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Macrophages polarize into distinct phenotypes in response to complex environmental cues. We found that the nuclear receptor PPARγ drove robust phenotypic changes in macrophages upon repeated stimulation with interleukin (IL)-4. The functions of PPARγ on macrophage polarization in this setting were independent of ligand binding. Ligand-insensitive PPARγ bound DNA and recruited the coactivator P300 and the architectural protein RAD21. This established a permissive chromatin environment that conferred transcriptional memory by facilitating the binding of the transcriptional regulator STAT6 and RNA polymerase II, leading to robust production of enhancer and mRNAs upon IL-4 re-stimulation. Ligand-insensitive PPARγ binding controlled the expression of an extracellular matrix remodeling-related gene network in macrophages. Expression of these genes increased during muscle regeneration in a mouse model of injury, and this increase coincided with the detection of IL-4 and PPARγ in the affected tissue. Thus, a predominantly ligand-insensitive PPARγ:RXR cistrome regulates progressive and/or reinforcing macrophage polarization.
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Epigénesis Genética/inmunología , Epigenómica/métodos , Regulación de la Expresión Génica/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , PPAR gamma/inmunología , Animales , Línea Celular , Células Cultivadas , Interleucina-4/inmunología , Interleucina-4/farmacología , Ligandos , Activación de Macrófagos/genética , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , PPAR gamma/genética , PPAR gamma/metabolismoRESUMEN
In mammals, the circadian clock coordinates cell physiological processes including inflammation. Recent studies suggested a crosstalk between these two pathways. However, the mechanism of how inflammation affects the clock is not well understood. Here, we investigated the role of the proinflammatory transcription factor NF-κB in regulating clock function. Using a combination of genetic and pharmacological approaches, we show that perturbation of the canonical NF-κB subunit RELA in the human U2OS cellular model altered core clock gene expression. While RELA activation shortened period length and dampened amplitude, its inhibition lengthened period length and caused amplitude phenotypes. NF-κB perturbation also altered circadian rhythms in the master suprachiasmatic nucleus (SCN) clock and locomotor activity behavior under different light/dark conditions. We show that RELA, like the clock repressor CRY1, repressed the transcriptional activity of BMAL1/CLOCK at the circadian E-box cis-element. Biochemical and biophysical analysis showed that RELA binds to the transactivation domain of BMAL1. These data support a model in which NF-kB competes with CRY1 and coactivator CBP/p300 for BMAL1 binding to affect circadian transcription. This is further supported by chromatin immunoprecipitation analysis showing that binding of RELA, BMAL1 and CLOCK converges on the E-boxes of clock genes. Taken together, these data support a significant role for NF-κB in directly regulating the circadian clock and highlight mutual regulation between the circadian and inflammatory pathways.
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Factores de Transcripción ARNTL/genética , Proteínas CLOCK/genética , Inflamación/genética , Factor de Transcripción ReIA/genética , Animales , Proteínas de Unión al Calcio/genética , Línea Celular Tumoral , Relojes Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Inflamación/patología , FN-kappa B/genética , Núcleo Supraquiasmático/metabolismoRESUMEN
INTRODUCTION: Dysferlin loss-of-function mutations cause muscular dystrophy, accompanied by impaired membrane repair and muscle weakness. Growth promoting strategies including insulin-like growth factor 1 (IGF-1) could provide benefit but may cause strength loss or be ineffective. The objective of this study was to determine whether locally increased IGF-1 promotes functional muscle hypertrophy in dysferlin-null (Dysf-/- ) mice. METHODS: Muscle-specific transgenic expression and postnatal viral delivery of Igf1 were used in Dysf-/- and control mice. Increased IGF-1 levels were confirmed by enzyme-linked immunosorbent assay. Testing for skeletal muscle mass and function was performed in male and female mice. RESULTS: Muscle hypertrophy occurred in response to increased IGF-1 in mice with and without dysferlin. Male mice showed a more robust response compared with females. Increased IGF-1 did not cause loss of force per cross-sectional area in Dysf-/- muscles. DISCUSSION: We conclude that increased local IGF-1 promotes functional hypertrophy when dysferlin is absent and reestablishes IGF-1 as a potential therapeutic for dysferlinopathies.
