Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity.

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Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity Thomas Chaillou1 and Johanna T. Lanner Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden

ABSTRACT: Reduced oxygen (O2) levels (hypoxia) are present during embryogenesis and exposure to altitude and in

pathologic conditions. During embryogenesis, myogenic progenitor cells reside in a hypoxic microenvironment, which may regulate their activity. Satellite cells are myogenic progenitor cells localized in a local environment, suggesting that the O2 level could affect their activity during muscle regeneration. In this review, we present the idea that O2 levels regulate myogenesis and muscle regeneration, we elucidate the molecular mechanisms underlying myogenesis and muscle regeneration in hypoxia and depict therapeutic strategies using changes in O2 levels to promote muscle regeneration. Severe hypoxia (£1% O2) appears detrimental for myogenic differentiation in vitro, whereas a 3–6% O2 level could promote myogenesis. Hypoxia impairs the regenerative capacity of injured muscles. Although it remains to be explored, hypoxia may contribute to the muscle damage observed in patients with pathologies associated with hypoxia (chronic obstructive pulmonary disease, and peripheral arterial disease). Hypoxia affects satellite cell activity and myogenesis through mechanisms dependent and independent of hypoxiainducible factor-1a. Finally, hyperbaric oxygen therapy and transplantation of hypoxia-conditioned myoblasts are beneficial procedures to enhance muscle regeneration in animals. These therapies may be clinically relevant to treatment of patients with severe muscle damage.—Chaillou, T. Lanner, J. T. Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity. FASEB J. 30, 3929–3941 (2016). www.fasebj.org KEY WORDS:

hypoxia

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muscle damage

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hyperbaric oxygen therapy

Oxygen (O2), which is necessary for life, is also a metabolic signal involved in several processes, including aerobic energy production, cellular homeostasis, and development. Changes in O2 levels can act as a physiologic or pathologic sensor to regulate these processes from embryogenesis to adulthood. A reduced level or tension of O2, called hypoxia, is observed in many situations, such as embryogenesis (developmental hypoxia) (1), exposure to high altitude (ambient hypoxia) (2), physical exercise (3), as well as pathologic conditions [e.g., chronic obstructive pulmonary disease (COPD), peripheral arterial disease and obstructive sleep apnea] (4–6).

ABBREVIATIONS: ATA, atmosphere absolute; bHLH, basic helix-loop-helix;

COPD, chronic obstructive pulmonary disease; DMOG, dimethyloxalylglycine; EDL, extensor digitorum longus; HBO, hyperbaric oxygen; HIF-1a, hypoxia-inducible factor-1a; ICD, intracellular domain; MHC, myosin heavy chain; miR, microRNA; MTOR, mechanical target of rapamycin; MRF, myogenic regulatory factor; MyoD, myogenic differentiation; Myf, myogenic factor; Pax, paired box; PHD, prolyl hydroxylase domain protein; PO2, O2 pressure 1

Correspondence: Department of Physiology and Pharmacology, Karolinska Institutet, Von Eulerväg 8, 171 77 Stockholm, Sweden. E-mail: th.chaillou@ gmail.com

doi: 10.1096/fj.201600757R

0892-6638/16/0030-3929 © FASEB

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ischemia

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cell transplantation

Chronic exposure to severe hypoxia has been shown to induce skeletal muscle adaptations, including metabolic changes (e.g., reduced oxidative capacity) (7, 8) and muscle atrophy [see Favier et al. (9) for a recent review]. In particular, a loss of skeletal muscle mass (i.e., muscle atrophy) has been observed in humans (10) and in rodents (11–13) in response to severe ambient hypoxia. A loss of muscle mass is the consequence of a negative balance between protein synthesis and protein breakdown. Muscle atrophy can also be the result of defective skeletal muscle regeneration after repetitive muscle injuries. Skeletal muscle is susceptible to several types of injury including direct trauma, crush, eccentric exercise, neurologic dysfunction, and innate genetic defects. In addition to atrophy, severe and repetitive muscle damage may reduce the quality of life and increase the risks of mortality and morbidity (14, 15). Muscle damage has been reported in patients with COPD (16) and during peripheral ischemia (17), 2 diseases associated with cellular hypoxia and muscle atrophy (16, 18). To date, the role played by hypoxic stress in skeletal muscle from patients with COPD and peripheral ischemia remains to be elucidated. Skeletal muscle regeneration is finely orchestrated by several cellular and molecular events, leading to the restoration of muscle mass, muscle vascularization, and

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innervation, as well as the recovery of contractile and metabolic properties of the muscle (19). The satellite cells, which are myogenic progenitor cells localized between the sarcolemma and the basal membrane of muscle fibers, play an essential role in skeletal muscle regeneration. These cells, which are quiescent under resting conditions, become activated after muscle damage and then proliferate, fuse, and differentiate to form new multinucleated myofibers or to repair existing injured fibers. For a more comprehensive review of satellite cell dynamics, the reader can refer to these recent reviews (20, 21). Moreover, it is well established that skeletal muscle regeneration shares some similarities with myogenesis, including several cellular events such as the activation and proliferation of myogenic cells, and the formation of myotubes (22). Over the past decade, it has been demonstrated that stem cells in both embryonic and adult organisms frequently occupy hypoxic microenvironments, so-called hypoxic niches (e.g., placental trophoblast stem cells and hematopoietic stem cells) (23, 24). A reduced oxygen level appears to be critical for regulating the activity of the stem cells, such as their self-renewal, proliferation, and differentiation (23). Myogenic progenitor cells are also present in a hypoxic microenvironment during embryonic development (1). Given that the satellite cells are considered to be the primary stem cells of skeletal muscle, it would not be surprising if the level of oxygen affects satellite cell activity during myogenesis and regeneration after muscle damage. Overall, a better understanding of how O2 levels affect satellite cell activity is necessary for the development of novel therapeutics to promote muscle regeneration. In the first part of this review, we present the idea that the level of oxygen regulates myogenesis and skeletal muscle regeneration. Then, we summarize the molecular mechanisms underlying myogenesis and muscle regeneration in hypoxia. Finally, we describe therapeutic strategies that use altered O2 levels (hypoxia and hyperbaric oxygen therapy) to promote skeletal muscle regeneration.

