Ectopic skeletal muscles derived from myoblasts implanted under the skin

1998 ◽  
Vol 111 (22) ◽  
pp. 3287-3297
Author(s):  
A. Irintchev ◽  
J.D. Rosenblatt ◽  
M.J. Cullen ◽  
M. Zweyer ◽  
A. Wernig
Keyword(s):  
Satellite Cells ◽  
Acetylcholine Receptors ◽  
De Novo ◽  
Primary Cultures ◽  
Trunk Muscles ◽  
Muscle Fibres ◽  
Lumbar Region ◽  
Primary Myoblasts ◽  
Alpha Bungarotoxin

We investigated the potential of cultured myoblasts to generate skeletal muscle in an ectopic site. Myoblasts from a clonal cell line or from expanded primary cultures were injected under the skin of the lumbar region of adult syngenic Balb/c mice. One to 7 weeks after injection, distinct muscles, of greater mass in mice injected with clonal myoblasts (6–78 mg, n=37) than in mice injected with primary myoblasts (1–7 mg, n=26), had formed between the subcutaneous panniculus carnosus muscle and the trunk muscles of host animals. These ectopic muscles exhibited spontaneous and/or electrically-evoked contractions after the second week and, when stimulated directly in vitro, isometric contractile properties similar to those of normal muscles. Histological, electron microscopical and tissue culture examination of these muscles revealed their largely mature morphology and phenotype. The fibres, most of which were branched, were contiguous, aligned and capillarised, exhibited normal sarcormeric protein banding patterns, and expressed muscle-specific proteins, including desmin, dystrophin, and isoforms of developmental and adult myosin heavy chain. Enveloping each fibre was a basal lamina, beneath which lay quiescent satellite cells, which could be stimulated to produce new muscle in culture. Presence of endplates (revealed by alpha-bungarotoxin and neurofilament staining), and the eventual loss of expression of neural cell adhesion molecule and extrasynaptic acetylcholine receptors, indicated that some fibres were innervated. That these muscle fibres were of implanted-cell origin was supported by the finding of Y-chromosome and a lack of dystrophin in ectopic muscles formed after subcutaneous injection of, respectively, male myoblasts into female mice and dystrophin-deficient (mdx) myoblasts into normal C57Bl/10 muscle. Our results demonstrate that an organised, functional muscle can be generated de novo from a disorganised mass of myoblasts implanted in an extramuscular subcutaneous site, whereby the host contributes significantly in providing support tissues and innervation. Our observations are also consistent with the idea that myogenic cells behave like tissue-specific stem cells, generating new muscle precursor (satellite) cells as well as mature muscle. Subcutaneous implantation of myoblasts may have a range of useful applications, from the study of myogenesis to the delivery of gene products.

scholarly journals Inflamma-miR-21 Negatively Regulates Myogenesis during Ageing

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 345 ◽  
Author(s):  
Maria Borja-Gonzalez ◽  
Jose C. Casas-Martinez ◽  
Brian McDonagh ◽  
Katarzyna Goljanek-Whysall
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Muscle Hypertrophy ◽  
Myogenic Potential ◽  
Age Related ◽  
Primary Myoblasts ◽  
Muscle Homeostasis ◽  
Old Mice

Ageing is associated with disrupted redox signalling and increased circulating inflammatory cytokines. Skeletal muscle homeostasis depends on the balance between muscle hypertrophy, atrophy and regeneration, however during ageing this balance is disrupted. The molecular pathways underlying the age-related decline in muscle regenerative potential remain elusive. microRNAs are conserved robust gene expression regulators in all tissues including skeletal muscle. Here, we studied satellite cells from adult and old mice to demonstrate that inhibition of miR-21 in satellite cells from old mice improves myogenesis. We determined that increased levels of proinflammatory cytokines, TNFα and IL6, as well as H2O2, increased miR-21 expression in primary myoblasts, which in turn resulted in their decreased viability and myogenic potential. Inhibition of miR-21 function rescued the decreased size of myotubes following TNFα or IL6 treatment. Moreover, we demonstrated that miR-21 could inhibit myogenesis in vitro via regulating IL6R, PTEN and FOXO3 signalling. In summary, upregulation of miR-21 in satellite cells and muscle during ageing may occur in response to elevated levels of TNFα and IL6, within satellite cells or myofibrillar environment contributing to skeletal muscle ageing and potentially a disease-related decline in potential for muscle regeneration.

scholarly journals A factor from neurons induces partial immobilization of nonclustered acetylcholine receptors on cultured muscle cells.