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Disferlina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Músculo Esquelético/metabolismo , Animales , Diafragma/metabolismo , Diafragma/patología , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/patología , Distrofias Musculares/genética , Tamaño de los ÓrganosRESUMEN
INTRODUCTION: Collagen cross-linking is a key parameter in extracellular matrix (ECM) maturation, turnover, and stiffness. We examined aspects of collagen cross-linking in dystrophin-deficient murine, canine, and human skeletal muscle. METHODS: DMD patient biopsies and samples from mdx mice and golden retriever muscular dystrophy dog samples (with appropriate controls) were analyzed. Collagen cross-linking was evaluated using solubility and hydroxyproline assays. Expression of the cross-linking enzyme lysyl oxidase (LOX) was determined by real-time polymerase chain reaction, immunoblotting, and immunofluorescence. RESULTS: LOX protein levels are increased in dystrophic muscle from all species evaluated. Dystrophic mice and dogs had significantly higher cross-linked collagen than controls, especially in the diaphragm. Distribution of intramuscular LOX was heterogeneous in all samples, but it increased in frequency and intensity in dystrophic muscle. CONCLUSION: These findings implicate elevated collagen cross-linking as an important component of the disrupted ECM in dystrophic muscles, and heightened cross-linking is evident in mouse, dog, and man. Muscle Nerve 54: 71-78, 2016.
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Diafragma/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Adolescente , Animales , Niño , Colágeno/metabolismo , Perros , Femenino , Humanos , Hidroxiprolina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Proteína-Lisina 6-Oxidasa/genética , ARN Mensajero/metabolismo , Vimentina/metabolismoRESUMEN
Clinical trials delivering high doses of adeno-associated viruses (AAVs) expressing truncated dystrophin molecules (microdystrophins) are underway for Duchenne muscular dystrophy (DMD). We examined the efficiency and efficacy of this strategy with 4 microdystrophin constructs (3 in clinical trials and a variant of the largest clinical construct), in a severe mouse model of DMD, using AAV doses comparable with those in clinical trials. We achieved high levels of microdystrophin expression in striated muscles with cardiac expression approximately 10-fold higher than that observed in skeletal muscle. Significant, albeit incomplete, correction of skeletal muscle disease was observed. Surprisingly, a lethal acceleration of cardiac disease occurred with 2 of the microdystrophins. The detrimental cardiac effect appears to be caused by variable competition (dependent on microdystrophin design and expression level) between microdystrophin and utrophin at the cardiomyocyte membrane. There may also be a contribution from an overloading of protein degradation. The significance of these observations for patients currently being treated with AAV-microdystrophin therapies is unclear since the levels of expression being achieved in the DMD hearts are unknown. However, these findings suggest that microdystrophin treatments need to avoid excessively high levels of expression in the heart and that cardiac function should be carefully monitored in these patients.
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Terapia Genética , Distrofia Muscular de Duchenne , Animales , Humanos , Masculino , Ratones , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Miocitos Cardíacos/metabolismo , Utrofina/genética , Utrofina/metabolismoRESUMEN
Cancer-induced muscle wasting reduces quality of life, complicates or precludes cancer treatments, and predicts early mortality. Herein, we investigated the requirement of the muscle-specific E3 ubiquitin ligase, MuRF1, for muscle wasting induced by pancreatic cancer. Murine pancreatic cancer (KPC) cells, or saline, were injected into the pancreas of WT and MuRF1-/- mice, and tissues analyzed throughout tumor progression. KPC tumors induced progressive wasting of skeletal muscle and systemic metabolic reprogramming in WT mice, but not MuRF1-/- mice. KPC tumors from MuRF1-/- mice also grew slower, and showed an accumulation of metabolites normally depleted by rapidly growing tumors. Mechanistically, MuRF1 was necessary for the KPC-induced increases in cytoskeletal and muscle contractile protein ubiquitination, and the depression of proteins that support protein synthesis. Together, these data demonstrate that MuRF1 is required for KPC-induced skeletal muscle wasting, whose deletion reprograms the systemic and tumor metabolome and delays tumor growth.
