JPFSM: Review Article

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1 J Phys Fitness Sports Med, 1(1): (2012) JPFSM: Review Article Roles played by protein metabolism and myogenic progenitor cells in exercise-induced muscle hypertrophy and their relation to resistance training regimens Naokata Ishii 1,2*, Riki Ogasawara 2, Koji Kobayashi 3 and Koichi Nakazato 3 1 Department of Life Science, Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Tokyo , Japan 2 Department of Environmental Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba , Japan 3 Graduate School of Health and Sport Sciences, Nippon Sport Science University, Fukazawa, Tokyo , Japan Received: March 23, 2012 / Accepted: April 2, 2012 Abstract To learn the mechanisms underlying resistance exercise-induced muscle hypertrophy, recent studies on muscle protein metabolism and myogenic progenitor cells were reviewed. Numerous studies have suggested that activation of the translation process plays a major role in a resistance exercise-induced increase in muscle protein synthesis, and also in muscle hypertrophy after a prolonged period of training. Among regulators of the translational activity, the mtorc1 signaling pathway has been shown to be important, although the relation between its upstream regulation and exercise regimen remains unclear. In addition, the muscle satellite cells play a part, even if not indispensable, in exercise-induced muscle hypertrophy, by supplying muscle fibers with new myonuclei. Middle to high exercise intensity has been regarded as essential for gaining muscle mass, because it causes the recruitment of large motor units with fast, type II muscle fibers, which are readily hypertrophied through activation of mtorc1 signaling. However, several studies have shown that low-intensity resistance exercises with either large exercise volume or prolonged contraction time effectively activate protein synthesis and induce muscle hypertrophy. These findings suggest that various strategies are possible in exercise regimens, and exercise intensity is not necessarily a primary factor for gaining muscular size. Keywords : mtor signaling, FOXO, exercise intensity, muscle satellite cells, myonuclei Introduction *Correspondence: ishii@idaten.c.u-tokyo.ac.jp Learning the mechanisms underlying resistance exercise-induced skeletal muscle hypertrophy is important in order to prescribe adequate exercise regimens for various subjects, each with different physical conditions. Skeletal muscle mass is regulated by the balance between muscle protein synthesis (MPS) and breakdown (MPB). The muscle net protein balance, i.e., MPS - MPB, has been shown to become transiently positive after a bout of resistance exercise mainly due to the increase in MPS; and this process has been regarded as an important factor in muscle hypertrophy after repeated bouts of resistance exercise 1). On the other hand, the size of muscle fiber may be limited by the number of myonuclei, suggesting that an increase in the size of muscle fiber beyond a certain extent requires an increase in the number of myonuclei through proliferation of the muscle satellite cells (Fig. 1). Aside from cellular and molecular mechanisms, numerous studies have reported the relation between resistance training protocol variables (e.g., intensity, contraction mode, and volume) and the magnitude of muscle hypertrophy. Based on these studies, some review articles have proposed training protocols that can be recommended for gaining muscular size 2-5). Although most of these articles have indicated that the intensity of exercise is a crucial factor for gaining muscular size and strength, there are many other variables to consider. For instance, resistance exercise training with restricted muscular blood flow has been shown to cause muscle hypertrophy even at low exercise intensity 6). Also, several studies have shown that low-intensity exercises with slow movement and maintained contractile force are effective for gaining muscular size 7,8). These studies provide us with an opportunity to revise the relation between training protocol variables and muscle hypertrophy. Recently, the cellular and molecular mechanisms underlying the regulation of muscular size are, at least partially, being uncovered. These mechanisms include the processes of gene transcription, signal transduction for the initiation of protein synthesis, and activation of some proteolytic enzymes. Several studies have investigated how training protocol variables affect these processes, and such studies can provide the basis for evaluating the effectiveness of a variety of training regimens used for gaining muscular size.

