Basic units of skeletal muscles

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Myofibres are basic units of skeletal muscles. They are formed of large multinucleate muscle stem cells. There are two kinds of myofibres, fast and slow contracting. The determination of different kinds depend upon the myosin heavy chains isoforms they express.MyHC content within an individual differs because myosin are encoded by different nuclei and maintained in nuclear domain. [14]

Fig 1. Differentiating capacity and typology of skeletal muscle stem cells

New muscle fibres are produced after existing ones are injured or are suffering from a disease. 'Satellite Cells' and myogenic progenitors which are present in basal lamina and muscle fibre membrane help in regeneration.[8] The first satellite cell was determined in 1961 in the tibialis anticus muscle of the frog.[14] They have their own membrane enclosed compartment and have more nucleus than cytoplasm. The molecular markers that are present in satellite cells are genes Pax7and Pax3, basic helix loop helix gene Myf5, and M- cadherin and caveloin-1[8]. Whenever a muscle is injured the satellite cells differentiate into myoblasts. [8]There are several pathways which trigger the activation of satellite cells. There are several factors involved, namely HGH, IGF, FGF and TGF beta family. [8][14][15]

After injury during regeneration HGF (hepatocyte growth factors) and protein levels are increased. When c- met an HGF receptor is expressed on C2C12 cells there is a generation of muscle precursor cells .It also activates the Ras Ral pathway which in turn send the satellite cells to the site of injury [1][14][15]

Fibroblasts growth factors (FGF) are large family of polypeptides growth factors. During muscle regeneration FGF6 expression is upregulated FGFR1 and FGFR4 are selectively expressed but when one of them is absent then muscle development is affected. For the growth of satellite cells FGFR4 is helpful while FGFR1 helps in maintaining the myoblasts which have already been formed [1] [14] [15]

The TGF BETA family are important cytokines which regulate the cell activity. Depending upon which state the satellite cells are in, the target genes are activated when TGF beta activates the phosphorylation of SMAD proteins which in turn is transferred into the nucleus. The TGF beta family has 3 members TGF beta1, TGFbeta2 and TGFbeta3.Recently a new member of the TGF beta family has been discovered which is called the myostatin (MSTN) or growth and differentiation factor 8 (GDF8).Few studies suggest that MSTN signalling can be achieved by binding of MSTN receptors to activin type 2. But a few experiments also show that when MSTN levels are high it inhibits the proliferation of C2C12 via cyclin cdk2 inactivation of retinoblastoma protein. Few studies also suggest that MSTN is mostly expressed in fast contracting muscles which contain low number of satellite cells rather than muscle mass. Thus all the studies together suggest that MSTN regulates myogenin by acting as an inhibitor to the satellite cell proliferation [1] [14] [15].

Figure 2

Asymmetric cell division of satellite cells during activation. This drawing represents the anatomy of a muscle fibre with adjacent small vessels. SCs and other myogenic cells are depicted. SC activation in vivo is followed by an asymmetric division, with Pax7, MyoD, and Myf5 being expressed in differentiating cells and Pax7 in cells returning to quiescence in order to maintain a pool of progenitors.

Courtesy: - Tedesco, F. S., A. Dellavalle, et al. (2010). [8]

Yajima et al suggested the presence of 'SIX' family genes which have six members control the growth of muscle satellite cells [7].During myogensis SIX1 and SIX4 are expressed in myoblasts and play important roles, removal of SIX1 and SIX4 genes lead to muscle hypoplasia. In an experiment when skeletal muscle was injured by cardio toxin SIX1 and SIX 4 positive cells were shown to be regenerating muscle. The quiescent muscle cells which have Pax7 and M - cadherin as molecular markers on them also possess SIX1 and Six4 genes. One of the interesting findings of this study is the growth of muscle satellite cells is negatively regulated when the amount of SIX proteins are above the normal level. But the presence of SIX5 protein acts as a cardinal repressor in the growth of activated satellite cells [7].