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Cancer-induced muscle wasting reduces quality of life, complicates or precludes cancer treatments, and predicts early mortality. Herein, we investigate the requirement of the muscle-specific E3 ubiquitin ligase, MuRF1, for muscle wasting induced by pancreatic cancer. Murine pancreatic cancer (KPC) cells, or saline, were injected into the pancreas of WT and MuRF1-/- mice, and tissues analyzed throughout tumor progression. KPC tumors induces progressive wasting of skeletal muscle and systemic metabolic reprogramming in WT mice, but not MuRF1-/- mice. KPC tumors from MuRF1-/- mice also grow slower, and show an accumulation of metabolites normally depleted by rapidly growing tumors. Mechanistically, MuRF1 is necessary for the KPC-induced increases in cytoskeletal and muscle contractile protein ubiquitination, and the depression of proteins that support protein synthesis. Together, these data demonstrate that MuRF1 is required for KPC-induced skeletal muscle wasting, whose deletion reprograms the systemic and tumor metabolome and delays tumor growth.
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Neoplasias Pancreáticas , Calidad de Vida , Animales , Ratones , Músculo Esquelético/metabolismo , Atrofia Muscular/genética , Páncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias PancreáticasRESUMEN
Ischemia/reperfusion (I/R) injury is a considerable insult to skeletal muscle, often resulting in prolonged functional deficits. The purpose of the current study was to evaluate the controlled release of the pro-regenerative growth factor, insulin-like growth factor-I (IGF-I), from a biodegradable polyethylene glycol (PEG)ylated fibrin gel matrix and the subsequent recovery of skeletal muscle from I/R. To accomplish this, the hind limbs of male Sprague-Dawley rats were subjected to 2-h tourniquet-induced I/R then treated with saline, bolus IGF-I (bIGF), PEGylated fibrin gel (PEG-Fib), or IGF-I conjugated PEGylated fibrin gel (PEG-Fib-IGF). Functional and histological evaluations were performed following 14 days of reperfusion, and muscles from 4-day reperfusion animals were analyzed by Western blotting and histological assessments. There was no difference in functional recovery between saline, bIGF, or PEG-Fib groups. However, PEG-Fib-IGF treatment resulted in significant improvement of muscle function and structure, as observed histologically. Activation of the PI3K/Akt pathway was significantly elevated in PEG-Fib-IGF muscles, compared to PEG-Fib treatment, at 4 days of reperfusion, suggesting involvement of the pathway PI3K/Akt as a mediator of the improved function. Surprisingly, myoblast activity was not evident as a result of PEG-Fib-IGF treatment. Taken together, these data give evidence for a protective role for the delivered IGF. These results indicate that PEG-Fib-IGF is a viable therapeutic technique in the treatment of skeletal muscle I/R injury.
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Factor I del Crecimiento Similar a la Insulina/uso terapéutico , Músculo Esquelético/irrigación sanguínea , Daño por Reperfusión/tratamiento farmacológico , Implantes Absorbibles , Animales , Portadores de Fármacos , Evaluación Preclínica de Medicamentos , Implantes de Medicamentos , Fibrina/administración & dosificación , Fibrina/análogos & derivados , Geles , Miembro Posterior/irrigación sanguínea , Inyecciones Intramusculares , Factor I del Crecimiento Similar a la Insulina/administración & dosificación , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Fosfatidilinositol 3-Quinasas/fisiología , Polietilenglicoles/administración & dosificación , Proteínas Proto-Oncogénicas c-akt/fisiología , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/patología , Transducción de Señal , TorniquetesRESUMEN
The muscular dystrophies (MDs) are genetic muscle diseases that result in progressive muscle degeneration followed by the fibrotic replacement of affected muscles as regenerative processes fail. Therapeutics that specifically address the fibrosis and failed regeneration associated with MDs represent a major unmet clinical need for MD patients, particularly those with advanced-stage disease progression. The current study investigated targeting NAD(P)H oxidase 4 (NOX4) as a potential strategy to reduce fibrosis and promote regeneration in disease-burdened muscle that models Duchenne muscular dystrophy (DMD). NOX4 was elevated in the muscles of dystrophic mice and DMD patients, localizing primarily to interstitial cells located between muscle fibers. Genetic and pharmacological targeting of NOX4 significantly reduced fibrosis in dystrophic respiratory and limb muscles. Mechanistically, NOX4 targeting decreased the number of fibrosis-depositing cells (myofibroblasts) and restored the number of muscle-specific stem cells (satellite cells) localized to their physiological niche, thereby rejuvenating muscle regeneration. Furthermore, acute inhibition of NOX4 was sufficient to induce apoptotic clearing of myofibroblasts within dystrophic muscle. These data indicate that targeting NOX4 is an effective strategy to promote the beneficial remodeling of disease-burdened muscle representative of DMD and, potentially, other MDs and muscle pathologies.
