Syndecan-3 is required for Notch processing by ADAM17/TACe and therefore regulates proliferation and viability of muscle satellite cells.
Skeletal muscle postnatal growth and repair depend on satellite cells and are regulated by molecular signals within the satellite cell niche. We investigated the molecular and cellular events that lead to altered myogenesis upon genetic ablation of Syndecan-3, a component of the satellite cell niche. In the absence of Syndecan-3, satellite cells stall in S phase, leading to reduced proliferation, increased cell death, delayed onset of differentiation, and markedly reduced numbers of Pax7+ satellite cells accompanied by myofiber hypertrophy and an increased number of centrally nucleated myofibers. We show that the aberrant cell cycle and impaired self-renewal of explanted Syndecan-3–null satellite cells are rescued by ectopic expression of the constitutively active Notch intracellular domain. Furthermore, we show that Syndecan-3 interacts with Notch and is required for Notch processing by ADAM17/tumor necrosis factor-α–converting enzyme (TACE) and signal transduction. Together, our data support the conclusion that Syndecan-3 and Notch cooperate in regulating homeostasis of the satellite cell population and myofiber size.
Muscle growth depends on the fusion of proliferate satellite cells to existing myofibers. We reported previously that 0–14 day intermittent feeding led to persistent retardation in myofiber hypertrophy. However, how satellite cells respond to such nutritional insult has not been adequately elucidated.
One-day-old broiler chicks were allocated to control (Con, ad libitum feeding), intermittent feeding (IF, feed provided on alternate days) and re-feeding (RF, 2 days ad libitum feeding after 12 days of intermittent feeding) groups. Chickens were killed on Day 15 and satellite cells were isolated. When cultured, satellite cells from the IF group demonstrated significant retardation in proliferation and differentiation potential, while RF partly restored the proliferation rate and differentiation potential of the satellite cells. Significant up-regulation of insulin like growth factor I receptor (IGF-IR) (P<0.05) and thyroid hormone receptor α (TRα) (P<0.05), and down-regulation of growth hormone receptor (GHR) (P<0.01) and IGF-I (P<0.01) mRNA expression was observed in freshly isolated IF satellite cells when compared with Con cells. In RF cells, the mRNA expression of IGF-I was higher (P<0.05) and of TRα was lower (P<0.01) than in IF cells, suggesting that RF restored the mRNA expression of TRα and IGF-I, but not of GHR and IGF-IR. The Bax/Bcl-2 ratio tended to increase in the IF group, which was reversed in the RF group (P<0.05), indicating that RF reduced the pro-apoptotic influence of IF. Moreover, no significant effect of T3 was detected on cell survival in IF cells compared with Con (P<0.001) or RF (P<0.05) cells.
These data suggest that early-age feed restriction inhibits the proliferation and differentiation of satellite cells, induces changes in mRNA expression of the GH/IGF-I and thyroid hormone receptors in satellite cells, as well as blunted sensitivity of satellite cells to T3, and that RF partially reverses these effects. Thus, a moderate nutritional strategy for feed restriction should be chosen in early chick rearing systems.
Feed restriction; Satellite cells; Proliferation; Gene expression; Chicken
Overloading healthy skeletal muscle produces myofiber hypertrophy and extracellular matrix remodeling, and these processes are thought to be interdependent for producing muscle growth. Inflammatory cytokine interleukin-6 (IL-6) gene expression is induced in overloaded skeletal muscle, and the loss of this IL-6 induction can attenuate the hypertrophic response to overload. Although the overload induction of IL-6 in skeletal muscle may be an important regulator of inflammatory processes and satellite cell proliferation, less is known about its role in the regulation of extracellular matrix remodeling. The purpose of the current study was to examine if overload-induced extracellular matrix remodeling, muscle growth, and associated gene expression were altered in mice that lack IL-6, when compared to wild-type mice.
Male C57/BL6 (WT) and C57/BL6 × IL-6-/- (IL-6-/-) mice (10 wks of age) were assigned to either a sham control or synergist ablation overload (OV) treatments for 3 or 21 days.
