Longer periods (e.g., 10–12 wk) of creatine (Cr) supplementation combined with heavy resistance training 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 increased 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). Although these earlier studies did not attempt to determine an underlying mechanism, it was speculated that Cr supplementation 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 demonstrated respective increases of 33%, 31%, and 36% for MHC Type I, IIa, and IIx mRNA, 17% and 16%, respectively, 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 suggested that the increases in MHC expression at the pre- and posttranslational level may possibly lead to increases in MHC synthesis as originally suggested by Ingwall and colleagues (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 colleagues (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 training 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 regulatory region of a specific DNA sequence that is present in the promoters and enhancers downstream of some muscle-specific genes such as MHC, myosin light chain, α-actin, troponin-I, and creatine kinase (CK), thereby activating their transcription (16). In general, Myo-D and Myf5 are involved 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 primarily 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 intracellular 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, promoting 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 potential 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 utilized the remaining muscle samples from our previous study (25) in an attempt to determine the effects of Cr supplementation 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 expression 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 expression and muscle strength following heavy resistance training seen in our previous study (25).
MATERIALS AND METHODS
In the present study, we used remaining muscle samples from our previous study (25) in which subjects signed university-approved informed consent 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, however, this study employed 22 untrained (no consistent, structured 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 training+Cr group [CRT, (N = 8)], or a resistance training+placebo group [PLC, (N = 8)]. Percutaneous 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 posttraining biopsy, attempts were made to extract tissue from approximately the same location by using the prebiopsy scar, depth markings on the needle, and a successive incision that was made approximately 0.5 cm to the former from medial to lateral. Muscle specimens were immediately frozen in liquid nitrogen and then stored at −70°C for later analysis.
During the course of the resistance-training period, subjects trained with 3 sets of 6–8 repetitions at a relative intensity of 85–90% of their one repetition maximum (1-RM) for 12 wk. Training sessions occurred 3 d·wk−1 on a Monday-Wednesday-Friday format for approximately 30 minutes per session employing the leg press, knee extension, and knee curl exercises. During each training session, 3 min of rest was allowed between each set and exercise. A 1-RM test was performed for each exercise 5 h before Monday’s training session every 2 wk. Due to the possibility of fatigue as a result of excessive trials (i.e., > 5 trials) during 1-RM testing, a goal of only five trials was set for all 1-RM testing sessions throughout the study. If necessary, after each testing session adjustments were made to the training loads so that subjects continued to train at the appropriate intensity. The CON group involved no resistance training or Cr supplementation during the course of the study. In addition to resistance training, CRT received 6 g·d−1 of Cr monohydrate (n[aminoiminomethyl]-N-methyl glycine) for 12 wk while PLC received the equal daily amount of dextrose as a placebo. The CRT and PLC supplements were provided in chewable tablet form (1 g/tablet) and matched in taste (Nutra Sense, Inc., Shawnee Mission, KS). Subjects were instructed to consume two tablets 3× d−1 throughout the duration of the study. The subjects’ diets were not standardized; however, with the exception of Cr supplementation all subjects were informed not to change their dietary habits during the course of the study.
Total RNA isolation.
Total cellular RNA was extracted from the homogenate of biopsy samples with a monophasic solution of phenol and guanidine isothiocyanate contained within the TRI-reagent (Sigma Chemical Co., St. Louis, MO). The RNA concentration was determined by optical density (OD) at 260 nm (by using an OD260 equivalent to 40 μg·μL−1), and the final concentration was adjusted to 1 μg·μL−1. In line with our previous work, this procedure yielded undegraded RNA, free of DNA and proteins as indicated by an OD260/OD280 ratio of approximately 2.0 (24,25). The RNA samples were stored at −70°C until later analysis.
Reverse transcription and cDNA synthesis.
Two μg of total skeletal muscle RNA were reverse transcribed to synthesize cDNA. A reverse transcription (RT) reaction mixture [2 μg of cellular RNA, 10× reverse transcription buffer (20 mM Tris-HCL, pH 8.3; 50 mM KCl; 2.5 mM MgCl2; 100 μg of bovine serum albumin per milliliter), a dNTP mixture containing 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.8 μM MgCl2, 1.0 u·μL−1 of rRNasin (ribonuclease inhibitor), 0.5 μg·μL−1 of oligo(dT)15 primer, and 25 u·μg−1 of AMV reverse transcriptase enzyme (Promega, Madison, WI)] was incubated at 42°C for 60 min, heated to 95°C for 10 min, and then quick chilled on ice. Starting template concentration was standardized by adjusting the RT reactions for all samples to 200 ng before PCR amplification (24,25).
