Exercise & Sport Sciences Reviews:
Postexercise Myogenic Gene Expression: Are Human Findings Lost During Translation?
Roberts, Michael D.1; Dalbo, Vincent J.2; Kerksick, Chad M.3
1Department of Biomedical Sciences, School of Veterinary Medicine, University of Missouri-Columbia, Columbia, MO; 2Central Queensland University, Institute of Health and Social Science Research, School of Medicine and Applied Sciences, Rockhampton, Queensland, Australia; and 3Department of Health and Exercise Sciences, University of Oklahoma, Norman, OK
Address for correspondence: Chad Kerksick, Ph.D., Department of Health and Exercise Science, University of Oklahoma, 1401 Asp Avenue, Room 104, Norman, OK 73019 (E-mail: firstname.lastname@example.org).
Accepted for publication: June 9, 2011.
Associate Editor: Stephen M. Roth, Ph.D., FACSM
Muscle progenitor cell (MPC) activity is exercise responsive. Post resistance-exercise alterations in myogenic messenger RNAs (mRNAs) found by us and others may initiate these events. However, these mRNA data in the absence of microscopic MPC activity data have limited this interpretation. Alternatively, with our acute exercise data as our basis, we propose that these genes may control other hypertrophic processes in postmitotic fibers.
Enthusiasm has surrounded satellite cells (also referred to as adult myogenic progenitor cells and will be termed as MPCs herein) since the discovery of these cells in frog skeletal muscle by Alexander Mauro in 1961 (17), and this enthusiasm has sprouted into a prevalent research topic, given that MPCs become depleted with aging and in some diseased states (i.e., muscular dystrophies) (28). Resistance exercise has been shown to stimulate MPC activity. For instance, 12 wk of moderate resistance training (i.e., 60%-80% one repetition max or 1RM) partially can recoup lost muscle mass in older people unaccustomed to the rigors of weightlifting; this being partially attributable to type II fiber MPCs returning to youth-like numbers in elder subjects following the training program (31). Recent data from Bamman's laboratory (21) also suggests that an individual's inherent capacity for MPC proliferation in response to months of structured resistance training predicts how well he/she accrues gains in skeletal muscle mass. Tarnopolsky (30) recently authored an elaborate review citing much of his own research, which demonstrates that resistance training is able to reverse age- and disease-associated muscle myopathies related to mitochondrial deoxyribonucleic (DNA) deficiencies. In short, Tarnopolsky claims that "DNA shifting" occurs when exercise-stimulated MPCs differentiate and donate their myonuclei (which lack the mutations or damaged DNA) to the preexisting fibers, which possess mitochondrial DNA deletions or mutations. It is in this simplistic yet complex physiological manner that exercise can be used medicinally to genetically reprogram faulty muscle fibers and delay physiological maladies.
"Myogenic" genes have been linked causally to increasing the proliferation or differentiation of MPCs (17); descriptions of many of these genes are provided in Table 1.
Human investigations examining the resistance exercise-induced alterations in myogenic messenger ribonucleic acid (mRNA) expression have provided invaluable data in regard to how skeletal muscle copes with mechanical loading. For instance, our laboratory (24,25,27) has addressed how acute resistance training transiently alters the mRNA expression of cell cycle regulators as well as myogenic regulatory factors (MRFs). However, limited data exist comparing postexercise MPC proliferation/differentiation patterns to the altered MRF and cell cycle mRNA expression patterns because of the multiple postexercise biopsy numbers and tissue amounts yielded for these analyses. To date, examining cross-sectional muscle sections for Pax-7+, N-cadherin+, c-met+, or other markers to deduce MPC activity is the gold standard technique for in vivo MPC activity, and these studies have undoubtedly yielded intriguing data. However, this technique is seemingly limited because of the fact that the analyzed specimens are in cross section (not in three dimension), multiple labeling may be needed to capture the entire MPC population, and satellite cell numbers per myofiber values are very low, making this parameter susceptible to statistical error (15). More importantly, no studies, to our knowledge, exist, which have examined the acute MPC response to a single bout of resistance training despite the fact that numerous publications have outlined the MRF and cell cycle mRNA expression patterns. Therefore, the purposes of the current review are the following: 1) to briefly summarize our recent work as well as the work of other laboratories with regard to how myogenic gene expression patterns respond to acute resistance training in humans, and 2) to discuss alternative functions of these genes n lieu of the postexercise time course manner in which our laboratory and others have found these mRNAs to be expressed. Although attempts have been made to include a majority of the pertinent studies illuminating these concepts, the authors apologize for excluding some of the literature, which may further detail the subject matter.