INTRAMUSCULAR OXYGEN PRESSURE AND EXPERIMENTAL APPROACHES TO MIMIC AMBIENT HYPOXIA Hypoxia is defined as reduced availability of oxygen. When studying hypoxia, it is important to determine where the hypoxic stress occurs and to define what it means at this level. The O2 pressure (PO2) dramatically decreases through its passage from air into the muscle. At sea level, PO2 is ;160 mmHg in the ambient air and decreases to ;100 mmHg in arterial blood and reaches ;35 mmHg in human skeletal muscle (25). Above sea level (e.g., on top of Mount Kilimanjaro at 5800 m), the PO2 is ;76 mmHg in ambient air. The altitude of Mount Kilimanjaro has been simulated in a laboratory environment, and the intramuscular PO2 estimated by using nuclear magnetic resonance spectrometry was ;23 mmHg in human calf muscles (25). In mice, exposure to a similar level of hypoxia (inspired O2 level of 10%) was shown to reduce skeletal muscle PO2 from ;50 to 20 mmHg (26). To reproduce ambient hypoxia experimentally, there are in principle 2 approaches; the most physiologic manner is to reduce 3930

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oxygen availability by decreasing the barometric pressure. This hypoxic condition, termed hypobaric hypoxia, can be reproduced in a laboratory environment by using a hypobaric chamber. The other alternative, called normobaric hypoxia, consists in decreasing the proportion of O2 available in the ambient air [i.e., reducing the inspired O2 fraction (FIO2)] without changing the barometric pressure. In this case, the patient inspires normobaric air, but the fraction of oxygen is reduced (,20.9%) (27). The concept of hypoxia becomes more complex when extrapolated to cell culture. For instance, standard cell culture gas that consists of 95% ambient air plus 5% CO2 (i.e., ;20% O2) has a PO2 of ;142 mmHg (28), which is remarkably higher than the O2 pressure reported at the intramuscular level in vivo (25, 26). Although the scientific community is aware of this aspect, the gaseous environment of cell culture is rarely modified from this standard, probably because of technical and financial aspects. In the rest of this article, a cell culture gas condition of 20% O2, which most likely mimics a hyperoxic rather than a normoxic condition, will be referred to as standard. Currently, for cell culture experiments,;3–6% O2 is considered a more physiologic normoxic environment for muscle cells (29–31).

EFFECTS OF LOW O2 LEVELS ON THE REGULATION OF MYOGENESIS Myogenesis during embryonic development Skeletal muscle fibers are formed by fusion of mesoderm progenitor cells called myoblasts during embryonic development [for review, see Yusuf and Brand-Saberi (22)]. More precisely, the somites formed by the segmentation of the paraxial mesoderm are partitioned into sclerotome and dermomyotome. The myotome, which is formed from the dermomyotome in a multicomplex process, is the first site where the skeletal muscle develops during embryogenesis. To investigate the presence of hypoxia during embryogenesis, Provot and colleagues (1) injected the hypoxic marker EF5 into pregnant female mice at embryonic d 12. They observed that EF5 bound mesenchymal condensations from mouse embryos, indicating that local hypoxia appears during embryonic development. In addition, the transcription factor hypoxiainducible factor (HIF)-1a, which is stabilized in hypoxia (in contrast to normoxia where it is degraded), was also detected in the somites, the transient structures that differentiate into several tissues, including skeletal muscle. This finding shows that low O2 levels occur during embryogenesis and that this environment participates in normal development (32). To examine whether the O2 level affects embryonic development and myogenesis, Hidalgo et al. (33) cultured Xenopus laevis embryos at different O2 levels. Control embryos were exposed to 21% O2, corresponding to normoxia, and hypoxic embryos were exposed to 10 and 5% O2 and were subjected to moderate and strong hypoxic stress, respectively. The examination of external embryo morphology indicated a decreased embryonic development rate in hypoxia (10 and 5% O2) compared with standard conditions. Moreover, the rate of lethality, determined through the depigmentation of embryos and the dissociation of tissues, was gradually increased by the

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CHAILLOU AND LANNER


degree of hypoxic stress. The hypoxia-induced embryo lethality also correlated with an increased number of apoptotic cells. These results indicate that hypoxia delays embryonic development and causes apoptosis and lethality after prolonged exposure. Furthermore, hypoxia does not impair somitogenesis but reduces the accumulation of myosin heavy chain (MHC), a marker of muscle maturation, suggesting that hypoxia impairs muscle formation during the early phase of vertebrate development. It is worth mentioning that the hypoxic conditions (5 and 10% O2) in this study cannot be directly compared with hypoxic stress in cell cultures of myogenic cells (29, 30), because different vertebrate organisms were used (Xenopus vs. mammalian organisms), and the gradient of oxygen pressure differs between an embryonic organism containing several layers of cells (embryo) and a monolayer of cells (myogenic cell culture); for an identical level of oxygen in the cell media, the PO2 is most probably lower in the deepest layers of the embryo compared with the monolayer of myogenic cells. Myogenesis: cell culture studies Myogenic proliferation The first studies examining the effects of a reduced oxygen level on myogenesis were published ;15 yr ago (29, 30). Chakravarthy and colleagues (30) showed remarkably higher proliferation of satellite cells from aged rats at ;3% O2 than in a standard condition (20% O2). In addition, enhanced satellite cell proliferation has been observed when cells were cultured at 6% O2 (29) or 5% O2 (28) as compared with standard conditions. However, a recent study did not observe any changes in the proliferation capacity of L6 myoblasts (rat myoblast cell line) when the cells were cultured at either 5 or 20% O2 (31). Altogether, a majority of reports suggests that a physiologic normoxic cellular environment (3–6% O2) is beneficial for the proliferation of primary myoblasts (i.e., satellite cells). The proliferative capacity of myoblasts is more controversial at lower oxygen levels. For instance, a reduced proliferative capacity was observed at 1% O2 in myoblasts derived from cell lines (rat L6 and mouse C2C12 myoblasts) (31, 34, 35) and in human primary myoblasts (35). However, the opposite result, (i.e., an increase proliferative capacity) was shown in bovine (1% O2) and mouse (2% O2) satellite cells (36, 37). This contrast suggests that the effect of the oxygen level on the proliferative capacity may depend on the cell type (cell line vs. primary myoblasts), the species (human vs. mouse and bovine), and the severity of the hypoxic stress. Furthermore, the window for the onset of the movement of the O2 level from beneficial to detrimental for satellite cell proliferation appears narrow, but it probably occurs between 1 and 2% O2 in human satellite cells (35, 38). Other factors that also may influence the proliferative capacity in response to various oxygen levels are the number of cell passages (38), the characteristics of the donor, and the specificity of the satellite cell population (37). For a more complete description, a detailed summary is presented in Table 1. OXYGEN LEVELS AND SATELLITE CELL ACTIVITY