Oligonucleotide primers for PCR.
The following 5' sense and 3' antisense oligonucleotide primers were used to isolate the mRNA expression of Myo-D (5' primer: bases 1239–1259, 3' primer: bases 1431–1411, GenEMBL AC X56677), myogenin (5' primer: bases 1986–2006, 3' primer: bases 2145–2125, GenEMBL AC X62155), MRF-4 (5' primer: bases 614–634, 3' primer: bases 1108–1088, GenEMBL AC XM006691), Myf5 (5' primer: bases 360–380, 3' primer: bases 853–833, GenEMBL AC X14894), and M-CK (5' primer: bases 336–356, 3' primer: bases 783–763, GenEMBL AC M14780). We have previously shown the Myo-D and myogenin primers to amplify respective PCR fragments of 504 and 495 base pairs (bp) (24), whereas the MRF-4, Myf5, and M-CK primers amplify fragments of 495, 494, 448 bp, respectively (Willoughby, D. S., unpublished observations, May 2002).
Relative control standard oligonucleotide primers for PCR.
Due to its consideration as a constitutively expressed “housekeeping gene,” glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an external reference standard for detecting the relative change in the quantity of MHC mRNA isoforms using PCR. For GAPDH mRNA (5' primer: bases 616–636, 3' primer: bases 1189–1169, GenEMBL AC NM 002046), we have previously shown these primers to amplify a PCR fragment of 574 bp (24,25).
Two hundred ng of cDNA were added to each of the six 25-μL PCR reactions for GAPDH, Myo-D, myogenin, MRF-4, MYF-5, and M-CK. Specifically, each PCR reaction contained the following mixtures: [10× PCR buffer, 0.2 μM dNTP mixture, 1.0 μM of a cocktail containing both the sense and antisense RNA oligonucleotide primers (Ransom Hill Biosciences, Ramona, CA), 2 mM MgCL2, 1.0 u·μL−1 of Taq DNA polymerase (Sigma), and nuclease-free dH2O]. Each PCR reaction was amplified with a thermal cycler (Bio Rad, Hercules, CA). The amplification profile involved a denaturation step at 95°C for 30 s, primer annealing at 55°C for 30 s, and extension at 72°C for 60 s (24,25). To help control for differences in amplification efficiency during thermocycling, all PCR reactions were prepared from the same stock solution. Also, the number of cycles was optimized at 25 so that the amplified signal was still on the linear portion of a plot with the yield expressed as a function of the absorbance at OD260 and the number of cycles for the four target amplifications (Fig. 1). The specificity of the PCR was demonstrated with an absolute negative control using a separate PCR reaction containing no cDNA. To assess reliability between amplifications, two separate PCR amplifications were performed for each sample to control for systemic differences between samples that could affect amplification efficiencies. Intra-assay coefficients of variation for the two PCR runs for all subjects were performed and resulted coefficients of variation of 3.14%, 3.24%, 3.96%, 3.65%, 3.94%, and 4.04%, respectively, for GAPDH, Myo-D, myogenin, MRF-4, Myf5, and M-CK mRNA.
The DNA within each amplified PCR reaction was purified of contaminants such as primer dimers and amplification primers using the Wizard PCR Preps DNA Purification System (Promega, Madison, WI). Aliquots of each remaining purified PCR reaction were used to quantify mRNA spectrophotometrically at a wavelength of OD260 at which point the mRNA concentration of each MHC isoform was calculated and normalized relative to GAPDH (24,25). It should be noted, however, that this method of PCR quantitation determines relative mRNA concentration only and should not be interpreted as absolute concentration values.
Myo-D, myogenin, MRF-4, and Myf5 quantification.
Muscle protein was isolated from the organic phase of the total RNA isolation using ethanol, isopropanol, and a 95% ethanol solution containing 0.3 M guanidine hydrochloride (24,25). The protein concentrations of Myo-D, myogenin, MRF-4, and Myf5 were determined in triplicate and the average concentration reported using an enzyme-linked immunoabsorbent assay (ELISA) using specific rabbit IgG polyclonal antibodies (Santa Cruz Biotech, Santa Cruz, CA). The anti-Myo-D antibody does not cross react with myogenin, MRF-4, or Myf5, and the myogenin antibody does not cross react with Myo-D, MRF-4, or Myf5. The secondary antibody immunoglobulin-G (IgG) was conjugated to the enzyme horseradish peroxidase (ICN Biomedical, Aurora, OH). Protein concentrations were determined at an optical density of 450 nm with a microplate reader (Bio Rad) and expressed relative to muscle wet weight. Intra-assay coefficients of variation were determined for each triplicate for all subjects and revealed coefficients of 3.58%, 4.31%, 3.95%, and 4.62%, respectively, for Myo-D, myogenin, MFR-4, and Myf5.