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Distrofia Muscular de Duchenne , Regeneración , Ratones , Animales , Ratones Endogámicos mdx , Distrofia Muscular de Duchenne/genética , Músculo Esquelético/patología , Fibrosis , NADPH Oxidasas , NADPH Oxidasa 4/genéticaRESUMEN
The potential use of the D2.mdx mouse (the mdx mutation on the DBA/2J genetic background) as a preclinical model of the cardiac aspects of Duchenne muscular dystrophy (DMD) has been criticized based on speculation that the DBA/2J genetic background displays an inherent hypertrophic cardiomyopathy (HCM) phenotype. Accordingly, the goal of the current study was to further examine the cardiac status of this mouse strain over a 12-month period to determine if observable signs of HCM develop, including histopathology and pathological enlargement of the myocardium. Previous reports have documented heightened TGFß signaling in the DBA2/J striated muscles, as compared to the C57 background, which, as expected, is manifested as increased cardiomyocyte size, wall thickness, and heart mass as compared to the C57 background. While normalized heart mass is larger in the DBA/2J mice, compared to age-matched C57/BL10 mice, both strains similarly increase in size from 4 to 12 months of age. We also report that DBA/2J mice contain equivalent amounts of left ventricular collagen as healthy canine and human samples. In a longitudinal echocardiography study, neither sedentary nor exercised DBA/2J mice demonstrated left ventricular wall thickening or cardiac functional deficits. In summary, we find no evidence of HCM, nor any other cardiac pathology, and thus propose that it is an appropriate background strain for genetic modeling of cardiac diseases, including the cardiomyopathy associated with DMD.
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Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.
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Relojes Circadianos , Enfermedades Musculares , Animales , Relojes Circadianos/genética , Ritmo Circadiano , Conectina/genética , Conectina/metabolismo , Ratones , Músculo Esquelético/metabolismo , Enfermedades Musculares/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Quinasas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
BACKGROUND AND OBJECTIVES: Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder with a well-characterized disease phenotype but considerable interindividual heterogeneity that is not well understood. The aim of this study was to evaluate the effects of dystrophin variations and genetic modifiers of DMD on rate and age of muscle replacement by fat. METHODS: One hundred seventy-five corticosteroid treated participants from the ImagingDMD natural history study underwent repeated magnetic resonance spectroscopy (MRS) of the vastus lateralis (VL) and soleus (SOL) to determine muscle fat fraction (FF). MRS was performed annually in most instances; however, some individuals had additional visits at 3 or 6 monthss intervals. FF changes over time were modeled using nonlinear mixed effects to estimate disease trajectories based on the age that the VL or SOL reached half-maximum change in FF (mu) and the time required for FF change (sigma). Computed mu and sigma values were evaluated for dystrophin variations that have demonstrated the ability to lead to a mild phenotype as well as compared between different genetic polymorphism groups. RESULTS: Participants with dystrophin gene deletions amenable to exon 8 skipping (n = 4) had minimal increases in SOL FF and had an increase in VL mu value by 4.4 years compared with a reference cohort (p = 0.039). Participants with nonsense variations within exons that may produce milder phenotypes (n = 11) also had minimal increases in SOL and VL FFs. No differences in estimated FF trajectories were seen for individuals amenable to exon 44 skipping (n = 10). Modeling of the SPP1, LTBP4, and thrombospondin-1 (THBS1) genetic modifiers did not result in significant differences in muscle FF trajectories between genotype groups (p > 0.05); however, trends were noted for the polymorphisms associated with long-range regulation of LTBP4 and THBS1 that deserve further follow-up. DISCUSSION: The results of this study link the historically mild phenotypes seen in individuals amenable to exon 8 skipping and with certain nonsense variations with alterations in trajectories of lower extremity muscle replacement by fat.