1981 ◽  
Vol 88 (2) ◽  
pp. 459-462 ◽  
Author(s):  
D Axelrod ◽  
H C Bauer ◽  
M Stya ◽  
C N Christian
Keyword(s):  
Conditioned Medium ◽  
Acetylcholine Receptors ◽  
Glioma Cell ◽  
Primary Cultures ◽  
Glioma Cell Line ◽  
Continuous Treatment ◽  
Hybrid Cells ◽  
Fluorescence Photobleaching Recovery ◽  
Photobleaching Recovery ◽  
Alpha Bungarotoxin

A factor or factors released by cultured NG108-15 neuroblastoma X glioma hybrid cells and added to the medium of rat myotube primary cultures was found to immobilize some of the previously mobile acetylcholine receptors in the myotube membrane. Partial receptor immobilization occurred within 3 h after the beginning of treatment with the NG108-15-conditioned medium factor and persisted for at least 24 h of continuous treatment. A similarly derived conditioned medium concentrate from the non-neuronal parent glioma cell line did not immobilize receptors, relative to untreated controls. Acetylcholine receptors were visualized by fluorescent alpha-bungarotoxin and their lateral motion was observed by the technique of fluorescence photobleaching recovery.

scholarly journals Activation of Peroxisome Proliferator-Activated Receptor Gamma by Human Cytomegalovirus for De Novo Replication Impairs Migration and Invasiveness of Cytotrophoblasts from Early Placentas

2009 ◽  
Vol 84 (6) ◽  
pp. 2946-2954 ◽  
Author(s):  
Benjamin Rauwel ◽  
Bernard Mariamé ◽  
Hélène Martin ◽  
Ronni Nielsen ◽  
Sophie Allart ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
De Novo ◽  
Present Report ◽  
Primary Cultures ◽  
First Trimester ◽  
Peroxisome Proliferator ◽  
Mobility Shift ◽  
Peroxisome Proliferator Activated Receptor ◽  
Human Trophoblast

ABSTRACT Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARγ) transcriptional activity in infected cells. We demonstrate that the PPARγ antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARγ, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARγ in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARγ human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.

scholarly journals Brain extract causes acetylcholine receptor redistribution which mimics some early events at developing neuromuscular junctions.

1982 ◽  
Vol 93 (2) ◽  
pp. 417-425 ◽  
Author(s):  
M M Salpeter ◽  
S Spanton ◽  
K Holley ◽  
T R Podleski
Keyword(s):  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Marked Change ◽  
Brain Extract ◽  
Bottom Surface ◽  
Culture Dish ◽  
Tissue Culture Dish ◽  
Em Autoradiography ◽  
Alpha Bungarotoxin

We studied the effect of rat brain extract on rat muscle cells in vitro by light and electron microscope (EM) autoradiography after labeling acetylcholine receptors (AChR's) with 125I-alpha-bungarotoxin. We found that: (a) In the absence of brain extract, peak site densities within AChR clusters usually do not exceed 4,000 sites/micrometer2. (b) Within hours after exposure to brain extract, AChR's redistribute to form clusters in which the peak site densities are greater than 10,000 sites/micrometer2. Receptor concentration within extract-induced clusters is thus within a factor of 2 of that at the neuromuscular junction (nmj). (c) In the absence of extract, the AChR's and AChR clusters are predominantly on the bottom surface of the myotubes (facing the tissue culture dish). After extract treatment, they are predominantly at the top surface. (d) Plasma membrane in regions of high-density AChR clusters is enriched in membrane with enhanced electron density and surface basal lamina whether or not cells are treated with extract. Extract causes an increase in both these specializations on the top surface of the myotubes. (e) Brain extract does not produce an overall increase in AChR site density or a marked change in degradation rate of receptors in either clustered or nonclustered regions. By producing AChR clusters with junctional site densities and enhanced surface specialization, and by causing an overall shift in AChR's distribution, brain extract mimics early events reported at developing neuromuscular junctions.

scholarly journals Interaction of di-iodinated 125I-labelled α-bungarotoxin and reversible cholinergic ligands with intact synaptic acetylcholine receptors on isolated skeletal-muscle fibres from the rat

1979 ◽  
Vol 181 (3) ◽  
pp. 545-557 ◽  
Author(s):  
P Darveniza ◽  
J A Morgan-Hughes ◽  
E J Thompson
Keyword(s):  
Skeletal Muscle ◽  
Dissociation Constant ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Muscle Fibres ◽  
First Order ◽  
Toxin Binding ◽  
Alpha Bungarotoxin ◽  
Skeletal Muscle Fibres

1. Intact synaptic acetylcholine receptors on freshly isolated rat skeletal-muscle fibres were characterized by their interaction with di-iodinated 125I-labelled alpha-bungarotoxin, acetylcholine and other cholinergic ligands at room temperature (22 deggrees C). 2. The time course and concentration dependence of 125I-labelled alpha-bungarotoxin association conformed to a bimolecular mechanism. In time-course experiments with different concentrations of 125I-labelled alpha-bungarotoxin (1.4–200 nM) the bimolecular-association rate constant, k + 1, was (2.27 +/- 0.49) × 10(4)M-1.S-1 (mean +/- S.D., N = 10). In concentration-dependence experiments, k + 1 was 2.10 × 10(4)M-1.S-1 and 1.74 × 10(4) M-1.S-1 with 10 and 135 min incubations respectively. In association experiments the first-order rate constant was proportional to the 125I-labelled alpha-bungarotoxin concentration. 125I-Labelled alpha-bungarotoxin dissociation was first order with a dissociation constant, k-1, less than or equal to 3 × 10(-6)S(-1) (half-life greater than or equal to 60 h.) The results indicated a single class of high-affinity toxin-binding sites at the end-plate with an equilibrium dissociation constant, Kd, equal to or less than 100 pM. The number of toxin-binding sites was (3.62 +/- 0.46) × 10(7) (mean +/- S.D., n = 22) per rat end-plate. 3. The apparent inhibitor dissociation constants, Ki, for reversible cholinergic ligands were determined by studying their effect at equilibrium on the rate of 125I-labelled alpha-bungarotoxin binding. There was heterogeneity of binding sites for cholinergic ligands, which were independent and non-interacting with antagonists. In contrast agonist affinity decreased with increasing receptor occupancy. Cholinergic ligands in excess inhibited over 90% of 125I-labelled alpha-bungarotoxin binding. 4. Cholinergic ligand binding was accompanied by an increase in entropy, which was greater for the agonist carbachol (delta So = +0.46 kJ.mol-1.K-1) than the antagonist tubocurarine (delta So = +0.26 kJ.mol-1.K-1). 5. The entropy and affinity changes that accompanied agonist binding suggested that agonists induced significant conformational changes in intact acetylcholine receptors. 6. The affinity and specificity of 125I-labelled alpha-bungarotoxin and tubocurarine binding to synaptic acetylcholine receptors from slow and fast muscle fibres were the same. 7. The study of binding only requires milligram amounts of tissue and may have application to other neurobiological studies and to the study of human neuromuscular disorders.

Regeneration of adult newt skeletal muscle tissue in vitro

Development ◽  
1983 ◽  
Vol 77 (1) ◽  
pp. 255-271
Author(s):  
Joan A. Schrag ◽  
Jo Ann Cameron
Keyword(s):  
Skeletal Muscle ◽  
Primary Cultures ◽  
Muscle Fibres ◽  
Myogenic Cells ◽  
Subsequent Formation ◽  
Myotube Formation ◽  
Adult Newt ◽  
Muscle Cultures ◽  
Minced Muscle

Explants and cells of forelimb muscle from adult Notophthalmus viridescens were cultured for periods up to 160 days in MEM-based medium supplemented with serum, hormones, andantibiotics. Explants which were not minced prior to culture contained muscle fibres withhealthy myonuclei and no evidence of dedifferentiation after four weeks. Explants which were minced prior to culture contained degenerated muscle fibres after 1 day and no evidence of dedifferentiation after four weeks. Mononucleated cells from both minced and non-minced explants proliferated. Cell proliferation and myotube formation was greater in the minced muscle cultures. Proliferation and fusion of myoblasts and subsequent formation ofmyofibrils were observed on the plate in primary cultures. Secondarily transferred cells proliferated and fused into myotubes. Although adult newt muscle does not contain satellite cells, myogenesis in this amphibian followed the same course as all other vertebrate skeletal muscle: proliferation of mononucleated myogenic cells, fusion of the myoblaststo form syncytia, and eventual accumulation of myofibrils. The ultimate source of the myogenic cells was not identified; however, the absence of dedifferentiation of the mature fibres and the occurrence of myogenesis in cultures of minced muscle explants demonstratedthat the regenerated muscle developed from a population of mononucleated cells whose origin did not depend upon dedifferentiation of intact fibres.

scholarly journals Agonist-induced myopathy at the neuromuscular junction is mediated by calcium.

1979 ◽  
Vol 82 (3) ◽  
pp. 811-819 ◽  
Author(s):  
J P Leonard ◽  
M M Salpeter
Keyword(s):  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Large Diameter ◽  
Mammalian Skeletal Muscle ◽  
Esterase Inhibitors ◽  
Bath Application ◽  
Esterase Inhibition ◽  
Muscle Necrosis ◽  
Alpha Bungarotoxin

Inactivation of cholinesterases at mammalian neuromuscular junctions (nmj) produces extensive muscle "necrosis." Fine-structurally, this myopathy begins near the nmj with an increase in large-diameter vesicles in the soleplasm, the dissolution of Z-disks, dilation of mitochondria, destruction of sarcoplasmic reticulum, and often a highly specific contracture of the muscle under the endplate. Since a Ca++-activated protease which specifically removes Z-disks is known to exist in mammalian skeletal muscle, we tested the possibility that the myopathy after esterase inactivation is due to the prolongation of acetylcholine lifetime and thus of Ca++ influx. We first produced the myopathy near endplates by inactivating esterases with diisopropylfluorophosphate (DFP) followed by nerve stimulation for 1--2 h in vitro. The myopathy was later mimicked by bath application of carbamylcholine without esterase inhibitors. This myopathy could be prevented by inactivating the acetylcholine receptors (AChR) with alpha-bungarotoxin (alpha-BGT) or by removing Ca++ from the bath with EGTA. These results favor the hypothesis that esterase inhibition leads to an agonist-induced myopathy, which is mediated by Ca++ and requires an intact AChR.

scholarly journals Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins fromBothrops marajoensis(Marajó Lancehead) Snake Venom

2016 ◽  
Vol 2016 ◽  
pp. 1-10
Author(s):  
Antonio Lisboa ◽  
Rodolfo Melaré ◽  
Junia R. B. Franco ◽  
Carolina V. Bis ◽  
Marta Gracia ◽  
...  
Keyword(s):  
Nerve Terminal ◽  
Neurotransmitter Release ◽  
Acetylcholine Receptors ◽  
Motor Nerve ◽  
Synaptic Proteins ◽  
Muscle Fibres ◽  
Motor Nerve Terminal ◽  
Transmission Electron ◽  
Skeletal Muscle Fibres

Neuromuscular preparations exposed toB. marajoensisvenom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposedin vitroto BmjeTX-I and BmjeTX-II toxins fromB. marajoensisvenom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

scholarly journals A Cystine-Rich Whey Supplement (Immunocal®) Provides Neuroprotection from Diverse Oxidative Stress-Inducing Agents In Vitro by Preserving Cellular Glutathione

2017 ◽  
Vol 2017 ◽  
pp. 1-15 ◽  
Author(s):  
Aimee N. Winter ◽  
Erika K. Ross ◽  
Vamsi Daliparthi ◽  
Whitney A. Sumner ◽  
Danielle M. Kirchhof ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
De Novo ◽  
Primary Cultures ◽  
Antioxidant Defenses ◽  
Nitric Oxide Donor ◽  
Neuroprotective Effects ◽  
Ht22 Cells ◽  
Principal Mechanism ◽  
Endogenous Antioxidant

Oxidative stress is a principal mechanism underlying the pathophysiology of neurodegeneration. Therefore, nutritional enhancement of endogenous antioxidant defenses may represent a viable treatment option. We investigated the neuroprotective properties of a unique whey protein supplement (Immunocal®) that provides an essential precursor (cystine) for synthesis of the endogenous antioxidant, glutathione (GSH). Primary cultures of rat cerebellar granule neurons (CGNs), NSC34 motor neuronal cells, or HT22 hippocampal cells were preincubated in medium containing Immunocal and then subsequently treated with agents known to induce oxidative stress. Immunocal protected CGNs against neurotoxicity induced by the Bcl-2 inhibitor, HA14-1, the nitric oxide donor, sodium nitroprusside, CuCl2, and AlCl3. Immunocal also significantly reduced NSC34 cell death due to either H2O2 or glutamate and mitigated toxicity in HT22 cells overexpressing β-amyloid1-42. The neuroprotective effects of Immunocal were blocked by inhibition of γ-glutamyl-cysteine ligase, demonstrating dependence on de novo GSH synthesis. These findings indicate that sustaining GSH with Immunocal significantly protects neurons against diverse inducers of oxidative stress. Thus, Immunocal is a nutritional supplement worthy of testing in preclinical animal models of neurodegeneration and in future clinical trials of patients afflicted by these diseases.

scholarly journals Evidence that coated vesicles transport acetylcholine receptors to the surface membrane of chick myotubes.

1984 ◽  
Vol 98 (2) ◽  
pp. 498-506 ◽  
Author(s):  
S Bursztajn ◽  
G D Fischbach
Keyword(s):  
Acetylcholine Receptors ◽  
Prolonged Exposure ◽  
Addition Reaction ◽  
Surface Membrane ◽  
Coated Vesicles ◽  
Intact Cells ◽  
Saline Extract ◽  
Permeabilized Cells ◽  
Alpha Bungarotoxin

Coated vesicles are present in the myoplasm of embryonic chick myotubes grown in vitro. They are most numerous beneath regions of the surface membrane that contain a high density of acetylcholine receptors (AChR). Prolonged exposure of myotubes to saline extract of chick brain increases the number of intracellular AChR and the number of coated vesicles. This suggests that coated vesicles contain AChR, and this hypothesis was tested with horseradish peroxidase-alpha-bungarotoxin (HRP-alpha BTX) conjugates. The conjugates enter saponin-permeabilized cells and, as judged by the inhibition of [125I] alpha BTX binding, they label the entire intracellular AChR pool. Approximately 50% of the coated vesicles contained HRP-alpha BTX reaction product. In addition, reaction product was detected in Golgi cisternae and along membranes that bound a subsurface tubulovesicular network. The majority of labeled vesicles are probably involved in exocytosis rather than endocytosis of AChR because very few coated vesicles were labeled when HRP-alpha BTX was added to the medium bathing intact cells. Moreover, inhibition of protein synthesis with puromycin resulted in a large decrease in the number of labeled vesicles. These results suggest that a subpopulation of coated vesicles ferry newly synthesized AChR to the cell surface.

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