Effect of creatine on contractile force and sensitivity in mechanically skinned single fibers from rat skeletal muscle

Robyn M. Murphy, D. George Stephenson, and Graham D. Lamb

Department of Zoology, La Trobe University, Melbourne, Victoria 3086, Australia

Submitted 10 June 2004 ; accepted in final form 22 July 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in Ca2+ sensitivity (+0.061 ± 0.004 and +0.049 ± 0.009 pCa units, respectively) and maximum Ca2+-activated force (to 104 ± 1 and 105 ± 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in Ca2+ sensitivity of the contractile apparatus, and maximum Ca2+-activated force was ~106 ± 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus.

ergogenic aid; muscle contraction; fatigue


THE INGESTION OF CREATINE (Cr) can result in improved performance during certain types of exercise (1, 4, 27, 29). It has been suggested that this ergogenic effect may be due to increased creatine phosphate (CrP) levels that enhance the capacity to either resynthesize ATP (4) or to maintain ATP stores by reducing the loss of adenine nucleotides (1), or enhance acid-base buffering (27).

Water accumulation and increases in muscle mass are often reported as side effects after Cr administration. One study showed that muscle intracellular volume increased 2.3 and 3.1% after 1 and 3 days of Cr supplementation, respectively (32). Longer periods (5 days to 6 wk) of Cr supplementation typically result in increases in fat-free body mass of 1–2 kg (7, 28, 29). This weight gain equates to a 3.5–7% increase in muscle water in a 70-kg person. Water moves into the muscle fibers after an increase in Cr content owing to the necessity to maintain osmotic balance. It has been suggested that since cellular swelling can act as an anabolic stimulator of protein synthesis (15), Cr supplementation may promote protein synthesis (32), and thereby muscle performance may be enhanced. Recently, however, it was demonstrated that muscle myofibrillar and sarcoplasmic protein synthesis were not different after Cr supplementation (20). This suggests that the effect of Cr ingestion on muscle mass is not through effects on muscle protein metabolism. Further consequences of the accumulation of cellular water with Cr have not been examined to date.

Studies have examined the effect of Cr supplementation on twitch and tetanic force production in human and rodent muscle (10, 16, 21, 23, 26, 30). In one study, no changes in peak tension, time to peak tension, or half-relaxation time during twitch or tetanic stimulations were observed in either soleus (SOL) or extensor digitorum longus (EDL) muscle in mice fed Cr compared with control mice (23). Other studies reported similar findings of no improvements in twitch or tetanic force production or half-relaxation time after Cr supplementation in various species and muscles (10, 16, 21, 26, 30). Given that improvements in muscle performance of only a few percent can be very important, small changes need to be measurable. One aspect that should be noted in the animal studies is that unpaired data sets were used (10, 21, 23, 26, 30). This inevitably limits the ability to detect small differences. Given this, the findings of the lack of effect of Cr supplementation on twitch and tetanic force production may not be conclusive.

As mentioned, the benefit of Cr supplementation on muscle performance is usually thought to be due to an increased ability to resynthesize ATP via increased CrP stores. However, studies in humans report that the intramuscular CrP-to-Cr (CrP/Cr) ratio actually falls after 5 days of Cr supplementation (6, 14, 22, 28). This means that the Cr concentration is increased more than the CrP concentration, which in fact should hinder, not aid, the ability of the Cr kinase reaction to rephosphorylate any ADP to ATP. In view of this, it seems that an alternate mechanism is needed to explain the observed improvement in performance with Cr supplementation.

One possibility is that the Cr uptake into muscle directly or indirectly affects force production by the contractile apparatus. Previously, it was shown that the addition of 50 mM Cr to a control solution resulted in a decrease in maximum Ca2+-activated force in rabbit single fibers (5, 11). Maximum Ca2+-activated force was not affected, however, by the addition of 25 mM Cr (11). Those two studies represented a nonphysiological change to the intracellular environment, because, as stated above, when Cr enters the muscle fiber, there is also an accumulation of water to maintain osmotic balance (32). The increase in cellular water is in fact a critical factor, because it means that the other cellular constituents are diluted and the ionic strength of the cytoplasm will decrease. There have been no studies to date that have measured the combined effects of Cr and the associated volume of water on the contractile apparatus in skeletal muscle.

