RESUMEN
WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.
Asunto(s)
Regulación Alostérica , Descubrimiento de Drogas , Inhibidores Enzimáticos , Proteómica , Helicasa del Síndrome de Werner , Animales , Femenino , Humanos , Masculino , Ratones , Regulación Alostérica/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/patología , Cisteína/efectos de los fármacos , Cisteína/metabolismo , Roturas del ADN de Doble Cadena/efectos de los fármacos , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inestabilidad de Microsatélites , Modelos Moleculares , Helicasa del Síndrome de Werner/antagonistas & inhibidores , Helicasa del Síndrome de Werner/química , Helicasa del Síndrome de Werner/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Muerte Celular/efectos de los fármacos , Adenosina Trifosfato/metabolismoRESUMEN
Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.
Asunto(s)
Complejos de Ubiquitina-Proteína Ligasa/fisiología , Línea Celular Tumoral , Humanos , Masculino , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Receptores Androgénicos/metabolismo , Ubiquitina/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismoRESUMEN
A structure-activity relationship has been developed around the meridianin scaffold for inhibition of Dyrk1a. The compounds have been focussed on the inhibition of kinase Dyrk1a, as a means to retain the transcription factor NFAT in the nucleus. NFAT is responsible for up-regulation of genes responsible for the induction of a slow, oxidative skeletal muscle phenotype, which may be an effective treatment for diseases where exercise capacity is compromised. The SAR showed that while strong Dyrk1a binding was possible with the meridianin scaffold the compounds have no effect on NFAT localisation, however, by moving from the indole to a 6-azaindole scaffold both potent Dyrk1a binding and increased NFAT residence time in the nucleus were obtained - properties not observed with the reported Dyrk1a inhibitors. One compound was shown to be effective in an ex vivo muscle fiber assay. The increased biological activity is thought to arise from the added interaction between the azaindole nitrogen and the lysine residue in the back pocket.
Asunto(s)
Núcleo Celular/metabolismo , Alcaloides Indólicos/química , Factores de Transcripción NFATC/metabolismo , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Compuestos Aza/química , Sitios de Unión , Núcleo Celular/efectos de los fármacos , Humanos , Alcaloides Indólicos/síntesis química , Alcaloides Indólicos/farmacología , Indoles/química , Concentración 50 Inhibidora , Ratones , Microscopía Confocal , Simulación del Acoplamiento Molecular , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Factores de Transcripción NFATC/genética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad , Regulación hacia Arriba/efectos de los fármacos , Quinasas DyrKRESUMEN
Controlled mechanical ventilation (CMV) is associated with the development of diaphragm atrophy and contractile dysfunction, and respiratory muscle weakness is thought to contribute significantly to delayed weaning of patients. Therefore, therapeutic strategies for preventing these processes may have clinical benefit. The aim of the current study was to investigate the role of the Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in CMV-mediated diaphragm wasting and weakness in rats. CMV-induced diaphragm atrophy and contractile dysfunction coincided with marked increases in STAT3 phosphorylation on both tyrosine 705 (Tyr705) and serine 727 (Ser727). STAT3 activation was accompanied by its translocation into mitochondria within diaphragm muscle and mitochondrial dysfunction. Inhibition of JAK signaling during CMV prevented phosphorylation of both target sites on STAT3, eliminated the accumulation of phosphorylated STAT3 within the mitochondria, and reversed the pathologic alterations in mitochondrial function, reduced oxidative stress in the diaphragm, and maintained normal diaphragm contractility. In addition, JAK inhibition during CMV blunted the activation of key proteolytic pathways in the diaphragm, as well as diaphragm atrophy. These findings implicate JAK/STAT3 signaling in the development of diaphragm muscle atrophy and dysfunction during CMV and suggest that the delayed extubation times associated with CMV can be prevented by inhibition of Janus kinase signaling.-Smith, I. J., Godinez, G. L., Singh, B. K., McCaughey, K. M., Alcantara, R. R., Gururaja, T., Ho, M. S., Nguyen, H. N., Friera, A. M., White, K. A., McLaughlin, J. R., Hansen, D., Romero, J. M., Baltgalvis, K. A., Claypool, M. D., Li, W., Lang, W., Yam, G. C., Gelman, M. S., Ding, R., Yung, S. L., Creger, D. P., Chen, Y., Singh, R., Smuder, A. J., Wiggs, M. P., Kwon, O.-S., Sollanek, K. J., Powers, S. K., Masuda, E. S., Taylor, V. C., Payan, D. G., Kinoshita, T., Kinsella, T. M. Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction.
