RESUMEN

Modulating autophagy and mitophagy, vital cellular quality control systems, offer therapeutic potential for critical illnesses. However, limited drug screening options hinder progress. We present a novel assay using the photoswitchable fluorescent reporter, mito-Kaede, to quantify mitophagy flux. Mito-Kaede's superior UV-induced photoconversion and brightness post-conversion make it ideal for prolonged mitochondrial dynamics tracking. Its specificity in responding to mitophagy, confirmed by parkin-knockout cells, adds value. When coupled with a custom fluid exchange system, enabling efficient medium changes, precise mitophagy observations become feasible. This mitophagy assay, alongside our methodological insights, can decipher mitophagy's role in pathology and supports drug screening efforts.

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Our method introduces a novel systematic approach for chronologically tracking the fluorescent decay of a photoactivatable fluorescent protein, mito-Kaede. This is combined with a fluid-exchange method to enable fixed-point observations before and after mitophagy stimulation.

Asunto(s)

Mitofagia , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Células HeLa , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Colorantes Fluorescentes/química

RESUMEN

BACKGROUND: Opioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway. METHODS: BV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells. RESULTS: Morphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects. CONCLUSION: Morphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

Asunto(s)

Infarto del Miocardio , Conocimientos, Actitudes y Práctica en Salud , Humanos , Infarto del Miocardio/terapia

Asunto(s)

Mitocondrias , Sepsis , Humanos , Sepsis/tratamiento farmacológico

RESUMEN

Sepsis remains a leading cause of mortality in critically ill patients. Muscle wasting is a major complication of sepsis and negatively affects clinical outcomes. Despite intense investigation for many years, the molecular mechanisms underlying sepsis-related muscle wasting are not fully understood. In addition, a potential role of muscle wasting in disease development of sepsis has not been studied. Myostatin is a myokine that downregulates skeletal muscle mass. We studied the effects of myostatin deficiency on muscle wasting and other clinically relevant outcomes, including mortality and bacterial clearance, in mice. Myostatin deficiency prevented muscle atrophy along with inhibition of increases in muscle-specific RING finger protein 1 (MuRF-1) and atrogin-1 expression and phosphorylation of signal transducer and activator of transcription protein 3 (STAT3; major players of muscle wasting) in septic mice. Moreover, myostatin deficiency improved survival and bacterial clearance of septic mice. Sepsis-induced liver dysfunction, acute kidney injury, and neutrophil infiltration into the liver and kidney were consistently mitigated by myostatin deficiency, as indicated by plasma concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and neutrophil gelatinase-associated lipocalin (NGAL) and myeloperoxidase activity in the organs. Myostatin deficiency also inhibited sepsis-induced increases in plasma high-mobility group protein B1 (HMGB1) and macrophage inhibitory cytokine (MIC)-1/growth differentiation factor (GDF)-15 concentrations. These results indicate that myostatin plays an important role not only in muscle wasting but also in other clinically relevant outcomes in septic mice. Furthermore, our data raise the possibility that muscle wasting may not be simply a complication, but myostatin-mediated muscle cachexia and related changes in muscle may actually drive the development of sepsis as well.NEW & NOTEWORTHY Muscle wasting is a major complication of sepsis, but its role in the disease development is not known. Myostatin deficiency improved bacterial clearance and survival and mitigated damage in the liver and kidney in septic mice, which paralleled prevention of muscle wasting. These results raise the possibility that muscle wasting may not simply be a complication of sepsis, but myostatin-mediated cachexic changes may have a role in impaired bacterial clearance and mortality in septic mice.

Asunto(s)

