RESUMO
The mechanism of pattern formation during limb muscle development remains poorly understood. The canonical view holds that naïve limb muscle progenitor cells (MPCs) invade a pre-established pattern of muscle connective tissue, thereby forming individual muscles. Here, we show that early murine embryonic limb MPCs highly accumulate pSMAD1/5/9, demonstrating active signaling of bone morphogenetic proteins (BMP) in these cells. Overexpression of inhibitory human SMAD6 (huSMAD6) in limb MPCs abrogated BMP signaling, impaired their migration and proliferation, and accelerated myogenic lineage progression. Fewer primary myofibers developed, causing an aberrant proximodistal muscle pattern. Patterning was not disturbed when huSMAD6 was overexpressed in differentiated muscle, implying that the proximodistal muscle pattern depends on BMP-mediated expansion of MPCs before their differentiation. We show that limb MPCs differentially express Hox genes, and Hox-expressing MPCs displayed active BMP signaling. huSMAD6 overexpression caused loss of HOXA11 in early limb MPCs. In conclusion, our data show that BMP signaling controls expansion of embryonic limb MPCs as a prerequisite for establishing the proximodistal muscle pattern, a process that involves expression of Hox genes.
Assuntos
Proteínas Morfogenéticas Ósseas , Músculo Esquelético , Animais , Humanos , Camundongos , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/fisiologia , Genes Homeobox , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Proteína Smad6/metabolismoRESUMO
Dystrophin is essential for muscle health: its sarcolemmal absence causes the fatal, X-linked condition, Duchenne muscular dystrophy (DMD). However, its normal, spatial organization remains poorly understood, which hinders the interpretation of efficacy of its therapeutic restoration. Using female reporter mice heterozygous for fluorescently tagged dystrophin (DmdEGFP), we here reveal that dystrophin distribution is unexpectedly compartmentalized, being restricted to myonuclear-defined sarcolemmal territories extending ~80 µm, which we called "basal sarcolemmal dystrophin units (BSDUs)." These territories were further specialized at myotendinous junctions, where both Dmd transcripts and dystrophin protein were enriched. Genome-level correction in X-linked muscular dystrophy mice via CRISPR/Cas9 gene editing restored a mosaic of separated dystrophin domains, whereas transcript-level Dmd correction, following treatment with tricyclo-DNA antisense oligonucleotides, restored dystrophin initially at junctions before extending along the entire fiber-with levels ~2% sufficient to moderate the dystrophic process. We conclude that widespread restoration of fiber dystrophin is likely critical for therapeutic success in DMD, perhaps most importantly, at muscle-tendon junctions.
Assuntos
Distrofina , Distrofia Muscular de Duchenne , Feminino , Camundongos , Animais , Distrofina/genética , Distrofina/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/metabolismo , Músculos/metabolismo , Edição de Genes , Resultado do Tratamento , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Modelos Animais de DoençasRESUMO
Limited knowledge exists regarding the chronic effect of muscular exercise on muscle function in a murine model of severe Duchenne muscular dystrophy (DMD). Here we determined the effects of 1 month of voluntary wheel running (WR), 1 month of enforced treadmill running (TR) and 1 month of mechanical overloading resulting from the removal of the synergic muscles (OVL) in mice lacking both dystrophin and desmin (DKO). Additionally, we examined the effect of activin receptor administration (AR). DKO mice, displaying severe muscle weakness, atrophy and greater susceptibility to contraction-induced functional loss, were exercised or treated with AR at 1 month of age and in situ force production of lower leg muscle was measured at the age of 2 months. We found that TR and OVL increased absolute maximal force and the rate of force development of the plantaris muscle in DKO mice. In contrast, those of the tibialis anterior (TA) muscle remained unaffected by TR and WR. Furthermore, the effects of TR and OVL on plantaris muscle function in DKO mice closely resembled those in mdx mice, a less severe murine DMD model. AR also improved absolute maximal force and the rate of force development of the TA muscle in DKO mice. In conclusion, exercise training improved plantaris muscle weakness in severely affected dystrophic mice. Consequently, these preclinical results may contribute to fostering further investigations aimed at assessing the potential benefits of exercise for DMD patients, particularly resistance training involving a low number of intense muscle contractions. KEY POINTS: Very little is known about the effects of exercise training in a murine model of severe Duchenne muscular dystrophy (DMD). One reason is that it is feared that chronic muscular exercise, particularly that involving intense muscle contractions, could exacerbate the disease. In DKO mice lacking both dystrophin and desmin, characterized by severe lower leg muscle weakness, atrophy and fragility in comparison to the less severe DMD mdx model, we found that enforced treadmill running improved absolute maximal force of the plantaris muscle, while that of tibialis anterior muscle remained unaffected by both enforced treadmill and voluntary wheel running. Furthermore, mechanical overloading, a non-physiological model of chronic resistance exercise, reversed plantaris muscle weakness. Consequently, our findings may have the potential to alleviate concerns and pave the way for exploring the prescription of endurance and resistance training as a viable therapeutic approach for the treatment of dystrophic patients. Additionally, such interventions may serve in mitigating the pathophysiological mechanisms induced by physical inactivity.