There are various model systems which have studied to gather information about the role of stem cells in muscle regeneration.[3] The non mammalian models which have studied are mostly 'Jelly Fish Muscle' model and the 'Amphibian Limb regeneration' model. The most commonly used animal models are chick and mouse model systems. Figec et al describes the embryonic origin of satellite cells using chick and mouse models systems [3].In chick the muscle progenitors and most of the satellite cells are derived from the dermomytome. In mouse, cells marked by Pax3 and Pax7 are formed in early myotome. They are maintained as growing population of cells and form the resident muscle progenitor cells. An emerging model to study muscle stem cells is 'Drosophila melanogaster'. In Drosophila the muscle stem cells are called adult muscle precursor cells (AMP) which have 'Twi' expression on them and can be easily identified. The mesoderm gives rise to the larval muscles which expresses the helix loop helix transcription factor (Twi factor). The body wall muscles arise from the mesodermal cells which have high levels of 'Twi'. At the end of larval life the AMP's grow and give rise to the adult fly musculature. [3]

Nicholas et al recently found and suggested that absence of CD34 on skeletal muscle satellite cells marks a reversible state of activation during injury [5].CD34 is found as a receptor on satellite cells of skeletal muscles. After injury CD34+ cells are activated to maintain a reserve during the course of regeneration , but the absence of Cd34 also shows a state of activation which is reversible .Cd34+/- equally take part in myogensis but CD34- cells show lower expression of Pax7, Pax3, Myf5, MyoD and c-met. Although the actual function of CD 34 is still unclear, it is a highly glycosylated siaolmucin receptor which plays a role in signalling, cellular adhesion and repulsion [5].

Filippin et al reviewed the role nitric oxide and repair of skeletal muscle injury. [4].Skeletal muscle undergoes a lot of injuries such as crush, bruises, freezing etc. These have profound effects on the normal functioning of the muscle. The repair of muscle takes place in three stages: degeneration and inflammation, regeneration of the muscle, fibrosis. After an injury the myogenic satellite cells are activated. They proliferate and differentiate and repair the damaged muscle fibres. Nitric oxide (NO), metalloproteinase (MMP) and hepatocyte growth factor (HGF) play an important role in the repair of skeletal muscle. During the release HGF from the extracellular matrix NO's role in it can be determined by administering l- NAME (an inhibitor of NO) before stretch treatment. In vivo the activation of satellite cells were prevented by l- NAME. This indicates that when muscle fibres are stretched it liberates HGF in an NO dependent manner. The release of No dependent HGF matrix is mediated by MMP which is a large family of zinc dependent endopeptidases and degrade several extracellular matrix proteins [4].

Chen et al has proposed that the growth of skeletal muscle satellite cells, myogenic progenitors cells are repressed by micro RNA1 and micro RNA206[2].During satellite cell differentiation micro RNA1 and micro RNA 206 are up regulated while during skeletal muscle regeneration they are down regulated . Over expression of micro RNA1 and micro RNA 206 prohibits the growth of potential satellite cells. They showed that Pax7 was one of the regulatory targets of micro RNA 1 and micro RNA206 during satellite cells proliferation and differentiation. During the progression from cell proliferation to differentiation the micro RNA play an important role by limiting Pax7 expression. All satellite cells express Pax7 and it initially activates Myf5 and MyoD for the satellite cells to differentiate and become myogenic progenitor cells. The activated MyoD in satellite cells up regulates micro RNA1 and micro RNA206 which in turn repress Pax7 post transcriptionally. Thus the repression of Pax7 protein levels is one of the mechanisms by micro RNA's regulate satellite cell activity [2].

Urish et al suggested that antioxidant levels represent a major determinant in the regenerative capacity of muscle stem cells. [16].Muscle derived stem cells (MDSC) during regeneration show a high resistance to oxidative stress. When muscle derive stem cells were exposed to hydrogen peroxide and TNF alpha they showed a greater rate of differentiation than the myoblasts. This shows that the muscle derived stem cells have a better ability to cope with oxidative stress. When myogenic cells are introduced to TNF alpha or IL 1 their differentiation is inhibited by activation of nuclear factor kappa B pathway. Inhibition causes the muscle progenitor cells to repair the damaged muscle fibres. This proves that the muscle derived stem cells can differentiate even if they are exposed to oxidative stress by modifying nuclear factor kappa B pathway activation [16].

