Skeletal muscle atrophy is associated with an increased expression of myostatin and impaired satellite cell function in the portacaval anastamosis rat

Proliferation and differentiation of satellite cells are critical in the regeneration of atrophied muscle following immobilization and aging. We hypothesized that impaired satellite cell function is responsible for the atrophy of skeletal muscle also seen in cirrhosis. Myostatin and insulin-like growth factor 1 (IGF1) have been identified to be positive and negative regulators, respectively, of satellite cell function. Using a rat model of cirrhosis [portacaval anastamosis (PCA)] and sham-operated controls, we examined the expression of myostatin, its receptor activinR2b, and its downstream messenger cyclin-dependent kinase inhibitor p21 (CDKI p21) as well as IGF1 and its receptor in the gastrocnemius muscle. Expression of PCNA, a marker of proliferation, and myogenic regulatory factors (myoD, myf5, and myogenin), markers of differentiation of satellite cells, were also measured. Real- time PCR for mRNA and Western blot assay for protein quantification were performed. PCA rats had lower body weight and gastrocnemius weight compared with sham animals (P < 0.05). PCNA and myogenic regulatory factors were lower in PCA rats (P < 0.05). Myostatin, activinR2b, and CDKI p21 were higher in the PCA animals (P < 0.05). The expression of IGF1 and its receptor was lower in liver and skeletal muscle of PCA animals (P < 0.05). These data suggest that skeletal muscle atrophy seen in the portacaval shunted rats is a consequence of impaired satellite cell proliferation and differentiation mediated, in part, by higher myostatin and lower IGF1 expression.     

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor.     

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P2 and S1P3. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P1 and S1P4 in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P4 receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P2 was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.     

Differential myogenicity of satellite cells isolated from extensor digitorum longus (EDL) and soleus rat muscles revealed in vitro

Following muscle damage, fast- and slow-contracting fibers regenerate, owing to the activation of their satellite cells. In rats, crush-induced regeneration of extensor digitorum longus (EDL, a fast muscle) and soleus (a slow muscle) present different characteristics, suggesting that intrinsic differences exist among their satellite cells. An in vitro comparative study of the proliferation and differentiation capacities of satellite cells isolated from these muscles is presented there. We observed several differences between soleus and EDL satellite cell cultures plated at high density on gelatin-coated dishes. Soleus satellite cells proliferated more actively and fused into myotubes less efficiently than EDL cells. The rate of muscular creatine kinase enzyme appeared slightly lower in soleus than in EDL cultures at day 11 after plating, when many myotubes were formed, although the levels of muscular creatine kinase mRNA were similar in both cultures. In addition, soleus cultures expressed higher levels of MyoD and myogenin mRNA and of MyoD protein than EDL satellite cell cultures at day 12. A clonal analysis was also carried out on both cell populations in order to determine if distinct lineage features could be detected among satellite cells derived from EDL and soleus muscles. When plated on gelatin at clonal density, cells from both muscles yielded clones within 2 weeks, which stemmed from 3–15 mitotic cycles and were classified into three classes according to their sizes. Myotubes resulting from spontaneous fusion of cells from the progeny of one single cell were seen regardless of the clone size in the standard culture medium we used. The proportion of clones showing myotubes in each class depended on the muscle origin of the cells and was greater in EDL- than in soleus-cell cultures. In addition, soleus cells were shown to improve their differentiation capacity upon changes in the culture condition. Indeed, the proportions of clones showing myotubes, or of cells fusing into myotubes in clones, were increased by treatments with a myotube-conditioned medium, with phorbol ester, and by growth on extra-cellular matrix components (Matrigel). These results, showing differences among satellite cells from fast and slow muscles, might be of importance to muscle repair after trauma and in pathological situations.     

Captopril treatment induces hyperplasia but inhibits myonuclear accretion following severe myotrauma in murine skeletal muscle

The role of ANG II in skeletal muscle and satellite cell regulation is largely unknown. Cardiotoxin (CTX) was used to investigate whether muscle injury activates a local ANG II signaling system. Following injury, immunohistochelmistry (IHC) analysis revealed a robust increase in the intensity of angiotensinogen and angiotensin type 1 (AT(1)) receptor expression. As regeneration proceeded, however, AT(1) and angiotensinogen were downregulated. Nuclear accretion and fiber formation were also assessed during muscle regeneration in mice treated with captopril (an angiotensin-converting enzyme inhibitor). When ANG II formation was blocked through the use of captopril, we observed a significantly reduced accretion of nuclei into myofibers (-25%), while tibialis anterior total fiber number was significantly increased +37%. This phenotype appeared to be due to alterations in satellite cell differentiation kinetics; captopril treatment led to sustained mRNA expression of markers associated with quiescence and proliferation (Myf5, Pax7) and simultaneously delayed or inhibited the expression of myogenin. IHC staining supported these findings, revealing that captopril treatment resulted in a strong trend (P = 0.06) for a decrease in the proportion of myogenin-positive myoblasts. Furthermore, these observations were associated with a delay in muscle fiber maturation; captopril treatment resulted in sustained expression of embryonic myosin heavy chain. Collectively, these findings demonstrate that localized skeletal muscle angiotensin signaling is important to muscle fiber formation, myonuclear accretion, and satellite cell function.     

Elevated satellite cell number in Duchenne muscular dystrophy

The regenerative potential of muscle tissue relies mostly on satellite cells situated between the muscular basal membrane and the sarcolemma. The regeneration of muscle tissue comprises proliferation, the propagation of satellite cells, and their subsequent differentiation with the expression of multiple muscle-specific proteins. However, in Duchenne muscular dystrophy (DMD), regeneration cannot compensate for the loss of muscle tissue. To examine the regenerative potential in DMD, satellite cell nuclei number and markers of differentiation in DMD muscle from various disease states were compared with control muscle. Differentiation of satellite cells is characterized by the helix-loop-helix factor myogenin, which is never co-expressed with Pax7, whereas MyoD1 and Myf5 are co-expressed with Pax7, with Myf5 being present even in muscle of controls. The results indicate that satellite cell number is elevated in DMD in comparison with control muscle, even in advanced stages of dystrophy, suggesting that exhaustion of satellite cells is not the primary cause for failed regeneration. The expression of myogenin is correlated neither with fibrosis nor with age. We suggest variable factors influencing the differentiation of satellite cells in DMD.     

PKCθ signaling is required for myoblast fusion by regulating the expression of caveolin-3 and β1D integrin upstream focal adhesion kinase

Fusion of mononucleated myoblasts to form multinucleated myofibers is an essential phase of skeletal myogenesis, which occurs during muscle development as well as during postnatal life for muscle growth, turnover, and regeneration. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, and recently focal adhesion kinase (FAK), has been proposed as a key mediator of myoblast fusion. Here we focused on the possible role of PKC, the PKC isoform predominantly expressed in skeletal muscle, in myoblast fusion. We found that the expression of PKC is strongly up-regulated following freeze injury-induced muscle regeneration, as well as during in vitro differentiation of satellite cells (SCs; the muscle stem cells). Using both PKC knockout and muscle-specific PKC dominant-negative mutant mouse models, we observed delayed body and muscle fiber growth during the first weeks of postnatal life, when compared with wild-type (WT) mice. We also found that myofiber formation, during muscle regeneration after freeze injury, was markedly impaired in PKC mutant mice, as compared with WT. This phenotype was associated with reduced expression of the myogenic differentiation program executor, myogenin, but not with that of the SC marker Pax7. Indeed in vitro differentiation of primary muscle-derived SCs from PKC mutants resulted in the formation of thinner myotubes with reduced numbers of myonuclei and reduced fusion rate, when compared with WT cells. These effects were associated to reduced expression of the profusion genes caveolin-3 and β1D integrin and to reduced activation/phosphorylation of their up-stream regulator FAK. Indeed the exogenous expression of a constitutively active mutant form of PKC in muscle cells induced FAK phosphorylation. Moreover pharmacologically mediated full inhibition of FAK activity led to similar fusion defects in both WT and PKC-null myoblasts. We thus propose that PKC signaling regulates myoblast fusion by regulating, at least in part, FAK activity, essential for profusion gene expression.     

