During each condition, preexercise/presupplement muscle biopsies were obtained from the vastus lateralis at a depth between 2 and 3 cm. The muscle samples were extracted under local anesthesia using 1% lidocaine after having the area shaved clean of leg hair and cleansed with an antiseptic soap. An incision approximately one-quarter of an inch was made using a sterile razor. A sterilized 5-mm Bergstrom biopsy needle with suction applied to its end was then inserted into the incision, suction was applied, and the muscle tissue was excised in a double-chop fasion. After collection of the sample, the muscle tissue was placed into a coded cryogenic tube and flash frozen in liquid nitrogen. Samples were then transferred for long-term storage into a −80°C ultralow freezer until follow-up analyses. It should be noted that postexercise biopsies were collected from the same area of the vastus lateralis by repeatedly reentering the same incision. Although repeated biopsies have been shown to elicit a satellite cell activation response in the muscle bed, the immunodetection of these events have been shown to be prolonged (i.e., >24 h after repeated biopsies) (17). Further, this same data suggest that eccentric exercise increases satellite cell activation (as determined by the presence of PCNA+ cells) by 163% at 6 h after exercise (P < 0.05), whereas a marker of T-cell infiltration modestly increased and was the only indicator of leukocyte presence in the muscle bed up to this time point after exercise. Hence, these data suggest that the influx of leukocytes is marginal up to 6 h after exercise and that the contamination of myogenic DNA/RNA/protein with leukocyte DNA/RNA/protein at our postexercise time points was likely minimal.
Upon arrival at each of the experimental conditions, participants were assigned to ingest one of three supplements, at random, under the supervision of the research staff. The supplement groups were as follows: a) 25 g of whey protein isolate (PRO), b) 25 g of maltodextrin (CHO), and c) water artificially sweetened with Splenda® (PLC). Each supplement was mixed with 350 mL (12 fl oz) of cold water and ingested 30 min before the start of the workout under the supervision of researchers.
Muscle total RNA isolation and determination.
Approximately 20 mg of muscle was homogenized using a tight-fitting pestle in proprietary Tri reagent (Sigma, St. Louis, MO) containing a monophasic solution of phenol and guanidine isothiocyanate. All muscle samples were preweighed, placed into an autoclaved microcentrifuge tube, and homogenized using a micropestle and 500 μL of Tri reagent per 25 mg of tissue. After homogenization, approximately 100 μL of chloroform was added to each sample, and the samples were vortexed for 15 s and allowed to stand at room temperature for 10 min. Samples were then centrifuged at 12,000 rpm at 4°C for 15 min. The upper aqueous phase was transferred into a new autoclaved microcentrifuge tube, and approximately 250 μL of 100% isopropanol per 500 μL of Tri reagent was used to precipitate the RNA from the aqueous phase. The RNA pellet was exposed to subsequent ethanol washes and finally dissolved in 80 μL of RNase-free water with repeated pipetting or vortexing. The diluted RNA samples were stored at −80°C until later analyses.
Total RNA concentrations for each sample were determined using a high-sensitivity RNA analysis kit with the Experion Automated Electrophoresis platform (Bio-Rad Laboratories, Hercules, CA). This method separates and quantitates mRNA ranging from 50 to 6000 nucleotides in length using a laser-excitable RNA stain and RNA ladder provided by the manufacturer. Furthermore, this procedure was found to yield undegraded RNA, free of DNA and proteins as indicated by prominent 28S and 18S ribosomal RNA bands given through visual electropherograms (data not shown). The preparation of reagents and the RNA ladder were performed according to the manufacturer's instructions. Furthermore, all RNA samples and the RNA ladder were thawed on ice during the assay to preserve mRNA integrity. A subset of samples was assayed in duplicate, and the average between-duplicate coefficient of variation (CV) was <5%.
After total RNA concentration determination, 200 ng of total skeletal muscle RNA was reverse transcribed to synthesize complementary DNA (cDNA). For each sample, a reverse transcription reaction mixture (40-μL total: 1) 200 ng of total cellular RNA diluted to 30 μL with RNase-free water; 2) 8 μL of 5× reverse transcription buffer, a dNTP mixture containing dATP, dCTP, dGTP, and dTTP, MgCl2, RNase inhibitor, and an oligo(dT)15 primer; and 3) 2 μL of MMLV reverse transcriptase enzyme; Bio-Rad Laboratories) was incubated at 42°C for 40 min, heated to 85°C for 5 min, and then quickly chilled on ice, yielding the cDNA product. Finally, 40 μL of RNase-free water was added to bring the cDNA solutions up to 80 μL, and cDNA solutions were subsequently frozen at −80°C until semiquantitative real-time polymerase chain reaction (PCR) was performed.