Plantaris muscle mass increased 59% in WT and 116% in IL-6-/- mice after 21d OV. Myofiber CSA was also increased by 21d OV in both WT and IL-6-/- mice. Overload induced a 2-fold greater increase in the volume of non-contractile tissue in IL-6-/- muscle as compared to WT. Overload also induced a significantly greater accumulation of hydroxyproline and procollagen-1 mRNA in IL-6-/- muscle, when compared to WT muscle after 21d OV. TGF-β and IGF-1 mRNA expression were also induced to a greater extent in IL-6-/- muscle when compared to WT muscle after 21d OV. There was no effect of IL-6 loss on the induction of myogenin, and cyclin D1 mRNA expression after 3d OV. However, MyoD mRNA expression in 3d OV IL-6-/- muscle was attenuated when compared to WT overload mice.
IL-6 appears to be necessary for the normal regulation of extracellular matrix remodeling during overload-induced growth.
functional overload; extracellular matrix; inflammation; transforming growth factor beta
Stem cell niche plays a critical role in regulating the behavior and function of adult stem cells that underlie tissue growth, maintenance, and regeneration. In the skeletal muscle, stem cells, called satellite cells, contribute to postnatal muscle growth and hypertrophy, and thus, meat production in agricultural animals. Satellite cells are located adjacent to mature muscle fibers underneath a sheath of basal lamina. Microenvironmental signals from extracellular matrix mediated by the basal lamina and from the host myofiber both impinge on satellite cells to regulate their activity. Furthermore, several types of muscle interstitial cells, including intramuscular preadipocytes and connective tissue fibroblasts, have recently been shown to interact with satellite cells and actively regulate the growth and regeneration of postnatal skeletal muscles. From this regard, interstitial adipogenic cells are not only important for marbling and meat quality, but also represent an additional cellular component of the satellite cell niche. At the molecular level, these interstitial cells may interact with satellite cells through cell surface ligands, such as delta-like 1 homolog (Dlk1) protein whose overexpression is thought to be responsible for muscle hypertrophy in callipyge sheep. In fact, extracellular Dlk1 protein has been shown to promote the myogenic differentiation of satellite cells. Understanding the cellular and molecular mechanisms within the stem cell niche that regulate satellite cell differentiation and maintain muscle homeostasis may lead to promising approaches to optimizing muscle growth and composition, thus improving meat production and quality.
delta-like 1 homolog; intramuscular adipose tissue; satellite cell; stem cell niche
Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively-isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.
Satellite cells of adult muscle are quiescent myogenic stem cells that can be induced to enter the cell cycle by an extract of crushed muscle (Bischoff, R. 1986. Dev. Biol. 115:140-147). Here, evidence is presented that the extract acts transiently to commit cells to enter the cell cycle. Satellite cells associated with both live and killed rat myofibers in culture were briefly exposed to muscle extract and the increase in cell number was determined at 48 h in vitro, before the onset of fusion. An 8-12-h exposure to extract with killed, but not live, myofibers was sufficient to produce maximum proliferation of satellite cells. Continuous exposure for over 40 h was needed to sustain proliferation of satellite cells on live myofibers. The role of serum factors was also studied. Neither serum nor muscle extract alone was able to induce proliferation of satellite cells. In the presence of muscle extract, however, satellite cell proliferation was directly proportional to the concentration of serum in the medium. These results suggest that mitogens released from crushed muscle produce long-lasting effects that commit quiescent satellite cells to divide, whereas serum factors are needed to maintain progression through the cell cycle. Contact with a viable myofiber modulates the response of satellite cells to growth factors.