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Distrofina , Distrofia Muscular de Duchenne , Humanos , Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico por imagen , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Exones , Imagen por Resonancia Magnética/métodos , Progresión de la EnfermedadRESUMEN
Skeletal muscle fibers are multinucleated cellular giants formed by the fusion of mononuclear myoblasts. Several molecules involved in myoblast fusion have been discovered, and finger-like projections coincident with myoblast fusion have also been implicated in the fusion process. The role of these cellular projections in muscle cell fusion was investigated herein. We demonstrate that these projections are filopodia generated by class X myosin (Myo10), an unconventional myosin motor protein specialized for filopodia. We further show that Myo10 is highly expressed by differentiating myoblasts, and Myo10 ablation inhibits both filopodia formation and myoblast fusion in vitro. In vivo, Myo10 labels regenerating muscle fibers associated with Duchenne muscular dystrophy and acute muscle injury. In mice, conditional loss of Myo10 from muscle-resident stem cells, known as satellite cells, severely impairs postnatal muscle regeneration. Furthermore, the muscle fusion proteins Myomaker and Myomixer are detected in myoblast filopodia. These data demonstrate that Myo10-driven filopodia facilitate multinucleated mammalian muscle formation.
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Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Mioblastos Esqueléticos/metabolismo , Miosinas/metabolismo , Seudópodos/metabolismo , Animales , Diferenciación Celular , Fusión Celular , Línea Celular , Proliferación Celular , Modelos Animales de Enfermedad , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Desarrollo de Músculos , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patología , Mioblastos Esqueléticos/patología , Miosinas/genética , Seudópodos/genética , Regeneración , Células Satélite del Músculo Esquelético/metabolismo , Células Satélite del Músculo Esquelético/patología , Factores de TiempoRESUMEN
Duchenne muscular dystrophy (DMD) is a devastating genetic muscle disease resulting in progressive muscle degeneration and wasting. Glucocorticoids, specifically prednisone/prednisolone and deflazacort, are commonly used by DMD patients. Emerging DMD therapeutics include those targeting the muscle-wasting factor, myostatin (Mstn). The aim of this study was to investigate how chronic glucocorticoid treatment impacts the efficacy of Mstn inhibition in the D2.mdx mouse model of DMD. We report that chronic treatment of dystrophic mice with prednisolone (Pred) causes significant muscle wasting, entailing both activation of the ubiquitin-proteasome degradation pathway and inhibition of muscle protein synthesis. Combining Pred with Mstn inhibition, using a modified Mstn propeptide (dnMstn), completely abrogates the muscle hypertrophic effects of Mstn inhibition independently of Mstn expression or SMAD3 activation. Transcriptomic analysis identified that combining Pred with dnMstn treatment affects gene expression profiles associated with inflammation, metabolism, and fibrosis. Additionally, we demonstrate that Pred-induced muscle atrophy is not prevented by Mstn ablation. Therefore, glucocorticoids interfere with potential muscle mass benefits associated with targeting Mstn, and the ramifications of glucocorticoid use should be a consideration during clinical trial design for DMD therapeutics. These results have significant implications for past and future Mstn inhibition trials in DMD.
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Glucocorticoides/metabolismo , Glucocorticoides/farmacología , Hipertrofia/metabolismo , Distrofias Musculares/tratamiento farmacológico , Miostatina/efectos de los fármacos , Miostatina/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/tratamiento farmacológico , Miostatina/genética , TranscriptomaRESUMEN
Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle degenerative disease caused by loss of dystrophin protein. DMD has no cure and few treatment options. Preclinical efforts to identify potential DMD therapeutics have been hampered by lack of a small animal model that recapitulates key features of the human disease. While the dystrophin-deficient mdx mouse on the C57BL/10 genetic background (B10.mdx) is mildly affected, a more severe muscle disease is observed when the mdx mutation is crossed onto the DBA/2J genetic background (D2.mdx). In this study, the functional and histological progression of the D2.mdx skeletal muscle pathology was evaluated to determine the distinguishing features of disease. Data herein details the muscular weakness and wasting exhibited by D2.mdx skeletal muscle, as well as severe histopathological features, which include the rapid progression of fibrosis and calcifications in the diaphragm and progressive fibrosis accumulation in limb muscles. Furthermore, a timeline of D2.mdx progression is provided that details distinct stages of disease progression. These data support the D2.mdx as a superior small animal model for DMD, as compared to the B10.mdx model. The insights provided in this report should facilitate the design of preclinical evaluations for potential DMD therapeutics.
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Ratones Endogámicos mdx , Músculo Esquelético/patología , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/patología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inflamación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/genética , Distribución Aleatoria , Manejo de Especímenes , TranscriptomaRESUMEN
Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder characterized by loss of motor neurons in the ventral horn of the spinal cord. Clinical features such as progressively lethal respiratory weakness and associated muscle wasting have been extensively studied but less attention has been given to gastrointestinal (GI) dysfunction, which is common symptomatology in SMA patients with 43% constipation, 15% abdominal pain, and 14% meteorism. In the current study, the PrP92-SMN mouse model of SMA was utilized, to complement previous studies in which cells of the Enteric Nervous system (ENS) were susceptible to Smn (survival motor neuron) deficiency and could possibly be the basis of the observed GI symptoms. Necropsy of our mouse model showed impairment in feces excretion and smaller bladder mass, compared to Wild-Type (WT) animals. Along with the reduction in bladder mass, we also observed a decrease in the size of smooth muscles, due to reduction in Cross-Sectional Area (CSA). Interstitial cells of Cajal (ICC) provide important regulatory functions in the GI tract. To investigate if ICC are implicated in Smn deficient-induced colonic dysmotility, we assessed ICC distribution and abundance, by c-Kit, a well-established marker. SMA mice exhibited fewer c-Kit positive cells with altered localization, compared to WT. In conclusion, the observed histopathological abnormalities of our mouse model, can be secondary to SMN deficiency and could possibly underlie the GI symptoms observed in SMA patients. Future therapeutic approaches for SMA, must address not only CNS symptoms, but also non-motor-neuron-related symptoms. The PrP92-SMN mouse model could be a useful model for assessing therapeutic rescue of GI dysfunction in SMA.
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In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor Bmal1. We identify a non-canonical E-box element in Bmal1 and demonstrate that is required for full MYOD1-responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and BMAL1:CLOCK work in a synergistic fashion through a tandem E-box to regulate the expression and amplitude of the muscle specific clock-controlled gene, Titin-cap (Tcap). In conclusion, these findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the regulation of molecular clock amplitude as well as synergistic regulation of clock-controlled genes in skeletal muscle.
