Unaccustomed or high-intensity exercise, especially when it involves eccentric contractions, can induce skeletal muscle damage and temporary impairment of muscle function (3,19). Exercise-induced muscle damage is characterized mainly by disruption of sarcomeres and sarcolemma, leading to protein leakage from myofibers, as illustrated by elevated levels of myoglobin and creatine (Cr) phosphokinase activity in plasma (4,29). Adult skeletal muscle has the remarkable capacity to regenerate after injury, mainly because of the satellite cells that represent a population of adult myoblasts that remain normally quiescent (24). Once activated after muscle injury, satellite cells rapidly proliferate, fuse, and differentiate to form new myofibers or to repair the damaged ones (2).
More than 30 yr ago, Ingwall et al. (20,21) reported that Cr stimulated incorporation of labeled amino acids into myosin heavy chain (MHC), the major myofibrillar protein, and stimulated muscle-specific protein synthesis in chicken myotubes in culture. However, in contrast to their reports, it was further found that Cr had no effect on myosin synthesis or on total protein synthesis (15). More recently, it was shown that the fusion of myogenic C2C12 (11) and satellite (35) cells is largely enhanced while creatine (Cr) is added to the culture medium during the differentiation phase. This was at least partly mediated by overexpression of insulin-like growth factor 1 and myogenic regulatory factors (MRF) (23). MRF are members of a family of basic helix-loop-helix proteins that act as transcription activators and regulate the transcription of some muscle-specific genes (31).
Creatine monohydrate (Cr) is one of the most commonly used supplements taken by athletes, recreational exercisers, and the elderly and children of both sexes. Cr loading has been widely studied because of its potential ergogenic effects in sports performance (for review, see ). In addition, recent studies have shown that Cr can enhance muscle functional capacity in patients with neuromuscular diseases, disuse atrophy, or muscular dystrophies (18,22,34). When combined with resistance training sessions, Cr activates human satellite cells at a higher level than resistance training alone (25) and accelerates muscle recovery after an immobilization period (18). These observations provide rationality for the use of Cr in patients in rehabilitation and could explain the beneficial effects of Cr in patients experiencing neuromuscular disease, disuse atrophy, and muscle dystrophies (33).
To date, the putative beneficial effects of Cr supplementation on recovery of muscle mass and/or phenotype after muscle injury remain to be examined in vivo. The current experiment was designed to test the hypothesis that Cr supplementation ingested during the regenerative period after extensive muscle injury could favor the recovery of both muscle mass and adult muscle phenotype, including recovery of protein involved in contractile function and metabolism.
MATERIALS AND METHODS
Female Wistar rats initially weighing 180-200 g were purchased from Charles River Laboratories (L'Arbresle, France). Animals were housed two per cage in a thermoneutral environment (22 ± 2°C) on a 12:12 h photoperiod and were provided with powder food and water ad libitum. Rat care and all experimental procedures used were in accordance with the policy statement of the American College of Sports Medicine on research with experimental animals. The study was approved by Ethical Committee for Animal experiments at the Centre de Recherches du Service de Santé des Armées de Grenoble, France.
Three days before myotoxic-induced degeneration of soleus (SOL) muscle, rats were randomly assigned to one of two groups, either supplemented (Cr) or not (N) with Cr (anhydrous Creatine®; Sigma Aldrich, Saint Quentin Fallavier, France) administered both in powder food (4.7%) and drinking water (1.4%). At day 0 of the experiment (D0), degeneration of the left SOL muscle was induced by notexin injection. As previously reported, the noninfiltrated right SOL muscle served as intact control muscle (14,27). Eight animals of each group were then anesthetized for tissue sampling and killed 1, 3, 7, 14, 21, 28, 35, and 42 d after initial muscle injury (D1, D3, D7, D14, D21, D28, D35, D42, respectively).