Statistical analyses were performed by utilizing separate 3 × 2 [group (CON, PLC, CRT)] × test (pretraining, posttraining) factorial analyses of variance (ANOVA) with repeated measures for each criterion variable. Further analysis of the main effects for group and test were performed by separate one-way ANOVA. Significant between-group differences were determined involving the Newman-Keuls post hoc test. However, to protect against Type I error, the conservative Hunyh-Feldt epsilon correction factor was used to evaluate observed within-group F-ratios. The effects of sample size were determined using the partial eta2 (η2) statistic. Statistical power was also determined. Bivariate correlations between M-CK mRNA and the mRNA and protein expression of Myo-D, myogenin, MRF-4, and Myf5 were performed with the Pearson product moment correlation coefficient procedure. A probability level of ≤ 0.05 was adopted throughout.
Myo-D, myogenin, MRF-4, Myf5, and M-CK mRNA expression.
Significant group × test interactions were located for Myo-D, myogenin, MRF-4, and M-CK mRNA (P < 0.05), whereas no significant interaction or main effects were noted for Myf5 (Table 1). Post hoc results of the main effects showed that for myogenin, MRF-4, and M-CK, CRT was significantly increased when compared with CON and PLC, whereas PLC was also greater than CON (P < 0.05). Myo-D was significantly increased in PLC and CRT when compared with CON (P < 0.05); however, PLC and CRT were not different from one another (Figs. 2 and 3).
Myo-D, myogenin, MRF-4, and Myf5 protein expression.
Significant group × test interactions were located for Myo-D, myogenin, and MRF-4 (P < 0.05), whereas no significant interaction or main effects were located for Myf5 (Table 1). Post hoc results of the main effects showed that for myogenin and MRF-4, CRT was significantly increased when compared with CON and PLC, whereas PLC was also greater than CON (P < 0.05). Myo-D was significantly increased in PLC and CRT when compared with CON (P < 0.05); however, PLC and CRT were not different from one another (Fig. 4).
Correlations between M-CK and Myo-D, myogenin, MRF-4, and Myf5.
Relative M-CK mRNA concentration was correlated with the relative mRNA and protein concentrations for Myo-D, myogenin, MRF-4, and Myf5 for the three groups. Significant correlations were shown to exist after the 12 wk of resistance training for CRT between M-CK and myogenin protein (r = 0.916, P = 0.029) and M-CK and MRF-4 protein (r = 0.883, P = 0.045).
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 expression 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·d−1. 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 supplemented Cr as a dosage of 6 g·d−1. 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 individuals 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 superior 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. Consequently, the respective changes in MHC isoform composition 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 hypertrophy 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 resistance 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 question is raised in regard to the present study; why were myogenin and MRF-4, but not Myo-D, preferentially increased with Cr supplementation?
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 affected 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 expression of the MHC Type IIx compared with Type I and IIa, whereas the corresponding IIx protein isoform was significantly decreased. It is apparent from previous research that the training load (6–8 RM) employed during heavy resistance 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 expression 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 enhancer 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. Despite 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 transcriptional 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 activity patterns (21) independent of the efficiency of transcriptional 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% increase in the expression of M-CK mRNA (Fig. 3), which was correlated with increases of 35% and 63% in the expression 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 resistance 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 supplementation group in our earlier study (25).
Because heavy resistance training stimulates muscle protein synthesis in humans, the amplified responses of myogenin 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 enhanced 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 supplementation may increase the mRNA template available for translation and muscle-specific protein synthesis (i.e., MHC isoforms and M-CK) in muscle undergoing hypertrophy resulting from alterations in transcriptional efficiency, transcriptional capacity, and/or mRNA stability that are dependent on the myogenic regulation of myogenin and MRF-4.
The results of this study suggest that 12 wk of Cr supplementation, 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). The results of the present study suggest that Cr conceivably operates as a transcriptional co-regulator in specifically regulating the expression of myogenin and MRF-4, and that the increased expression of these two MRF are correlated to increases in the expression of M-CK gene expression. As a result, the effects of Cr supplementation on myogenin and MRF-4 expression, along with subsequent increases in M-CK expression, apparently lead to increases in MHC gene expression within skeletal muscle that occurs during heavy resistance training.
This study was supported in part by a donation of creatine monohydrate and placebos from NutraSense, Inc., Shawnee Mission, KS.
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