RESUMO
Muscle atrophy is an extrapulmonary complication of acute exacerbations (AE) in chronic obstructive pulmonary disease (COPD). The endogenous production and therapeutic application of glucocorticoids (GCs) have been implicated as drivers of muscle loss in AE-COPD. The enzyme 11 ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) activates GCs and contributes toward GC-induced muscle wasting. To explore the potential of 11ßHSD1 inhibition to prevent muscle wasting here, the objective of this study was to ascertain the contribution of endogenous GC activation and amplification by 11ßHSD1 in skeletal muscle wasting during AE-COPD. Emphysema was induced by intratracheal (IT) instillation of elastase to model COPD in WT and 11ßHSD1/KO mice, followed by vehicle or IT-LPS administration to mimic AE. µCT scans were obtained prior and at study endpoint 48 h following IT-LPS, to assess emphysema development and muscle mass changes, respectively. Plasma cytokine and GC profiles were determined by ELISA. In vitro, myonuclear accretion and cellular response to plasma and GCs were determined in C2C12 and human primary myotubes. Muscle wasting was exacerbated in LPS-11ßHSD1/KO animals compared with WT controls. RT-qPCR and western blot analysis showed elevated catabolic and suppressed anabolic pathways in muscle of LPS-11ßHSD1/KO animals relative to WTs. Plasma corticosterone levels were higher in LPS-11ßHSD1/KO animals, whereas C2C12 myotubes treated with LPS-11ßHSD1/KO plasma or exogenous GCs displayed reduced myonuclear accretion relative to WT counterparts. This study reveals that 11ß-HSD1 inhibition aggravates muscle wasting in a model of AE-COPD, suggesting that therapeutic inhibition of 11ß-HSD1 may not be appropriate to prevent muscle wasting in this setting.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1 , Enfisema , Doença Pulmonar Obstrutiva Crônica , Animais , Humanos , Camundongos , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Glucocorticoides/farmacologia , Lipopolissacarídeos , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Atrofia Muscular/prevenção & controle , Doença Pulmonar Obstrutiva Crônica/complicaçõesRESUMO
INTRODUCTION: Physical inactivity significantly contributes to loss of muscle mass and performance in bed-bound patients. Loss of skeletal muscle mitochondrial content has been well-established in muscle unloading models, but the underlying molecular mechanism remains unclear. We hypothesized that apparent unloading-induced loss of muscle mitochondrial content is preceded by increased mitophagy- and decreased mitochondrial biogenesis-signaling during the early stages of unloading. METHODS: We analyzed a comprehensive set of molecular markers involved in mitochondrial-autophagy, -biogenesis, -dynamics, and -content, in the gastrocnemius muscle of C57BL/6J mice subjected to 0- and 3-days hind limb suspension, and in biopsies from human vastus lateralis muscle obtained before and after 7 days of one-leg immobilization. RESULTS: In both mice and men, short-term skeletal muscle unloading results in molecular marker patterns indicative of increased receptor-mediated mitophagy and decreased mitochondrial biogenesis regulation, before apparent loss of mitochondrial content. DISCUSSION: These results emphasize the early-onset of skeletal muscle disuse-induced mitochondrial remodeling.
Assuntos
Elevação dos Membros Posteriores , Mitocôndrias Musculares/metabolismo , Mitofagia/genética , Músculo Esquelético/metabolismo , Biogênese de Organelas , Adolescente , Adulto , Animais , Moldes Cirúrgicos , Expressão Gênica , Humanos , Imobilização , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Musculares/patologia , Mitofagia/fisiologia , Músculo Esquelético/patologia , Músculo Quadríceps/metabolismo , Músculo Quadríceps/patologia , Suporte de Carga , Adulto JovemRESUMO
Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism.
Assuntos
Infecções por Coxsackievirus/metabolismo , Enterovirus Humano B , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas Musculares/metabolismo , Miocardite/metabolismo , NF-kappa B/metabolismo , Doença Aguda , Animais , Infecções por Coxsackievirus/patologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Miocardite/patologia , Miocardite/virologia , PPAR gama/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Pulmonary inflammation in response to respiratory infections can evoke muscle wasting. Increased activity of the ubiquitin (Ub)-proteasome system (UPS) and the autophagy lysosome pathway (ALP) have been implicated in inflammation-induced muscle atrophy. Since poly-Ub conjugation is required for UPS-mediated proteolysis and has been implicated in the ALP, we assessed the effect of impaired ubiquitin conjugation on muscle atrophy and recovery following pulmonary inflammation, and compared activation and suppression of these proteolytic systems to protein synthesis regulation. METHODS: Pulmonary inflammation was induced in mice by an intratracheal instillation of LPS. Proteolysis (UPS and ALP) and synthesis signaling were examined in gastrocnemius muscle homogenates. Ub-conjugation-dependency of muscle atrophy and recovery was addressed using Ub-K48R (K48R) mice with attenuated poly-ubiquitin conjugation, and compared to UBWT control mice. RESULTS: Pulmonary inflammation caused a decrease in skeletal muscle mass which was accompanied by a rapid increase in expression of UPS and ALP constituents and reduction in protein synthesis signaling acutely after LPS. Muscle atrophy was attenuated in K48R mice, while ALP and protein synthesis signaling were not affected. Muscle mass recovery starting 72 h post LPS, correlated with reduced expression of UPS and ALP constituents and restoration of protein synthesis signaling. K48R mice however displayed impaired recovery of muscle mass. CONCLUSION: Pulmonary inflammation-induced muscle atrophy is in part attributable to UPS-mediated proteolysis, as activation of ALP- and suppression of protein synthesis signaling occur independently of poly-Ub conjugation during muscle atrophy. Recovery of muscle mass following pulmonary inflammation involves inverse regulation of proteolysis and protein synthesis signaling, and requires a functional poly-Ub conjugation.
