??4 Integrin Marks Interstitial Myogenic Progenitor Cells in Adult Murine Skeletal Muscle

Skeletal muscle growth and its regeneration following injury rely on myogenic progenitor cells, a heterogeneous population that includes the satellite cells and other interstitial progenitors. The present study demonstrates that surface expression of β4 integrin marks a population of vessel-associated interstitial muscle progenitor cells. Muscle β4 integrin–positive cells do not express myogenic markers upon isolation. However, they are capable of undergoing myogenic specification in vitro and in vivo: β4 integrin cells differentiate into multinucleated myotubes in culture dishes and contribute to muscle regeneration upon delivery into diseased mice. Subfractionation of β4 integrin–expressing cells based on CD31 expression does not further enrich for myogenic precursors. These findings support the expression of β4 integrin in interstitial, vessel-associated cells with myogenic activity within adult 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.     

Signals from damaged but not undamaged skeletal muscle induce myogenic differentiation of rat bone-marrow-derived mesenchymal stem cells

The regenerative capacity of skeletal muscle has been usually attributed to resident satellite cells, which, upon activation by local or distant stimuli, initiate a myogenic differentiation program. Although recent studies have revealed that bone-marrow-derived progenitor cells may also participate in regenerative myogenesis, the signals and mechanisms involved in this process have not been elucidated. This study was designed to investigate whether signals from injured rat skeletal muscle were competent to induce a program of myogenic differentiation in expanded cultures of rat bone-marrow-derived mesenchymal stem cells (MSC). We observed that the incubation of MSC with a conditioned medium prepared from chemically damaged but not undamaged muscle resulted in a time-dependent change from fibroblast-like into elongated multinucleated cells, a transient increase in the number of MyoD positive cells, and the subsequent onset of myogenin, alpha-actinin, and myosin heavy chain expression. These results show that damaged rat skeletal muscle is endowed with the capacity to induce myogenic differentiation of bone-marrow-derived mesenchymal progenitors.     

The role of TGF‐β1 during skeletal muscle regeneration

The injury of adult skeletal muscle initiates series of well‐coordinated events that lead to the efficient repair of the damaged tissue. Any disturbances during muscle myolysis or reconstruction may result in the unsuccessful regeneration, characterised by strong inflammatory response and formation of connective tissue, that is, fibrosis. The switch between proper regeneration of skeletal muscle and development of fibrosis is controlled by various factors. Amongst them are those belonging to the transforming growth factor β family. One of the TGF‐β family members is TGF‐β1, a multifunctional cytokine involved in the regulation of muscle repair via satellite cells activation, connective tissue formation, as well as regulation of the immune response intensity. Here, we present the role of TGF‐β1 in myogenic differentiation and muscle repair. The understanding of the mechanisms controlling these processes can contribute to the better understanding of skeletal muscle atrophy and diseases which consequence is fibrosis disrupting muscle function.     

肌卫星细胞在失重肌萎缩中的可塑性变化及机制

肌卫星细胞在骨骼肌生长发育和出生后骨骼肌损伤修复中起着重要的作用,但是有关肌萎缩中肌卫星细胞的可塑性变化、作用及其机制尚不清楚.本研究采用小鼠尾悬吊模拟失重效应诱导失重肌萎缩,动态分析了失重肌萎缩发生过程中不同类型肌纤维的肌卫星细胞数量和增殖、分化潜能可塑性的改变,发现在失重肌萎缩过程中,处于安静状态的肌卫星细胞显著增多、激活增殖的肌卫星细胞显著减少,而具有成肌分化潜能的肌卫星细胞有持续减少趋势.此外,在失重肌萎缩比目鱼肌单根肌纤维移出的体外培养中,证明了失重肌萎缩肌纤维肌卫星细胞可塑性降低的特征性变化.进一步,通过对比分析Smad3基因敲除及其同窝野生型小鼠,在失重肌萎缩中肌卫星细胞可塑性的差异性变化,揭示了Smad3在调控失重肌萎缩肌卫星细胞可塑性变化中的关键作用.     