In the present study, mechanically skinned single fibers from rat skeletal muscle were used to measure the performance of the contractile apparatus (18, 25). Fibers from SOL and EDL muscles were studied to obtain fibers at the two extremes of fiber types (i.e., predominantly slow oxidative and fast glycolytic, respectively) and therefore bracket the spectrum of fiber types. We were able to examine separately the direct effects on the contractile apparatus of increasing Cr concentration as well as decreasing ionic strength (mimicking the changes seen with acute Cr supplementation) and increasing Cr and CrP concentrations with no change in ionic strength (mimicking the changes seen with longer term Cr supplementation). In addition, to determine whether Cr per se has an effect on the contractile apparatus, we examined the effect of increasing Cr concentration alone. Our approach allowed the detection of even small differences (in the order of 1–2%) in muscle performance because the same fiber was used for both control and test experiments. In addition, the cytoplasmic environment of the fiber could be readily manipulated to study individual factors or combinations of factors as desired. We hypothesized that an improvement in muscular performance after Cr supplementation might be due to a decrease in the ionic strength of the intracellular environment rather than any direct effect of Cr itself.


    METHODS
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Control solutions. All chemicals were obtained from Sigma (St. Louis, MO). Heavily Ca2+-buffered solutions of varying pCa (pCa = –log10 [Ca2+]) were prepared from two main solutions, high-relaxing (type I, pCa >9) and high-activating (type II, pCa ~4.5) solutions, based on those described previously (9). The type I solution contained (in mM) 64 K+, 100 Na+, 25 EGTA, 1 free Mg2+ (10.3 total Mg2+), 9 total ATP, 19 Cr, 40 CrP, 1 Pi, and 60 HEPES at pH 7.10 ± 0.01. The type II solution was made similarly, but with 24 mM total Ca2+ (pCa ~4.5) and 9.25 total Mg2+ to maintain the free Mg2+ concentration at 1 mM. The type I and type II solutions were mixed to give a series of solutions with progressively increasing concentrations of Ca2+ (pCa 6.7–4.5). These were then split in two to prepare the control and test solutions for each experiment, thereby producing two solution sets of matched pCa, pH, and osmolality. In addition, Na+-based type I and II solutions were prepared in which all K+ in the type I and II solutions described above was replaced with Na+. As for the K+-based solutions, a set of matched solutions was prepared by mixing the Na+ type I and type II solutions to give a series of solutions of increasing concentrations of Ca2+.

Mechanically skinned fiber preparation. Adult Long Evans hooded rats, ages 24–28 wk, were killed by an overdose of halothane, as approved by the Animal Ethics Committee at La Trobe University. The EDL and SOL muscles were carefully removed, blotted dry on filter paper, and pinned at resting length under paraffin oil. Single muscle fibers were dissected, mechanically skinned, mounted on a force transducer (AME875; SensoNor, Horten, Norway), and stretched to 120% of their resting length. Each fiber was then placed into a 2-ml bath containing the high-relaxing solution (pCa >9) to equilibrate for 2 min before being activated in solutions of higher Ca2+ concentration (see below). Experiments were performed at room temperature (24 ± 1°C).

Ca2+ activation of contractile apparatus. The force-pCa relationship for each fiber was determined by exposing the fiber to a sequence of solutions at progressively higher concentrations of Ca2+, allowing the fiber to reach close to a steady-state force level in each solution before moving to the next. The final solution was the high-activating (pCa ~4.5) solution, and the force elicited in that solution was defined as the maximum Ca2+-activated force. In each experiment, the same fiber was used to measure one or more test conditions bracketed by control measurements. Between each of the activation sequences, the fiber was relaxed in the high-relaxing solution (pCa >9) for 1 min. All results are reported on fibers for which the decline in the maximum Ca2+-activated force over the full sequence of controls and tests was <10%.