Atrofia Muscular/genética , Miostatina/deficiencia , Miostatina/genética , Sepsis/genética , Lesión Renal Aguda/genética , Animales , Caquexia/genética , Caquexia/prevención & control , Lipocalina 2/sangre , Hepatopatías/etiología , Hepatopatías/genética , Pruebas de Función Hepática , Masculino , Ratones , Proteínas Musculares/biosíntesis , Proteínas Musculares/genética , Atrofia Muscular/prevención & control , Infiltración Neutrófila/genética , Fosforilación , Factor de Transcripción STAT3/biosíntesis , Factor de Transcripción STAT3/genética , Sepsis/microbiología , Sepsis/mortalidad , Análisis de Supervivencia , Proteínas de Motivos Tripartitos/biosíntesis , Proteínas de Motivos Tripartitos/genética , Ubiquitina-Proteína Ligasas/biosíntesis , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Pulmonary immunosuppression often occurs after burn injury (BI). However, the reasons for BI-induced pulmonary immunosuppression are not clearly understood. Neutrophil recruitment and neutrophil extracellular trap (NET) formation (NETosis) are important components of a robust pulmonary immune response, and we hypothesized that pulmonary inflammation and NETosis are defective after BI. To test this hypothesis, we established a mouse model with intranasal LPS instillation in the presence or absence of BI (15% of body surface burn) and determined the degree of immune cell infiltration, NETosis, and the cytokine levels in the airways and blood on d 2. Presence of LPS recruited monocytes and large numbers of neutrophils to the airways and induced NETosis (citrullinated histone H3, DNA, myeloperoxidase). By contrast, BI significantly reduced LPS-mediated leukocyte recruitment and NETosis. This BI-induced immunosuppression is attributable to the reduction of chemokine (C-C motif) ligand (CCL) 2 (monocyte chemoattractant protein 1) and CCL3 (macrophage inflammatory protein 1α). BI also suppressed LPS-induced increase in IL-17A, IL-17C, and IL-17E/IL-25 levels in the airways. Therefore, BI-mediated reduction in leukocyte recruitment and NETosis in the lungs are attributable to these cytokines. Regulating the levels of some of these key cytokines represents a potential therapeutic option for mitigating BI-mediated pulmonary immunosuppression.-Sakuma, M., Khan, M. A. S., Yasuhara, S., Martyn, J. A., Palaniyar, N. Mechanism of pulmonary immunosuppression: extrapulmonary burn injury suppresses bacterial endotoxin-induced pulmonary neutrophil recruitment and neutrophil extracellular trap (NET) formation.

Asunto(s)

Quemaduras/fisiopatología , Trampas Extracelulares/inmunología , Terapia de Inmunosupresión , Lipopolisacáridos/toxicidad , Infiltración Neutrófila/inmunología , Neutrófilos/inmunología , Neumonía/inmunología , Animales , Trampas Extracelulares/metabolismo , Ratones , Neutrófilos/metabolismo , Neutrófilos/patología , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/patología

RESUMEN

Mitochondrial dysfunction is associated with metabolic alterations in various disease states, including major trauma (e.g., burn injury). Metabolic derangements, including muscle insulin resistance and hyperlactatemia, are a clinically significant complication of major trauma. Coenzyme Q10 (CoQ10) is an essential cofactor for mitochondrial electron transport, and its reduced form acts as a lipophilic antioxidant. Here, we report that burn injury induces impaired muscle insulin signaling, hyperlactatemia, mitochondrial dysfunction (as indicated by suppressed mitochondrial oxygen consumption rates), morphological alterations of the mitochondria (e. g., enlargement, and loss of cristae structure), mitochondrial oxidative stress, and disruption of mitochondrial integrity (as reflected by increased mitochondrial DNA levels in the cytosol and circulation). All of these alterations were significantly alleviated by CoQ10 treatment compared with vehicle alone. These findings indicate that CoQ10 treatment is efficacious in protecting against mitochondrial dysfunction and insulin resistance in skeletal muscle of burned mice. Our data highlight CoQ10 as a potential new strategy to prevent mitochondrial damage and metabolic dysfunction in burn patients.

Asunto(s)

Quemaduras/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Transducción de Señal , Ubiquinona/análogos & derivados , Animales , Masculino , Ratones , Ubiquinona/metabolismo

