In Duchenne muscular dystrophy (DMD), sarcolemma fragility and myofiber necrosis produce cellular debris that attract inflammatory cells. Macrophages and T-lymphocytes infiltrate muscles in response ...to damage-associated molecular pattern signalling and the release of TNF-α, TGF-β and interleukins prevent skeletal muscle improvement from the inflammation. This immunological scenario was extended by the discovery of a specific response to muscle antigens and a role for regulatory T cells (Tregs) in muscle regeneration. Normally, autoimmunity is avoided by autoreactive T-lymphocyte deletion within thymus, while in the periphery Tregs monitor effector T-cells escaping from central regulatory control. Here, we report impairment of thymus architecture of mdx mice together with decreased expression of ghrelin, autophagy dysfunction and AIRE down-regulation. Transplantation of dystrophic thymus in recipient nude mice determine the up-regulation of inflammatory/fibrotic markers, marked metabolic breakdown that leads to muscle atrophy and loss of force. These results indicate that involution of dystrophic thymus exacerbates muscular dystrophy by altering central immune tolerance.
Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of ...adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.
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•Single-cell RNA-seq reveals an altered cell landscape in dystrophic skeletal muscle•Classification of interstitial stem cell states from healthy and dystrophic muscle•Adipo-regulatory cells (Aregs) block adipogenesis through GDF10 secretion•The amount of Aregs decreases in dystrophic muscle, thereby increasing fat depots
Camps et al. describe a cell population that blocks fat turnover in the skeletal muscle by single-cell analysis. Furthermore, they show that this population acts through GDF10 secretion and is reduced in dystrophic muscle. This could be an essential turning point in the progressive deterioration of muscular dystrophy patients.
Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead ...express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.
The definition of cell metabolic profile is essential to ensure skeletal muscle fiber heterogeneity and to achieve a proper equilibrium between the self-renewal and commitment of satellite stem ...cells. Heme sustains several biological functions, including processes profoundly implicated with cell metabolism. The skeletal muscle is a significant heme-producing body compartment, but the consequences of impaired heme homeostasis on this tissue have been poorly investigated. Here, we generate a skeletal-muscle-specific feline leukemia virus subgroup C receptor 1a (FLVCR1a) knockout mouse model and show that, by sustaining heme synthesis, FLVCR1a contributes to determine the energy phenotype in skeletal muscle cells and to modulate satellite cell differentiation and muscle regeneration.
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•FLVCR1a loss in skeletal muscle impairs tissue features and physical performance•FLVCR1a tunes heme synthesis, impacting myofiber identity and mitochondrial function•By sustaining heme synthesis, FLVCR1a controls satellite cell differentiation
Controlling metabolic pathways vital for muscle function and regeneration is a promising strategy to counteract muscle disease. Here, Mistretta and Fiorito et al. analyze the consequences of FLVCR1a deficiency in skeletal muscle and demonstrate that, by sustaining heme synthesis, FLVCR1a shapes myofiber energy phenotype and regulates satellite cell differentiation.
α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG ...glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP -ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a "glycan remodelling" process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase.
In recent years, numerous reports have identified in mouse different sources of myogenic cells distinct from satellite cells that exhibited a variable myogenic potential in vivo. Myogenic stem cells ...have also been described in humans, although their regenerative potential has rarely been quantified. In this study, we have investigated the myogenic potential of human muscle–derived cells based on the expression of the stem cell marker CD133 as compared to bona fide satellite cells already used in clinical trials. The efficiency of these cells to participate in muscle regeneration and contribute to the renewal of the satellite cell pool, when injected intramuscularly, has been evaluated in the Rag2−/− γC−/− C5−/− mouse in which muscle degeneration is induced by cryoinjury. We demonstrate that human muscle–derived CD133+ cells showed a much greater regenerative capacity when compared to human myoblasts. The number of fibers expressing human proteins and the number of human cells in a satellite cell position are all dramatically increased when compared to those observed after injection of human myoblasts. In addition, CD133+/CD34+ cells exhibited a better dispersion in the host muscle when compared to human myoblasts. We propose that muscle-derived CD133+ cells could be an attractive candidate for cellular therapy.
Nutritional compounds can exert both anti-inflammatory and anti-oxidant effects. Since these events exacerbate the pathophysiology of muscular dystrophies, we investigated nutraceutical ...supplementation as an adjuvant therapy in dystrophic patients, to low costs and easy route of administration. Moreover, this treatment could represent an alternative therapeutic strategy for dystrophic patients who do not respond to corticosteroid treatment.
A 24 weeks randomized double-blind placebo-controlled clinical study was aimed at evaluating the safety and efficacy of daily oral administration of flavonoids- and omega3-based natural supplement (FLAVOMEGA) in patients affected by muscular dystrophy with recognized muscle inflammation.
We screened 60 patients diagnosed for Duchenne (DMD), Facioscapulohumeral (FSHD), and Limb Girdle Muscular Dystrophy (LGMD). Using a computer-generated random allocation sequence, we stratified patients in a 2:1:1 ratio (DMD:FSHD:LGMD) to one of two treatment groups: continuous FLAVOMEGA, continuous placebo. Of 29 patients included, only 24 completed the study: 15 were given FLAVOMEGA, 14 placebo.
FLAVOMEGA was well tolerated with no reported adverse events. Significant treatment differences in the change from baseline in 6 min walk distance (6MWD; secondary efficacy endpoint) (
= 0.033) and in isokinetic knee extension (
= 0.039) (primary efficacy endpoint) were observed in LGMD and FSHD subjects. Serum CK levels (secondary efficacy endpoint) decreased in all FLAVOMEGA treated groups with significant difference in DMD subjects (
= 0.039).
Although the small number of patients and the wide range of disease severity among patients reduced statistical significance, we obtained an optimal profile of safety and tolerability for the compound, showing valuable data of efficacy in primary and secondary endpoints.
NCT03317171 Retrospectively registered 25/10/2017.
Duchenne muscular dystrophy (DMD) is an X-linked syndrome that affects skeletal and cardiac muscle and is caused by mutation of the dystrophin gene. Induced pluripotent stem cells (iPSCs) were ...generated from dermal fibroblasts by electroporation with episomal vectors containing the reprogramming factors (OCT4, SOX2, LIN28, KLF4, and l-MYC). The donor carried an out-of-frame deletion of exons 45–50 of the dystrophin gene. The established iPSC line exhibited normal morphology, expressed pluripotency markers, had normal karyotype and possessed trilineage differentiation potential.
Duchenne muscular dystrophy (DMD) is a debilitating fatal X‐linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, ...insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store‐operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti‐IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) and removal of intracellular Ca2+. Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.
Synopsis
IGF2R is over‐expressed in Duchenne Muscular Dystrophy (DMD) and mdx muscles. Blockade of IGF2R rescued the dystrophic muscle phenotype, ameliorated vascular architecture defects and improved muscle force.
IGF2R expression is increased in dystrophic muscles and binds to CD20.
Blockade of IGF2R facilitates IGF2‐IGF1R interactions and activates CD20 phosphorylation, promoting the entrance of Ca2+ ions in the sarcoplasm.
Increasing levels of Ca2+ ions regulate calcineurin/CaMKII pathway and activates SERCA1 leading to in vitro premature myogenic differentiation and in vivo increased force production and vasculature remodeling.
Blockade of IGF2R reestablished the correct oscillating pattern of Ca2+ ion levels in the microenvironment of myofibrils and protects from muscular dystrophy acting on different mechanisms of dystrophic muscles.
Despite this variety of mechanisms of action, monoclonal antibodies can be used in DMD to normalize the over‐expression of IGF2R and the availability of muscle IGFs.
IGF2R is over‐expressed in Duchenne Muscular Dystrophy (DMD) and mdx muscles. Blockade of IGF2R rescued the dystrophic muscle phenotype, ameliorated vascular architecture defects and improved muscle force.
Duchenne muscular dystrophy (DMD) is a common X-linked disease characterized by widespread muscle damage that invariably leads to paralysis and death. There is currently no therapy for this disease. ...Here we report that a subpopulation of circulating cells expressing AC133, a well-characterized marker of hematopoietic stem cells, also expresses early myogenic markers. Freshly isolated, circulating AC133(+) cells were induced to undergo myogenesis when cocultured with myogenic cells or exposed to Wnt-producing cells in vitro and when delivered in vivo through the arterial circulation or directly into the muscles of transgenic scid/mdx mice (which allow survival of human cells). Injected cells also localized under the basal lamina of host muscle fibers and expressed satellite cell markers such as M-cadherin and MYF5. Furthermore, functional tests of injected muscles revealed a substantial recovery of force after treatment. As these cells can be isolated from the blood, manipulated in vitro, and delivered through the circulation, they represent a possible tool for future cell therapy applications in DMD disease or other muscular dystrophies.
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