Experimental conditions. The first set of experiments examined the effect of increasing Cr concentration on the force-pCa relationship of the contractile apparatus by using the heavily Ca2+-buffered solutions described. Cr accumulation in muscle cells in vivo is accompanied by an increase in cellular water. This would result in osmolality being maintained and ionic strength being decreased (formal ionic strength, {Gamma}/2 = {Sigma}Cizi2, where Ci and zi are the concentration and charge, respectively, of the ith ionic species). These conditions were mimicked in this first set of experiments. Control solutions contained 19 mM Cr and 40 mM CrP (CrP/Cr ratio = 2.1:1). In the test solutions, Cr concentration was increased by 10 and 14 mM, with concomitant decreases in the ionic strength and the concentration of other major constituents by 3.4% (Cr10-Osm) and 5% (Cr14-Osm), respectively. The final concentrations of Cr were ~28 and 32 mM in the Cr10-Osm and Cr14-Osm solutions, respectively. Details of the solutions are given in Table 1. Dilution of the solutions was achieved by the addition of water, with the volume determined as the amount required to maintain osmolality of the solution after the addition of Cr. In the Cr10-Osm and Cr14-Osm solutions, the CrP/Cr ratio decreased from 2.1:1 to 1.4:1 and 1.2:1, respectively, similar to that which would be expected in vivo immediately after the accumulation of Cr into a muscle fiber. In addition, the effect of a 14 mM increase in Cr concentration alone (Cr14) was tested (i.e., no accompanying addition of water and therefore no change in ionic strength). The final Cr concentration in this solution was 33 mM (see Table 1). In matched pairs of solutions, Cr14 solutions were prepared by adding 14 mM Cr to one half of the heavily Ca2+-buffered solutions of varying Ca2+ concentrations. The other halves of these solutions were used for the control conditions.


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Table 1. Final Cr and CrP concentrations, CrP/Cr ratio, and formal ionic strength of solutions

 
In the second set of experiments, the effect of increasing total Cr (TCr = CrP + Cr) concentration by 6 (TCr6) and 9 mM (TCr9) plus water (i.e., at constant ionic strength) was examined. These conditions mimicked the intracellular environment after longer term Cr supplementation, in which some of the Cr has been phosphorylated to CrP and ionic strength is largely restored. The TCr6 and TCr9 solutions were prepared by adding a volume of TCr stock solution ([CrP] = 82 mM, [Cr] = 55 mM, CrP/Cr = 1.5:1) to one half of the matched heavily Ca2+-buffered solutions. The TCr stock solution was prepared to be able to make solutions with raised concentrations of Cr and CrP and with the final CrP/Cr ratio of ~2:1, similar to the control solutions. With this procedure, sufficient water was added with the neutral Cr and charged CrP species such that there was no difference in final ionic strength between the TCr6 and TCr9 solutions and the control solutions (see Table 1). In addition, pCa, pH, or osmolality (290 ± 8 mosmol/kg solvent) of the final TCr6 and TCr9 solutions was unchanged compared with the matched control solutions. Details of the solutions are shown in Table 1. The CrP used was Na2CrP, so to ensure that the concentration of Na+ was the same in both the control and TCr6 and TCr9 solutions, the same volume of the appropriate mix of Na+-based type I and type II solutions was added to the matched control solutions.

Analysis. Each activation sequence from each fiber was analyzed individually. For all sequences, the force achieved at each Ca2+ concentration was expressed relative to the maximum Ca2+-activated force obtained for that sequence. The force-pCa data were then fitted with a Hill curve based on the equation below (GraphPad Prism 4; GraphPad Software, San Diego, CA) to obtain the pCa50 (Ca2+ concentration expressed in pCa units giving 50% of maximum force) and the Hill coefficient (nH)

where F(pCa) is the relative force. Results are expressed as means ± SE of n observations. Statistical significance was determined at the 95% confidence level using two-tailed Student's t-test for paired samples.


