Differentiation Of Muscle Satellite Cells Research Articles

Versican, Matrix-Gla Protein, and Death-Associated Protein Expression Affect Muscle Satellite Cell Proliferation and Differentiation

Versican, Matrix-Gla Protein, and Death-Associated Protein Expression Affect Muscle Satellite Cell Proliferation and Differentiation

Different regulation role of myostatin in differentiating pig ADSCs and MSCs into adipocytes

Myostation (MSTN), which is primarily expressed in muscle, plays an important role in myogenic and adipogenic cells. However, there is little information about whether MSTN displays different roles between adipose-derived stem cells (ADSCs) and muscle satellite cells (MSCs). The two kinds of cells can both exist in the muscle and differentiate into adiposities. In this research, we isolated ADSCs and MSCs from porcine fat tissues and semitendinosus muscle, respectively, to investigate the effect of MSTN on the adipogenesis of those cells. ADSCs and MSCs were treated with recombinant human MSTN during the induction of adipogenesis or before the induction of differentiation. Then, we evaluated adipogenesis by Oil Red O staining and assessed the expression patterns of adipocyte-specific fatty acid binding protein (aP2) and peroxisome proliferator-activated receptor (PPAR) γ using real-time polymerase chain reaction methods. Our results indicated that the treatment with MSTN before or during the induction of differentiation in MSCs could both inhibit the adipogenesis. However, the treatment with MSTN only during the induction of differentiation in ADSCs could suppress the adipogenesis. Those results showed that MSTN had different roles in the adipogenesis of ADSCs and MSCs. It can shed new light on the origin of adipocyte located in muscle.

Activity of bilateral posterior cricoarytenoid muscle satellite cell after denervation or reinnervation with ansa in dogs

To investigate the activity of bilateral posterior cricoarytenoid muscle satellite cell after denervation or reinnervation with ansa cervicalis. Twenty four dogs were randomly divided into 3 groups. The bilateral laryngeal recurrent nerves were cut in group one in all dogs. The bilateral laryngeal recurrent nerves were anastomosed with ansa cervicalis after incision in group two in all dogs. The dogs in group three were used as control. Nine weeks after surgery, the electromyography was used to test the regeneration of the nerve. The posterior cricoarytenoid muscles biopsy were collected. The expression of mRNA of Myogenin, Myf5, and Pax7 was assayed by realtime RT-PCR after total RNA isolation. Two dogs died after surgery in incision and anastomose group. The electromyography suggested that the RLN of all dogs had denervated in the incision group and had reinnervated in the anastomose group after 9 weeks. Myogenin mRNA from RLN incision dogs PCA muscles had greater expression versus controls (Z = 1.42, P < 0.01) or anastomosed dogs (Z = 1.38, P < 0.01). Myf5 mRNA expression from RLN incision dogs PCA muscles had significant increase versus control dogs (Z = 1.66, P < 0.01) or anastomosed dogs (Z = 1.69, P < 0.01). Pax7 mRNA expression from RNL incision dogs had significant increase compared with control (Z = 1.66, P < 0.01) or anastomosed animals (Z = 1.42, P < 0.05). There was no significant difference in Myogenin (Z = 1.34, P > 0.05), Myf5 (Z = 0.54, P > 0.05) and Pax (Z = 0.54, P > 0.05) mRNA expression between controls and anastomosed animals. The bilateral denervation of RLN cause significantly increasing in dog PCA muscle satellite cell proliferation and differentiation. The bilateral reinnervation of RLN cause PCA muscle satellite cell come back nonproliferative, quiescent state in dog.

P21 ErbB3 binding protein-1 (Ebp1) contributes to the control of proliferation and differentiation in adult muscle satellite cells

P21 ErbB3 binding protein-1 (Ebp1) contributes to the control of proliferation and differentiation in adult muscle satellite cells

TGF-β isoforms inhibit IGF-1-induced migration and regulate terminal differentiation in a cell-specific manner

