A role for Galgt1 in skeletal muscle regeneration
© Singhal and Martin; licensee BioMed Central. 2015
Received: 12 August 2014
Accepted: 22 December 2014
Published: 27 January 2015
Cell surface glycans are known to play vital roles in muscle membrane stability and muscle disease, but to date, roles for glycans in muscle regeneration have been less well understood. Here, we describe a role for complex gangliosides synthesized by the Galgt1 gene in muscle regeneration.
Cardiotoxin-injected wild type (WT) and Galgt1 −/− muscles, and mdx and Galgt1 −/− mdx muscles, were used to study regeneration in response to acute and chronic injury, respectively. Muscle tissue was analyzed at various time points for morphometric measurements and for gene expression changes in satellite cell and muscle differentiation markers by quantitative real-time polymerase chain reaction (qRT-PCR). Primary cell cultures were used to measure growth rate and myotube formation and to identify Galgt1 expression changes after cardiotoxin by fluorescence-activated cell sorting (FACS). Primary cell culture and tissue sections were also used to quantify satellite cell apoptosis.
A query of a microarray data set of cardiotoxin-induced mouse muscle gene expression changes identified Galgt1 as the most upregulated glycosylation gene immediately after muscle injury. This was validated by qRT-PCR as a 23-fold upregulation in Galgt1 expression 1 day after cardiotoxin administration and a 16-fold upregulation in 6-week-old mdx muscles. These changes correlated with increased expression of Galgt1 protein and GM1 ganglioside in mononuclear muscle cells. In the absence of Galgt1, cardiotoxin-induced injury led to significantly reduced myofiber diameters after 14 and 28 days of regeneration. Myofiber diameters were also significantly reduced in Galgt1-deficient mdx mice compared to age-matched mdx controls, and this was coupled with a significant increase in the loss of muscle tissue. Cardiotoxin-injected Galgt1 −/− muscles showed reduced gene expression of the satellite cell marker Pax7 and increased expression of myoblast markers MyoD, Myf5, and Myogenin after injury along with a tenfold increase in apoptosis of Pax7-positive muscle cells. Cultured primary Galgt1 −/− muscle cells showed a normal growth rate but demonstrated premature fusion into myofibers, resulting in an overall impairment of myofiber formation coupled with a threefold increase in muscle cell apoptosis.
These experiments demonstrate a role for Galgt1 in skeletal muscle regeneration and suggest that complex gangliosides made by Galgt1 modulate the survival and differentiation of satellite cells.
KeywordsDuchenne muscular dystrophy Muscle regeneration Ganglioside Satellite cell Apoptosis
Skeletal muscle displays a remarkable potential to regenerate after injury, and satellite cells, the predominant stem cells in adult skeletal muscle, play an essential role in this process [1-3]. Satellite cells normally reside in the niche between the skeletal myofiber membrane and its surrounding basal lamina , which is composed predominantly of laminin 211 (α2, β1, γ1) and collagen IV (α1, α2) [4-8]. When a myofiber is injured beyond repair due to membrane rupture, mechanical insult, or aberrant calcium homeostasis, the resulting necrosis of skeletal myofibers is coupled with myofiber clearance by invading immune cells and the stimulation of asymmetric satellite cell division to generate myoblasts that will ultimately fuse to form new skeletal myofibers and new satellite cells that will replenish the muscle stem cell niche. In response to acute injury, newly regenerated contractile skeletal myofibers are formed over a period of about 2 weeks [2,9-12]. Certain compounds, including the snake peptide cardiotoxin (CTX), can mimic the aberrant calcium homeostasis that occurs during muscle damage and be used to synchronize the induction of muscle regeneration [13,14].
In the muscular dystrophies, impaired regeneration, coupled with chronic muscle damage, can lead to muscle wasting, which involves the loss of muscle tissue and its replacement with extracellular matrix or fat . Muscle wasting in the muscular dystrophies drives the development of muscle weakness and ultimately death. Muscle wasting, which likely occurs due to an imbalance between muscle degeneration and regeneration, is a hallmark of Duchenne muscular dystrophy (DMD). DMD is caused by mutations or deletions in the dystrophin (DMD) gene that give rise to an absence of dystrophin protein expression in skeletal and cardiac muscle [16,17]. The most commonly used DMD animal model is the mdx mouse [18-20]. The mdx mouse muscle undergoes chronic cycles of degeneration coupled with muscle regeneration. These cycles begin at about 3 weeks of age and peak at 4–6 weeks of age, when a severe period of muscle damage occurs. This is followed by a more subdued, but chronic, disease process throughout the remainder of the mouse’s lifespan . Introduction of secondary gene deletion or transgene overexpression has implicated a number of additional genes, including utrophin, telomerase, integrin α7, sarcospan, Galgt2, and Cmah, amongst others, as modulators of mdx disease severity [22-32].
The important cellular mechanisms involved in muscle regeneration, satellite cell activation and differentiation, myoblast migration and fusion, and myofiber growth are mediated by signaling molecules including Akt and Wnt kinases, mTOR, Notch receptors, and their down-stream signals [33-37]. While at least some cell signaling processes at the membrane involved in muscle regeneration are well described, roles for cell surface glycans, with the exception of syndecan proteoglycans and glycosaminoglycan endosulfatases [38-44], remain poorly understood. This stands in contrast with the large number of studies demonstrating roles for glycans in mediating muscle membrane stability and causing muscle disease [31,32,45-47]. Complex gangliosides are good candidates for mediating aspects of muscle regeneration. Gangliosides have been shown to be able to modulate cell-cell interactions, cell signal transduction, and the activity of membrane ion channels in numerous cell types, including myoblasts [48-55]. GM2, GD2, and all down-stream complex gangliosides, including GM1, require the enzymatic activity encoded by the Galgt1 gene for their production [56,57]. Galgt1 has clear roles in mediating the binding of complex gangliosides to endogenous sialic acid-binding lectins that are known to control important aspects of nervous system development, for example axon guidance, axon stability, and axonal regeneration [53,57-60]. In this study, we demonstrate dynamic and pronounced changes in Galgt1 expression during skeletal muscle regeneration and demonstrate a role for this gene in the regeneration process.