    RESULTS
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To maintain cellular osmolality, the accumulation of Cr into muscle cells results in an accompanying increase in muscle water. A consequence of water being drawn into the cell is a concomitant decrease in the concentration of other cellular constituents and, importantly, a decrease in the ionic strength of the myoplasm. The first part of the first experiment examined the effects of increasing both Cr concentration and water content on the contractile apparatus of mechanically skinned single fibers from rat SOL and EDL muscle. Figure 1 shows the steady-state force response from a SOL fiber over a range of Ca2+ concentrations, using heavily Ca2+-buffered solutions, when exposed to control and test (Cr10-Osm) solutions. Hence, the same fiber was used as its own control. This is indicated in Fig. 1 by the two Cr10-Osm force-pCa staircases bracketed by two control staircases. For each fiber, the isometric force generated under both the control and test conditions was then plotted against pCa, and each individual force-pCa data set was fitted with a Hill curve. The Hill plots for the two control sequences and the two test (Cr10-Osm) sequences for the SOL fiber in Fig. 1 are shown in Fig. 2. The relative maximum Ca2+-activated force in Cr10-Osm and Cr14-Osm conditions and the mean change ({Delta}) in pCa50 and in nH are given in Table 2. Compared with control conditions, maximum Ca2+-activated force in the Cr10-Osm conditions increased to 106 ± 1% in SOL fibers and 106 ± 2% in EDL fibers. In the Cr14-Osm conditions, maximum Ca2+-activated force increased to 104 ± 1% in SOL fibers and 105 ± 1% in EDL fibers compared with control conditions. Compared with the addition of 10 mM Cr (Cr10-Osm), adding 14 mM Cr (Cr14-Osm) would result in a greater decrease in ionic strength of the solutions (see Table 1). The increase in maximum Ca2+-activated force would be expected to be greater under the Cr14-Osm conditions, although in fact the increase in maximum force appeared to be slightly greater under Cr10-Osm compared with Cr14-Osm conditions (see Table 2). This small difference, however, was evidently due to the particular subpopulation of fibers sampled, because in each case, when any given fiber was examined in both of the two test solutions (i.e., Cr10-Osm and Cr14-Osm), maximum Ca2+-activated force was always greater in the Cr14-Osm solution compared with the Cr10-Osm solution (+1.3 ± 0.4%, P < 0.05, n = 7).



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Fig. 1. Force-pCa behavior for a soleus fiber exposed to control and test (increased contents of creatine and water) conditions. A skinned soleus fiber was exposed to stepwise increasing concentrations of Ca2+ [denoted by arrows: 1 (pCa >9, no force), 2 (pCa 6.45), 3 (pCa 5.95), 4 (pCa 5.78), 5 (pCa 5.64), 6 (pCa 5.50), and 7 (pCa 4.5, maximum Ca2+-activating solution)] under control conditions (single horizontal bar) and test conditions [10 mM creatine (Cr) plus addition of water to maintain osmolality to give a 3.4% dilution of other constituents, Cr10-Osm; double horizontal bar]. The force produced is shown for 2 control activation sequences bracketing 2 Cr10-Osm sequences. The top dashed line (A) indicates the steady-state force response at maximum Ca2+-activated force, and the bottom dashed line (B) indicates the steady-state force response at pCa 5.64. In both traces, more force was produced in the 2 Cr10-Osm responses compared with their control responses. There was a greater difference between the 2 conditions at pCa 5.64 (B) than at maximum Ca2+-activated force (A), indicating an increase in Ca2+ sensitivity. Maximum force in control solution = 0.26 mN; time for each series (solution 1 back to solution 1) = 160 s.