Real-time PCR to detect postexercise expression of genes of interest
Forward and reverse oligonucleotide primer pairs were constructed using the commercially available Beacon Designer software (Bio-Rad Laboratories) and synthesized (Integrated DNA Technologies, Coralville, IA) (Table 2). Beta-actin was used as an internal reference for detecting relative change in the quantity of target mRNA because of its consideration as a constitutively expressed housekeeping gene after resistance exercise (16). Two microliters of cDNA was added to each of the seven separate PCR reactions for CDK4, CYCLIN D1, mechano-growth factor (MGF), myogenic differentiation factor 1 (MYOD), P21CIP1, P27KIP1, and beta-actin. Each PCR reaction contained the following mixtures: 12.5 μL of SYBR Green Supermix (Bio-Rad Laboratories; 100 mM of KCl mixture, 40 mM of Tris-HCl, 0.4 mM of each deoxynucleoside triphosphate, 50 U·μL−1 of iTaq DNA polymerase, 6.0 mM of MgCl2, SYBR Green I, 20 nM of fluorescein), 1.5 μL of forward and reverse primers, and 7.5 μL of nuclease-free dH2O. The PCR reactions were amplified with a thermal cycler (Bio-Rad Laboratories), whereby the amplification sequence involved an initial 3-min cycle at 95°C to activate the Taq polymerase followed by a 40-cycle period with a denaturation step at 95°C for 10 s and a primer annealing or extension step at 55°C for 30 s. All assays were performed in duplicate, and the critical threshold coefficient of variation (CV) values for each gene were as follows: CDK4 = 0.8%, CYCLIN D1 = 1.0%, MGF = 1.1%, MYOD = 1.0%, P21CIP1 = 0.8%, and P27KIP1 = 1.0%. Further, the plate-to-plate CV value for beta-actin critical threshold values using a control cDNA sample was <1.0%.
Gene expression data were calculated using the Pfaffl (27) method (i.e., 2−ΔΔCT assuming 100% primer binding efficiency), where
Further, gene expression values were expressed as percent change from 0, which was the value given to preexercise gene expression values, as in previous literature (15). As an example, if the expression of a gene before exercise carried an arbitrary value of 10 and this value was reduced to 1 after exercise, this would be expressed as follows:
Muscle DNA determination.
To determine muscle DNA concentrations, we allocated a modified protocol published by Haddad and Adams (7). Before homogenization, all muscle samples were visibly cleansed of blood or leukocyte and fat tissue and blotted dry. Each specimen was then weighed, and ∼20 mg of muscle was homogenized on ice with 400 μL of homogenizing buffer (250 mM of sucrose, 100 mM of KCl, 5 mM of ethylenediaminetetraacetic acid, 10 mM of Tris-HCl, pH 6.8) using a tight-fitting pestle. For muscle DNA determination, 30 μL of homogenate was assayed with 1 mL of a fluorometric dye (Hoechst 33258 dye; Sigma), and the fluorescent signal was detected using a single cuvette-based fluorometer (Bio-Rad Laboratories). The buffer for this assay contains a high salt concentration (2 M of NaCl, 10 mM of Tris-HCl, pH 7.4, 1 mM of ethylenediaminetetraacetic acid), and under the high salt condition, the dye binds only to the DNA and not to the RNA component of the homogenate. Further, this is reported to have a high affinity for AT-rich sequences in the minor groove of double-stranded DNA and, when excited at 360 nm, emits a fluorescent signal that is detectable with a 460-nm filter. Before assaying batch samples, a new standard curve was generated using calf thymus DNA provided by the manufacturer (note, R 2 values for each standard curve exceeded 0.99). The average duplicate CV values for all samples were <10%.
Because of the large variance commonly attributed to molecular variables (i.e., interindividual differences in DNA, mRNA, and protein concentrations), the Shapiro-Wilk statistic was performed for each dependent variable to ensure a normality in distribution existed. For normally distributed molecular data, one-way repeated-measure ANOVA were used to determine main and interactive effects, whereas dependent t-tests with Bonferroni corrections were used to compared corresponding between-condition time points.
