Baixe Artigo creatina e outras Notas de estudo em PDF para Nutrição, somente na Docsity! BASIC SCIENCES Original Investigations Effects of Oral Creatine and Resistance Training on Myogenic Regulatory Factor Expression DARRYN S. WILLOUGHBY1 and JOHN M. ROSENE2 1Department of Kinesiology, Texas Christian University, Fort Worth, TX; and 2Department of Kinesiology and Leisure Science, University of Hawaii at Manoa, Honolulu, HI ABSTRACT WILLOUGHBY, D. S., and J. M. ROSENE. Effects of Oral Creatine and Resistance Training on Myogenic Regulatory Factor Expression. Med. Sci. Sports Exerc., Vol. 35, No. 6, pp. 923–929, 2003. Purpose: This study examined 12 wk of creatine (Cr) supplementation and heavy resistance training on skeletal muscle creatine kinase (M-CK) mRNA expression and the mRNA and protein expression of the myogenic regulatory factors Myo-D, myogenin, MFR-4, and Myf5. Methods: Twenty-two untrained males were randomly assigned to either a control (CON), placebo (PLC), or Cr (CRT) group in a double-blind fashion. Muscle biopsies were obtained before and after training. PLC and CRT trained thrice weekly using 3 sets of 6–8 repetitions at 85–90% 1-RM on the leg press, knee extension, and knee curl exercises. CRT ingested 6 g·d1 of Cr for 12 wk while PLC consumed the equal amount of placebo. Results: After training, M-CK mRNA expression, as well as myogenin and MRF-4 mRNA and protein expression, were found to be significantly greater for CRT compared with PLC and CON, whereas PLC was also significantly different from CON (P 0.05). For Myo-D mRNA and protein, both CRT and PLC were significantly different from CON (P 0.05), but CRT and PLC were not different from one another. No significant differences were located for Myf5 mRNA or protein (P 0.05). M-CK mRNA was correlated with myogenin (r 0.916) and MRF-4 (r 0.883) protein (P 0.05). Conclusion: When combined with heavy resistance training, Cr supplementation increases M-CK mRNA expression, likely due to concomitant increases in the expression of myogenin and MRF-4. Therefore, increases in myogenin and MRF-4 mRNA and protein may play a role in increasing myosin heavy chain expression, already shown to occur with Cr supplementation. Key Words: DNA BINDING PROTEIN, TRANSCRIPTION FACTOR, GENE EXPRES- SION, TRANSLATION Longer periods (e.g., 10–12 wk) of creatine (Cr) sup-plementation combined with heavy resistance train-ing have recently been implicated as providing an ergogenic benefit, primarily as a result of apparent increases in the size of Type I, IIa, and IIab muscle fibers (19), the mRNA (Type I, IIa, and IIx), and protein (Type I and IIx) expression of the myosin heavy chain (MHC) isoforms (25), and the expression of the myogenic regulatory factor (MRF) MRF-4 (10). Interestingly, a series of earlier studies showed that Cr supplementation provided to cultured myocytes in- creased the rate of myosin synthesis without any effects on degradation (13–15). In addition, Cr supplementation was shown to selectively stimulate MHC synthesis and was suggested to play a role in muscle hypertrophy (14). Al- though these earlier studies did not attempt to determine an underlying mechanism, it was speculated that Cr supple- mentation may actually increase MHC synthesis by playing a role as a transcriptional co-regulator, or that it may act to alter the levels of charged tRNAs or amino acid pools which may be specific for myofibrillar protein synthesis (15). Our recent work (25) provides some support to the earlier work of Ingwall and colleagues (13–15). Even though our study involved humans and employed 12 wk of heavy resistance training combined with oral Cr supplementation, we dem- onstrated respective increases of 33%, 31%, and 36% for MHC Type I, IIa, and IIx mRNA, 17% and 16%, respec- tively, for MHC Type I and IIx protein, 58% in myofibrillar protein content, and 65% for muscle strength; all of these variables were statistically superior when compared with the control and placebo groups. Even though our work sug- gested that the increases in MHC expression at the pre- and posttranslational level may possibly lead to increases in Address for correspondence: Darryn S. Willoughby, Ph.D., FACSM, De- partment of Kinesiology, Texas Christian University, TCU Box 297730, Fort Worth, TX 76129; E-mail: d.willoughby@tcu.edu. Submitted for publication August 2002. Accepted for publication January 2003. 