Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats

Skeletal muscle regeneration after injury involves various processes, such as infiltration by inflammatory cells, the proliferation of satellite cells and fusion to myotubes. The c-ski nuclear protein has been implicated in the control of cell proliferation and/or terminal differentiation in the growth of skeletal muscle. However, there have been no reports concerning the involution of c-ski in the regeneration of injured skeletal muscle in mammals. A possible role for c-ski in the proliferation of myogenic cells in rat skeletal muscle during regeneration has been investigated with the assistance of in vitro experiments with L6 skeletal muscle cells. The expression levels of c-ski mRNA in regenerating tissues increased to approximately threefold that of intact tissues at 2 days after injury and decreased to normal levels at 2 weeks after injury. Many mononuclear cells among the Ski-positive cells expressed desmin and proliferating cell nuclear antigen, indicating that Ski-producing cells include the proliferating myogenic cells. The proliferation of L6 cells was significantly retarded by expression of the antisense ski gene. The results of the present study reveal that the c-ski gene plays an important role in the proliferation of myogenic cells in the regeneration of injured skeletal muscle.     

Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration.

Myogenesis in the embryo and the adult mammal consists of a highly organized and regulated sequence of cellular processes to form or repair muscle tissue that include cell proliferation, migration, and differentiation. Data from cell culture and in vivo experiments implicate both FGFs and HGF as critical regulators of these processes. Both factors require heparan sulfate glycosaminoglycans for signaling from their respective receptors. Since syndecans, a family of cell-surface transmembrane heparan sulfate proteoglycans (HSPGs) are implicated in FGF signaling and skeletal muscle differentiation, we examined the expression of syndecans 1-4 in embryonic, fetal, postnatal, and adult muscle tissue, as well as on primary adult muscle fiber cultures. We show that syndecan-1, -3, and -4 are expressed in developing skeletal muscle tissue and that syndecan-3 and -4 expression is highly restricted in adult skeletal muscle to cells retaining myogenic capacity. These two HSPGs appear to be expressed exclusively and universally on quiescent adult satellite cells in adult skeletal muscle tissue, suggesting a role for HSPGs in satellite cell maintenance or activation. Once activated, all satellite cells maintain expression of syndecan-3 and syndecan-4 for at least 96 h, also implicating these HSPGs in muscle regeneration. Inhibition of HSPG sulfation by treatment of intact myofibers with chlorate results in delayed proliferation and altered MyoD expression, demonstrating that heparan sulfate is required for proper progression of the early satellite cell myogenic program. These data suggest that, in addition to providing potentially useful new markers for satellite cells, syndecan-3 and syndecan-4 may play important regulatory roles in satellite cell maintenance, activation, proliferation, and differentiation during skeletal muscle regeneration.     

INDUCTION OF HEPATOCYTE GROWTH FACTOR IN THE LIVER, KIDNEY AND LUNG FOLLOWING TOTAL BODY IRRADIATION IN RAT

Hepatocyte factor (HGF) has been shown to have a pleiotropic function to act as a potent organotropic factor in the regeneration of injury in various organs, including the liver, kidney and lung. To examine the involvement of HGF in radiation injury, the authors analysed the changes in HGF mRNA and HGF protein levels in the rat organs (liver, lung, kidney) and plasma following 6 Gy of total body irradiation. Expression of HGF mRNA in the liver and kidney increased 6–48 h after total body irradiation and returned to previous values 1 week later. HGF protein levels in lung and liver showed 1.3-2 fold elevations 1–2 weeks after irradiation (P< 0.05). HGF levels in plasma stayed at undetectable levels up to 1 month after total body irradiation. The labelling index determined 2 weeks and 1 month after total body irradiation indicated no enhancement of regeneration. Thus, total body irradiation induced transient HGF elevation in these organs without enhancement of regeneration     

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.     