Rats were anesthetized with sodium pentobarbital (60 mg·kg−1) and left SOL muscle degeneration was induced by notexin injection (0.2 mL, 10 μg·mL−1) isolated from snake venom (Notechis scutatus, Latoxan, Valence, France) directly into the belly of the muscle surgically exposed as previously described (14). Because the blank surgery did not induce any specific alteration in muscle tissue, no surgical procedure was done on the right SOL, which served as an intact control. The effects of Cr supplementation were then studied in regenerating SOL muscle (Reg), in comparison with contralateral intact noninjured muscles (Int).
Animals were anesthetized with sodium pentobarbital (90 mg·kg−1) administered intraperitoneally. Regenerated (i.e., left) and intact (i.e., right) SOL muscles were excised, cleaned of adipose and connective tissues, and weighed. Muscles were immediately frozen in liquid nitrogen. All samples were stored at −80°C until analyses were performed.
Serial transverse sections (12 μm thick) were cut from the midbelly portion of SOL in a cryostat microtome maintained at −20°C and stained with hematein, eosin, and safran (HES) to visualize nucleus, cytoplasm, and connective tissue at each time of recovery. To determine the total number of myofibers, photographs of the entire muscle were taken at low magnification. Moreover, 20 to 30 photographs at high magnification, covering the entire muscle section, were used to determine the cross-sectional area of at least 1000 fibers in each muscle sample at D42, for both Int and Reg muscles. Analyses were performed with a light microscope computerized image analysis system (Lucia 5; Laboratory Imaging, Prague, Czech Republic).
Analysis of muscle Cr content.
Approximately 10-15 mg of muscle was used for spectrophotometric determination of free and total Cr (TCr = Cr phosphate + free Cr). Muscle was extracted in 0.25 N HClO4 and neutralized with 1 N KOH. To hydrolyze creatine phosphate (PCr), 2 N HCl was added to the supernatant, which was then heated for 15 min at 60°C. The reaction was stopped on ice, and the supernatant was neutralized with 2 N NaOH. TCr was immediately determined enzymatically using a spectrophotometric method, as previously described (16) (Creatinine PAP; Boehringer Mannheim, Germany, from which the creatininase was omitted). Free Cr was determined by the same method omitting the step of PCr hydrolysis. PCr content was then calculated as the difference between TCr content and free Cr.
Analysis of the distribution of MHC isoforms.
Muscles were subjected to the analysis of MHC isoforms as described previously (32). Myosin was extracted from small sections of muscles in seven volumes of buffer solution (0.3 M NaCl, 0.1 M NaH2PO4, 0.05 M Na2HPO4, 0.01 M Na4P2O7, 1 mM MgC12·6H2O, 10 mM EDTA, 1.4 mM 2-β-mercaptoethanol, pH = 6.5). Electrophoresis was performed using a Mini Protean II system (Bio-Rad, Marne-la-Coquette, France) with 8% and 4% acrylamide-bis (50:1) separating and stacking gels, respectively, containing 0.4% sodium dodecyl sulfate (SDS). Myofibril samples were denatured with the SDS incubation medium, according to the method of Laemmli. Gels were run at constant voltage (72 V) for 31 h and then silver-stained (1). The MHC protein isoform bands were scanned and quantified using a densitometer GS 800 driven by Quantify One 4.6.1 (Bio-Rad).
Protein isolation and immunoblot analyses.
A range of 10 to 20 mg of muscle was lysed with the appropriate buffer, and homogenates were all centrifuged at 15,000g for 15 min at 4°C. Protein content was determined using the bicinchronic acid method (Roche/Hitachi 912 Instrument; Roche Diagnostic, Mannheim, Germany). Equal amounts of muscle protein (1, 12, 50, and 75 μg for MHC, proliferator cell nuclear antigen (PCNA), myogenin, and MyoD, respectively) were separated on SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes (Hybond C-extra; Amersham Pharmacia Biotech, Orsay, France), which means that lane loading was normalized for total muscle protein content. A standardized amount of protein prepared from intact SOL was also applied on each gel to serve as an internal standard for comparison across blots. Membranes were incubated overnight with the appropriate target antibody and then 2 h with the corresponding horseradish peroxidase-conjugated antibody. Washed blots were subjected to the ECL Western blotting detection reagent kit (ECL®; Amersham Pharmacia Biotech) and then exposed to x-ray film (Hyperfilm ECL; Amersham Pharmacia Biotech). The relative protein expression was determined by the ratio of sample band intensity to internal standard band intensity by densitometry, using the densitometer system described previously.
