Caveolin-3 in skeletal muscle fibres of Duchenne muscular dystrophy and mdx mouse

AbstractDuchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

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Co-Transplantation of Bone Marrow-MSCs and Myogenic Stem/Progenitor Cells from Adult Donors Improves Muscle Function of Patients with Duchenne Muscular Dystrophy

Cells ◽

10.3390/cells9051119 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1119

Author(s):

Aleksandra Klimczak ◽

Agnieszka Zimna ◽

Agnieszka Malcher ◽

Urszula Kozlowska ◽

Katarzyna Futoma ◽

...

Keyword(s):

Skeletal Muscle ◽

Bone Marrow ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Progenitor Cells ◽

Muscle Function ◽

Progenitor Cell ◽

Genetic Disorder ◽

Cell Populations ◽

Muscle Fibres

Duchenne muscular dystrophy (DMD) is a genetic disorder associated with a progressive deficiency of dystrophin that leads to skeletal muscle degeneration. In this study, we tested the hypothesis that a co-transplantation of two stem/progenitor cell populations, namely bone marrow-derived mesenchymal stem cells (BM-MSCs) and skeletal muscle-derived stem/progenitor cells (SM-SPCs), directly into the dystrophic muscle can improve the skeletal muscle function of DMD patients. Three patients diagnosed with DMD, confirmed by the dystrophin gene mutation, were enrolled into a study approved by the local Bioethics Committee (no. 79/2015). Stem/progenitor cells collected from bone marrow and skeletal muscles of related healthy donors, based on HLA matched antigens, were expanded in a closed MC3 cell culture system. A simultaneous co-transplantation of BM-MSCs and SM-SPCs was performed directly into the biceps brachii (two patients) and gastrocnemius (one patient). During a six-month follow-up, the patients were examined with electromyography (EMG) and monitored for blood kinase creatine level. Muscle biopsies were examined with histology and assessed for dystrophin at the mRNA and protein level. A panel of 27 cytokines was analysed with multiplex ELISA. We did not observe any adverse effects after the intramuscular administration of cells. The efficacy of BM-MSC and SM-SPC application was confirmed through an EMG assessment by an increase in motor unit parameters, especially in terms of duration, amplitude range, area, and size index. The beneficial effect of cellular therapy was confirmed by a decrease in creatine kinase levels and a normalised profile of pro-inflammatory cytokines. BM-MSCs may support the pro-regenerative potential of SM-SPCs thanks to their trophic, paracrine, and immunomodulatory activity. Both applied cell populations may fuse with degenerating skeletal muscle fibres in situ, facilitating skeletal muscle recovery. However, further studies are required to optimise the dose and timing of stem/progenitor cell delivery.

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Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy

Clinical Chemistry ◽

10.1093/clinchem/47.3.451 ◽

2001 ◽

Vol 47 (3) ◽

pp. 451-458 ◽

Cited By ~ 31

Author(s):

Angelika Hammerer-Lercher ◽

Petra Erlacher ◽

Reginald Bittner ◽

Rudolf Korinthenberg ◽

Daniela Skladal ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Blot Analysis ◽

Cardiac Troponin ◽

Western Blot Analysis ◽

Clinical Evidence ◽

Mdx Mouse ◽

Muscle Biopsies

Abstract Background: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD). Methods: Plasma from 14 patients (mean age, 7.5 years; range, 5.7–19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6). Results: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 μg/L; range, 0.06–0.16 μg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763–5030 U/L) with age (median, 7.5 years; range, 6.8–10.9 years; r = −0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms. Conclusions: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.

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The D2.mdx mouse as a preclinical model of the skeletal muscle pathology associated with Duchenne muscular dystrophy

Scientific Reports ◽

10.1038/s41598-020-70987-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

David W. Hammers ◽

Cora C. Hart ◽

Michael K. Matheny ◽

Lillian A. Wright ◽

Megan Armellini ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Pathology ◽

Mdx Mouse ◽

Preclinical Model

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Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs

Blood ◽

10.1182/blood-2004-06-2247 ◽

2004 ◽

Vol 104 (13) ◽

pp. 4311-4318 ◽

Cited By ~ 58

Author(s):

Chiara Dell'Agnola ◽

Zejing Wang ◽

Rainer Storb ◽

Stephen J. Tapscott ◽

Christian S. Kuhr ◽

...

Keyword(s):

Skeletal Muscle ◽

Bone Marrow ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Cell Transplantation ◽

Hematopoietic Cell ◽

Mdx Mouse ◽

Hematopoietic Stem ◽

Bone Marrow Derived Cells ◽

Donor Muscle

Abstract Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene on the X-chromosome that result in skeletal and cardiac muscle damage and premature death. Studies in mice, including the mdx mouse model of DMD, have demonstrated that circulating bone marrow–derived cells can participate in skeletal muscle regeneration, but the potential clinical utility of treating human DMD by allogeneic marrow transplantation from a healthy donor remains unknown. To assess whether allogeneic hematopoietic cell transplantation (HCT) provides clinically relevant levels of donor muscle cell contribution in dogs with canine X-linked muscular dystrophy (c-xmd), 7 xmd dogs were given hematopoietic cell (HC) transplants from nonaffected littermates. Compared with the pretransplantation baseline, the number of dystrophin-positive fibers and the amount of wild-type dystrophin RNA did not increase after HCT, with observation periods ranging from 28 to 417 days. Similar results were obtained when the recipient dogs were given granulocyte colony-stimulating factor (G-CSF) after their initial transplantation to mobilize the cells. Despite successful allogeneic HCT and a permissive environment for donor muscle engraftment, there was no detectable contribution of bone marrow–derived cells to either skeletal muscle or muscle precursor cells assayed by clonal analyses at a level of sensitivity that should detect as little as 0.1% donor contribution.

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Changes in Myonuclear Number During Postnatal Growth –Implications for AAV Gene Therapy for Muscular Dystrophy

Journal of Neuromuscular Diseases ◽

10.3233/jnd-210683 ◽

2021 ◽

pp. 1-8

Author(s):

Jennifer Morgan ◽

Francesco Muntoni

Keyword(s):

Skeletal Muscle ◽

Gene Therapy ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Growth ◽

Postnatal Growth ◽

Muscle Fibres ◽

Postnatal Life ◽

Adult Skeletal Muscle ◽

Dividing Cells

Adult skeletal muscle is a relatively stable tissue, as the multinucleated muscle fibres contain post-mitotic myonuclei. During early postnatal life, muscle growth occurs by the addition of skeletal muscle stem cells (satellite cells) or their progeny to growing muscle fibres. In Duchenne muscular dystrophy, which we shall use as an example of muscular dystrophies, the muscle fibres lack dystrophin and undergo necrosis. Satellite-cell mediated regeneration occurs, to repair and replace the necrotic muscle fibres, but as the regenerated muscle fibres still lack dystrophin, they undergo further cycles of degeneration and regeneration. AAV gene therapy is a promising approach for treating Duchenne muscular dystrophy. But for a single dose of, for example, AAV coding for dystrophin, to be effective, the treated myonuclei must persist, produce sufficient dystrophin and a sufficient number of nuclei must be targeted. This latter point is crucial as AAV vector remains episomal and does not replicate in dividing cells. Here, we describe and compare the growth of skeletal muscle in rodents and in humans and discuss the evidence that myofibre necrosis and regeneration leads to the loss of viral genomes within skeletal muscle. In addition, muscle growth is expected to lead to the dilution of the transduced nuclei especially in case of very early intervention, but it is not clear if growth could result in insufficient dystrophin to prevent muscle fibre breakdown. This should be the focus of future studies.

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Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy

The Journal of Physiology ◽

10.1113/jp271418 ◽

2016 ◽

Vol 594 (11) ◽

pp. 3095-3110 ◽

Cited By ~ 33

Author(s):

Jessica R. Terrill ◽

Gavin J. Pinniger ◽

Jamie A. Graves ◽

Miranda D. Grounds ◽

Peter G. Arthur

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Muscle Function ◽

Mdx Mouse ◽

Skeletal Muscle Function

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In situ localisation of single-stranded DNA breaks in nuclei of a subpopulation of cells within regenerating skeletal muscle of the dystrophic mdx mouse

Journal of Cell Science ◽

10.1242/jcs.102.3.653 ◽

1992 ◽

Vol 102 (3) ◽

pp. 653-662 ◽

Cited By ~ 1

Author(s):

G.R. Coulton ◽

B. Rogers ◽

P. Strutt ◽

M.J. Skynner ◽

D.J. Watt

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Dna Polymerase ◽

Muscle Regeneration ◽

Regenerative Process ◽

Muscle Fibres ◽

Dna Breaks ◽

Expression Of Genes ◽

Muscle Lesions

Degeneration of muscle fibres during the early stages of Duchenne Muscular Dystrophy (DMD) is accompanied by muscle fibre regeneration where cell division and myoblast fusion to form multinucleate myotubes within the lesions appear to recapitulate the events of normal muscle development. The mechanisms that govern the expression of genes regulating differentiation of myoblasts in regenerating skeletal muscle are of great interest for the development of future therapies designed to stimulate muscle regeneration. We show here that single-stranded breaks in DNA are localised in nuclei, using an exogenously applied medium containing labelled deoxynucleotides and the Klenow fragment of DNA polymerase I. The nuclei of a sub-population of cells lying in the inflammatory infiltrate of lesions in the skeletal muscle of the muscular dystrophic mouse (mdx), a genetic homologue of DMD, were labelled in this fashion. By contrast, labelled cells were completely absent from the muscles of normal non-myopathic animals (C57BL/10) and non-lesioned areas of mdx muscles. Cells expressing the muscle-specific regulatory gene, myogenin, were also found within mononucleate cells and myotubes within similar mdx muscle lesions. While we cannot yet say that the cells labelled by the DNA polymerase reaction are in fact differentiating, they were found only in significant numbers within mdx muscle lesions where new muscle fibres appear, providing strong circumstantial evidence that they are intimately associated with the regenerative process. Using a range of nucleases and different DNA polymerases, we show that the DNA polymerase-labelling reaction observed was DNA-dependent and most probably due to infilling of naturally occurring single-stranded gaps in DNA. Since the regenerative process in human Duchenne Muscular Dystrophy is apparently less effective than that seen in mdx mice, continued study of single-stranded DNA breaks may help to elucidate further the mechanisms controlling the expression of genes that characterise the myogenic process during skeletal muscle regeneration. Such findings might be applied in the development of future therapies designed to stimulate muscle regeneration in human dystrophies.

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Lipocalin 2 Influences Bone and Muscle Phenotype in the MDX Mouse Model of Duchenne Muscular Dystrophy

International Journal of Molecular Sciences ◽

10.3390/ijms23020958 ◽

2022 ◽

Vol 23 (2) ◽

pp. 958

Author(s):

Marco Ponzetti ◽

Argia Ucci ◽

Antonio Maurizi ◽

Luca Giacchi ◽

Anna Teti ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Bone Loss ◽

Mouse Model ◽

Mononuclear Cells ◽

Serum Levels ◽

Mdx Mouse ◽

Mdx Mice ◽

Muscle Fibres ◽

Lipocalin 2

Lipocalin 2 (Lcn2) is an adipokine involved in bone and energy metabolism. Its serum levels correlate with bone mechanical unloading and inflammation, two conditions representing hallmarks of Duchenne Muscular Dystrophy (DMD). Therefore, we investigated the role of Lcn2 in bone loss induced by muscle failure in the MDX mouse model of DMD. We found increased Lcn2 serum levels in MDX mice at 1, 3, 6, and 12 months of age. Consistently, Lcn2 mRNA was higher in MDX versus WT muscles. Immunohistochemistry showed Lcn2 expression in mononuclear cells between muscle fibres and in muscle fibres, thus confirming the gene expression results. We then ablated Lcn2 in MDX mice, breeding them with Lcn2−/− mice (MDXxLcn2−/−), resulting in a higher percentage of trabecular volume/total tissue volume compared to MDX mice, likely due to reduced bone resorption. Moreover, MDXxLcn2−/− mice presented with higher grip strength, increased intact muscle fibres, and reduced serum creatine kinase levels compared to MDX. Consistently, blocking Lcn2 by treating 2-month-old MDX mice with an anti-Lcn2 monoclonal antibody (Lcn2Ab) increased trabecular volume, while reducing osteoclast surface/bone surface compared to MDX mice treated with irrelevant IgG. Grip force was also increased, and diaphragm fibrosis was reduced by the Lcn2Ab. These results suggest that Lcn2 could be a possible therapeutic target to treat DMD-induced bone loss.

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