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Musculoskeletal Regeneration 2016; 2: e1193. doi: 10.14800/mr.1193; © 2016 by Zhuohui Gan http://www.smartscitech.com/index.php/mr REVIEW Hypoxia in skeletal muscles: from physiology to gene expression Zhuohui Gan Department of Bioengineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0412, USA Correspondence: Zhuohui Gan E-mail: [email protected] Received: January 16, 2016 Published online: February 29, 2016 Hypoxia is studied as a common clinical factor and a condition associated with a number of diseases. The responses induced by hypoxia in tissue are hypoxia severity and duration dependent. Compared with other tissues, skeletal muscles are relatively tolerant to hypoxia due to its low oxygen demand and its adaptation to hypoxia during physical exercise. This review focuses on molecular responses of skeletal muscles to hypoxia, with an emphasis on signaling pathway. Based on published works, hypoxia in skeletal muscle induces a number of responses involving energy metabolism, redox metabolism and angiogenesis. Signaling pathways including PI3K/Akt signaling, AMPK/mTOR signaling, HIF signaling as well as Notch signaling are widely involved in these responses. Energy metabolism associated signaling pathways such as PI3K/Akt signaling, AMPK/mTOR signaling are active during hypoxia in skeletal muscles. Keywords: hypoxia; skeletal muscle; mitochondria; metabolism; signaling pathway; angiogenesis; PI3K/Akt signaling To cite this article: Zhuohui Gan. Hypoxia in skeletal muscles: from physiology to gene expression. Musculoskelet Regen 2016; 2: e1193. doi: 10.14800/mr.1193. Copyright: © 2016 The Authors. Licensed under a Creative Commons Attribution 4.0 International License which allows users including authors of articles to copy and redistribute the material in any medium or format, in addition to remix, transform, and build upon the material for any purpose, even commercially, as long as the author and original source are properly cited or credited. Introduction Hypoxia is identified as a clinical factor widely associated with a number of pathological, physiological or environmental conditions. Diseases such as anemias, shock, acute respiratory distress syndrome can induce a whole body or tissue hypoxia due to the insufficient oxygen availability under these pathophysiological conditions [1-4]. Hypoxia is also identified as an important clinical factor in tumor metastasis as well as a role involved in normal tissue development and differentiation [5-7]. Additionally, exposure to high altitude is an environmental condition which induces a whole body hypoxia due to the decreased barometric pressure and hence a decrease in arterial oxygen pressure [8, 9]. Another situation that potentially results in hypoxia is physical exercise, where the exercising skeletal muscle undergoes oxygen tension due to the rapid oxygen consumption [10-12]. Therefore, hypoxia is widely studied as a common clinical factor and a condition widely associated with lethal diseases. It is worth noticing that responses to hypoxia are dependent on the severity and duration of hypoxia. For example, insulin resistance is widely reported accompanying hypoxic exposure. Baum et al. observed insulin resistance in dogs exposed to 8% O2 for 30 minutes but not dogs exposed to 10% or 12% O2 for 30 minutes [13]. Similarly, the decreased blood glucose is observed in subjects exposed to 8% Page 1 of 9 Musculoskeletal Regeneration 2016; 2: e1193. doi: 10.14800/mr.1193; © 2016 by Zhuohui Gan http://www.smartscitech.com/index.php/mr O2 for 15-30 minutes but not 12% O2 [14]. These differential responses suggest a dependency of hypoxia responses on hypoxia level. On the other hand, the hypoxic duration also affects hypoxia responses. For example, the insulin resistance which can’t be observed in 30 minutes of exposure to 10% O2 is observed in 24 hours of exposure to 10% O2 [15]. Longer exposure such as 4 weeks exposure to 10% O2 can then lead to an increase in insulin sensitivity [15]. Similarly, the changes on blood glucose also indicate a temporal feature. The glucose level