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1.
Int J Mol Sci ; 21(5)2020 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-32155735

RESUMEN

Sarcoglycanopathies are rare limb girdle muscular dystrophies, still incurable, even though symptomatic treatments may slow down the disease progression. Most of the disease-causing defects are missense mutations leading to a folding defective protein, promptly removed by the cell's quality control, even if possibly functional. Recently, we repurposed small molecules screened for cystic fibrosis as potential therapeutics in sarcoglycanopathy. Indeed, cystic fibrosis transmembrane regulator (CFTR) correctors successfully recovered the defective sarcoglycan-complex in vitro. Our aim was to test the combined administration of some CFTR correctors with C17, the most effective on sarcoglycans identified so far, and evaluate the stability of the rescued sarcoglycan-complex. We treated differentiated myogenic cells from both sarcoglycanopathy and healthy donors, evaluating the global rescue and the sarcolemma localization of the mutated protein, by biotinylation assays and western blot analyses. We observed the additive/synergistic action of some compounds, gathering the first ideas on possible mechanism/s of action. Our data also suggest that a defective α-sarcoglycan is competent for assembly into the complex that, if helped in cell traffic, can successfully reach the sarcolemma. In conclusion, our results strengthen the idea that CFTR correctors, acting probably as proteostasis modulators, have the potential to progress as therapeutics for sarcoglycanopathies caused by missense mutations.


Asunto(s)
Aminopiridinas/farmacología , Benzodioxoles/farmacología , Fibras Musculares Esqueléticas/efectos de los fármacos , Mutación , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Sarcoglicanopatías/tratamiento farmacológico , Sarcoglicanos/metabolismo , Fibrosis Quística , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Combinación de Medicamentos , Células HEK293 , Humanos , Fibras Musculares Esqueléticas/metabolismo , Sarcoglicanopatías/genética , Sarcoglicanopatías/metabolismo , Sarcoglicanopatías/patología , Sarcoglicanos/genética
2.
Curr Opin Pharmacol ; 68: 102332, 2023 02.
Artículo en Inglés | MEDLINE | ID: mdl-36566666

RESUMEN

Fibrosis, defined as an excessive accumulation of extracellular matrix, is the end point of a defective regenerative process, unresolved inflammation and/or chronic damage. Numerous muscle disorders (MD) are characterized by high levels of fibrosis associated with muscle wasting and weakness. Fibrosis alters muscle homeostasis/regeneration and fiber environment and may interfere with gene and cell therapies. Slowing down or reversing fibrosis is a crucial therapeutic goal to maintain muscle identity in the context of therapies. Several pathways are implicated in the modulation of the fibrotic progression and multiple therapeutic compounds targeting fibrogenic signals have been tested in MDs, mostly in the context of Duchenne Muscular Dystrophy. In this review, we present an up-to-date overview of pharmacotherapies that have been tested to reduce fibrosis in the skeletal muscle.


Asunto(s)
Músculo Esquelético , Distrofia Muscular de Duchenne , Humanos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Distrofia Muscular de Duchenne/metabolismo , Matriz Extracelular/metabolismo , Fibrosis
3.
Front Cell Dev Biol ; 10: 952041, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36200044

RESUMEN

Skeletal muscle is a highly plastic tissue composed of a number of heterogeneous cell populations that, by interacting and communicating with each other, participate to the muscle homeostasis, and orchestrate regeneration and repair in healthy and diseased conditions. Although muscle regeneration relies on the activity of muscle stem cells (MuSCs), many other cellular players such as inflammatory, vascular and tissue-resident mesenchymal cells participate and communicate with MuSCs to sustain the regenerative process. Among them, Fibro-Adipogenic Progenitors (FAPs), a muscle interstitial stromal population, are crucial actors during muscle homeostasis and regeneration, interacting with MuSCs and other cellular players and dynamically producing and remodelling the extra-cellular matrix. Recent emerging single-cell omics technologies have resulted in the dissection of the heterogeneity of each cell populations within skeletal muscle. In this perspective we have reviewed the recent single-cell omics studies with a specific focus on FAPs in mouse and human muscle. More precisely, using the OutCyte prediction tool, we analysed the "virtual" secretome of FAPs, in resting and regenerating conditions, to highlight the potential of RNAseq data for the study of cellular communication.

4.
J Cachexia Sarcopenia Muscle ; 13(3): 1771-1784, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35319169

RESUMEN

BACKGROUND: Fibrosis is defined as an excessive accumulation of extracellular matrix (ECM) components. Many organs are subjected to fibrosis including the lung, liver, heart, skin, kidney, and muscle. Muscle fibrosis occurs in response to trauma, aging, or dystrophies and impairs muscle function. Fibrosis represents a hurdle for the treatment of human muscular dystrophies. While data on the mechanisms of fibrosis have mostly been investigated in mice, dystrophic mouse models often do not recapitulate fibrosis as observed in human patients. Consequently, the cellular and molecular mechanisms that lead to fibrosis in human muscle still need to be identified. METHODS: Combining mass cytometry, transcriptome profiling, in vitro co-culture experiments, and in vivo transplantation in immunodeficient mice, we investigated the role and nature of nonmyogenic cells (fibroadipogenic progenitors, FAPs) from human fibrotic muscles of healthy individuals (FibMCT ) and individuals with oculopharyngeal muscular dystrophy (OPMD; FibMOP ), as compared with nonmyogenic cells from human nonfibrotic muscle (MCT ). RESULTS: We found that the proliferation rate of FAPs from fibrotic muscle is 3-4 times higher than those of FAPs from nonfibrotic muscle (population doubling per day: MCT 0.2 ± 0.1, FibMCT 0.7 ± 0.1, and FibMOP 0.8 ± 0.3). When cocultured with muscle cells, FAPs from fibrotic muscle impair the fusion index unlike MCT FAPs (myoblasts alone 57.3 ± 11.1%, coculture with MCT 43.1 ± 8.9%, with FibMCT 31.7 ± 8.2%, and with FibMOP 36.06 ± 10.29%). We also observed an increased proliferation of FAPs from fibrotic muscles in these co-cultures in differentiation conditions (FibMCT +17.4%, P < 0.01 and FibMOP +15.1%, P < 0.01). This effect is likely linked to the increased activation of the canonical TGFß-SMAD pathway in FAPs from fibrotic muscles evidenced by pSMAD3 immunostaining (P < 0.05). In addition to the profibrogenic TGFß pathway, we identified endothelin as a new actor implicated in the altered cross-talk between muscle cells and fibrotic FAPs, confirmed by an improvement of the fusion index in the presence of bosentan, an endothelin receptor antagonist (from 33.8 ± 10.9% to 52.9 ± 10.1%, P < 0.05). CONCLUSIONS: Our data demonstrate the key role of FAPs and their cross-talk with muscle cells through a paracrine signalling pathway in fibrosis of human skeletal muscle and identify endothelin as a new druggable target to counteract human muscle fibrosis.


Asunto(s)
Adipogénesis , Distrofia Muscular Oculofaríngea , Animales , Endotelinas/metabolismo , Retroalimentación , Fibrosis , Humanos , Ratones , Fibras Musculares Esqueléticas , Músculo Esquelético/patología , Distrofia Muscular Oculofaríngea/metabolismo , Factor de Crecimiento Transformador beta/metabolismo
5.
Hum Gene Ther ; 31(3-4): 233-240, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31880951

RESUMEN

The adeno-associated virus (AAV) vector is an efficient tool for gene delivery in skeletal muscle. AAV-based therapies show promising results for treatment of various genetic disorders, including muscular dystrophy. These dystrophies represent a heterogeneous group of diseases affecting muscles and typically characterized by progressive skeletal muscle wasting and weakness and the development of fibrosis. The tropism of each AAV serotype has been extensively studied using systemic delivery routes, but very few studies have compared their transduction efficiency through direct intramuscular injection. Yet, in some muscular dystrophies, where only a few muscles are primarily affected, a local intramuscular injection to target these muscles would be the most appropriate route. A comprehensive comparison between different recombinant AAV (rAAV) serotypes is therefore needed. In this study, we investigated the transduction efficiency of rAAV serotypes 1-10 by local injection in skeletal muscle of control C57BL/6 mice. We used a CMV-nls-LacZ reporter cassette allowing nuclear expression of LacZ to easily localize targeted cells. Detection of ß-galactosidase activity on muscle cryosections demonstrated that rAAV serotypes 1, 7, 8, 9, and 10 were more efficient than the others, with rAAV9 being the most efficient in mice. Furthermore, using a model of human muscle xenograft in immunodeficient mice, we observed that in human muscle, rAAV8 and rAAV9 had similar transduction efficiency. These findings demonstrate for the first time that the human muscle xenograft can be used to evaluate AAV-based therapeutical approaches in a human context.


Asunto(s)
Dependovirus/genética , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Músculo Esquelético/metabolismo , Transducción Genética , Animales , Dependovirus/clasificación , Femenino , Expresión Génica , Genes Reporteros , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Humanos , Inyecciones Intramusculares , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Serogrupo , Transgenes
6.
PLoS One ; 14(5): e0211522, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31048846

RESUMEN

Xenotransplantation of human cells into immunodeficient mouse models is a very powerful tool and an essential step for the pre-clinical evaluation of therapeutic cell- and gene- based strategies. Here we describe an optimized protocol combining immunofluorescence and real-time quantitative PCR to both quantify and visualize the fate and localization of human myogenic cells after injection in regenerating muscles of immunodeficient mice. Whereas real-time quantitative PCR-based method provides an accurate quantification of human cells, it does not document their specific localization. The addition of an immunofluorescence approach using human-specific antibodies recognizing engrafted human cells gives information on the localization of the human cells within the host muscle fibres, in the stem cell niche or in the interstitial space. These two combined approaches offer an accurate evaluation of human engraftment including cell number and localization and should provide a gold standard to compare results obtained either using different types of human stem cells or comparing healthy and pathological muscle stem cells between different research laboratories worldwide.


Asunto(s)
Mioblastos/citología , Mioblastos/metabolismo , Animales , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/metabolismo , Masculino , Ratones , Ratones SCID , Modelos Teóricos , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Reacción en Cadena de la Polimerasa , Células Madre/citología , Células Madre/metabolismo
7.
Nat Commun ; 10(1): 1974, 2019 04 29.
Artículo en Inglés | MEDLINE | ID: mdl-31036801

RESUMEN

Caveolin-3 is the major structural protein of caveolae in muscle. Mutations in the CAV3 gene cause different types of myopathies with altered membrane integrity and repair, expression of muscle proteins, and regulation of signaling pathways. We show here that myotubes from patients bearing the CAV3 P28L and R26Q mutations present a dramatic decrease of caveolae at the plasma membrane, resulting in abnormal response to mechanical stress. Mutant myotubes are unable to buffer the increase in membrane tension induced by mechanical stress. This results in impaired regulation of the IL6/STAT3 signaling pathway leading to its constitutive hyperactivation and increased expression of muscle genes. These defects are fully reversed by reassembling functional caveolae through expression of caveolin-3. Our study reveals that under mechanical stress the regulation of mechanoprotection by caveolae is directly coupled with the regulation of IL6/STAT3 signaling in muscle cells and that this regulation is absent in Cav3-associated dystrophic patients.


Asunto(s)
Caveolas/metabolismo , Caveolina 3/genética , Caveolina 3/metabolismo , Interleucina-6/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/metabolismo , Factor de Transcripción STAT3/metabolismo , Línea Celular , Humanos , Interleucina-6/genética , Mecanotransducción Celular , Fibras Musculares Esqueléticas/patología , Mutación/genética , Factor de Transcripción STAT3/genética
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