Mammalian skeletal muscles show substantial heterogeneity as a result of differing proportions of distinct fiber types within different muscles. The functional and phenotypic characteristics of fiber types are closely related to the distinct myosin heavy chain (MHC) isoforms expressed by those fibers (30). Mammalian single-fiber analysis has revealed the existence of pure muscle fibers (expressing a single MHC isoform) and hybrid muscle fibers (expressing 2 or more MHC isoforms) (23). The following pure fiber types are based on human MHC isoform expression patterns: 1 slow-Type I expressing MHCI, and 2 fast-type IIA with MHCIIa and type IIX/IID with MHCIIx/IId (29,32). However, adult rat limb muscles express 4 different MHC isoforms: Types I, IIa, IIx/IId, and IIb in fiber types I, IIA, IIX/IID, and IIB, respectively (2). The differential expression of MHC isoforms in different muscles is reflected in their functional responses, such as contractile and metabolic behavior. These responses also can be affected by a variety of stimuli, including chronic stimulation, removal of a synergist muscle, endurance exercise, and heavy resistance training (13,22,24,31,35). It is generally accepted that skeletal muscle can adapt to resistance training (RT) via both a quantitative mechanism, based on changes in muscle mass and fiber size, and a qualitative mechanism, based on changes in fiber type and MHC content (30). In this context, many quantitative histochemical and biochemical studies have reported that resistance training promotes significant alteration in fiber MHC content, enhancing physical performance (4,15,31).
In addition, Green et al. (14) suggested that the intracellular energy state of muscle fibers might influence patterns of MHC isoform expression. They reported that the intracellular phosphorylation potential (IPP), defined as the adenosine triphosphate (ATP)/adenosine diphosphate (ADPfree) ratio, was diminished during prolonged chronic low-frequency stimulation (CLFS). They further reported that this decrease preceded transitions in fiber type. Similarly, Conjard et al. (6) demonstrated a positive correlation between the ATP/ADPfree ratio and MHC-based fiber type in normal and chronically stimulated rodent muscles. The relationship between MHC isoform expression and the ATP/ADPfree ratio suggests that a persistent change in the IPP may act as an important physiological signal, contributing to transitions in fiber type. Collectively, the results of those studies indicate that the energetic state of muscle fibers can influence changes in the MHC-based phenotype. Consistent with this hypothesis, several studies have shown that when rats are fed with β-guanidinopropionic acid (β-GPA), a competitive inhibitor of creatine (Cr) uptake into muscle, they exhibit lower intramuscular concentrations of ATP and phosphocreatine (PCr), a lower ATP/ADPfree ratio, and associated fast-to-slow fiber-type transitions (9,26).
Considering that Cr supplementation increases total creatine (TCr) and PCr concentrations in rodent (8) and human (16) muscles, its use during resistance training could increase the efficiency of high-energy phosphate shuttling (38). This, consequently, could attenuate contraction-induced reductions in the IPP and influence MHC-based fiber-type transitions. Although the evidence suggests that Cr supplementation can have a profound impact on the skeletal muscle fiber phenotype as a result of an influence on the IPP, it remains unknown whether Cr supplementation influences the pattern of fiber type and MHC isoform expression in muscles subjected to resistance training. The purpose of this study was to test the hypothesis that Cr supplementation during resistance training would influence the pattern of slow-twitch muscle MHC isoform expression. We investigated the soleus muscle because it is highly recruited in our model of resistance training and it possesses a mixed slow-twitch MHC-based phenotype. The soleus muscle also presents lower TCr content and high creatine transporter protein (CreaT) expression when compared to glycolytic muscle, indicating a significant capability for creatine transport (20).
