses were normalized to the average control force response before and after Pi treatment. Maximum Ca2+-activated force in pCa 4.5 and strontium (pSr 5.4) exposure are shown.

 
Figure 3 also illustrates the mean ST data showing the effect of Pi on caffeine-induced Ca2+ release in the absence (Fig. 3A) and in the presence (Fig. 3B) of CP for different exposure times (10 s to 30 s). It can be seen that the effect of phosphate on the area of the caffeine response was initiated rapidly (within 10 s) but then did not change with longer exposure times (up to 30 s shown). Exposure to Pi for up to 1 min (data not shown) did not cause any further reduction in Ca2+ release from the SR [mean area after Pi exposure for 1 min was 103 ± 27% (n = 4; 0 mM CP) and 104 ± 14 (n = 4; 10 mM CP) of bracketing controls]. Importantly, the effect of Pi on ST fibers was significantly less at both 20- and 30-s exposures than for FT fibers, with an approximately twofold greater effect of Pi being observed in FT fibers.

Phosphate Equilibration Under Paraffin Oil Because the SR leaks Ca2+ when fibers are equilibrated in the presence of EGTA (1 mM), the SR Ca2+ content must rapidly decline from the initial level set by the designated load period. In particular, this means that the SR Ca2+ content in ST fibers may be greatly reduced after the 30-s equilibration period, and this may underlie the reduced effect of Pi on the response to caffeine seen in these fibers, given that one may expect that the probability of Ca2+-Pi precipitation should then decrease with decreasing SR luminal [Ca2+]. To test this, we created a closed system around the fiber by using paraffin oil. Under these conditions, we found that the SR of both FT and ST fibers did not lose Ca2+ over the 30-s equilibration period (<10% in every fiber examined as determined by the change in the area of the force response to caffeine compared with the preequilibration control). Therefore, after a 10-s dip in the 50Pi/0CP solution (1 mM EGTA), fibers were immersed in paraffin oil for a further 20 s (30 s equilibration in total). The area of the response to caffeine was still reduced significantly more in FT than in ST fibers (P < 0.001, one-way ANOVA; see Table 2). Importantly, these data confirm that our original observations in FT fibers (33) were not simply an artifact of net losses or gains in SR Ca2+.


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  		Table 2. Effect of 50 mM Pi exposure on the peak caffeine-induced force response in fast-twitch and slow-twitch fibers equilibrated in the presence and absence of paraffin oil

 
Effect of Pi on ST Muscle With Different SR Ca2+ Contents To determine whether the starting SR Ca2+ content influenced the effect of Pi in ST fibers, we loaded ST fibers for 20 s, 30 s, and 2 min, respectively. From Fig. 1, a 20-s load equates to ~50% of the maximum SR Ca2+ content. Consistent SR Ca2+ contents at lower load times were difficult to obtain because of the rapid loading rate of ST muscle. Thus we only examined 20- and 30-s load times in ST fibers. This is still approximately up to 1.5-fold the endogenous proportion found in FT fibers (see Fig. 1; see also Ref. 37) but, nevertheless, should give some indication whether starting SR Ca2+ content is important.

Figure 5 shows the mean data of the area of the caffeine-induced force response in both FT and ST muscles equilibrated with 50 mM Pi/0 mM CP for 30 s after a given load period. Again, ST fibers still appear to be substantially less affected by Pi treatment than FT fibers. Moreover, this effect of Pi on Ca2+ release appears to decrease with decreasing SR Ca2+ content in ST fibers (although this was not significant).


Figure 5
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  		Fig. 5. Effect of Pi on caffeine-induced calcium release in ST fibers at different SR Ca2+ contents. ST fibers were loaded at different times [2 min (n = 13), 30 s (n = 5), and 20s (n = 3)] to vary the SR Ca2+ content and were then exposed to a 50Pi/0CP solution for 30 s (see METHODS). The mean (±SE) relative area of the caffeine-induced force response as a percentage of bracketing control responses is shown. It can be seen that the area of the response to caffeine for FT fibers (n = 7) was significantly smaller compared with all other treatments for ST fibers (*P < 0.01 for each comparison, one-way ANOVA).