0195-9131/03/3506-0923 MEDICINE & SCIENCE IN SPORTS & EXERCISE® Copyright © 2003 by the American College of Sports Medicine DOI: 10.1249/01.MSS.0000069746.05241.F0 923 MHC synthesis as originally suggested by Ingwall and col- leagues (13–15), we did not attempt to elucidate a possible mechanism for the increased MHC expression as a result of oral Cr supplementation. Since our previous study (25), a more recent investigation has shown that the role of myogenic regulatory factors (MRF) may, at least in part, provide some explanation as to a possible mechanism for the increase in MHC expression seen in our study. In this recent study by Hespel and col- leagues (11), 4 wk of Cr supplementation combined with resistance training was effective in increasing the protein content of MRF-4. Also, those authors showed that even though myogenin was also increased with training, it was not preferentially affected by Cr supplementation. Skeletal muscle Type I, IIa, and IIx MHC mRNA expression has been shown to be regulated at the pretranslational level by MRF (3). As such, we have recently shown that 6 h after a single session of heavy resistance exercise, MHC Type I, IIa, and IIx MHC mRNA and Myo-D and myogenin mRNA and protein expression were all significantly increased (25). In addition, Type I and IIa MHC mRNA were correlated with myogenin mRNA and protein, whereas Type IIx MHC mRNA was correlated with Myo-D mRNA (24). Therefore, Cr supplementation combined with heavy resistance train- ing may have a profound effect on MHC isoform expression. The MRF, which include Myo-D, myogenin, MRF-4, and Myf5, are members of a family of basic helix-loop-helix (bHLH) proteins that function as transcription activators due to their inherent properties as DNA-binding proteins and, as a result, initiate transcription and regulate gene expression by binding as either homo- or hetero-dimers in the regula- tory region of a specific DNA sequence that is present in the promoters and enhancers downstream of some muscle-spe- cific genes such as MHC, myosin light chain, -actin, tro- ponin-I, and creatine kinase (CK), thereby activating their transcription (16). In general, Myo-D and Myf5 are in- volved in the determination of myoblasts, and myogenin and MRF-4 are involved in the differentiation of adult fibers (16). Additionally, Myo-D and myogenin have also been implicated in regulating muscle fiber type, as myogenin and MRF-4 expression is higher in slow-twitch fibers (21) and correlated with Type I and IIa MHC mRNA (24), whereas Myo-D expression is higher in fast-twitch fibers (16) and correlated with Type IIx MHC mRNA (24). Active uptake of Cr in skeletal muscle is facilitated by a Na-dependent transporter against a concentration gradient (6) mediated by a Cr transport protein (22). This is true of Cr produced in vivo, as well as Cr that has been consumed orally. The cytosolic phosphocreatine (PCr) pool is primar- ily considered to function as an energy store that buffers changes in ATP levels during periods of muscle exercise by replenishing hydrolyzed ATP via the dimeric muscle CK (M-CK) isozyme. Creatine kinase has been implicated in affecting cell growth (1), and the content and activity of CK is dose dependent on the CK substrate Cr. As a result, Cr supplementation is thought to possibly enhance muscular strength, power, and high-intensity exercise performance by increasing the total intramuscular Cr pool (9). The intracel- lular increase in Cr up-regulates the expression of the Cr transport protein (22) and M-CK isozyme (18). This process is known to aid in energy transfer from the mitochondria to the contractile proteins by the PCr shuttle (2) which up- regulates MHC (25) and myosin expression (13–15), thereby stimulating myofibrillar protein synthesis, promot- ing muscle hypertrophy, and increasing muscle strength (19,25). In light of previous research, it is evident that the exact mechanisms of how Cr supplementation increases muscle strength and hypertrophy are unclear in relation to its po- tential to function as a transcriptional co-regulator of M-CK and MHC gene expression. It is possible that increases in the expression of the MRF may also facilitate the up-regulation in the expression of muscle specific-genes such as MHC and M-CK, thereby facilitating respective increases in muscle mass and strength. Therefore, in the present study we uti- lized the remaining muscle samples from our previous study (25) in an attempt to determine the effects of Cr supple- mentation after 12 wk of heavy resistance training on the mRNA expression of M-CK, as well as the mRNA and protein expression of Myo-D, myogenin, MRF-4, and Myf5. We hypothesized that heavy resistance training combined with Cr supplementation would facilitate increases in MRF expression that would be related to an increase in the ex- pression of M-CK. As a result, we speculated that any increases in MRF and M-CK expression seen in this study may conceivably play a role in the increased MHC expres- sion and muscle strength following heavy resistance training seen in our previous study (25). MATERIALS AND METHODS Experimental design. In the present study, we used remaining muscle samples from our previous study (25) in which subjects signed university-approved informed con- sent documents, approval was granted by the Institutional Review Board for Human Subjects, and all experimental procedures conformed to the ethical consideration of the Helsinki Code. As a result, the specific, detailed methods and procedures are outlined previously (25). In brief, how- ever, this study employed 22 untrained (no consistent, struc- tured