Altered expression of genes regulating skeletal muscle mass in the portacaval anastomosis rat

We examined the temporal relationship between portacaval anastomosis (PCA), weight gain, changes in skeletal muscle mass and molecular markers of protein synthesis, protein breakdown, and satellite cell proliferation and differentiation. Male Sprague-Dawley rats with end to side PCA (n=24) were compared with sham-operated pair-fed rats (n=24). Whole body weight, lean body mass, and forelimb grip strength were determined at weekly intervals. The skeletal muscle expression of the ubiquitin proteasome system, myostatin, its receptor (the activin 2B receptor) and its signal, cyclin-dependent kinase inhibitor (CDKI) p21, insulin-like growth factor (IGF)-I and its receptor (IGF-I receptor-alpha), and markers of satellite cell proliferation and differentiation were quantified. PCA rats did not gain body weight and had lower lean body mass, forelimb grip strength, and gastrocnemius muscle weight. The skeletal muscle expression of the mRNA of ubiquitin proteasome components was higher in PCA rats in the first 2 wk followed by a lower expression in the subsequent 2 wk (P<0.01). The mRNA and protein of myostatin, activin 2B receptor, and CDKI p21 were higher, whereas IGF-I and its receptor as well as markers of satellite cell function (proliferating nuclear cell antigen, myoD, myf5, and myogenin) were lower at weeks 3 and 4 following PCA (P < 0.05). We conclude that PCA resulted in uninhibited proteolysis in the initial 2 wk. This was followed by an adaptive response in the later 2 wk consisting of an increased expression of myostatin that may have contributed to reduced muscle protein synthesis, impaired satellite cell function, and lower skeletal muscle mass.     

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.     

Brain-derived Neurotrophic Factor Regulates Satellite Cell Differentiation and Skeltal Muscle Regeneration

In adult skeletal muscle, brain-derived neurotrophic factor (BDNF) is expressed in myogenic progenitors known as satellite cells. To functionally address the role of BDNF in muscle satellite cells and regeneration in vivo, we generated a mouse in which BDNF is specifically depleted from skeletal muscle cells. For comparative purposes, and to determine the specific role of muscle-derived BDNF, we also examined muscles of the complete BDNF^−/− mouse. In both models, expression of the satellite cell marker Pax7 was significantly decreased. Furthermore, proliferation and differentiation of primary myoblasts was abnormal, exhibiting delayed induction of several markers of differentiation as well as decreased myotube size. Treatment with exogenous BDNF protein was sufficient to rescue normal gene expression and myotube size. Because satellite cells are responsible for postnatal growth and repair of skeletal muscle, we next examined whether regenerative capacity was compromised. After injury, BDNF-depleted muscle showed delayed expression of several molecular markers of regeneration, as well as delayed appearance of newly regenerated fibers. Recovery of wild-type BDNF levels was sufficient to restore normal regeneration. Together, these findings suggest that BDNF plays an important role in regulating satellite cell function and regeneration in vivo, particularly during early stages.     

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.     

Changes in proliferation,differentiation, fibroblast growth factor 2 responsiveness and expression of syndecan‐4 and glypican‐1 with turkey satellite cell age

Myogenic satellite cells are heterogeneous multipotential stem cells that are required for muscle repair, maintenance, and growth. The membrane‐associated heparan sulfate proteoglycans syndecan‐4 and glypican‐1 differentially regulate satellite cell proliferation, differentiation, fibroblast growth factor 2 (FGF2) signal transduction, and expression of the myogenic regulatory factors MyoD and myogenin. The objective of the current study was to determine the effect of age on syndecan‐4 and glypican‐1 satellite cell populations, proliferation, differentiation, FGF2 responsiveness, and expression of syndecan‐4, glypican‐1, MyoD, and myogenin using satellite cells isolated from the pectoralis major muscle of 1‐day‐old, 7‐week‐old and 16‐week‐old turkeys. Proliferation was significantly reduced in the 16‐week‐old satellite cells, while differentiation was decreased in the 7‐week‐old and the 16‐week‐old cells beginning at 48 h of differentiation. Fibroblast growth factor 2 responsiveness was highest in the 1‐day‐old and 7‐week‐old cells during proliferation; during differentiation there was an age‐dependent response to FGF2. Syndecan‐4 and glypican‐1 satellite cell populations decreased with age, but syndecan‐4 and glypican‐1 were differentially expressed with age during proliferation and differentiation. MyoD and myogenin mRNA expression was significantly decreased in 16‐week‐old cells compared to the 1‐day‐old and 7‐week‐old cells. MyoD and myogenin protein expression was higher during proliferation in the 16‐week‐old cells and decreased with differentiation. These data demonstrate an age‐dependent effect on syndecan‐4 and glypican‐1 satellite cell subpopulations, which may be associated with age‐related changes in proliferation, differentiation, FGF2 responsiveness, and the expression of the myogenic regulatory factors MyoD and myogenin.