Satellite cells express the trophic factor IGF-I in regenerating skeletal muscle

The expression of insulin-like growth factor I (IGF-I, somatomedin C) was studied in regenerating skeletal muscle. Irreversible damage to skeletal muscle cells was induced in the extensor digitorum longus muscle (EDL) of adult rats by ischaemia, preceded by glycogen depletion, and the regeneration process was studied for periods up to 14 days after injury. The IGF-I was demonstrated by indirect immunofluorescence. Immunoreactivity against ribonucleotide reductase (RR) was used as a marker for DNA synthesis, that is, cell proliferation. Increased IGF-I immunoreactivity could be demonstrated within 24h after injury in satellite cells, intramuscular nerves and in blood vessels. The IGF-I immunoreactivity remained virtually unchanged in the contralateral, undamaged EDL. An increasing number of satellite cells, expressing high IGF-I immunoreactivity, could be demonstrated in the injured EDL, and within 72 h myoblasts, expressing high IGF-I and RR immunoreactivity, were formed. Small immature muscle cells, displaying high IGF-I immunoreactivity, were observed 4 days after injury. Increased IGF-I immunoreactivity was still obvious in the regenerated muscle cells 14 days after injury while RR immunoreactivity was seen only in scattered satellite cells. It is concluded that IGF-I may act as a trophic factor during regeneration of skeletal muscle after injury.     

Evidence for distinct fast and slow myogenic cell lineages in human foetal skeletal muscle

To analyse the myogenic cell lineages in human foetal skeletal muscle, muscle cell cultures were prepared from different foetal stages of development. The in vitro muscle cell phenotype was defined by staining the myotubes with antibodies to fast and slow skeletal muscle type myosin heavy chains using immunoperoxidase or double immunofluorescence procedures. The antibodies to fast skeletal muscle myosin heavy chains stained nearly all myotubes dark in cell cultures prepared from quadriceps muscles at 10–18 weeks of gestation. The antibodies to slow skeletal muscle myosin heavy chains, in contrast, stained only 10–40% of the myotubes very dark. The remaining myotubes were further subdivided into two populations, one of which was unstained while the other stained with variable intensity for slow myosin heavy chain. The slow myosin heavy chain staining was not influenced by the nature of the substratum used to culture these cells, although the growth of muscle cell cultures was greatly improved on matrigel-coated dishes. The presence of both slow and fast myosin heavy chains was detected even when myotubes were grown on uncoated petri dishes. The myotube diversity was further investigated by analysing the clonal populations of human foetal skeletal muscle cells in vitro. When cultured at clonal densities, two types of myogenic clones were identified by their differential staining with antibodies to slow myosin heavy chain. As was the case with the high density muscle cell cultures, virtually all myotubes in both groups of clones stained with antibodies to fast myosin heavy chains. Antibodies to slow myosin heavy chains stained nearly all myotubes dark in one group of myogenic clones, but only a subset of the myotubes stained dark for slow myosin heavy chain in the second group of clones. The proportion of slow myosin heavy chain positive myotubes in this group varied in different clones. The myogenic diversity was thus apparent in both high density and clonal human muscle cell cultures, and myogenic cells retained their ability to modify their muscle cell phenotype. This revised version was published online in September 2006 with corrections to the Cover Date.     

Regenerating skeletal muscle cells express insulin-like growth factor I

The expression of the trophic peptide insulin-like growth factor I (IGF-I; somatomedin C) was investigated in the regenerating soleus muscle of mice after injury by the snake venom taipoxin. No specific IGF-I immunoreactivity was observed in muscle cells during control conditions. Within 2 days after taipoxin injection, IGF-I immunoreactivity could be demonstrated in activated satellite cells. Myoblasts and myotubes expressed high IGF-I immunoreactivity. The IGF-I immunoreactivity was strictly cytoplasmatic and obviously associated with polyribosomes. No vesicular or membraneous IGF-I immunoreactivity could be demonstrated. It is concluded that IGF-I is synthesized in myogenic cells during skeletal muscle regeneration. It is suggested that IGF-I exerts its effects on skeletal muscle mainly by autocrine mechanisms.     