Asunto(s)
Diafragma/metabolismo , Quinasas Janus/metabolismo , Respiración Artificial/efectos adversos , Transducción de Señal/fisiología , Animales , Interleucina-6/metabolismo , Masculino , Mitocondrias/metabolismo , Debilidad Muscular/metabolismo , Atrofia Muscular/metabolismo , Estrés Oxidativo/fisiología , Fosforilación/fisiología , Proteolisis , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMEN
Intermittent claudication is a form of exercise intolerance characterized by muscle pain during walking in patients with peripheral artery disease (PAD). Endothelial cell and muscle dysfunction are thought to be important contributors to the etiology of this disease, but a lack of preclinical models that incorporate these elements and measure exercise performance as a primary end point has slowed progress in finding new treatment options for these patients. We sought to develop an animal model of peripheral vascular insufficiency in which microvascular dysfunction and exercise intolerance were defining features. We further set out to determine if pharmacological activation of 5'-AMP-activated protein kinase (AMPK) might counteract any of these functional deficits. Mice aged on a high-fat diet demonstrate many functional and molecular characteristics of PAD, including the sequential development of peripheral vascular insufficiency, increased muscle fatigability, and progressive exercise intolerance. These changes occur gradually and are associated with alterations in nitric oxide bioavailability. Treatment of animals with an AMPK activator, R118, increased voluntary wheel running activity, decreased muscle fatigability, and prevented the progressive decrease in treadmill exercise capacity. These functional performance benefits were accompanied by improved mitochondrial function, the normalization of perfusion in exercising muscle, increased nitric oxide bioavailability, and decreased circulating levels of the endogenous endothelial nitric oxide synthase inhibitor asymmetric dimethylarginine. These data suggest that aged, obese mice represent a novel model for studying exercise intolerance associated with peripheral vascular insufficiency, and pharmacological activation of AMPK may be a suitable treatment for intermittent claudication associated with PAD.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Dieta Alta en Grasa , Activadores de Enzimas/administración & dosificación , Obesidad/complicaciones , Enfermedades Vasculares Periféricas/fisiopatología , Esfuerzo Físico/fisiología , Envejecimiento , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Apolipoproteínas E/fisiología , Arginina/análogos & derivados , Arginina/sangre , Cilostazol , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Humanos , Claudicación Intermitente/complicaciones , Claudicación Intermitente/tratamiento farmacológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fatiga Muscular/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Óxido Nítrico Sintasa de Tipo III/metabolismo , Enfermedades Vasculares Periféricas/etiología , Inhibidores de Fosfodiesterasa 3/administración & dosificación , Tetrazoles/administración & dosificación , VasodilatadoresRESUMEN
Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.
Asunto(s)
Caquexia/metabolismo , Neoplasias del Colon/complicaciones , Interleucina-6/sangre , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Fosforilación Oxidativa , Tejido Adiposo/patología , Aldehídos/metabolismo , Animales , Peso Corporal , Caquexia/etiología , Caquexia/patología , Caquexia/fisiopatología , Catalasa/genética , Citocromos c/metabolismo , ADN Mitocondrial/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , GTP Fosfohidrolasas/genética , Expresión Génica/genética , Genes APC , Miembro Posterior/metabolismo , Miembro Posterior/patología , Miembro Posterior/fisiopatología , Interleucina-6/genética , Canales Iónicos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Fibras Musculares de Contracción Rápida/metabolismo , Fibras Musculares de Contracción Lenta/metabolismo , Músculo Esquelético/fisiopatología , Estrés Oxidativo/fisiología , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Factor de Transcripción STAT3/metabolismo , Sirtuina 1/genética , Succinato Deshidrogenasa/metabolismo , Superóxido Dismutasa/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción , Proteína Desacopladora 3RESUMEN
Interleukin 6 (IL-6) has received significant attention for its regulatory role in muscle wasting during cachexia. This review examines the role of circulating IL-6 for decreasing muscle mass during cancer and emphasizes some of the indirect actions of IL-6 that may cause muscle wasting.