2 84 JPFSM: Ishii N, et al. This brief review summarizes the cellular and molecular events that may be related to exercise-induced muscle hypertrophy, i.e., protein metabolism, cell signaling and activation of myogenic progenitor cells, with special reference to their relation to resistance training regimens. It would help us to reconsider the principles operating in the regimens of conventional resistance training, and also the effects of some novel types of training without sufficient evidence at either the cellular or molecular level. Resistance exercise training and muscle protein metabolism 1. Muscle protein metabolism 1-1. Cell signaling in muscle protein synthesis Theoretically, MPS can be regulated in three processes: the number of myonuclei (DNA content), the transcription of DNA, and the translation of mrna (Fig. 1). Among these processes, the translation of mrna has been shown to play a crucial role in an exercise-induced increase in MPS 9-11). The mammalian target of rapamycin complex 1 (mtorc1) signaling pathway is considered as a key regulator of translation initiation and has been shown to be important in muscle hypertrophy 12,13). Resistance exercise has been shown to activate mtorc1 signaling, although its upstream regulation has not been fully understood 14-16) [Fig. 2; for more detail, see recent reviews 14,16-18) ]. Activation (phosphorylation) of mtorc1 stimulates MPS through phosphorylation of its downstream targets, eukaryotic initiation factor 4E binding protein (4E-BP1) and p70 ribosomal protein S6 kinase (p70s6k). Multisite phosphorylation of the translational repressor 4E-BP1 results in its dissociation from eukaryotic initiation factor 4E (eif4e), thereby allowing eif4e to assemble with ei- F4G, and facilitate the recruitment of other translation initiation factors to form the eif4f complex 19). On the other hand, activation of p70s6k by mtorc1-mediated phosphorylation promotes the translation of mrna through several substrates, such as SKAR, eef2k, eef4b, PDCD4, CCTβ, CBP80, and rps6, a component of the 40S ribosome 20). Increases in the phosphorylated forms of 4E-BP1 and p70s6k after a bout of resistance exercise have been shown to positively correlate with MPS 21-23), and also with the magnitude of muscle hypertrophy after repeated bouts of exercise 13,24,25). Resistance exercise also activates extracellular signalregulated kinase (ERK) 1/2 and its downstream substrate, p90 ribosomal S6 kinase (p90rsk) 22,26-29). The ERK1/2 signaling pathway is also known to play a regulatory role in translation initiation, either dependently or independently of the mtorc1 signaling pathway 12,30). Therefore, quantifying several signal transduction substrates in both mtorc1 and ERK1/2 signaling pathways is thought to be an effective way to predict protein synthesis activity Cell signaling in muscle protein breakdown Currently, three proteolytic systems are known to be involved in the skeletal muscle protein breakdown: lysosomal proteases (cathepsins), the cytosolic calciumdependent calpain system, and the ATP-dependent ubiquitin-proteasome system (UPS). Among these systems, UPS plays an important role in MPB and is thought to be regulated by the muscle-specific ubiquitin ligases (E3): muscle atrophy F-box (MAFbx/Atrogin-1) and musclespecific RING finger-1 (MuRF1) 31,32). Either unloading or aging causes muscle atrophy through activation of MPB by elevated expressions of Atrogin-1 and MuRF1 33,34). In addition, elevated expressions of Atrogin-1 and MuRF1 mrna are associated with several diseases, e.g., sepsis 35), cancer 36), AIDS 37), diabetes 38), and renal failure 39), which eventually cause the loss of muscle mass. The expression of ubiquitin ligases is regulated by the Forkhead transcription factor (FOXO). When protein synthesis is activated, FOXO is simultaneously phosphorylated through protein kinase B (Akt/PKB) pathway and is kept localized in the cytosolic compartment (Fig. 3). On the contrary, the dephosphorylated form of FOXO moves from the cytosol into the nucleus, and upregulates the transcription of ubiquitin ligase genes 40). A pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) induces Satellite cell activation DNA transcription Satellite cell proliferation mrna translation Myonulear number / DNA content Muscle protein synthesis Muscle protein breakdown Net protein balance Muscle fiber size Fig. 1 Major events involved in the regulation of skeletal muscle size.

