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 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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