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

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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Faculty Opinions recommendation of microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717397850.793153110 ◽

2012 ◽

Author(s):

Michael Rudnicki ◽

Alessandra Pasut

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Skeletal Muscle Regeneration

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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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Faculty Opinions recommendation of microRNA-206 promotes skeletal muscle regeneration and delays progression of Duchenne muscular dystrophy in mice.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717397850.792802850 ◽

2012 ◽

Author(s):

Aaron Russell

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Skeletal Muscle Regeneration

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Reparative dysfunction during muscle regeneration in Duchenne muscular dystrophy: therapeutic approaches to modulating the muscle environment and muscle stem cells

10.32469/10355/76151 ◽

2019 ◽

Author(s):

◽

Michael Everette Nance

Keyword(s):

Skeletal Muscle ◽

Stem Cells ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Genetic Mutation ◽

Critical Determinant ◽

Muscle Stem Cells ◽

Dystrophin Protein

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Duchenne muscular dystrophy (DMD) is a lethal muscular dystrophy resulting from functional loss of the dystrophin protein, a critical sub-sarcolemmal protein involved in membrane stability. While reparative dysfunction is thought to be a critical determinant of disease progression in humans, regeneration is not significantly impaired in the murine muscular dystrophy (mdx) model. Furthermore, it is not well understood if reparative dysfunction is related to inherent defects in stem cells or chronic alterations in the muscle environment due to disease related remodeling. To address these observed discrepancies, we adapted a whole muscle transplant model to study the in vivo regeneration of intact pieces of skeletal muscle from normal and dystrophic dogs (cDMD), a physiological and clinically relevant model to humans. Regeneration in cDMD muscle grafts was significantly attenuated compared to normal and predisposed to the development of skeletal muscle tumors. We used an adeno-associated virus (AAV) expressing a micro-dystrophin protein to specifically rescue the muscle environment by preventing fiber damage while retaining dystrophin-null SCs. AAV.micro-dystrophin rescued the environment by improving fibrosis, stiffness, and fiber orientation, which significantly improved early muscle regeneration but not late regeneration (2 greater than and less than 4 months post-transplant) via enhancing muscle stem cells differentiation. We next developed Cre- and CRISPR-cas9 gene editing strategies to test the ability of AAV serotype 9 to transduce and treat the genetic mutation in muscle stem cells. We observed efficient SC transduction when used as a single vector expressing Cre. Dual-vector CRISPR-cas9 SC transduction was inefficient and likely related to the requirement for two vectors, promoter usage, and mechanistic differences between Cre-recombination and CRISPR genome editing.

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

Neuromuscular Disorders ◽

10.1016/s0960-8966(97)87201-x ◽

1997 ◽

Vol 7 (6-7) ◽

pp. 436

Author(s):

Y. Hagiwara ◽

Y. Nishina ◽

M. Imamura ◽

M. Yoshida ◽

T. Kikuchi ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mdx Mouse ◽

Muscle Fibres ◽

Skeletal Muscle Fibres

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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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The PKA-p38MAPK-NFAT5-Organic Osmolytes Pathway in Duchenne Muscular Dystrophy: From Essential Player in Osmotic Homeostasis, Inflammation and Skeletal Muscle Regeneration to Therapeutic Target

Biomedicines ◽

10.3390/biomedicines9040350 ◽

2021 ◽

Vol 9 (4) ◽

pp. 350

Author(s):

Sandrine Herbelet ◽

Caroline Merckx ◽

Boel De Paepe

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Protein Kinase ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Janus Kinase ◽

Skeletal Muscle Regeneration ◽

Organic Osmolytes ◽

Nuclear Factor ◽

Osmotic Homeostasis

In Duchenne muscular dystrophy (DMD), the absence of dystrophin from the dystrophin-associated protein complex (DAPC) causes muscle membrane instability, which leads to myofiber necrosis, hampered regeneration, and chronic inflammation. The resulting disabled DAPC-associated cellular pathways have been described both at the molecular and the therapeutical level, with the Toll-like receptor nuclear factor kappa-light-chain-enhancer of activated B cells pathway (NF-ƘB), Janus kinase/signal transducer and activator of transcription proteins, and the transforming growth factor-β pathways receiving the most attention. In this review, we specifically focus on the protein kinase A/ mitogen-activated protein kinase/nuclear factor of activated T-cells 5/organic osmolytes (PKA-p38MAPK-NFAT5-organic osmolytes) pathway. This pathway plays an important role in osmotic homeostasis essential to normal cell physiology via its regulation of the influx/efflux of organic osmolytes. Besides, NFAT5 plays an essential role in cell survival under hyperosmolar conditions, in skeletal muscle regeneration, and in tissue inflammation, closely interacting with the master regulator of inflammation NF-ƘB. We describe the involvement of the PKA-p38MAPK-NFAT5-organic osmolytes pathway in DMD pathophysiology and provide a clear overview of which therapeutic molecules could be of potential benefit to DMD patients. We conclude that modulation of the PKA-p38MAPK-NFAT5-organic osmolytes pathway could be developed as supportive treatment for DMD in conjunction with genetic therapy.

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The Immune System in Duchenne Muscular Dystrophy Pathogenesis

Biomedicines ◽

10.3390/biomedicines9101447 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1447

Author(s):

Luana Tripodi ◽

Chiara Villa ◽

Davide Molinaro ◽

Yvan Torrente ◽

Andrea Farini

Keyword(s):

Skeletal Muscle ◽

T Cells ◽

Immune System ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Regeneration ◽

Point Of View ◽

Central Tolerance ◽

Immune System Activation ◽

Killer T Cells

Growing evidence demonstrates the crosstalk between the immune system and the skeletal muscle in inflammatory muscle diseases and dystrophic conditions such as Duchenne Muscular Dystrophy (DMD), as well as during normal muscle regeneration. The rising of inflammation and the consequent activation of the immune system are hallmarks of DMD: several efforts identified the immune cells that invade skeletal muscle as CD4+ and CD8+ T cells, Tregs, macrophages, eosinophils and natural killer T cells. The severity of muscle injury and inflammation dictates the impairment of muscle regeneration and the successive replacement of myofibers with connective and adipose tissue. Since immune system activation was traditionally considered as a consequence of muscular wasting, we recently demonstrated a defect in central tolerance caused by thymus alteration and the presence of autoreactive T-lymphocytes in DMD. Although the study of innate and adaptive immune responses and their complex relationship in DMD attracted the interest of many researchers in the last years, the results are so far barely exhaustive and sometimes contradictory. In this review, we describe the most recent improvements in the knowledge of immune system involvement in DMD pathogenesis, leading to new opportunities from a clinical point-of-view.

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Cellular senescence-mediated exacerbation of Duchenne muscular dystrophy

Scientific Reports ◽

10.1038/s41598-020-73315-6 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Hidetoshi Sugihara ◽

Naomi Teramoto ◽

Katsuyuki Nakamura ◽

Takanori Shiga ◽

Taku Shirakawa ◽

...

Keyword(s):

Skeletal Muscle ◽

Body Weight ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Muscle Strength ◽

Progenitor Cells ◽

Satellite Cells ◽

Muscle Regeneration ◽

Mesenchymal Progenitor Cells

Abstract Duchenne muscular dystrophy (DMD) is a progressive disease characterised by chronic muscle degeneration and inflammation. Our previously established DMD model rats (DMD rats) have a more severe disease phenotype than the broadly used mouse model. We aimed to investigate the role of senescence in DMD using DMD rats and patients. Senescence was induced in satellite cells and mesenchymal progenitor cells, owing to the increased expression of CDKN2A, p16- and p19-encoding gene. Genetic ablation of p16 in DMD rats dramatically restored body weight and muscle strength. Histological analysis showed a reduction of fibrotic and adipose tissues invading skeletal muscle, with increased muscle regeneration. Senolytic drug ABT263 prevented loss of body weight and muscle strength, and increased muscle regeneration in rats even at 8 months—the late stage of DMD. Moreover, senescence markers were highly expressed in the skeletal muscle of DMD patients. In situ hybridization of CDKN2A confirmed the expression of it in satellite cells and mesenchymal progenitor cells in patients with DMD. Collectively, these data provide new insights into the integral role of senescence in DMD progression.

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