 
Pi-Assisted SR Ca2+ Loading The differences in the effect of Pi observed between FT and ST fibers in the present study may reflect some difference in the way Pi accumulates and precipitates with Ca2+ in the SR in these fiber types. To test this, we exploited an old trick used for Ca2+ loading the SR of SR vesicle preparations, that is, by Pi assistance (18, 20) (see METHODS). Figure 6 shows the mean data of these experiments. What was striking was the extent to which FT fibers could exceed their endogenous maximum SR Ca2+ content (initially determined at 2-min loading pCa 6.7) by as much as 13-fold in the presence of Pi, whereas for ST fibers, Ca2+ loading in the presence of Pi only doubled the endogenous maximum SR Ca2+ content. These data indicate that Pi-assisted loading was markedly different between the fiber types.


Figure 6
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  		Fig. 6. Pi-assisted SR Ca2+ loading in skinned FT and ST fibers. The SR of both FT and ST fibers was initially exposed to the load solution (see Table 1) for 2 min to maximally load the SR with Ca2+ (refer to Fig. 1). The area of the force response to caffeine subsequently obtained after a 2-min load was the response to which all subsequent responses to caffeine following Pi-assisted loading were normalized. The graph shows the normalized area of the caffeine-induced force response after fibers were exposed to a 50 mM Pi/10 mM CP solution with 1 mM total EGTA, pCa 6.7 (e.g., modified load solution) for different times. The data were analyzed by using a two-way ANOVA (overall P value <0.001; *P < 0.05, Bonferroni post hoc test).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results indicate that 1) myoplasmic phosphate inhibits SR Ca2+ release in both FT and ST skeletal muscle but with an ~2-fold greater effect in FT fibers; 2) the effect of Pi in either fiber type is not different when the cytoplasmic [ADP] is kept very low by the presence of 10 mM CP; 3) the difference in the total SR Ca2+ contents between FT and ST fibers cannot account for the greater effects of Pi on SR Ca2+ release observed in FT fibers; and 4) Pi-assisted Ca2+-loading of the SR results in a substantially increased SR Ca2+ content, well above the maximum endogenous content for FT fibers but not for ST fibers. These results show that ST fibers are more resistant to the effects of Pi on Ca2+ release and that this is possibly due to a reduced ability of the SR to accumulate Ca2+-Pi precipitate.

Effects of Pi on SR Ca2+ Release in FT and ST Muscle We have previously shown (33) and confirm here that Pi reduces Ca2+ release from the SR in FT fibers consistent with the formation of a Ca2+-Pi precipitate. We also describe a reduced effect of Pi on Ca2+ release in ST fibers. Furthermore, we show that the effect of Pi in both FT and ST fibers was not altered when the free cytoplasmic [ADP] was buffered to very low levels by the presence of CP [consistent with previous work by Dutka et al. (11)].

The difference in the effect of Pi between these two fiber types may be attributed to the known differences in the total endogenous SR Ca2+ contents [11 and 21 mM for FT and ST fibers, respectively, normalized to SR volume (16)]. It is well known that the ability of Pi to form a precipitate with Ca2+ is a function of both free [Ca2+] and [Pi]. The solubility product of Ca2+-Pi has been estimated to be around 5–6 mM2 (8, 15). Intuitively, one would first expect that the formation of a Ca2+-Pi precipitate would occur at much lower [Pi] and more rapidly in ST fibers given the higher total endogenous SR Ca2+ content. However, it is apparent that, even when Ca2+ content differences are corrected for (see Fig. 5), Ca2+ release in ST fibers remained less susceptible to the effects of Pi. If anything, as the Ca2+ content in ST fibers approached that of FT fibers in the present study, the effect of Pi continued to decrease (as one would predict; see Fig. 5). Thus it appears that the different SR Ca2+ contents could not simply account for the reduced effect of Pi seen in ST fibers observed here.

Another possibility that may explain the different efficacy of Pi on Ca2+ release between FT and ST fibers is that Pi is less permeable to the SR of ST fibers. We have previously suggested (33) that one possible pathway for Pi entry into the SR in FT fibers is the passive influx into the SR through a nonspecific anion channel. Laver et al. (23) recently identified this channel as possibly being the small Cl– channel. Fryer et al. (17) also showed that there may be alternative pathways for Pi entry through an ATP-dependent Pi transporter. However, at present, there has been no previous examination of the presence, density, or function of either small Cl– channels or Pi pumps in ST fibers, so we do not know anything about the putative role of these channels and pumps in ST fibers. Nevertheless, its seems unlikely that the differences in the effects of Pi on Ca2+ release in FT and ST fibers reported here are due to differences in the relative permeability of the SR to Pi; Pi still prevented some Ca2+ release in ST, albeit to a smaller extent than FT fibers, which indicates that the SR of ST fibers must be reasonably permeable to Pi. Furthermore, oxalate, which is functionally similar to Pi, was also found to be equally permeable to the SR of both FT and ST fibers from human muscle (36).