3 JPFSM: Muscle hypertrophy and training regimens 85 muscle atrophy through the upregulation of protein degradation. This process is possibly mediated with the phosphorylation of nuclear factor kappa B (NFκB) inhibitor (IκB), which allows NFκB to separate from IκB and initiate the transcription of MuRF1 gene 41). Also, TNF-α may stimulate phosphorylation of p38, which then promotes the expression of Atrogin-1 41). 2. Resistance training protocol variables and muscle protein metabolism 2-1. High intensity is not essential for muscle protein synthesis It has been widely accepted that resistance training at an intensity ranging from 70 to 90% of 1-repetition maximum (1RM) or from 6 to 12 RM is optimal for gaining Resistance exercise Mechanical stress Growth factor Nutrient sensing mtorc1 FOXO 4E-BP1 p70s6k Atrogin-1 MuRF1 mrna translation MPS MPB Fig. 2 A simplified schematic representation of how resistance exercise increases muscle protein synthesis (MPS) and breakdown (MPB). Resistance exercise increases both MPS and MPB through mtorc1 and FOXO signaling pathway, respectively. We depicted rough outline to help the reader to understand the whole context. For more details, please refer to the recent review articles 14,16-18). Moderate intensity Higher intensity Myostatin Satellite cell proliferation PI3K IGF-1 PI3K-P Muscle fiber damages Akt Akt-P FOXO-P FOXO mtorc1 mtorc1-p Myonuclear number p70s6k p70s6k-p Atrogin-1 MuRF1 Muscle protein synthesis Muscle protein breakdown Fig. 3 Intensity-dependent changes in muscle protein synthesis and breakdown in eccentric exercise. IGF-1, insulin-like growth factor-1; PI3K and PI3K-P, phosphoinositide 3-kinase and its phosphorylated form; Akt and Akt-P, protein kinase B and its phosphorylated form. Based on Ochi et al. 83).

4 86 JPFSM: Ishii N, et al. matched 22). More recently, they also reported that lowintensity (30% 1RM) resistance exercise with slow lifting movements (6-s concentric and 6-s eccentric actions without pause, 3 sets) effectively caused an increase in MPS, when the exercise was performed until failure. The effect was much greater than that of exercise with the same intensity and volume (number of repetitions) but shorter movement duration (1-s concentric and 1-s eccentric actions) 8). These studies suggest that either mechanical impulse (contractile force x time) or muscle fatigue is an important factor in the activation of MPS. It has been shown that both resistance exercise-induced phosphorylation of signal transduction substrates and muscle fiber hypertrophy occur mainly in type II fibers 61-63). Therefore, recruiting type II muscle fibers appears to be an indispensable factor in resistance exercise regimens for gaining muscular size. Henneman s size principle states that smaller motor units are predominantly recruited and the larger motor units are additionally recruited with increasing force production 64). Because type II fibers are mainly involved in large motor units with a high recruitment threshold, they are not readily recruited by low-intensity resistance exercise. However, fatiguing exercise at low intensity has also been shown to eventually cause the recruitment of large motor units as the ability of muscle to generate force progressively declines and approaches the given exercise intensity 65-68). According to the above arguments, it is suggested that repeated bouts of low-intensity resistance exercise effectively causes muscle hypertrophy if each exercise is performed until failure. In fact, Tanimoto & Ishii reported that low-intensity (~50% 1RM) resistance exercise with slow lifting movement and maintained force generation (3-s concentric and 3-s eccentric actions) performed until volitional fatigue induced muscle hypertrophy comparable in magnitude to that induced by high-intensity (~80% 1RM) exercise with conventional movement speed (1-s concentric and 1-s eccentric actions). In addition, lowintensity resistance exercise with conventional movement speed (same exercise intensity and volume as those for the exercise with slow movement) did not cause muscle hypertrophy 7). As already mentioned, both low-intensity exercise with large exercise volume and that with slow movement and long contraction time cause acute increases in MPS. This suggests that a low-intensity exercise regimen with conventional movement speed is also effective for gaining muscular mass if it eventually causes strong muscular fatigue. To test this, we recently examined the long-term effect of