If data exhibited a nonnormal distribution (i.e., skewness and/or kurtosis > 1.96 or Shapiro-Wilk statistic P < 0.05) and standard transformation approaches were not possible, a nonparametric approach as has been previously reported (4,35) was used to analyzed changes in gene expression. The Kruskal-Wallis statistic was used to detect between-condition differences at differing time points with nonnormally distributed data. If this statistic yielded a significant P value, then Mann-Whitney U statistics were used as a post hoc measure to determine which condition(s) was significantly different. The Friedman test was used to detect changes in nonnormally distributed data among all condition over time. If the Friedman statistic yielded a P < 0.05, then the Wilcoxon signed rank tests were used as a post hoc measure to determine which time points were significantly different. Significance for all statistical analyses was determined using an alpha level of 0.05.
For muscle total RNA (Fig. 3), a Kruskal-Wallis test determined that there were no between-condition differences before (P = 0.99), 2 h (P = 0.49), or 6 h (P = 0.31) after exercise. Likewise, a Friedman test determined that there was no significant increase among all conditions over time (P = 0.50).
Normality distribution analyses revealed that muscle [DNA] and total [RNA] were nonnormally distributed. Preexercise and postexercise muscle [DNA] values are presented in Figure 3. A Kruskal-Wallis test determined that there were no between-condition differences before (P = 0.52), 2 h (P = 0.94), or 6 h (P = 0.25) after exercise. A Friedman test determined that there was a significant increase among all conditions over time (P = 0.004). A follow-up Wilcoxon signed rank test determined that there was no change in muscle DNA 2 h postexercise (P = 0.31) but a significant increase in muscle DNA 6 h (P = 0.002) postexercise during all conditions. Within-group Wilcoxon signed rank tests revealed that muscle DNA increased at 6 h postexercise within the PRO (P = 0.007) and PLC (P = 0.028) groups.
Postexercise changes in the expression of targeted genes.
Normality distribution statistics revealed that the expression patterns of all analyzed genes were nonnormally distributed. Percent changes in CDK4 mRNA expression after exercise are presented in Figure 4. A Kruskal-Wallis test determined that there were no between-condition differences 2 h (P = 0.27) or 6 h (P = 0.55) after exercise. A Friedman test determined that there was a significant increase in CDK4 mRNA expression among all conditions over time (P < 0.001). A follow-up Wilcoxon signed rank test examining all groups determined that the change in CDK4 mRNA expression increased 6 h after exercise across all conditions (P < 0.001). Within-group Wilcoxon signed rank tests determined that CDK4 mRNA expression increased 6 h postexercise within the PRO group (P = 0.009) and the PLC group (P = 0.013).
Percent changes in CYCLIN D1 mRNA expression after exercise are presented in Figure 4. A Kruskal-Wallis test determined that between-condition differences approached significance at 2 h (P = 0.08) but was not present 6 h (P = 0.57) after exercise. A follow-up Mann-Whitney U test revealed that CYCLIN D1 mRNA expression tended to be greater during the PRO versus CHO condition 2 h after exercise (P = 0.063). A Friedman test determined that there was no significant change in CYCLIN D1 mRNA expression among all conditions over time (P = 0.74), although the trend difference between PRO and CHO at 2 h postexercise obviated the need for within-group analyses. In this regard, within-group Friedman tests determined that the change in CYCLIN D1 mRNA expression approached significance over time within the CHO group only (P = 0.06). Follow-up within-CHO Wilcoxon signed rank tests determined that there was a significant decrement in the mRNA expression of CYCLIN D1 2 h postexercise (P = 0.007).
Percent changes in MGF mRNA expression after exercise are presented in Figure 5. A Kruskal-Wallis test determined that there were no between-condition differences 2 h (P = 0.27) or 6 h (P = 0.55) after exercise. A Friedman test determined that there were no significant change in MGF mRNA expression among all conditions over time (P = 0.44).
Percent changes in MYOD mRNA expression after exercise are presented in Figure 6. A Kruskal-Wallis test determined that there were no between-condition differences 2 h (P = 0.75) or 6 h (P = 0.28) after exercise. A Friedman test determined that there was a significant increase in MYOD mRNA expression among all conditions over time (P = 0.015). A follow-up Wilcoxon signed rank test determined that there was an increase in MYOD mRNA expression 6 h (P = 0.001) postexercise during all conditions. Within-group Wilcoxon signed rank tests determined that the change in MYOD mRNA expression increased 6 h postexercise within the PRO group (P = 0.001) and the CHO group (P = 0.007).