Asunto(s)
Caquexia/metabolismo , Interleucina-6/metabolismo , Músculo Esquelético/metabolismo , Caquexia/sangre , Caquexia/etiología , Ejercicio Físico/fisiología , Glucógeno/metabolismo , Humanos , Hipertrofia , Inflamación/sangre , Inflamación/metabolismo , Interleucina-6/sangre , Neoplasias/complicaciones , Factores de Riesgo , Transducción de SeñalRESUMEN
Muscle weakness ensues when serum testosterone declines with age in men. Testosterone's female counterpart, estrogen, also has been implicated in age-related strength loss, but these results are less conclusive. Our working hypothesis is that estrogens do benefit muscle strength, and that the underlying mechanism involves estrogen receptors to improve muscle quality more so than quantity.
Asunto(s)
Envejecimiento/fisiología , Estrógenos/fisiología , Fuerza Muscular/fisiología , Animales , Espectroscopía de Resonancia por Spin del Electrón , Estradiol/fisiología , Femenino , Humanos , Menopausia/fisiología , Ratones , Debilidad Muscular , Músculo Esquelético/metabolismo , Miosinas/fisiología , Miosinas/ultraestructura , Ratas , Receptores de Estrógenos/metabolismoRESUMEN
Interleukin-6 (IL-6) is necessary for cachexia in Apc ( Min/+ ) mice, but the mechanisms inducing this myofiber wasting have not been established. The purpose of this study was to examine gastrocnemius muscle wasting in the Apc ( Min/+ ) mouse and to determine IL-6 regulated mechanisms contributing to muscle loss. Gastrocnemius type IIB mean fiber cross-sectional area (CSA) from Apc ( Min/+ ) mice decreased 32% between 13 and 22 weeks of age. Apc ( Min/+ ) mice lacking IL-6 did not have type IIB fiber atrophy, while overexpression of circulating IL-6 exacerbated the loss of type IIB fiber CSA in Apc ( Min/+ ) mice. Muscle Atrogin-I mRNA expression was induced at least ninefold at 18 and 22 weeks of age compared to 13-week-old mice. Atrogin-I gene expression was also induced by overexpression of circulating IL-6. These data suggest that high circulating IL-6 levels induce type IIB fiber CSA loss in Apc ( Min/+ ) mice, and circulating IL-6 is sufficient to regulate Atrogin-I gene expression in cachectic mice.
Asunto(s)
Caquexia/fisiopatología , Interleucina-6/fisiología , Proteínas Musculares/biosíntesis , Atrofia Muscular/fisiopatología , Proteínas Ligasas SKP Cullina F-box/biosíntesis , Animales , Caquexia/patología , Expresión Génica , Interleucina-6/sangre , Pólipos Intestinales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Proteínas Musculares/metabolismo , Fuerza Muscular/fisiología , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Atrofia Muscular/patología , Miofibrillas/patologíaRESUMEN
BACKGROUND: The loss of dystrophin compromises muscle cell membrane stability and causes Duchenne muscular dystrophy and/or various forms of cardiomyopathy. Increased expression of the dystrophin homolog utrophin by gene delivery or pharmacologic up-regulation has been demonstrated to restore membrane integrity and improve the phenotype in the dystrophin-deficient mdx mouse. However, the lack of a viable therapy in humans predicates the need to explore alternative methods to combat dystrophin deficiency. We investigated whether systemic administration of recombinant full-length utrophin (Utr) or DeltaR4-21 "micro" utrophin (muUtr) protein modified with the cell-penetrating TAT protein transduction domain could attenuate the phenotype of mdx mice. METHODS AND FINDINGS: Recombinant TAT-Utr and TAT-muUtr proteins were expressed using the baculovirus system and purified using FLAG-affinity chromatography. Age-matched mdx mice received six twice-weekly intraperitoneal injections of either recombinant protein or PBS. Three days after the final injection, mice were analyzed for several phenotypic parameters of dystrophin deficiency. Injected TAT-muUtr transduced all tissues examined, integrated with members of the dystrophin complex, reduced serum levels of creatine kinase (11,290+/-920 U versus 5,950+/-1,120 U; PBS versus TAT), the prevalence of muscle degeneration/regeneration (54%+/-5% versus 37%+/-4% of centrally nucleated fibers; PBS versus TAT), the susceptibility to eccentric contraction-induced force drop (72%+/-5% versus 40%+/-8% drop; PBS versus TAT), and increased specific force production (9.7+/-1.1 N/cm(2) versus 12.8+/-0.9 N/cm(2); PBS versus TAT). CONCLUSIONS: These results are, to our knowledge, the first to establish the efficacy and feasibility of TAT-utrophin-based constructs as a novel direct protein-replacement therapy for the treatment of skeletal and cardiac muscle diseases caused by loss of dystrophin.
Asunto(s)
Distrofina/deficiencia , Distrofia Muscular Animal/tratamiento farmacológico , Distrofia Muscular de Duchenne/tratamiento farmacológico , Proteínas Recombinantes de Fusión/uso terapéutico , Utrofina/uso terapéutico , Animales , Creatina Quinasa/sangre , Distrofina/genética , Productos del Gen tat/genética , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Distrofia Muscular Animal/patología , Distrofia Muscular de Duchenne/patología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Utrofina/genética , Utrofina/metabolismoRESUMEN
Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc(Min/+) mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc(Min/+) mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc(Min/+) mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc(Min/+) mice were randomly assigned to control (n = 20) or exercise (n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc(Min/+) mice were used to quantify markers of polyp inflammation, apoptosis, and beta-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. beta-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc(Min/+) mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.
Asunto(s)
Poliposis Adenomatosa del Colon/genética , Poliposis Adenomatosa del Colon/patología , Condicionamiento Físico Animal/fisiología , Animales , Apoptosis/fisiología , Western Blotting , Ciclooxigenasa 2/biosíntesis , Ciclooxigenasa 2/genética , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Inflamación/patología , Inflamación/fisiopatología , Macrófagos/fisiología , Masculino , Ratones , Ratones Noqueados , Infiltración Neutrófila/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/fisiología , Proteína X Asociada a bcl-2/biosíntesis , Proteína X Asociada a bcl-2/genética , beta Catenina/biosíntesis , beta Catenina/genéticaRESUMEN
Force loss in skeletal muscle exposed to eccentric contraction is often attributed to injury. We show that EDL muscles from dystrophin-deficient mdx mice recover 65% of lost force within 120 min of eccentric contraction and exhibit minimal force loss when the interval between contractions is increased from 3 to 30 min. A proteomic screen of mdx muscle identified an 80% reduction in the antioxidant peroxiredoxin-2, likely due to proteolytic degradation following hyperoxidation by NADPH Oxidase 2. Eccentric contraction-induced force loss in mdx muscle was exacerbated by peroxiredoxin-2 ablation, and improved by peroxiredoxin-2 overexpression or myoglobin knockout. Finally, overexpression of γcyto- or ßcyto-actin protects mdx muscle from eccentric contraction-induced force loss by blocking NADPH Oxidase 2 through a mechanism dependent on cysteine 272 unique to cytoplasmic actins. Our data suggest that eccentric contraction-induced force loss may function as an adaptive circuit breaker that protects mdx muscle from injurious contractions.
Asunto(s)
Distrofina/metabolismo , Contracción Muscular/fisiología , Músculo Esquelético/metabolismo , Peroxirredoxinas/metabolismo , Animales , Distrofina/deficiencia , Immunoblotting , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Contracción Muscular/genética , Peroxirredoxinas/genéticaRESUMEN
Aging can alter the skeletal muscle growth response induced by overload. The initiation of overload induces muscle extracellular matrix expansion, increased cellularity, and inflammatory gene expression, which are all related to processes important for myofiber growth. These remodeling processes are also biological targets of testosterone. It is not certain how aging affects the inflammatory response to functional overload and whether anabolic steroid administration can alter this response. The effect of anabolic steroid administration on inflammatory processes during functional overload is not known. The purpose of this study was to determine if age altered the skeletal muscle inflammatory response at the onset of functional overload and whether anabolic steroid administration would modulate this response in young or older animals. Five-month and 25 month F344 x BRN rats were given nandrolone decanoate (ND) (6 mg/kg bw/wk) or sham injections for 3 weeks, and then the soleus muscle was overloaded (OV) for 3 days by synergist ablation. ND alone induced a 230% increase in ED1(+) cells in 5 month muscle. Three days of OV had no effect on ED1(+) cell number at either age. OV combined with ND induced a 90% increase in ED2(+) cells in 5 month muscle, while there was no effect of either treatment alone at this age. In 25 month muscle, OV induced a 40% increase in ED2(+) cells. Regardless of age, OV induced muscle TNF-alpha mRNA expression (300%) and IL-6 mRNA expression (900%). ND attenuated OV-induced IL-6 mRNA but not TNF-alpha expression in both age groups. The overload induction of IL-1beta mRNA was 3-fold greater in 25 month muscle (1400%), compared to 5 month muscle (400%). ND administration ablated the overload IL-1beta mRNA induction in 25 month muscle. Anabolic steroid administration can suppress inflammatory cytokine gene expression at the onset of overload and this effect is age dependent.
Asunto(s)
Envejecimiento/inmunología , Anabolizantes/farmacología , Músculo Esquelético/inmunología , Nandrolona/análogos & derivados , Envejecimiento/efectos de los fármacos , Animales , Peso Corporal/fisiología , Núcleo Celular/inmunología , Núcleo Celular/patología , Citocinas/inmunología , Matriz Extracelular/inmunología , Matriz Extracelular/patología , Inmunohistoquímica/métodos , Macrófagos/inmunología , Macrófagos/patología , Masculino , Fibras Musculares Esqueléticas/inmunología , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Nandrolona/farmacología , Nandrolona Decanoato , Tamaño de los Órganos/fisiología , ARN Mensajero/análisis , Ratas , Ratas Endogámicas F344 , Células Satélite del Músculo Esquelético/inmunología , Células Satélite del Músculo Esquelético/patología , Testosterona/inmunologíaRESUMEN
PURPOSE: The primary objective of this study was to determine whether strength loss and recovery after eccentric contractions are impaired in healthy and dystrophic female mice with low levels of ovarian hormones. METHODS: Female C57BL/6 (wild-type) or mdx mice were randomly assigned to ovarian-intact (Sham) and ovariectomized (Ovx) groups. Anterior crural muscles were tested for susceptibility to injury from 150 or 50 eccentric contractions in wild-type and mdx mice, respectively. An additional experiment challenged mdx mice with a 2-wk treadmill running protocol followed by an eccentric contraction injury to posterior crural muscles. Functional recovery from injury was evaluated in wild-type mice by measuring isometric torque 3, 7, 14, or 21 d after injury. RESULTS: Ovarian hormone deficiency in wild-type mice did not affect susceptibility to injury because the â¼50% isometric torque loss after eccentric contractions did not differ between Sham and Ovx mice (P = 0.121). Similarly, in mdx mice, hormone deficiency did not affect the percent of preinjury isometric torque lost by anterior crural muscles after eccentric contractions (P = 0.952), but the percent of preinjury torque in posterior crural muscles was lower in Ovx than in Sham mice (P = 0.014). Recovery from injury in wild-type mice was affected by hormone deficiency. Sham mice recovered preinjury isometric strength by 14 d (96% ± 2%), whereas Ovx mice maintained deficits at 14 and 21 d after injury (80% ± 3% and 84% ± 2%, P < 0.001). CONCLUSIONS: Ovarian hormone status did not affect the vulnerability of skeletal muscle to strength loss after eccentric contractions. However, ovarian hormone deficiency did impair the recovery of muscle strength in female mice.
Asunto(s)
Hormonas/metabolismo , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Ovario/metabolismo , Animales , Creatina Quinasa/sangre , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Contracción Muscular/fisiología , Proteínas Musculares/metabolismo , Ovariectomía , Distribución Aleatoria , Recuperación de la Función/fisiología , TorqueRESUMEN
Numerous human diseases can lead to atrophy of skeletal muscle, and loss of this tissue has been correlated with increased mortality and morbidity rates. Clinically addressing muscle atrophy remains an unmet medical need, and the development of preclinical tools to assist drug discovery and basic research in this effort is important for advancing this goal. In this report, we describe the development of a bioluminescent gene reporter rat, based on the zinc finger nuclease-targeted insertion of a bicistronic luciferase reporter into the 3' untranslated region of a muscle specific E3 ubiquitin ligase gene, MuRF1 (Trim63). In longitudinal studies, we noninvasively assess atrophy-related expression of this reporter in three distinct models of muscle loss (sciatic denervation, hindlimb unloading and dexamethasone-treatment) and show that these animals are capable of generating refined detail on in vivo MuRF1 expression with high temporal and anatomical resolution.
Asunto(s)
Mediciones Luminiscentes/métodos , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Femenino , Genes Reporteros , Suspensión Trasera , Proteínas Musculares/genética , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/patología , Ratas , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Facioscapulohumeral muscular dystrophy (FSHD) is an enigmatic disease associated with epigenetic alterations in the subtelomeric heterochromatin of the D4Z4 macrosatellite repeat. Each repeat unit encodes DUX4, a gene that is normally silent in most tissues. Besides muscular loss, most patients suffer retinal vascular telangiectasias. To generate an animal model, we introduced a doxycycline-inducible transgene encoding DUX4 and 3' genomic DNA into a euchromatic region of the mouse X chromosome. Without induction, DUX4 RNA was expressed at low levels in many tissues and animals displayed a variety of unexpected dominant leaky phenotypes, including male-specific lethality. Remarkably, rare live-born males expressed DUX4 RNA in the retina and presented a retinal vascular telangiectasia. By using doxycycline to induce DUX4 expression in satellite cells, we observed impaired myogenesis in vitro and in vivo. This mouse model, which shows pathologies due to FSHD-related D4Z4 sequences, is likely to be useful for testing anti-DUX4 therapies in FSHD.
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Genes Dominantes , Genes Ligados a X , Proteínas de Homeodominio/genética , Distrofia Muscular Facioescapulohumeral/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Eucromatina/genética , Proteínas de Homeodominio/metabolismo , Masculino , Ratones , Ratones Transgénicos , Distrofia Muscular Facioescapulohumeral/patología , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Retina/metabolismo , Retina/patologíaRESUMEN
PURPOSE: We tested the hypothesis that low-intensity vibration training in mice improves contractile function of hindlimb skeletal muscles and promotes exercise-related cellular adaptations. METHODS: We subjected C57BL/6J mice to 6 wk, 5 d·wk, 15 min·d of sham or low-intensity vibration (45 Hz, 1.0g) while housed in traditional cages (Sham-Active, n = 8; Vibrated-Active, n = 10) or in small cages to restrict physical activity (Sham-Restricted, n = 8; Vibrated-Restricted, n = 8). Contractile function and resistance to fatigue were tested in vivo (anterior and posterior crural muscles) and ex vivo on the soleus muscle. Tibialis anterior and soleus muscles were evaluated histologically for alterations in oxidative metabolism, capillarity, and fiber types. Epididymal fat pad and hindlimb muscle masses were measured. Two-way ANOVAs were used to determine the effects of vibration and physical inactivity. RESULTS: Vibration training resulted in a 10% increase in maximal isometric torque (P = 0.038) and 16% faster maximal rate of relaxation (P = 0.030) of the anterior crural muscles. Posterior crural muscles were unaffected by vibration, except greater rates of contraction in Vibrated-Restricted mice compared with Vibrated-Active and Sham-Restricted mice (P = 0.022). Soleus muscle maximal isometric tetanic force tended to be greater (P = 0.057), and maximal relaxation was 20% faster (P = 0.005) in vibrated compared with sham mice. The restriction of physical activity induced muscle weakness but was not required for vibration to be effective in improving strength or relaxation. Vibration training did not affect muscle fatigability or any indicator of cellular adaptation investigated (P ≥ 0.431). Fat pad but not hindlimb muscle masses were affected by vibration training. CONCLUSION: Vibration training in mice improved muscle contractility, specifically strength and relaxation rates, with no indication of adverse effects to muscle function or cellular adaptations.