low-intensity, exhaustive exercise (30% 1RM, 4 sets, repetition until failure in each set) on muscular size. This exercise regimen caused muscle hypertrophy, the magnitude of which was similar to that caused by a typical, high-intensity regimen (75% 1RM, 10 reps 3 sets) (Ogasawara et al. in preparation). These results indicate thatl exercise intensity is not necessarily a primary varimuscular size 2,42,43). Therefore, most studies on the effect of resistance exercise on MPS have used this range of intensity, and have reported increases in the phosphorylated forms of signal transduction substrates and activation of MPS 12,44-54). In agreement with previous studies showing that low-intensity (e.g., <50% 1RM) resistance training is not sufficient to cause muscle hypertrophy 2,42,43), several studies have reported that low-intensity exercise is substantially ineffective to activate MPS 23,55,56). However, Kumar et al. recently reported that MPS increased with exercise intensity of up to 60% 1RM, whereas no further increase in MPS occurred at higher intensity (75 and 90% 1RM), even when the exercise volume was matched 23). This study implies that repeated bouts of resistance exercise at 60% 1RM can cause muscle hypertrophy comparable in magnitude to that induced by resistance training at higher intensity (70-85% 1RM), because acute changes in muscle protein synthesis have been shown to correlate with changes in muscle size after a period of training 57). Low-intensity (30-50% 1RM) resistance exercise with restricted muscular blood flow has been shown to readily fatigue the working muscle 58,59), and cause muscle hypertrophy and strength gain after a period of training 6,60). Fujita et al. reported that this type of exercise effectively stimulated mtorc1 signaling pathway and MPS, while a normal exercise (without blood flow restriction) of the same intensity and volume did not 55). These studies imply that high-intensity is not prerequisite to stimulate MPS and to gain muscular size Low-intensity exhaustive training stimulates muscle protein synthesis Exercise volume (e.g., weight repetitions sets) is also an important variable in resistance training regimens. Generally, using multiple sets has been believed to be superior to a single set for muscle hypertrophy. Burd et al. reported that the increase in MPS was much larger and lasted longer after an exercise session with multiple sets (3 sets) than after that with a single set 21). Furthermore, Terzis et al. reported that phosphorylated forms of both p70s6k and its downstream target rps6 increased with exercise volume (1 set < 3 sets < 5 sets), though MPS was not measured 24). On the other hand, phosphorylation of extracellular signal-regulated kinase (ERK1/2) and its downstream substrate p90rsk was not affected by the increase in exercise volume in both studies. These studies suggest that the mtorc1 signaling pathway is more responsive to exercise volume than the ERK1/2 signaling pathway. Burd et al. recently reported that low-intensity (30% 1RM) resistance exercise caused an increase in MPS similar to high-intensity (90% 1RM) exercise, when the exercise was performed until repetition failure 22). However, the low-intensity exercise was far less effective than the high-intensity exercise when the exercise volume was

5 JPFSM: Muscle hypertrophy and training regimens 87 able in exercise regimens for gaining muscular size Mode of muscle contraction may not be a primary determinant for muscle protein synthesis Resistance training protocol variables include the mode of muscle contraction, i.e., isotonic, isometric, isokinetic, concentric, eccentric, etc. However, contraction modespecific effects on MPS and muscular size are unknown. Up to present, a limited number of studies have separately investigated the effects of concentric and eccentric actions under nominally isotonic conditions. Generally, eccentric training performed at high intensities has been shown to be more effective for gaining muscle mass than concentric training 69), but no difference in musclehypertrophic effect has been reported between these two types of training with the same exercise volume (weight x repetitions) 70). Moore et al. reported that MPS increased more rapidly after eccentric exercise than after concentric exercise, but no substantial difference in the magnitude of MPS was observed between these two types of contraction mode 71). Cuthbertson et al. also reported no significant difference in MPS between concentric and eccentric contractions 72). However, it should also be noted that high-intensity eccentric exercise gives rise to strong mechanical stress, which may cause some additional effects specific to eccentric actions 69). Thus, high-intensity eccentric exercise may be useful as a part of periodized training regimens, because performing the same regimen of resistance training is known to cause a gradual decline in the acute changes in signal transduction substrates and MPS after a bout of exercise 51,73,74), and also in the rate of muscle growth 75,76). 