scholarly journals Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice.

1991 ◽  
Vol 115 (6) ◽  
pp. 1685-1694 ◽  
Author(s):  
K Ohlendieck ◽  
K P Campbell
Keyword(s):  
Skeletal Muscle ◽  
Immunoblot Analysis ◽  
Protein Product ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Normal Density ◽  
Mouse Muscle ◽  
Glycoprotein Complex ◽  
Associated Proteins ◽  
Dy Mouse

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.

scholarly journals Several dystrophin-glycoprotein complex members are present in crude surface membranes but they are sodium dodecyl sulphate invisible in KCl-washed microsomes from mdx mouse muscle

2010 ◽  
Vol 15 (1) ◽  
Author(s):  
Stéphanie Daval ◽  
Chantal Rocher ◽  
Yan Cherel ◽  
Elisabeth Rumeur
Keyword(s):  
Muscular Dystrophy ◽  
Core Protein ◽  
Surface Membrane ◽  
Sodium Dodecyl ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Mouse Muscle ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Dystrophin Glycoprotein

AbstractThe dystrophin-glycoprotein complex (DGC) is a large trans-sarcolemmal complex that provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. In skeletal muscle, it consists of the dystroglycan, sarcoglycan and cytoplasmic complexes, with dystrophin forming the core protein. The DGC has been described as being absent or greatly reduced in dystrophin-deficient muscles, and this lack is considered to be involved in the dystrophic phenotype. Such a decrease in the DGC content was observed in dystrophin-deficient muscle from humans with muscular dystrophy and in mice with X-linked muscular dystrophy (mdx mice). These deficits were observed in total muscle homogenates and in partially membrane-purified muscle fractions, the so-called KCl-washed microsomes. Here, we report that most of the proteins of the DGC are actually present at normal levels in the mdx mouse muscle plasma membrane. The proteins are detected in dystrophic animal muscles when the immunoblot assay is performed with crude surface membrane fractions instead of the usually employed KCl-washed microsomes. We propose that these proteins form SDS-insoluble membrane complexes when dystrophin is absent.

scholarly journals Nav1.4 Deregulation in Dystrophic Skeletal Muscle Leads to Na+ Overload and Enhanced Cell Death

2008 ◽  
Vol 132 (2) ◽  
pp. 199-208 ◽  
Author(s):  
Carole Hirn ◽  
George Shapovalov ◽  
Olivier Petermann ◽  
Emmanuelle Roulet ◽  
Urs T. Ruegg
Keyword(s):  
Skeletal Muscle ◽  
Cell Death ◽  
Early Death ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Strongly Correlated ◽  
Muscle Degeneration ◽  
Voltage Gated ◽  
Voltage Gated Sodium Channels ◽  
Associated Proteins

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca2+ concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na+ concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Nav1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na+ concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Nav1.4, leading to an increased Na+ concentration under the sarcolemma. Moreover, the distribution of Nav1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin α-1, thus suggesting that syntrophin is an important linker between dystrophin and Nav1.4. Additionally, we show that these modifications of Nav1.4 gating properties and increased Na+ concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na+ overload can be reversed by 3 nM tetrodotoxin, a specific Nav1.4 blocker.

Talin, vinculin and DRP (utrophin) concentrations are increased at mdx myotendinous junctions following onset of necrosis

1994 ◽  
Vol 107 (6) ◽  
pp. 1477-1483 ◽  
Author(s):  
D.J. Law ◽  
D.L. Allen ◽  
J.G. Tidball
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Cytoskeletal Proteins ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Myotendinous Junction ◽  
Glycoprotein Complex ◽  
Transmembrane Glycoprotein ◽  
Muscle Necrosis ◽  
Myotendinous Junctions

Duchenne muscular dystrophy (DMD) and the myopathy seen in the mdx mouse both result from absence of the protein dystrophin. Structural similarities between dystrophin and other cytoskeletal proteins, its enrichment at myotendinous junctions, and its indirect association with laminin mediated by a transmembrane glycoprotein complex suggest that one of dystrophin's functions in normal muscle is to form one of the links between the actin cytoskeleton and the extracellular matrix. Unlike Duchenne muscular dystrophy patients, mdx mice suffer only transient muscle necrosis, and are able to regenerate damaged muscle tissue. The present study tests the hypothesis that mdx mice partially compensate for dystrophin's absence by upregulating one or more dystrophin-independent mechanisms of cytoskeleton-membrane association. Quantitative analysis of immunoblots of adult mdx muscle samples showed an increase of approximately 200% for vinculin and talin, cytoskeletal proteins that mediate thin filament-membrane interactions at myotendinous junctions. Blots also showed an increase (143%) in the dystrophin-related protein called utrophin, another myotendinous junction constituent, which may be able to substitute for dystrophin directly. Muscle samples from 2-week-old animals, a period immediately preceding the onset of muscle necrosis, showed no significant differences in protein concentration between mdx and controls. Quantitative analyses of confocal images of myotendinous junctions from mdx and control muscles show significantly higher concentrations of talin and vinculin at the myotendinous junctions of mdx muscle. These findings indicate that mdx mice may compensate in part for the absence of dystrophin by increased expression of other molecules that subsume dystrophin's mechanical function.