Assuntos
Desmina , Distrofina , Músculo Esquelético , Condicionamento Físico Animal , Corrida , Animais , Masculino , Camundongos , Desmina/genética , Desmina/metabolismo , Distrofina/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Contração Muscular , Força Muscular , Músculo Esquelético/fisiologia , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Corrida/fisiologiaRESUMO
Pax7 expression marks stem cells in developing skeletal muscles and adult satellite cells during homeostasis and muscle regeneration. The genetic determinants that control the entrance into the myogenic program and the appearance of PAX7+ cells during embryogenesis are poorly understood. SIX homeoproteins are encoded by the sine oculis-related homeobox Six1-Six6 genes in vertebrates. Six1, Six2, Six4 and Six5 are expressed in the muscle lineage. Here, we tested the hypothesis that Six1 and Six4 could participate in the genesis of myogenic stem cells. We show that fewer PAX7+ cells occupy a satellite cell position between the myofiber and its associated basal lamina in Six1 and Six4 knockout mice (s1s4KO) at E18. However, PAX7+ cells are detected in remaining muscle masses present in the epaxial region of the double mutant embryos and are able to divide and contribute to muscle growth. To further characterize the properties of s1s4KO PAX7+ cells, we analyzed their transcriptome and tested their properties after transplantation in adult regenerating tibialis anterior muscle. Mutant stem cells contribute to hypotrophic myofibers that are not innervated but retain the ability to self-renew.
Assuntos
Proteínas de Homeodomínio/metabolismo , Fator de Transcrição PAX7/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Fator de Transcrição PAX7/genética , Células-Tronco/citologia , Células-Tronco/metabolismo , Transativadores/genéticaRESUMO
OBJECTIVE: This study was undertaken to determine whether a low residual quantity of dystrophin protein is associated with delayed clinical milestones in patients with DMD mutations. METHODS: We performed a retrospective multicentric cohort study by using molecular and clinical data from patients with DMD mutations registered in the Universal Mutation Database-DMD France database. Patients with intronic, splice site, or nonsense DMD mutations, with available muscle biopsy Western blot data, were included irrespective of whether they presented with severe Duchenne muscular dystrophy (DMD) or milder Becker muscular dystrophy (BMD). Patients were separated into 3 groups based on dystrophin protein levels. Clinical outcomes were ages at appearance of first symptoms; loss of ambulation; fall in vital capacity and left ventricular ejection fraction; interventions such as spinal fusion, tracheostomy, and noninvasive ventilation; and death. RESULTS: Of 3,880 patients with DMD mutations, 90 with mutations of interest were included. Forty-two patients expressed no dystrophin (group A), and 31 of 42 (74%) developed DMD. Thirty-four patients had dystrophin quantities < 5% (group B), and 21 of 34 (61%) developed BMD. Fourteen patients had dystrophin quantities ≥ 5% (group C), and all but 4 who lost ambulation beyond 24 years of age were ambulant. Dystrophin quantities of <5%, as low as <0.5%, were associated with milder phenotype for most of the evaluated clinical outcomes, including age at loss of ambulation (p < 0.001). INTERPRETATION: Very low residual dystrophin protein quantity can cause a shift in disease phenotype from DMD toward BMD. ANN NEUROL 2021;89:280-292.