Anti-ganglioside GM1 antibody was purchased from Millipore (345757). Rabbit polyclonal antibody to Galgt1 peptide CQVRAVDLTKAFDAEE was made in our lab by immunizing rabbits with KLH-conjugated peptide, after which antibody was purified over peptide-conjugated resin as previously described . Anti-mouse Pax7 antibody was a gift from Dr. Michael Rudnicki (Ottawa Health Research Institute). Anti-mouse integrin α7 conjugated to fluorescein isothiocyanate (FITC) was purchased from MBL International (K0046-4) and R & D Systems (FAB3518F). Anti-mouse CD11b conjugated to FITC and Rat anti-Ertr7 were gifts from Dr. Jill Rafael-Fortney (The Ohio State University). Rat anti-mouse Ly-6A/E conjugated to FITC (Sca1, 553335), rat anti-mouse CD45 conjugated to PE-Cy7 (552848), rat anti-mouse CD31 conjugated to APC (551262), and rat anti-mouse CD16/CD32 Fc block (553142) were purchased from BD Biosciences. All secondary antibodies conjugated to fluorophores were purchased from Jackson ImmunoResearch. Rhodamine-conjugated α-bungarotoxin was purchased from Life Technologies. Sections from normal human and Duchenne muscular dystrophy muscle biopsies from clinical specimens archived as part of the United Dystrophinopathy Project were obtained in accordance with approval from the Institutional Review Board.
All animal experiments were conducted after approval from the Institutional Animal Use and Care Committee (IACUC) at The Research Institute at Nationwide Children’s Hospital. Mice lacking Galgt1 (Galgt1 −/− ) were obtained from Consortium for Functional Glycomics (www.functionalglycomics.org) and were originally made by Proia and colleagues [57,62]. mdx and wild type (C57Bl/6) mice were purchased from Jackson Laboratories. Galgt1 −/− mdx mice were obtained by interbreeding of Galgt1 −/− mice with mdx mice. Six-week-old, 3-month-old, and 6-month-old animals were used for wild type (WT), Galgt1 −/− , mdx, and Galgt1 −/− mdx experiments as indicated.
Cardiotoxin-induced muscle regeneration
Two-month-old animals were used for cardiotoxin injection experiments. Cardiotoxin, from Naja mossambica mossambica venom, was purchased from Sigma-Aldrich (C9759). It was diluted to a 10-μM concentration in phosphate-buffered saline (PBS) and injected intramuscularly into the gastrocnemius, tibialis anterior, or quadriceps muscles in a volume of 50 μl (gastroc or quad) or 25 μl (tibialis anterior (TA)). Muscles were collected 1, 4, 7, 14, and 28 days after cardiotoxin injection, snap frozen in liquid nitrogen-cooled isopentane, and sectioned at 8 μm in cross section on a cryostat for histological analysis. Mock-injected muscles from control animals were harvested at these same time points as controls.
Muscles were dissected from tendon to tendon, mounted on OCT, and snap frozen in liquid nitrogen-cooled isopentane. Muscles were cross-sectioned on a cryostat at 8-μm thickness and mounted on slides for staining. For immunostaining, sections were blocked in 3 mg/ml bovine serum albumin (BSA) in PBS. Primary antibodies were diluted in 3 mg/ml BSA/PBS and sections incubated overnight at 4°C. After washing in PBS, sections were stained with a secondary antibody, added at a dilution of 1:250, for 1 h. After washing, slides were mounted in Prolong Gold Antifade with 4’,6-diamidino-2-phenylindole (DAPI) and visualized on a Zeiss Axiophot epifluorescence microscope using fluorescein-, rhodamine-, or DAPI-specific optics. For hematoxylin and eosin (H & E) staining, cryostat-cut 8-μm muscle cross sections were first fixed in 10% neutral-buffered formalin. After washing in tap water, slides were stained with Gill’s 3 Hematoxylin for 2 min, washed in tap water, then bluing agent, and again in tap water. Sections were then stained in eosin for 1 min and excess stain removed by dipping in 30% ethanol, after which the sections were dehydrated in 70%, 90%, 95%, and 100% ethanol. The sections were cleared in xylene and mounted in the xylene-based mounting media Cytoseal.
Quantification of muscle morphometry
Myofiber diameter and central nuclei measurements were carried out and quantified using Zeiss AxioVision Rel.4.8 software as previously described . H & E images were photographed at × 20 magnification for such measurements. Briefly, muscle sections from at least four separate planes of the injected muscle were counted in total and averaged to create single average data points for each measure, and the average of these measures from all animals per time point or condition was then used to determine the average myofiber diameter, the percentage of myofibers with central nuclei, the number of myofibers per unit area, or the percentage of non-muscle area. For CTX experiments comparing wild type and Galgt1 −/− muscles, and for experiments comparing mdx and Galgt1 −/− mdx muscles, six muscles were averaged per condition, with 1,600–3,600 total myofibers counted per condition.
Fluorescence Activated Cell Sorting (FACS)
The limb muscles of three WT mice were injected with 10-μM cardiotoxin and compared to WT animals injected with an equivalent volume of PBS. One day after injection, the muscles were removed, minced, and digested with 5 mg/ml collagenase IV and 1.2 U/ml Dispase for 30 min at 37°C. The reaction was stopped by dilution with DMEM, filtration of cells through a 70-μm membrane and then a 40-μm cell strainer, and centrifugation in horse serum to remove enzymes from the cells. Cells were stained after washing in 0.5% BSA/PBS. Cells were blocked with Mouse BD Fc Block (BD Pharmingen 553141) at a concentration of 2 μl/106 cells on ice for 10 min followed by staining with fluorophore-conjugated primary antibody at a concentration of 2 μl/106 cells for 10 min on ice. Cells were then washed, resuspended, and filtered in fluorescence-activated cell sorting (FACS) tubes. Propidium iodide (PI, 2 μg/ml) was added just before sorting to identify dead cells. Cells were first gated based on side scatter and forward scatter then based on the height and width. PI-positive (dead) cells were removed prior to further analysis. Cells were sorted for Sca1+ and Sca1− fractions and each fraction was further sorted for CD31+, CD45+, and CD31−CD45−. Fractions were collected and analyzed for Galgt1 mRNA using quantitative real-time polymerase chain reaction (qRT-PCR) as previously described . FACS analysis was repeated with gating for CD31−APC and CD45−PE Cy7 and the CD31−CD45− fractions, with sorting for integrin α7 against side scatter. The integrin α7+CD31−CD45−, integrin α7−CD31−CD45−, integrin α7−CD31+CD45−, and integrin α7−CD31−CD45+ fractions were collected and analyzed for Galgt1 mRNA using qRT-PCR as before .
Semi-quantitative real-time polymerase chain reaction (qRT-PCR)
Gastrocnemius muscles were homogenized in TRIzol (Invitrogen) and total RNA was extracted and purified using either RNeasy Mini or Micro kits (Qiagen) according to the manufacturer’s instructions. The quality of RNA was determined by electrophoresis using 6000 Nano LabChip kit on Bioanalyzer 2100 (Agilent). The samples without RNA degradation were used to synthesize cDNA using High Capacity Reverse Transcriptase kit (Applied Biosystems). Taqman ABI 7500 sequence detection system (Applied Biosystems) was used for real-time qPCR measurements on the samples using the delta-delta CT method . qRT-PCR probes for Galgt1 and 18S RNA have been described in previous publications [32,65]. Primers for dystrophin (Mm01216926_m1), Pax7 (Mm00834079_m1), Myf5 (Mm00435125_m1), MyoD (Mm00440387_m1), Myogenin (Mm00446194_m1), and Myh3 (Mm01332463_m1) were purchased from Applied Biosystems. 18S was used as an internal control. Measures shown are replicates of three to four muscles per condition.
Terminal deoxynucleotidyl transferase (TUNEL) staining was performed according to the manufacturer’s instructions using the Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay (Life Technologies, C10245). For Pax7-TUNEL co-staining, TUNEL staining was done prior to Pax7 staining, which was done as previously described . Replicates shown are averages of six muscles per condition for tissue staining or of three to six replicates per condition for cell culture experiments.
Muscle cell isolation
Gastrocnemius, quadriceps, tibialis anterior (TA), and triceps were dissected from WT and Galgt1 −/− mice as previously described . The skeletal muscles were collected in ice-cold sterile PBS and dissected muscles were minced in a sterile laminar-flow hood using aseptic conditions. The minced muscles were digested in a solution containing 1.2 U/ml Dispase and 5 mg/ml collagenase IV. The minced tissue was incubated with enzymes at 37°C for 30 min with intermittent pipetting every 10 min, followed by addition of DMEM/F12 media + 10% heat-inactivated horse serum + 1% penicillin-streptomycin solution. The solution was then filtered through 70-μm cell strainer and then a 40-μm cell strainer. Cells were then centrifuged at 1,500 rpm at 4°C for 10 min. The cell pellet was resuspended in 0.5% BSA/PBS and overlayed on 100% heat-inactivated horse serum and centrifuged at 220 × g at 4°C for 10 min. The pellet was then washed in 0.5% BSA/PBS and centrifuged at 1,500 rpm at 4°C for 10 min and resuspended in growth media (DMEM/F-12 + 10% FBS + 4% chick embryo extract + 1% P/S). Cells were pre-plated on uncoated tissue culture plates, with muscle-cell-enriched supernatant removed after fibroblasts were allowed to adhere for 20 min. The pre-plating step was repeated if needed to obtain pure muscle cell cultures. Purified muscle cells were then plated on 0.1% collagen-1-coated tissue culture plates. As in our previous studies , such cultures typically showed an excess of 90% positive staining for c-met, a muscle cell marker.
Muscle cell growth rate and fusion assays
To measure the growth rate of isolated muscle cells, cells extracted and purified from WT and Galgt1 −/− skeletal muscles were grown in the growth media (DMEM/F-12 + 10% FBS + 4% chick embryo extract + 1% P/S) and were counted at three different time points (1, 2, and 3 days post-plating). For the muscle cell fusion assay, cells were grown in the growth media till they reached 100% confluence and were then switched to fusion media (×1 DMEM/F-12 + 2% horse serum + 1% P/S). Skeletal myofibers were analyzed at four time points—Day 0 (just before addition of fusion media), Day 1 (1 day after addition of fusion media), Day 3, and Day 6. Sixteen equivalent fields of view were evaluated per well with at least three wells evaluated per time point and per genotype. Data shown are averages of six experiments per condition, normalized to baseline cell number on Day 0 in each instance.
All determinations of significance were done by ANOVA with post hoc Bonferroni analysis and/or unpaired t-tests. A P value of less than or equal to 0.05 was considered significant.
Galgt1 and GM1 expression are increased in regenerating skeletal muscle
We next stained skeletal muscles taken at different days after CTX injection with an antibody to Galgt1 protein or with an antibody to GM1, a complex ganglioside that requires Galgt1 activity for its expression  (Figure 1C). GM1 staining was chosen to reflect Galgt1 activity as antibodies to GM2 and GD2 can also cross-react with glycans made by Galgt2, another glycosyltransferase in the Galgt1 gene family, while antibodies to GM1 do not . As shown previously, antibodies to Galgt1 protein and GM1 ganglioside both stained the neuromuscular junction (NMJ) in control muscle (uninjected Day 0) . This was confirmed by co-staining of NMJs with rhodamine α bungarotoxin (data not shown). On Day 1 after CTX injection, Galgt1 and GM1 staining were both increased on intramuscular mononuclear cells. This staining of mononuclear cells was also present on Day 4 and Day 7, though here, myofiber staining was also evident. By Day 14 and Day 28, both GM1 and Galgt1 staining were primarily localized to regions including the NMJ, just as at Day 0 (Figure 1C). Thus, Galgt1 protein and GM1, a ganglioside product of Galgt1 activity, were both transiently elevated in muscles after CTX addition, much as would be expected given the dramatic increase in Galgt1 gene expression.