The following antibodies were used: mouse monoclonal antibodies against slow type Iβ MHC (1:200 dilution, NCL-MHCS; Novocastra, Newcastle upon Tyne, United Kingdom), myogenin (1:500, sc-12732 [F5D], Santa Cruz Biotechnology®, Heidelberg, Germany), PCNA (1:500, Ab-1 Clone PC10; NeoMarkers Interchim, Montluçon, France), and MyoD (1:500, no. 554130 clone MoAb 5.8A; BD Pharmingen, BD Biosciences, Pont de Claix, France). Incubation with horseradish peroxidase-conjugated goat antimouse IgG antibody was used as secondary antibody (1:4000, 1:2000, 1:2500, and 1:5000 dilutions for MHC, myogenin, PCNA, and MyoD, respectively, sc-2005; Santa Cruz Biotechnology).
Activities of metabolic enzymes.
Frozen tissue samples were weighed and placed into an ice-cold homogenization buffer (30 mg wet mass·mL−1) containing: 5 mM HEPES (pH 8.7), 1 mM EGTA, 1 mM dithiothreitol, 5 mM MgCl2, and 0.1% Triton. Samples were homogenized using a microglass hand homogenizer and were incubated for 60 min at 0°C to ensure complete enzyme extraction. Citrate synthase (CS) and lactate dehydrogenase (LDH) activities were measured at 30°C (pH 7.5) using coupled enzyme systems as previously described (6). The LDH isoenzyme profile was determined using agarose gel electrophoresis (Sigma LDH Reagent Kit; Sigma) at 200 V for 90 min followed by image analysis using the densitometer system.
All data are presented as means ± SEM. Data were analyzed using a three-way ANOVA to determine the main statistical effects of time, injury and Cr supplementation, and interactions between those factors. When appropriate, differences between groups were tested with a Newman-Keuls post hoc test, especially to compare values measured during muscle regeneration with those observed in contralateral noninjured muscles. Statistical significance was accepted at P < 0.05. When no effect of time was observed within intact muscles, results were reported with values of intact muscles pooled.
Body and Muscle Mass
The mean body mass of rats was similar in the two groups when muscle degeneration was induced by notexin injection. There was a global effect of time (P < 0.001) on the body mass of animals. Consistent with the expected growth rate of animals, the body mass increased regularly from D1 to D42 both in Cr-treated and nontreated rats.
Mass of both intact and regenerated muscles of Cr- and nontreated animals are reported Table 1. The three-way ANOVA revealed neither main effect of Cr supplementation nor interaction between Cr and time or between Cr and injury. By contrast, there were main effects of time (P < 0.001) and injury (P < 0.001), with a strong interaction between those factors (P < 0.001) indicating that only regenerated muscle mass varied along time without differences in intact muscle mass. As a consequence, the regenerated muscle mass was normalized and expressed as the ratio of the mass of regenerated muscle to the mass of contralateral intact muscle (Fig. 1). Relative muscle mass was first increased at D1 after notexin-induced injury (P < 0.001), then markedly decreased until D7 when regenerated muscle mass represented only 66% and 74% of intact muscle mass (P < 0.001) for Cr-treated and nontreated rats, respectively. Relative muscle mass then only slowly increased from D7 to D42. However, regenerated muscles did not fully recover mass values similar to intact muscles, even 42 d after initial injury (12% and 17% less than intact muscles of Cr-treated and nontreated rats, respectively, P < 0.01).