Assuntos
Distrofina/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Adolescente , Corticosteroides/uso terapêutico , Adulto , Idade de Início , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Western Blotting , Criança , Estudos de Coortes , Progressão da Doença , Distrofina/genética , Humanos , Masculino , Limitação da Mobilidade , Mortalidade , Distrofia Muscular de Duchenne/terapia , Ventilação não Invasiva/estatística & dados numéricos , Oxidiazóis/uso terapêutico , Fenótipo , Modelos de Riscos Proporcionais , Estudos Retrospectivos , Índice de Gravidade de Doença , Fusão Vertebral/estatística & dados numéricos , Volume Sistólico , Traqueostomia/estatística & dados numéricos , Capacidade Vital , Adulto JovemRESUMO
INTRODUCTION/AIMS: Respiratory status is a key determinant of prognosis in patients with Duchenne muscular dystrophy (DMD). We aimed to evaluate the determinants of diaphragm ultrasound and its performance in predicting restrictive respiratory patterns in DMD. METHODS: This was a retrospective study of DMD patients followed in our center and admitted for an annual checkup from 2015 to 2018. We included DMD patients who underwent diaphragm ultrasound and pulmonary functional tests. RESULTS: This study included 74 patients with DMD. The right diaphragm thickening fraction (TF) was significantly associated with age (P = .001), Walton score (P = .012), inspiratory capacity (IC) (P = .004), upright forced vital capacity (FVC) (P < .0001), supine FVC (P = .038), and maximal inspiratory pressure (MIP) (P = .002). Right diaphragm excursion was significantly associated with age (P < .0001), steroid use (P = .008), history of spinal fusion (P < .0001), body mass index (BMI) (P = .002), Walton score (P < .0001), IC (P < .0001), upright FVC (P < .0001), supine FVC (P < .0001), and MIP (P < .0001). A right diaphragm TF >28% and a right diaphragm excursion>25.4 mm were associated with an FVC >50% with, respectively, an area under the curve (AUC) of 0.95 (P = .001) and 0.93 (P < .001). A left diaphragm TF >26.8% and a left diaphragm excursion >21.5 mm were associated with an FVC >50% with, respectively, an AUC of 0.95 (P = .011) and 0.97 (P < .001). DISCUSSION: Diaphragm excursion and diaphragm TF can predict restrictive pulmonary insufficiency in DMD.
Assuntos
Diafragma , Distrofia Muscular de Duchenne , Diafragma/diagnóstico por imagem , Humanos , Testes de Função Respiratória , Estudos Retrospectivos , Capacidade VitalRESUMO
AIMS: To estimate the effect of prophylactic angiotensin-converting enzyme inhibitors (ACEi) on survival in Duchenne muscular dystrophy (DMD). METHODS AND RESULTS: We analysed the data from the French multicentre DMD Heart Registry (ClinicalTrials.gov: NCT03443115). We estimated the association between the prophylactic prescription of ACEi and event-free survival in 668 patients aged 8 to 13 years, with normal left ventricular function, using (i) a Cox model with intervention as a time-dependent covariate, (ii) a propensity-based analysis comparing ACEi treatment vs. no treatment, and (iii) a set of sensitivity analyses. The study outcomes were overall survival and hospitalizations for heart failure (HF) or acute respiratory failure. Among the 668 patients included in the DMD Heart Registry, 576 (mean age 6.1 ± 2.8 years) were eligible for this study, of whom 390 were treated with ACEi prophylactically. Death occurred in 53 patients (13.5%) who were and 60 patients (32.3%) who were not treated prophylactically with ACEi, respectively. In a Cox model with intervention as a time-dependent variable, the hazard ratio (HR) associated with ACEi treatment was 0.49 [95% confidence interval (CI) 0.34-0.72] and 0.47 (95% CI 0.31-0.17) for overall mortality after adjustment for baseline variables. In the propensity-based analysis, 278 patients were included in the treatment group and 834 in the control group, with 18.5% and 30.4% 12-year estimated probability of death, respectively. ACEi were associated with a lower risk of death (HR 0.39; 95% CI 0.17-0.92) and hospitalization for HF (HR 0.16; 95% CI 0.04-0.62). All other sensitivity analyses yielded similar results. CONCLUSION: Prophylactic ACEi treatment in DMD was associated with a significantly higher overall survival and lower rates of hospitalization for HF.