Following muscle injury, the damaged tissue and influx of inflammatory cells stimulate the secretion of growth factors and cytokines to initiate repair processes. This release of chemotactic signaling factors activates resident precursor cells and stimulates their mobilization and migration to the site of injury where terminal differentiation can occur. The three transforming growth factor-β (TGF-β) isoforms, and insulin-like growth factor-1 (IGF-1) are among the known regulatory factors released following muscle damage. We investigated the effect of recombinant active TGF-β1, -β2, -β3 and IGF-1 on C2C12 skeletal muscle satellite cell and P19 embryonal carcinoma cell terminal differentiation and migration. C2C12 myoblast fusion as well as P19 embryoid body formation and myogenic differentiation was assessed following 72h TGF-β treatment (5ng/ml), whereas the effect of the TGF-β isoforms on migration was determined following 7h incubation. Our results showed that TGF-β decreases C2C12 myoblast fusion in an isoform-independent manner, whereas in the P19 cell lineage, results demonstrate that TGF-β1 specifically and significantly increased P19 embryoid body formation, but not expression of Connexin-43 or Myosin Heavy Chain. IGF-1 significantly increased migration compared to TGF-β isoforms, which, on their own, had no significant effect on the mobilization of either C2C12 or P19 cells. TGF-β isoforms decreased IGF-1-induced migration of both cell lineages. By distinguishing the factors involved in, and the molecular signals required for, myoblast recruitment during repair processes, strategies can be developed towards improved cell-mediated therapies for muscle injury.

MicroRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7

microRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7

MicroRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7

Skeletal muscle satellite cells are adult stem cells responsible for postnatal skeletal muscle growth and regeneration. Paired-box transcription factor Pax7 plays a central role in satellite cell survival, self-renewal, and proliferation. However, how Pax7 is regulated during the transition from proliferating satellite cells to differentiating myogenic progenitor cells is largely unknown. In this study, we find that miR-1 and miR-206 are sharply up-regulated during satellite cell differentiation and down-regulated after muscle injury. We show that miR-1 and miR-206 facilitate satellite cell differentiation by restricting their proliferative potential. We identify Pax7 as one of the direct regulatory targets of miR-1 and miR-206. Inhibition of miR-1 and miR-206 substantially enhances satellite cell proliferation and increases Pax7 protein level in vivo. Conversely, sustained Pax7 expression as a result of the loss of miR-1 and miR-206 repression elements at its 3' untranslated region significantly inhibits myoblast differentiation. Therefore, our experiments suggest that microRNAs participate in a regulatory circuit that allows rapid gene program transitions from proliferation to differentiation.

Six family genes control the proliferation and differentiation of muscle satellite cells

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4 +/− Six5 − /− mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.

BMP signalling permits population expansion by preventing premature myogenic differentiation in muscle satellite cells

Satellite cells are the resident stem cells of adult skeletal muscle, supplying myonuclei for homoeostasis, hypertrophy and repair. In this study, we have examined the role of bone morphogenetic protein (BMP) signalling in regulating satellite cell function. Activated satellite cells expressed BMP receptor type 1A (BMPR-1A/Alk-3) and contained phosphorylated Smad proteins, indicating that BMP signalling is operating during proliferation. Indeed, exogenous BMP4 stimulated satellite cell division and inhibited myogenic differentiation. Conversely, interfering with the interactions between BMPs and their receptors by the addition of either the BMP antagonist Noggin or soluble BMPR-1A fragments, induced precocious differentiation. Similarly, blockade of BMP signalling by siRNA-mediated knockdown of BMPR-1A, disruption of the intracellular pathway by either Smad5 or Smad4 knockdown or inhibition of Smad1/5/8 phosphorylation with Dorsomorphin, also caused premature myogenic differentiation. BMP signalling acted to inhibit the upregulation of genes associated with differentiation, in part, through regulating Id1. As satellite cells differentiated, Noggin levels increased to antagonise BMP signalling, since Noggin knockdown enhanced proliferation and impeded myoblast fusion into large multinucleated myotubes. Finally, interference of normal BMP signalling after muscle damage in vivo perturbed the regenerative process, and resulted in smaller regenerated myofibres. In conclusion, BMP signalling operates during routine satellite cell function to help coordinate the balance between proliferation and differentiation, before Noggin is activated to antagonise BMPs and facilitate terminal differentiation.

Influence of platelet-rich plasma on proliferation and osteogenic differentiation of skeletal muscle satellite cells: An in vitro study

Platelet-rich plasma (PRP) is a new application of tissue engineering and a developing area for researchers and clinicians. The aim of this study was to assess the effect of PRP on the proliferation and osteogenic differentiation of skeletal muscle satellite cell (MSC) population and the ability of PRP to induce the production of some osteogeneic-related factors in vitro. The PRP was obtained from Sprague-Dawley rats using 2 centrifugation techniques. Primary cultures of rat MSCs were exposed to various concentrations of PRP (0.16 × 10(8), 0.625 × 10(8), and 2.5 × 10(8) thrombocytes/carrier) on MSC proliferation using an MTT proliferation assay. Alkaline phosphatase (ALP) activity, Alizarin red S (AR) staining, calcium analyses and real-time reverse-transcription polymerase chain reaction (RT-PCR) of osteogenic-related genes were performed to study the effect of PRP on osteogenic differentiation of cultured MSCs population. The platelet concentration and growth factors (GFs) in our PRP preparations were significantly higher than in the whole blood. PRP showed a dose-dependent stimulation of cell proliferation. The maximum effect was achieved with a concentration of 0.625 × 10(8) thrombocytes/carrier. ALP activity, AR staining, and calcium analyses showed enhanced cell osteogenic differentiation in the PRP group. The real-time RT-PCR results showed that PRP up-regulated osteocalcin at day 14 and type I collagen and osteopontin at day 7 compared with the control group. The results of this study suggest that PRP containing osteoinductive GFs stimulates cell proliferation and osteogenic differentiation of rat-derived MSCs in vitro.

High glucose induces differentiation and adipogenesis in porcine muscle satellite cells via mTOR

The present study investigated whether the mammalian target of rapamycin (mTOR) signal pathway is involved in the regulation of high glucose-induced intramuscular adipogenesis in porcine muscle satellite cells. High glucose (25 mM) dramatically increased intracellular lipid accumulation in cells during the 10-day adipogenic differentiation period. The expressions of CCAAT/enhancer binding protein-alpha (C/EBP-alpha) and fatty acid synthase (FAS) protein were gradually enhanced during the 10-day duration while mTOR phosphorylation and sterol-regulatory-element-binding protein (SREBP)-1c protein were induced on day 4. Moreover, inhibition of mTOR activity by rapamycin resulted in a reduction of SREBP-1c protein expression and adipogenesis in cells. Collectively, our findings suggest that the adipogenic differentiation of porcine muscle satellite cells and a succeeding extensive adipogenesis, which is triggered by high glucose, is initiated by the mTOR signal pathway through the activation of SREBP-1c protein. This process is previously uncharacterized and suggests a cellular mechanism may be involved in ectopic lipid deposition in skeletal muscle during type 2 diabetes.

MicroRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7

microRNA-1 and microRNA-206 regulate skeletal muscle satellite cell proliferation and differentiation by repressing Pax7

S-adenosylmethionine stimulates fatty acid metabolism-linked gene expression in porcine muscle satellite cells

Evidence indicates that both S-adenosylmethionine (SAMe) metabolism and intramuscular fat are associated with insulin resistance and type II diabetes. However, it is still unknown whether SAMe have effects on intramuscular adipogenesis. The present study investigated the roles of SAMe in the adipogenic differentiation of porcine muscle satellite cells. Cells isolated from neonatal pig muscle were treated with different concentrations of SAMe (0, 0.5 and 1.0 mM) for 24 h, induced for a 9-day adipogenic differentiation and were finally stained by oil red O staining. The adipocyte determination and differentiation factor-1 (ADD1) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein were stimulated by SAMe treatment in a dose-dependent manner. Lipoprotein lipase (LPL) mRNA and protein were enhanced in 1.0 mM treatment group, compared with the control. No significant difference was observed in the intracellular lipid content among treatments. These results provide evidence that SAMe may be associated with intramuscular adipogenesis and indicate a novel action of SAMe in fat metabolism.

NET37, a Nuclear Envelope Transmembrane Protein with Glycosidase Homology, Is Involved in Myoblast Differentiation

The nuclear lamina and its associated proteins are important for nuclear structure and chromatin organization and also have been implicated in the regulation of cell signaling and gene expression. In this study we demonstrate that the lamina-associated nuclear envelope transmembrane protein NET37 is required for myogenic differentiation of C2C12 cells. NET37, a member of glycosidase family 31, is highly expressed in mouse skeletal muscle and is strongly up-regulated during C2C12 differentiation. By protease mapping we show that its glycosidase homology domain is located in the lumen of the nuclear envelope/endoplasmic reticulum. When NET37 is depleted from proliferating myoblasts by RNAi, myogenic differentiation is significantly impaired, and there is a concomitant delay in up-regulation of the late myogenic transcription factor myogenin. We expressed silencing-resistant NET37 mutated at a conserved residue in the glycosidase domain and found that this predicted catalytically inactive protein is unable to support myogenesis in cells depleted of wild type NET37. Therefore, the enzymatic function of NET37 appears to be important for myogenic differentiation. C2C12 cells depleted of NET37 have reduced activation of Akt after shifting to differentiation medium and are defective in insulin like growth factor-II (IGF-II) secretion, an autocrine/paracrine factor involved in Akt activation. We also observed that pro-IGF-II co-immunoprecipitates with NET37. Based on our results, we propose that NET37 has a role in IGF-II maturation in the secretory pathway during myoblast differentiation. The localization of NET37 at the nuclear envelope raises the possibility that it may coordinate myogenic events between the nuclear interior and the endoplasmic reticulum lumen via transmembrane communication.

MEK5 and ERK5 are mediators of the pro-myogenic actions of IGF-2

During the differentiation of muscle satellite cells, committed myoblasts respond to specific signalling cues by exiting the cell cycle, migrating, aligning, expressing muscle-specific genes and finally fusing to form multinucleated myotubes. The predominant foetal growth factor, IGF-2, initiates important signals in myogenesis. The aim of this study was to investigate whether ERK5 and its upstream MKK activator, MEK5, were important in the pro-myogenic actions of IGF-2. ERK5 protein levels, specific phosphorylation and kinase activity increased in differentiating C2 myoblasts. ERK5-GFP translocated from the cytoplasm to the nucleus after activation by upstream MEK5, whereas phospho-acceptor site mutated (dominant-negative) ERK5AEF-GFP remained cytoplasmic. Exogenous IGF-2 increased MHC levels, myogenic E box promoter-reporter activity, ERK5 phosphorylation and kinase activity, and rapidly induced nuclear localisation of ERK5. Transfection with antisense Igf2 decreased markers of myogenesis, and reduced ERK5 phosphorylation, kinase and transactivation activity. These negative effects of antisense Igf2 were rescued by constitutively active MEK5, whereas transfection of myoblasts with dominant-negative MEK5 blocked the pro-myogenic action of IGF-2. Our findings suggest that the MEK5-ERK5 pathway is a novel key mediator of IGF-2 action in myoblast differentiation.

Satellite Cell Differentiation in Goat Skeletal Muscle Single Fiber Culture

Satellite cells are skeletal muscle progenitor/stem cells that reside between the basal lamina and plasma membranes of skeletal muscle fibers in vivo and can give rise to both myogenic and adipogenic cells. The isolated skeletal muscle single fiber culture is an in vitro model that has the advantage of enabling the researcher to monitor the differentiation of satellite cells in a closer microenvironment found in living muscle in vivo. The aim of the present study was to establish single fiber culture of goat skeletal muscle and monitor satellite cell differentiation in this system. Fine single fibers were isolated from goat intercostal muscle by enzymatic treatment, and satellite cells on the fibers were analyzed immunocytochemically. Satellite cells on freshly isolated fibers were positive for Pax7, but negative for MyoD and myogenin, indicating that they are quiescent. As the culture period progressed, the satellite cells became positive for myogenic markers MyoD and myogenin. The satellite cells that detached from the fiber and migrated onto the culture dish were positive for both MyoD and myogenin, and some of them formed multinucleated myosin heavy chain-positive myotubes. In addition, hypercontraction of isolated fine fibers induced adipogenic differentiation of satellite cells. The present study is the first to describe establishment of a goat skeletal muscle single fiber culture that can be used as a model system for further studies on satellite cell behavior in ruminants.