Histological and Morphological Aspects of Regenerated Muscles
Histological aspects of intact muscles were similar in nontreated (Fig. 2A) and Cr-treated animals (Fig. 2B). At D7 of regeneration, all necrotic fibers appeared to have been replaced by regenerated myofibers (Fig. 2C and D). The size of these fibers was still smaller than the noninjured fibers, and most of the regenerating fibers showed central nuclei. By 21 d after injury, the regeneration process was in progress and numerous small diameter fibers were present (Fig. 2E and F). Extracellular spaces were large both in nontreated and Cr-treated animals, likely related to fibrosis as highlighted by safran staining. By 42 d after toxin injection, myofibers remained irregular with variable diameters and large space between fibers (Fig. 2G and H). The histological aspects of regenerated muscles were similar in nontreated and Cr-treated rats, all along the recovery period. The mean fiber cross-sectional area and the number of fibers per cross-section of muscles at D42 are given Table 2. The two-way ANOVA revealed a strong effect of injury (P < 0.001) and no effect of Cr supplementation for both variables. The mean fiber cross-sectional area was strongly decreased (P < 0.001) and the number of fibers increased (P < 0.001) in regenerated muscles compared with intact ones.
TCr and PCr Contents
The three-way ANOVA revealed main effects of time (P < 0.001), injury (P < 0.001), and Cr supplementation (P < 0.001 and P < 0.05 for TCr and PCr, respectively), with a strong interaction between injury and time for TCr only (P < 0.001). The duration of Cr treatment had no effect on TCr and PCr contents within intact muscles, so we decided to present graphically the results of all intact muscles pooled (Fig. 3). Cr supplementation significantly increased TCr content of intact muscles (+19 ± 8%, P < 0.01) and only tended to increase the PCr content (+11 ± 10%, NS). By contrast, TCr and PCr contents were strongly decreased early after initial injury (global effect of injury, P < 0.001) while they progressively recovered during muscle regeneration (global effect of time, P < 0.001). Recovery was enhanced in Cr-treated rats (global effect of Cr supplementation, P < 0.001 and P < 0.05 for TCr and PCr, respectively). At D35, TCr and PCr contents of Cr-treated rats were similar in regenerated and intact muscles, whereas they remained lower in regenerated than in intact muscles of nontreated animals (P < 0.05).
Proliferative and Differentiation Markers of the Regenerative Process
PCNA protein expression is a marker of cell proliferation. Whereas PCNA protein expression was very low in intact muscles, it was markedly increased in regenerating muscles (global effect of injury, P < 0.001) with the highest levels at D3 and then progressively returned to basal values at D14 (global effect of time of recovery, P < 0.001; Fig. 4A). There were no differences between Cr-treated and nontreated animals. Because its expression did not vary along time within intact muscles, the results of intact muscles of either nontreated or Cr-treated rats were pooled for graphical representation (Fig. 4A).
MyoD expression is commonly used as a reliable marker of satellite cell activation. MyoD protein levels markedly increased in regenerated muscles (global effect of injury, P < 0.001) with a peak as soon as D1 after muscle injury (Fig. 4B). Then MyoD protein expression slowly decreased, with higher levels in regenerated than those in intact muscles until D3 and D7, for nontreated and Cr-treated rats, respectively (P < 0.05). There was no significant effect of Cr supplementation on MyoD protein expression during the early step of muscle regeneration. In intact muscles, MyoD protein expression was similar whatever the time of observation, so that we have pooled the results of all intact muscles of either nontreated or Cr-treated rats for graphical representation (Fig. 4B).
Myogenin is a protein known to be expressed during the terminal differentiation program. As expected, myogenin protein levels were higher in regenerated than that in intact muscles (global effect of injury, P < 0.001). There was a marked effect of time (global effect, P < 0.001), with an interaction with muscle injury (P < 0.001). As a result, the increased myogenin expression in regenerated muscles was only observed at D3 (P < 0.001, in comparison with intact muscles). Thereafter, myogenin protein levels decreased to values observed in intact muscles as soon as D7. Moreover, there was no effect of Cr supplementation on myogenin protein levels, all along the regenerative process. Because its expression did not vary along time within intact muscles, we decided to pool the results of intact muscles of either nontreated or Cr-treated rats for graphical representation (Fig. 4C).