Assuntos
Insuficiência Cardíaca , Distrofia Muscular de Duchenne , Antagonistas de Receptores de Angiotensina/uso terapêutico , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Criança , Pré-Escolar , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/prevenção & controle , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Sistema de Registros , Resultado do Tratamento , Função Ventricular EsquerdaRESUMO
Postnatal growth of skeletal muscle largely depends on the expansion and differentiation of resident stem cells, the so-called satellite cells. Here, we demonstrate that postnatal satellite cells express components of the bone morphogenetic protein (BMP) signaling machinery. Overexpression of noggin in postnatal mice (to antagonize BMP ligands), satellite cell-specific knockout of Alk3 (the gene encoding the BMP transmembrane receptor) or overexpression of inhibitory SMAD6 decreased satellite cell proliferation and accretion during myofiber growth, and ultimately retarded muscle growth. Moreover, reduced BMP signaling diminished the adult satellite cell pool. Abrogation of BMP signaling in satellite cell-derived primary myoblasts strongly diminished cell proliferation and upregulated the expression of cell cycle inhibitors p21 and p57 In conclusion, these results show that BMP signaling defines postnatal muscle development by regulating satellite cell-dependent myofiber growth and the generation of the adult muscle stem cell pool.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Western Blotting , Proteínas Morfogenéticas Ósseas/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
SH3 and cysteine-rich domain-containing protein 3 (STAC3) is an essential component of the skeletal muscle excitation-contraction coupling (ECC) machinery, though its role and function are not yet completely understood. Here, we report 18 patients carrying a homozygous p.(Trp284Ser) STAC3 variant in addition to a patient compound heterozygous for the p.(Trp284Ser) and a novel splice site change (c.997-1G > T). Clinical severity ranged from prenatal onset with severe features at birth, to a milder and slowly progressive congenital myopathy phenotype. A malignant hyperthermia (MH)-like reaction had occurred in several patients. The functional analysis demonstrated impaired ECC. In particular, KCl-induced membrane depolarization resulted in significantly reduced sarcoplasmic reticulum Ca2+ release. Co-immunoprecipitation of STAC3 with CaV 1.1 in patients and control muscle samples showed that the protein interaction between STAC3 and CaV 1.1 was not significantly affected by the STAC3 variants. This study demonstrates that STAC3 gene analysis should be included in the diagnostic work up of patients of any ethnicity presenting with congenital myopathy, in particular if a history of MH-like episodes is reported. While the precise pathomechanism remains to be elucidated, our functional characterization of STAC3 variants revealed that defective ECC is not a result of CaV 1.1 sarcolemma mislocalization or impaired STAC3-CaV 1.1 interaction.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Hipertermia Maligna/genética , Miotonia Congênita/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Adolescente , Cálcio/metabolismo , Criança , Pré-Escolar , Acoplamento Excitação-Contração , Feminino , Predisposição Genética para Doença , Humanos , Lactente , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta , Masculino , Hipertermia Maligna/etiologia , Hipertermia Maligna/metabolismo , Miotonia Congênita/complicações , Miotonia Congênita/metabolismo , Linhagem , Fenótipo , Ligação Proteica , Transporte Proteico , Retículo Sarcoplasmático/metabolismo , Índice de Gravidade de Doença , Sequenciamento do Exoma , Adulto JovemRESUMO