RESUMEN

INTRODUCTION: Burn injury (BI) leads to both systemic and neuro-inflammation and is associated with muscle wasting and weakness, which increase morbidity and mortality. Disuse atrophy is concomitantly present in BI patients. Most studies have focused on muscle with little attention to role of central nervous system (CNS) in the neuromuscular changes. We tested the hypothesis that BI-induced muscle wasting stems from CNS microglia activation and cytokines and chemokine release, which is associated with spinal ventral horn motor neuron degeneration. METHODS: Body surface (35%) BI, immobilization alone (Immob), BI with immobilization (BI + Immob), or Sham BI were administered to mice. Spinal cord (L3-L4 segments) and skeletal muscle tissues were harvested on days 7 and 14 after perturbations to examine microglia, motor neuron, and skeletal muscle changes. RESULTS: BI and BI + Immob significantly (P < 0.05) activated microglia, evidenced by its increased density around motor neurons, upregulated neuroinflammation-marker, translocator protein 18 kDa expression and inflammatory cytokines (interleukin-1ß, tumor necrosis factor-α) and/or chemokines (CXCL2) expression at days 7 and 14. Ventral horn motor neurons apoptosis and downregulation were observed at both periods after BI and was significantly magnified by concomitant BI + Immob. BI and more prominently BI + Immob disintegrated and fragmented the pretzel-shaped synapse and was associated with significantly decreased gastrocnemius, tibialis, and soleus muscle masses. CONCLUSION: BI induces microglia proliferation and activation (cytokine and chemokine release), degeneration of ventral horn motor neurons and muscle mass loss, all of which were accentuated by concomitant immobilization. The mechanisms connecting microglia activation and motor neuron degeneration to muscle mass loss require further delineation.

Asunto(s)

Quemaduras/fisiopatología , Microglía/citología , Neuronas Motoras/patología , Atrofia Muscular/fisiopatología , Animales , Apoptosis , Citocinas/metabolismo , Modelos Animales de Enfermedad , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/fisiopatología , Unión Neuromuscular/fisiopatología , Médula Espinal/fisiopatología

RESUMEN

Metabolic derangements are a clinically significant complication of major trauma (e.g., burn injury) and include various aspects of metabolism, such as insulin resistance, muscle wasting, mitochondrial dysfunction and hyperlactatemia. Nonetheless, the molecular pathogenesis and the relation between these diverse metabolic alterations are poorly understood. We have previously shown that burn increases farnesyltransferase (FTase) expression and protein farnesylation and that FTase inhibitor (FTI) prevents burn-induced hyperlactatemia, insulin resistance, and increased proteolysis in mouse skeletal muscle. In this study, we found that burn injury activated mTORC1 and hypoxia-inducible factor (HIF)-1α, which paralleled dysfunction, morphological alterations (i.e., enlargement, partial loss of cristae structure) and impairment of respiratory supercomplex assembly of the mitochondria, and ER stress. FTI reversed or ameliorated all of these alterations in burned mice. These findings indicate that these burn-induced changes, which encompass various aspects of metabolism, may be linked to one another and require protein farnesylation. Our results provide evidence of involvement of the mTORC1-HIF-1α pathway in burn-induced metabolic derangements. Our study identifies protein farnesylation as a potential hub of the signaling network affecting multiple aspects of metabolic alterations after burn injury and as a novel potential molecular target to improve the clinical outcome of severely burned patients.

Asunto(s)

Quemaduras/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Mitocondrias/metabolismo , Músculos/patología , Prenilación de Proteína , Animales , Modelos Animales de Enfermedad , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Estrés del Retículo Endoplásmico , Redes y Vías Metabólicas , Ratones Endogámicos C57BL , Multimerización de Proteína

RESUMEN

The role of interleukin-6 (IL-6) in physiological processes and disease is poorly understood. The hypothesis tested in this study was that selective alpha7 acetylcholine receptor (α7AChR) agonist, GTS-21, releases IL-6 in association with myonuclear accretion and enhances insulin signaling in muscle cells, and improves survival of burn injured (BI) mice. The in vitro effects of GTS-21 were determined in C2C12 myoblasts and 7-day differentiated myotubes (myotubes). The in vivo effects of GTS-21 were tested in BI wild-type (WT) and IL-6 knockout (IL6KO) mice. GTS-21 dose-dependently (0 µM, 100 µM, and 200 µM) significantly increased IL-6 levels in myoblasts and myotubes at 6 and 9 h. GTS-21-induced IL-6 release in myotubes was attenuated by methyllycaconitine (α7AChR antagonist), and by Stat-3 or Stat-5 inhibitors. GTS-21 increased MyoD and Pax7 protein expression, myonuclear accretion, and insulin-induced phosphorylation of Akt, GSK-3ß, and Glut4 in myotubes. The glucose levels of burned IL6KO mice receiving GTS-21 decreased significantly compared with sham-burn IL6KO mice. Superimposition of BI on IL6KO mice caused 100% mortality; GTS-21 reduced mortality to 75% in the IL6KO mice. The 75% mortality in burned WT mice was reduced to 0% with GTS-21. The in vitro findings suggest that GTS-21-induced IL-6 release from muscle is mediated via α7AChRs upstream of Stat-3 and -5 pathways and is associated with myonuclear accretion, possibly via MyoD and Pax7 expression. GTS-21 in vivo improves survival in burned WT mice and IL6KO mice, suggesting a potential therapeutic application of α7AChR agonists in the treatment of BI.

Asunto(s)

Compuestos de Bencilideno/farmacología , Quemaduras/tratamiento farmacológico , Interleucina-6/biosíntesis , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Mioblastos Esqueléticos/metabolismo , Piridinas/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Animales , Quemaduras/genética , Quemaduras/metabolismo , Línea Celular , Interleucina-6/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/patología , Proteínas Musculares/genética , Mioblastos Esqueléticos/patología , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo

RESUMEN

Inflammation and apoptosis develop in skeletal muscle after major trauma, including burn injury, and play a pivotal role in insulin resistance and muscle wasting. We and others have shown that inducible nitric oxide synthase (iNOS), a major mediator of inflammation, plays an important role in stress (e.g., burn)-induced insulin resistance. However, it remains to be determined how iNOS induces insulin resistance. Moreover, the interrelation between inflammatory response and apoptosis is poorly understood, although they often develop simultaneously. Nuclear factor (NF)-κB and p53 are key regulators of inflammation and apoptosis, respectively. Sirt1 inhibits p65 NF-κB and p53 by deacetylating these transcription factors. Recently, we have shown that iNOS induces S-nitrosylation of Sirt1, which inactivates Sirt1 and thereby increases acetylation and activity of p65 NF-κB and p53 in various cell types, including skeletal muscle cells. Here, we show that iNOS enhances burn-induced inflammatory response and apoptotic change in mouse skeletal muscle along with S-nitrosylation of Sirt1. Burn injury induced robust expression of iNOS in skeletal muscle and gene disruption of iNOS significantly inhibited burn-induced increases in inflammatory gene expression and apoptotic change. In parallel, burn increased Sirt1 S-nitrosylation and acetylation and DNA-binding capacity of p65 NF-κB and p53, all of which were reversed or ameliorated by iNOS deficiency. These results indicate that iNOS functions not only as a downstream effector but also as an upstream enhancer of burn-induced inflammatory response, at least in part, by Sirt1 S-nitrosylation-dependent activation (acetylation) of p65 NF-κB. Our data suggest that Sirt1 S-nitrosylation may play a role in iNOS-mediated enhanced inflammatory response and apoptotic change, which, in turn, contribute to muscle wasting and supposedly to insulin resistance after burn injury.

Asunto(s)

Apoptosis/fisiología , Quemaduras/patología , Inflamación/patología , Músculo Esquelético/patología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Sirtuina 1/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Animales , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/genética

RESUMEN

INTRODUCTION: Muscle wasting (MW) in catabolic conditions (e.g., burn injury [BI]) is a major risk factor affecting prognosis. Activation of interleukin-1ß (IL-1ß)/nuclear factor-kappa B (NF-κB), interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3), and/or forkhead box O transcriptional factor (FOXO)-mediated gene transcription pathways is the pivotal trigger of inflammatory response-induced protein catabolic processes in muscle. The α7 acetylcholine receptors (α7AChRs) are upregulated in macrophages and peripheral tissues including skeletal muscle during MW conditions. Stimulation of α7AChRs mitigates inflammatory responses. Hypothesis tested is that attenuation of inflammation by α7AChR stimulation with specific α7AChR agonist, GTS-21, will reverse BI-induced body mass and MW by modulating inflammatory and proteolytic signals. METHODS: Body surface area (30%) BI or sham BI mice were treated with GTS-21 or saline. Tibialis anterior (TA) muscle was harvested at 6 h, day 1 or 3 to examine inflammatory and proteolytic signals. RESULTS: GTS-21 significantly ameliorated the BI-induced increased expression of inflammatory cytokines IL-6, IL-1ß, C-X-C motif chemokine ligand 2 (6 h), phosphorylated STAT3, and NF-κB (day 1) in TA muscle. GTS-21 also significantly inhibited BI-induced increase of MuRF1 and FOXO1 (day 1). Consistent with the cytokine and inflammatory mediator changes, BI-induced body weight and TA muscle mass loss at day 3 were mitigated by GTS-21 treatment. The beneficial effect of GTS-21 on BI changes was absent in methyllycaconitine (α7AChR antagonist)-treated wild-type and α7AChR knockout mice. CONCLUSION: GTS-21 stimulation of α7AChRs, by modulating multiple molecular signals related to inflammation and proteolysis, attenuates protein wasting, evidenced by maintenance of body weight and attenuation of distant muscle mass loss after BI. GTS-21 can be a novel, potent therapeutic option for reversal of BI-induced MW.