 


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Fig. 2. Effect on contractile apparatus of increasing Cr concentration by 10 mM and adding water to result in a 3.4% decrease in ionic strength. Force-pCa relationships are shown for the soleus fiber shown in Fig. 1 exposed to a series of solutions with increasing concentrations of Ca2+, under control conditions and test conditions (Cr10-Osm), and expressed relative to the average maximum Ca2+-activated force in the control solutions. For both conditions there are 2 sets of measurements that were very similar and overlie each other in the plot. The pCa50 (Ca2+ concentration in pCa units at which half-maximum force is reached) and Hill coefficient (nH) for each response are given at bottom. The Cr10-Osm conditions resulted in a shift to the left in the force-pCa curve compared with the control curve, indicating an increase in Ca2+ sensitivity; i.e., a greater relative force was elicited at the same absolute Ca2+ concentration. In addition, the maximum Ca2+-activated force produced in the Cr10-Osm conditions was higher than in the control conditions.

 

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Table 2. Summary of effects of addition of water and Cr, or addition of Cr alone, on contractile apparatus properties

 
In both Cr10-Osm and Cr14-Osm solutions, the force-pCa relationship was shifted to the left (i.e., shifted to a lower Ca2+ concentration) in SOL and EDL fibers (Fig. 2). The mean {Delta}pCa50 was +0.044 ± 0.003 and +0.034 ± 0.005 in SOL and EDL fibers, respectively, with Cr10-Osm conditions and +0.061 ± 0.004 and +0.049 ± 0.009, respectively, in Cr14-Osm conditions (Table 2). The Hill plot for SOL fibers became slightly less steep, as indicated by the small decrease in nH (–1.4 ± 0.3 and –0.4 ± 0.1 for Cr10-Osm and Cr14-Osm, respectively). In EDL fibers, the steepness of the force-pCa relationship was not significantly affected in either solution (Table 2).

To determine whether the effects seen in the Cr10-Osm and Cr14-Osm solutions were due to effects of Cr alone (rather than to the decrease in ionic strength), we examined another condition. In this case, the concentration of Cr was increased by 14 mM (Cr14) without an accompanying increase in water (see METHODS). Given that Cr entering a muscle cell will always take water with it, the Cr14 solutions do not mimic the physiological situation. The conditions nevertheless enabled the direct effects of Cr per se on the contractile apparatus to be examined. The associated 14 mosmol/kg solvent increase in osmolality would not be expected to alter the contractile properties of the fibers (19). Importantly, there is no change in the ionic strength between the control and Cr14 solutions. As shown in Table 2, the effects of the Cr14 conditions were in the opposite direction to those seen in the Cr10-Osm and Cr14-Osm conditions. In both SOL and EDL fibers, maximum Ca2+-activated force decreased to 97 ± 1% compared with the control conditions, and {Delta}pCa50 decreased slightly (–0.026 ± 0.004 and –0.018 ± 0.005 in SOL and EDL fibers, respectively; Table 2). There was no change observed in nH in either fiber type. These results show that Cr per se has a slightly inhibitory effect on Ca2+-activated force, which contrasts with the substantial increase in maximum force and Ca2+ sensitivity observed when both Cr and water are present. Thus the increase in Cr concentration was not the cause of the changes in the latter case.