A population of myogenic stem cells that survives skeletal muscle aging

Age-related decline in integrity and function of differentiated adult tissues is widely attributed to reduction in number or regenerative potential of resident stem cells. The satellite cell, resident beneath the basal lamina of skeletal muscle myofibers, is the principal myogenic stem cell. Here we have explored the capacity of satellite cells within aged mouse muscle to regenerate skeletal muscle and to self-renew using isolated myofibers in tissue culture and in vivo. Satellite cells expressing Pax7 were depleted from aged muscles, and when aged myofibers were placed in culture, satellite cell myogenic progression resulted in apoptosis and fewer total differentiated progeny. However, a minority of cultured aged satellite cells generated large clusters of progeny containing both differentiated cells and new cells of a quiescent satellite-cell-like phenotype characteristic of self-renewal. Parallel in vivo engraftment assays showed that, despite the reduction in Pax7(+) cells, the satellite cell population associated with individual aged myofibers could regenerate muscle and self-renew as effectively as the larger population of satellite cells associated with young myofibers. We conclude that a minority of satellite cells is responsible for adult muscle regeneration, and that these stem cells survive the effects of aging to retain their intrinsic potential throughout life. Thus, the effectiveness of stem-cell-mediated muscle regeneration is determined by both extrinsic environmental influences and diversity in intrinsic potential of the stem cells themselves.     

Satellite cells of skeletal muscle fibers in human progressive muscular dystrophy

Summary In biopsy specimens of five cases of human progressive muscular dystrophy studied with the electron microscope a special kind of cell located between the two layers of the surface membrane complex of the muscle fiber is described. Based on the fact that they hypertrophy when the muscle fiber is damaged, and especially when it is regenerating, it is suggested that they might perform a trophic function, acting as an intermediate cell between the tissue space and the muscle fiber.

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Satellitenzellen an Skeletmuskelfasern bei der progressiven Muskeldystrophie des Menschen

Zusammenfassung Auf Grund elektronenmikroskopischer Untersuchungen von Muskelbiopsien von fünf Fällen menschlicher progressiver Muskeldystrophie wird eine besondere Zellart beschrieben, die zwischen den beiden Lagern des oberflächlichen Membrankomplexes der Muskelfasern gelegen ist. Da diese Zellen hypertrophieren, wenn die Muskelfaser geschädigt ist, besonders aber, wenn sie regeneriert, wird angenommen, daß sie eine trophische Funktion besitzen und als celluläre Vermittler zwischen Gewebsraum und Muskelfaser dienen.

     

Myogenic progenitor cells express filamin C in developing and regenerating skeletal muscle

The regenerative capacity of skeletal muscle is due to the myogenic progenitor cell population that is resident in adult skeletal muscle. To enhance our understanding of this cell population, we examined the temporal-spatial expression pattern for filamin C during murine embryogenesis, adult muscle regeneration and in selected myopathic models of human disease. Using in situ hybridization, we observed filamin C to be restricted to mesodermal lineages including the developing heart and skeletal muscle during embryogenesis. Following cardiotoxin-induced muscle injury of adult skeletal muscle, filamin C expression was dynamically regulated in activated myogenic progenitor cells and newly regenerated myotubes. This expression pattern was further supported using RT-PCR analysis of filamin C expression in differentiating C2C12 myotubes. These results support the paradigm that the regulatory mechanisms of muscle regeneration largely recapitulate the fundamental events observed during muscle development and that filamin C may function in signal transduction or cellular migration of the myogenic progenitor cell population.     