To date, most studies have been performed on human subjects to determine the benefit of training for specific athletic performances. However, human studies are influenced by the motivation of individuals during training and physical tests. Our animal model provides a method of training independent of subject volition and also ensures total creatine dose intake. In humans, food intake and creatine supplementation may be affected by individual behavior. In an animal study, these confounds are removed by administering supplemental creatine by oral gavage. Also, assessment of muscle composition and morphology in humans is limited to small muscle biopsies and indirect methods of determining muscle mass. Using our model, we can isolate single muscles and perform analysis on whole muscle preparations. A single prior study applied an animal model to investigating the effects of Cr supplementation on MHC isoform profile in slow- and fast-twitch muscle during voluntary running (10). The authors suggested that a greater average IPP in the plantaris of the Cr-consuming voluntary running group contributed to the maintenance of a faster MHC phenotypic profile. To our knowledge, the present study provides the first data on the effects of creatine on a slow-twitch muscle MHC isoform profile during resistance training. We are not interested in discussing the relationship between Cr supplementation and IPP during resistance training. The main focus of our study was to examine the effects of Cr supplementation on patterns of MHC isoform expression in the slow-twitch muscle during resistance training. We used a rodent model to investigate the muscle adaptation, allowing greater control of variables that can affect the phenomenon investigated and ensure a reliable response when these same relationships are tested in human subjects.
Experimental Approach to the Problem
An animal model was utilized to examine the MHC isoform expression in the slow-twitch muscle after 5 weeks of resistance training with and without creatine supplementation. The intensity and volume of training were the same in the groups with and without creatine supplementation, so the only difference between groups was the creatine treatment. We felt that this protocol provided an effective way to investigate the effects of creatine supplementation independently from overload training adjustments. Two days after the final day of training, morphological and biochemical analyses were performed on a single isolated muscle. The muscle weight was normalized by muscle weight/body weight ratio. The animal model provided the unique and accurate way to isolate single muscles and perform analysis on whole muscle preparations, reflecting the total muscle response.
Animals and Experimental Groups
Male Wistar rats (80 days old, 250-300 g) were obtained from the Multidisciplinary Center for Biological Investigation (CEMIB, UNICAMP, Campinas, São Paulo, Brazil). They were housed in collective polypropylene cages (4 animals per cage) covered with metallic grids, in a temperature-controlled room (22-24°C) under a 12-hour light-dark cycle, and provided with unlimited access to standard rat chow and water. We used the independent variables Creatine and Training to examine the effects of both, isolated or associated, on the muscle parameters (dependent variables). For this purpose, rats were randomly divided into 4 groups: Nontrained with no creatine supplementation (CO, n = 8), nontrained with creatine supplementation (CR, n = 8), trained with no creatine supplementation (TR, n = 8), and trained with creatine supplementation (TRCR, n = 8). This experiment was approved by the Biosciences Institute Ethics Committee, UNESP, Botucatu, SP, Brazil (Protocol No. 017/06-CEEA) and was conducted in conformance with the policy statement of the American College of Sports Medicine on research with experimental animals.
CR and TRCR groups were given creatine monohydrate daily (Sigma, C-3630, St. Louis, MO, USA) and water as a vehicle, via gavage. Groups CO and TR received water only via gavage. Creatine supplementation began 5 days before initiation of the training protocol and was kept up until the end of the experiment. Animals were weighed daily to prepare creatine solution; creatine intake per animal was 0.5 g/kg−1/d−1 (18), which exceeds the amount necessary to elevate muscle levels of creatine in humans.
TR and TRCR groups were submitted to a high-intensity resistance training program for 5 weeks (5 days/week), similar to that described by Cunha et al. (7) (Table 1). Before the initial training program, animals performed a 1-week pretraining (once a day) to familiarize them with the water and exercise. In this phase, the rats individually performed sessions of jumping into a 38-cm-deep vat of water at 28 to 32°C (Figure 1). Animals jumped to the water surface to breathe without needing any direct stimulus to complete the jumping sessions. The depth was appropriate to allow each animal to breathe on the surface of the water during successive jumps. A jump was counted when the animal reached the surface and returned to the bottom of the vat, a movement performed repeatedly. The adaptation protocol consisted of increasing the number of sets (2 to 4) and repetitions (5 to 10) with 40-second rests between each set, carrying an overload of 50% body weight strapped to a vest on the animal's chest (Figure 1). After the adaptation period, TR and TRCR groups began the resistance training program, which consisted of 4 sets of 10 jumps with overload equivalent to 50% body weight (first and second weeks), 60% (third and fourth weeks), and 70% (fifth wk), respectively (Table 1). Sessions were performed between 2 and 3 pm.