After short-term (5- to 10-day) Cr supplementation protocols, increases in intramuscular TCr stores are typically in the order of 15–25% in human and rodent muscle (2, 3, 6, 12, 14, 2224, 28). Once Cr enters the muscle fibers, it takes time for it to be phosphorylated to CrP (6, 12, 14, 22, 28). This results in there being an initial decrease in the CrP/Cr ratio after Cr supplementation, which is only restored toward original levels over longer periods of time. In the second set of experiments, solutions were prepared to mimic the myocellular environment after increases in both Cr and CrP concentrations and the reestablishment of the initial CrP/Cr ratio (and hence the ionic strength), as is sometimes reported to occur with longer-term low-dose Cr supplementation (6). Solutions were prepared with increases in TCr concentration of either 6 (TCr6) or 9 mM (TCr9), and the final CrP/Cr ratios were 2:1 and 1.9:1, respectively, close to control conditions (see Table 1). These changes equated to 11 and 15% increases in TCr concentration and therefore conservatively mimicked the range observed in vivo after Cr supplementation. The isometric forces generated at different pCa values in a SOL fiber under both control and TCr6 conditions were fitted with Hill curves and are shown in Fig. 3. The {Delta}pCa50, {Delta}nH, and maximum Ca2+-activated force in TCr6 and TCr9 conditions for all fibers are given in Table 3. Maximum Ca2+-activated force in the TCr6 conditions increased to 106 ± 1% in SOL and EDL fibers and to 104 ± 1% in SOL fibers in TCr9 conditions. There was virtually no change in the pCa50 in TCr6 solutions, whereas in TCr9 solutions, pCa50 increased slightly in SOL fibers (+0.028 ± 0.002). The nH became slightly shallower in SOL fibers in both the TCr6 and TCr9 solutions (–0.4 ± 0.1 and –0.5 ± 0.1, respectively) and was not affected in EDL fibers in TCr6 conditions (Table 3). Overall, these results indicate that when there is an increase in the contents of CrP and Cr, some potentiating action remains even though the CrP/Cr ratio and the ionic strength were returned to original values. These effects are seen as an increase in maximum Ca2+-activated force and a slight increase in Ca2+ sensitivity of the contractile apparatus of the fibers.



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Fig. 3. Effect on contractile apparatus of a 6 mM increase in total Cr (TCr) concentration with the creatine phosphate (CrP)-to-Cr (CrP/Cr) ratio and ionic strength kept approximately constant. Force-pCa relationships are shown for a soleus fiber exposed to a series of solutions with increasing concentrations of Ca2+, under control conditions and test conditions (addition of 6 mM TCr, TCr6; final CrP/Cr = 1.9:1), and expressed relative to the average maximum Ca2+-activated force in the control conditions. The pCa50 and nH for each case are given at bottom. Maximum Ca2+-activated force was higher in TCr6 compared with control conditions. When control and TCr6 conditions are compared, there is virtually no change in the Ca2+ sensitivity; i.e., a similar relative force was elicited at the same absolute Ca2+ concentration.

 

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Table 3. Summary of effects of addition of 6 or 9 mM TCr at a constant CrP/Cr ratio on contractile apparatus properties

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we used mechanically skinned single fibers from rat SOL and EDL muscles to examine the individual direct effects on the contractile apparatus of increasing Cr concentration, increasing Cr concentration plus decreasing ionic strength, and increasing Cr and CrP concentrations with no change in ionic strength.

Using changes similar to those occurring in muscle with short-term Cr supplementation studies, we determined the effect of increasing Cr concentration and decreasing ionic strength on the contractile apparatus of rat skeletal muscle fibers. We found that increasing the Cr concentration of a muscle fiber, along with an appropriate increase in cellular water to maintain osmolality, resulted in an increase in the Ca2+ sensitivity as well as the maximum Ca2+-activated force in both SOL and EDL fibers. When Cr was added to the muscle cytoplasmic environment without the addition of water, however, the changes were in the reverse direction. The major difference between these two conditions was the ionic strength, which was decreased when water was added (Cr10-Osm and Cr14-Osm compared with Cr14). These results show that there is actually a small detrimental effect of Cr itself on the contractile apparatus of skeletal muscle but that this effect is reversed when osmolality is maintained by the addition of water, and force responses are instead potentiated.