Galgt1 and GM1 expression are increased in dystrophic skeletal muscle
Galgt1-deficient muscles show altered regeneration in acute and chronic muscle injury models
Galgt1 is overexpressed in muscle cells following muscle injury
Regenerating Galgt1 −/− muscle has altered expression of satellite and myoblast cell markers
Myoblast cell fusion is accelerated in time but reduced in extent in the absence of Galgt1
Increased apoptosis in satellite cells in Galgt1 −/− muscle in vitro and in vivo
Here we have described a new role for complex gangliosides made by Galgt1, a gene encoding a β1,4GalNAc glycolipid glycosyltransferase, in muscle regeneration. Galgt1 expression was dramatically elevated in the first few days after induction of acute muscle regeneration by cardiotoxin and was elevated in mdx muscles, which undergo chronic regeneration as well. During regeneration, elevated Galgt1 expression was identified in muscle cells, but not in intramuscular fibroblasts, immune cells, or mesenchymal stem cells. Moreover, studies of muscle cells isolated from normal and Galgt1-deficient muscles demonstrated that Galgt1 is required for proper muscle cell differentiation and survival; regenerating Galgt1 −/− muscles showed a deficit in gene expression for the satellite cell marker Pax7 and increased gene expression for myoblast markers MyoD, Myf5, and Myogenin, cultured muscle cells from Galgt1 −/− mice showed premature fusion into myofibers which resulted in less robust myofiber formation over time, and regenerating muscles from Galgt1 −/− mice showed an order of magnitude increase in the apoptosis of Pax7-positive cells during the regeneration process. The end result, both for acute and chronic muscle regeneration models, was a deficit in regeneration with increased populations of smaller caliber myofibers. In Galgt1 −/− dystrophin-deficient (mdx) muscles, this was also coupled with increased loss of muscle tissue. Further work will be needed to more carefully analyze the possible mechanisms of action, but this study clearly shows that Galgt1 plays a role in the regenerative process in skeletal muscle and identifies complex gangliosides made by Galgt1 as potential targets for therapeutic interventions to stimulate aspects of muscle regeneration.
Our findings are consistent with previous studies implicating other types of glycans, including glycosaminoglycans and N-linked glycans, in satellite cell function during muscle regeneration. The results reported here showing reduced Pax7 expression and increased MyoD expression in Galgt1 −/− muscle, for example, are similar to the studies of Denis and colleagues on Mgat5-deficient mice, which had reduced satellite cell number with a bias towards increased MyoD expression at the expense of Pax7 . Mgat5 affects the formation of β1,6-GlcNAc-branched N-linked glycans and would not directly affect the biosynthesis of gangliosides made by Galgt1; nevertheless, it is possible that these pathways might intersect through a less-direct mechanism. Emerson and colleagues have shown that inhibition of heparin sulfate 6-O-endosulfatases (Sulfs) in Sulf1/Sulf2 double knockout mice increases the number of Pax7-positive satellite cells as well as increasing fibroblast growth factor (FGF) signaling . Ai and colleagues have further shown a complex differential regulation of canonical and non-canonical Wnt signaling pathways in Sulf1/2 double mutant mice, with a bias towards premature differentiation of satellite cells and formation of myofibers with smaller overall area after cardiotoxin-induced injury , akin to what we observed in these studies in Galgt1-deficient mice. Work by Olwin, Brandan, Velleman, and others suggests that mice or cells lacking the proteoglycans Syndecan-3 or Syndecan-4 show impairment of satellite cell division and/or differentiation, including altered induction of MyoD expression and localization as well as impaired myofiber formation [38,39,42,71,72]. These syndecans could impact signaling of multiple regeneration signaling pathways, including NotchR, FGF, and Wnt . Gangliosides, through their ability to orient proteins in lipid rafts on the cell membrane, might interact with any or all of these other glycan-dependent pathways to regulate their ability to govern various signaling functions in muscle cells. Future work will be required to delineate the possible pathways by which complex gangliosides, N-linked glycans, and glycosaminoglycans might intersect to modulate these vital regenerative signaling pathways.
A number of studies support such roles for complex gangliosides in muscle cell function. Gangliosides are regulated differentially through the various stages of muscle cell differentiation [73-75]. Since GD1a and GM1 expression are both increased in cultured myoblasts just before fusion , and as they both require Galgt1 for their biosynthesis , their elevated expression likely arises from increased Galgt1 gene expression. GM1 has also been shown to be present on the lamellipodia of myoblasts, and affecting the clustering and dispersal of lipid rafts has been shown to be important for myoblast fusion [76-78]. Studies on the fusion-resistant fu-1 rat myoblast cell line have demonstrated that there is a consistent decrease in the levels of glycosphingolipids in these cells and that they have lost the ability to differentiate and express the markers of differentiation [75,79,80]. Glycosylation inhibitors like tunicamycin also impair myoblast cell fusion , and no myoblast fusion is observed in certain lectin-resistant muscle cell mutants [79,80,82]. Many other studies suggest that gangliosides can alter intracellular signaling by kinases [49,50,52,53,83,84], including kinases known to affect muscle regeneration such as Akt [85,86] and mTOR . Modulation of these and other signaling pathways would also impact muscle cell growth or differentiation. One current obstacle to muscle cell therapies is the inefficient muscle engraftment of transplanted stem or induced pluripotent stem cells (iPS) . This too would be an avenue worth exploring as regards Galgt1 function as a potential stimulator of muscle regeneration, as it appears unlikely that the logarithmic increase in Galgt1 gene expression seen in muscle cells a day after injury in vivo would be maintained once these cells were isolated and amplified for prolonged periods in culture.
This work demonstrates a role for Galgt1 during muscle regeneration. Galgt1 gene expression is dramatically elevated in both acute and chronic mouse models of muscle injury, as is GM1, a ganglioside that requires Galgt1 activity for its biosynthesis. Loss of Galgt1 in mice leads to impaired muscle regeneration in both acute and chronic muscle injury models, and loss of Galgt1 is coupled to premature differentiation and increased apoptosis of satellite cells in vitro and in vivo. These data support the notion that complex gangliosides generated by Galgt1 play modulatory roles during muscle regeneration and might be therapeutic targets in myopathies where muscle regeneration is impaired or insufficient.
Duchenne muscular dystrophy
Terminal deoxynucleotidyl transferase
Fluorescence-activated cell sorting
Induced pluripotent stem cells
Bovine serum albumin
Hematoxylin and eosin
quantitative Real-time Polymerase Chain Reaction
We would like to acknowledge and thank Michael Rudnicki (Ottawa Health Research Institute) for providing us with Pax7 antibody, Jill Rafael-Fortney (Ohio State University) for providing Ertr7 and CD11b antibody, Federica Montanaro, Christopher Penton, and Cynthia McAllister (The Research Institute at Nationwide Children’s Hospital) for their help with FACS analysis, and Kathleen Jacobs (UC San Diego) for assistance with Microarray data analysis. This work was supported by NIH grant R01 AR049722 to PTM.
- Bischoff R, Franzini-Armstrong C. Satellite cells. In: Engel AG, Franzini-Armstrong C, editors. Myology. 3rd ed. New York: McGraw-Hill; 2004. p. 66.Google Scholar
- Wang YX, Rudnicki MA. Satellite cells, the engines of muscle repair. Nat Rev Mol Cell Biol. 2012;13:127–33.Google Scholar
- Seale P, Sabourin LA, Girgis-Gabardo A, Mansouri A, Gruss P, Rudnicki MA. Pax7 is required for the specification of myogenic satellite cells. Cell. 2000;102:777–86.View ArticlePubMedGoogle Scholar
- Chiu AY, Sanes JR. Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle. Dev Biol. 1984;103:456–67.View ArticlePubMedGoogle Scholar
- Engvall E, Earwicker D, Haaparanta T, Ruoslahti E, Sanes JR. Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits. Cell Regul. 1990;1:731–40.PubMed CentralPubMedGoogle Scholar
- Patton BL, Miner JH, Chiu AY, Sanes JR. Distribution and function of laminins in the neuromuscular system of developing, adult, and mutant mice. J Cell Biol. 1997;139:1507–21.View ArticlePubMed CentralPubMedGoogle Scholar
- Patton BL, Connoll AM, Martin PT, Cunningham JM, Mehta S, Pestronk A, et al. Distribution of ten laminin chains in dystrophic and regenerating muscles. Neuromuscul Disord. 1999;9:423–33.View ArticlePubMedGoogle Scholar
- Singhal N, Martin PT. Role of extracellular matrix proteins and their receptors in the development of the vertebrate neuromuscular junction. Dev Neurobiol. 2011;71:982–1005.View ArticlePubMed CentralPubMedGoogle Scholar
- Rudnicki MA, Le Grand F, McKinnell I, Kuang S. The molecular regulation of muscle stem cell function. Cold Spring Harb Symp Quant Biol. 2008;73:323–31.View ArticlePubMedGoogle Scholar
- Zammit PS, Partridge TA, Yablonka-Reuveni Z. The skeletal muscle satellite cell: the stem cell that came in from the cold. J Histochem Cytochem. 2006;54:1177–91.View ArticlePubMedGoogle Scholar
- Le Grand F, Rudnicki MA. Skeletal muscle satellite cells and adult myogenesis. Curr Opin Cell Biol. 2007;19:628–33.View ArticlePubMed CentralPubMedGoogle Scholar
- Camboni M, Hammond S, Martin LT, Martin PT. Induction of a regenerative microenvironment in skeletal muscle is sufficient to induce embryonal rhabdomyosarcoma in p53-deficient mice. J Pathol. 2012;226:40–9.View ArticlePubMedGoogle Scholar
- Ownby CL, Fletcher JE, Colberg TR. Cardiotoxin 1 from cobra (Naja naja atra) venom causes necrosis of skeletal muscle in vivo. Toxicon. 1993;31:697–709.View ArticlePubMedGoogle Scholar
- Lin Shiau SY, Huang MC, Lee CY. Mechanism of action of cobra cardiotoxin in the skeletal muscle. J Pharmacol Exp Ther. 1976;196:758–70.PubMedGoogle Scholar
- Emery AE. The muscular dystrophies. Lancet. 2002;359:687–95.View ArticlePubMedGoogle Scholar
- Deconinck N, Dan B. Pathophysiology of Duchenne muscular dystrophy: current hypotheses. Pediatr Neurol. 2007;36:1–7.View ArticlePubMedGoogle Scholar
- Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell. 1987;50:509–17.View ArticlePubMedGoogle Scholar
- Bulfield G, Siller WG, Wight PA, Moore KJ. X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc Natl Acad Sci U S A. 1984;81:1189–92.View ArticlePubMed CentralPubMedGoogle Scholar
- Tanabe Y, Esaki K, Nomura T. Skeletal muscle pathology in X chromosome-linked muscular dystrophy (mdx) mouse. Acta Neuropathol. 1986;69:91–5.View ArticlePubMedGoogle Scholar
- Cullen MJ, Jaros E. Ultrastructure of the skeletal muscle in the X chromosome-linked dystrophic (mdx) mouse. Comparison with Duchenne muscular dystrophy. Acta Neuropathol. 1988;77:69–81.View ArticlePubMedGoogle Scholar