Myogenic differentiation The influence of oxygen levels on the differentiation of myoblasts into myotubes has been extensively investigated, and the main results are summarized in Table 1. Several studies have demonstrated that a low oxygen level (#1% O2) has deleterious effects on myogenic differentiation (31, 34, 35, 39–43). For instance, Di Carlo et al. (34) showed an inhibition of myotube formation and a reduced expression of MHC at 1% O2 compared with standard conditions. C2C12 myoblasts cultured at 1% O2 for 48 h and then shifted to standard conditions (20% O2) were able to differentiate properly. Thus, hypoxia-induced inhibition of myogenic differentiation is not permanent when standard oxygen levels are introduced. Yun and colleagues (41) investigated whether the degree of hypoxia influences C2C12 myoblast differentiation. Three oxygen levels were studied: physiologic hypoxia (2% O2), pathologic hypoxia (0.5% O2), and extreme hypoxia (0.01% O2). They demonstrated that the alteration of myoblast differentiation by hypoxia was inversely correlated to the level of O2. Reduced capacity to differentiate at low O2 level (#1% O2) has also been reported in human and mouse primary myoblasts (35, 39, 42, 43). Intriguingly, Kook et al. (36) observed that hypoxia (1% O2) promoted the differentiation of primary myoblasts from bovine, whereas the differentiation of C2C12 myoblasts was compromised at this O2 level. Although the mechanisms explaining these results remain to be elucidated, it seems that the myogenic cells from bovine have a better capacity to differentiate at low O2 levels. A few studies have investigated myogenic differentiation under physiologic cellular normoxia (3–6% O2) (30, 31, 44). Differentiation of primary myoblasts from aged rats was enhanced at 3% O2 compared with 20% O2 (30). Similar results were observed in L6 myoblasts culture at 5% O2 (31), whereas a slight decrease in differentiation was reported in mouse primary myoblasts cultured at the same O2 level (44). The differences between these 2 studies may result from technical variations in culture conditions. In summary, the effects of low O2 levels on myogenic differentiation are not completely established, and the discrepancies in the literature are certainly related to several parameters, including the duration and levels of O2, the type of myogenic cells (cell line vs. primary cells, species, for example) and the technical procedures. However, the majority of the studies support the idea that a severe reduction in oxygen levels (#1% O2) inhibits myogenic differentiation in primary and cell line–derived myoblasts. Moreover, it seems that the formation of myotubes is more effective at physiologic normoxia (3–6% O2) than in standard conditions (20% O2).

SKELETAL MUSCLE REGENERATION AND EFFECTS OF LOW O2 LEVELS Skeletal muscle is a heterozygous tissue composed of multinucleated fibers that have distinct contractile and metabolic properties. Mature skeletal muscle fibers are mitotically inactive cells. They are constituted of contractile myofibrils that fill the sarcoplasm, pushing the nuclei


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TABLE 1. Effects of hypoxia on myogenesis in vitro Species

Cell type

O2 level

Main effect

Reference

1% O2

Membrane damage and slight reduced viability of myotubes ↑ Proliferation and differentiation ↑ Proliferation ↓ Proliferation and differentiation ↓ Proliferation and differentiation at 1% O2. ↑ differentiation but no change in proliferation at 5% O2 ↑ Proliferation. No effect on differentiation ↑ Proliferation of SC and myofiber survival ↑ Proliferation and slight ↓ differentiation ↓ Differentiation ↓ Proliferation and differentiation ↓ Differentiation and ↑ proliferation in response to IGF-II ↓ Differentiation inversely correlated to % O2 ↑ Proliferation and differentiation ↑ Proliferation ↓ Differentiation and maintenance of the cells in an undifferentiated state ↑ Quiescence and self-renewal of SC. Hypoxia-conditioned myoblasts have better transplanting efficiency. ↓ Differentiation Delayed embryonic development, reduced accumulation of MHC in somites

84

Rat

L6

Aged rat Rat Rat and human Rat

SC Myogenic progenitor cell L6 and human SC L6

Human

Primary myoblasts

2% O2

Mouse

SC and myofiber

6% O2

Mouse

Primary and H-2K myoblasts

5% O2

Mouse Mouse Mouse

Primary myoblasts C2C12 C2C12

1% O2 1% O2 1% O2

Mouse

C2C12

Bovine Mouse Mouse

SC SC SC and C2C12

1% O2 2% O2 1% O2

Mouse

SC

1% O2

Mouse Xenopus

SC and C2C12 Embryo

3% O2 5% O2 1% O2 1 and 5% O2

2, 0.5 and 0.01% O2

0.5% O2 10, and 5% O2

30 28 35 31 38 29 44 39 34 40 41 36 37 42 59

43 33

SC, satellite cell; MHC, myosin heavy chain.

against the sarcolemma. The satellite cells, also referred to as myogenic progenitor cells, are located between the sarcolemma and the basal membrane of muscle fibers. Satellite cells are quiescent under baseline conditions. However, in response to muscle damage, they have the ability to proliferate, fuse and differentiate to form new multinucleated muscle fibers or repair existing injured fibers (20, 21). This process replicates several aspects and cellular events observed during myogenesis (22). Although skeletal muscle regeneration and myogenesis share some similarities, these 2 processes are not identical. In particular, skeletal muscle regeneration is a dynamic and complex process that is governed by the myogenic cells, but it is also dependent on other parameters, including muscle vascularization and innervation, as well as the inflammatory response.