The goal of regenerative medicine is to restore form and function to damaged and aging tissues. Adult stem cells, present in tissues such as skeletal muscle, comprise a reservoir of cells with a remarkable capacity to proliferate and repair tissue damage. Muscle stem cells, known as satellite cells, reside in a quiescent state in an anatomically distinct compartment, or niche, ensheathed between the membrane of the myofiber and the basal lamina. Recently, procedures for isolating satellite cells were developed and experiments testing their function upon transplantation into muscles revealed an extraordinary potential to contribute to muscle fibers and access and replenish the satellite cell compartment. However, these properties are rapidly lost once satellite cells are plated in culture. Accordingly, elucidating the role of extrinsic factors in controlling muscle stem cell fate, in particular self-renewal, is critical. Through careful design of bioengineered culture platforms, analysis of specific proteins presented to stem cells is possible. Critical to the success of the approach is single cell analysis, as more rapidly proliferating progenitors may mask the behavior of stem cells that proliferate slowly. Bioengineering approaches provide a potent means of gaining insight into the role of extrinsic factors in the stem cell microenvironment on stem cell function and the mechanisms that control their diverse fates. Ultimately, the multidisciplinary approach presented here will lead to novel therapeutic strategies for degenerative diseases.
As the resident stem cells of skeletal muscle, satellite cells are activated by extracellular cues associated with local damage. Once activated, satellite cells will re-enter the cell cycle to proliferate and supply a population of myoblasts, which will repair or replace damaged myofibers by differentiating and fusing either with an existing myofiber or with each other. There is also evidence that the orientation of cell division with respect to the myofiber may indicate or convey asymmetry in the two daughter cells. Our recent studies with time-lapse imaging of myofiber-associated satellite cells in vitro have yielded new data on the timing and orientation of satellite cell divisions, and revealed persistent differences in the behavior of daughter cells from planar versus vertical divisions.
We analyzed 244 individual fiber-associated satellite cells in time-lapse video from 24 to 48 hours after myofiber harvest. We found that initial cell division in fiber culture is not synchronous, although presumably all cells were activated by the initial trauma of harvest; that cell cycling time is significantly shorter than previously thought (as short as 4.8 hours, averaging 10 hours between the first and second divisions and eight hours between the second and third); and that timing of subsequent divisions is not strongly correlated with timing of the initial division. Approximately 65% of first and 80% of second cell divisions occur parallel to the axis of the myofiber, whereas the remainder occur outside the plane of the fiber surface (vertical division). We previously demonstrated that daughter cells frequently remain associated with each other after division or reassociate after a brief separation, and that unrelated cells may also associate for significant periods of time. We show in this paper that daughter cells resulting from a vertical division remain associated with one another several times longer than do daughters from a horizontal division. However, the total average time of association between sister cells is not significantly different from the total average time of association between unrelated cells.
These longitudinal characterizations of satellite cell behavior shortly after activation provide new insights into cell proliferation and association as a function of relatedness, and indicate significant and consistent heterogeneity within the population based on these metrics.
Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of external lamina. Although their activation and recruitment to cycle enable muscle repair and adaptation, the activation signal is not known. Evidence is presented that nitric oxide (NO) mediates satellite cell activation, including morphological hypertrophy and decreased adhesion in the fiber-lamina complex. Activation in vivo occurred within 1 min after injury. Cell isolation and histology showed that pharmacological inhibition of nitric oxide synthase (NOS) activity prevented the immediate injury-induced myogenic cell release and delayed the hypertrophy of satellite cells in that muscle. Transient activation of satellite cells in contralateral muscles 10 min later suggested that a circulating factor may interact with NO-mediated signaling. Interestingly, satellite cell activation in muscles of mdx dystrophic mice and NOS-I knockout mice quantitatively resembled NOS-inhibited release of normal cells, in agreement with reports of displaced and reduced NOS expression in dystrophin-deficient mdx muscle and the complete loss of NOS-I expression in knockout mice. Brief NOS inhibition in normal and mdx mice during injury produced subtle alterations in subsequent repair, including apoptosis in myotube nuclei and myotube formation inside laminar sheaths. Longer NOS inhibition delayed and restricted the extent of repair and resulted in fiber branching. A model proposes the hypothesis that NO release mediates satellite cell activation, possibly via shear-induced rapid increases in NOS activity that produce “NO transients.”