Insulin-like growth factor-1 (IGF-1) and leucine activate pig myogenic satellite cells through mammalian target of rapamycin (mTOR) pathway

Myogenic satellite cells are adult stem cells and have important roles in skeletal muscle growth, repair, and regeneration. Both insulin-like growth factor-1 (IGF-1) and leucine stimulate skeletal muscle growth, which link to the activation and proliferation of myogenic satellite cells in skeletal muscle. Mammalian target of rapamycin (mTOR) signaling is one of the main signaling pathways controlling protein synthesis and cell proliferation. Thus, IGF-1 and leucine may stimulate activation of myogenic satellite cells through mTOR signaling. In this study, myogenic satellite cells were isolated from 6-month-old pigs and subjected to IGF-1 and leucine treatments. Both IGF-1 and leucine upregulated mTOR signaling in myogenic satellite cells. The phosphorylation of mTOR at Ser(2448) increased 83.8 +/- 7.7% by IGF-1 (P < 0.05) and 83.4 +/- 5.7% by leucine (P < 0.05). The downstream targets of mTOR, S6 kinase, and 4E-binding protein 1 (4EBP1) were also phosphorylated due to IGF-1 and leucine treatments. Treatment with IGF-1 and leucine induced the phosphorylation of tuburin (TSC2), a key mediator upstream of mTOR signaling, by 272.8 +/- 26.4% and 94.2 +/- 28.7%, respectively. Treatment of cells with both IGF-1 and leucine did not show synergistic effect on mTOR signaling. Inhibition of mTOR by rapamycin abolished the protein synthesis and cell proliferation stimulated by both IGF-1 and leucine. In summary, our data showed that in preliminary cultured myogenic satellite cells mTOR signaling was activated due to IGF-1 and leucine treatments, and this mTOR activation is necessary for the activation of myogenic satellite cells.     

鸡骨骼肌卫星细胞的分离培养、鉴定及生物学特性研究

采用I型胶原酶和胰蛋白酶二步消化,经体外培养获得鸡骨骼肌卫星细胞,并通过检测肌卫星细胞特异基因的表达进行鉴定。结果表明：二步消化法适用于鸡骨骼肌卫星细胞的分离和获取,此方法分离得到的鸡骨骼肌卫星细胞表达卫星细胞特异的标志基因desmin和Pax7,并具有良好的增殖和分化能力,为鸡骨骼肌细胞增殖、分化和再生机制的研究提供技术平台。      

Up-regulation of type XIX collagen in rhabdomyosarcoma cells accompanies myogenic differentiation

Rhabdomyosarcomas are known to recapitulate some of the early events in skeletal muscle embryogenesis, and cultures derived from these tumors have been extensively used to elucidate processes associated with the differentiation of primitive mesenchymal cells. These neoplasms have also provided important systems for studying different collagen types. This aspect is particularly relevant to type XIX collagen, which was originally identified from rhabdomyosarcoma cDNA clones. Although this collagen has been localized in vivo to basement membrane zones in a wide variety of tissues, including skeletal muscle, the tumor cells appear to be a unique source of its expression in vitro. We have found that one particular cell line-derived from a peritesticular embryonal rhabdomyosarcoma-produced relatively large amounts of type XIX collagen, especially in those rare instances in which these cells appear to spontaneously differentiate. To characterize this phenomenon, tumor cells were grown under conditions known to induce differentiation in normal myoblast cultures. In response to this treatment, the typical tumor cell morphology consistently and reproducibly switched from polygonal to round/spindle-shaped with the subsequent appearance of some structures resembling myotubes. Concurrently, the cultures commenced a dramatic up-regulation of type XIX collagen and skeletal muscle myosin heavy chain and alpha-actinin in a time-dependent fashion, whereas protein and mRNA levels of other matrix proteins were either decreased or unchanged. Moreover, immunocytochemical analysis revealed that only a subpopulation of the cells was responsible for the increased synthesis of type XIX collagen, alpha-actinin, and myosin, and that the same cells which stained positive for the collagen also stained positive for the muscle proteins. Taken together, the results suggested that type XIX collagen may be involved in the initial stages of skeletal muscle cell differentiation.     

A novel lncRNA promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1

Myogenesis, the process of skeletal muscle formation, is a highly coordinated multistep biological process. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are emerging as a gatekeeper in myogenesis. Up to now, most studies on muscle development-related lncRNAs are mainly focussed on humans and mice. In this study, a novel muscle highly expressed lncRNA, named lnc23, localized in nucleus, was found differentially expressed in different stages of embryonic development and myogenic differentiation. The knockdown and over-expression experiments showed that lnc23 positively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Then, TMT 10-plex labelling quantitative proteomics was performed to screen the potentially regulatory proteins of lnc23. Results indicated that lnc23 was involved in the key processes of myogenic differentiation such as cell fusion, further demonstrated that down-regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells. Furthermore, RNA pulldown/LC-MS and RIP experiment illustrated that PFN1 was a binding protein of lnc23. Further, we also found that lnc23 positively regulated the protein expression of RhoA and Rac1, and PFN1 may negatively regulate myogenic differentiation and the expression of its interacting proteins RhoA and Rac1. Hence, we support that lnc23 may reduce the inhibiting effect of PFN1 on RhoA and Rac1 by binding to PFN1, thereby promoting myogenic differentiation. In short, the novel identified lnc23 promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.     