3. Resistance exercise and muscle protein breakdown So far, most studies on changes in protein metabolism after resistance exercise have mainly investigated MPS and MPS-related signal transduction, because it has been shown that MPB does not change considerably after exercise. Phillips et al. reported that the rate of MPB increased after a bout of resistance exercise in sedentary subjects 51). Yang et al. and Louis et al. also showed that the expression of MuRF1 mrna increased after one bout of highintensity (65~70% 1RM) resistance exercise in sedentary men 52,118). Leger et al. showed that an 8-wk resistance training period caused increases in FOXO1, Atrogin-1 and MuRF1 77). These studies indicate that resistance exercise causes both acute and chronic increases in MPB. On the other hand, Phillips et al. showed that the rate of MPB did not change after a bout of resistance exercise in trained subjects 51). Mascher et al. showed that the expression of Atrogin-1 mrna was reduced by 32% after the second session of training when compared to that after the initial session of training 78). Karagounis et al. investigated the effects of three bouts of resistance exercise on TNF-α and IKK using a rat training model, and showed that the expression of these proteins markedly increased in response to the initial bout, whereas the responses to subsequent bouts of exercise declined gradually 79). Zanchi et al. also showed that a 12-wk resistance training period caused decreases in atrogin-1 and MuRF1 in rats 80). These studies suggest that repeated bouts of resistance exercise diminish the acute increase in MPB after exercise. High-intensity eccentric exercise is known to cause structural damage in muscle fibers (Z-line streaming and sarcomere disruption), inflammatory reactions and MPB 81). Willoughby et al. investigated changes in the expression of atrogin-1 protein after two successive bouts of 150% 1RM eccentric training, and showed that the increase in atrogin-1 protein after the 2 nd bout was diminished as compared to that after the 1 st bout of training 82). These studies suggest that eccentric exercise promotes MPB within muscle fibers subjected to intense mechanical stress, but some adaptation takes place gradually with the succession of training. On the other hand, our recent study with the rat eccentric training model has shown that changes in both MPS and MPB are strongly dependent on exercise intensity (Fig. 3). Eccentric contractions with slow stretch and moderate contractile force activated the signal transduction towards MPS; whereas those with rapid stretch and large contractile force worked oppositely to activate the signal transduction towards MPB 83). Therefore, the speed of eccentric actions would be an important factor even in conventional resistance training regimens for optimizing protein metabolism. Myogenic cells and their contribution to muscle hypertrophy 1. Satellite cells are major myogenic progenitor cells in the adult skeletal muscle Skeletal muscle adapts to the environment with dynamic changes in its architecture. In response to anabolic stimulus or damage, myogenic cells within muscle are activated and play a role to form a new architecture. It has been reported that several types of myogenic progenitor cells exist, such as muscle satellite cells, bone marrow stem cells, skeletal muscle side population cells, and mesoangioblasts 84). The satellite cells, especially, are major myogenic progenitor cells in adult muscle. Satellite cells are quiescent cells located between the sarcolemma and the basal lamina 85). When muscle is injured or subjected to mechanical stress, satellite cells are activated to enter the cell proliferation cycle, differentiate, and finally fuse with muscle fiber 86). This process has been regarded as a key event in muscle regeneration and hypertrophy. Up to present, two strategies have been taken to examine relationships between resistance exercise training and satellite cell activation/proliferation. One is immunohistochemical observation to identify satellite cells and myonuclei. The other is to measure expressions of myogenic mrnas in exercised muscle specimens