Asunto(s)

Compuestos de Bencilideno/uso terapéutico , Quemaduras/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/prevención & control , Atrofia Muscular/metabolismo , Atrofia Muscular/prevención & control , Piridinas/uso terapéutico , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidores , Animales , Quemaduras/complicaciones , Quemaduras/metabolismo , Immunoblotting , Inflamación/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Atrofia Muscular/etiología

RESUMEN

INTRODUCTION: Skeletal muscle wasting and weakness with mitochondrial dysfunction (MD) are major pathological problems in burn injury (BI) patients. Fibrinogen levels elevated in plasma is an accepted risk factor for poor prognosis in many human diseases, and is also designated one of damage-associated molecular pattern (DAMPs) proteins. The roles of upregulated fibrinogen on muscle changes of critical illness including BI are unknown. The hypothesis tested was that BI-upregulated fibrinogen plays a pivotal role in the inflammatory responses and MD in muscles, and that DAMPs inhibitor, glycyrrhizin mitigates the muscle changes. METHODS: After third degree BI to mice, fibrinogen levels in the plasma and at skeletal muscles were compared between BI and sham-burn (SB) mice. Fibrinogen effects on inflammatory responses and mitochondrial membrane potential (MMP) loss were analyzed in C2C12 myotubes. In addition to survival, the anti-inflammatory and mitochondrial protective effects of glycyrrhizin were tested using in vivo microscopy of skeletal muscles of BI and SB mice. RESULTS: Fibrinogen in plasma and its extravasation to muscles significantly increased in BI versus SB mice. Fibrinogen applied to myotubes evoked inflammatory responses (increased MCP-1 and TNF-α; 32.6 and 3.9-fold, respectively) and reduced MMP; these changes were ameliorated by glycyrrhizin treatment. In vivo MMP loss and superoxide production in skeletal muscles of BI mice were significantly attenuated by glycyrrhizin treatment, together with improvement of BI survival rate. CONCLUSIONS: Inflammatory responses and MMP loss in myotubes induced by fibrinogen were reversed by glycyrrhizin. Anti-inflammatory and mitochondrial protective effect of glycyrrhizin in vivo leads to amelioration of muscle MD and improvement of BI survival rate.

Asunto(s)

Quemaduras/sangre , Quemaduras/tratamiento farmacológico , Quemaduras/metabolismo , Fibrinógeno/metabolismo , Ácido Glicirrínico/uso terapéutico , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Animales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Fibrinógeno/farmacología , Masculino , Ratones , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Oxidative stress induces mitochondrial dysfunction and facilitates apoptosis, tissue damage or metabolic alterations following infection. We have previously discovered that the Pseudomonas aeruginosa (PA) quorum sensing (QS)-excreted small volatile molecule, 2-aminoacetophenone (2-AA), which is produced in infected human tissue, promotes bacterial phenotypes that favor chronic infection, while also dampening the pathogen­induced innate immune response, thus compromising muscle function and promoting host tolerance to infection. In this study, murine whole-genome expression data have demonstrated that 2-AA affects the expression of genes involved in reactive oxygen species (ROS) homeostasis, thus producing an oxidative stress signature in skeletal muscle. The results of the present study demonstrated that the expression levels of genes involved in apoptosis signaling pathways were upregulated in the skeletal muscle of 2-AA-treated mice. To confirm the results of our transcriptome analysis, we used a novel high-resolution magic-angle-spinning (HRMAS), proton (1H) nuclear magnetic resonance (NMR) method and observed increased levels of bisallylic methylene fatty acyl protons and vinyl protons, suggesting that 2-AA induces skeletal muscle cell apoptosis. This effect was corroborated by our results demonstrating the downregulation of mitochondrial membrane potential in vivo in response to 2-AA. The findings of the present study indicate that the bacterial infochemical, 2-AA, disrupts mitochondrial functions by inducing oxidative stress and apoptosis signaling and likely promotes skeletal muscle dysfunction, which may favor chronic/persistent infection.