Regeneration of adult injured skeletal muscle is due to activation of satellite cells, a population of stem cells resident beneath the basal lamina. Thus, information on soluble factors affecting satellite cell activation, as well as migration towards injury and fusion into new myofibers are essential. Here, we show that globular adiponectin (gAd), positively affects several features of muscle satellite cells. gAd activates satellite cells to exit quiescence and increases their recruitment towards myotubes. gAd elicits in satellite cells a specific motility program, involving activation of the small GTPase Rac1, as well as expression of Snail and Twist transcription factors driving a proteolytic motility, useful to reach the site of injury. We show that satellite cells produce autocrine full length adiponectin (fAd), which is converted to gAd by activated macrophages. In turns, gAd concurs to attract to the site of injury both satellite cells and macrophages and induces myogenesis in muscle satellite cells. Thus, these findings add a further role for gAd in skeletal muscle, including the hormone among factors participating in muscle regeneration.
Myoblast proliferation and differentiation are essential for normal skeletal muscle growth and repair. Muscle recovery is dependent on the quiescent population of muscle stem cells - satellite cells. During muscle injury, satellite cells become mitotically active and begin the repair process by fusing with each other and/or with myofibers. Aging, prolonged inactivity, obesity, cachexia and other muscle wasting diseases are associated with a decreased number of quiescent and proliferating satellite cells, which impedes the repair process.
A high-content/high-throughput platform was developed and utilized for robust phenotypic evaluation of human primary satellite cells in vitro for the discovery of chemical probes that may improve muscle recovery. A 1600 compound pilot screen was developed using two highly annotated small molecule libraries. This screen yielded 15 dose responsive compounds that increased proliferation rate in satellite cells derived from a single obese human donor. Two of these compounds remained dose responsive when counter-screened in 3-donor obese superlot. The Alk-5 inhibitor LY364947, was used as a positive control for assessing satellite cell proliferation/delayed differentiation. A multivariate approach was utilized for exploratory data analysis to discover proliferation vs. differentiation-dependent changes in cellular phenotype. Initial screening efforts successfully identified a number of phenotypic outcomes that are associated with desired effect of stimulation of proliferation and delayed differentiation.
High-content screening; LY364947; muscle atrophy; phenotypic high-content analysis and satellite cell proliferation.
The capacity of muscle to grow or to regenerate after damage is provided by adult stem cells, so called satellite cells, which are located under the basement lamina of each myofiber. Upon activation satellite cells enter the cell cycle, proliferate and differentiate into myoblasts, which fuse to injured myofibers or form new fibers. These processes are tightly controlled by many growth factors.
Here we investigate the role of bone morphogenetic proteins (BMPs) during satellite cell differentiation. Unlike the myogenic C2C12 cell line, primary satellite cells do not differentiate into osteoblasts upon BMP signaling. Instead BMP signaling inhibits myogenic differentiation of primary satellite cells ex vivo. In contrast, inhibition of BMP signaling results in cell cycle exit, followed by enhanced myoblast differentiation and myotube formation. Using an in vivo trauma model we demonstrate that satellite cells respond to BMP signals during the regeneration process. Interestingly, we found the BMP inhibitor Chordin upregulated in primary satellite cell cultures and in regenerating muscles. In both systems Chordin expression follows that of Myogenin, a marker for cells committed to differentiation.
Our data indicate that BMP signaling plays a critical role in balancing proliferation and differentiation of activated satellite cells and their descendants. Initially, BMP signals maintain satellite cells descendants in a proliferating state thereby expanding cell numbers. After cells are committed to differentiate they upregulate the expression of the BMP inhibitor Chordin thereby supporting terminal differentiation and myotube formation in a negative feedback mechanism.
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.
Activation of adult myoblasts called satellite cells during muscle degeneration is an important aspect of muscle regeneration. Satellite cells are believed to be the only myogenic stem cells in adult skeletal muscle and the source of regenerating muscle fibers. Upon activation, satellite cells proliferate, migrate to the site of degeneration, and become competent to fuse and differentiate. We show here that the transcription factor polyomavirus enhancer activator 3 (PEA3) is expressed in adult myoblasts in vitro when they are proliferative and during the early stages of differentiation. Overexpression of PEA3 accelerates differentiation, whereas blocking of PEA3 function delays myoblast fusion. PEA3 activates gene expression following binding to the ets motif most efficiently in conjunction with the transcription factor myocyte enhancer factor 2 (MEF2). In vivo, PEA3 is expressed in satellite cells only after muscle degeneration. Taken together, these results suggest that PEA3 is an important regulator of activated satellite cell function.
Growth, repair, and regeneration of adult skeletal muscle depends on the persistence of satellite cells: muscle stem cells resident beneath the basal lamina that surrounds each myofiber. However, how the satellite cell compartment is maintained is unclear. Here, we use cultured myofibers to model muscle regeneration and show that satellite cells adopt divergent fates. Quiescent satellite cells are synchronously activated to coexpress the transcription factors Pax7 and MyoD. Most then proliferate, down-regulate Pax7, and differentiate. In contrast, other proliferating cells maintain Pax7 but lose MyoD and withdraw from immediate differentiation. These cells are typically located in clusters, together with Pax7−ve progeny destined for differentiation. Some of the Pax7+ve/MyoD−ve cells then leave the cell cycle, thus regaining the quiescent satellite cell phenotype. Significantly, noncycling cells contained within a cluster can be stimulated to proliferate again. These observations suggest that satellite cells either differentiate or switch from terminal myogenesis to maintain the satellite cell pool.
stem; skeletal muscle regeneration; Pax7; MyoD; myogenin
SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.
Skeletal muscle repair and regeneration requires the activity of satellite cells, a population of myogenic stem cells scattered throughout the tissue and activated to proliferate and differentiate in response to myotrauma or disease. While it seems likely that satellite cells would need to navigate local muscle tissue to reach damaged areas, relatively little data on such motility exist, and most studies have been with immortalized cell lines. We find that primary satellite cells are significantly more motile than myoblast cell lines, and that adhesion to laminin promotes primary cell motility more than fourfold over other substrates. Using timelapse videomicroscopy to assess satellite cell motility on single living myofibers, we have identified a requirement for the laminin-binding integrin α7β1 in satellite cell motility, as well as a role for hepatocyte growth factor in promoting directional persistence. The extensive migratory behavior of satellite cells resident on muscle fibers suggests caution when determining, based on fixed specimens, whether adjacent cells are daughters from the same mother cell. We also observed more persistent long-term contact between individual satellite cells than has been previously supposed, potential cell-cell attractive and repulsive interactions, and migration between host myofibers. Based on such activity, we assayed for expression of “pathfinding” cues, and found that satellite cells express multiple guidance ligands and receptors. Together, these data suggest that satellite cell migration in vivo may be more extensive than currently thought, and could be regulated by combinations of signals, including adhesive haptotaxis, soluble factors, and guidance cues. Stem Cells 2009;27:2527–2538
Adult stem cells; Cell migration; Muscle stem cells; Integrins; Satellite cells
Myofibers are the functional contractile units of skeletal muscle. Mononuclear satellite cells located between the basal lamina and the plasmalemma of the myofiber are the primary source of myogenic precursor cells in postnatal muscle. This chapter describes protocols used in our laboratory for isolation, culturing and immunostaining of single myofibers from mouse skeletal muscle. The isolated myofibers are intact and retain their associated satellite cells underneath the basal lamina. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. Myofibers are cultured in dishes coated with Vitrogen collagen and satellite cells remain associated with the myofibers undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL). Different from the FDB myofibers, the longer EDL myofibers tend to tangle and break if cultured together; therefore, EDL myofibers are cultured individually. These myofibers are cultured in dishes coated with Matrigel. The satellite cells initially remain associated with the myofiber and later migrate away to its vicinity, resulting in extensive cell proliferation and differentiation. These culture protocols allow studies on the interplay between the myofiber and its associated satellite cells.