- De la Porte S, Morin S, Koenig J. Characteristics of skeletal muscle in mdx mutant mice. Int Rev Cytol. 1999;191:99–148.View ArticlePubMedGoogle Scholar
- Deconinck AE, Rafael JA, Skinner JA, Brown SC, Potter AC, Metzinger L, et al. Utrophin-dystrophin-deficient mice as a model for Duchenne muscular dystrophy. Cell. 1997;90:717–27.View ArticlePubMedGoogle Scholar
- Grady RM, Teng H, Nichol MC, Cunningham JC, Wilkinson RS, Sanes JR. Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin: a model for Duchenne muscular dystrophy. Cell. 1997;90:729–38.View ArticlePubMedGoogle Scholar
- Rooney JE, Welser JV, Dechert MA, Flintoff-Dye NL, Kaufman SJ, Burkin DJ. Severe muscular dystrophy in mice that lack dystrophin and alpha7 integrin. J Cell Sci. 2006;119:2185–95.View ArticlePubMedGoogle Scholar
- Sacco A, Mourkioti F, Tran R, Choi J, Llewellyn M, Kraft P, et al. Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice. Cell. 2010;143:1059–71.View ArticlePubMed CentralPubMedGoogle Scholar
- Peter AK, Miller G, Crosbie RH. Disrupted mechanical stability of the dystrophin-glycoprotein complex causes severe muscular dystrophy in sarcospan transgenic mice. J Cell Sci. 2007;120:996–1008.View ArticlePubMedGoogle Scholar
- Rafael JA, Tinsley JM, Potter AC, Deconinck AE, Davies KE. Skeletal muscle-specific expression of a utrophin transgene rescues utrophin-dystrophin deficient mice. Nat Genet. 1998;19:79–82.View ArticlePubMedGoogle Scholar
- Tinsley J, Deconinck N, Fisher R, Kahn D, Phelps S, Gillis JM, et al. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat Med. 1998;4:1441–4.View ArticlePubMedGoogle Scholar
- Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ, Kaufman SJ. Enhanced expression of the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. J Cell Biol. 2001;152:1207–18.View ArticlePubMed CentralPubMedGoogle Scholar
- Nguyen HH, Jayasinha V, Xia B, Hoyte K, Martin PT. Overexpression of the cytotoxic T cell GalNAc transferase in skeletal muscle inhibits muscular dystrophy in mdx mice. Proc Natl Acad Sci U S A. 2002;99:5616–21.View ArticlePubMed CentralPubMedGoogle Scholar
- Martin PT, Xu R, Rodino-Klapac LR, Oglesbay E, Camboni M, Montgomery CL, et al. Overexpression of Galgt2 in skeletal muscle prevents injury resulting from eccentric contractions in both mdx and wild-type mice. Am J Physiol Cell Physiol. 2009;296:C476–88.View ArticlePubMed CentralPubMedGoogle Scholar
- Chandrasekharan K, Yoon JH, Xu Y, de Vries S, Camboni M, Janssen PM, et al. A human-specific deletion in mouse Cmah increases disease severity in the mdx model of Duchenne muscular dystrophy. Sci Transl Med. 2010;2:42ra54.View ArticlePubMed CentralPubMedGoogle Scholar
- Conboy IM, Rando TA. The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev Cell. 2002;3:397–409.View ArticlePubMedGoogle Scholar
- Luo D, Renault VM, Rando TA. The regulation of Notch signaling in muscle stem cell activation and postnatal myogenesis. Semin Cell Dev Biol. 2005;16:612–22.View ArticlePubMedGoogle Scholar
- von Maltzahn J, Bentzinger CF, Rudnicki MA. Wnt7a-Fzd7 signalling directly activates the Akt/mTOR anabolic growth pathway in skeletal muscle. Nat Cell Biol. 2012;14:186–91.View ArticleGoogle Scholar
- Bentzinger CF, von Maltzahn J, Dumont NA, Stark DA, Wang YX, Nhan K, et al. Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength. J Cell Biol. 2014;205:97–111.View ArticlePubMed CentralPubMedGoogle Scholar
- von Maltzahn J, Chang NC, Bentzinger CF, Rudnicki MA. Wnt signaling in myogenesis. Trends Cell Biol. 2012;22:602–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Cornelison DD, Filla MS, Stanley HM, Rapraeger AC, Olwin BB. Syndecan-3 and syndecan-4 specifically mark skeletal muscle satellite cells and are implicated in satellite cell maintenance and muscle regeneration. Dev Biol. 2001;239:79–94.View ArticlePubMedGoogle Scholar
- Cornelison DD, Wilcox-Adelman SA, Goetinck PF, Rauvala H, Rapraeger AC, Olwin BB. Essential and separable roles for Syndecan-3 and Syndecan-4 in skeletal muscle development and regeneration. Genes Dev. 2004;18:2231–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Tanaka KK, Hall JK, Troy AA, Cornelison DD, Majka SM, Olwin BB. Syndecan-4-expressing muscle progenitor cells in the SP engraft as satellite cells during muscle regeneration. Cell Stem Cell. 2009;4:217–25.View ArticlePubMed CentralPubMedGoogle Scholar
- Pisconti A, Cornelison DD, Olguin HC, Antwine TL, Olwin BB. Syndecan-3 and Notch cooperate in regulating adult myogenesis. J Cell Biol. 2010;190:427–41.View ArticlePubMed CentralPubMedGoogle Scholar
- Pisconti A, Bernet JD, Olwin BB. Syndecans in skeletal muscle development, regeneration and homeostasis. Muscles Ligaments Tendons J. 2012;2:1–9.PubMed CentralPubMedGoogle Scholar