6 88 JPFSM: Ishii N, et al. Fig. 4 Myogenic markers for myogenesis Frequently used markers for proliferation and differentiation of myogenic cells are shown. Please refer to articles by others for detailed information. (Fig. 4). Although both are definitely effective strategies, this review will mainly focus on immunohistochemical studies with human subjects. 2. Effect of resistance training on satellite cell number Satellite cells provide muscle fibers with additional nuclei so as to increase the capacity of muscle fibers for hypertrophy. Acute effects of resistance exercise on the number of satellite cells have been examined in young and old subjects. A single bout of strenuous eccentric exercise increased N-CAM positive satellite cells in young (20-35 years) and older (60-75 years) subjects 87-89). The magnitude of increase in young men (3.2±1.3 fold) was higher than in older participants (1.3±0.6 fold) when expressed relative to pre-exercise numbers 88). Electrical stimulation of the gastrocnemius muscle also increased the number of activated satellite cells (+CD56+Ki67) within 48 hours in young (20±5 years) subjects 90). Combined exercise with cycle ergometer ( < 62% of workload capacity) and leg press machine (55-70% 1RM) activated satellite cells (+DLK1+CD56) within 9 hours in young subjects 91). Taken together, one bout of resistance exercise induces the activation and proliferation of satellite cells, especially in younger subjects. Interestingly, several studies reported that satellite cell proliferation after resistance training was attenuated by anti-inflammatory medication 92,93), suggesting that inflammatory reaction plays a part in the activation of satellite cells. Repeated bouts of resistance training also cause chronic increases in the number of satellite cells. The phenomenon, unlike the acute response, appears to be independent of age and gender. Kadi and Thornell showed that a 12- wk strength training (shoulder press, rowing, triceps and latissimus pulldown; RM) induced concomitant increases in muscle fiber cross sectional area (36%), myonuclear number (70%), and satellite cells (+CD56) (46%) in female trapezius muscle 94). Several studies demonstrated that more than 9 weeks of resistance training increased the number of satellite cells (electron microscopy or +Pax7) in both younger and older subjects, regardless of gender difference 95-98). Notably, Verdijk et al. showed that, in elderly adults (72±2 years), a 12-wk resistance training period increased type II muscle fiber cross-sectional area and the number of adjacent satellite cells (+CD56) 99). This might be related to the aging-specific atrophy of type II muscle fibers and the decrease of subsidiary satellite cells in the elderly 88,100). 3. Effect of endurance training on satellite cell number In comparison to resistance training, responses of satellite cells to endurance exercise training have not been well documented. Charifi et al. showed that a 12-wk endurance training period (cycle ergometer; 65-95% of peak oxygen consumption) elicited an increase in satellite cell (+CD56) number in vastus lateralis in elderly subjects (73±3 years). Their research also showed no significant increases in

7 JPFSM: Muscle hypertrophy and training regimens 89 the number of myonuclei and the muscle cross sectional area 101). Verney et al. examined the effects of a combination of resistance training for the upper limb muscles (20-10 RM) and endurance training with the lower limb (cycle ergometer; HRmax). After a 14-wk training period, the numbers of NCAM-positive satellite cells increased in both upper and lower limb muscles, whereas the numbers of myonuclei were unchanged 102). Recently, Snijders et al. reported that a 6-month endurance-type exercise did not alter the number of satellite cells in muscles of type 2 diabetes patients 103). Although further studies are needed, endurance exercise appears to stimulate, to some extent, the proliferation of satellite cells in healthy subjects. It should also be noted that endurance exercise does not increase the number of myonuclei. 