Suppression of macrophage functions impairs skeletal muscle regeneration with severe fibrosis

When damaged, skeletal muscle regenerates. In the early phases of regeneration, inflammatory cells such as neutrophils/granulocytes and macrophages infiltrate damaged muscle tissue. To reveal the roles of macrophages during skeletal muscle regeneration, we injected an antibody, AFS98 that blocks the binding of M-CSF to its receptor into normal mice that received muscle damages. Anti-M-CSF receptor administration suppressed macrophage but not neutrophil infiltration. Histological study indicated that suppression of macrophages function leads to the incomplete muscle regeneration. In addition FACS and immunohistochemical study showed that the acute lack of macrophages delayed proliferation and differentiation of muscle satellite cells in vivo. Furthermore, mice injected with the anti-M-CSF receptor antibody exhibited not only adipogenesis, but also significant collagen deposition, i.e., fibrosis and continuous high expression of connective tissue growth factor. Finally we indicate that these fibrosis markers were strongly enriched in CD90(+) cells that do not include myogenic cells. These results indicate that macrophages directly affect satellite cell proliferation and that a macrophage deficiency severely impairs skeletal muscle regeneration and causes fibrosis.

Osterix overexpression enhances osteoblast differentiation of muscle satellite cells in vitro

Muscle satellite cells have long been considered a distinct myogenic lineage responsible for postnatal growth, repair and maintenance of skeletal muscle. Recent studies have demonstrated that they are multi-potential. Osterix (Osx), a novel zinc-finger-containing transcription factor of the sp family, is required for osteoblast differentiation and bone formation. It was hypothesized that Osx overexpression would enhance osteoblast differentiation of muscle satellite cells in vitro. Recombinant adenovirus-mediated Osx gene (Ad-Osx) was constructed and used to transfect muscle satellite cells. Osx overexpression inhibited myogenesis, as demonstrated by suppression of MyoD and myogenin mRNA levels and reduced myotube formation. Muscle satellite cells transduced with Ad-Osx exhibited apparent osteoblast differentiation as determined by the expression of related osteoblastic genes, increased activity of alkaline phosphatase and the formation of mineralized nodules. These results confirmed the ability of Osx to enhance osteoblast differentiation of muscle satellite cells in vitro, and the competence of muscle satellite cells as promising seed cells for bone tissue engineering.

Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD(-/-) myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD(-/-) myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD(-/-) myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD(-/-) myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD(-/-) myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD(-/-) myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD(-/-) myoblasts. Therefore, MyoD(-/-) myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.

The effects of whey protein on myostatin and cell cycle-related gene expression responses to a single heavy resistance exercise bout in trained older men

Myostatin decreases muscle mass and this is accomplished, in part, by inhibiting muscle satellite cell proliferation and differentiation by regulating the expression of cell cycle-related proteins (e.g. p21 and cdk2) and myogenic regulatory factors (e.g. myogenin and MyoD). The purpose of this investigation was to determine whether protein ingestion before and after a resistance exercise (RE) bout affects myostatin and cell cycle-related gene expression. Strength-trained middle-aged to older men were divided into a protein group (61.4 +/- 4.3 years, n = 9) or a placebo group (62.1 +/- 4.2 years, n = 9). Muscle biopsies from the vastus lateralis muscle were taken at rest and 1 and 48 h after a 5 x 10 repetition leg press RE bout. Protein (15 g whey) or non-caloric placebo was taken immediately before and after the RE bout. mRNA expression levels of myostatin and related genes (AcvrIIb, FLRG, p21, p27, cdk2, myogenin and MyoD) were determined by Taqman probe-based real-time RT-PCR and normalized to GAPDH mRNA. Myostatin mRNA decreased after a RE bout, but only in the placebo group (P < or = 0.05). Conversely, myostatin-binding protein FLRG and cell-cycle kinase cdk2 mRNA increased only in the protein group (P < or = 0.05). p21 mRNA was increased at 1 h post-RE in placebo (P < or = 0.05) and tended to be increased in the protein group (P = 0.08). Myostatin, its binding protein and cell cycle-related gene expressions are affected by single RE bout and these responses are further modified by whey protein intake. Therefore, controlling nutrition intake is important when studying gene expression responses to exercise.