Postnatal blockade of the activin type IIB receptor (ActRIIB) represents a promising therapeutic strategy for counteracting dystrophic muscle wasting. However, its impact on muscle function and bioenergetics remains poorly documented in physiologic conditions. We have investigated totally noninvasively the effect of 8-wk administration of either soluble ActRIIB signaling inhibitor (sActRIIB-Fc) or vehicle PBS (control) on gastrocnemius muscle force-generating capacity, energy metabolism, and anatomy in dystrophic mdx mice using magnetic resonance (MR) imaging and dynamic [31P]-MR spectroscopy ([31P]-MRS) in vivo ActRIIB inhibition increased muscle volume (+33%) without changing fiber-type distribution, and increased basal animal oxygen consumption (+22%) and energy expenditure (+23%). During an in vivo standardized fatiguing exercise, maximum and total absolute contractile forces were larger (+40 and 24%, respectively) in sActRIIB-Fc treated animals, whereas specific force-generating capacity and fatigue resistance remained unaffected. Furthermore, sActRIIB-Fc administration did not alter metabolic fluxes, ATP homeostasis, or contractile efficiency during the fatiguing bout of exercise, although it dramatically reduced the intrinsic mitochondrial capacity for producing ATP. Overall, sActRIIB-Fc treatment increased muscle mass and strength without altering the fundamental weakness characteristic of dystrophic mdx muscle. These data support the clinical interest of ActRIIB blockade for reversing dystrophic muscle wasting.-Béchir, N., Pecchi, E., Vilmen, C., Le Fur, Y., Amthor, H., Bernard, M., Bendahan, D., Giannesini, B. ActRIIB blockade increases force-generating capacity and preserves energy supply in exercising mdx mouse muscle in vivo.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Metabolismo Energético/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/patologia , Animais , Peso Corporal/fisiologia , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos mdx , Modelos Animais , Condicionamento Físico Animal/métodosRESUMO
INTRODUCTION: The effect of constitutive inactivation of the gene encoding myostatin on the gain in muscle performance during postnatal growth has not been well characterized. METHODS: We analyzed 2 murine myostatin knockout (KO) models, (i) the Lee model (KOLee ) and (ii) the Grobet model (KOGrobet ), and measured the contraction of tibialis anterior muscle in situ. RESULTS: Absolute maximal isometric force was increased in 6-month-old KOLee and KOGrobet mice, as compared to wild-type mice. Similarly, absolute maximal power was increased in 6-month-old KOLee mice. In contrast, specific maximal force (relative maximal force per unit of muscle mass was decreased in all 6-month-old male and female KO mice, except in 6-month-old female KOGrobet mice, whereas specific maximal power was reduced only in male KOLee mice. CONCLUSIONS: Genetic inactivation of myostatin increases maximal force and power, but in return it reduces muscle quality, particularly in male mice. Muscle Nerve 55: 254-261, 2017.