Differential expression of hepatocyte growth factor in liver, kidney, lung, and spleen following burn in rats

Hepatocyte growth factor (HGF) plays a role as an organotropic factor for regeneration of injured organs. HGF is synthesized as an inactive single-chain precursor which is then converted to a biologically active heterodimeric form by proteolytic processing. Burn is the insult that results in hypovolemia which causes systemic organ injury. In this study, we investigated the induction and activation of HGF in various rat organs following burn trauma. Tissue HGF content determined as the total amount of the single-chain and heterodimeric form increased significantly in liver, lung, spleen, and kidney 12 h after burn. Molecular analysis revealed that HGF in these four organs of control rats was the single-chain precursor. In the burned rats, HGF was the single-chain form in the liver and lung, whereas heterodimeric HGF was detected in the spleen and kidney. Tissue protein content, an index of tissue injury, decreased significantly in the spleen and kidney, indicating that tissue damage was severe in these two organs. These results suggest that burn induces the production of HGF in various organs, and that the induced HGF is activated according to the severity of tissue damage caused by burn.     

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.     

AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.     

A Novel Isoform of Met Receptor Tyrosine Kinase Blocks Hepatocyte Growth Factor/Met Signaling and Stimulates Skeletal Muscle Cell Differentiation

Hepatocyte growth factor (HGF) and its receptor, Met, regulate skeletal muscle differentiation. In the present study, we identified a novel alternatively spliced isoform of Met lacking exon 13 (designated Δ13Met), which is expressed mainly in human skeletal muscle. Alternative splicing yielded a truncated Met having extracellular domain only, suggesting an inhibitory role. Indeed, Δ13Met expression led to a decrease in HGF-induced tyrosine phosphorylation of Met and ERK phosphorylation, as well as cell proliferation and migration via sequestration of HGF. Interestingly, in human primary myoblasts undergoing differentiation, Δ13Met mRNA and protein levels were rapidly increased, concomitantly with a decrease in wild type Met mRNA and protein. Inhibition of Δ13Met with siRNA led to a decreased differentiation, whereas its overexpression potentiated differentiation of human primary myoblasts. Furthermore, in notexin-induced mouse injury model, exogenous Δ13Met expression enhanced regeneration of skeletal muscle, further confirming a stimulatory role of the isoform in muscle cell differentiation. In summary, we identified a novel alternatively spliced inhibitory isoform of Met that stimulates muscle cell differentiation, which confers a new means to control muscle differentiation and/or regeneration.     

肝细胞生长因子mRNA在成年小鼠和人胎儿不同器官中的表达

本实验从成年小鼠和胎龄４－５月的人胎儿不同器官中分离总ＲＮＡ。经斑点印迹分析显示,肝细胞生长因子（ＨＧＦ）ｍＲＮＡ在成年ＫＭ小鼠多种器官中表达,其表达水平由高到低依次为：肺、肝、肾、卵巢、睾丸、大脑和胃;在脾、心、骨髓、小肠和骨骼肌组织中以ＨＧＦｍＲＮＡ。在胎龄４－５月的人胎儿中,ＨＧＦｍＲＮＡ表达水平由高到低依次为：大脑、肝、腮腺、胃、小肠、肾、心和骨骼肌;肺和脾组织为阴性。由此可见,ＨＧＦ在成     

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway

Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.     

Emerin increase in regenerating muscle fibers

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

Sequence of IGF-I, IGF-II, and HGF expression in regenerating skeletal muscle

Various cytokines are thought to play a role in muscle regeneration, however, the interaction and mechanisms of action of these cytokines remains largely unknown. In this study, we investigated the role of HGF, IGF-I, and IGF-II during myogenesis using the regeneration model of skeletal muscle as well as myoblast culture. RT-PCR analysis revealed that HGF and IGF-I expressions were markedly upregulated, in regenerating muscle. In contrast, there was no significant difference in IGF-II expression between normal and regenerating muscle. Immunohistochemical analysis demonstrated that HGF was expressed mostly by myocytes during the early stages of muscle regeneration. Additionally, HGF inhibited the formation of myotubes by myoblasts, but promoted cellular proliferation. Otherwise, IGF-I and IGF-II were expressed by myocytes through the early to middle stages of muscle regeneration. The addition of HGF to myoblast growing in vitro significantly increased the number of cells. These findings indicate that these three cytokines have pleiotropic effects in regenerating skeletal muscle.     