Percent changes in P21CIP1 mRNA expression after exercise are presented in Figure 7. A Kruskal-Wallis test determined that there were no between-condition differences 2 h (P = 0.53) or 6 h (P = 0.23) after exercise. A Friedman test determined that there was a significant increase in P21CIP1 mRNA expression among all conditions over time (P < 0.001). Wilcoxon signed rank tests determined that there was a significant increase in P21CIP1 mRNA expression 2 h (P < 0.001) and 6 h (P < 0.001) postexercise during all conditions. Within-group Wilcoxon signed rank tests determined P21CIP1 mRNA expression increased at 2 and 6 h postexercise within the all supplement groups (P < 0.001).
Percent changes in P27KIP1 mRNA expression after exercise are presented in Figure 7. A Kruskal-Wallis test determined that there were no between-condition differences 2 h (P = 0.74) or 6 h (P = 0.39) after exercise. A Friedman test determined that there was a significant decrease in P27KIP1 mRNA expression among all conditions over time (P < 0.001). A follow-up Wilcoxon signed rank test determined that there was a significant decrease in P27KIP1 mRNA expression 2 h (P = 0.001) and 6 h (P < 0.001) postexercise during all conditions. Within-group Wilcoxon signed rank tests determined that P27KIP1 mRNA expression decreased 6 h postexercise (P = 0.008) in the PRO group as well as 2 h (P = 0.013) and 6 h (P = 0.007) postexercise within the CHO group.
Although various studies have microscopically demonstrated that resistance training acutely increases satellite cell number by 100%-200% 24 h after exercise (5,6,17,24), limited research has investigated the impact of preexercise macronutrient ingestion on satellite cell activity. Hulmi et al. (11) recently determined that CDK2 mRNA increases 300% 48 h after resistance exercise when 15 g of whey was administered before and after one bout of resistance exercise compared with a placebo/exercise group, which experienced a 50% increase in CDK2 mRNA after exercise (P > 0.05). However, these authors also reported that the expression patterns of P21CIP1, P27KIP1, MYOGENIN, and MYOD were unaltered 48 h after exercise. Conversely, our results demonstrate that, independent of preexercise macronutrient ingestion, an acute bout of conventional resistance training (i.e., nine sets of lower-body exercise training using a lifting intensity of 80% 1RM) with or without preexercise nutrient ingestion in college-aged men 1) increased muscle [DNA] 6 h postexercise (+40%, P < 0.05), 2) increased CDK4 expression 6 h postexercise (+86%, P < 0.05), 3) increased MYOD expression 6 h postexercise (+98%, P < 0.05), 4) decreased P27KIP1 expression 2 h (−35%, P < 0.05) and 6 h (−59%, P < 0.001) postexercise, and 5) increased P21CIP1 expression substantially 2 and 6 h postexercise (+1.250% and +4.670%, respectively, P < 0.001). These divergent findings are seemingly due to the fact that the postexercise nadir of cell cycle-regulating gene expression occurs transiently (i.e., 6-24 h) after exercise.
In the current study, we assessed the amount of DNA present in muscle homogenates using a highly sensitive fluorometric method that has been similarly performed in rodents in to examine satellite cell activity (1). Likewise, one bout of simulated resistance exercise in rats has been shown to increase muscle DNA by 20% (P < 0.05) at 12 and 36 h after exercise in rats (34), this finding being attributed to an increase in satellite cell proliferation. Interestingly, although exercise increased muscle DNA 6 h after exercise, neither whey protein nor CHO feedings before exercise enhanced this response when compared with a noncaloric placebo. In this regard, Olsen et al. (25) demonstrated in humans that 16 wk of resistance training and concomitant protein supplementation (20 g per session) did not experience an increase satellite cell number when compared with a nonsupplemented/nonexercising cohort, further confirming that protein ingestion may be limited in its ability to impact satellite cell activity. Therefore, our findings in concert with those of Olsen et al. (25) demonstrate that protein ingestion surrounding resistance training seemingly operates through other anabolic mechanisms (i.e., mTORC1-stimulated postmitotic myofibrillar protein synthesis ) to stimulate muscle hypertrophy.
The concomitant increases in DNA and the differential expression of genes that regulate satellite cell activity in the current investigation seemingly indicate that that satellite cell proliferation, not differentiation, predominantly occurs inside skeletal muscle at early postexercise time points. The significant increase in CDK4 gene expression at 6 h after exercise when all conditions were collapsed suggests that exercise may induce the overexpression of G1 phase cyclins to increase satellite cell activity. In this regard, an increase in CDK4 enzyme activity has been associated with an increase in myoblast proliferation and a decrease in differentiation in culture (36). The significant decrement in P27KIP1 gene expression at both postexercise time points in the current study also supports the aforementioned hypotheses, given that the ectopic overexpression of p27kip1 in murine satellite cells decreases the IGF-1-mediated induction in satellite cell proliferation (3). As mentioned, there was substantial increase in P21CIP1 expression at 2 and 6 h after exercise. The protein encoded by this gene is classified as a CDK inhibitor that binds to and inhibits CDK-cyclin complexes in proliferating cells (30), although others have suggested that the protein encoded by this gene assists in satellite cell proliferation (10), whereas others have shown it to be highly up-regulated (i.e., 6-fold) immediately after eccentric exercise (4). Therefore, the notion that p21 enhances satellite cell activity (vs an inhibition hypothesis) inherently makes sense, given that satellite cell proliferation and P21CIP1 mRNA expression seemingly increase in a parallel fashion after resistance training. Finally, the 6-h postexercise increase in MYOD gene expression across all conditions is in agreement with other investigations (28,29,35). MYOD is classified as a basic helix-loop-helix transcription factor that acts in a heterodimer complex to induce differentiation in a subset of proliferating myoblasts when the addition of a myonuclear domain is warranted. Because no research has indicated that postmitotic fiber myonuclear number increases after one bout of resistance training, which would indicate that differentiation and fusion had occurred, it is surprising that our findings indicate that this gene is up-regulated transiently after exercise. Nonetheless, future investigations should examine why MYOD mRNA expression increases without differentiation in response to an acute bout resistance exercise.
It is atypical that exercise elicited a decrease in the postexercise expression of MGF mRNA 2 h after exercise within the CHO condition and a nonstatistical decrease in this gene 2 and 6 h postexercise during the PRO and PLC conditions. Likewise, it is unusual that exercise did not induce an increase in CYCLIN D1 gene expression within or across all conditions. Past research has demonstrated that exercise dramatically increases the expression of the MGF variant in younger men up to 2.5 h after resistance exercise (8), albeit others have demonstrated that this gene is significantly increased during more prolonged postexercise time points (i.e., 24 h) (22). From a methodological perspective, we allocated the same primer sequences as the aforementioned study to probe the transcripts of this gene, and our PCR melt curve analysis (data not shown) revealed that one gene product was being amplified. Likewise, our RNA analyses using a highly sensitive and reproducible automated microfluidic chip electrophoresis platform indicated that our RNA was of sufficient quantity and quality (12), further making our findings inexplicable. It remains speculative that MGF and CYCLIN D1 may be under translational and not transcriptional control after one exercise bout (i.e., an increase in the translational efficiency of IGF-1 or an increase in protein content without increases in mRNA after mechanical loading, as postulated by Adams and Haddad (1)). In this regard, a summation of exercise bouts may lead to the eventual increased expression of MGF and CYCLIN D1, as evidenced during the early postoperative phases of a synergist ablation rat model (1). Nonetheless, these hypotheses are limited because of the methodological constraints in the current study, and more research is needed to clarify the association between MGF and CYCLIN D1 expression in relation to satellite cell activity after exercise.
In conclusion, this investigation suggests that an acute bout of conventional resistance training with or without preexercise nutrient ingestion in college-aged men increases markers of satellite cell activity up to 6 h after exercise. Furthermore, although past evidence suggests that macronutrient consumption before exercise stimulates an increase in postexercise net muscle protein synthesis (32), this investigation suggests that 25 g of whey protein or CHO ingestion does not affect the genetic expression of select cell cycle regulators and/or muscle DNA up to 6 h after exercise. Future studies should examine other genes and proteins that regulate the cell cycle (i.e., HGF, bFGF, CDK2/6, cyclin A/E, and Delta and Notch isoforms as examples) as well as use immunohistochemistry methods with the intent of determining the early postexercise mitotic events that occur in response to exercise in healthy and diseased (i.e., sarcopenic, dystrophic, and cachexic) populations.
Funding disclosure: This project was internally funded using preexisting laboratory funds.
The authors thank the subjects who participated in this study as well as all laboratory assistants who assisted with data collection and analysis. They also thank Dr. Fadia Haddad from The University of California, Irvine, for graciously volunteering her expertise in assisting with specimen analysis. Supplements and reagents were partially funded using monies from a Young Investigator Grant awarded by the National Strength and Conditioning Foundation to the corresponding author (CK).
The results of the present study do not constitute endorsement by the American College of Sports Medicine.
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Keywords:©2010The American College of Sports Medicine
CYCLINS; CYCLIN-DEPENDENT KINASES; CDK INHIBITORS; RESISTANCE EXERCISE; SKELETAL MUSCLE