Assuntos
Contração Muscular/genética , Força Muscular/genética , Músculo Esquelético/fisiologia , Doenças Musculares/patologia , Miostatina/deficiência , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Knockout , Doenças Musculares/genética , Miostatina/genética , Fatores SexuaisRESUMO
Because it leads to a rapid and massive muscle hypertrophy, postnatal blockade of the activin type IIB receptor (ActRIIB) is a promising therapeutic strategy for counteracting muscle wasting. However, the functional consequences remain very poorly documented in vivo. Here, we have investigated the impact of 8-wk ActRIIB blockade with soluble receptor (sActRIIB-Fc) on gastrocnemius muscle anatomy, energy metabolism, and force-generating capacity in wild-type mice, using totally noninvasive magnetic resonance imaging (MRI) and dynamic(31)P-MRS. Compared with vehicle (PBS) control, sActRIIB-Fc treatment resulted in a dramatic increase in body weight (+29%) and muscle volume (+58%) calculated from hindlimb MR imaging, but did not alter fiber type distribution determined via myosin heavy chain isoform analysis. In resting muscle, sActRIIB-Fc treatment induced acidosis and PCr depletion, thereby suggesting reduced tissue oxygenation. During an in vivo fatiguing exercise (6-min repeated maximal isometric contraction electrically induced at 1.7 Hz), maximal and total absolute forces were larger in sActRIIB-Fc treated animals (+26 and +12%, respectively), whereas specific force and fatigue resistance were lower (-30 and -37%, respectively). Treatment with sActRIIB-Fc further decreased the maximal rate of oxidative ATP synthesis (-42%) and the oxidative capacity (-34%), but did not alter the bioenergetics status in contracting muscle. Our findings demonstrate in vivo that sActRIIB-Fc treatment increases absolute force-generating capacity and reduces mitochondrial function in glycolytic gastrocnemius muscle, but this reduction does not compromise energy status during sustained activity. Overall, these data support the clinical interest of postnatal ActRIIB blockade.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Peso Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Condicionamento Físico Animal , Proteínas Recombinantes de Fusão/farmacologia , Animais , Glicólise , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Camundongos , Mitocôndrias Musculares/metabolismo , Contração Muscular/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Cadeias Pesadas de Miosina/efeitos dos fármacos , Cadeias Pesadas de Miosina/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Isótopos de FósforoRESUMO
Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.
Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Mutação de Sentido Incorreto , Miopatias da Nemalina/genética , Substituição de Aminoácidos , Animais , Povo Asiático/genética , Estudos de Coortes , Mutação da Fase de Leitura , Genes Recessivos , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Proteínas Musculares/genética , Miopatias da Nemalina/etnologia , Miopatias da Nemalina/patologia , Linhagem , Polimorfismo de Nucleotídeo Único , Índice de Gravidade de Doença , Peixe-Zebra/genéticaRESUMO
Myostatin regulates skeletal muscle size via the activin receptor IIB (ActRIIB). However, its effect on muscle energy metabolism and energy-dependent muscle function remains largely unexplored. This question needs to be solved urgently since various therapies for neuromuscular diseases based on blockade of ActRIIB signaling are being developed. Here, we show in mice, that 4-month pharmacological abrogation of ActRIIB signaling by treatment with soluble ActRIIB-Fc triggers extreme muscle fatigability. This is associated with elevated serum lactate levels and a severe metabolic myopathy in the mdx mouse, an animal model of Duchenne muscular dystrophy. Blockade of ActRIIB signaling downregulates porin, a crucial ADP/ATP shuttle between cytosol and mitochondrial matrix leading to a consecutive deficiency of oxidative phosphorylation as measured by in vivo Phosphorus Magnetic Resonance Spectroscopy ((31)P-MRS). Further, ActRIIB blockade reduces muscle capillarization, which further compounds the metabolic stress. We show that ActRIIB regulates key determinants of muscle metabolism, such as Pparß, Pgc1α, and Pdk4 thereby optimizing different components of muscle energy metabolism. In conclusion, ActRIIB signaling endows skeletal muscle with high oxidative capacity and low fatigability. The severe metabolic side effects following ActRIIB blockade caution against deploying this strategy, at least in isolation, for treatment of neuromuscular disorders.
Assuntos
Receptores de Activinas Tipo II/antagonistas & inibidores , Fragmentos Fc das Imunoglobulinas/farmacologia , Músculos/fisiopatologia , Distrofia Muscular Animal/fisiopatologia , Animais , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Porinas/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn(-/-) mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn(-/-) mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn(-/-) mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn(-/-) mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.
Assuntos
Metabolismo Energético , Contração Muscular , Músculo Esquelético/metabolismo , Miostatina/metabolismo , Resistência Física , Animais , Genótipo , Glicólise , Ácido Láctico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias Musculares/metabolismo , Fadiga Muscular , Miostatina/deficiência , Miostatina/genética , Consumo de Oxigênio , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Fenótipo , Fosfopiruvato Hidratase/metabolismo , Corrida , Transdução de Sinais , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Although it is well established that the lack of myostatin (Mstn) promotes skeletal muscle hypertrophy, the corresponding changes regarding force generation have been studied mainly in vitro and remain conflicting. Furthermore, the metabolic underpinnings of these changes are very poorly documented. To clarify this issue, we have investigated strictly noninvasively in vivo the impact of the lack of Mstn on gastrocnemius muscle function and energetics in Mstn-targeted knockout (Mstn-/-) mice using ¹H-magnetic resonance (MR) imaging and ³¹P-MR spectroscopy during maximal repeated isometric contractions induced by transcutaneous electrostimulation. In Mstn-/- animals, although body weight, gastrocnemius muscle volume, and absolute force were larger (+38, +118, and +34%, respectively) compared with wild-type (Mstn+/+) mice, specific force (calculated from MR imaging measurements) was significantly lower (-36%), and resistance to fatigue was decreased. Besides, Mstn deficiency did not affect phosphorylated compound concentrations and intracellular pH at rest but caused a large increase in ATP cost of contraction (up to +206% compared with Mstn+/+) throughout the stimulation period. Further, Mstn deficiency limits the shift toward oxidative metabolism during muscle activity despite the fact that oxidative ATP synthesis capacity was not altered. Our data demonstrate in vivo that the absence of Mstn impairs both mechanical performance and energy cost of contraction in hypertrophic muscle. These findings must be kept in mind when considering Mstn as a potential therapeutic target for increasing muscle mass in patients suffering from muscle-wasting disorders.
Assuntos
Metabolismo Energético/fisiologia , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Miostatina/genética , Miostatina/metabolismo , Condicionamento Físico Animal/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Fenômenos Biomecânicos/genética , Estimulação Elétrica , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Atrofia Muscular/fisiopatologiaRESUMO
Background Chronic respiratory failure and heart involvement may occur in Duchenne muscular dystrophy. We aimed to assess the prognostic value of the right ventricular (RV) systolic dysfunction in patients with Duchenne muscular dystrophy. Methods and Results We studied 90 genetically proven patients with Duchenne muscular dystrophy from 2010 to 2019, to obtain respiratory function and Doppler echocardiographic RV systolic function. Prognostic value was assessed in terms of death and cardiac events. The median age was 27.5 years, and median forced vital capacity was at 10% of the predicted value: 83 patients (92%) were on home mechanical ventilation. An RV systolic dysfunction was found in 46 patients (51%). In patients without RV dysfunction at inclusion, a left ventricular systolic dysfunction at inclusion was associated with a higher risk of developing RV dysfunction during follow-up with an odds ratio of 4.5 (P=0.03). RV systolic dysfunction was significantly associated with cardiac events, mainly acute heart failure (62%) and cardiogenic shock (23%). In a multivariable Cox model, the adjusted hazard ratio was 4.96 (95% CI [1.09-22.6]; P=0.04). In terms of death, we found a significant difference between patients with RV dysfunction versus patients without RV dysfunction in the Kaplan-Meier curves (log-rank P=0.045). Conclusions RV systolic dysfunction is frequently present in patients with Duchenne muscular dystrophy and is associated with increased risk of cardiac events, irrespective of left ventricular dysfunction and mechanical ventilation. Registration URL: https://www.clinicaltrials.org; unique identifier: NCT02501083.
Assuntos
Cardiomiopatias , Distrofia Muscular de Duchenne , Disfunção Ventricular Esquerda , Disfunção Ventricular Direita , Adulto , Humanos , Cardiomiopatias/complicações , Ecocardiografia Doppler , Coração , Distrofia Muscular de Duchenne/complicações , Prognóstico , Volume Sistólico , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/complicações , Disfunção Ventricular Direita/etiologia , Disfunção Ventricular Direita/complicações , Função Ventricular DireitaRESUMO
BACKGROUND: Drug repurposing could provide novel treatment options for Duchenne muscular dystrophy. Because tamoxifen-an oestrogen receptor regulator-reduced signs of muscular pathology in a Duchenne muscular dystrophy mouse model, we aimed to assess the safety and efficacy of tamoxifen in humans as an adjunct to corticosteroid therapy over a period of 48 weeks. METHODS: We did a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial at 12 study centres in seven European countries. We enrolled ambulant boys aged 6·5-12·0 years with a genetically confirmed diagnosis of Duchenne muscular dystrophy and who were on stable corticosteroid treatment for more than 6 months. Exclusion criteria included ophthalmological disorders, including cataracts, and haematological disorders. We randomly assigned (1:1) participants using an online randomisation tool to either 20 mg tamoxifen orally per day or matched placebo, stratified by centre and corticosteroid intake. Participants, caregivers, and clinical investigators were masked to treatment assignments. Tamoxifen was taken in addition to standard care with corticosteroids, and participants attended study visits for examinations every 12 weeks. The primary efficacy outcome was the change from baseline to week 48 in scores on the D1 domain of the Motor Function Measure in the intention-to-treat population (defined as all patients who fulfilled the inclusion criteria and began treatment). This study is registered with ClinicalTrials.gov (NCT03354039) and is completed. FINDINGS: Between May 24, 2018, and Oct 14, 2020, 95 boys were screened for inclusion, and 82 met inclusion criteria and were initially enrolled into the study. Three boys were excluded after initial screening due to cataract diagnosis or revoked consent directly after screening, but before randomisation. A further boy assigned to the placebo group did not begin treatment. Therefore, 40 individuals assigned tamoxifen and 38 allocated placebo were included in the intention-to-treat population. The primary efficacy outcome did not differ significantly between tamoxifen (-3·05%, 95% CI -7·02 to 0·91) and placebo (-6·15%, -9·19 to -3·11; 2·90% difference, -3·02 to 8·82, p=0·33). Severe adverse events occurred in two participants: one participant who received tamoxifen had a fall, and one who received placebo suffered a panic attack. No deaths or life-threatening serious adverse events occurred. Viral infections were the most common adverse events. INTERPRETATION: Tamoxifen was safe and well tolerated, but no difference between groups was reported for the primary efficacy endpoint. Slower disease progression, defined by loss of motor function over time, was indicated in the tamoxifen group compared with the placebo group, but differences in outcome measures were neither clinically nor statistically significant. Currently, we cannot recommend the use of tamoxifen in daily clinical practice as a treatment option for boys with Duchenne muscular dystrophy due to insufficient clinical evidence. FUNDING: Thomi Hopf Foundation, ERA-Net, Swiss National Science Foundation, Duchenne UK, Joining Jack, Duchenne Parent Project, Duchenne Parent Project Spain, Fondation Suisse de Recherche sur les Maladies Musculaires, Association Monegasque contre les Myopathies.
Assuntos
Distrofia Muscular de Duchenne , Masculino , Animais , Camundongos , Humanos , Distrofia Muscular de Duchenne/tratamento farmacológico , Método Duplo-Cego , Modelos Animais de Doenças , Reposicionamento de Medicamentos , EtnicidadeRESUMO
Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN(-/-)) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN(-/-) mice that can be induced by training. To this end, we subjected MSTN(-/-) mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN(-/-) mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN(-/-) mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile.
Assuntos
Hipertrofia/fisiopatologia , Fibras Musculares Esqueléticas/fisiologia , Miostatina/deficiência , Condicionamento Físico Animal , Indutores da Angiogênese/metabolismo , Animais , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Citoplasma/metabolismo , Citoplasma/fisiologia , Tolerância ao Exercício , Glicólise , Hipertrofia/genética , Hipertrofia/metabolismo , Masculino , Metaboloma , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/metabolismo , Miostatina/genética , Miostatina/metabolismo , Tamanho do Órgão , Oxirredução , Fenótipo , EnsinoRESUMO
Myostatin, a member of the TGF-beta family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells.