RESUMO
Gene editing is often touted as a permanent method for correcting mutations, but its long-term benefits in Duchenne muscular dystrophy (DMD) may depend on sufficiently high editing efficiencies to halt muscle degeneration. Here, we explored the persistence of dystrophin expression following recombinant adeno-associated virus serotype 6 (rAAV6):CRISPR-Cas9-mediated multi-exon deletion/reframing in systemically injected 2- and 11-week-old dystrophic mice and show that induction of low dystrophin levels persists for several months in cardiomyocytes but not in skeletal muscles, where myofibers remain susceptible to necrosis and regeneration. Whereas gene-correction efficiency in both muscle types was enhanced with increased ratios of guide RNA (gRNA)-to-nuclease vectors, obtaining high dystrophin levels in skeletal muscles via multi-exon deletion remained challenging. In contrast, when AAV-microdystrophin was codelivered with editing components, long-term gene-edited dystrophins persisted in both muscle types. These results suggest that the high rate of necrosis and regeneration in skeletal muscles, compared with the relative stability of dystrophic cardiomyocytes, caused the rapid loss of edited genomes. Consequently, stable dystrophin expression in DMD skeletal muscles will require either highly efficient gene editing or the use of cotreatments that decrease skeletal muscle degeneration.
Assuntos
Distrofina/genética , Edição de Genes , Vetores Genéticos/administração & dosagem , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/prevenção & controle , Distrofia Muscular de Duchenne/prevenção & controle , Miocárdio/metabolismo , Animais , Sistemas CRISPR-Cas , Dependovirus/genética , Modelos Animais de Doenças , Distrofina/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , RNA Guia de Sistemas CRISPR-CasRESUMO
Oxidative stress and inflammatory processes strongly contribute to pathogenesis in Duchenne muscular dystrophy (DMD). Based on evidence that excess iron may increase oxidative stress and contribute to the inflammatory response, we investigated whether deferoxamine (DFX), a potent iron chelating agent, reduces oxidative stress and inflammation in the diaphragm (DIA) muscle of mdx mice (an experimental model of DMD). Fourteen-day-old mdx mice received daily intraperitoneal injections of DFX at a dose of 150 mg/kg body weight, diluted in saline, for 14 days. C57BL/10 and control mdx mice received daily intraperitoneal injections of saline only, for 14 days. Grip strength was evaluated as a functional measure, and blood samples were collected for biochemical assessment of muscle fiber degeneration. In addition, the DIA muscle was removed and processed for histopathology and Western blotting analysis. In mdx mice, DFX reduced muscle damage and loss of muscle strength. DFX treatment also resulted in a significant reduction of dystrophic inflammatory processes, as indicated by decreases in the inflammatory area and in NF-κB levels. DFX significantly decreased oxidative damage, as shown by lower levels of 4-hydroxynonenal and a reduction in dihydroethidium staining in the DIA muscle of mdx mice. The results of the present study suggest that DFX may be useful in therapeutic strategies to ameliorate dystrophic muscle pathology, possibly via mechanisms involving oxidative and inflammatory pathways.
Assuntos
Desferroxamina/farmacologia , Inflamação/prevenção & controle , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Animais , Western Blotting , Peso Corporal/efeitos dos fármacos , Desferroxamina/administração & dosagem , Diafragma/efeitos dos fármacos , Diafragma/metabolismo , Feminino , Inflamação/metabolismo , Injeções Intraperitoneais , Quelantes de Ferro/administração & dosagem , Quelantes de Ferro/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Força Muscular/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/prevenção & controle , Distrofia Muscular de Duchenne/metabolismo , NF-kappa B/metabolismoRESUMO
Receptor-activator of NF-κB, its ligand RANKL, and the soluble decoy receptor osteoprotegerin are the key regulators of osteoclast differentiation and bone remodeling. Although there is a strong association between osteoporosis and skeletal muscle atrophy/dysfunction, the functional relevance of a particular biological pathway that synchronously regulates bone and skeletal muscle physiopathology still is elusive. Here, we show that muscle cells can produce and secrete osteoprotegerin and pharmacologic treatment of dystrophic mdx mice with recombinant osteoprotegerin muscles. (Recombinant osteoprotegerin-Fc mitigates the loss of muscle force in a dose-dependent manner and preserves muscle integrity, particularly in fast-twitch extensor digitorum longus.) Our data identify osteoprotegerin as a novel protector of muscle integrity, and it potentially represents a new therapeutic avenue for both muscular diseases and osteoporosis.
Assuntos
Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/prevenção & controle , Osteoprotegerina/metabolismo , Animais , Linhagem Celular , Fragmentos Fc das Imunoglobulinas/metabolismo , Técnicas In Vitro , Inflamação/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculos/efeitos dos fármacos , Músculos/metabolismo , Músculos/patologia , Músculos/fisiopatologia , Distrofia Muscular Animal/fisiopatologiaRESUMO
Duchenne muscular dystrophy is a fatal muscle wasting disease that is characterized by a deficiency in the protein dystrophin. Previously, we reported that the expression of hematopoietic prostaglandin D synthase (HPGDS) appeared in necrotic muscle fibers from patients with either Duchenne muscular dystrophy or polymyositis. HPGDS is responsible for the production of the inflammatory mediator, prostaglandin D(2). In this paper, we validated the hypothesis that HPGDS has a role in the etiology of muscular necrosis. We investigated the expression of HPGDS/ prostaglandin D(2) signaling using two different mouse models of muscle necrosis, that is, bupivacaine-induced muscle necrosis and the mdx mouse, which has a genetic muscular dystrophy. We treated each mouse model with the HPGDS-specific inhibitor, HQL-79, and measured both necrotic muscle volume and selected cytokine mRNA levels. We confirmed that HPGDS expression was induced in necrotic muscle fibers in both bupivacaine-injected muscle and mdx mice. After administration of HQL-79, necrotic muscle volume was significantly decreased in both mouse models. Additionally, mRNA levels of both CD11b and transforming growth factor beta1 were significantly lower in HQL-79-treated mdx mice than in vehicle-treated animals. We also demonstrated that HQL-79 suppressed prostaglandin D(2) production and improved muscle strength in the mdx mouse. Our results show that HPGDS augments inflammation, which is followed by muscle injury. Furthermore, the inhibition of HPGDS ameliorates muscle necrosis even in cases of genetic muscular dystrophy.
Assuntos
Modelos Animais de Doenças , Oxirredutases Intramoleculares/antagonistas & inibidores , Lipocalinas/antagonistas & inibidores , Distrofia Muscular Animal/prevenção & controle , Anestésicos Locais/toxicidade , Animais , Western Blotting , Bupivacaína/toxicidade , Citocinas/genética , Humanos , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/genética , Lipocalinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Camundongos Transgênicos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/enzimologia , Músculo Esquelético/patologia , Distrofia Muscular Animal/induzido quimicamente , Distrofia Muscular Animal/enzimologia , Necrose , Piperidinas/farmacologia , Prostaglandina D2/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaAssuntos
Acetilcisteína/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Animais , Caveolina 3/metabolismo , Modelos Animais de Doenças , Distroglicanas/metabolismo , Distrofina/genética , Distrofina/metabolismo , Sequestradores de Radicais Livres/farmacologia , Camundongos , Camundongos Endogâmicos mdx , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Distrofia Muscular Animal/etiologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/prevenção & controle , Mutação/genéticaRESUMO
Duchenne muscular dystrophy (DMD) is a severe degenerative muscle disease caused by a mutation in the gene encoding dystrophin, a protein linking the cytoskeleton to the extracellular matrix. In this study we investigated whether the antioxidant N-acetylcysteine (NAC) provided protection against dystrophic muscle damage in the mdx mouse, an animal model of DMD. In isolated mdx muscles, NAC prevented the increased membrane permeability and reduced the force deficit associated with stretch-induced muscle damage. Three-week-old mdx mice were treated with NAC in the drinking water for 6 weeks. Dihydroethidium staining showed that NAC treatment reduced the concentration of reactive oxygen species (ROS) in mdx muscles. This was accompanied by a significant decrease in centrally nucleated fibres in muscles from NAC-treated mdx mice. Immunoblotting showed that NAC treatment decreased the nuclear protein expression of NF-kappaB, a transcription factor involved in pro-inflammatory cytokine expression. Finally, we show that NAC treatment reduced caveolin-3 protein levels and increased the sarcolemmal expression of beta-dystroglycan and the dystrophin homologue, utrophin. Taken together, our findings suggest that ROS play an important role in the dystrophic pathogenesis, both in terms of activating damage pathways and in regulating the expression of some dystrophin-associated membrane proteins. These results offer the prospect that antioxidants such as NAC could have therapeutic potential for DMD patients.
Assuntos
Acetilcisteína/uso terapêutico , Sequestradores de Radicais Livres/uso terapêutico , Músculo Esquelético/fisiopatologia , Distrofia Muscular Animal/prevenção & controle , Acetilcisteína/farmacologia , Animais , Caveolina 3/metabolismo , Modelos Animais de Doenças , Distroglicanas/metabolismo , Distrofina/genética , Distrofina/metabolismo , Sequestradores de Radicais Livres/farmacologia , Camundongos , Camundongos Endogâmicos mdx , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/etiologia , Distrofia Muscular Animal/fisiopatologia , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/prevenção & controle , Mutação/genética , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The mdx mouse lacks dystrophin and is a model of human Duchenne muscular dystrophy. Single mdx muscle fibres were isolated and subjected to a series of stretched (eccentric) contractions while measuring intracellular calcium concentration ([Ca(2+)](i)) with fluo-3 and confocal microscopy. Following the stretched contractions there was a slow rise in resting [Ca(2+)](i) and after 30 min both the [Ca(2+)](i) during a tetanus (tetanic [Ca(2+)](i)) and the tetanic force were reduced. Two blockers of stretch-activated channels, streptomycin and the spider venom toxin GsMTx4, prevented the rise of resting [Ca(2+)](i) and partially prevented the decline of tetanic [Ca(2+)](i) and force. Reducing extracellular calcium to zero also prevented the rise in resting [Ca(2+)](i) and prevented some of the decline in tetanic [Ca(2+)](i) and force. Patch-clamping experiments identified a stretch-activated channel in both wild-type and mdx myotubes which was blocked by GsMTx4. These data suggest that blockers of stretch-activated channels can ameliorate the force reduction following stretched contractions by reducing the influx of Ca(2+) into the muscle. We therefore tested whether in intact mdx mice streptomycin, added to the drinking water, was capable of reducing muscle damage. mdx mice show a period of muscle damage from 20 to 40 days of life and fibres which regenerate from this damage display central nuclei. We measured the frequency of central nuclei in control mdx mice compared to streptomycin-treated mdx mice and showed that the incidence of central nuclei was significantly reduced by streptomycin treatment. This result suggests that blockers of stretch-activated channels may protect against muscle damage in the intact mdx mouse.