In FT fibers, 8 min of Pi-assisted Ca2+ loading increased the SR Ca2+ content by as much as 13 times above the endogenous maximum Ca2+ content, which indicates a substantial capacity of the SR to store Ca2+-Pi (Fig. 6). These results are similar to the observations of Fryer et al. (17), except that the increased Ca2+ content achieved under our conditions was substantially higher than the threefold increase reported by Fryer et al. (17). This difference is likely simply due to the different free Ca2+ between our loading conditions and theirs (pCa 6.7 vs. 7.15). Interestingly, in ST fibers, this Pi-assisted Ca2+ loading only doubled the Ca2+ content. The reduced capacity to load Ca2+ in the presence of Pi in ST fibers may be due to differences in the final equilibrium state between the amount of Pi and Ca2+ uptake (and loss) from the SR and Ca2+-Pi precipitation in the SR lumen compared with FT fibers. This could be a consequence (in part) to the presence of different isoforms of calsequestrin in FT and ST SR. Furthermore, the relatively smaller SR volume of rat ST compared with FT fibers may place a simple constraint on the amount of Ca2+-Pi precipitate that can accumulate. Interestingly, this may not be the case for human skeletal muscle, in which maximum SR Ca2+ storage capacity appears to be similar for both fiber types (36). Nevertheless, this striking difference between FT and ST fibers observed here suggests that Ca2+-Pi precipitation is reduced in ST fibers.

Physiological Relevance FT and ST fibers perform different roles in the body. Typically, FT fibers are found in muscle groups that are required to provide both rapid contractions and large force outputs, whereas ST fibers are predominantly associated with muscles that perform long-duration activity (such as breathing and posture). It is evident then that these two major fiber types would have different sensitivities to the many metabolites thought to be involved in fatigue and, thus, differences in fatigability.

ST fibers already have a reduced rate of Ca2+ release, force production, and relaxation rate (3, 5). As mentioned above, much of this pattern of activity can be explained by the differences in the density and/or function of the dihydropyridine receptors, ryanodine receptors, and SR Ca2+-ATPases (10, 13, 39) as well as the contractile apparatus (35). We show in the present study that elevated myoplasmic Pi has a markedly reduced effect on SR Ca2+ handling in ST fibers compared with FT fibers. This reduced (but not absent) effect of Pi in ST fibers may have an important functional role in controlling the activity of ST fibers during normal activity and also prevent the development of fatigue. It is interesting that the SR of ST fibers is essentially full (15). This would be expected to restrict Ca2+ uptake, because the SR Ca2+-ATPases would be prevented from pumping Ca2+ across such a large gradient. It is known that the dynamic range of myoplasmic Pi between rest and activity is smaller in ST than in FT fibers (~6–30 mM in ST and ~1–50 mM in FT fibers). A reduced sensitivity to Pi in ST fibers (for the reasons described above) would have two major effects. The first is that the effective reduction in the free [Ca2+] in the SR (when some Pi enters the SR and precipitates with Ca2+) would allow Ca2+ uptake, which would otherwise be impossible if the SR was completely full. The second is that the seemingly reduced capacity to accumulate Ca2+- Pi precipitate in the SR of ST fibers would minimize any Pi-induced reduction in the free Ca2+ available for release. We know in FT fibers that the amount of Ca2+ released per action potential is relatively fixed and not affected until the free Ca2+ in the SR is substantially reduced [< 200 µM (35)], and this is true even when the SR Ca2+ content is well above the normal endogenous content. In ST fibers, perhaps the initial extra reserve of Ca2+ ensures that even with some Pi entry into the SR, the free Ca2+ available for release remains relatively unaffected. Thus some reduction in SR Ca2+ in ST fibers would still allow sufficient Ca2+ release to initiate contraction. In FT fibers, however, the greater dynamic range of Pi coupled to a smaller total SR Ca2+ content greatly decreases the amount of free releasable Ca2+ sufficiently to reduce force output and contribute to fatigue.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the School of Molecular and Biomedical Sciences start-up fund and by National Health and Medical Research Council of Australia Grant 349456.


   ACKNOWLEDGMENTS
 
We thank the School of Molecular and Biomedical Sciences, University of Adelaide, and the National Health and Medical Research Council of Australia for financial support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. S. Posterino, Discipline of Physiology, School of Molecular and Biomedical Sciences, Univ. of Adelaide, Adelaide 5005, SA, Australia (e-mail: giuseppe.posterino{at}adelaide.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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 DISCUSSION
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