Many published studies (2, 22, 2729) have examined the effect of Cr supplementation on muscle performance and metabolism. In all cases, attempts were made to increase intramuscular TCr stores by the oral administration of Cr. Although it is accepted that TCr stores are often increased, outcomes are perturbed by factors such as variations in initial TCr content (14) and fiber or muscle type (23, 24). Nevertheless, when TCr stores are increased in the order of 15% or more, ergogenic effects are seen with certain exercise regimes (for review, see Ref. 27). Improvements in muscle performance have been attributed to factors including an enhanced capacity to either resynthesize ATP via increased CrP levels or maintain ATP stores via a reduced loss of adenine nucleotides, or an enhanced acid-base buffering (27). Because the accumulation of Cr into a muscle fiber is accompanied by an increase in muscle cell water (32), there would in fact always be a decrease in cytoplasmic ionic strength after Cr supplementation. The accumulation of water in the muscle is reported as an increase in fat-free body mass (32). This increase in fat-free body mass is unlikely to be due to an increase in muscle protein given that myofibrillar and sarcoplasmic protein contents are not affected by Cr supplementation (20). We have shown that decreasing ionic strength by 3.4 or 5%, equivalent to that estimated as a 1- to 2-kg increase in fat-free body mass in a 70-kg man, results in an increase in the Ca2+ sensitivity as well as in the maximum Ca2+-activated force in both EDL and SOL fibers from rat skeletal muscle. It was previously shown in mechanically skinned single fibers from EDL and SOL muscle that a 40% decrease in ionic strength resulted in an increased Ca2+ sensitivity (+0.38 pCa units) and maximum Ca2+-activated force (+20%) of the fibers (8). Consistent with this, in the present study the decrease in ionic strength was ~10 times less (3.4 and 5%) and the observed shift of +0.047 in the pCa50 ~10 times smaller than that seen by Fink et al. (8). In other words, the changes we found are well accounted for by the known effects of changes in ionic strength on the contractile apparatus. In the present study, we found an increase in Ca2+ sensitivity ({Delta}pCa approximately +0.05; note this is in a log scale) of ~10% and an increase of ~6% in maximum Ca2+-activated force. Such improvements in muscle performance could be highly beneficial in terms of an individual's competitive performance. These findings suggest that increasing Cr concentration and water content in muscle cells would result in two benefits: 1) an increase in tetanic peak force (produced by the increase in maximum Ca2+-activated force) and 2) an increase in Ca2+ sensitivity (i.e., at a given Ca2+ concentration, more force would be produced, see pCa 6.0 to pCa 5.8 in Fig. 2).

Previous studies in which the effects of Cr supplementation on tetanic force production were measured found no effect in human and rodent models (10, 16, 21, 23, 26, 30). However, a number of limitations existed in those studies. First, the protocols relied on the individual responding to the Cr ingestion by increasing Cr stores in the muscle. Intracellular TCr content was measured in only some of the studies (23, 26), so it is uncertain whether the Cr concentration was indeed increased in the others (10, 16, 21, 30). In humans there is a variable response by individuals to Cr supplementation (14), and in animals the uptake of Cr into muscle seems to be fiber type dependent (23, 24). A second major limitation in the animal studies was the necessity to use unpaired sampling sets, making it hard to see small differences (10, 21, 23, 26, 30). In rats, 10-day Cr supplementation resulted in a tendency for tetanic force to increase when normalized to weight (501 ± 49 and 547 ± 16 g/g in control and Cr-fed rats, respectively) of SOL muscle (30). Although those results were not significant, the results from the present study indicate that increases in muscle Cr concentration and water content produce only a small increase in maximum Ca2+-activated force, which would translate into tetanic force being augmented.

During fatigue, sarcoplasmic reticulum Ca2+ release may be reduced, resulting in lower cytoplasmic Ca2+ concentrations. In the present study, we saw an increase in Ca2+ sensitivity when the Cr concentration and water content were both raised (e.g., Cr10-Osm conditions) compared with control conditions. These findings suggest that more force would be produced at the lower Ca2+ concentrations occurring during fatigue. This is shown in Fig. 2, where for a given concentration of Ca2+, more force was produced. For example, at pCa 5.78, ~19% of maximum force was produced under control conditions and ~35% of maximum force was produced in the Cr10-Osm conditions (i.e., almost double the force).