weight training or Cr supplementation for at least 6 months before beginning the study) male subjects with an average (SD) age of 20.41 (1.73) yr, height of 180.44 (3.72) cm, and body mass of 85.49 (14.28) kg. Subjects were randomly assigned, in a double-blind fashion, to either a control group [CON, (N 6)] involving no resistance training, a resistance trainingCr group [CRT, (N 8)], or a resistance trainingplacebo group [PLC, (N 8)]. Per- cutaneous muscle biopsies were obtained before and after the 12-wk period. Muscle samples were taken from the middle portion of the right vastus lateralis muscle at the midpoint between the patella and the greater trochanter of the femur at a depth between 1 and 2 cm. For the posttrain- ing biopsy, attempts were made to extract tissue from ap- proximately the same location by using the prebiopsy scar, 924 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org DISCUSSION A notable outcome of the present study was that Cr supplementation and 12 wk of heavy resistance training (85–90% 1-RM) significantly increased the mRNA and protein expression of both myogenin and MRF-4, and that the protein expression of myogenin (r 0.916) and MRF-4 (r 0.883) was significantly correlated to the mRNA ex- pression of M-CK. Our results are in disagreement with those of Hespel and colleagues (11), who showed that Cr supplementation combined with 4 wk of resistance training (60% 1-RM) increased MRF-4 protein expression, whereas myogenin expression was increased only in the placebo group. Our results could have differed from the work of Hespel and colleagues (11) due to the fact that in their study Cr was supplemented at a dosage of 5 g·d1. In addition, the muscle was immobilized for 2 wk before training after which point the duration of resistance training was 4 wk and the training intensity was 60% of the 1-RM using only the knee extension exercise. In our previous study (25), we supple- mented Cr as a dosage of 6 g·d1. Also, we did not previously immobilize the muscle before training and subjects trained for 12 wk at an intensity of 85–90% of the 1-RM using the leg press, knee extension, and knee curl exercises. The human vastus lateralis muscle of untrained individ- uals has been shown to be composed of approximately 42%, 32%, and 20%, respectively, for Type I, IIa, and IIb muscle fibers (17). In our previous study (25), we used untrained subjects and showed that Cr supplementation induced su- perior changes, compared with the control and placebo groups in MHC isoforms, such that after 12 wk of heavy resistance training and Cr supplementation vastus lateralis biopsies contained approximately 42%, 44%, and 14% for Type I, IIa, and IIb MHC protein, respectively. Conse- quently, the respective changes in MHC isoform composi- tion for the control and placebo groups were 37%, 35%, and 27% and 38%, 42%, and 20% for Type I, IIa, and IIb (25). Resistance training is known to result primarily in the hy- pertrophy of Type I and IIa muscle fibers (5), primarily as a result of the recruitment of both Type I and II motor units (7). It has recently been shown that 12 wk of heavy resis- tance training combined with Cr supplementation induced superior increases in Type I, IIa, and IIab muscle fiber cross-sectional areas (19). Because Myo-D is associated with Type IIb muscle fibers and the Type IIx MHC isoform, and myogenin and MRF-4 are associated with Type I and IIa muscle fibers and the Type I and IIa MHC isoforms (12), it is conceivable that the expression of Myo-D, myogenin, and MRF-4 were all increased with 12 wk of heavy resistance training due to the continual recruitment of primarily Type I and IIa motor units. In light of this assumption, the ques- tion is raised in regard to the present study; why were myogenin and MRF-4, but not Myo-D, preferentially in- creased with Cr supplementation? FIGURE 3—Expression of mRNA for M-CK. *Significant group test interaction; ‡significantly different from CON; †significantly dif- ferent from PLC (P < 0.05). FIGURE 2—Expression of mRNA for (A) Myo-D, (B) myogenin, (C) MRF-4, and (D) Myf5. *Significant group test interaction; ‡significantly different from CON; †signifi- cantly different from PLC (P < 0.05). CREATINE AND TRANSCRIPTION FACTORS Medicine & Science in Sports & Exercise 927 This question may be answered based on the premise that the mRNA and protein expression of Myo-D in the present study was significantly increased with heavy resistance training; however, the increases were not preferentially af- fected by Cr supplementation (Figs. 2 and 4). Other studies have shown that resistance exercise increases the expression of Myo-D (11,24). In our previous study (25), the creatine group underwent a smaller increase in the mRNA expres- sion of the MHC Type IIx compared with Type I and IIa, whereas the corresponding IIx protein isoform was signifi- cantly decreased. It is apparent from previous research that the training load (6–8 RM) employed during heavy resis- tance training results in