Xiaoyun Wu et al have described the presence of muscle derived stem cells (MDSC) which are a small population of stem cells which differentiate more slowly [13]. This shows a separate entity of stem cells which is present within the satellite cell population. In earlier studies three populations of muscle derived stem cells (MDSC) have been isolated based on their adhesive and proliferative properties. These populations are: 1) rapidly adhering cell population (RAC) 2) slowly adhering cell population (SAC) and another population of proliferating cells which is derived from primary SAC. These cells proliferate for a longer time in vivo and have an extended capacity of self renewal and multipotent differentiation. This suggests that these cells are a new type of pluripotent muscle derived stem cells (MDSC) [13]. Currently most extensive technique used to isolate MDSC is a marker profile independent method which is based upon the adhesion characteristics know as modified preplate technique. It is identified by using immuno staining for certain stem cell markers and also by using fluorescent activated cell sorting (FACS). It expresses CD31 and CD56 and less of CD10 and Sca1. MDSC play a very important role in healing, after a skeletal muscle injury. They survive for a longer time and directly participate in myofibre regeneration. In conclusion it can be said that MDSCs are readily available which show great future in cell and gene therapy applications [13].

Jackson et al suggest two therapeutic applications using muscle derived stem cells (MDSC) [ 17].One is tissue engineering where cells are loaded on to a scaffold and allowed to differentiate into the appropriate cell type for the tissue which is to be made. The second is however to promote the regenerative capacity of stem and progenitor cells in vivo to boost the inner mechanisms of tissue regeneration [17].

Fig. 3 Isolation of MDSCs. various populations of muscle-derived cells were isolated based on their adhesion characteristics. The first cells to adhere during the early stages of the preplate technique, within minutes to hours of seeding, are known as rapidly adhering cells (RAC) and have been shown to be comprised of mostly fibroblastic-like and myoblast cells. Other populations of slowly adhering cells (SAC), containing MDSCs, are obtained in later preplates within several days of seeding.

(Courtesy: - Wu, X., S. Wang, et al. (2010).)[13]

Shea et al has also illustrated the role of RTK inhibitor sprouty1 (spry1) in the functioning of adult stem cells.[9] They showed that interruption of Spry1 in adult Pax7+ cells prevent their quiescence which results in prevention of replacement of satellite cells to their homeostatic levels after an injury. Spry1 regulates quiescence and homeostasis of the stem cell pool.Pax3, Pax7 , Myf5, MyoD are expressed on satellite cells during their development.Spry1 is especially expressed on Pax7+ cells in an uninjured muscle which suggests that Spry1 is a marker of Pax7+ satellite cells. Stem cells travel through a growth factor mediated checkpoint before returning to quiescence .Interruption of Spry 1 in Pax7+ satellite cells does not affect the genesis of satellite cells but during regeneration, Pax7+ satellite cells do not come back to their homeostatic condition. Thus it can be said that spry genes may regulate the extrinsic signals in the regenerating tissue to decide the nemesis of stem cells for tissue homeostasis and repair. [9][10]

In summary, stem cells in the muscle tissue are controlled by a number of growth factors such as Pax3 Pax7, Myf5, MyoD, and m-cadherin. They differentiate into mature myotubes. The satellite cells are highly propagating and they fuse with the residing muscle fibres to form new muscle fibres. Due to the satellite cells dysfunction it can lead to muscular disorders such as muscular dystrophy or myopathy. The question that can be investigated in future is the role of other circulating cells, non satellite cells which are present in skeletal muscle and have a myogenic potential. Even if these cells do not play a role in muscle regeneration it will be helpful to illustrate the likely outcome of those cells. [14][15]

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