- Langsdorf A, Do AT, Kusche-Gullberg M, Emerson Jr CP, Ai X. Sulfs are regulators of growth factor signaling for satellite cell differentiation and muscle regeneration. Dev Biol. 2007;311:464–77.View ArticlePubMedGoogle Scholar
- Tran TH, Shi X, Zaia J, Ai X. Heparan sulfate 6-O-endosulfatases (Sulfs) coordinate the Wnt signaling pathways to regulate myoblast fusion during skeletal muscle regeneration. J Biol Chem. 2012;287:32651–64.View ArticlePubMed CentralPubMedGoogle Scholar
- Godfrey C, Foley AR, Clement E, Muntoni F. Dystroglycanopathies: coming into focus. Curr Opin Genet Dev. 2011;21:278–85.View ArticlePubMedGoogle Scholar
- Stalnaker SH, Aoki K, Lim JM, Porterfield M, Liu M, Satz JS, et al. Glycomic analyses of mouse models of congenital muscular dystrophy. J Biol Chem. 2011;286:21180–90.View ArticlePubMed CentralPubMedGoogle Scholar
- Inamori K, Yoshida-Moriguchi T, Hara Y, Anderson ME, Yu L, Campbell KP. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE. Science. 2012;335:93–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Furukawa K, Aixinjueluo W, Kasama T, Ohkawa Y, Yoshihara M, Ohmi Y, et al. Disruption of GM2/GD2 synthase gene resulted in overt expression of 9-O-acetyl GD3 irrespective of Tis21. J Neurochem. 2008;105:1057–66.View ArticlePubMed CentralPubMedGoogle Scholar
- Julien S, Bobowski M, Steenackers A, Le Bourhis X, Delannoy P. How do gangliosides regulate RTKs signaling? Cell. 2013;2:751–67.View ArticleGoogle Scholar
- Igarashi Y, Nojiri H, Hanai N, Hakomori S. Gangliosides that modulate membrane protein function. Methods Enzymol. 1989;179:521–41.View ArticlePubMedGoogle Scholar
- Nagai Y. Functional roles of gangliosides in bio-signaling. Behav Brain Res. 1995;66:99–104.View ArticlePubMedGoogle Scholar
- Hakomori S, Igarashi Y. Functional role of glycosphingolipids in cell recognition and signaling. J Biochem. 1995;118:1091–103.PubMedGoogle Scholar
- Lopez PH, Schnaar RL. Gangliosides in cell recognition and membrane protein regulation. Curr Opin Struct Biol. 2009;19:549–57.View ArticlePubMed CentralPubMedGoogle Scholar
- Rebbaa A, Hurh J, Yamamoto H, Kersey DS, Bremer EG. Ganglioside GM3 inhibition of EGF receptor mediated signal transduction. Glycobiology. 1996;6:399–406.View ArticlePubMedGoogle Scholar
- Muthing J, Maurer U, Weber-Schurholz S. Glycosphingolipids of skeletal muscle: II. Modulation of Ca2(+)-flux in triad membranes by gangliosides. Carbohydr Res. 1998;307:147–57.View ArticlePubMedGoogle Scholar
- Takamiya K, Yamamoto A, Furukawa K, Yamashiro S, Shin M, Okada M, et al. Mice with disrupted GM2/GD2 synthase gene lack complex gangliosides but exhibit only subtle defects in their nervous system. Proc Natl Acad Sci U S A. 1996;93:10662–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Sheikh KA, Sun J, Liu Y, Kawai H, Crawford TO, Proia RL, et al. Mice lacking complex gangliosides develop Wallerian degeneration and myelination defects. Proc Natl Acad Sci U S A. 1999;96:7532–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Pan B, Fromholt SE, Hess EJ, Crawford TO, Griffin JW, Sheikh KA, et al. Myelin-associated glycoprotein and complementary axonal ligands, gangliosides, mediate axon stability in the CNS and PNS: neuropathology and behavioral deficits in single- and double-null mice. Exp Neurol. 2005;195:208–17.View ArticlePubMed CentralPubMedGoogle Scholar
- Schnaar RL. Brain gangliosides in axon-myelin stability and axon regeneration. FEBS Lett. 2010;584:1741–7.View ArticlePubMed CentralPubMedGoogle Scholar
- Schnaar RL, Gerardy-Schahn R, Hildebrandt H. Sialic acids in the brain: gangliosides and polysialic acid in nervous system development, stability, disease, and regeneration. Physiol Rev. 2014;94:461–518.View ArticlePubMedGoogle Scholar
- Xia B, Hoyte K, Kammesheidt A, Deerinck T, Ellisman M, Martin PT. Overexpression of the CT GalNAc transferase in skeletal muscle alters myofiber growth, neuromuscular structure, and laminin expression. Dev Biol. 2002;242:58–73.View ArticlePubMedGoogle Scholar
- Liu Y, Wada R, Kawai H, Sango K, Deng C, Tai T, et al. A genetic model of substrate deprivation therapy for a glycosphingolipid storage disorder. J Clin Invest. 1999;103:497–505.View ArticlePubMed CentralPubMedGoogle Scholar
- Singhal N, Xu R, Martin PT. Distinct contributions of Galgt1 and Galgt2 to carbohydrate expression and function at the mouse neuromuscular junction. Mol Cell Neurosci. 2012;51:112–26.View ArticlePubMed CentralPubMedGoogle Scholar
- Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 2001;25:402–8.View ArticlePubMedGoogle Scholar
- Yoon JH, Chandrasekharan K, Xu R, Glass M, Singhal N, Martin PT. The synaptic CT carbohydrate modulates binding and expression of extracellular matrix proteins in skeletal muscle: partial dependence on utrophin. Mol Cell Neurosci. 2009;41:448–63.View ArticlePubMed CentralPubMedGoogle Scholar