Evaluation of the Maturation Step of the Regenerative Process
MHC protein expression.
Although MHC is one of the most abundant proteins in skeletal muscle, MHC-Iβ represents at least 90% of total MHC within adult intact SOL muscle of rats. The time course of expression of this specific isoform during regeneration after injury is viewed as a marker of contractile phenotype maturation.
There was a global effect of injury (P < 0.001) and time (P < 0.001) on the distribution of MHC isoforms, with a strong interaction between these factors (P < 0.001) for all adult (MHC-I, MHC-IIA, MHC-IIX, MHC-IIB) and immature (MHC-emb, MHC-neo) isoforms (Fig. 5). In fact, the time course of expression of MHC isoforms during the recovery process was consistent with previous observations (14): 1) the reexpression of immature isoforms between D1 and D14 or D21 for MHC-emb and MHC-neo, respectively; 2) the de novo expression of the fast isoforms MHC-IIB and MHC-IIX until D28 and D35, respectively; 3) the MHC-IIA isoform, which is the only rapid isoform expressed in adult SOL muscle of rats, peaked at D14 (P < 0.05 between regenerated and intact muscles), and then decreased after D21 to return to basal values at D28; 4) the relative amount of MHC-Iβ was recovered from day 28. Cr supplementation had no effect on the MHC isoform transitions observed during the regeneration process after notexin-induced injury.
In this study, protein expression of MHC-Iβ was analyzed by Western blot and reported to a determined amount of proteins. There was a global effect of time of recovery (P < 0.001) and injury (P < 0.001) on MHC-Iβ expression, with a strong interaction between those factors (P < 0.001; Fig. 6). MHC-Iβ protein levels were very low in regenerated compared with intact muscles at D7 (P < 0.001) and then progressively increased throughout the recovery time. MHC-Iβ protein levels were similar in regenerated and intact muscles of nontreated rats from D21. Although there was no global effect of Cr supplementation, MHC-Iβ levels remained low in regenerated muscles of Cr-treated rats at D28 (P < 0.01). Interestingly, MHC-Iβ protein levels further increased between D28 and D42 after injury (P < 0.05), with higher levels in regenerated than in intact muscles both in Cr-treated and nontreated rats (P < 0.05). In intact muscles, MHC-Iβ protein expression was similar whatever the time of observation, so that we have pooled the results of all intact muscles of either nontreated or Cr-treated rats for graphical representation (Fig. 6).
LDH activity was strongly affected by both injury and time (global effects, P < 0.001) with a strong interaction between these factors (Fig. 7A). Total LDH activity strongly decreased at D1, and thereafter rapidly increased (P < 0.05 between D7 and D3 and between D14 and D7), so that total LDH activity in regenerated muscles represented only 73% of LDH activity measured in intact muscles (P < 0.001). From D14 to D42, total LDH activity slowly increased with levels at D42 similar to those in intact muscles.
Considering specific activities of LDH, both H- and M-LDH activities were markedly decreased at D1, but only M isozyme of lactate dehydrogenase (M-LDH), which is the glycolytic isoform of LDH, strongly recovered between D1 and D3 (P < 0.001) to reach similar levels as in intact muscles as early as D3. On the contrary, the oxidative isoform of LDH, namely H-LDH, only slightly and progressively increased (P < 0.05) to reach lower levels than that in intact muscles at D42 (74 ± 5%, P < 0.001 and 85 ± 8%, P < 0.05 for nontreated and Cr-treated rats, respectively; Fig. 7A).
CS activity was markedly affected by injury and time of recovery (global effect, P < 0.001, with interaction between these factors; Fig. 7B). CS activity strongly decreased at D1, and thereafter rapidly increased (P < 0.05 between D3 and D7 and between D7 and D14), so that CS activity was similar in regenerated and intact muscles 14 d after initial injury.
There were no effects of Cr supplementation on the recovery of total, specific LDH, or CS activities.
