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Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.
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Actinina/genética , Músculo Esquelético/fisiologia , Anaerobiose , Animais , Animais Recém-Nascidos , Atletas , Calcineurina/metabolismo , Dependovirus/metabolismo , Regulação para Baixo/genética , Estudo de Associação Genômica Ampla , Heterozigoto , Homozigoto , Humanos , Camundongos Endogâmicos C57BL , Fadiga Muscular , Fibras Musculares Esqueléticas/metabolismo , Tamanho do Órgão , OxirreduçãoRESUMO
Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Distrofina/farmacologia , Éxons/efeitos dos fármacos , Morfolinos/farmacologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , Modelos Animais de Doenças , Distrofina/genética , Éxons/genética , Terapia Genética/métodos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genéticaRESUMO
Repair of skeletal muscle after sarcolemmal damage involves dysferlin and dysferlin-interacting proteins such as annexins. Mice and patient lacking dysferlin exhibit chronic muscle inflammation and adipogenic replacement of the myofibers. Here, we show that similar to dysferlin, lack of annexin A2 (AnxA2) also results in poor myofiber repair and progressive muscle weakening with age. By longitudinal analysis of AnxA2-deficient muscle we find that poor myofiber repair due to the lack of AnxA2 does not result in chronic inflammation or adipogenic replacement of the myofibers. Further, deletion of AnxA2 in dysferlin deficient mice reduced muscle inflammation, adipogenic replacement of myofibers, and improved muscle function. These results identify multiple roles of AnxA2 in muscle repair, which includes facilitating myofiber repair, chronic muscle inflammation and adipogenic replacement of dysferlinopathic muscle. It also identifies inhibition of AnxA2-mediated inflammation as a novel therapeutic avenue for treating muscle loss in dysferlinopathy.
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Anexina A2/metabolismo , Anexina A2/fisiologia , Adipogenia , Animais , Anexina A2/genética , Disferlina , Inflamação/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/terapia , Miofibrilas/fisiologia , Sarcolema/metabolismoRESUMO
A common null polymorphism (R577X) in ACTN3 causes α-actinin-3 deficiency in â¼ 18% of the global population. There is no associated disease phenotype, but α-actinin-3 deficiency is detrimental to sprint and power performance in both elite athletes and the general population. However, despite considerable investigation to date, the functional consequences of heterozygosity for ACTN3 are unclear. A subset of studies have shown an intermediate phenotype in 577RX individuals, suggesting dose-dependency of α-actinin-3, while others have shown no difference between 577RR and RX genotypes. Here, we investigate the effects of α-actinin-3 expression level by comparing the muscle phenotypes of Actn3(+/-) (HET) mice to Actn3(+/+) [wild-type (WT)] and Actn3(-/-) [knockout (KO)] littermates. We show reduction in α-actinin-3 mRNA and protein in HET muscle compared with WT, which is associated with dose-dependent up-regulation of α-actinin-2, z-band alternatively spliced PDZ-motif and myotilin at the Z-line, and an incremental shift towards oxidative metabolism. While there is no difference in force generation, HET mice have an intermediate endurance capacity compared with WT and KO. The R577X polymorphism is associated with changes in ACTN3 expression consistent with an additive model in the human genotype-tissue expression cohort, but does not influence any other muscle transcripts, including ACTN2. Overall, ACTN3 influences sarcomeric composition in a dose-dependent fashion in mouse skeletal muscle, which translates directly to function. Variance in fibre type between biopsies likely masks this phenomenon in human skeletal muscle, but we suggest that an additive model is the most appropriate for use in testing ACTN3 genotype associations.
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Actinina/genética , Dosagem de Genes , Músculo Esquelético/metabolismo , Resistência Física/genética , Polimorfismo Genético , Actinina/deficiência , Actinina/metabolismo , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Heterozigoto , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Condicionamento Físico Animal , Sarcômeros/metabolismoRESUMO
Fibro/adipogenic progenitors (FAPs) are skeletal muscle stromal cells that support regeneration of injured myofibers and their maintenance in healthy muscles. FAPs are related to mesenchymal stem cells (MSCs/MeSCs) found in other adult tissues, but there is poor understanding of the extent of similarity between these cells. Using single-cell RNA sequencing (scRNA-seq) datasets from multiple mouse tissues, we have performed comparative transcriptomic analysis. This identified remarkable transcriptional similarity between FAPs and MeSCs, confirmed the suitability of PDGFRα as a reporter for FAPs, and identified extracellular proteolysis as a new FAP function. Using PDGFRα as a cell surface marker, we isolated FAPs from healthy and dysferlinopathic mouse muscles and performed scRNA-seq analysis. This revealed decreased FAP-mediated Wnt signaling as a potential driver of FAP dysfunction in dysferlinopathic muscles. Analysis of FAPs in dysferlin- and dystrophin-deficient muscles identified a relationship between the nature of muscle pathology and alteration in FAP gene expression.
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Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.
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Macrófagos , Transcriptoma , Camundongos , Animais , Humanos , Camundongos Endogâmicos C57BL , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , FibroseRESUMO
The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.
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Aside from myofibers, numerous mononucleated cells reside in the skeletal muscle. These include the mesenchymal cells called fibro-adipogenic progenitors (FAPs), that support muscle development and regeneration in adult muscles. Recent evidence shows that defects in FAP function contributes to chronic muscle diseases and targeting FAPs offers avenues for treating these diseases.
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Adipogenia , Células-Tronco Mesenquimais , Adulto , Diferenciação Celular , Humanos , Desenvolvimento Muscular , Músculo EsqueléticoRESUMO
Mutations in the Anoctamin 5 (Ano5) gene that result in the lack of expression or function of ANO5 protein, cause Limb Girdle Muscular Dystrophy (LGMD) 2L/R12, and Miyoshi Muscular Dystrophy (MMD3). However, the dystrophic phenotype observed in patient muscles is not uniformly recapitulated by ANO5 knockout in animal models of LGMD2L. Here we describe the generation of a mouse model of LGMD2L generated by targeted out-of-frame deletion of the Ano5 gene. This model shows progressive muscle loss, increased muscle weakness, and persistent bouts of myofiber regeneration without chronic muscle inflammation, which recapitulates the mild to moderate skeletal muscle dystrophy reported in the LGMD2L patients. We show that these features of ANO5 deficient muscle are not associated with a change in the calcium-activated sarcolemmal chloride channel activity or compromised in vivo regenerative myogenesis. Use of this mouse model allows conducting in vivo investigations into the functional role of ANO5 in muscle health and for preclinical therapeutic development for LGMD2L.
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Anoctaminas/genética , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Animais , Canais de Cloreto/genética , Modelos Animais de Doenças , Camundongos , Camundongos Knockout , Debilidade Muscular/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação , FenótipoRESUMO
Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-ß activity. While anti-TGF-ß therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-ß activity in the severe, but not in the mild, dystrophic models. Increased TGF-ß activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-ß signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-ß as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.
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Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Fator de Crescimento Transformador beta/metabolismo , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Desenvolvimento Muscular/fisiologia , Regeneração/fisiologiaRESUMO
Muscle loss due to fibrotic or adipogenic replacement of myofibers is common in muscle diseases and muscle-resident fibro/adipogenic precursors (FAPs) are implicated in this process. While FAP-mediated muscle fibrosis is widely studied in muscle diseases, the role of FAPs in adipogenic muscle loss is not well understood. Adipogenic muscle loss is a feature of limb girdle muscular dystrophy 2B (LGMD2B) - a disease caused by mutations in dysferlin. Here we show that FAPs cause the adipogenic loss of dysferlin deficient muscle. Progressive accumulation of Annexin A2 (AnxA2) in the myofiber matrix causes FAP differentiation into adipocytes. Lack of AnxA2 prevents FAP adipogenesis, protecting against adipogenic loss of dysferlinopathic muscle while exogenous AnxA2 enhances muscle loss. Pharmacological inhibition of FAP adipogenesis arrests adipogenic replacement and degeneration of dysferlin-deficient muscle. These results demonstrate the pathogenic role of FAPs in LGMD2B and establish these cells as therapeutic targets to ameliorate muscle loss in patients.
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Tecido Adiposo/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Células-Tronco/citologia , Adipócitos/patologia , Adipogenia/efeitos dos fármacos , Tecido Adiposo/patologia , Adolescente , Idade de Início , Animais , Anexina A2/metabolismo , Estudos de Casos e Controles , Disferlina/genética , Venenos Elapídicos/toxicidade , Feminino , Fibrose , Humanos , Técnicas In Vitro , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Índice de Gravidade de Doença , Células-Tronco/efeitos dos fármacos , Tiofenos/farmacologia , Adulto JovemRESUMO
Muscle function is regulated by Ca2+, which mediates excitation-contraction coupling, energy metabolism, adaptation to exercise, and sarcolemmal repair. Several of these actions rely on Ca2+ delivery to the mitochondrial matrix via the mitochondrial Ca2+ uniporter, the pore of which is formed by mitochondrial calcium uniporter (MCU). MCU's gatekeeping and cooperative activation are controlled by MICU1. Loss-of-protein mutation in MICU1 causes a neuromuscular disease. To determine the mechanisms underlying the muscle impairments, we used MICU1 patient cells and skeletal muscle-specific MICU1 knockout mice. Both these models show a lower threshold for MCU-mediated Ca2+ uptake. Lack of MICU1 is associated with impaired mitochondrial Ca2+ uptake during excitation-contraction, aerobic metabolism impairment, muscle weakness, fatigue, and myofiber damage during physical activity. MICU1 deficit compromises mitochondrial Ca2+ uptake during sarcolemmal injury, which causes ineffective repair of the damaged myofibers. Thus, dysregulation of mitochondrial Ca2+ uptake hampers myofiber contractile function, likely through energy metabolism and membrane repair.
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Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Debilidade Muscular/metabolismo , Sarcolema/patologia , Síndrome de Emaciação/metabolismo , Adolescente , Adulto , Animais , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Transporte de Cátions/deficiência , Membrana Celular/metabolismo , Citosol/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Homeostase , Humanos , Masculino , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Modelos Biológicos , Contração Muscular , Debilidade Muscular/complicações , Debilidade Muscular/patologia , Músculo Esquelético/metabolismo , Atrofia Muscular/complicações , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Sarcolema/metabolismo , Tétano , Síndrome de Emaciação/complicações , Síndrome de Emaciação/patologiaRESUMO
The originally published version of this Article contained an error in Figure 6. In panel b, the top graph (BrdU 21-24d) and the bottom graph (BrdU 28-31d) were inadvertently swapped. This error has now been corrected in both the PDF and HTML versions of the Article.
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In the original version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to include support from the CRI Light Microscopy and Image Analysis Core.
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Strain and physical trauma to mechanically active cells, such as skeletal muscle myofibers, injures their plasma membranes, and mitochondrial function is required for their repair. We found that mitochondrial function was also needed for plasma membrane repair in myoblasts as well as nonmuscle cells, which depended on mitochondrial uptake of calcium through the mitochondrial calcium uniporter (MCU). Calcium uptake transiently increased the mitochondrial production of reactive oxygen species (ROS), which locally activated the guanosine triphosphatase (GTPase) RhoA, triggering F-actin accumulation at the site of injury and facilitating membrane repair. Blocking mitochondrial calcium uptake or ROS production prevented injury-triggered RhoA activation, actin polymerization, and plasma membrane repair. This repair mechanism was shared between myoblasts, nonmuscle cells, and mature skeletal myofibers. Quenching mitochondrial ROS in myofibers during eccentric exercise ex vivo caused increased damage to myofibers, resulting in a greater loss of muscle force. These results suggest a physiological role for mitochondria in plasma membrane repair in injured cells, a role that highlights a beneficial effect of ROS.
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Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Oxirredução , Transdução de Sinais , Actinas/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Condicionamento Físico Animal , Espécies Reativas de Oxigênio/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and progressive weakness. There is considerable inter-patient variability in disease onset and progression, which can confound the results of clinical trials. Here we show that a common null polymorphism (R577X) in ACTN3 results in significantly reduced muscle strength and a longer 10 m walk test time in young, ambulant patients with DMD; both of which are primary outcome measures in clinical trials. We have developed a double knockout mouse model, which also shows reduced muscle strength, but is protected from stretch-induced eccentric damage with age. This suggests that α-actinin-3 deficiency reduces muscle performance at baseline, but ameliorates the progression of dystrophic pathology. Mechanistically, we show that α-actinin-3 deficiency triggers an increase in oxidative muscle metabolism through activation of calcineurin, which likely confers the protective effect. Our studies suggest that ACTN3 R577X genotype is a modifier of clinical phenotype in DMD patients.