decreases in 2 hours of exposure to 11% O2 and then recovers to the normal level after 4 hours of exposure [16]. Though changes on gene expression under hypoxia, especially in the early stage of hypoxia, are poorly studied, the existing studies suggest a similar dynamic feature. For example, the expression of glucose transporter 4 (GLUT4) is increased after mice one-day exposed to 10% O2 and recovers to the normal level after 4 weeks of exposure. [15] . In summary, hypoxia responses show a dynamic feature which is hypoxic-duration and hypoxic-severity dependent. Compared with other tissues such as brain, heart, which are oxygen-sensitive, skeletal muscles are relatively tolerant to hypoxia, probably due to the energy buffering system, the relatively less demand of oxygen and its accustom to hypoxia during physical exercise [17-19]. However, hypoxia still induces a number of responses in skeletal muscles. Chronic hypoxia reduces fiber area to improve oxygen diffusion into muscle cells and muscle mass to decrease oxygen demand, along with a developed muscle fiber atrophy due to the inhibition of protein synthesis caused by energy stress under hypoxia [20]. Acute hypoxia induced by exercise can lead to skeletal muscle vasodilation and hence maintain the delivery of O2 as a compensatory response [21]. As a metabolically active tissue, a rapid switch from aerobic to anaerobic metabolism is observed in skeletal muscles during hypoxic exposure [22-24]. Hypoxia studies of skeletal muscles also report impairments of mitochondrial functions and increased production of reactive oxygen species [19, 23, 25-27]. Recent studies suggest that these responses are associated with signaling pathways such as 5'-AMP-activated protein kinase / mammalian target of rapamycin (AMPK/mTOR) signaling, hypoxia inducible factor (HIF) signaling and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling [28-32]. To further our understanding of the effects of hypoxia on skeletal muscles, this review briefly summaries the changes on biological systems including redox metabolism, energy metabolism and angiogenesis in skeletal muscles under hypoxia and the involved signaling pathways in these changes. The effects of hypoxia on biological systems in skeletal muscles I. Changes in mitochondria and redox metabolism during hypoxia It is well-known that hyperoxia induces oxidative stress, while hypoxia is also demonstrated as a trigger of oxidative stress due to the increase in the generation of reactive oxygen species (ROS) [33-35]. Mitochondria is the most important source of ROS due to the leakage of electron in the respiratory chain at complex I and complex III [36]. The impairment of mitochondrial respiratory chain increases electron leakage and hence results in an increase in mitochondrial production of reactive oxygen species (ROS) [37, 38] . A decrease in mitochondrial density and an impairment of the activity and coupling of mitochondrial respiratory chain, accompanying with an increased production of mitochondrial ROS, are observed in skeletal muscles in chronic obstructive pulmonary disease (COPD) patients who suffer from chronic hypoxia [36]. Consistently, Magalhaes et al. report a functional impairment of mitochondrial complex V in skeletal muscles during an acute hypoxia induced by eccentric exercise [12]. A switch of mitochondrial complex IV subunits COX4-1 and COX4-2 is discovered by Fukuda et al. which contributes to the change of complex IV activity under chronic hypoxia [39]. Similarly, a decrease in oxygen consumption through mitochondrial state III respiratory in skeletal muscles from rats exposed to high altitude for 30 days is found [40]. These results suggest an impairment of mitochondrial function along with an increase in ROS in skeletal muscles exposed to either chronic or acute hypoxia. In Horscroft et al.’s review regarding hypoxia-induced changes in skeletal muscles, he points out that mass-specific mitochondrial function is decreased prior to mass-specific mitochondrial density, implicating intra-mitochondrial changes in the response to environmental hypoxia. [23]. Recently, Jacobs et al. reported an increase in the mitochondrial volume density in skeletal muscle after 28 days of exposure to high altitude [41], where the mitochondrial function is not affected through either complex I or complex II [42] . This result partially agrees with Horscroft’s conclusion. Along with the changes on mitochondria, the production of nitric oxide (NO) is increased quickly either in oxygen sensitive tissue such as brain or oxygen tolerant tissue such as skeletal muscle during ischemia [43, 44]. As a vasodilator, the increase of NO production plays a role in vasodilation. Joyner et al. suggests that NO played the role by linking to a β-adrenergic receptor mechanism in hypoxia caused by a low intensity exercise [21]. Though the synthesis of NO is increased in the acute hypoxia, the hypoxia study in smooth muscle by McQuillan et al. suggests a decrease in mRNA transcription of endothelial NO synthase (eNOS) following a decrease in eNOS mRNA half-life after 24 hours of hypoxia [45]. This inconsistence suggests the complexity of the relationship between mRNA Page 2 of 9 Musculoskeletal Regeneration 2016; 2: e1193. doi: 10.14800/mr.1193; © 2016 by Zhuohui Gan http://www.smartscitech.com/index.php/mr expression and protein synthesis. Studies in brain suggest the potential role of NO on the activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) under hypoxia [46]. Besides NO, increases in the activity of anti-oxidant enzymes such as superoxide dismutase (SOD) which removes superoxide and catalase (CAT) which removes hydrogen peroxide as well as pro-oxidant enzymes such as NADPH oxidase are also reported [47]. Thus, the redox system is active under hypoxia as both the pro-oxidants and the anti-oxidants are responded. II. Changes in energy metabolism during hypoxia Due to the shortage of O2 supply, ATP production through oxidative phosphorylation in mitochondria is decreased during hypoxia [48]. A rapid switch from aerobic to anaerobic metabolism such as glycolysis is observed during hypoxic exposure [22-24, 49]. The expression of glucose transporters such as GLUT1 and GLUT3, glycolytic enzymes and lactate dehydrogenase are increased [23, 48, 50]. It is believed that the changes of blood glucose due to the inhibition of oxidative phosphorylation cause the up-regulation of GLUT-mediated glucose transport [51]. Briefly, more GLUTs are translocated to plasma membrane and transporters pre-exiting in the plasma membrane are activated [51], while prolonged exposure to hypoxia suggest an enhancement of transcription of GLUT1 and GLUT4 [16, 51, 52]. These responses contribute to the optimization of the ATP synthesis in the face of the down-regulated oxidative metabolism under the hypoxic environment. Hypoxia inducible factor (HIF-1) is suggested as a primary regulator of the stimulated expression of GLUT and glycolytic enzymes under hypoxia [53-55]. Blood insulin, which is highly relevant to blood glucose [14], is increased its level and decreased its sensitivity in skeletal muscles under hypoxia [56]. Published works indicate a relationship between insulin resistance and a decrease in Akt activity in PI3K signaling pathway [56-59]. Besides glycolysis, the increased pyruvate dehydrogenase kinase is considered as another way to compensate the decreased ATP production [55]. This response might be tissue specific since, in cardiac muscles, a decrease in pyruvate metabolism is reported [17]. Furthermore, Norrbom et al. report an enhancement of fatty acid metabolism in skeletal muscles during acute hypoxia induced by exercise [60], while Morash et al. report a reduction of fatty acid oxidation in skeletal muscle during hypoxia [17]. These results suggest a potential role of fatty acid metabolism in energy rebalance during hypoxia. III.Changes in angiogenesis during hypoxia A number of studies have indicated that hypoxia is a trigger of angiogenesis [28, 61-63]. In brain, significant increase in angiogenesis is found after mice exposed to moderate hypoxia for 3 weeks [64]. Though Lundby et al. suggest that hypoxia does not appear to promote angiogenesis in skeletal muscles at rest [4], skeletal muscles