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Adaptación Fisiológica , Contracción Muscular/fisiología , Fuerza Muscular/fisiología , Músculo Esquelético/fisiología , Condicionamiento Físico Animal/métodos , Vibración , Animales , Biomarcadores/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora , Fatiga Muscular/fisiología , Músculo Esquelético/anatomía & histología , Músculo Esquelético/metabolismo , Condicionamiento Físico Animal/instrumentación , Distribución AleatoriaRESUMEN
PURPOSE: We tested the hypothesis that low-intensity exercise in mdx mice improves plantar flexor muscle contractile function, resistance to fatigue, and mitochondrial adaptations without exacerbating muscular dystrophy. METHODS: We subjected mdx mice to 12 wk of voluntary low-resistance wheel running (Run, n = 17) or normal cage activities (sedentary (Sed), n = 16) followed by in vivo analyses for plantar flexor torque generation and fatigue resistance or running capacity on a treadmill. Gastrocnemius muscles were further evaluated for exercise-induced mitochondrial adaptations and fiber type distribution and central nuclei. t-tests were used to determine differences between the Sed and Run groups. RESULTS: Plantar flexor submaximal isometric torques and maximal isometric torque at multiple ankle joint angles and resistance to fatigue were greater in Run compared with Sed mdx mice (P G 0.05). Citrate synthase and A-hydroxyacyl-CoA dehydrogenase enzyme activities and cytochrome c oxidase IV protein expression in gastrocnemius muscles were greater in Run than in Sed mdx mice(P e 0.04), along with a trend of fiber type transformation from Type IIb to Type IIx fibers. Exercise training in mdx mice did not elevate serum creatine kinase levels but led to a significant reduction of centrally nucleated myofibers. CONCLUSIONS: Voluntary low-resistance wheel running in mdx mice can result in skeletal muscle adaptation, leading to improved contractile function and reduced fatigability,with no indication that exercise was detrimental. This study supports the need for further investigation of low-intensity exercise as an early therapeutic intervention in ambulatory boys with Duchenne muscular dystrophy.
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Músculo Esquelético/fisiología , Distrofia Muscular Animal/fisiopatología , Condicionamiento Físico Animal/fisiología , Animales , Articulación del Tobillo/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos mdx , Mitocondrias/enzimología , Actividad Motora/fisiología , Contracción Muscular/fisiología , Fatiga Muscular/fisiología , Músculo Esquelético/anatomía & histología , Músculo Esquelético/metabolismo , Carrera/fisiologíaRESUMEN
BACKGROUND: Cachexia involves unintentional body weight loss including diminished muscle and adipose tissue mass and is associated with an underlying disease. Systemic overexpression of IL-6 accelerates cachexia in the Apc(Min/+) mouse, but does not induce wasting in control C57BL/6 mice. With many chronic diseases, chronic inflammation and metabolic dysfunction can be improved with moderate exercise. A direct effect of regular moderate exercise on the prevention of IL-6-induced cachexia in the Apc(Min/+) mouse has not been investigated. The purpose of this study was to assess the effects of exercise on the development of cachexia in the Apc(Min/+) mouse. METHODS: Mice were randomly assigned to moderate treadmill exercise (18 m/min, 1 h, 6 days/week, 5% grade) or cage control (CC) groups from 6 to 14 weeks of age. At 12 weeks of age, mice were electroporated with either IL-6-containing or control plasmid into the quadriceps muscle. Mice were killed after 2 weeks of systemic IL-6 overexpression or control treatment. RESULTS: IL-6 overexpression induced an 8% loss in body weight in CC mice, which was significantly attenuated by exercise. IL-6 overexpression in CC mice increased fasting insulin and triglyceride levels, which were normalized by exercise, and associated with increased oxidative capacity, an induction of AKT signaling, and a repression of AMPK signaling in muscle. These exercise-induced changes occurred despite elevated inflammatory signaling in skeletal muscle. CONCLUSION: We conclude that moderate-intensity exercise can attenuate IL-6-dependent cachexia in Apc(Min/+) mice, independent of changes in IL-6 concentration and muscle inflammatory signaling. The exercise effect was associated with improved insulin sensitivity and improved energy status in the muscle.