At the end of the experiment animals were anesthetized with pentobarbital sodium (40 mg/kg IP) and sacrificed by decapitation. The right soleus muscle was removed and its weight was normalized to body weight (muscle weight/body weight ratio). Muscle water content was obtained by calculating the ratio between the wet and dry weight (wet muscle weight to dry muscle weight ratio) of a fraction of the middle portion of the muscle, weighed before and after 48-hour dehydration at 80°C. Measuring total wet and dry muscle weight in a similar manner to our study is not possible in humans. With our model, we can isolate individual muscles and examine their total intramuscular water content.
Left soleus muscle (SOL) was collected and the middle portion was frozen in liquid nitrogen at −156°C. Samples were kept at -80°C until use. Histological sections (10-μm thick) were obtained in a cryostat (JUNG CM1800, Leica, Germany) at −24°C and submitted to hematoxylin and eosin stain for morphological analysis (Figure 2A). To determine the muscle fiber-type composition, myofibrillar adenosine triphosphatase (mATPase) histochemistry was performed using preincubation at pH 4.4. Analyses revealed the existence of pure muscle fibers (I Type I and IIA Type IIA) and hybrid muscle fibers (IC Type IC and IIC Type IIC) based on their staining intensities (34) (Figure 2B). Muscle fiber-type percentages were determined using Image Analysis System Software (Leica QWin Plus, Germany). Note that the morphological analysis showed muscle fibers with polygonal or rounded aspect and peripheral nucleus, surrounded by an endomysium of loose connective tissue. Muscle fiber was distributed in fascicles surrounded by perimysium (Figure 2A).
Myosin heavy chain isoform analysis was performed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in duplicate (maximum 5% of variation). Eight histological sections (12-μm thick) were collected from each sample and placed in a solution (0.5 mL) containing glycerol 10% (w/vol), 2-mercaptoethanol 5% (vol/vol), and sodium dodecylsulfate (SDS) 2.3% (w/vol) in a Tris/HCl buffer 0.9% (pH 6.8) (w/vol). The final solution was shaken for 1 minute and heated for 10 minutes at 60°C. Small amounts (8 μL) of the extracts were submitted to electrophoresis reaction (SDS-PAGE 7-10%), using a 4% stacking gel, for 19 to 21 hours at 120V. The gels were stained with Coomassie Blue (2) and used to identify the MHC isoforms according to their molecular weight showing bands at the MHCI and MHCII levels. One sample of the extensor digitorum longus (EDL) muscle was used as comparative control because it consists of 90% MHCII and 10% MHCI (Figure 3). The gels were photographed and images were captured by VDS Software (Pharmacia Biotech, Piscataway, NJ, USA). Finally, densitometry was performed using Image Master VDS Software (version 3.0), which determined the relative MHC isoform content.
Statistical analyses were performed using a software package (SPSS for Windows, version 13.0). To ensure that the data were stable, the statistic procedure was accomplished after the preliminary study of the variable related to normality and equality of variance among all groups, with statistical power of 80% for the comparisons realized. Fiber-type frequency data were analyzed using the Goodman Test for contrasts intermultinomial and intramultinomial populations (11,12) to assess differences among all groups. Statistical comparisons among the groups were made using analysis of variance (ANOVA) for the 2-factor model (40) for body weight, food intake, muscle weight, water content, and MHC isoforms content values. When significant main effects were revealed, specific differences were assessed using Tukey's post hoc comparisons. Data are expressed as mean ± SD. Differences were considered significant with a p value of ≤0.05.
Body Weight, Food Intake, Water Retention, and Muscle Weight
The growth progression of the groups throughout the experiment period and the final average body weight are shown in Figure 4 and Table 2, respectively. Confirming that animals initiated the experiment with similar health status and physical activity levels, no statistical difference was observed in the initial body weight among all groups (Table 2). After 5 weeks of experiment the body weight gains (Δ%) were 28.4%, 31.8%, 24.7%, and 28.2% in the CO, CR, TR, and TRCR groups, respectively (Table 2). Those values were not statistically (p > 0.05) different among all groups. Although no statistical difference has been observed in body weight percentages variation (Δ%), all groups demonstrated significant (p < 0.05) weight gain by the end of the experiment (Table 2 and Figure 4).