Satellite cells; skeletal muscle; myofiber isolation; single myofiber culture; flexor digitorum brevis; extensor digitorum longus; mouse; Vitrogen collagen; Matrigel
Unlike normal mature limb skeletal muscles, in which satellite cells are quiescent unless the muscle is injured, satellite cells in mammalian adult extraocular muscles (EOM) are chronically activated. This is evidenced by hepatocyte growth factor, the myogenic regulatory factor, Pax-7, and the cell-cycle marker, Ki-67, localized to the satellite cell position using serial sections and the positional markers laminin and dystrophin. Bromodeoxyuridine (brdU) labeling combined with dystrophin immunostaining showed brdU-positive myonuclei, presumably the result of fusion of activated satellite cells into existing myofibers. One new myonucleus was added to every 1000 myofibers in cross-section using a 12-hour brdU-labeling paradigm. The EOM thus appear to retain a stable nuclear population by an opposing process of apoptosis that results in myonuclear removal as visualized by terminal deoxynucleotidyltransferase-mediated nick end labeling (TUNEL). Activated caspase-3 was present in localized cytoplasmic domains extending from 10 to 210 μm within individual myofibers, suggesting segmental cytoplasmic reorganization. Understanding the cellular mechanisms that maintain this process of continuous myonuclear addition and removal in normal adult EOM may suggest new hypotheses to explain the preferential involvement or sparing of these muscles in skeletal muscle disease.
apoptosis; extraocular muscles; mitosis; myofibers; satellite cells; ANOVA, analysis of variance; brdU, bromodeoxyuridine; EOM, extraocular muscles; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
Satellite cells, as stem cells of adult skeletal muscle, are tightly associated with the differentiated muscle fibers and remain quiescent in the absence of muscle damage. In response to an injury, the quiescent satellite cell is activated by soluble factors, including FGFs released from injured myofibers. Using immunostaining, we here first show that TRPC1 channels are highly expressed in satellite cells attached to muscle fibers. Since CD34, a traditional stem cell marker, was recently found to be expressed in skeletal muscle satellite cells we labeled living satellite cells in their physiological niche associated with host FDB fibers using anti-CD34-FITC antibody. We then monitored intra-cellular calcium in anti-CD34-FITC labeled satellite cells attached to muscle fibers using the calcium sensitive dye X rhod-1 which has little fluorescence cross talk with FITC. FGF2 increased intracellular calcium in satellite cells, which was antagonized by the TRPC channel blocker SKF 96365. Immunostaining showed that NFATc3 is highly expressed in satellite cells, but not in host FDB fibers. Elevation of intracellular calcium by FGF2 is accompanied by nuclear translocation of NFATc3 and NFATc2 and by an increase in the number of MyoD positive cells per muscle fiber, both of which were attenuated by TRPC blocker SKF 96365. Our results suggest a novel pathway of satellite cell activation where FGF2 enhances calcium influx through a TRPC channel, and the increased cytosolic calcium leads to both NFATc3 and NFATc2 nuclear translocation and enhanced number of MyoD positive satellite cells per muscle fiber.
TRPC; FGF2; satellite cell; calcium; NFATc3; NFATc2
Skeletal muscle injury activates adult myogenic stem cells, known as satellite cells, to initiate proliferation and differentiation to regenerate new muscle fibers. The skeletal muscle–specific microRNA miR-206 is upregulated in satellite cells following muscle injury, but its role in muscle regeneration has not been defined. Here, we show that miR-206 promotes skeletal muscle regeneration in response to injury. Genetic deletion of miR-206 in mice substantially delayed regeneration induced by cardiotoxin injury. Furthermore, loss of miR-206 accelerated and exacerbated the dystrophic phenotype in a mouse model of Duchenne muscular dystrophy. We found that miR-206 acts to promote satellite cell differentiation and fusion into muscle fibers through suppressing a collection of negative regulators of myogenesis. Our findings reveal an essential role for miR-206 in satellite cell differentiation during skeletal muscle regeneration and indicate that miR-206 slows progression of Duchenne muscular dystrophy.
Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.
Skeletal muscle; satellite cells; stem cells; collagen; Matrigel; myofiber isolation; flexor digitorum brevis; extensor digitorum longus; diaphragm; masseter; extraocular; mouse; immunostaining; Pax7
We previously reported that leukocyte specific β2 integrins contribute to hypertrophy after muscle overload in mice. Because intercellular adhesion molecule-1 (ICAM-1) is an important ligand for β2 integrins, we examined ICAM-1 expression by murine skeletal muscle cells after muscle overload and its contribution to the ensuing hypertrophic response. Myofibers in control muscles of wild type mice and cultures of skeletal muscle cells (primary and C2C12) did not express ICAM-1. Overload of wild type plantaris muscles caused myofibers and satellite cells/myoblasts to express ICAM-1. Increased expression of ICAM-1 after muscle overload occurred via a β2 integrin independent mechanism as indicated by similar gene and protein expression of ICAM-1 between wild type and β2 integrin deficient (CD18-/-) mice. ICAM-1 contributed to muscle hypertrophy as demonstrated by greater (p<0.05) overload-induced elevations in muscle protein synthesis, mass, total protein, and myofiber size in wild type compared to ICAM-1-/- mice. Furthermore, expression of ICAM-1 altered (p<0.05) the temporal pattern of Pax7 expression, a marker of satellite cells/myoblasts, and regenerating myofiber formation in overloaded muscles. In conclusion, ICAM-1 expression by myofibers and satellite cells/myoblasts after muscle overload could serve as a mechanism by which ICAM-1 promotes hypertrophy by providing a means for cell-to-cell communication with β2 integrin expressing myeloid cells.
Repair of adult skeletal muscle depends on satellite cells, myogenic stem cells located between the basal lamina and the plasmalemma of the myofiber. Standardized protocols for the isolation and culture of satellite cells are key tools for understanding cell autonomous and extrinsic factors that regulate their performance. Knowledge gained from such studies can contribute important insights to developing strategies for the improvement of muscle repair following trauma and in muscle wasting disorders. This chapter provides an introduction to satellite cell biology and further describes the basic protocol used in our laboratory to isolate and culture satellite cells from adult skeletal muscle. The cell culture conditions detailed herein support proliferation and differentiation of satellite cell progeny and the development of reserve cells, which are thought to reflect the in vivo self-renewal ability of satellite cells. Additionally, this chapter describes our standard immunostaining protocol that allows the characterization of satellite cell progeny by the temporal expression of characteristic transcription factors and structural proteins associated with different stages of myogenic progression. While emphasis is given here to the isolation and characterization of satellite cells from mouse hindlimb muscles, the protocols are suitable for other muscle types (such as diaphragm and extraocular muscles) and for muscles from other species, including chicken and rat. Altogether, the basic protocols described are straightforward and facilitate the study of diverse aspects of skeletal muscle stem cells.
Skeletal muscle; satellite cell; stem cell; myogenesis; Pronase; gelatin; Matrigel; Pax7; MyoD; myogenin
Skeletal muscle is formed during development by the progressive specification, proliferation, migration, and fusion of myoblasts to form terminally differentiated, contractile, highly patterned myofibers. Skeletal muscle is repaired or replaced postnatally by a similar process, involving a resident myogenic stem cell population referred to as satellite cells. In both cases, the activity of the myogenic precursor cells in question is regulated by local signals from the environment, frequently involving other, non-muscle cell types. However, while the majority of studies on muscle development were done in the context of the whole embryo, much of the current work on muscle satellite cells has been done in vitro, or on satellite cell-derived cell lines. While significant practical reasons for these approaches exist, it is almost certain that important influences from the context of the injured and regenerating muscle are lost, while potential tissue culture artifacts are introduced. This review will briefly address extracellular influences on satellite cells in vivo and in vitro that would be expected to impinge on their activity.
MUSCLE REGENERATION; SATELLITE CELLS; STEM CELLS