MPC ACTIVITY AFTER ACUTE RESISTANCE EXERCISE IN HUMANS
Exercise scientists have determined that resistance training likely stimulates adulthood myogenesis in humans as evidenced by the 24-h posteccentric exercise increases in MPC proliferation as well as chronic (weeks/months) increases in MPC and postmitotic myonuclear number. With regard to the former, Crameri et al. (6) microscopically demonstrated that one bout of three different eccentric leg extensor resistance exercises (totaling 210 repetitions) stimulated a 146%-192% increase in satellite cell number in younger male subjects 2-8 d after exercise. A subsequent study performed by Dreyer et al. (7) demonstrated that one bout of 92 eccentric leg extensions increased satellite cell number per muscle fiber in young men by 141% and in old men by 51%, with this increase being statistically greater in the former group. Similarly, recent data from O'Reilly et al. (19) demonstrated that 300 repetitions of eccentric leg extensor exercises increased satellite cell number by 138%, 147%, and 118% compared with baseline at 1, 3, and 5 d after the bout, respectively. Paulsen et al. (20) employed double-labeling immunofluorescent microscopy techniques to demonstrate that one bout of 70 repetitions of eccentric arm flexions may increase satellite cell differentiation (or the "fusion of myoblasts") in a subset of individuals. As mentioned, however, no studies to date have examined MPC activity acutely (1-12 h after exercise) after a conventional resistance training bout, which obviates the question as to whether MPCs are needed during the initial stages of muscle hypertrophy or repair in novice trainees. We have used a sensitive fluorometric assay and discovered that muscle DNA concentration increased 40% by 6 h after exercise (24). However, this methodology is limited by the potential of non-MPCs (e.g., infiltrating leukocytes after exercise) to obscure the measurement; note, however, that infiltrating leukocytes likely do not infiltrate muscle until later postexercise time points (i.e., 24-48 h), making this an intriguing finding (16). All this evidence suggests that one bout of rigorous eccentric loading stimulates MPC proliferation in human skeletal muscle, although it is uncertain as to whether these resident stem cells are required to facilitate recovery from one bout of "normal" resistance exercise-induced trauma.
MRF AND CELL CYCLE mRNA EXPRESSION OCCURS "OUT OF TURN" AFTER RESISTANCE EXERCISE
Widespread studies examining how resistance exercise affects the expression of myogenic genes are presented in Table 2.
Data from our group at the University of Oklahoma (24) have determined that one bout of conventional lower-body resistance exercise causes the following alterations in human vastus lateralis skeletal muscle: a) increased cdk4 mRNA expression 6 h after exercise (+86%, P < 0.05), b) increased MyoD mRNA expression 6 h after exercise (+98%, P < 0.05), c) decreased p27 mRNA expression 2 h (−35%, P < 0.05) and 6 h after exercise (−59%, P < 0.001), d) substantially increased p21mRNA expression at 2 h (+1250%, P < 0.001) and 6 h after exercise and (+4670%, P < 0.001). We initially hypothesized that the altered expression of MRF and cell cycle mRNAs immediately after a resistance training stimulus was largely due to increases in MPC activity (Figure).