scholarly journals ProNGF/p75NTR Axis Drives Fiber Type Specification by Inducing the Fast-Glycolytic Phenotype in Mouse Skeletal Muscle Cells

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2232
Author(s):  
Valentina Pallottini ◽  
Mayra Colardo ◽  
Claudia Tonini ◽  
Noemi Martella ◽  
Georgios Strimpakos ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Myogenic Differentiation ◽  
Fiber Type ◽  
Pcr Analysis ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Muscle Physiology

Despite its undisputable role in the homeostatic regulation of the nervous system, the nerve growth factor (NGF) also governs the relevant cellular processes in other tissues and organs. In this study, we aimed at assessing the expression and the putative involvement of NGF signaling in skeletal muscle physiology. To reach this objective, we employed satellite cell-derived myoblasts as an in vitro culture model. In vivo experiments were performed on Tibialis anterior from wild-type mice and an mdx mouse model of Duchenne muscular dystrophy. Targets of interest were mainly assessed by means of morphological, Western blot and qRT-PCR analysis. The results show that proNGF is involved in myogenic differentiation. Importantly, the proNGF/p75NTR pathway orchestrates a slow-to-fast fiber type transition by counteracting the expression of slow myosin heavy chain and that of oxidative markers. Concurrently, proNGF/p75NTR activation facilitates the induction of fast myosin heavy chain and of fast/glycolytic markers. Furthermore, we also provided evidence that the oxidative metabolism is impaired in mdx mice, and that these alterations are paralleled by a prominent buildup of proNGF and p75NTR. These findings underline that the proNGF/p75NTR pathway may play a crucial role in fiber type determination and suggest its prospective modulation as an innovative therapeutic approach to counteract muscle disorders.

scholarly journals In vitro and in vivo analysis of human fibroblast reprogramming and multipotency

2015 ◽  
Vol 20 (3) ◽  
Author(s):  
Rongqing Pang ◽  
Xiangqing Zhu ◽  
Jia Geng ◽  
Yongyun Zhang ◽  
Qiang Wang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Human Fibroblasts ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Therapeutic Effects ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Activity Measurements ◽  
Human Dystrophin

AbstractMultipotent stem cells have potential therapeutic roles in the treatment of Duchenne muscular dystrophy (DMD). However, the limited access to stem cell sources restricts their clinical application. To address this issue, we established a simple in vitro epigenetic reprogramming technique in which skin fibroblasts are induced to dedifferentiate into multipotent cells. In this study, human fibroblasts were isolated from circumcised adult foreskin and were reprogrammed by co-culture for 72 h with fish oocyte extract (FOE) in serum-free medium. The cells were then observed and analyzed by immunofluorescence staining, flow cytometry and in vitro differentiation assays. Then FOE-treated human fibroblasts were transplanted by tail vein injection into irradiated mdx mice, an animal model of DMD. Two months after injection, the therapeutic effects of FOE-treated fibroblasts on mdx skeletal muscle were evaluated by serum creatine kinase (CK) activity measurements and by immunostaining and RT-PCR of human dystrophin expression. The results indicated that the reprogrammed fibroblasts expressed higher levels of the pluripotent antigen markers SSEA-4, Nanog and Oct-4, and were able to differentiate in vitro into adipogenic cells, osteoblastic cells, and myotube-like cells. Tail vein injection of FOE-treated fibroblasts into irradiated mdx mice slightly reduced serum CK activity and the percentage of centrally nucleated myofibers two months after cell transplantation. Furthermore, we confirmed human dystrophin protein and mRNA expression in mdx mouse skeletal muscle. These data demonstrated that FOE-treated fibroblasts were multipotent and could integrate into mdx mouse myofibers through the vasculature.

Pharmacological rescue of the dystrophin-glycoprotein complex in Duchenne and Becker skeletal muscle explants by proteasome inhibitor treatment

2006 ◽  
Vol 290 (2) ◽  
pp. C577-C582 ◽  
Author(s):  
Stefania Assereto ◽  
Silvia Stringara ◽  
Federica Sotgia ◽  
Gloria Bonuccelli ◽  
Aldobrando Broccolini ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscular Dystrophy ◽  
Proteasome Inhibitor ◽  
Becker Muscular Dystrophy ◽  
Culture Conditions ◽  
Mdx Mouse ◽  
Glycoprotein Complex ◽  
Dystrophin Glycoprotein Complex ◽  
Novel Method ◽  
Dystrophin Glycoprotein

In this report, we have developed a novel method to identify compounds that rescue the dystrophin-glycoprotein complex (DGC) in patients with Duchenne or Becker muscular dystrophy. Briefly, freshly isolated skeletal muscle biopsies (termed skeletal muscle explants) from patients with Duchenne or Becker muscular dystrophy were maintained under defined cell culture conditions for a 24-h period in the absence or presence of a specific candidate compound. Using this approach, we have demonstrated that treatment with a well-characterized proteasome inhibitor, MG-132, is sufficient to rescue the expression of dystrophin, β-dystroglycan, and α-sarcoglycan in skeletal muscle explants from patients with Duchenne or Becker muscular dystrophy. These data are consistent with our previous findings regarding systemic treatment with MG-132 in a dystrophin-deficient mdx mouse model (Bonuccelli G, Sotgia F, Schubert W, Park D, Frank PG, Woodman SE, Insabato L, Cammer M, Minetti C, and Lisanti MP. Am J Pathol 163: 1663–1675, 2003). Our present results may have important new implications for the possible pharmacological treatment of Duchenne or Becker muscular dystrophy in humans.

scholarly journals Clonal Isolation of Muscle-Derived Cells Capable of Enhancing Muscle Regeneration and Bone Healing

2000 ◽  
Vol 150 (5) ◽  
pp. 1085-1100 ◽  
Author(s):  
Joon Yung Lee ◽  
Zhuqing Qu-Petersen ◽  
Baohong Cao ◽  
Shigemi Kimura ◽  
Ron Jankowski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Stem Cell ◽  
Bone Healing ◽  
Muscle Regeneration ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Hematopoietic Stem ◽  
Isolation And Characterization ◽  
Osteogenic Lineage

Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin+ cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34+ cells and Bcl-2+ cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34+Bcl-2+ enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.

Association of dystrophin-related protein with dystrophin-associated proteins in mdx mouse muscle

Nature ◽  
1992 ◽  
Vol 360 (6404) ◽  
pp. 588-591 ◽  
Author(s):  
Kiichiro Matsumura ◽  
James M. Ervasti ◽  
Kay Ohlendieck ◽  
Steven D. Kahl ◽  
Kevin P. Campbell
Keyword(s):  
Mdx Mouse ◽  
Related Protein ◽  
Mouse Muscle ◽  
Associated Proteins

scholarly journals Extensive but Coordinated Reorganization of the Membrane Skeleton in Myofibers of Dystrophic (mdx) Mice

1999 ◽  
Vol 144 (6) ◽  
pp. 1259-1270 ◽  
Author(s):  
McRae W. Williams ◽  
Robert J. Bloch
Keyword(s):  
Skeletal Muscle ◽  
Membrane Proteins ◽  
Muscle Fibers ◽  
Membrane Skeleton ◽  
Confocal Imaging ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Intracellular Membrane ◽  
Dystrophic Muscle ◽  
Dihydropyridine Receptors

We used immunofluorescence techniques and confocal imaging to study the organization of the membrane skeleton of skeletal muscle fibers of mdx mice, which lack dystrophin. β-Spectrin is normally found at the sarcolemma in costameres, a rectilinear array of longitudinal strands and elements overlying Z and M lines. However, in the skeletal muscle of mdx mice, β-spectrin tends to be absent from the sarcolemma over M lines and the longitudinal strands may be disrupted or missing. Other proteins of the membrane and associated cytoskeleton, including syntrophin, β-dystroglycan, vinculin, and Na,K-ATPase are also concentrated in costameres, in control myofibers, and mdx muscle. They also distribute into the same altered sarcolemmal arrays that contain β-spectrin. Utrophin, which is expressed in mdx muscle, also codistributes with β-spectrin at the mutant sarcolemma. By contrast, the distribution of structural and intracellular membrane proteins, including α-actinin, the Ca-ATPase and dihydropyridine receptors, is not affected, even at sites close to the sarcolemma. Our results suggest that in myofibers of the mdx mouse, the membrane- associated cytoskeleton, but not the nearby myoplasm, undergoes widespread coordinated changes in organization. These changes may contribute to the fragility of the sarcolemma of dystrophic muscle.

Upregulation of store-operated Ca2+ entry in dystrophic mdx mouse muscle

2010 ◽  
Vol 299 (1) ◽  
pp. C42-C50 ◽  
Author(s):  
Joshua N. Edwards ◽  
Oliver Friedrich ◽  
Tanya R. Cully ◽  
Frederic von Wegner ◽  
Robyn M. Murphy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Muscle Fibers ◽  
Mdx Mouse ◽  
Cell Necrosis ◽  
Wild Type ◽  
Dystrophic Muscle ◽  
Adult Skeletal Muscle ◽  
Mouse Muscle ◽  
Threefold Increase ◽  
Tubular System

Store-operated Ca2+ entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca2+ from the transverse tubule into the junctional cleft during periods of depleting Ca2+ release. In dystrophic muscle fibers, SOCE may be a source of Ca2+ overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca2+ release. To examine this, Ca2+ in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca2+ from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca2+ transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca2+ concentrations ([Ca2+]SR). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca2+ into the fiber for an equivalent depletion of [Ca2+]SR via SOCE, it remains unclear whether this is deleterious.

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