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Péptidos y Proteínas de Señalización del Ritmo Circadiano/biosíntesis , Regulación de la Expresión Génica , Músculo Esquelético/enzimología , Proteína MioD/metabolismo , Factores de Transcripción ARNTL/metabolismo , Animales , Proteínas CLOCK/metabolismo , Relojes Circadianos , Conectina/metabolismo , Ratones Endogámicos C57BLRESUMEN
Lack of sarcolemma-localized neuronal nitric oxide synthase mu (nNOSµ) contributes to muscle damage and fatigue in dystrophic muscle. In this study, we examined the effects of compensating for lack of nNOSµ with a phosphodiesterase type 5 (PDE5) inhibitor in mdx mice following downhill running and endurance training. Dystrophic mice (mdx) were treated with sildenafil citrate and compared with untreated mdx and wild-type mice after an acute bout of downhill running and during a progressive low-intensity treadmill running program (5 days/wk, 4 wk). Magnetic resonance imaging (MRI) and spectroscopy (MRS) transverse relaxation time constant (T2) of hindlimb and forelimb muscles were measured as a marker of muscle damage after downhill running and throughout training. The MRI blood oxygenation level dependence (BOLD) response and 31phosphorus MRS (31P-MRS) data were acquired after stimulated muscle contractions. After downhill running, the increase in T2 was attenuated (P < 0.05) in treated mdx and wild-type mice compared with untreated mdx. During training, resting T2 values did not change in wild-type and mdx mice from baseline values; however, the running distance completed during training was greater (P < 0.05) in treated mdx (>90% of target distance) and wild-type (100%) than untreated mdx (60%). The post-contractile BOLD response was greater (P < 0.05) in treated mdx that trained than untreated mdx, with no differences in muscle oxidative capacity, as measured by 31P-MRS. Our findings indicate that PDE5 inhibition reduces muscle damage after a single bout of downhill running and improves performance during endurance training in dystrophic mice, possibly because of enhanced microvascular function. NEW & NOTEWORTHY This study examined the combined effects of PDE5 inhibition and exercise in dystrophic muscle using high-resolution magnetic resonance imaging and spectroscopy. Our findings demonstrated that sildenafil citrate reduces muscle damage after a single bout of downhill running, improves endurance-training performance, and enhances microvascular function in dystrophic muscle. Collectively, the results support the combination of exercise and PDE5 inhibition as a therapeutic approach in muscular dystrophies lacking nNOSµ.
Asunto(s)
Músculo Esquelético/efectos de los fármacos , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Inhibidores de Fosfodiesterasa 5/farmacología , Condicionamiento Físico Animal/fisiología , Animales , Entrenamiento Aeróbico/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Contracción Muscular/fisiología , Sarcolema/efectos de los fármacos , Citrato de Sildenafil/farmacologíaRESUMEN
This study investigated the effect of age on recovery of skeletal muscle from an ischemia-reperfusion (I/R)-induced injury. Young (6 mo old) and old (24-27 mo old) Sprague-Dawley rats underwent a 2-h bout of hindlimb ischemia induced by a pneumatic tourniquet (TK). The TK was released to allow reperfusion of the affected limb, and animals were divided into 7- and 14-day recovery groups. Maximum plantar flexor force production was assessed in both 7- and 14-day recovery groups of both ages, followed by histological evaluation. Subsequent analysis of IGF-I gene expression and intracellular signaling in 7-day recovery muscles was performed by RT-PCR and Western blotting, respectively. Old rats had significantly greater deficits in force production and exhibited more evidence of histological pathology than young at both 7 and 14 days postinjury. In addition, old rats demonstrated an attenuated upregulation of IGF-I mRNA and induction of proanabolic signaling compared with young in response to injury. We conclude that aged skeletal muscle exhibits more damage and/or defective regeneration following I/R and identify an age-associated decrease in local IGF-I responsiveness as a potential mechanism for this phenomenon.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/metabolismo , Fuerza Muscular , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatología , Regeneración , Daño por Reperfusión/metabolismo , Daño por Reperfusión/fisiopatología , Factores de Edad , Animales , Modelos Animales de Enfermedad , Miembro Posterior , Factor I del Crecimiento Similar a la Insulina/genética , Masculino , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/patología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Recuperación de la Función , Daño por Reperfusión/etiología , Daño por Reperfusión/genética , Transducción de Señal , Factores de Tiempo , Torniquetes/efectos adversosRESUMEN
Muscle cells are designed to generate force and movement. There are three types of mammalian muscles-skeletal, cardiac, and smooth. Skeletal muscles are attached to bones and move them relative to each other. Cardiac muscle comprises the heart, which pumps blood through the vasculature. Skeletal and cardiac muscles are known as striated muscles, because the filaments of actin and myosin that power their contraction are organized into repeating arrays, called sarcomeres, that have a striated microscopic appearance. Smooth muscle does not contain sarcomeres but uses the contraction of filaments of actin and myosin to constrict blood vessels and move the contents of hollow organs in the body. Here, we review the principal molecular organization of the three types of muscle and their contractile regulation through signaling mechanisms and discuss their major structural and functional similarities that hint at the possible evolutionary relationships between the cell types.