- Martin PT, Golden B, Okerblom J, Camboni M, Chandrasekharan K, Xu R, et al. A comparative study of N-glycolylneuraminic acid (Neu5Gc) and cytotoxic T Cell (CT) carbohydrate expression in normal and dystrophin-deficient dog and human skeletal muscle. PLoS One. 2014;9:e88226.View ArticlePubMed CentralPubMedGoogle Scholar
- Chen J, Zhao P, Massaro D, Clerch LB, Almon RR, DuBois DC, et al. The PEPR GeneChip data warehouse, and implementation of a dynamic time series query tool (SGQT) with graphical interface. Nucleic Acids Res. 2004;32:D578–81.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhao P, Iezzi S, Carver E, Dressman D, Gridley T, Sartorelli V, et al. Slug is a novel downstream target of MyoD. Temporal profiling in muscle regeneration. J Biol Chem. 2002;277:30091–101.View ArticlePubMedGoogle Scholar
- Martin PT, Scott LJ, Porter BE, Sanes JR. Distinct structures and functions of related pre- and postsynaptic carbohydrates at the mammalian neuromuscular junction. Mol Cell Neurosci. 1999;13:105–18.View ArticlePubMedGoogle Scholar
- Cheung P, Pawling J, Partridge EA, Sukhu B, Grynpas M, Dennis JW. Metabolic homeostasis and tissue renewal are dependent on beta1,6GlcNAc-branched N-glycans. Glycobiology. 2007;17:828–37.View ArticlePubMedGoogle Scholar
- Casar JC, Cabello-Verrugio C, Olguin H, Aldunate R, Inestrosa NC, Brandan E. Heparan sulfate proteoglycans are increased during skeletal muscle regeneration: requirement of syndecan-3 for successful fiber formation. J Cell Sci. 2004;117:73–84.View ArticlePubMedGoogle Scholar
- Song Y, McFarland DC, Velleman SG. Fibroblast growth factor 2 and protein kinase C alpha are involved in syndecan-4 cytoplasmic domain modulation of turkey myogenic satellite cell proliferation. Comp Biochem Physiol A Mol Integr Physiol. 2012;161:44–52.View ArticlePubMedGoogle Scholar
- Leskawa KC, Erwin RE, Buse PE, Hogan EL. Glycosphingolipid biosynthesis during myogenesis of rat L6 cells in vitro. Mol Cell Biochem. 1988;83:47–54.View ArticlePubMedGoogle Scholar
- Leskawa KC, Hogan EL. Regulation of glycolipid synthesis during differentiation of clonal murine muscle cells. Mol Cell Biochem. 1990;96:163–73.View ArticlePubMedGoogle Scholar
- Cambron LD, Leskawa KC. Glycosphingolipids during skeletal muscle cell differentiation: comparison of normal and fusion-defective myoblasts. Mol Cell Biochem. 1994;130:173–85.View ArticlePubMedGoogle Scholar
- Mukai A, Kurisaki T, Sato SB, Kobayashi T, Kondoh G, Hashimoto N. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells. Exp Cell Res. 2009;315:3052–63.View ArticlePubMedGoogle Scholar
- Lee PM, Ketis NV, Barber KR, Grant CW. Ganglioside headgroup dynamics. Biochim Biophys Acta. 1980;601:302–14.View ArticlePubMedGoogle Scholar
- Ollmann M, Galla HJ. Ganglioside headgroups decrease lipid order in reconstituted phosphatidylcholine liposomes. FEBS Lett. 1985;179:173–6.View ArticlePubMedGoogle Scholar
- Kaufman SJ, Parks CM. Loss of growth control and differentiation in the fu-1 variant of the L8 line of rat myoblasts. Proc Natl Acad Sci U S A. 1977;74:3888–92.View ArticlePubMed CentralPubMedGoogle Scholar
- Gilfix B, Rogers J, Sanwal BD. Isolation and characterization of rat skeletal myoblast mutants resistant to lectins. Mol Cell Biol. 1983;3:2166–71.PubMed CentralPubMedGoogle Scholar
- Gilfix BM, Sanwal BD. Inhibition of myoblast fusion by tunicamycin and pantomycin. Biochem Biophys Res Commun. 1980;96:1184–91.View ArticlePubMedGoogle Scholar
- Spearman MA, Wayne S, Jamieson JC, Wright JA, Gospodarek E. Studies on glycosyltransferases in fusion-defective conA-resistant L6 rat myoblast cell lines. Int J Biochem. 1989;21:531–4.View ArticlePubMedGoogle Scholar
- Newburn EN, Duchemin AM, Neff NH, Hadjiconstantinou M. GM1 ganglioside enhances Ret signaling in striatum. J Neurochem. 2014;130:541–54.View ArticlePubMedGoogle Scholar
- Bremer EG, Schlessinger J, Hakomori S. Ganglioside-mediated modulation of cell growth. Specific effects of GM3 on tyrosine phosphorylation of the epidermal growth factor receptor. J Biol Chem. 1986;261:2434–40.PubMedGoogle Scholar
- Bodine SC, Stitt TN, Gonzalez M, Kline WO, Stover GL, Bauerlein R, et al. Akt/mTOR pathway is a crucial regulator of skeletal muscle hypertrophy and can prevent muscle atrophy in vivo. Nat Cell Biol. 2001;3:1014–9.View ArticlePubMedGoogle Scholar
- Risson V, Mazelin L, Roceri M, Sanchez H, Moncollin V, Corneloup C, et al. Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy. J Cell Biol. 2009;187:859–74.View ArticlePubMed CentralPubMedGoogle Scholar
- Li Y, Huang X, Zhang J, Li Y, Ma K. Synergistic inhibition of cell migration by tetraspanin CD82 and gangliosides occurs via the EGFR or cMet-activated Pl3K/Akt signalling pathway. Int J Biochem Cell Biol. 2013;45:2349–58.View ArticlePubMedGoogle Scholar
- Rinaldi F, Perlingeiro RC. Stem cells for skeletal muscle regeneration: therapeutic potential and roadblocks. Transl Res. 2014;163:409–17.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.