TAM kinase signaling is indispensable for proper skeletal muscle regeneration in mice

Skeletal muscle regeneration following injury results from the proliferation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Infiltrating macrophages play an essential role in the process partly by clearing the necrotic cell debris, partly by producing cytokines that guide myogenesis. Infiltrating macrophages are at the beginning pro-inflammatory, but phagocytosis of dead cells induces a phenotypic change to become healing macrophages that regulate inflammation, myoblast fusion and growth, fibrosis, vascularization and return to homeostasis. The TAM receptor kinases Mer and Axl are known efferocytosis receptors in macrophages functioning in tolerogenic or inflammatory conditions, respectively. Here we investigated their involvement in the muscle regeneration process by studying the muscle repair following cardiotoxin-induced injury in Mer^−/− mice. We found that Axl was the only TAM kinase receptor expressed on the protein level by skeletal muscle and C2C12 myoblast cells, while Mer was the dominant TAM kinase receptor in the CD45⁺ cells, and its expression significantly increased during repair. Mer ablation did not affect the skeletal muscle weight or structure, but following injury it resulted in a delay in the clearance of necrotic muscle cell debris, in the healing phenotype conversion of macrophages and consequently in a significant delay in the full muscle regeneration. Administration of the TAM kinase inhibitor BMS-777607 to wild type mice mimicked the effect of Mer ablation on the muscle regeneration process, but in addition, it resulted in a long-persisting necrotic area. Finally, in vitro inhibition of TAM kinase signaling in C2C12 myoblasts resulted in decreased viability and in impaired myotube growth. Our work identifies Axl as a survival and growth receptor in the mouse myoblasts, and reveals the contribution of TAM kinase-mediated signaling to the skeletal muscle regeneration both in macrophages and in myoblasts.Subject terms: Mechanisms of disease, Immunological disorders     

大鼠心肌虚血再灌流后肝细胞再生因子的表达变化

肝细胞再生因子（hepatocyte growth factor, HGF）对多种细胞都具有促进增殖及运动、抗凋亡的作用,对组织器官的发育形成也起到重要作用．在肝脏、肾脏、肺、心脏等器官受损之后的修复过程中,有积极的促进再生的作用．本研究采用了心虚血再灌流大鼠模型,发现心肌细胞受损伤后 6 h 血清中HGF水平显著增高．在比较了肾脏、肺、肝脏、脾脏等组织提取液中HGF的含量之后,发现心虚血再灌流手术后,肾脏、肺、肝脏中HGF的含量变化不明显,而脾脏的提取液中HGF的含量增加显著．对脾脏组织的连续切片进行HGF与血管内皮细胞的特异性标志物von Willanbrand Factor (vWF)免疫组织化学染色研究,发现手术后脾脏中产生HGF的细胞主要为血管内皮细胞．此项研究首次阐明组织器官受损后,远端组织器官的血管内皮细胞能够增加HGF的合成和分泌,增加的HGF通过体液循环到达受损组织器官,促进其修复再生．     

Activation of insulin-like growth factor II expression during skeletal muscle regeneration in the rat: correlation with myotube formation.

Insulin-like growth factors (IGFs) are important stimulators of proliferation and differentiation of cultured myoblasts. It has previously been shown that IGF-I is induced during muscle regeneration in rodents, however, little is known about the expression of IGF-II. Therefore, two in vivo models were used to analyze IGF-II mRNA expression during skeletal muscle regeneration in the rat: injection of the snake venom notexin and induction of ischemia. During the regeneration process the levels of both IGF-I and IGF-II mRNA were transiently induced, as analyzed by solution hybridization. Both IGF-I-like immunoreactivity and IGF-II-like immunoreactivity were found to be present during muscle regeneration. In a time course study, induction of IGF-II was preceded by IGF-I, both at the mRNA and protein levels. Using alpha- and beta-actin as markers for different stages of skeletal muscle differentiation, together with the immunohistochemistry data, it is concluded that the expression of IGF-I and IGF-II occurs at different differentiation stages, and that IGF-II appears concomitant to the formation of myotubes. These results suggest that each IGF has a distinct role during the differentiation of muscle cells.