Asunto(s)

Acetofenonas/metabolismo , Apoptosis , Interacciones Huésped-Patógeno , Músculo Esquelético/microbiología , Estrés Oxidativo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiología , Animales , Regulación de la Expresión Génica , Humanos , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Infecciones por Pseudomonas/genética , Infecciones por Pseudomonas/patología , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

BACKGROUND: It has been known that skeletal muscles show atrophic changes after prolonged sedation or general anesthesia. Whether these effects are due to anesthesia itself or disuse during anesthesia has not been fully clarified. Autophagy dysregulation has been implicated in muscle-wasting conditions. This study tested the hypothesis that the magnitude of skeletal muscle autophagy is affected by both anesthesia and immobility. METHODS: The extent of autophagy was analyzed chronologically during general anesthesia. In vivo microscopy was performed using green fluorescent protein-tagged LC3 for the detection of autophagy using sternomastoid muscles of live mice during pentobarbital anesthesia (n = 6 and 7). Western blotting and histological analyses were also conducted on tibialis anterior muscles (n = 3 to 5). To distinguish the effect of anesthesia from that due to disuse, autophagy was compared between animals anesthetized with pentobarbital and those immobilized by short-term denervation without continuation of anesthesia. Conversely, tibialis anterior and sternomastoid muscles were electrically stimulated during anesthesia. RESULTS: Western blots and microscopy showed time-dependent autophagy up-regulation during pentobarbital anesthesia, peaking at 3 h (728.6 ± 93.5% of basal level, mean ± SE). Disuse by denervation without sustaining anesthesia did not lead to equivalent autophagy, suggesting that anesthesia is essential to cause autophagy. In contrast, contractile stimulation of the tibialis anterior and sternomastoid muscles significantly reduced the autophagy up-regulation during anesthesia (85% at 300 min). Ketamine, ketamine plus xylazine, isoflurane, and propofol also up-regulated autophagy. CONCLUSIONS: Short-term disuse without anesthesia does not lead to autophagy, but anesthesia with disuse leads to marked up-regulation of autophagy.

Asunto(s)

Anestesia , Autofagia/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Trastornos Musculares Atróficos/patología , Animales , Desnervación , Estimulación Eléctrica , Hipnóticos y Sedantes , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Contracción Muscular/efectos de los fármacos , Pentobarbital , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

BACKGROUND: The acetylcholinesterase inhibitor, pyridostigmine, is prophylactically administered to mitigate the toxic effects of nerve gas poisoning. The authors tested the hypothesis that prolonged pyridostigmine administration can lead to neuromuscular dysfunction and even down-regulation of acetylcholine receptors. METHODS: Pyridostigmine (5 or 25 mg·kg·day) or saline was continuously administered via osmotic pumps to rats, and infused for either 14 or 28 days until the day of neuromuscular assessment (at day 14 or 28), or discontinued 24 h before neuromuscular assessment. Neurotransmission and muscle function were examined by single-twitch, train-of-four stimulation and 100-Hz tetanic stimulation. Sensitivity to atracurium and acetylcholine receptor number (quantitated by I-α-bungarotoxin) provided additional measures of neuromuscular integrity. RESULTS: Specific tetanic tensions (Newton [N]/muscle weight [g]) were significantly (P < 0.05) decreased at 14 (10.3 N/g) and 28 (11.1 N/g) days of 25 mg·kg·day pyridostigmine compared with controls (13.1-13.6 N/g). Decreased effective dose (0.81-1.05 vs. 0.16-0.45 mg/kg; P < 0.05) and decreased plasma concentration (3.02-3.27 vs. 0.45-1.37 µg/ml; P < 0.05) of atracurium for 50% paralysis (controls vs. 25 mg·kg·day pyridostigmine, respectively), irrespective of discontinuation of pyridostigmine, confirmed the pyridostigmine-induced altered neurotransmission. Pyridostigmine (25 mg·kg·day) down-regulated acetylcholine receptors at 28 days. CONCLUSIONS: Prolonged administration of pyridostigmine (25 mg·kg·day) leads to neuromuscular impairment, which can persist even when pyridostigmine is discontinued 24 h before assessment of neuromuscular function. Pyridostigmine has the potential to down-regulate acetylcholine receptors, but induces neuromuscular dysfunction even in the absence of receptor changes.

Asunto(s)

Inhibidores de la Colinesterasa/toxicidad , Regulación hacia Abajo/efectos de los fármacos , Enfermedades Neuromusculares/inducido químicamente , Bromuro de Piridostigmina/toxicidad , Receptores Colinérgicos/efectos de los fármacos , Animales , Inhibidores de la Colinesterasa/administración & dosificación , Esquema de Medicación , Masculino , Bromuro de Piridostigmina/administración & dosificación , Ratas , Ratas Sprague-Dawley , Cloruro de Sodio/administración & dosificación , Factores de Tiempo

RESUMEN

Skeletal muscle wasting is an exacerbating factor in the prognosis of critically ill patients. Using a systemic burn injury model in mice, we have established a role of autophagy in the resulting muscle wasting that is distant from the burn trauma. We provide evidence that burn injury increases the autophagy turnover in the distal skeletal muscle by conventional postmortem tissue analyses and by a novel in vivo microscopic method using an autophagy reporter gene (tandem fluorescent LC3). The effect of tadalafil, a phosphodiesterase 5 inhibitor (PDE5I), on burn-induced skeletal muscle autophagy is documented and extends our published results that PDE5Is attenuates muscle degeneration in a muscular dystrophy model. We also designed a translational experiment to examine the impact of PDE5I on whole body and demonstrated that PDE5I administration lessened muscle atrophy, mitigated microcirculatory disturbance, and improved the survival rate after burn injury.

Asunto(s)

Autofagia/efectos de los fármacos , Quemaduras/patología , Carbolinas/farmacología , Microcirculación/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/patología , Inhibidores de Fosfodiesterasa 5/farmacología , Animales , Western Blotting , Quemaduras/tratamiento farmacológico , Quemaduras/fisiopatología , ADN/biosíntesis , ADN/genética , Genes Reporteros , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Flujo Sanguíneo Regional , Sobrevida , Tadalafilo , Cicatrización de Heridas/efectos de los fármacos

RESUMEN

BACKGROUND: Nerve-stimulated fade in muscle is generally accepted as a prejunctional phenomenon mediated by block of prejunctional acetylcholine receptors (AChRs) at the nerve terminal, whereas decrease of twitch tension is considered a postjunctional effect due to block of muscle AChRs. Using ligands with specific pre- or postjunctional effects only, we tested the hypothesis that fade is not necessarily a prejunctional phenomenon. METHODS: Neuromuscular function in rats was evaluated after IM (2.5 U) or IV (12.0 U) injection of botulinum toxin (Botx), or IV (250 µg/kg) α-bungarotoxin (α-BTX) alone. The acute neuromuscular effects of IV 2 mg/kg dihydro-ß-erythroidine (DHßE), alone and in combination with α-BTX, were also tested. Botx decreases vesicular release of ACh, and α-BTX binds to postjunctional nicotinic AChRs only, whereas DHßE binds specifically to prejunctional α3ß2 AChRs only. In view of the lack of acute effects of Botx even at 2 hours after IV injection, its neuromuscular effects were also evaluated at 24 hours after IM injection (0.6 U) and compared with IM injection of α-BTX (25 µg/kg) or saline also given 24 hours earlier. The sciatic nerve-tibialis muscle preparation, during train-of-four and tetanic stimulation, was used to test neuromuscular effects in vivo. RESULTS: IV and IM Botx had no observable neuromuscular effects at 2 hours. IV α-BTX caused twitch depression within a few minutes, and significant fade (P = 0.002) at 75% of baseline twitch tension; these effects persisted until the end of the observation period of 2 hours. IV DHßE alone caused no significant change in single twitch (P = 0.899) or train-of-four ratio (P = 0.394), but significantly enhanced the fade of IV α-BTX (P = 0.001 at 75% of baseline twitch tension). IM Botx or α-BTX, at 24 hours after their injection, resulted in a significant decrease of single twitch and tetanic tensions (P < 0.0001), but Botx did not cause fade, whereas α-BTX caused significant (P < 0.0001) fade at 24 hours. The tibialis muscle weights and protein expression of α1 subunit of AChR (Western blots) did not differ between Botx, α-BTX and saline-injected groups at 24 hours but increased in denervated muscle (positive control). CONCLUSIONS: Botx-induced decreased ACh release in and of itself does not cause fade but does cause decrease of absolute tensions. Decrease of available (functional) postjunctional AChRs by α-BTX did induce fade. The prejunctional fade effects of DHßE on α3ß2 AChRs become manifest only when the margin of safety was decreased by concomitant administration of α-BTX. Thus, fade during repetitive stimulation is not always a prejunctional phenomenon and may also reflect the decreased margin of safety of neurotransmission, which can be due to a pure postjunctional AChRs block or to a combination of both pre- and postjunctional AChRs block. Block of prejunctional α3ß2 AChRs alone is not necessary and sufficient to cause fade.