Effects of low O2 levels on the regenerative response Several in vitro studies provide solid data showing that the oxygen level regulates myogenesis (31, 34, 35, 39–43). The question arises as to whether low oxygen availability can affect skeletal muscle regeneration in vivo. To answer this 3932

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question, we recently compared the regenerative response of the soleus muscle after notexin injection in rats exposed to either normoxia or hypobaric hypoxia (PO2 = 79 mmHg, simulated altitude of 5500 m) (7, 45). The snake venom notexin is a phospholipase A2 toxin that causes severe myotoxic muscle damage (46). Our results revealed that hypoxia delayed the formation and growth of new myofibers during the first week after injury, but did not affect the final recovery of muscle mass and the fiber crosssectional area after 4 wk (45). Our results also indicate that hypoxia delays the recovery of the oxidative capacity of regenerated muscle without affecting the maturation of its contractile phenotype (i.e., the transition from fast to slow MHC isoforms observed during muscle regeneration) (7). Similarly, it was demonstrated that the formation of myotubes, which was repressed during the early phase of differentiation at low O2 level (0.5% O2) in vitro, was able to progress during the late phase (12 d) (41). These findings support the hypothesis that reduced muscle oxygenation delays but does not prevent myogenic differentiation and muscle regenerative response. In our model, muscle degeneration was induced by notexin injection, and muscle regeneration was investigated in response to ambient hypoxia (7, 45). Thus, hypoxia was not the stimulus that triggered muscle injury, but a factor that modulated the


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regenerative response. Even if the use of myotoxin does not seem to severely affect the microvasculature structure in regenerating skeletal muscle (46), it may influence the capacity to supply oxygen from the capillaries to the muscle in animals housed in normoxia. Additional experiments are needed to determine to which extent the injured fibers are exposed to low O2 levels in this model under normoxic conditions. A recent study proposed another approach to induce either transient or prolonged hypoxia during skeletal muscle regeneration in rats (47). Six days after injury of the quadriceps muscle caused by a needle, a cocktail of mitochondrion-targeted RNAs was injected at the injury site. This administration induced a robust and transient restoration of mitochondrial respiratory capacity in the injured muscles, leading to cellular hypoxia (evidenced by the formation of intracellular adducts of pimonidazole and by elevated levels of HIF-1a protein). In this study, clusters of satellite cells were observed within hypoxic areas between muscle fibers, suggesting that the recovery of mitochondria activity in injured fibers creates a transient hypoxic microenvironment beneficial for satellite cell proliferation. However, it is not clear whether hypoxia is the stimulus leading to myogenic proliferation or is only the consequence of the restoration of mitochondrial respiratory capacity. In this study, dimethyloxalylglycine (DMOG) was also used to stabilize HIF-1a in injured fibers and mimic prolonged hypoxia. HIF-1a stabilization compromised the induction of 2 myogenic regulators, myogenic factor (Myf)-5 and myogenin. Although HIF-1a stabilization through DMOG treatment most probably does not completely mimic a hypoxic stress, these results are consistent with our findings and provide further evidence that prolonged hypoxia has a negative effect on myogenic differentiation during muscle regeneration (45). Do low O2 levels per se induce skeletal muscle regeneration? The presented findings above provide strong evidence that the myogenic differentiation in vitro is impaired at a low O2 level (#1% O2) (35, 39, 42, 43) (Table 1), whereas severe hypobaric hypoxia delays the formation and growth of new myofibers in rats during notexin-induced muscle regeneration (45). To our knowledge, no studies have determined the effects of hypoxia on skeletal muscle regeneration after severe injury in humans. As mentioned, hypoxic stress appears to be a metabolic sensor that modulates the regenerative response in animals, but whether hypoxia itself is a stimulus that triggers muscle damage is poorly documented. Cumulative data indicate that severe hypoxia induces skeletal muscle atrophy in both humans and rodents that mainly appear to be the result of inactivated anabolic pathways and activation of proteolytic systems (see ref. 9 for a recent review). In our recent studies, we did not observe any signs of regeneration in skeletal muscles (control soleus and plantaris muscles) from rats exposed to severe hypobaric hypoxia (7, 8, 13, 45), suggesting that hypoxia per se does not trigger muscle regeneration in animals. OXYGEN LEVELS AND SATELLITE CELL ACTIVITY

Recently, Mancinelli et al. (48) investigated whether hypobaric hypoxia could affect satellite cell activity in climbers. Muscles biopsies were obtained from 6 climbers before and after an expedition at high altitude (.5000 m above sea level). The satellite cells were properly isolated from the 6 biopsy samples before the expedition, but surprisingly only the muscle biopsy from 1 climber provided satellite cells after the expedition. This single subject showed that the ability of the myogenic cells to proliferate and differentiate was lower after than before the expedition. The proportion of myogenic cells in single muscle fibers was also lower after than before the expedition. To date, it remains to be clarified whether the decreased myogenic capacity observed in this study was the consequence of exposure to extreme altitude or the result of other stresses of mountaineering (e.g., extreme physical exercise, cold climate, and altered food intake). Altogether, there is currently no robust evidence showing that low O2 levels per se induce skeletal muscle regeneration.

MYOGENESIS AND MUSCLE REGENERATION IN DISEASES RELATED TO PATHOLOGIC HYPOXIA Severe pathologic hypoxia occurs in several diseases, and the reduction of oxygen supply may be a contributing factor to muscle damage and atrophy. In this section, we focus our attention on peripheral arterial disease and COPD, 2 diseases that lead to ischemia (a restriction in blood supply to tissues) and hypoxemia (an abnormally low concentration of oxygen in the blood), respectively (4, 49). The main results are summarized in Table 2. In the model of hindlimb ischemia, the ligation of the femoral artery prevents the blood supply to the hindlimb muscles and leads to acute skeletal muscle injury (17, 50). In this model of peripheral arterial disease, the stress induced by the deprivation of O2 and nutrients are the main factors inducing muscle fiber damage. Recently, Majmundar et al. (43) showed that protein expression of 2 markers of skeletal muscle differentiation, myogenic differentiation 1 (MyoD) and myogenin, was reduced in ischemic extensor digitorum longus (EDL) muscles, when compared with nonischemic EDL muscles. This finding suggests that the absence of blood perfusion impedes the differentiation of myogenic progenitor cells in the injured muscle. In contrast, once the revascularization process is restored and perfusion of the limb is recovered, muscle regeneration occurs (50, 51). Although the ischemia-associated factors that prevent myogenic differentiation remain to be clarified (chronic inflammation, oxidative stress, lack of nutrient, and O2 supplies) cellular hypoxic stress may act as a negative regulator of myotube formation in this model. Loss of skeletal muscle mass is commonly observed in patients with COPD (16, 52). In atrophic muscles from patients with COPD, senescent satellite cells and exhausted regenerative capacity has been observed (16, 53). Furthermore, primary culture of satellite cells from patients with COPD showed a reduced accumulation of MHC into newly formed myotubes (53) and the presence of smaller myotubes (54). A reduced expression of myogenin protein was also found in skeletal muscle from


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cachectic patients with COPD compared with the expression in controls (55), and MyoD protein levels were lower in cachectic than noncachectic patients with COPD (56). Intriguingly, it was recently reported that patients with COPD with preserved muscle mass were subjected to increased muscle regenerative events (16). Collectively, these finding indicate that myogenic differentiation and skeletal muscle regenerative capacity are impaired in patients with COPD, which could contribute to the progression of muscle atrophy in this population. Thus, low O2 levels might be included among the factors (i.e., physical inactivity, systemic inflammation, oxidative stress, glucocorticoids, nutritional deficiencies, and hypoxemia/ hypoxia) that contribute to the impaired regenerative capacity observed in patients with COPD, leading to atrophy (57). Currently, the potential impact of low O2 levels on the regenerative response in patients with COPD remains to be further investigated. MOLECULAR MECHANISMS UNDERLYING SATELLITE CELL ACTIVITY AND MYOGENESIS IN HYPOXIA The activity of satellite cells and myogenesis is orchestrated by several signaling pathways and transcription factors, controlling satellite cell/myoblast activation, selfrenewal, proliferation, fusion into multinucleated myotubes, and finally the maturation of skeletal muscle fibers. During quiescence and self-renewal, satellite cells express the transcription factor paired-box (Pax)-7, and then start to express the myogenic regulatory transcription factors (MRFs), including Myf5, MyoD, myogenin, and muscle regulatory factor (MRF)-4 (see refs. 20, 21 for more detailed information). The MRFs are basic helix-loop-helix (bHLH) transcription factors that form heterodimers with the ubiquitous bHLH protein E2A, leading to the activation of gene expression through binding to the E-box sequence in the regulatory regions of target genes. Myf5 is highly expressed upon activation and proliferation, along with MyoD. MyoD remains upregulated during early differentiation, and myogenin, followed by MRF4, becomes activated during late differentiation. In addition to the regulation of the MRFs, several lines of evidence indicate that hypoxia affects signaling pathways involved in the control of myogenesis and satellite cell activity (31, 39, 40, 42, 45, 58, 59). The major molecular mechanisms underlying satellite cell activity and myogenesis in hypoxia are illustrated in Fig. 1. Myogenic regulatory factors Several in vitro studies have provided strong evidence that the expression of MRFs is impaired during myogenic differentiation in hypoxia (34, 35, 39, 41, 43, 59). In particular, cell culture experiments revealed that hypoxia (1% O2) decreases the protein expression of Myf5, MyoD, myogenin, and MRF4 during differentiation of C2C12 myoblasts (34, 36). Furthermore, the hypoxia-induced inhibition of myogenic differentiation was reversed by ectopic expression of MyoD, myogenin, and E2A (41), which supports 3934

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the idea that hypoxia impedes myogenic differentiation through the repression of MRF expression. In addition, hypoxia-induced MyoD degradation appears to occur through the activation of the ubiquitin-proteasome pathway (34). The degradation of MyoD results in the repression of myogenin, cyclin-dependent kinase inhibitor p21, and retinoblastoma protein, which prevents both cell withdraw and the terminal differentiation. In addition to the defective expression of MRFs, a low O2 level also results in reduced MHC expression, indicating that hypoxia exerts a negative effect on the terminal maturation of myotubes (34, 41, 43). We have reported that hypobaric hypoxia repressed the early regenerative response after extensive injury induced by notexin (45). Our results revealed that hypoxia attenuates the increase in MyoD and myogenin protein expression during the first week of regeneration. This finding is consistent with the delayed formation and growth of new muscle fibers in response to hypoxia. As mentioned earlier, muscle damage is induced by muscle ischemia, and skeletal muscle is able to regenerate once the blood perfusion is recovered (50, 51). Recent findings indicates that MyoD and myogenin protein expression is reduced in EDL muscle after the ligation of the femoral artery, strongly suggesting that muscle ischemia impairs myogenic differentiation (43). Mechanisms dependent on HIF-1a Regulation of HIF-1a Since its discovery in 1992, HIF-1 has been described as the master regulator of hypoxia-mediated cellular adaptations (see ref. 60 for a review). HIF-1 is a heterodimeric protein composed of a constitutively expressed HIF-1b subunit and an O2-regulated HIF-1a subunit. HIF-1a protein is ubiquitously expressed but is unstable in the presence of oxygen. In normoxic conditions, the degradation of HIF1a protein is mediated by hydroxylation of its proline residues by prolyl hydroxylase domain proteins, resulting in its binding to the von Hippel-Lindau tumor-suppressor protein, which interacts with elongin C and thereby recruits an E3 ubiquitin-protein ligase that targets HIF-1a for ubiquitination and degradation by the proteasome. Under hypoxic conditions, the hydroxylation reaction is inhibited, which prevents HIF-1a ubiquitination and proteasomal degradation. Thereafter, HIF-1a accumulates and translocates to the nucleus where it binds to HIF-1b to form the active transcription factor HIF-1. The heterodimeric protein HIF-1 then activates the expression of O2sensitive genes through binding to the hypoxia-responsive element in the promoter regions of target genes. Role of HIF-1a during myogenesis under standard conditions In contrast to other tissues, skeletal muscle contains a significant amount of HIF-1a in normoxic conditions, which becomes higher in response to hypoxia (61). In cell culture studies, it has been reported that HIF-1a protein is


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Figure 1. Molecular mechanisms involved in regulating satellite cell activity and myogenesis in hypoxia. Black arrows: direct activation of a signaling pathway or increased expression of a protein. Blunt red arrows: direct inhibition of a signaling pathway or decreased expression of a protein/micro-RNA. Blue arrows: general stimulation of a biologic process. The line between HIF-1a and Notch pathway indicated a direct interaction.

highly expressed in myotubes exposed to standard conditions, as well as at high O2 levels (42% O2), but HIF-1a accumulation was exclusively observed in the cytoplasm (62). Under reduced O2 levels (3% O2), HIF-1a was highly expressed and accumulated in the nucleus. Another study showed that the expression of HIF-1a protein is negligible in C2C12 myoblasts during proliferation, and then increases upon the early phase of differentiation (63). The ubiquitin-proteasome–dependent pathway has been suggested to induce HIF-1a degradation during myoblasts proliferation under standard conditions, whereas the heat shock protein HSP90 could promote HIF-1a stabilization during the differentiation phase (63). These researchers also showed an impaired myotube formation and reduced expression of MHC in myotubes transfected with HIF-1a small-interfering RNA. This finding suggests that HIF-1a is necessary for myogenic differentiation under standard conditions. In a recent study, Majmundar et al. (58) showed OXYGEN LEVELS AND SATELLITE CELL ACTIVITY

that C2C12 cell differentiation is inhibited by hypoxia (0.5% O2), which is partially prevented by the inhibition of Hif1a. Altogether, it seems that HIF-1a distinctly regulates myogenesis in standard conditions compared with hypoxic conditions (58, 63).

Role of HIF-1a during muscle development and muscle regeneration after ischemic injury Majmundar et al. (58) recently explored the role of HIF-1a in murine skeletal muscle development and skeletal muscle regeneration after ischemic injury. They demonstrated that the deletion of the Hif1a gene specifically in Pax3+ presomitic mesoderm cells (which differentiate in several tissues, including skeletal muscle) did not affect the embryonic and fetal skeletal muscle formation. However, structural defects were observed in tissues derived from


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Pax3+ somatic cells (e.g., altered rib bone calcification). Thus, HIF-1a appears to be essential for normal embryonic development, but not for embryonic myogenesis. The femoral artery ligation model was used to induce muscle damage and regeneration in mice. They observed that HIF-1a protein accumulated in ischemic EDL muscles 2 d after the ligation and then declined as blood flow recovered, suggesting that hypoxic stress transiently occurs in muscles in this model. Furthermore, tamoxifen-induced specific ablation of Hif1a in satellite cells was shown to increase the density of centrally nucleated fibers (d 7 after injury) and enhanced the growth of regenerated muscle fibers (d 14 after injury). These results indicate that loss of HIF1a derived from satellite cells accelerates features of skeletal muscle regeneration (i.e., fiber formation and growth), indicative that HIF1a regulates skeletal myogenesis in vivo in adult mice. Notably, the observed effects of Hif1a deletion in satellite cells differs considerably from the effects of global Hif1a deletion. EDL muscles with global Hif1a depletion display impaired reperfusion after femoral artery ligation, which confirms the established role of global Hif1a expression in revascularization after ischemic injury (43, 55). Thus, whereas HIF-1a expression in skeletal muscle originating from satellite cells constrains muscle regeneration, its expression in other tissues (e.g., endothelial or inflammatory cells) is necessary for revascularization and blood reperfusion, promoting skeletal muscle regeneration after ischemic injury.

Signaling pathways dependent on HIF-1a To explore the mechanisms behind the HIF-1a-induced impairment of regeneration in ischemic muscles, Majmundar et al. (58) exposed C2C12 myoblasts to hypoxia. They revealed that the inhibition of C2C12 myoblast differentiation in hypoxia (0.5% O2) was partially prevented by Hif1a inhibition. Furthermore, they demonstrated that HIF-1a impairs myogenesis in hypoxia through the inhibition of the canonical Wnt signaling, which is well-established to promote muscle regeneration (64). Moreover, Gustafsson and colleagues (42) have reported that hypoxia inhibits myogenic differentiation and maintains myogenic cells in an undifferentiated state through the activation of Notch signaling pathway. They showed that HIF-1a interacts with the Notch 1 intracellular domain (ICD) in response to hypoxia, leading to HIF-1a stabilization. Thereafter, the Notch 1-ICD/HIF-1a complex is recruited to Notch-responsive promoters and activates the transcription of Notch downstream genes (e.g., Hey and Hes genes). In addition to Wnt and Notch pathways, HIF-1a has also been shown to regulate the activity of ERK 1/2 MAPK in hypoxia (40). In this study, C2C12 myoblasts were examined under standard condition and hypoxia (1% O2). It was shown that IGF-II treatment causes myogenic differentiation in standard conditions. In contrast, IGF-II does not induce cell differentiation in hypoxia, but promotes cell proliferation. This study demonstrated that hypoxia promotes myoblast proliferation in response to IGF-II 3936

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through the activation ERK 1/2 in an HIF-1a-dependent manner. Mechanisms independent of HIF-1a PI3K/AKT/MTOR pathway Recent studies support the idea that hypoxia also blocks myogenic differentiation through HIF-1a-independent mechanisms. For instance, knockdown HIF-1a using lentivirus shRNA in differentiating myoblasts at 0.5% O2 showed that hypoxia-induced repression of MyoD expression was HIF-1a independent, whereas hypoxiainduced decrease in myogenin expression was HIF-1a dependent (43). Furthermore, it was demonstrated that hypoxia-induced inhibition of the phosphatidylinositol 3-kinase (PI3K)/AKT/MTOR pathway, which participates to myogenic differentiation (65, 66), can also occur in a HIF-1a-independent manner. The hypoxia-induced inhibition of the PI3K/AKT/MTOR pathway was shown to be the result of reduced IGF-I receptor sensitivity to growth factors (i.e., IGF-I) (62, 63). The hypoxia-induced repression of the PI3K/AKT/MTOR pathway has been confirmed by others studying myogenic differentiation at low O2 levels (31, 40). In addition, we reported that the impaired formation and growth of new myofibers after extensive injury in hypoxia was associated with a blunted MTOR activation (45). Specifically, we showed that hypoxia-induced inhibition of MTOR pathway is independent of AKT, but coincides with increased AMPK activity, an endogenous repressor of MTOR. Thus, it appears that hypoxia disrupts the energetic status in the early regenerative phase, leading to the activation of AMPK and the repression of MTOR signaling. p38 MAPK Ren and colleagues (67) have reported that the p38 MAPK pathway, which has been shown to play a central role in myogenesis, is severely impaired in differentiating myoblasts in response to hypoxia (40). Their experimental data indicate that hypoxia-induced repression of the p38 MAPK pathway contributes to the conversion of myogenic action (myogenic differentiation) of IGF-II into mitogenic action (myoblast proliferation). Bhlhe40 Recently, the blunted myogenic differentiation in hypoxia has been associated with an increased expression of the bHLH transcription factor Bhlhe40 (39). Bhlhe40 overexpression in standard culture conditions mimics the negative effect of hypoxia on myogenesis and, vice versa, Bhlhe40 knockdown enhances myogenic differentiation under hypoxia. The induction of Bhlhe40 expression in hypoxia is independent of HIF-1a, but involves the p53 signaling pathway. Bhlhe40 was shown to repress myogenic differentiation by binding to the E-box sequences of myogenin promoter, leading to reduced binding affinity


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and transcriptional activity of MyoD on myogenin. Altogether, these findings indicate that Bhlhe40 plays a crucial role in mediating the deleterious effect of hypoxia on myogenic differentiation. Pax7 In addition to its role in regulation myogenic differentiation, hypoxia has been proposed to maintain quiescence and promote self-renewal of satellite cells through Pax7 upregulation (59). Hypoxic culture conditions were shown to activate Notch1 (i.e., increase Notch1 ICD expression), leading to reduced expression of miR-1 and miR-206 through the Notch target genes Hes and Hey, which resulted in Pax7 upregulation. As mentioned in the Signaling pathways dependent on HIF-1a section, Gustafsson et al. (42) observed that HIF-1a interacts with Notch 1 ICD in response to hypoxia, but in contrast to Liu et al (59), they did not find that hypoxia increases the expression of Notch1 ICD. The discrepancy between these 2 studies may result from differences in the cell culture procedures and conditions. THERAPEUTIC STRATEGIES USING CHANGES IN O2 LEVEL TO PROMOTE SKELETAL MUSCLE REGENERATION Skeletal muscle damage occurs in a range of situations, including direct trauma, crush, physical exercise (e.g., eccentric contractions), pathologic diseases (e.g., ischemia, neurologic dysfunction), and innate genetic defects (e.g., dystrophies) (19). Novel therapeutic interventions are of medical importance to improve muscle function and the quality of life for patients afflicted with disease-induced muscle damage. Rehabilitation of muscle injury in athletes could also be promoted by enhanced therapeutic interventions. In this section, we will focus our attention on therapeutic strategies using changes in O2 level (hypoxia and hyperbaric oxygen therapy) to promote skeletal muscle regeneration. Transplantation of hypoxiaconditioned myoblasts Previously in this review, we highlighted data showing that a low O2 level impairs myoblast differentiation and the formation of myotubes, whereas proliferation of primary myoblasts appeared to be enhanced by hypoxia (36, 37). Along those lines, Urbani et al. (37) recently showed that the proliferative capacity of satellite cells with high myogenic potential was greater in hypoxia (2% O2) than in standard conditions. To determine whether hypoxiaconditioned satellite cells could promote muscle regeneration after extensive injury, an identical number of satellite cells cultured in either hypoxia or standard conditions was transplanted into cardiotoxin-injured muscle. This experiment showed that the conditions of satellite cell before culturing (hypoxia vs. standard condition) did not affect the regenerative response of injured muscle. The researchers OXYGEN LEVELS AND SATELLITE CELL ACTIVITY

proposed that a moderately low level of O2 could promote the expansion of satellite cells while maintaining their intrinsic myogenic potential, thereby leading to a higher transplantation capacity. Recently, Liu and colleagues (59) transplanted either hypoxia-conditioned primary myoblasts (1% O2) or nonconditioned myoblasts into dystrophic muscles from mdx mice, which lack expression of dystrophin. The amount of injected myoblasts was identical in the 2 experimental conditions. They observed that the number of transplanted myogenic cells that expressed dystrophin was higher in muscles injected with hypoxia-conditioned myoblasts than in muscles injected with myoblasts cultured in standard conditions. This result indicates that hypoxia-conditioned myoblasts improves the grafting efficiency in dystrophic muscle. Furthermore, transplanted myogenic cells derived from hypoxia-conditioned myoblasts exhibited an increased self-renewal capacity and a better ability to occupy the satellite cell compartment than myoblasts cultured in standard conditions. These 2 studies show that culturing myoblasts in hypoxic conditions has the potential for enhance regeneration of injured muscle after cell transplantation. Hyperbaric oxygen therapy Human studies Hyperbaric oxygen (HBO) therapy involves inhalation of 100% O2 at barometric pressures higher than 1 atmosphere absolute (ATA) (68). HBO treatment has been used to treat diseases and illnesses, such as decompression sickness, carbon monoxide poisoning, cerebral infarction, chronic ulcers of the lower limbs, autism, and burn wounds (69–73). In the early 1980s, HBO therapy gained popularity in treating elite athletes based on its suggested beneficial effects on bone, tendon, and muscle after a sports injury (74). Despite a growing interest in sport medicine in the 1990s and 2000s, there are limited studies looking at the effects of HBO therapy on muscle recovery after injury in human (75–77). In these studies, muscle damage was induced after eccentric repetitions, and the patients were treated daily with HBO consisting of 60–100 min of exposure to 2–2.5 ATA at 100% O2. Data showed that HBO does not promote muscle recovery after eccentric exercise (75, 76), but slightly enhances the recovery of muscle torque a few days after the eccentric repetitions (77). Even if the use of HBO therapy for the treatment of sport injuries has been suggested in the scientific literature, human studies do not demonstrate any substantial effect of HBO on the recovery of muscle function and regeneration after damage (Table 2). However, these human studies have been limited due to small sample sizes and the type of muscle injury (injury induced by eccentric exercise). The main limitation of eccentric exercise is that it does not lead to massive muscle damage, but induces delayed-onset muscle soreness, which is the consequence of microstructural damage. More recently, animal models of severe muscle injury have been used to understand whether HBO is an effective therapy to improve muscle recovery and regeneration after extensive injury.


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TABLE 2. Effects of O2 levels and diseases associated with hypoxia on skeletal muscle regeneration in vivo Hypoxia/HBO

Species

Hypoxia

Rat

HBO

Regeneration model

O2 level

Human

Soleus regeneration after notexin injection Muscle fibers and SC

Hypobaric hypoxia (PO2 = 79 mmHg) COPD

Mouse

Hindlimb ischemia

Hypoxic stress induced by ischemia

Rat

Tibialis anterior regeneration after injection with bupivacaine hydrochloride Tibialis anterior regeneration after injection with cardiotoxin Gastrocnemius regeneration after hindlimb ischemia Stretch injury of the tibialis anterior

Normal air at 1.25 ATA for 4 wk

Rat

Mouse Rabbit Rat

Rat

Rabbit

Human Human Human

Soleus regeneration after injection with bupivacaine hydrochloride EDL regeneration after injection with bupivacaine hydrochloride Muscle degeneration induced by extremity lengthening (distraction osteogenesis) Eccentric exercise (elbow flexors) Eccentric exercise (elbow flexors) Eccentric exercise (quadriceps muscle)

Main effect

Reference

Delayed formation and growth of news fibers ↑ Regenerative events in patients with preserved muscle mass. Senescent SC and exhausted regenerative capacity in patients with atrophic muscles ↓ MRF expression compared with nonischemic muscles ↑ Skeletal muscle regeneration in the early phase after injury