The significant (p < 0.05) increase of body weight observed in the TR and TRCR groups throughout the experiment indicated that the training model used, although intense, did not subject the animals to a state of overtraining (Table 2 and Figure 4). Both Cr supplementation and resistance training alone or associated did not affect body weight gain in the CR, TR, and TRCR groups compared to the CO group (Figure 4). The data show that the increase in body weight reflected only the somatic growth of animals. Consistent with no significant (p > 0.05) change in body weight, Cr supplementation did not promote significant (p > 0.05) alteration in food intake in the CR and TRCR groups compared to the CO group (Figure 5). Furthermore, no statistical differences (p > 0.05) were observed in soleus muscle weight, muscle weight/body weight ratio, and intramuscular water content (wet muscle weight/dry muscle weight ratio) among the groups (data not shown).
MHC Content and Fiber-Type Frequency
The representative SDS-PAGE gel used to quantify MHC isoforms is shown in Figure 3, and the corresponding data are summarized in Table 3. After 5 weeks of experimentation, the Cr supplementation promoted a significant (p < 0.02) increase in MHCI content and reduction in MHCII in the CR group compared to the CO group. Inversely, the TR group showed a significant (p < 0.02) increase in MHCII content and a decrease in MHCI in relation to the CO group (Table 3). When combined, both Cr supplementation and resistance training did not promote significant changes (p > 0.05) in MHCI and MHCII content of the TRCR group in relation to the CO group (Table 3).
The advantages of our study compared with previous work in this area include the full range of histochemical fiber types and relative MHC content to validate the histochemical data. Fiber-type frequency for each group is shown in Figure 6. The significant increase of MHCI content and reduction of MHCII in the CR group reflected in a significant (p < 0.05) increase of type I fibers and reduction of type IIA fibers compared to the CO group (Figure 6). However, the increase of MHCII and reduction MHCI in the TR group were not associated with significant (p > 0.05) change in percentages of muscle fiber type (Figure 6). The Cr supplementation in conjunction with resistance training promoted a significant (p < 0.05) increase in the percentage of type I fibers and reduction of type IIa fibers in the TRCR group compared to the CO group (Figure 6).
Although it is easy to study resistance training in humans, it is difficult to determine the phenotypic muscle responses to this training. This limitation is primarily a result of the invasive nature of muscle biopsies and the risks inherent in using human subjects. Considering the heterogeneity of muscle fibers in different muscle regions, a small muscle sample cannot accurately reflect the total muscle response. To circumvent these problems, Tamaki et al. (37) suggested a weightlifting protocol designed to induce hypertrophy in rat limb muscles. Here, we used a model of weightlifting in a liquid medium, suggested as a variation of the model proposed by Tamaki et al. No statistical differences were observed in the body weight gain and food intake among all groups (Figures 4 and 5). The lack of variation in weight gain and food intake indicated that the independent variables (training and creatine) used in the present study did not interfere with developmental aspects of the animals. Our animal model provides the advantage of the ability to perform analysis on whole muscle preparations, providing a more extensive examination of muscle phenotype adaptations during training. During the training of our subjects, the muscle response was not affected by lifting technique, motivation, food, creatine intake, or any other psychological parameters. With these variables controlled, the purpose of this study was to test the hypothesis that Cr supplementation would influence patterns of slow-twitch muscle MHC isoform expression during resistance training. The major finding of this study was that creatine supplementation may have the potential to abolish exercise-induced MHC isoform transitions from slow MHCI to faster MHCII in slow-twitch muscle. This is consistent with an antagonistic action of both creatine and resistance training on MHC-isoform changes. A direct statistical analysis supports this interpretation. As compared to the CO group, the CR group exhibited greater MHCI and lower MHCII content, whereas the TR group exhibited lower MHCI and greater MHCII content (Table 3). Furthermore, compared to the CO group, the application of both creatine and resistance training inhibited significant changes in the MHC content in the TRCR group (Table 3). Several studies have reported a positive correlation between MHC isoform expression and the ATP/ADPfree ratio in normal and CLFS muscle (6,14), suggesting that a persistent depression in the energetic state may act as a potential physiological signal, contributing to MHC-based fast-to-slow fiber type transitions in fast-twitch muscle (6,14). In our study, exercise-induced MHCI to MHCII isoform transitions (Table 3) demonstrate that, in slow-twitch muscle, contrary to what is observed in electrically stimulated fast-twitch muscle (25), the energetic state cannot induce the MHC-based fast-to-slow transition. Although measurements of the ATP/ADPfree ratio have not been performed in our study, Conjard et al. (6) demonstrated that ATP and PCr contents were approximately doubled in type IIB fibers as compared to type I fibers. They also noted a 2-fold increase in the ATP/ADPfree ratio. In addition, Sant'Ana et al. (28) and Sahlin et al. (27) observed that human type II fibers contain 10% more ATP and 20% more PCr than type I fibers. Thus, it seems reasonable that in slow muscle that contains less PCr, has a lower ATP/ADPfree ratio, and does not express MHCIIb, the energetic state may not be a determining factor for the MHC-based fiber transition.
Based on the knowledge that creatine supplementation increases TCr and PCr concentrations in rodent (8) and human (16) muscles, their use during resistance training could increase the efficiency of high-energy phosphate shuttling (38) and, consequently, attenuate contraction-induced reductions in the energetic state and influence MHC-based transitions in fiber type. In the present study, the exercise-induced slow-to-fast phenotypic transition in the TR group compared to the CO group was abolished in the TRCR group (Table 3), suggesting that creatine could influence the MHC isoform profile during resistance training. In addition, Cr supplementation alone demonstrated a greater MHCI and lower MHCII content in the CR group compared to the CO group (Table 3). Our study disagrees with Brannon et al. (3), who did not show significant alterations in MHC isoforms distribution in the soleus and plantaris muscles of rats supplemented with creatine with and without high-intensity exercise. Despite contradicting Brannon et al. (3), the increased MHCI and reduced MHCII seen in our study are consistent with greater fatigue resistance (19) and improved oxidative capacity of the creatine-fed rat soleus muscle (3). Collectively, these results demonstrate that creatine maintains a slow phenotype in slow muscle, accepting the hypothesis that creatine could influence the MHC isoform profile during resistance training. Although the molecular events that underlie our findings remain unknown, these observations raise the question of what signals and cellular conditions initiate MHC isoform profile changes in different muscles.
Consistent with previous studies in which MHC isoform modulation preceded fiber transition (4,36), the increase of MHCI and reduction of MHCII in the CR group were reflected in a significant increase of type I fibers and a reduction of type II fibers compared to the CO group (Figure 6). However, the increase of MHCII and reduction of MHCI in the TR group were not associated with a significant change in the relative proportion of muscle fiber types (Figure 6). Corroborating our findings, Harber et al. (15) demonstrated adjustments in MHC isoform distribution in human muscle subjected to circuit weight training but without significant alterations in fiber type. The authors suggest a change in proportion of MHC isoforms prior to muscle fiber modulation, as observed in nontrained individuals after 2 weeks of RT (33) and nontrained elderly subjects after 12 weeks of RT (80% 1 repetition maximum) (39). The advantages of this kind of study, compared with previous work in this area, include an examination of the full range of histochemical fiber types and relative MHC content to validate the histochemical data. To date, most studies used changes in the MHC content to predict alterations in fiber frequency. Our findings, together with those of others (15,33,39), show that the exercise-induced quantitative changes in MHC isoform expression may not reflect changes in muscle fiber contents. The exercise-induced MHCI-to-MHCII transition was consistent with the findings of Andersen et al. (1), which demonstrated a decrease in percentage of fibers containing MHCI and an increase in percentage of fibers containing MHCIIa resulting from sprint training in humans. Contrary to previous resistance-training studies that showed an MHCIIb-to-MHCIIa transition within the fast fiber population (4,15), we found that exercise induced an MHCI to MHCII conversion. However, most of these studies examined predominantly glycolytic muscles (e.g., vastus lateralis) that contain a high proportion of MHCIIb. The soleus muscle investigated in this study does not express MHCIIb or MHCIId because it is made up of mainly type I (90%) and type IIA fibers (10%). To our knowledge, we are showing for the first time that there is a shift from MHCI to MHCII in oxidative muscles subjected to resistance training. This transition may enhance the ability of this muscle to supply overload increases during training sessions.
In this context, Gallo et al. (10) observed an MHCIIb-to-MHCIIx and -MHCIIa transition in the rat plantaris muscle during voluntary running, without significant changes in soleus muscle MHC isoform expression. According to those authors, the contractile properties-based recruitment pattern of the muscle during activity may be crucial to promote changes in the MHC content. This recruitment condition may not have been met in the slow-twitch soleus muscle during voluntary running. Our training model promoted substantial recruitment of the soleus muscle, which could explain the exercise-induced MHC isoform transition, contrary to that observed by Gallo et al. (10). Our results reveal new insight into the changes in contractile properties of slow-twitch muscles recruited during resistance training, showing that these muscles should not be discarded in the preparation of training programs because they can adapt to promote performance. In addition, Gallo et al. (10) demonstrated that the exercise-induced MHCIIb-to-MHCIIx and -MHCIIa transition was attenuated when exercise was combined with Cr supplementation, resulting in a faster MHC-based phenotype in the plantaris muscle. In our study, the significant MHCII increase and MHCI reduction of the TR group, as compared to the CO group, was not observed in the TRCR group (Table 3). Although the exercise-induced MHC isoform transition in our study was opposite (slow-to-fast) that observed by Gallo et al. (10) (fast-to-slow), the mechanism of action of creatine was similar. Collectively, our results show that creatine may have the potential to abolish exercise-induced MHC isoform transitions from slow-to-faster in slow-twitch muscle and fast to slower in fast-twitch plantaris muscle (10). Because postural maintenance requires tonic contractile activity, the potential of creatine to abolish exercise-induced MHC isoform transition from slow-to-fast in postural soleus muscles might represent a pathway to the development of muscle endurance. Thus, our results suggest that creatine supplementation might be a suitable strategy for fast-to-slow fiber conversion, improving fatigue resistance in endurance athletes.
In conclusion, creatine supplementation abolished exercise-induced MHC isoform transitions from slow MHCI to faster MHCII in slow-twitch soleus muscle. This was consistent with an antagonistic action of both creatine (lower MHCII and greater MHCI) and resistance training (greater MHCII and lower MHCI). Functional studies in humans should be conducted in the future to investigate the events that mediate creatine-induced phenotypic profile transitions during exercise training. Although the molecular events that underlie our findings remain unknown, the stimulation of a transcriptional pathway (e.g., calcineurin), a calcium-dependent protein phosphatase that stimulates slow fiber-specific gene expression (5,21), might occur as a result of increased availability of creatine. This stimulation may be associated with an altered ATP/ADP ratio or altered cytosolic Ca+2 concentration during or after contractile activity. Alternatively, the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) during muscle repair might facilitate the fast-to-slow fiber transition in slow soleus muscle (17), suggesting that it might be stimulated by the increase of muscle creatine.
The results of the present study suggest that slow-twitch muscles supporting resistance training can change its contractile properties to enhance performance. In addition to the changes observed in the primary muscles recruited during resistance training, the strength and conditioning professionals may benefit from application of training protocols involving the recruitment of postural muscles (e.g., soleus) because it may represent an additional benefit to promote athletic performance. The administration of creatine alone can promote increase of MHCI content in slow-twitch muscle, enhancing fatigue resistance of skeletal muscle. Besides, Cr supplementation also might be a suitable strategy to abolish exercise-induced isoform transition from slow MHCI to faster MHCII, maintaining a slow phenotype in slow muscle, which may be favorable to maintenance of muscle oxidative capacity of endurance athletes.
Grant support: FAPESP, Proc. 04/08627-3. This work is part of the MSc thesis presented by AF Aguiar to Universidade Estadual Paulista Júlio de Mesquita Filho, UNESP, Instituto de Biociências in 2007.
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