4. Roles played by satellite cells and myonuclei in muscle hypertrophy Although the myonuclear domain, i.e., cytoplasmic volume per myonucleus, has been regarded as important in the regulation of myonuclear number, numerous studies have shown that the size of myonuclear domain is variable, depending on muscle fiber type, age, and muscle usage 104,105). In addition, there have been several papers describing that satellite cells are not indispensable for muscle hypertrophy 106,107). For example, satellite cell-depleted mice showed a double increase in muscle size after a 2-wk functional overload 107). Hypertrophy caused by Akt-activation in mouse skeletal muscle was associated with neither satellite cell activation nor newly myonuclear incorporation 108). For humans, Petrella et al. showed a significantly larger myofiber area per myonucleus in extreme responders, i.e., individuals who showed marked muscle hypertrophy after resistance training 109). Thus, the size of the myonuclear domain may not be defined initially, so that it may not be a major reason why the activation of satellite cells occurs along with muscle hypertrophy. However, muscle hypertrophy is actually accompanied by a proliferation of satellite cells 94). Kosek et al. showed that resistance training-induced muscle fiber hypertrophy was superior in young men than in young women and older adults 110). Petrella et al. found that the number of satellite cells (+NCAM) and myonuclei increased only in young men. These studies suggest that the ability of myofiber to incorporate new myonuclei is important to achieve potent hypertrophy 111). Petrella et al. also showed that extreme responders had more satellite cells before resistance exercise. After resistance exercise, the extreme responders exhibited significant increases in the numbers of myonuclei and satellite cells along with an increase in the fiber area per myonucleus 109). Based on these results, the authors argued that individuals with a greater basal number of satellite cells achieved potent muscle growth after a period of training, because they had a higher ability to expand the satellite cell pool 109). Thus, the activation of satellite cells possibly enhances the growth of muscle fibers, and is important especially in the later phase of muscle hypertrophy 112,113). Satellite cells definitely play an indispensable role in the muscle regeneration process. They are able to regenerate damaged muscle fibers and have been the target for cell grafting therapy 114,115). Muscles lacking satellite cells could not regenerate normal muscle fibers seven days after 1.2 % BaCl 2 induced injury 107). Since strenuous exercise is considered to cause micro traumas in muscle fibers, satellite cells might be essential especially in heavy resistance training to induce potent hypertrophy. Apart from the notion of myonuclear domain, the number of myonuclei may be directly related to the activation of protein synthesis. By using C2C12 cells, Nader et al 116) showed that serum-stimulated muscle growth was associated with significant increases in retinoblastoma protein (Rb) phosphorylation and cyclin D1 protein. Cyclin D1 was detected in the nuclei of myotubes. These cell cycle regulating proteins increased ribosomal RNA through its upstream binding factor (UBF), and eventually activated the translation process. Interestingly, these events occurring in the growth of myotube were blocked with rapamycin, suggesting that mtorc1 signaling regulates the expression of cell cycle regulating proteins within myonuclei. Another study showed that cell cycle regulators such as p21 and Rb, were detected in the myonuclei of mature muscle fibers in vivo 117). A similar association of increased cell cycle regulators with hypertrophy has previously been shown in heart muscles 118). Therefore, an increased number of myonuclei could enhance translation activity through increased expression of cell cycle regulators, when mtorc1 signaling is activated by resistance exercise. Summary and conclusion To summarize the present review, Fig. 5 illustrates an overview of the relation between some variables in a resistance exercise regimen and protein metabolism. Up to the present, numerous studies have suggested that activation of the translation process plays a major role in a resistance exercise-induced increase in muscle protein synthesis, and also in muscle hypertrophy after a prolonged period of training. Among regulators of the translational activity, the mtorc1 signaling pathway has been shown to be important in exercise-induced muscle hypertrophy, although the relation between its upstream regulation and exercise regimen remains unclear. Compared to muscle protein synthesis, muscle protein breakdown may play a minor role, because it does not show considerable changes after exercise. However, strenuous resistance exercise such as heavy eccentric exercise has been shown to stimulate protein degradation, due possibly to structural damage to the working muscle fibers. On the other hand, it has also been shown that eccentric exercise with moderate intensity suppresses protein degradation through