Asunto(s)

Estimulación Eléctrica , Monitoreo Intraoperatorio , Unión Neuromuscular/efectos de los fármacos , Periodo Refractario Electrofisiológico/efectos de los fármacos , Toxinas Biológicas , Acetilcolina/metabolismo , Anestesia , Animales , Toxinas Botulínicas , Bungarotoxinas , Dihidro-beta-Eritroidina , Masculino , Contracción Muscular/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Colinérgicos/efectos de los fármacos , Receptores Colinérgicos/metabolismo , Transmisión Sináptica/efectos de los fármacos

RESUMEN

INTRODUCTION: Immobilization by casting induces disuse muscle atrophy (DMA). METHODS: Using wild type (WT) and caspase-3 knockout (KO) mice, we evaluated the effect of caspase-3 on muscle mass, apoptosis, and inflammation during DMA. RESULTS: Caspase-3 deficiency significantly attenuated muscle mass decrease [gastrocnemius: 28 ± 1% in KO vs. 41 ± 3% in WT; soleus: 47 ± 2% in KO vs. 56 ± 2% in WT; (P < 0.05)] and gastrocnemius twitch tension decrease (23 ± 4% in KO vs. 36 ± 3% in WT, P < 0.05) at day 14 in immobilized vs. contralateral hindlimb. Lack of caspase-3 decreased immobilization-induced increased apoptotic myonuclei (3.2-fold) and macrophage infiltration (2.2-fold) in soleus muscle and attenuated increased monocyte chemoattractant protein-1 mRNA expression (2-fold in KO vs. 18-fold in WT) in gastrocnemius. CONCLUSIONS: Caspase-3 plays a key role in DMA and associated decreased tension, presumably by acting on the apoptosis and inflammation pathways.

Asunto(s)

Apoptosis/genética , Caspasa 3/metabolismo , Inflamación/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Animales , Caspasa 3/genética , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Inmovilización , Inflamación/genética , Inflamación/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/patología , Atrofia Muscular/genética , Atrofia Muscular/patología

RESUMEN

OBJECTIVES: Recent studies suggest that activation of glycogen synthase kinase (GSK)-3ß may be involved in burn injury-induced metabolic derangements and protein breakdown in skeletal muscle. However, the mechanism for GSK-3ß activation after burn injury is unknown. To investigate the role of inducible nitric oxide synthase (iNOS) in this scenario, a major mediator of inflammation, we examined the effects of a specific inhibitor for iNOS, L-NIL, on GSK-3ß activity in skeletal muscle of burned rats. MATERIALS/METHODS: Full-thickness third degree burn injury comprising 40% of total body surface area was produced under anesthesia in male Sprague-Dawley rats (160-190g) by immersing the back of the trunk for 15s and the abdomen for 8s in 80°C water. Burned and sham-burned rats were treated with L-NIL (60mg/kg BW, b.i.d., IP) or phosphate-buffered saline for three days. GSK-3ß activity in skeletal muscle was evaluated by immune complex kinase assay, and by phosphorylation status of GSK-3ß and its endogenous substrate, glycogen synthase. RESULTS: GSK-3ß activity was increased in a time-dependent manner in skeletal muscle after burn injury, concomitant with the induction of iNOS expression. iNOS inhibitor, L-NIL, reverted the elevated GSK-3ß activity in skeletal muscle of burned rats, although L-NIL did not alter GSK-3ß activity in sham-burned rats. CONCLUSIONS: Our results clearly indicate that iNOS plays an important role in burn injury-induced GSK-3ß activation in skeletal muscle. These findings suggest that iNOS may contribute to burn injury-induced metabolic derangements, in part, by activating GSK-3ß.

Asunto(s)

Quemaduras/enzimología , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Lisina/análogos & derivados , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/enzimología , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Animales , Quemaduras/metabolismo , Activación Enzimática/efectos de los fármacos , Glucógeno Sintasa/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Lisina/farmacología , Masculino , Músculo Esquelético/lesiones , Músculo Esquelético/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fosforilación , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley