Myogenic satellite cells usually quiescent at its niche that is between sacropina and basal lamina. They are activated to repair and regenerate myofibers responding to growth factors and cytokines released by inflammatory cells after muscle injury. Different regulators control satellite cell activities at different stages of cell cycling via regulate the expression of Paired box transcription factors and myogenic determination factors. Although NO, HGF, IGF, VDGF and cytokines IL-6 TGF myostain have been determinate in control satellite cells after injury, some of the detail pathway is still remain unclear.
Myogenic satellite cells are pluripotent cells which were identified by Alexander Mauro in 1961 and named due to its peripheral location. Satellite cells are located between muscle fiber plasma membrane and basal lamina during quiescent state. Mysatellit cells have the potential to develop into myblast cell which will further differentiate on to mytuble forming new myfibers. In respond to the stressors such as trauma and excises, satellite cells will exit in quiescent state , activate , proliferate ,and differentiate to myoblasts which then migrate to the site of injury , fuse with muscle fibers to repair them or and fuse each other to form new myofibers. The newly developed myofibers are defined by the centered nuclus.
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After skeletal muscular injury, infiltration of immune cells, start pahcytes the debisis of
The infiltration of immune cells are pathcytes and were believed to contribute in the second injury. However, they are important in muscular regeneration after injury by secret growth factor and cytokines. A few studies indicated that the depression of immune cells will slow the muscle repairing. There is a correlation of decrease in the number of active satellite cells.
The disrupted niche together with cytokines secreted by immune cells after skeletal muscle injury promotes activation, proliferation and differentiation of satellite cells that will regenerate myofibers.
Satellite cells could lead to muscle hypertrophy by many modulator induced by exercise and injury. Impaired activity of satellite cells also appears in diseases related to muscle hypotrophy. In term of skeletal muscle injury, growth factor and cytokines released via inflammatory response would be the predominate factors regulating satellite cells.
The satellite cell activation is induced by combining the disruption of niche and releasing cytokines via inflammatory response.
The number of satellite cells depend on the severity of trauma (Ioannis Stratos, 2009).
These progenitor cells are subject to influences from local environment including diffusible factors from interstitial cells, the vasculature, and the nervous system. In response to muscle injury, satellite cells become activated, fuse with muscle fibers, and promote muscle repair. Furthermore, activated satellite cells from undamaged parts of the muscle migrate into the site of injury. Currently, it is difficult to find out an injury-dependent proliferation pattern of activated satellite cells. Several molecules, like Pax-7, M-cadherin, MyoD and Myf5, are expressed during satellite cell activation, which are also up-regulated after muscle injury. Therefore, the present observation of muscle regeneration is based on the proliferation of activated satellite cells.
Paired box transcription factor Pax7 and Pax3 and myogenic determination factor MFS
Satellite cells have been identified by electron microscopy for many years. More recently, several antigens are used to identify satellite cells, including receptor of hepatocyte growth factors (c-met), hematopoietic progenitor cell antigen CD34, syndencan 4, muscle type cadherin (M-cadherin), intergin Î±7 and integrin Î²1, that most of the antigens are not exclusively expressed in myogenic cell line. The paired box transcription factor Pax7, selectively expressed in satellite cell, was currently used as a identifying marker in immunolabeling myofibers (Seale et al., 2000). The activated satellite cells express the muscle regulator factors MRF at different stages of cell cycling, including myogenic determination factor 1 MyoD, myogenic factor 5 Myf5, myogenin and myogenic regulatory factor 4 MRF4. In addition, the markers are not specific markers and the expression is dependent on muscle types, stages and specieces. PAX 3 is only expressed in some muscles. Satellite cells express different markers during cell cycling. For example, quiescent satellite cells (Pax7+,Myod-) during activation and proliferation, up-regulate Myod expression , becoming Pax7+Myod+ myoblasts . Then the cells undergoing differentiation will down-regulate Pax7 and become Pax7-myod+. Human cells usually express CD56 instead of CD34 in mice cells.
Pax7 belongs to the family of Pax transcription factors, containing one paired and one DNA binding homeodomain and involved in cell type and organ determination during development. Pax7 knockout mice showed a dramatic decrease in number of satellite cells during early postnatal period (Oustanina, 2004). Thus, Pax7 is possibly involved in supporting satellite cell survival. After activation , Pax7 expressing satellite cells undergoes asymmetric division, which means one daughter cell remains quiescent or self-renew , while another daughter cell expressing the myogenic regulatory factors, MyoD and Myf5, becomes proliferative, followed by up-regulation of myogenin and myogenic regulatory factor 4 MRF4 and down-regulation of Pax 7 . Mater myogenic master transcription factor Myod regulates cell differentiation by inducing later cell cycle regulators to withdraw cells from cell cycling ( Yablonka- reuveni, Z., et al , 2008).
Stem Cell Niche
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Marked to Standard
Ohlstein defined stem cell niche as "a specific location in a tissue where stem cells can reside for an indefinite period of time and produce progeny cells while self`-renewing". Niches differ among various tissue, and muscle satellite cells are located between sacromenia and basal laminar. Sacronimia is a membrane surrounding myofibers. The electrical, mechanical and chemical signals could change satellite cell behavior. On the other side, basal lamina is a major component of the extracellular matrix ECM and contains mainly of lamina, collage , and proteoglycans. The micro capillary delivery nourishes immune cells after injury. Therefore, the signals from myofibers, ECM and circulation system together regulate satellite cell quiescence, activation and proliferation (shihuan kuang , 2007). Satellite cells are maintained and protected at quiescent statues within the niche. The down regulation of genes encodes adhesion molecules and change in transcription of extracellular matrix component will affect responses to growth factor. The proteinase activity is inhibited in quiescent satellite cells to avoid breakdown of niche (Giorgia pallafacchina). Satellite cell niche will degenerate during muscle injury via infiltration of inflammatory cells.
The process of muscle repair
Process of muscle repair could be divided into three overlap stages that are destruction, repair and remolding phases. The degree of inflammation response is dependent on the severity of muscle injury.
Once trauma occurs, an immediate "damage control" process is initiated. Platelets accumulated and adhered at disrupted vasculature become activated to form platelet clot to control haemorrhage. The accurate inflammation response is triggered at the same time and initiated the cascades that recruit immune cells. Vasodilation is induced by cytokines and mediators released by damaged tissue and platelet and together they will promote chemotaxis or haptotaxis migration of immune cells to injured skeletal muscle. Blood-bone neutrophils and monocytes infiltrate into the site of injury by attracting them via selectin interaction between endothelial and immune cells. The accumulation of neutrophils reaches the peak concentration within 24 hours, releasing more pro inflammatory cytokines to attract more inflammatory cells (reference).
At regeneration phase, neutrophils and macrophages phagocyte cellular debris, switching secretion from a pro- to anti-inflammatory cytokines to prevent muscle cells from apoptosis; the secretion of growth factors and cytokines promotes satellite cell activation and growth to facilitate vascular and muscle fiber repair. During degenerative phase, the increase in inflammatory response actives satellite cells, and they continue to proliferate and differentiate, ultimately reconstituting the health myofiber population (carine smith 2008, and shu).
Inflammatory cells help muscle regeneration that involved in the activation of satellite cells. Neutrophils and macrophages secreted pro-inflammation cytokines cause necrosis and the secondary injury to muscle tissue. However, the switch from pro-inflammation to anti inflammation cytokines will promote muscle regeneration and remodeling. The subtype macrophages ED2 with CD68-/168+ are defined as anti-inflammatory promoting the regeneration of muscles. There are a few growth factors and cytokines, releasing by immune cells or myofibers, involved in the regulation of satellite cells by up or down regulation of muscle specific genes. Most of the factors are secreted by active immune cells or muscle cells under stress. The signal form vascular or motor neurons might also involve in produce of growth factor (reference).
Satellite cell activity regulator
The cytokines and growth factors are dominant regulators in skeletal muscle injury. Although a few cytokines have been indicated in regulating satellite cell activity, the detailed molecular pathways still remain unclear.
NO is catalyzed by nitric oxide synthase isoform iNOS mainly in macrophages and nNOS in endothelium cells. During acute inflammation, the reputed endothelium cells and infiltrated macrophages release iNOS and nNOS that produce large amount of NO to increase vascular permeability and vasodilatation, and to modulate chemotaxis and cytotoxicity of inflamatoty cells (Kumar, V., Filippin, L.I. ).
NO plays a role in active satellite cells by active Matrix metalloproteinases MMPS to degraded extracellular matrix liberating stored growth factor HGF. MMPs are secreted by several cell types (fibroblasts, macrophage, neutrophils) in response to growth factors, cytokines, phagocytosis and physical stress. Yamada et al showed that the activation of MMP2 mediates HFG expression is NO dependent manner in rate satellite cells culture. The MMP 2 did not convert into active form when added NO synthase inhibitor L-NAME and very little HGF produced at the culture of NOC-7-treated MMP2 with tissue inhibitor-1 of metalloproteinases ( M. Yamada, 2008). According to Barnes (2009) study, MMP 2 and MMP9 showed an increase in activity with decrease in tissue inhibitor of metalloproteinase-2 TIMP-2 after mice Tibialis anterior muscles injury by applying a cold steel probe (-79Â°C). Thus, MMP2 also contributes in muscle trauma. NO mediated MMP activation certainly partially contributes in HGF librating and satellite cell activation.
Hepatocyte growth factor HGF
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Allen and Anderson showed, after injury the increase in producing in NO will induce bound HGF releasing from extracellular matrix (). It is a rapid process within minutes and the amount of HGF releasing is proportional to the degree of injury (). HGF is also secreted by myogenic cells, inflammatory cells and satellite cells in paracrine and autocrine manner, which will continue to active satellite cells and stimulate satellite cell migration to the site of the injury (Bischoff R., 1997 and Arnold L, 2007). The released free activated HGF then binds to the c-met receptor on satellite cells plasma membrane initiating a cascade of signaling events that promote the activation and proliferation of satellite cells (Allen et al .1995 & Tatsumi et al. 2002).
According to a recent study by Yamada, the activation of HGF in satellite cell is in a dose-dependent manner, that the low concentration of HGF promotes the activation, while the high concentration will inhibit satellite cell proliferation and subsequently return to the quiescence state. When active satellite cells were cultured with 10-500ng/ml HGF, which is much higher than the optimum culture concentration of 2.5ng/ml, the percentage of active cells decreased compared to the control cultures (). The result of RT-PCR demonstrated an increase in myostain mRNA expression at high concentration of HGF culture.
The possible explanation is the dual thresholds of HFG concentration. The low concentration threshold is associated with high affinity c-met receptor (). Following mechanical stimulation, CA2+ influx through a stretch-activated -Ca2+ channel, calcium -calmodulin formation, promoting NO production (). NO radicals will active matrix metalloproteinase MMP, resulting in rapid releasing of HGF from its extracellular tethering (). However, Satellite cells might also express the unknown low affinity receptors, which only activate when the high threshold is reached after a time lag of present high concentration of HGF (). The low affinity receptors might involve signaling myostatin expression which quiescence the satellite cell (Yamada, M and et al, 2009).
Insulin like growth factor IGF1, IGF2
IGF probably is the most well studied growth factors for muscle hypertrophy. It increases protein turn-over and reduces protein degradation. Circulating IGF 1 is mainly produced in liver and it can also produce myogenic satellite cells and immune cells in a paracrine/autocrine fashion after injury (). In vitro studies showed the ability of IGF-1 and 2 to promote myogenic satellite cell proliferation, differentiation and fusion to myofibers. These studies support that the expression of IGF-1 induces muscle hypertrophy in transgenic mice, which correlates with increase in DNA content (Adms and Mccue 1998). MIGF also helps in repair process by suppressing the pro-inflammation cytokines such as TNF-Î±, IL-1Î², NFÎºB, which decrease significantly in mIGF 1 transgenic mice comparing to WT mice after 5 days of injury and there is no difference at day 2 (Laura Pelosi ,2007).
The molecular pathway involved in IGF-I-induced muscle fibers growth and hypertrophy has been shown to require protein kinase B (PKB), which is also known as Akt-mediated activation of mammalian target of rapamycin (mTOR) and downstream activation of protein synthesis via p70-S6-kinase (Rommel et al., 2001). The two main pathway up-regulated by IGF are mitogen-activated protein kinase (MAPK) pathway, which increases the mygenic DNA transcription and phosphyatidylinositol 3- kinase(PI3K) pathway followed by anti-apoptotic Akt activation. Chakravarthy demonstrated that PI3K pathway up-regulated during satellite proliferation (ä¹¦). More recent studies using MKR transgenic mice that express a muscle specific dominant negative IGF-IR in notexin-induced injury showed an delay in muscle regeneration and also a decrease in the diameter of myofiber in MKR mice (Lisa Heron-Milhavet, 2010). The number of myogenin positive cells also reduced significantly (PÂ <Â 0.0001) indicating that the decrease in myoblast differencation (Lisa Heron-Milhavet, 2010).
(The study conducted by Bischoff () demonstrated the satellite cells remained quiescence when exposed to normal muscle extract or crushed extract from other tissue but activated when exposed to crushed muscle tissue. This indicated that the component in degraded muscle tissue will unregulated satellite cell activate.)
Cytokines are short acting soluble mediators of inflammation and immune response. Cytokines promote leukocytes recruitment and acute response, regulate lymphocytes growth, and activate inflammation cells (). Cytokines also have growth promoting function and chemotaxis on satellite cells (). Cytokine such as TNF and IL-6, promote cells to exist G0 to enter the cell cycle usually followed by growth factors acting at G1 on cell proliferation (Joseph G. Cannon, 1998).
TNF-Î± is a pro-inflammatory cytokine involved in muscle wasting in some chronic diseases (). It is secreted after muscle injury and perturbs muscle regeneration by promoting satellite cells proliferation and preventing them from differentiation ().
TNF-Î± regulates satellite cell activity via JNK pathway by JNK1 in C2 cell culture (Alter, 2008). TNF-Î± treated C2 cells show an increased number of cells in S phase, but not in JNK1 null cells. TNF-Î± inhibits cell differentiation both in wild type and JNK2 null cells but not in JNK 1 null (Alter, 2008). Thus, JNK1 is a downstream mediator of TNF-Î±. Adding pharmacological inhibitor of JNK, SP600125 partially rescued the differentiation of JNK1 null in the presence of TNF (Alter, 2008).
Low level of autocrine TNF-Î± induces the p38 MAPK pathway, which is necessary for myoblast differentiation in cell culture and for the regeneration in injured muscle of mouse model (). P38 MAPK pathway stimulates Myod transcription and P21, which help cells exit cell cycle to terminal differentiation (Chen, S. E., 2007). In contrast, in addition to p38, high level of TNF-Î± also induces the JNK pathway, which may be dominant over p38 and promote proliferation of myoblasts (Alter, 2008)
IL-6 like cytokines
IL-6 is a cytokine secreted by macrophages in response to muscle injuring leading to inflammation and by muscle cells in response to muscle contraction (Febbraio MA, Pedersen BK , 2005). IL-6 binds to its receptor, type I cytokine receptor, sharing gp130 with IL-6 family cytokines and LIF receptors (). Suggesting IL-6 family initiates the same signaling pathway via JAK/STAT (Heinrich PC, 1998; D. Kamimura., 2003). The number of Myod positive cells is severely reduced in IL-6âˆ’/âˆ’ comparing to wild type mice; however, the BrdU-positive cells, which indicates cells in S phase, appeared in both IL-6âˆ’/âˆ’ and wild type with a decline in IL-6 absent cells (). Together, these demonstrate that IL-6 is critical for satellite cell proliferation but not for satellite cell activation (Antonio L. Serrano, 2008).
Transforming growth factors TGF (anti)
The transforming growth factor Î² acts as an anti-proliferating factor in satellite cells through inhibiting transcription of Mydo family members. As the muscle regulatory factors (MRF'S) such as Myf5, mydo, mygenin and MRF4, are the key factors for successful differentiation ().
There are three isoform 1, 2 and 3, and the effect on satellite cell shows an isoform independent manner in C2C12 myoblast cell culture treated with TGF-Î² (Schabort, 2008). TGF-Î² showed an increase in Myod degradation and nuclear localization with no change in Myod expression level, suggesting that TGF-Î² works on reducing the transport of Myod into nuclei and the stability of Myod (Schabort, 2008). C2C12 cell line is very sensitive to the concentration of TGF-Î² as low as 1 ng/mL (). TGF-Î² will effectively block myotube formation and Myosin Heavy Chain (MHC) expression via the mediator Smad3, as impaired Smad3 signaling suppressed myogenic differentiation at present of TGF-Î² (). TGF -Î² could inhibit cell differentiation by preventing cell from entering cell cycle (Liu, D., 2001). There is a decrease in the expression of p21 in response to the treatment with TGF-Î², which causes the failure of cells to entre G0 (). Failure to exit the cell cycle would prevent the myblasts from entering differentiation (). In addition, the C2C12 cell line could not represent the response in primary satellite cell culture due to variable TGF-Î² receptor expression (Schabort, 2008).
Myostain, also known as growth differentiation factor 8 (GDF8), is a member of the TGF-Î² family. Of all the growth factors studied so far, only myostain have been well documented to have the powerful signals that block satellite cells activation, proliferation and differentiation. Myostatin knockout mice show an increased satellite cell proliferation compared with wild-type controls (Wagner et al. 2005). Recently, myostain has also been shown to inhibit protein synthesis by suppressing Akt, which is correlated with muscle hypertrophy (). Therefore, myostain have dual inhibition on both differentiation and Akt-induced protein synthesis (Michiko, Yamada).
In term of satellite cells, myostain arrests cell cycling and is involved in down-regulating synthesis and activity of Pax 7 and MyoD, which are important in satellite cell proliferation and differentiation (). Myostain inhibits cell proliferation via up-regulation of p21, a suppressor of cycling-dependent kinase CDK25 (Michiko, Yamada). Myostatin treatment severely down-regulates the expression of Pax7 in actively growing primary myoblast culture as determined by Western blot analysis (). The level of Pax7 expression is higher in the myostatin-null primary myoblasts when compared to wild-type control (Craig McFarlane, 2008). Myostatin-treated myoblasts also show a down-regulation of MyoD occurred at RNA level (Langley, 2002).
Satellite cells repopulate myofibers in responding to growth factor and cytokines. The growth factor HGF, IGF and VEGF basically involve activation of satellite cells to enter the cell cycle. However, cytokines either enhance or inhibit proliferation and differentiation to induce satellite cells finally differentiate in to myofibers . pro inflammation cytokines mainly promotes satellite cell activation and proliferation , while anti inflammation cytokines surepress satellite cell activity or induce the terminal diffeernation to form myotube. This process is delicate and involves multipathways which might interact with each other. Some of the regulators such as HGF, are dose sensitive, suggesting that different receptors and pathways active at different concentration to act as negative control. In addition, the study on my cell lines might not represent the satellite cell activity. Thus, further more studies need be conducted on primary cell cultures.