8 90 JPFSM: Ishii N, et al. Variables in exercise regimen Contraction mode (concentric/eccentric) Intensity Volume Mechanical impulse (force x time) Upstream factors Fiber damages Type II fiber recruitment Muscle fatigue Satellite cell activation Cell signaling FOXO signaling mtorc1 signaling Increased myonuclei Ubiquitin ligases Translation of mrna Cell cycle regulators Protein metabolism Muscle protein breakdown < Muscle protein synthesis Muscle fiber hypertrophy Fig. 5 Schematic presentation of the relations between variables in resistance exercise regimens and muscle protein metabolism. phosphorylation of FOXO. Middle to high exercise intensity has been regarded as essential for gaining muscle mass, because it causes the recruitment of large motor units with fast, type II muscle fibers, which are readily hypertrophied through activation of mtorc1 signaling. In contrast, several studies have shown that low-intensity resistance exercises with either large exercise volume or large mechanical impulse (force x time) effectively activate protein synthesis and induce muscle hypertrophy. Even at low intensity, these types of exercise give rise to strong muscular fatigue, and eventually cause an additional recruitment of type II fibers. Both high-intensity resistance exercise and low-intensity endurance-oriented exercise have been shown to activate the muscle satellite cells. The satellite cells play a part, even if not indispensable, in exercise-induced muscle hypertrophy, by supplying muscle fibers with new myonuclei. An increased number of myonuclei may enhance the translation process through mtorc1-mediated expression of cell cycle regulator proteins. In conclusion, several approaches are possible to activate mtorc1 signaling and muscle protein synthesis; and exercise intensity may not necessarily be a primary variable for gaining muscle mass. References 1) Burd NA, Tang JE, Moore DR, Phillips SM Exercise training and protein metabolism: influences of contraction, protein intake, and sex-based differences. J Appl Physiol 106: ) Burd NA, Andrews RJ, West DW, Little JP, Cochran AJ, Hector AJ, Cashaback JG, Gibala MJ, Potvin JR, Baker SK, Phillips SM Muscle time under tension during resistance exercise stimulates differential muscle protein sub-fractional synthetic responses in men. J Physiol 590: ) Tanimoto M, Ishii N Effects of low-intensity resistance exercise with slow movement and tonic force generation on muscular function in young men. J Appl Physiol 100: ) ACSM American College of Sports Medicine position stand. Progression models in resistance training for healthy adults. Med Sci Sports Exerc 41: ) Baechle TR, Earle RW Essentials of Strength Training and Conditioning-3rd Edition: Human Kinetics. 6) Bird SP, Tarpenning KM, Marino FE Designing resistance training programmes to enhance muscular fitness: a review of the acute programme variables. Sports Med 35: ) Hass CJ, Feigenbaum MS, Franklin BA Prescription of resistance training for healthy populations. Sports Med 31: ) Takarada Y, Takazawa H, Sato Y, Takebayashi S, Tanaka Y, Ishii N Effects of resistance exercise combined with moderate vascular occlusion on muscular function in humans. J Appl Physiol 88: ) Bolster DR, Kimball SR, Jefferson LS Translational control mechanisms modulate skeletal muscle gene expression during hypertrophy. Exerc Sport Sci Rev 31: ) Kimball SR, Jefferson LS Control of translation initiation through integration of signals generated by hormones, nutrients, and exercise. J Biol Chem 285: ) Kimball SR Regulation of global and specific mrna translation by amino acids. J Nutr 132: ) Drummond MJ, Fry CS, Glynn EL, Dreyer HC, Dhanani S, Timmerman KL, Volpi E, Rasmussen BB Rapamycin administration in humans blocks the contraction-induced increase in skeletal muscle protein synthesis. J Physiol 587: ) Baar K, Esser K Phosphorylation of p70(s6k) correlates with increased skeletal muscle mass following resistance exercise. Am J Physiol 276: C ) West DW, Burd NA, Staples AW, Phillips SM Human

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