Satellite cells isolated from aged or dystrophic muscle exhibit a reduced capacity to promote angiogenesis in vitro

Deficits in skeletal muscle function exist during aging and muscular dystrophy, and suboptimal function has been related to factors such as atrophy, excessive inflammation and fibrosis. Ineffective muscle regeneration underlies each condition and has been attributed to a deficit in myogenic potential of resident stem cells or satellite cells. In addition to reduced myogenic activity, satellite cells may also lose the ability to communicate with vascular cells for coordination of myogenesis and angiogenesis and restoration of proper muscle function. Objectives of the current study were to determine the angiogenic-promoting capacity of satellite cells from two states characterized by dysfunctional skeletal muscle repair, aging and Duchenne muscular dystrophy. An in vitro culture model composed of satellite cells or their conditioned media and rat adipose tissue microvascular fragments (MVF) was used to examine this relationship. Microvascular fragments cultured in the presence of rat satellite cells from adult muscle donors (9–12 month of age) exhibited greater indices of angiogenesis (endothelial cell sprouting, tubule formation and extensive branching) than MVF co-cultured with satellite cells from aged muscle donors (24 month of age). We sought to determine if the differential degree of angiogenesis we observed in the co-culture setting was due to soluble factors produced by each satellite cell age group. Similar to the co-culture experiment, conditioned media produced by adult satellite cells promoted greater angiogenesis than that of aged satellite cells. Next, we examined differences in angiogenesis-stimulating ability of satellite cells from 12 mo old MDX mice or age-matched wild-type mice. A reduction in angiogenesis activity of media conditioned by satellite cells from dystrophic muscle was observed as compared to healthy muscle. Finally, we found reduced gene expression of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in both aged and dystrophic satellite cells compared to their adult and normal counterparts, respectively. These results indicate that functional deficits in satellite cell activities during aging and diseased muscle may extend to their ability to communicate with other cells in their environment, in this case cells involved in angiogenesis.     

The impact of aging on mitochondrial function and biogenesis pathways in skeletal muscle of sedentary high- and low-functioning elderly individuals

Age-related loss of muscle mass and strength (sarcopenia) leads to a decline in physical function and frailty in the elderly. Among the many proposed underlying causes of sarcopenia, mitochondrial dysfunction is inherent in a variety of aged tissues. The intent of this study was to examine the effect of aging on key groups of regulatory proteins involved in mitochondrial biogenesis and how this relates to physical performance in two groups of sedentary elderly participants, classified as high- and low-functioning based on the Short Physical Performance Battery test. Muscle mass was decreased by 38% and 30% in low-functioning elderly (LFE) participants when compared to young and high-functioning elderly participants, respectively, and positively correlated to physical performance. Mitochondrial respiration in permeabilized muscle fibers was reduced (41%) in the LFE group when compared to the young, and this was associated with a 30% decline in cytochrome c oxidase activity. Levels of key metabolic regulators, SIRT3 and PGC-1α, were significantly reduced (50%) in both groups of elderly participants when compared to young. Similarly, the fusion protein OPA1 was lower in muscle from elderly subjects; however, no changes were detected in Mfn2, Drp1 or Fis1 among the groups. In contrast, protein import machinery components Tom22 and cHsp70 were increased in the LFE group when compared to the young. This study suggests that aging in skeletal muscle is associated with impaired mitochondrial function and altered biogenesis pathways and that this may contribute to muscle atrophy and the decline in muscle performance observed in the elderly population.     

In vitro developmental expression of dystroglycan and laminin-alpha2 in human skeletal muscle

The alpha-subunit of dystroglycan, a member of the dystrophin associated protein complex, binds to extracellular laminin-alpha2, while its beta-subunit interacts with cytoskeletal dystrophin. The exact biological role of dystroglycan, especially during human skeletal muscle development, has not been fully explored. Here, we analysed the distribution and expression characteristics of both dystroglycan subunits and laminin-alpha2 in primary human skeletal muscle cells. During development, expression levels of all three proteins increased with differentiation. The proteins were relocated from the sarcoplasm to the sarcolemma. The size of alpha-dystroglycan decreased from 150-220 kDa at the proliferation stage to 100-120 kDa at the late developmental stage. Both alpha- and beta-dystroglycan were involved in forming a complex with their respective partners laminin-alpha2 and dystrophin/utrophin. Our data show that, during development, cells may employ tightly regulated post-translational species-specific modification to produce different isoforms of alpha-dystroglycan to participate in appropriate functions.     

Reduced troponin I phosphorylation and increased Ca2+-dependent ATP-consumption in triton X-skinned fiber preparations from Gαq overexpressor mice

Mechanical stress leads to satellite cell activation, which is an important event in the development, growth, and remodeling of postnatal skeletal muscle. Although there is a considerable knowledge on the events involved in skeletal muscle regeneration and development, the precise role of mechanical stress on activation of satellite cells remains unclear. Previously, satellite cells were isolated from adult bovine muscle and it was shown that the cells are multipotent, i.e., capable of proliferating and to differentiating into both myoblasts and adipocytes. This study investigated the cellular mechanisms by which cyclic mechanical stretching modulates the proliferation and differentiation of adult bovine satellite cells. The application of cyclic stretch induced the proliferation of satellite cells and inhibited their differentiation into myotubes. This response is believed to be closely related to the stretch-mediated changes in the expression of myogenic and cell cycle regulatory factors. Cyclic stretching increased the level of extracellular signal-regulated kinase (ERK) phosphorylation, whereas a specific ERK inhibitor (PD98058) blocked the stretch-mediated inhibition of myogenesis in a dose-dependent manner. Overall, this study demonstrates for the first time that cyclic mechanical stretch induces the proliferation of bovine satellite cells and suppresses their myogenic differentiation through the activation of ERK.     

Myogenic potential of human alveolar mucosa derived cells

Difficulties related to the obtainment of stem/progenitor cells from skeletal muscle tissue make the search for new sources of myogenic cells highly relevant. Alveolar mucosa might be considered as a perspective candidate due to availability and high proliferative capacity of its cells. Human alveolar mucosa cells (AMC) were obtained from gingival biopsy samples collected from 10 healthy donors and cultured up to 10 passages. AMC matched the generally accepted multipotent mesenchymal stromal cells criteria and possess population doubling time, caryotype and immunophenotype stability during long-term cultivation. The single myogenic induction of primary cell cultures resulted in differentiation of AMC into multinucleated myotubes. The myogenic differentiation was associated with expression of skeletal muscle markers: skeletal myosin, skeletal actin, myogenin and MyoD1. Efficiency of myogenic differentiation in AMC cultures was similar to that in skeletal muscle cells. Furthermore, some of differentiated myotubes exhibited contractions in vitro. Our data confirms the sufficiently high myogenic potential and proliferative capacity of AMC and their ability to maintain in vitro proliferation-competent myogenic precursor cells regardless of the passage number.     

Aging affects the in vivo regenerative potential of human mesoangioblasts

Sarcopenia is the age‐related loss of muscle mass, strength, and function. Although the role of human satellite cells (SCs) as adult skeletal muscle stem cells has been deeply investigated, little is known about the impact of aging on muscle interstitial stem cells. Here, we isolated the non‐SC CD56^– fraction from human muscle biopsies of young and elderly subjects. The elderly interstitial cell population contained a higher number of CD15⁺ and PDGFRα⁺ cells when compared to young samples. In addition, we found that the CD56^–/ALP⁺ cells were well represented as a multipotent stem cell population inside the CD56^– fraction. CD56^–/ALP⁺/CD15^– cells were clonogenic, and since they were myogenic and expressed NG2, α‐SMA and PDGFRβ can be considered mesoangioblasts (MABs). Interestingly, elderly MABs displayed a dramatic impairment in the myogenic differentiation ability in vitro and when transplanted in dystrophic immunodeficient Sgcb‐null Rag2‐null γc‐null mice. In addition, elderly MABs proliferated less, but yet retained other multilineage capabilities. Overall, our results indicate that aging negatively impacted on the regenerative potential of MABs and this should be carefully considered for potential therapeutic applications of MABs.     

Differentiation of stem cells isolated from rat skeletal muscles towards cardiomyocytes: The effect of an inhibitor of DNA methylation 5-azacytidine

The effects of the inhibitor of DNA methylation 5-azacytidine on stem (satellite) cells isolated from fetal and definitive skeletal muscles of rats lead to the expression of marker genes of cardiomyogenesis (Gata4, Nkx2.5, connexin-43, n-cadherin, as well as Cacna 1 c encoding the cardiac subunit of the L-type Ca²⁺-channel). Through comparative analysis of the dynamics of expression of key markers of cardiomyogenesis, it was established that satellite cells isolated from fetal muscles have a more expressive potency to cardiomyocyte differentiation in vitro (expression of specific marker genes) compared to cells from muscles of adult animals. In the process of induced cardiomyocyte differentiation, the expression of MyoD and m-cadherin marker genes of skeletal muscle differentiation was not detected, suggesting that in the experiments presented the program of myogenic differentiation of skeletal muscles is inhibited.