In support of previous results provided mostly through in vitro experiments (23,35), the present study was designed to test the hypothesis that Cr supplementation may have beneficial effects on the recovery of adult muscle properties after extensive muscle injury. The main results are the following: 1) although injury induced an early and strong decrease of TCr and PCr contents within regenerated muscles, Cr supplementation ensured recovery of TCr and PCr concentrations faster than that in control nontreated rats; 2) whether animals were supplemented with Cr, the mass of regenerated muscles remained still lower than that of intact ones, even 42 d after injury; 3) the MHC profile of regenerated muscles was recovered 35 d after injury, both in Cr-treated and nontreated rats; 4) a full recovery of CS activity was observed from D14, whereas the specific H-LDH activity remained lower in the regenerated than in the intact muscles until 42 d, without any differences between Cr-treated and nontreated animals. Taken together, the present results show that Cr supplementation had no beneficial effects on the time course of recovery of skeletal muscle mass and phenotype after notexin-induced injury in rats.
To assess the effects of Cr on the time course of recovery of adult muscle properties, we used a well-defined model of muscle injury in rats, known to cause a rapid and extensive myofiber necrosis sparing satellite cells, followed by a complete and synchronous regenerative process (9,17). Using this model of muscle injury, it was shown that the full recovery of muscle mass is delayed beyond 42 d, a result that underlines the critical need to develop means to accelerate and improve the muscle regeneration process. With this intent, we used a Cr supplementation protocol expected to ensure Cr loading within muscles. The total amount of Cr daily ingested in the present study was higher than 5 g·kg−1, a dosage previously shown to increase free Cr (30%), PCr (15%), and TCr (20%) levels in rat SOL muscle after 5 d of Cr feeding (26). In the present experiment, we verified that Cr supplementation successfully increased Cr content within intact muscles (31 ± 8%, 11 ± 10%, and 19 ± 8% for free Cr, PCr, and TCr, respectively). More interestingly, although notexin-induced muscle injury led to a strong decrease of TCr and PCr contents, we observed a fast and full recovery of TCr content within regenerated muscles only in Cr-treated rats. Seven days after the initial injury, TCr and PCr contents in regenerated muscles of Cr-treated rats indeed reached physiological levels similar to those observed in intact muscles of nontreated rats, whereas 35 d after injury, Cr contents were not fully restored in nontreated rats. These findings suggest a fast recovery of the sodium-dependent Cr transporter, the major route of Cr entry into myofibers (8). Once reexpressed in sarcolemma of regenerative myofibers, the Cr uptake rate is expected to be enhanced by dietary Cr supplementation, at least partly because intracellular Cr concentration is low during the first steps of muscle regeneration (7).
We failed to accelerate the recovery of adult muscle phenotype by Cr supplementation, either for the fast recovery of oxidative capacities, as estimated by the level of CS and H-LDH activities, or recovery of the slow MHC profile. The time course of MHC-Iβ expression and relative distribution of MHC isoforms were similar in regenerated muscles from Cr-treated and nontreated rats. These results might be surprising considering that Cr supplementation has been previously described to increase the expression of specific target genes, especially those encoding MHC isoforms (11,20,30,36). However, recent evidences indicate that Cr does not induce any direct anabolic effect on protein synthesis (10,15,22,27) but most probably reduces the rate of protein breakdown (27).
Although the Cr supplementation was efficient to increase TCr and PCr contents in regenerated muscles, the recovery of muscle mass has not improved in Cr-treated rats, whenever the time after injury. The analysis of the mean fiber cross-sectional area and the number of fibers per cross-section revealed that this incomplete recovery of regenerated muscle mass was clearly due to the atrophy of muscle fibers, which appeared similar in Cr-treated and nontreated rats. By contrast, the number of fibers per cross-section was strongly increased in regenerated muscles in the same extent in Cr-treated and nontreated rats, suggesting that the recruitment and the proliferation of satellite cells were similar in both groups and were not the limited step for the recovery of muscle mass.