exhibit the capacity to generate new capillaries as an adaptation to exercise training [65] . Angiogenesis associated with hypoxia is suggested as a complex process that has been tested in both in vitro and in vivo studies [61]. HIF-1 is found as an important regulator which affects angiogenesis in several ways including angiogenic factors, proangiogenic chemokines and their receptors, leading to neovascularization and protection against hypoxic injury [66-68]. Among those angiogenic factors, vascular endothelial growth factor (VEGF) is known as the most prominent proangiogenic factor that can be activated by [69, 70] hypoxia . Recent studies suggest peroxisome-proliferator-activated receptor-c coactivator-1α (PGC-1α) as an independent regulator of VEGF expression, angiogenesis in cultured muscle cells and angiogenesis in in vivo skeletal muscle [62]. Besides VEGF, the strong increase of Erythropoietin (EPO), which is required for the formation of red blood cells, can be another contributor for the angiogenesis during hypoxia [71]. Additionally, hypoxia has been shown to increase the rate of collagen synthesis. Exposure of mice to hypoxia induced up-regulation of collagen type I and type III expression [72]. Transcriptional studies indicate that a number of genes involved in angiogenesis have been shown to increase by hypoxia [73]. For example, Myllyharju et al. shows an increase in mRNA expression of proteins in collagen chains such as Proα1(I), proα1(III), and α2(IV) and vertebrate collagen P4Hs I and II after hypoxic exposure [1]. LOX which are likely involved in extracellular matrix synthesis are also strongly increased its gene expression during hypoxia [74, 75]. The role of signaling pathways in hypoxia responses I. AMPK/mTOR signaling AMPK is an important energy sensor. Stimuli either inhibiting ATP synthesis or accelerating ATP consumption can activate AMPK, including hypoxia which induces an energy stress [76, 77]. The activation of AMPK can repress fatty acid synthesis while enhance fatty acid uptake, fatty acid oxidation and glucose uptake [76, 78, 79]. In skeletal muscles, changes induced by AMPK activation can be acute through the direct phosphorylation of metabolic enzymes, or chronic, through the control of gene expression [30]. In our transcriptomic study on hours of hypoxia in mouse skeletal muscles (results not published yet), the gene changes are enriched in AMPK/mTOR signaling pathway, suggesting the involvement of AMPK/mTOR signaling in gene responses. Peroxisome proliferator-activated receptor α (PPARα) is critical for muscle endurance since it is an important Page 3 of 9 Musculoskeletal Regeneration 2016; 2: e1193. doi: 10.14800/mr.1193; © 2016 by Zhuohui Gan http://www.smartscitech.com/index.php/mr regulator of fatty acid oxidation. In skeletal muscles, a decrease in PPARα expression is found during hypoxia [17]. Li et al. suggests that the hypoxia-induced increase in PPARα was AMPK dependent [80]. Additionally, PGC-1α, the co-activator of PPARα and the master regulator of mitochondrial biogenesis, has been shown an increased gene expression induced by the activation of AMPK in skeletal muscle [27]. The AMPK-dependent phosphorylation of PGC-1α initiates gene regulatory functions of AMPK in skeletal muscles including the expression of glucose transporter 4 (GLUT4), mitochondrial genes, and PGC-1α itself [27]. PGC-1α and its co-activator can induce expression of E2-related factors NRF-1 and NRF-2, which can enhance the expression of nuclear-encoded subunits of respiratory chain [81]. Furthermore, they also act on the promoters of genes encoding transcription factors for mtDNA such as mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M) and mitochondrial transcription factor B2 (TFB2M) [82, 83]. Consistently, after muscle cells exposed to hypoxia for 5 days, a significant increase in NRF-2α expression and a decrease in TFB2M expression are observed [27]. Additionally, chronic hypoxia studies also suggest that the activation of AMPK is essential for mitochondrial biogenesis in muscle tissue [84, 85]. In addition to the direct regulation on mitochondrial