To determine whether the benefits we observed in Cr10-Osm and Cr14-Osm conditions could be attributable to Cr per se, we investigated the effect of increasing the concentration of Cr by 14 mM in the cytoplasmic environment without altering the concentrations of other constituents or total water content. The 14 mM increase in Cr concentration gave a final concentration of 33 mM Cr (see Table 1). Although this manipulation would not occur in a muscle cell (because osmotic equilibrium is not maintained), it enabled examination of the effect of Cr per se on muscle contractile apparatus performance. In this set of experiments we found the effects to be in the opposite direction to those of the Cr-plus-water experiments (Cr10-Osm and Cr14-Osm). Other investigators (5, 11) previously reported a decrease in Ca2+ sensitivity and maximum Ca2+-activated force in rabbit fast twitch muscle when the Cr concentration was 50 mM compared with 0 mM. Interestingly, no difference was detected at 25 mM Cr compared with 0 mM Cr (11). These previous findings and our present data suggest that a small deleterious effect on Ca2+ sensitivity and maximum Ca2+-activated force might occur when Cr concentrations are greater than ~33 mM. The improvements in Ca2+ sensitivity and maximum Ca2+-activated force when an increase in Cr concentration is accompanied by the accumulation of water to maintain osmotic balance are clearly not the consequence of increasing Cr stores per se. It is unclear why Cr at concentrations greater than 33 mM has a deleterious effect on maximum Ca2+-activated force. It is unlikely that this effect is related to a decreased ability of the Cr kinase to rephosphorylate ADP to ATP because previous investigators (5) reported that even when Cr kinase was inactivated, 50 mM Cr was deleterious to force production.

Studies in which maintenance doses of Cr supplementation were used for 6–8 wk reported the CrP/Cr ratio either returning to presupplementation levels (6) or remaining lower than the initial level (28). In the present study, when both the Cr and CrP contents were increased (TCr6 and TCr9), the ionic strength and CrP/Cr ratio were set at levels similar to the control conditions. We found that the apparent Ca2+ sensitivity was virtually unchanged with 6 mM additional TCr but increased slightly with 9 mM additional TCr (see Table 3). Importantly, maximum Ca2+-activated force increased to 104–106% of that in the control solution. Given that ionic strength was not different in the control and test solutions, this improvement in maximum force must be due to some other factor. One possibility is that it is the result of improved buffering of ATP in localized regions of high ATP usage, in particular in the vicinity of the myosin heads during prolonged activation. Even though the CrP/Cr ratio is not much different in the TCr6 and TCr9 solutions than in the control solution, the absolute amounts are increased, and this would mean that there would be increased diffusion of CrP into any local regions where it had been depleted. Thus the TCr6 and TCr9 solutions would help ensure better buffering of CrP and ATP in localized regions where ATP is being used at a high rate (17) and where the buildup of metabolites could be retarding force production. This effect could also be even more important during times of increased ATP usage with active shortening of the fiber (31). This could explain the improvement in muscle peak torque production in humans after the ingestion of Cr compared with the ingestion of a placebo (13).

In summary, findings of the present study suggest that after short-term Cr supplementation, the decrease in ionic strength that accompanies the accumulation of intramuscular water will result in beneficial effects on the contractile apparatus. These effects are unlikely to be due to the increase in Cr concentration alone, which in fact seems to be detrimental to force production. Evidently, the deleterious effects on muscle contractile properties of increased Cr per se are normally completely offset by the benefits associated with the accompanying decrease in ionic strength that occurs in vivo. These effects offer an explanation for the erogenic effect of Cr supplementation that is observed even before intramuscular CrP content has increased substantially. After longer term Cr supplementation, when both the CrP/Cr ratio and the ionic strength have returned toward the presupplementation values, a beneficial effect on maximal force production persists. This effect is possibly due to the increase in intramuscular CrP content and the associated enhancement in ATP rephosphorylation capacity in localized regions of high ATP usage.


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 ABSTRACT
 METHODS
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 DISCUSSION
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This work was supported by National Health & Medical Research Council of Australia Grant 280623 and by the Australian Research Council.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. M. Murphy, Dept. of Zoology, La Trobe Univ., Melbourne, Victoria 3086, Australia (E-mail: r.murphy{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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