Although it has been shown that Cr affects satellite cell proliferation and differentiation in cell culture (35) and that Cr-induced hypertrophy of C2C12 cells was, at least partly, mediated by overexpression of IGF-1 and MRF (23), the present results failed to show any positive effect of Cr supplementation on the early step of muscle regeneration in rats. We used PCNA expression to examine the cellular activity during the proliferating step (13,27). In our conditions, PCNA expression could be relevant to cellular events related to proliferation of satellite cells and/or infiltrating nonmyogenic cells. Moreover, MyoD, which augments during the proliferative step, is representative of the myogenic differentiation (31). PCNA levels increased from D1 to D14 and such an increase was only partially associated with increased levels of MyoD, which likely means that only a part of the proliferating cells has myogenic capacities. In fact, the peak of PCNA observed at D3 was more likely due to infiltrating inflammatory cells, as highlighted by the histological aspect of regenerating muscles (Fig. 2). Nevertheless, the time course of proliferation as assessed by PCNA expression was not affected by Cr supplementation. In particular, there was no additional wave of proliferating cells in regenerated muscles of Cr-treated rats at D3. In a recent study, no effects of Cr on PCNA and MyoD mRNA levels were observed after a resistance exercise session consisting of mainly eccentric muscle contractions in humans (11). Thus, a clear discrepancy exists between in vitro and in vivo studies, possibly due to larger changes in TCr and/or PCr cell content in vitro than in vivo (12,23). The concentration of Cr in the medium of cell culture studies (typically 5 mM) indeed leads to TCr content within myotubes of approximately 700 mmol·kg−1 (12), which strongly exceeds the concentration of Cr within muscles during the in vivo experiment.
Muscle regeneration after extensive muscle injury consists on an initial mitotic phase, which provides new myonuclei required to increase protein synthesis during the subsequent phase of differentiation and maturation. In another model that requires a strong increase in protein synthesis for muscle hypertrophic adaptation, namely, the model of compensatory overload by ablation of synergistic muscles, Young and Young (37) also failed to find any effect of Cr supplementation on SOL muscle mass or performance. Many other previous studies evidenced increased muscle mass and strength in response to Cr supplementation, but mostly in parallel with a physical training program (5). Hespel et al. (18) reported that Cr improved muscle recovery in humans during the rehabilitation phase after an immobilization period of 2 wk. Muscle biopsies taken in those patients showed changes in the MRF profile with Cr suggesting a potential relationship with a faster muscle regrowth. Recently, a higher level of satellite cell activation has been observed in humans supplemented with Cr and submitted to a training program of resistance exercises in comparison to a group performing the same program but receiving a placebo (25). These previous findings disagree by some aspects with the present results. However, rats of the present study were maintained at rest, and the combined effects of contractile activity and Cr supplementation are questioned.
We previously showed that increasing physical activity during muscle regeneration was able to ensure the early and full recovery of muscle mass (28). Thus, it would be of interest to test the hypothesis that providing Cr supplementation in exercising rats would be able to improve the recovery of muscle mass after extensive injury because of the conjunction of Cr loading and direct stimuli related to physical exercise on protein synthesis. Further studies are clearly necessary to test such hypothesis.
In conclusion, oral Cr supplementation was able to increase TCr and PCr contents within both intact and regenerated muscles of rats but was ineffective in accelerating the time course of recovery of skeletal muscle mass and phenotype after extensive injury.
No funding was received for this work from the National Institutes of Health (NIH), Wellcome Trust, and Howard Hughes Medical Institute (HHMI). This experiment was supported by grants from Agence franç aise de lutte contre le dopage (AFLD).
Results of the present study do not constitute endorsement by ACSM.
We are thankful to Mrs Josiane Denis, Valérie Leroux, and Antonia Alonso for protein assays and Mrs Rachel Chapot and Nadine Simler for technical assistance.
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Keywords:©2009The American College of Sports Medicine
MUSCLE INJURY; MUSCLE MASS; MUSCLE PHENOTYPE; MRF; MHC; OXIDATIVE ENZYMES