bioenergetics and metabolic bioenergetics, Benziane et al. reported an AMPK-dependent activation of an energy-consuming ion pumping process, where cyanide-induced artificial anoxia induced an activation of AMPK and hence promoted translocation of the Na+, K+-ATPase α1-subunit to the plasma membrane in muscle cells [77]. In summary, as an energy sensor, AMPK plays an important role in hypoxia responses, where metabolism and gene expression are widely regulated. II. Insulin/PI3K signaling Insulin resistance is widely reported during hypoxia [13, 15, . This might be relevant to the changes on blood glucose during hypoxia as the blood insulin is highly relevant to blood glucose [14]. Previous studies suggest that this resistance is associated with changes on Akt activity in PI3K/Akt signaling pathway [57-59]. Hypoxia can inhibit PI3K/Akt pathway in a predominantly HIF-1α-independent manner [89]. The deprivation of oxygen affects Akt activity by reducing insulin-like growth factor I receptor sensitivity to growth factors and hence contributes to insulin resistance under hypoxia [89]. D’Hulst et al. further points out that though insulin resistance is observed in skeletal muscles, the development of whole body insulin intolerance is not affected by the defect of insulin signaling in skeletal muscles [16] . This suggests that the changes on PI3K/Akt signaling are not skeletal muscle specific. 16, 52, 86-88] The changes on PI3K/Akt signaling play a role of hypoxia responses. Majmunda et al. suggest that Akt could be a key molecular link between oxygen and skeletal muscle differentiation [89]. Recently, heme oxygenase 1 (HO-1), which plays a protective role against insulin resistance, is found to increase its expression by PI3K/Akt signaling through an activation of nuclear translocation of NRF-2 during hypoxia [90]. PI3K/Akt signaling is also reported as a regulator of HIF-1 activation evidenced by the up-regulation of HIF-1 transcription by PI3K/NF-κb [91] and the repression of the activation of HIF-1 by targeting PI3K/Akt [92]. In our transcriptomic study on hours of hypoxia in mouse skeletal muscles (results not published yet), insulin/PI3K signaling is identified as an important nodal signaling pathway. These in vivo studies suggest insulin/PI3K signaling could play a big role in hypoxia responses. III. HIF signaling HIFs, whose protein level is increased by the inhibition of its degradation when O2 supply is insufficient, play an important role in hypoxia responses [29]. The discovered members of HIF family include HIF-1α, HIF-2α and HIF-3α. HIF-1α is considered as the dominant member, while HIF-3α negatively regulates HIF-1α and HIF-2α due to the lack of C-terminal transactivation [93]. Previous studies indicate that one hour of systemic exposure to 6% O2 is sufficient to increase HIF-1α protein level in most organs including skeletal muscles [94]. However, the HIF-1α protein level in skeletal muscles exposed to altitude (~11-12% O2), either acutely or chronically, is barely modified [95]. Lundby et al. summarize published work on altitude hypoxia and conclude that HIF-1 signaling is only activated to a minor degree by hypoxia in skeletal muscles [4]. The difference in hypoxic severity, severe hypoxia (6% O2) vs. moderate hypoxia (~12% O2), may be a cause of the different conclusions. On the other hand, at to the gene expression, Slivka reports that exercise but not one hour of hypobaric hypoxia exposure (~13% O2) plays a role on gene expression of HIF-1α in skeletal muscles [96] . Furthermore, Robach et al. suggests that HIF-1α protein expression is probably not regulated at the transcriptional level since high altitude only permanently up-regulates mRNA level of HIF-1α but not protein level of HIF-1α in skeletal muscles [97]. In our transcriptomic study on hours of hypoxia in mouse skeletal muscles, mRNA of HIF-1α and HIF-2α is not changed in up to 6 hours of severe hypoxic exposure (8% O2), though the protein level of HIF-1α is supposed to be increased. This result partially agrees with above argument. As an important transcription factor induced by hypoxia, HIFs regulate a number