RESUMO
Duchenne muscular dystrophy (DMD) is a devastating monogenic skeletal muscle-wasting disorder. Although many pharmacological and genetic interventions have been reported in preclinical studies, few have progressed to clinical trials with meaningful benefit. Identifying therapeutic potential can be limited by availability of suitable preclinical mouse models. More rigorous testing across models with varied background strains and mutations can identify treatments for clinical success. Here, we report the generation of a DMD mouse model with a CRISPR-induced deletion within exon 62 of the dystrophin gene (Dmd) and the first generated in BALB/c mice. Analysis of mice at 3, 6 and 12â months of age confirmed loss of expression of the dystrophin protein isoform Dp427 and resultant dystrophic pathology in limb muscles and the diaphragm, with evidence of centrally nucleated fibers, increased inflammatory markers and fibrosis, progressive decline in muscle function, and compromised trabecular bone development. The BALB/c.mdx62 mouse is a novel model of DMD with associated variations in the immune response and muscle phenotype, compared with those of existing models. It represents an important addition to the preclinical model toolbox for developing therapeutic strategies.
Assuntos
Modelos Animais de Doenças , Distrofina , Camundongos Endogâmicos BALB C , Músculo Esquelético , Distrofia Muscular de Duchenne , Animais , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/genética , Distrofina/metabolismo , Distrofina/genética , Músculo Esquelético/patologia , Músculo Esquelético/metabolismo , Camundongos Endogâmicos mdx , Camundongos , Éxons/genética , Masculino , Fibrose , FenótipoRESUMO
Exercise induces signaling networks to improve muscle function and confer health benefits. To identify divergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Ca2+ handling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the divergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function.
Assuntos
Proteínas Quinases Ativadas por AMP , Proteínas Adaptadoras de Transdução de Sinal , Exercício Físico , Músculo Esquelético , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Humanos , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Fosforilação , Transdução de SinaisRESUMO
The AMP-activated protein kinase (AMPK) αßγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2ß2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2ß2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating ß2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2ß2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2ß1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2ß2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body.
Assuntos
Proteínas Quinases Ativadas por AMP , Diabetes Mellitus Tipo 2 , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/metabolismo , Camundongos , Músculo Esquelético/metabolismo , FosforilaçãoRESUMO
BACKGROUND: Oxidative stress is implicated in the pathophysiology of Duchenne muscular dystrophy (DMD, caused by mutations in the dystrophin gene), which is the most common and severe of the muscular dystrophies. To our knowledge, the distribution of iron, an important modulator of oxidative stress, has not been assessed in DMD. We tested the hypotheses that iron accumulation occurs in mouse models of DMD and that modulation of iron through the diet or chelation could modify disease severity. METHODS: We assessed iron distribution and total elemental iron using LA-ICP-MS on skeletal muscle cross-sections of 8-week-old Bl10 control mice and dystrophic mdx mice (with moderate dystrophy) and dystrophin/utrophin-null mice (dko, with severe dystrophy). In addition, mdx mice (4 weeks) were treated with either an iron chelator (deferiprone 150 mg/kg/day) or iron-enriched feed (containing 1% added iron as carbonyl iron). Immunoblotting was used to determine the abundance of iron- and mitochondria-related proteins. (Immuno)histochemical and mRNA assessments of fibrosis and inflammation were also performed. RESULTS: We observed a significant increase in total elemental iron in hindlimb muscles of dko mice (+50%, P < 0.05) and in the diaphragm of mdx mice (+80%, P < 0.05), with both tissues exhibiting severe pathology. Iron dyshomeostasis was further evidenced by an increase in the storage protein ferritin (dko: +39%, P < 0.05) and ferroportin compared with Bl10 control mice (mdx: +152% and dko: +175%, P < 0.05). Despite having features of iron overload, dystrophic muscles had lower protein expression of ALAS-1, the rate-limiting enzyme for haem synthesis (dko -44%, P < 0.05), and the haem-containing protein myoglobin (dko -54%, P < 0.05). Deferiprone treatment tended to decrease muscle iron levels in mdx mice (-30%, P < 0.1), which was associated with lower oxidative stress and fibrosis, but suppressed haem-containing proteins and mitochondrial content. Increasing iron via dietary intervention elevated total muscle iron (+25%, P < 0.05) but did not aggravate the pathology. CONCLUSIONS: Muscles from dystrophic mice have increased iron levels and dysregulated iron-related proteins that are associated with dystrophic pathology. Muscle iron levels were manipulated by iron chelation and iron enriched feed. Iron chelation reduced fibrosis and reactive oxygen species (ROS) but also suppressed haem-containing proteins and mitochondrial activity. Conversely, iron supplementation increased ferritin and haem-containing proteins but did not alter ROS, fibrosis, or mitochondrial activity. Further studies are required to investigate the contribution of impaired ferritin breakdown in the dysregulation of iron homeostasis in DMD.
Assuntos
Sobrecarga de Ferro , Distrofia Muscular de Duchenne , Animais , Deferiprona , Distrofina/genética , Ferritinas , Fibrose , Heme/metabolismo , Ferro/metabolismo , Quelantes de Ferro , Sobrecarga de Ferro/etiologia , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Gastrointestinal (GI) dysfunction is an important, yet understudied condition associated with Duchenne muscular dystrophy (DMD), with patients reporting bloating, diarrhea, and general discomfort, contributing to a reduced quality of life. In the mdx mouse, the most commonly used mouse model of DMD, studies have confirmed GI dysfunction (reported as altered contractility and GI transit through the small and large intestine), associated with increased local and systemic inflammation. Sulforaphane (SFN) is a natural isothiocyanate with anti-inflammatory and anti-oxidative properties via its activation of Nrf2 signalling that has been shown to improve aspects of the skeletal muscle pathology in dystrophic mice. Whether SFN can similarly improve GI function in muscular dystrophy was unknown. Video imaging and spatiotemporal mapping to assess gastrointestinal contractions in isolated colon preparations from mdx and C57BL/10 mice revealed that SFN reduced contraction frequency when administered ex vivo, demonstrating its therapeutic potential to improve GI function in DMD. To confirm this in vivo, four-week-old male C57BL/10 and mdx mice received vehicle (2% DMSO/corn oil) or SFN (2 mg/kg in 2% DMSO/corn oil) via daily oral gavage five days/week for 4 weeks. SFN administration reduced fibrosis in the diaphragm of mdx mice but did not affect other pathological markers. Gene and protein analysis revealed no change in Nrf2 protein expression or activation of Nrf2 signalling after SFN administration and oral SFN supplementation did not improve GI function in mdx mice. Although ex vivo studies demonstrate SFN's therapeutic potential for reducing colon contractions, in vivo studies should investigate higher doses and/or alternate routes of administration to confirm SFN's potential to improve GI function in DMD.
Assuntos
Gastroenteropatias/tratamento farmacológico , Isotiocianatos/farmacologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Sulfóxidos/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Colo/patologia , Diafragma/patologia , Modelos Animais de Doenças , Fibrose/metabolismo , Gastroenteropatias/metabolismo , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
A modified Chromium 10x droplet-based protocol that subsamples cells for both short-read and long-read (nanopore) sequencing together with a new computational pipeline (FLAMES) is developed to enable isoform discovery, splicing analysis, and mutation detection in single cells. We identify thousands of unannotated isoforms and find conserved functional modules that are enriched for alternative transcript usage in different cell types and species, including ribosome biogenesis and mRNA splicing. Analysis at the transcript level allows data integration with scATAC-seq on individual promoters, improved correlation with protein expression data, and linked mutations known to confer drug resistance to transcriptome heterogeneity.
Assuntos
Sequenciamento por Nanoporos/métodos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Processamento Alternativo , Animais , Éxons , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Splicing de RNA , RNA Mensageiro , TranscriptomaRESUMO
OBJECTIVES: Preferential damage to fast, glycolytic myofibers is common in many muscle-wasting diseases, including Duchenne muscular dystrophy (DMD). Promoting an oxidative phenotype could protect muscles from damage and ameliorate the dystrophic pathology with therapeutic relevance, but developing efficacious strategies requires understanding currently unknown biological roles for dystrophin and utrophin in dystrophic muscle adaptation and plasticity. METHODS: Combining whole transcriptome RNA sequencing and mitochondrial proteomics with assessments of metabolic and contractile function, we investigated the roles of dystrophin and utrophin in fast-to-slow muscle remodeling with low-frequency electrical stimulation (LFS, 10 Hz, 12 h/d, 7 d/wk, 28 d) in mdx (dystrophin null) and dko (dystrophin/utrophin null) mice, two established preclinical models of DMD. RESULTS: Novel biological roles in adaptation were demonstrated by impaired transcriptional activation of estrogen-related receptor alpha-responsive genes supporting oxidative phosphorylation in dystrophic muscles. Further, utrophin expression in dystrophic muscles was required for LFS-induced remodeling of mitochondrial respiratory chain complexes, enhanced fiber respiration, and conferred protection from eccentric contraction-mediated damage. CONCLUSIONS: These findings reveal novel roles for dystrophin and utrophin during LFS-induced metabolic remodeling of dystrophic muscle and highlight the therapeutic potential of LFS to ameliorate the dystrophic pathology and protect from contraction-induced injury with important implications for DMD and related muscle disorders.
Assuntos
Adaptação Fisiológica/fisiologia , Distrofina/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Utrofina/metabolismo , Animais , Distrofina/genética , Masculino , Engenharia Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Mitocôndrias/metabolismo , Contração Muscular , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Utrofina/genéticaRESUMO
Skeletal muscle has a remarkable capacity to regenerate following injury, a property conferred by a resident population of muscle stem cells (MuSCs). In response to injury, MuSCs must double their cellular content to divide, a process requiring significant new biomass in the form of nucleotides, phospholipids, and amino acids. This new biomass is derived from a series of intracellular metabolic cycles and alternative routing of carbon. In this review, we examine the link between metabolism and skeletal muscle regeneration with particular emphasis on the role of the cellular microenvironment in supporting the production of new biomass and MuSC proliferation.