Intermediate filament proteins in TPA-treated skeletal muscle cells in culture

Summary The cocarcinogenic phorbol ester 13-tetradecanoyl-O-phorbol acetate selectively and reversibly inhibits the ongoing differentiation programme of chick muscle cells in culture. 13-tetradecanoyl-O-phorbol acetate promptly blocks spontaneous contractions in mature myotubes and induces them to retract, forming giant myosacs and concurrently stress fibre-like structures are assembled. Using indirect immunofluorescence to localise desmin, the muscle specific intermediate filament protein, it was shown that its distribution is longitudinally oriented in mature myotubes. In myosacs, desmin has a reticular pattern although not as linearly oriented as in control myotubes. Using gel electrophoresis of control and 13-tetradecanoyl-O-phorbol acetate treated cell extracts, three major protein bands were observed with molecular weight of 43, 50 and 55 kDa. They migrate as actin, desmin and vimentin, respectively. The 50 kDa and 55 kDa proteins were expressed more in 13-tetradecanoyl-O-phorbol acetate-treated cells. The 50 kDa band was confirmed as desmin by immunoblotting using anti-chicken desmin antibody. Two-dimensional gel electrophoresis analysis showed the appearance of more acidic isoforms of the 50 and 55 kDa proteins 13-tetradecanoyl-O-phorbol in acetate-treated cells. The 43 kDa protein was seen as three distinct isoforms in control cells and as only two isoforms in 13-tetradecanoyl-O-phorbol acetate-treated cells.     

Expression of P-glycoprotein in normal muscle cells and myogenic tumors.

We have analyzed the expression of the multidrug resistance (mdr-1) gene product, P-glycoprotein, by immunohistochemical staining of frozen tissue sections of human normal muscle fibers and 31 tissue specimens of cases of myogenic sarcomas. The objective of this study was to further characterize what appears to be a variety of responses to therapy in like-appearing but distinct tumors. We have used two mouse monoclonal antibodies that recognize two different epitopes of P-glycoprotein. Mouse monoclonal antibody HYB-241 detects an extracellular epitope of P-glycoprotein, whereas C219 detects a carboxy-terminal intracellular epitope and has recently been reported to cross-react with the mdr-3 gene product. Differential epitope expression was observed among normal muscle fibers with the two antibodies used. Smooth-muscle cells were unreactive for the two antibodies, whereas cardiac and a subgroup of skeletal muscle fibers were intensely stained by C219, but not by HYB-241. P-glycoprotein expression was observed in 23% of the 31 myogenic sarcomas analyzed. Our study was conducted mainly using adult myogenic sarcomas (28 out of 31 cases), with a few cases (three out of 31 cases) of childhood sarcomas. Nineteen tumors were leiomyosarcomas, seven cases were embryonal rhabdomyosarcomas, and five cases were rhabdomyosarcomas. We have considered expression of the mdr-1-coded P-glycoprotein when we observed either HYB-241 and C219 staining, or just HYB-241 immunoreactivities. Although P-glycoprotein expression can now be detected in human sarcomas, further studies are needed, mainly comparing tumor samples before, during, and after therapy, to establish the possible significance of the P-glycoprotein expression in clinical drug resistance.     

Hierarchization of myogenic and adipogenic progenitors within human skeletal muscle

Skeletal muscle cells constitute a heterogeneous population that maintains muscle integrity through a high myogenic regenerative capacity. More unexpectedly, this population is also endowed with an adipogenic potential, even in humans, and intramuscular adipocytes have been found to be present in several disorders. We tested the distribution of myogenic and adipogenic commitments in human muscle-derived cells to decipher the cellular basis of the myoadipogenic balance. Clonal analysis showed that adipogenic progenitors can be separated from myogenic progenitors and, interestingly, from myoadipogenic bipotent progenitors. These progenitors were isolated in the CD34(+) population on the basis of the expression of CD56 and CD15 cell surface markers. In vivo, these different cell types have been found in the interstitial compartment of human muscle. In vitro, we show that the proliferation of bipotent myoadipogenic CD56(+)CD15(+) progenitors gives rise to myogenic CD56(+)CD15(-) progenitors and adipogenic CD56(-)CD15(+) progenitors. A cellular hierarchy of muscle and fat progenitors thus occurs within human muscle. These results provide cellular bases for adipogenic differentiation in human skeletal muscle, which may explain the fat development encountered in different muscle pathological situations.     

Involvement of Wnt4 signaling during myogenic proliferation and differentiation of skeletal muscle.

The direct effects of Wnt4 on myogenic proliferation and differentiation of skeletal muscle precursors are examined. Wnt4 cDNA was misexpressed in the presumptive limb fields on the right side of stage 16 chick embryos. Muscle development was evaluated at stage 37 with hematoxylin-eosin staining and immunohistochemical staining for fast and slow types of the myosin heavy chain (MyHC). Overexpression of Wnt4 resulted in up-regulation of Pax7 and MyoD1 expression. The muscle mass showed a significant increase compared with that of the control limb. The area for fast MyHC-expressing cells showed a significant increase, whereas a slight decrease was observed for slow MyHC-expressing cells. Wnt4 acted as a stimulator during myogenic proliferation and differentiation, especially, for fast-type muscle in C2C12 cells. The present results are identical to those of myostatin knockout, suggesting that Wnt4 is acting against myostatin as an antagonizing signal for myostatin.     

Incorporation of leucine into human skeletal muscle proteins.

The in vitro incorporation of labelled leucine into human skeletal muscle proteins was studied with the aim to elucidate the relationship between the amino acid tissue pools and protein biosynthesis. The distribution volumes of leucine and cycloleucine in skeletal muscle tissue were similar but the equilibration time was shorter for leucine than for cycloleucine. The cellular uptake of leucine and cycloleucine was competitively inhibited by increased concentration of amino acids in the medium indicating an active transport. Optimal stimulation for incorporation of leucine into proteins was obtained at an amino acid concentration in the medium corresponding to 10 times that of normal human plasma. The incorporation of 14C-leucine into skeletal muscle proteins was linear before the total pool of free intracellular 14C-leucine and the incorporation rate of leucine calculated from the specific activity in the medium versus the amino acid concentration in the medium were different in the same experiment indicating a re-utilization of amino acids released at protein degradation. The results are compatible with the hypothesis that the proteolytically released amino acids have a competitive advantage for incorporation as compared with extra- and intracellular free amino acids. It is concluded that the amino acid pool which is in the immediate continuity with the protein biosynthesis sites equilibrates rapidly with the extracellular amino acid pool.     

Bacteroides loeschei PK1295 cells express two distinct adhesins simultaneously.

Bacteroides loeschei synthesizes two distinct adhesins that mediate its coaggregation with Streptococcus sanguis 34 and Actinomyces israeli PK14. Streptococcal adhesin-specific and actinomyces adhesin-specific monoclonal antibodies were used to prepare antibody-coated 5- or 10-nm gold particles. These were used in immunoelectron microscopic studies to establish that essentially all bacteroides cells in a population express both adhesins. In general, the two sizes of gold particles representing each type of adhesin appeared to be spatially separated on neighboring fimbriae of B. loeschei. Deposition of antibody-coated gold particles, representing both types of adhesin, at or near the same fimbria was observed less frequently.     

Immunological capabilities of skeletal muscle cells.

Muscle is the target of immunological injury in several muscle diseases. It is important therefore to understand the immunological capabilities of muscle cells themselves. Although it is conventional to discuss the effects of the immune system on other cells, tissues or organs, the system's boundaries cannot be sharply drawn, and in an increasing number of ways, the immunological capabilities of non-immune tissues are recognized as determining the course of immune-inflammatory processes. Muscle cells have an inherent ability to express and respond to a variety of immunologically relevant surface molecules, cytokines, and chemokines under inflammatory conditions. The ability of muscle cells to process and present antigens to the immune cells is currently debated; thus, this review is aimed at examining the immunological capabilities of skeletal muscle cells in vitro and in vivo.     

Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis

Transforming growth factor-beta1 (TGF-beta1) is thought to play a crucial role in fibrotic diseases. This study demonstrates for the first time that TGF-beta1 stimulation can induce myoblasts (C2C12 cells) to express TGF-beta1 in an autocrine manner, down-regulate the expression of myogenic proteins, and initiate the production of fibrosis-related proteins in vitro. Direct injection of human recombinant TGF-beta1 into skeletal muscle in vivo stimulated myogenic cells, including myofibers, to express TGF-beta1 and induced scar tissue formation within the injected area. We also observed the local expression of this growth factor by myogenic cells, including regenerating myofibers, in injured skeletal muscle. Finally, we demonstrated that TGF-beta1 gene-transfected myoblasts (CT cells) can differentiate into myofibroblastic cells after intramuscular transplantation, but that decorin, an anti-fibrosis agent, prevents this differentiation process by blocking TGF-beta1. In summary, these findings indicate that TGF-beta1 is a major stimulator that plays a significant role in both the initiation of fibrotic cascades in skeletal muscle and the induction of myogenic cells to differentiate into myofibroblastic cells in injured muscle.     

香烟对小鼠C2C12成肌细胞分化的影响

目的:探讨香烟烟雾浓缩物(CSC)对小鼠骨骼肌C2C12成肌细胞分化的影响。方法:小鼠骨骼肌成肌细胞C2C12传代后分为对照组、0.3%CSC孵育组、3%CSC孵育组,细胞在含有马血清的DMEM培养基第6-7d可分化成熟的肌细胞。细胞分别在第1d、第3d在相应组中加入CSC,并在第6d进行各项检测:通过光学显微镜来观察细胞形态的变化,通过酶联免疫检测仪检测细胞内谷胱甘肽(GSH)和肌酸磷酸激酶(CK)的浓度,应用Western印迹检测肌球蛋白重链(MYHC)的蛋白表达,通过凝胶迁移率阻滞实验来检测转录因子核因子-κB(NF-κB)的活性。结果:在细胞形态上0.3%和3%的CSC能够抑制骨骼肌肌管的形成;0.3%和3%CSC处理组细胞中GSH和CK的含量较对照组明显减少,在0.3%和3%的CSC孵育组中MYHC蛋白表达明显减少,0.3%和3%的CSC孵育组中NF-κB的活性较对照组明显增强。结论:CSC一方面加重细胞氧化应激,使细胞的GSH减少,同时导致细胞的炎症反应增加,导致NF-κB的活性增加。二者相互作用引起骨骼肌细胞的分化明显减弱。     

Influence of neural and humoral beta-adrenoceptor stimulation on dynamic myogenic microvascular reactivity in cat skeletal muscle.

Analysis of myogenic microvascular reactivity in terms of its recently described prominent dynamic component was performed before and during graded sympathetic stimulation and catecholamine infusion. Phenoxybenzamine and propranolol were used to differentiate between alpha- and beta-adrenoceptor effects. The study first confirmed previous findings of a beta-adrenergic inhibitory component in the neural control of microvascular resistance which attenuated the alpha-adrenergic constriction. The results concerning the interaction between adrenergic and myogenic control mechanisms corroborated the conclusion that the sympathoadrenal system, via its beta-adrenergic link, exerts effective inhibitory action on myogenic excitatory reactions. As regards the neural control, its beta-adrenergic component seemed to quite precisely compensate for the reinforcing effect on the myogenic constrictor response which results from increased vascular tone per se (in this case caused by alpha-adrenergic constriction), interpreted as a physical 'gain' effect inherent in the inverse fourth power relationship between radius and resistance. The latter complicating factor, which implies non-linearity in integrated peripheral resistance control, was thus revealed only after beta-blockade, but not on the vascular bed with intact adrenoceptors, where a given transmural pressure stimulus evoked an almost equally large myogenic constrictor response irrespective of the prevailing level of vascular tone. The beta-inhibitory action of blood-borne noradrenaline was similar to the neural one, whereas that of adrenaline was more effective, causing decline of myogenic reactivity below control.     

Glutathione depletion impairs myogenic differentiation of murine skeletal muscle C2C12 cells through sustained NF-kappaB activation

Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.