of genes containing a hypoxia response element (HRE) in their regulatory sequences. Page 4 of 9 Musculoskeletal Regeneration 2016; 2: e1193. doi: 10.14800/mr.1193; © 2016 by Zhuohui Gan http://www.smartscitech.com/index.php/mr Genome-wide chromatin immunoprecipitation assay identified several hundreds of genes as targets of HIFs [98, 99], which play roles in the reduction in oxygen consumption, enhancement of oxygen delivery, regulation of inflammation, angiogenesis and extracellular matrix homoeostasis in hypoxic conditions [1]. Lee et al. summarizes that the target genes of HIF-1α are especially enriched in functions associated with angiogenesis, cell proliferation/survival, and glucose/iron metabolism [100]. In our transcriptomic study, HIF signaling pathway is identified as one of the gene responses-enriched pathways in skeletal muscles after hours of systemic hypoxic exposure. These results indicate the role of HIF signaling pathway in gene regulation in skeletal muscles during hypoxia. IV. VEGF signaling VEGF plays an important role in angiogenesis, which directly participates in angiogenesis by recruiting endothelial cells into hypoxic and avascular area and stimulating their proliferation [101]. Hypoxia stimulates the secretion of VEGF, which protects against hypoxia injury [102]. HIF-1α plays a role of the increased VEGF as HIF-1α is identified as a trigger of the transcription of VEGF [73]. The expression of VEGF gene is critically important for angiogenesis in skeletal muscles because the deletion of VEGF gene in the mouse has been shown to greatly reduce skeletal muscle capillarity [26]. An increase in the mRNA level of VEGF is found during hypoxia induced by exercise in skeletal muscles [103] . However, in our transcriptomic study when mice are exposed to acute severe hypoxia, the mRNA level of VEGF is decreased in skeletal muscles. This suggests that the gene responses of VEGF could be stimulus-dependent. How the changes on mRNAs translate into the functional responses is not clear yet, especially for those temporary changes during an acute stimulus. Additional mechanisms that can signal VEGF activation in skeletal muscles during hypoxia include inflammation, which is possibly linked to oxidative stress, or an energy stress reflected by AMP kinase activity [26]. V. PGC signaling PGC-1α, a potent metabolic sensor and regulator, is reported as a transcriptional co-activator which could be induced by hypoxia [104]. Shoag’s study shows a powerful induction of PGC-1α gene expression in muscles during exercise which does not appear to require HIF activity [104]. This induction of VEGF expression by PGC-1α is also demonstrated as HIF-independent [104]. However, O’Hagan finds the PGC-1α dependent induction of HIF target genes is regulated by HIF-1α protein stabilization but not HIF-1α mRNA level [105]. Recently, Baresic et al. investigated the effect of PGC-1α on transcriptional network and suggests that ERR-α and the activator protein 1 complex (AP-1) play a major role in regulating the PGC-1α-controlled gene program of the hypoxia response [106]. Several studies have been done to discover the cause of PGC-1α induction during hypoxia. Jager et al. suggest that AMPK phosphorylates PGC-1α directly during hypoxia [76]. Insulin signaling is found to inhibit PGC-1α activity through Akt [107]. A more recent study indicates that PGC-1α expresses its truncated forms which confer angiogenic specificity to the PGC-1α-mediated hypoxia response in skeletal muscle cells [108]. The role of PGC-1α during hypoxia is widely studied. PGC-1α transcriptionally activates the nuclear respiratory factors NRF-1 and NRF-2, which are known to be important for mitochondrial biogenesis [60], while Arany et al. suggest that PGC-1α impacts mitochondrial biogenesis through its co-activator oestrogen related receptor α (ERR-α) [62]. Kallio et al. suggests a switch in fuel consumption to fatty acids with the expression of PGC-1α [109]. In addition, PGC-1α impacts the fiber types and oxidative capacity of muscle fibers. The study by Lin et al. shows a 10% conversion of type II fibers to type I fibers in PGC-1a transgenic mice [60]. VI. Notch signaling Notch signaling is reported as a highly evolutionarily conserved pathway that regulates many aspects of cellular differentiation [110]. Pilar et al. suggests an inhibition of the differentiation in myogenic cells under hypoxia, with the stimulated expression of Notch downstream genes such as Hes1 and Hey2 after 4 hours of hypoxia [110]. Deregulated Notch signaling has been linked to tumor development, though Notch can act either as an oncogene or as a tumor-suppressor depending on the cell type [5]. Different from Pilar’s results, Koning et al. reports an increase in proliferation rate of cultured muscle stem cells (human satellite cells) under hypoxia [111]. The gene expression of transcription factors Pax7, Myf5 and Myod as well as gene expression of structural proteins Myl1 and Myl3 in cultured muscle stem cells are also up-regulated by hypoxia [111]. Our transcriptomic study on hours of hypoxia in in vivo skeletal muscles suggests Notch signaling as a pathway enriched with gene changes, where Hes1 and Hey1 are significantly down-regulated. This suggests the gene regulation in Notch signaling pathway under hypoxic could be tissue specific. The relationship between changes on gene expression and the potential phenotype is not clear yet due to the complexity of the translation of gene changes, especially for acute studies. Summary Page 5 of 9 Musculoskeletal Regeneration 2016; 2: e1193. doi: 10.14800/mr.1193; © 2016 by Zhuohui Gan http://www.smartscitech.com/index.php/mr Though skeletal muscles are relatively tolerant to hypoxia, the current published data suggest that hypoxia induces a number of responses in skeletal muscles including energy rebalance, redox metabolism and others. These hypoxia responses are dynamic. The hypoxia severity and duration play a role on the kinetics of hypoxia responses. Previous studies indicate that signaling pathways such as PI3K/Akt signaling, AMPK/mTOR signaling, HIF signaling, PGC signaling are widely involved in hypoxic responses in skeletal muscles, accompanying with transcriptional changes. The systemic relationship between transcriptional responses and physiological responses is still unclear and could be a challenging and interesting research direction. Conflicting interests The author has declared that no conflict of interests exists. 6. Jin XL. The effect of hypoxia on differentiation from myeloid-like cells to osteoclasts in mouse. Bone 2010; 47:S364-S364. 7. Matsuo-Takasaki M, Zhao Y, Ohneda O. Effects of hypoxia on neural differentiation of mouse embryonic stem cells. Differentiation 2010; 80:S43-S43. 8. Kuo NT, Benhayon D, Przybylski RJ, Martin RJ, LaManna JC. Time course of the hypobaric hypoxia-induced vascular endothelial growth factor (VEGF) mRNA and protein in adult mouse brain. Faseb Journal 1997; 11:1697-1697. 9. Magalhaes J, Ascensao A, Soares JMC, Ferreira R, Neuparth MJ, Marques F, et al. Acute and severe hypobaric hypoxia increases oxidative stress and impairs mitochondrial function in mouse skeletal muscle. 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AKT: protein kinase B; AMPK: 5'-AMP-activated protein kinase; AP-1: activator; protein 1; CAT: catalase; COPD: chronic obstructive pulmonary disease; eNOS: endothelial nitric oxide synthase; EPO: erythropoietin; ERK: extracellular; signal-regulated kinase; ERR-α: oestrogen related receptor α; GLUT: glucose transporter; HO-1: heme oxygenase 1; HRE: hypoxia response element; JNK: c-jun N-terminal kinase; MAPK: mitogen-activated protein kinase; mTOR: mammalian target of rapamycin; NF-κb: nuclear factor κb; NO: nitric oxide; NRF: nuclear respiratory factor; PI3K: phosphatidylinositol 3-kinase; PGC: peroxisome-proliferator-activated receptor-c coactivator; ROS: reactive oxygen species; SOD: superoxide dismutase; TFAM: mitochondrial transcription factor A; TFB1M: mitochondrial transcription factor B1; TFB2M: mitochondrial transcription factor B2; VEGF: vascular endothelial growth factor. 12. Magalhaes J, Fraga M, Lumini-Oliveira J, Goncalves I, Costa M, Ferreira R, et al. 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