45 16

43 85

100% O2 at 2.5 ATA, 2 h/d, 5 d/wk for 2 wk

↑ SC proliferation and myofiber maturation

81

100% O2 at 3 ATA, 1 h/d for 2 wk

↑ Blood flow and regeneration of ischemic muscle ↑ Functional recovery and regeneration of the injured muscle ↑ Functional recovery of regenerating muscle but not the recovery of muscle size ↑ Recovery of muscle function and fiber size, with a greater effect at 3 ATA Overcome the detrimental effects of distraction to skeletal muscle and preserve muscle ultrastructure No reduction of delayed onset muscle soreness No effect on recovery after eccentric exercise ↑ Muscle torque recovery

82

.95% O2 at 2.5 ATA, 1 h/d for 5 d 100% O2 at 3 ATA, 1 h/d for 25 d 100% O2 at 3 ATA, 60 min/d, or 100% O2 at 2 ATA, 90 min/d for 25 d 100% O2 at 2.5 ATA, 120 min/d for 20 d

100% O2 at 2.5 ATA, 60 min/d for 6 d 100% O2 at 2.5 ATA, 100 min/d for 4 d 100% O2 at 2 ATA, 60 min/d for 5 d

78 80

79

86

75 76 77

SC, satellite cell.

Animal studies It has been demonstrated that HBO therapy promotes the recovery of muscle function after injury induced by muscle stretch (78) or by injection with bupivacaine hydrochloride (79, 80). More precisely, specific force production in soleus and EDL muscles previously injured by myotoxin was higher after HBO therapy (100% O2 at 3 ATA for 60 min per day) as compared with standard conditions (79, 80). These 2 studies also showed a beneficial effect of HBO treatment on muscle fiber growth in regenerating skeletal muscle after myotoxic injury, as evidenced by a higher relative cross-sectional area (CSA of regenerating fibers/ CSA of contralateral intact fibers) of regenerating fibers in 3938

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rats treated with HBO (79, 80). The effects of HBO therapy on satellite cell activity and regeneration of rat tibialis anterior muscle after injury by cardiotoxin was recently investigated (81). It was found that HBO treatment (100% O2 at 2.5 ATA for 120 min, 5 d/wk during 2 wk) promotes the recovery of muscle function (maximum tetanic force) and muscle fiber size (larger fiber CSA). In addition, the number of proliferating (Pax7+/MyoD+) and differentiating (Pax72/MyoD+) satellite cells, as well as the mRNA expression of MyoD were increased after HBO therapy. This finding provides evidence that HBO treatment increases satellite cell proliferation and muscle fiber maturation in extensively injured rat muscle. The effect of HBO therapy has also been studied in the tibialis anterior muscle


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after ligation of the femoral artery, a model of peripheral arterial disease (82). HBO therapy (100% O2 at 3 ATA for 60 min, daily for 2 wk) improved the blood flow and regeneration of ischemic muscles after femoral artery ligation. HBO therapy must be performed under strictly controlled circumstances because improper therapy conditions are associated with severe complications, such as barotraumatic lesions, oxygen toxicity of the lungs and the central nervous system, and ocular effects (83). Although the mechanisms behind the effective regenerative response of HBO therapy remain elusive, it appears to be a promising treatment for improving muscle function in patients with peripheral arterial disease.

of hypoxia-conditioned myoblasts is beneficial in patients with myopathy or severe muscle damage remains to be elucidated. Several lines of evidence demonstrate that HBO therapy promotes skeletal muscle regeneration after extensive injury in animals. To date, the molecular mechanisms through which HBO enhances skeletal muscle repair remain to be determined. The effect of HBO therapy in athletes after eccentric exercise is negligible, probably because exercise induces microstructural lesions rather than severe muscle damage. Although the use of HBO therapy requires caution, because it can induce severe complications, this therapy appears clinically relevant for treating patients with extensive muscle damage.

CONCLUSIONS AND PERSPECTIVES

ACKNOWLEDGMENTS

A reduced O2 level is naturally occurring during embryogenesis and appears to be necessary for normal development (32). Cell culture studies have demonstrated that an O2 level of 3–6% is a proficient condition to promote myogenic differentiation and myotube formation, whereas most of the studies indicate that a more severe hypoxic environment (#1% O2) has negative effects on myogenic differentiation and myotube formation. Results from animal models have provided evidence that hypoxic stress impairs the regenerative capacity of injured muscle. Although other parameters should not be excluded, hypoxic stress may be a factor contributing to skeletal muscle damage in patients who have peripheral arterial disease or COPD. Currently, the potential impact of low O2 levels on skeletal muscle damage remains to be further investigated. Numerous factors and signaling pathways have been proposed to modulate satellite cell activity and myogenesis in hypoxia. Hypoxia impairs the expression of the MRFs, leading to the inhibition of myotube growth. Moreover, recent findings indicate that the transcription factor HIF-1a negatively regulates myogenic differentiation in hypoxia and adult myogenesis in ischemic skeletal muscles. However, HIF-1a is most likely necessary in endothelial and inflammatory cells for revascularization and blood reperfusion of the regenerative muscle after ischemic injury. The inhibition of myogenesis in hypoxia appears to be mediated by HIF-1a-dependent mechanisms, which involve the Wnt signaling pathway and the Notch signaling pathway. In addition, hypoxia represses myogenic differentiation through the repression of the PI3K/ AKT/MTOR pathway and the induction of Bhlhe40 by p53 signaling pathway. Some therapeutic interventions involving changes in O2 level have been shown to promote skeletal muscle repair in animal models. One strategy, which consists of culturing satellite cells at a low O2 level (1–2% O2), seems to be beneficial in enhancing muscle regeneration after cell transplantation (37, 59). Proliferation of myogenic cells appears to be the most efficient at 3–6% O2. Hence, culturing myoblasts at this O2 level may be a more relevant strategy to improve the grafting efficiency in regenerating muscle. However, whether transplantation

This work was supported by a postdoctoral fellowship from the Wenner–Gren Foundations (Stockholm, Sweden; to T.C.). The authors declare no conflicts of interest.

OXYGEN LEVELS AND SATELLITE CELL ACTIVITY

AUTHOR CONTRIBUTIONS T. Chaillou and J. T. Lanner wrote the paper.

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Regulation of myogenesis and skeletal muscle regeneration: effects of oxygen levels on satellite cell activity Thomas Chaillou and Johanna T. Lanner FASEB J 2016 30: 3929-3941 originally published online September 6, 2016 Access the most recent version at doi:10.1096/fj.201600757R

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. The FASEB Journal Vol.30, No.12 , pp:3929-3941, October, 2017

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