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Actinina/genética , Calcineurina/genética , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/genética , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Actinina/deficiência , Animais , Calcineurina/metabolismo , Modelos Animais de Doenças , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Estudos Longitudinais , Masculino , Camundongos , Camundongos Endogâmicos mdx , Camundongos Knockout , Fibras Musculares Esqueléticas/patologia , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/mortalidade , Distrofia Muscular de Duchenne/patologia , Mutação , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fenótipo , Transdução de Sinais , Análise de SobrevidaRESUMO
OBJECTIVE: To identify changes in skeletal muscle microRNA expression after endurance exercise and associate the identified microRNAs with mRNA and protein expression to disease-specific pathways in polymyositis (PM) and dermatomyositis (DM) patients. METHODS: Following a parallel clinical trial design, patients with probable PM or DM, exercising less than once a week, and on stable medication for at least one month were randomized into two groups at Karolinska University Hospital: a 12-week endurance exercise group (n = 12) or a non-exercised control group (n = 11). Using an Affymetrix microarray, microRNA expression was determined in paired muscle biopsies taken before and after the exercise intervention from 3 patients in each group. Ingenuity pathway analysis with a microRNA target filter was used to identify microRNA transcript targets. These targets were investigated at the mRNA (microarray) and protein (mass spectrometry) levels in patients. RESULTS: Endurance exercise altered 39 microRNAs. The microRNAs with increased expression were predicted to target transcripts involved in inflammatory processes, metabolism, and muscle atrophy. Further, these target transcripts had an associated decrease in mRNA expression in exercised patients. In particular, a decrease in the NF-κB regulator IKBKB was associated with an increase in its target microRNA (miR-196b). At the protein level, there was an increase in mitochondrial proteins (AK3, HIBADH), which were associated with a decrease in microRNAs that were predicted to regulate their expression. CONCLUSION: Improvement in disease phenotype after exercise is associated with increasing microRNAs that target and downregulate immune processes at the transcript level, as well as decreasing microRNAs that target and upregulate mitochondrial content at the protein level. Therefore, microRNAs may improve disease by decreasing immune responses and increasing mitochondrial biogenesis. TRIAL REGISTRATION: ClinicalTrials.gov NCT01184625.
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Exercício Físico , MicroRNAs/genética , Miosite/genética , Resistência Física/fisiologia , Humanos , Miosite/fisiopatologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.Exon skipping is a strategy for the treatment of Duchenne muscular dystrophy, but has variable efficacy. Here, the authors show that dystrophin restoration occurs preferentially in areas of myofiber regeneration, where antisense oligonucleotides are stored in macrophages and delivered to myoblasts and newly formed myotubes.
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Distrofina/genética , Macrófagos/metabolismo , Morfolinos/uso terapêutico , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Animais , Modelos Animais de Doenças , Éxons , Técnicas de Transferência de Genes , Terapia Genética , CamundongosRESUMO
Dystrophin deficiency is the genetic basis for Duchenne muscular dystrophy (DMD), but the cellular basis of progressive myofiber death in DMD is not fully understood. Using two dystrophin-deficient mdx mouse models, we find that the mitochondrial dysfunction is among the earliest cellular deficits of mdx muscles. Mitochondria in dystrophic myofibers also respond poorly to sarcolemmal injury. These mitochondrial deficits reduce the ability of dystrophic muscle cell membranes to repair and are associated with a compensatory increase in dysferlin-mediated membrane repair proteins. Dysferlin deficit in mdx mice further compromises myofiber cell membrane repair and enhances the muscle pathology at an asymptomatic age for dysferlin-deficient mice. Restoring partial dystrophin expression by exon skipping improves mitochondrial function and offers potential to improve myofiber repair. These findings identify that mitochondrial deficit in muscular dystrophy compromises the repair of injured myofibers and show that this repair mechanism is distinct from and complimentary to the dysferlin-mediated repair of injured myofibers.
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Membrana Celular/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular , Disferlina/deficiência , Disferlina/genética , Distrofina/antagonistas & inibidores , Distrofina/genética , Distrofina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Interleucina-1beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Contração Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Mioblastos/citologia , Mioblastos/metabolismo , Oligodesoxirribonucleotídeos Antissenso/metabolismo , Ácido Pirúvico/farmacologia , Imagem com Lapso de TempoRESUMO
BACKGROUND: Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials. METHODS: In the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays. RESULTS: The triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression. CONCLUSIONS: While we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.