decreases in the MHC Type IIx protein isoform (5,25). Because the Type I and IIa MHC isoforms were increased in our previous study (25), and Myo-D expression seems to preferentially occur in Type IIx MHC mRNA (24) and in Type IIb muscle fibers (12), the results from the present study suggest that Myo-D expres- sion is not as responsive to Cr as myogenin and MRF-4. Therefore, Cr does not appear to serve as a transcriptional co-regulator of Myo-D expression. It has been shown that all four MRF bind the same sequence within the enhancer of the M-CK gene; however, MRF-4 does not transactivate the enhancer as effectively as myogenin (4). Additionally, myogenin regulates binding of the M-CK en- hancer for transcriptional activation. That MRF-4 binds the M-CK enhancer without activating transcription as effectively as myogenin suggests domains in addition to those required for DNA binding are important for transcriptional activation. De- spite the fact that MRF-4 functions as a positive transcriptional regulator, it is conceivable that Cr may function as a specific transcriptional co-regulator that interacts and affects the tran- scriptional efficiency of MRF-4 (8). The ability of myogenin and MRF-4 to discriminate between muscle-specific enhancers of target genes suggests that MRF-4 may selectively regulate unique sets of muscle-specific genes such as M-CK due to different developmental hypertrophic pathways or neural ac- tivity patterns (21) independent of the efficiency of transcrip- tional activation. Therefore, our present results indicate that Cr supplementation and heavy resistance training seem to provide some additional impetus for increasing myogenin and MRF-4 expression that appears to up-regulate the expression of the M-CK gene over that of Myo-D and Myf5. In regard to Myf5, based on the results of the present study, and those of Hespel and colleagues (11), its expression does not appear to be affected by resistance training. This is likely due to the fact that Myf5 plays a significant role in myoblast determination and is typically only expressed in low levels in adult muscle (20). In the present study, when compared with the placebo and control groups, we showed that Cr supplementation and heavy resistance training preferentially induced a 64% in- crease in the expression of M-CK mRNA (Fig. 3), which was correlated with increases of 35% and 63% in the ex- pression of myogenin (r 0.916) and MRF-4 (r 0.883) protein, respectively (Fig. 4). In addition, myogenin and MRF-4 mRNA expression were increased by 61% and 65%, respectively (Fig. 2), but neither was correlated to M-CK. The role of M-CK is to catalyze the biochemical reactions involved in ATP resynthesis that would occur during heavy resistance training. An up-regulation in M-CK expression, which is strictly predicated on transcriptional activity, is initiated by MRF and would be beneficial in potentially enhancing muscular strength and performance during resis- tance training. As a result, the enhanced transcriptional activity of the M-CK gene observed in the present study, potentially facilitated by increases in myogenin and MRF-4, conceivably may have contributed, at least in part, to the 65% increase in strength observed in the Cr supplementa- tion group in our earlier study (25). FIGURE 4—Expression of protein for (A) Myo-D, (B) myogenin, (C) MRF-4, and (D) Myf5. *Significant group test interaction; ‡significantly different from CON; †signifi- cantly different from PLC (P < 0.05). 928 Official Journal of the American College of Sports Medicine http://www.acsm-msse.org Because heavy resistance training stimulates muscle pro- tein synthesis in humans, the amplified responses of myo- genin and MRF-4 mRNA and protein expression as a result of Cr supplementation seem to have increased transcription of the M-CK gene. In addition, the elevations in myogenin and MRF-4 expression could have also facilitated an en- hanced transcription of Type I, IIa, and IIx MHC mRNA molecules based on observations from our previous studies (24,25), or from an increased rate of translation of each molecule of MHC mRNA (23). Therefore, Cr supplemen- tation may increase the mRNA template available for trans- lation and muscle-specific protein synthesis (i.e., MHC iso- forms and M-CK) in muscle undergoing hypertrophy resulting from alterations in transcriptional efficiency, tran- scriptional capacity, and/or mRNA stability that are depen- dent on the myogenic regulation of myogenin and MRF-4. The results of this study suggest that 12 wk of Cr sup- plementation, in conjunction with heavy resistance training, increases the mRNA expression of M-CK by way of a pretranslational mechanism, likely due to the concomitant increases in the expression of myogenin and MRF-4. The increases in myogenin, MRF-4, and M-CK expression may also offer a possible mechanism as to the increases in MHC isoform mRNA expression and muscle strength observed in our previous study (25). 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