RESUMEN
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
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
Controlling neuropathic pain is an unmet medical need and we set out to identify new therapeutic candidates. AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation and can partition into the CNS. Recent data strongly implicate activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. We hypothesized that AV411 might be effective in the treatment of neuropathic pain and, hence, tested whether it attenuates the mechanical allodynia induced in rats by chronic constriction injury (CCI) of the sciatic nerve, spinal nerve ligation (SNL) and the chemotherapeutic paclitaxel (Taxol). Twice-daily systemic administration of AV411 for multiple days resulted in a sustained attenuation of CCI-induced allodynia. Reversal of allodynia was of similar magnitude to that observed with gabapentin and enhanced efficacy was observed in combination. We further show that multi-day AV411 reduces SNL-induced allodynia, and reverses and prevents paclitaxel-induced allodynia. Also, AV411 cotreatment attenuates tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses were also performed. Overall, the results indicate that AV411 is effective in diverse models of neuropathic pain and support further exploration of its potential as a therapeutic agent for the treatment of neuropathic pain.