Figure. Enzymes and ...Image Tools
Because Grounds et al. (9) asserted that MPC proliferation occurs 24-48 h after myogenic stimulus, we were next interested in how repeated exercise bouts affected the prolonged expression of these mRNAs in younger versus older male subjects during the 24-48 h after exercise time points after three sequential lower body resistance training bouts that occurred on a Monday, Wednesday, and Friday (26). Contrary to our hypothesis, we discovered that there were no alterations in proliferating cell nuclear antigen protein or the mRNA concentrations of cdk2, cdk4, cyclin D1, p21, or p27 during the 24-48 h after exercise time points after three consecutive resistance exercise bouts. There was a modest, but significant, increase in MyoD mRNA after the first training bout in young men, but this increase was less than we had observed in our previous study (24) demonstrating that this gene peaked 6 h after exercise. Furthermore, data from our second study were in agreement with previous data from our group whereby no alterations in the 24-h postexercise mRNA levels of p21 or myogenin occurred in young and older male subjects after one lower-body exercise bout (25).
It remains possible that the mRNA levels of MRFs and cell cycle genes occur early after exercise and precede changes in protein levels, which, in turn, confer changes in MPC proliferation and differentiation at later postexercise time points (>24 h). However, there is a striking molecular disconnect between exercise-induced MRF and cell cycle mRNA expression patterns and MPC activity after exercise, the most notable being the aforementioned 12.5-fold increase in p21 mRNA 2 h after a resistance training bout, whereas G1-phase mRNAs (cdk4 and cyclin D1) remain statistically unchanged at this time point (24). Given that p21 purportedly confers MPC differentiation by halting the cell cycle, it is confounding that the swift elevation in this mRNA precedes mRNAs that putatively regulate MPC proliferation. Likewise, it is unlikely that myoblast differentiation occurs after one conventional exercise bout, despite the fact that we have shown mRNAs linked to differentiation (i.e., p21 and MyoD) increase within a 6-h postexercise window.
MYOGENIC mRNAs: INITIATORS OF MYOGENESIS OR POSTMITOTIC MYOFIBER PROTEIN SYNTHESIS?
Our work demonstrated that the mRNA expression of p21 peaks 50-fold 6 h after a resistance training bout (24). The notion that MPC nuclei contribute to approximately 4%-8% of the total nuclei pool in mammalian skeletal muscle, compared with postmitotic myofiber nuclei that contribute approximately 50%-70% of muscle bed nuclei (27), suggests that postmitotic fibers may be expressing transcripts, such as p21, which are increased during postresistance exercise time points. In support of this hypothesis, Chen et al. (5) have argued that if cell cycle gene expression changes originate from a subset of exercise-responsive MPCs, then they must be expressing those genes very highly for them to be consistently detectable in whole muscle homogenate.
Various animal models have been used to demonstrate that MRF mRNAs are expressed in both MPCs and mature myofibers. For instance, Kami et al. (11) determined that skeletal muscle crushing in rodents induces the swift expression of the c-jun and c-fos proto-oncogene mRNAs within hours of injury followed by the subsequent increase in myogenin mRNA expression. These events were confined to satellite cell myonuclei as defined by double-labeling adjacent muscle sections with desmin and laminin antibodies. However, other studies have used in situ hybridization techniques to determine that MyoD (13), myogenin (11), and MRF4 (13) mRNA transcripts are expressed in postmitotic fibers as well as MPCs after muscle damage protocols in rodents. Charge et al. (4) used a novel transgenic approach (MD6.0-lacZ transgenic mice whereby a 6 kb proximal enhancer/promoter region of MyoD drives lacZ) to demonstrate that β-galactosidase accumulates in adult muscle fibers and correlates with fiber innervation and maturation. Lowe and Alway (14) demonstrated that stretch-overloaded anterior latissimus dorsi muscles devoid of most MPCs via gamma irradiation were capable of increasing the mRNA expression of MRF4, MyoD, and myogenin mRNAs up to fourfold as assessed by Northern blotting methods.
Given that the aforementioned animal models convincingly demonstrate that MRF mRNA transcripts can be expressed by postmitotic myofibers, the hypothetical functions of these genes in postmitotic fibers after resistance training pose intriguing questions. It is well known that resistance training acutely increases muscle protein synthesis, which peaks at approximately 4-16 h and can remain elevated up to 48 h after exercise (29). Psilander et al. (22) have suggested that MRFs may act in a hierarchical fashion to "regulate hypertrophy" by increasing the mRNA expression of structural proteins (i.e., myosin light chain, troponin, and desmin) in postmitotic fibers. This is a sound hypothesis because postmitotic myofiber structural and contractile proteins contain canonical E box sequences in which MRFs bind to and affect gene transcription rates (1).
Interestingly, Nader et al. (18) reported that differentiated myotubes exhibit a rapamycin-sensitive increased expression of cyclin D1 protein and cyclin D1/cdk 4 kinase activity in culture when treated with mitogenic media. This hallmark study demonstrated a cyclin D1-mediated increase in postmitotic myotube ribosomal RNA content and hypertrophy. These authors also postulated that p21 also may function as a catalyst in postmitotic myotubes to form the cyclin D1/cdk 4 enzyme complex. Although there is a paucity of literature to complement the aforementioned data, evidence from one other cell culture siRNA model suggests that p21 increases protein synthesis in postmitotic mesangial cells that are costimulated with high glucose media and insulin-like growth factor 1 (IGF-1) (8). Taking all of these data into consideration suggests that the immediate postexercise increases in MRF and cell cycle mRNAs that we have observed may occur solely in postmitotic fibers with the purpose of increasing the mRNA expression of genes required for muscle architecture as well as ribosomal mRNAs needed to increase translational capacity. Likewise, our findings (26) that these mRNAs all return to basal levels 24-48 h after exercise (when postexercise MPC activity is presumed to take place) further supports the hypothesis that these genes prominently function to alter the phenotype of postmitotic fibers.
Our laboratory and others have demonstrated that resistance exercise in humans alters the mRNA expression patterns of myogenic genes within a 0- to 12-h postexercise window (24,27). The lack of microscopic MPC data within the 0- to 12-h postexercise window makes it difficult to determine how MRF and cell cycle mRNAs affect MPC activity. Data presented herein demonstrate that some of these genes orchestrate the mRNA expression of structural and ribosomal proteins. Likewise, the finding that protein synthesis occurs in other cell lines in a p21-dependent manner suggests that MRF and cell cycle mRNAs may act in an acute postexercise fashion to increase transcriptional and translational processes in postmitotic myofibers. Future experiments can be conducted to confirm our hypotheses including the following: 1) using a conventional resistance training model in humans to microscopically examine how MPC activity is affected in concert with MRF and cell cycle mRNA/protein expression patterns (i.e., determine the cellular localization of the aforementioned genes/proteins), 2) using a conventional resistance training model in rodents to determine how knocking down MRF and/or cell cycle mRNAs affects MPC and/or muscle protein synthesis activities, and/or 3) using an inducible MRF/cell cycle construct in myotubes to examine how muscle protein synthesis activities are affected. In conclusion, the functional role of resistance exercise-inducible MRFs, cell cycle regulators, and/or other myogenic genes such as Notch-related transcripts serve in response to exercise will continue to prove to be elusive until more mechanistic animal and cell culture models demonstrate how these genes affect postmitotic myofiber properties.
We would like to thank Kyle Sunderland, Chris Poole, Tyler Kirby, Dr. Chris Lockwood, Scott Hassell, and Dr. Dan Feeback for help in critiquing this manuscript and/or obtaining data presented from our laboratory in this manuscript. Also, we would like to thank Drs. Frank Booth and Mike Bemben for insightful comments, Dr. Stephen Roth for editorial critiques, and the reviewers who greatly impacted the final draft of this manuscript. In general, the authors recognize the work of other researchers, which could not be cited because of the reference limitations. Costs for this article were graciously provided by the Graduate College at the University of Oklahoma. The National Strength and Conditioning Foundation provided funds for the aforementioned data generated from the Applied Biochemistry and Molecular Physiology Laboratory (University of Oklahoma) through a doctoral research grant award endowed to M.D.R. as well as young investigator grant to C.M.K.
The work cited from our laboratory in this manuscript was supported partially by the National Strength and Conditioning Foundation.
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resistance exercise; gene expression; myogenesis; satellite cell; post mitotic myofiber
©2011 The American College of Sports Medicine
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