Assuntos
Pneumopatias/complicações , Pneumopatias/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Poliubiquitina/metabolismo , Animais , Inflamação/complicações , Inflamação/metabolismo , Inflamação/patologia , Pneumopatias/patologia , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Atrofia Muscular/patologia , Recuperação de Função FisiológicaRESUMO
Muscle wasting impairs physical performance, increases mortality and reduces medical intervention efficacy in chronic diseases and cancer. Developing proficient intervention strategies requires improved understanding of the molecular mechanisms governing muscle mass wasting and recovery. Involvement of muscle protein- and myonuclear turnover during recovery from muscle atrophy has received limited attention. The insulin-like growth factor (IGF)-I signaling pathway has been implicated in muscle mass regulation. As glycogen synthase kinase 3 (GSK-3) is inhibited by IGF-I signaling, we hypothesized that muscle-specific GSK-3ß deletion facilitates the recovery of disuse-atrophied skeletal muscle. Wild-type mice and mice lacking muscle GSK-3ß (MGSK-3ß KO) were subjected to a hindlimb suspension model of reversible disuse-induced muscle atrophy and followed during recovery. Indices of muscle mass, protein synthesis and proteolysis, and post-natal myogenesis which contribute to myonuclear accretion, were monitored during the reloading of atrophied muscle. Early muscle mass recovery occurred more rapidly in MGSK-3ß KO muscle. Reloading-associated changes in muscle protein turnover were not affected by GSK-3ß ablation. However, coherent effects were observed in the extent and kinetics of satellite cell activation, proliferation and myogenic differentiation observed during reloading, suggestive of increased myonuclear accretion in regenerating skeletal muscle lacking GSK-3ß. This study demonstrates that muscle mass recovery and post-natal myogenesis from disuse-atrophy are accelerated in the absence of GSK-3ß.
Assuntos
Diferenciação Celular , Quinase 3 da Glicogênio Sintase/metabolismo , Desenvolvimento Muscular , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Atrofia Muscular/enzimologia , Regeneração , Animais , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Atrofia Muscular/genética , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologiaRESUMO
BACKGROUND: Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis. METHODS: We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls. RESULTS: We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1ß. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1. CONCLUSION: The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD.
Assuntos
Brônquios/patologia , Células Epiteliais/patologia , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Dinâmica Mitocondrial/fisiologia , Renovação Mitocondrial/fisiologia , Fumar/efeitos adversos , Adulto , Idoso , Brônquios/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Células Cultivadas , Citocinas/metabolismo , Dinaminas , Células Epiteliais/metabolismo , Feminino , GTP Fosfo-Hidrolases/metabolismo , Humanos , Técnicas In Vitro , Masculino , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Quinases/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Fatores de Risco , Superóxido Dismutase/metabolismo , Fatores de TempoRESUMO
Palmitate activates the NF-κB pathway, and induces accumulation of lipid metabolites and insulin resistance in skeletal muscle cells. Little information is available whether and how these processes are causally related. Therefore, the objectives were to investigate whether intra-cellular lipid metabolites are involved in FA-induced NF-κB activation and/or insulin resistance in skeletal muscle and to investigate whether FA-induced insulin resistance and NF-κB activation are causally related. Inhibiting DGAT or CPT-1 by using, respectively, amidepsine or etomoxir increased DAG accumulation and sensitized myotubes to palmitate-induced insulin resistance. While co-incubation of palmitate with etomoxir increased NF-κB transactivation, co-incubation with amidepsine did not, indicating that DAG accumulation is associated with insulin resistance but not with NF-κB activation. Furthermore, pharmacological or genetic inhibition of the NF-κB pathway could not prevent palmitate-induced insulin resistance. In conclusion, we have demonstrated that activation of the NF-κB pathway is not required for palmitate-induced insulin resistance in skeletal muscle cells.
Assuntos
Resistência à Insulina/fisiologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiologia , NF-kappa B/metabolismo , Palmitatos/farmacologia , Animais , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/fisiopatologia , Diglicerídeos/metabolismo , Ácidos Graxos não Esterificados/sangue , Humanos , Camundongos , Músculo Esquelético/citologia , Oxirredução , Palmitatos/metabolismoRESUMO
BACKGROUND: Brown adipose tissue (BAT) has been primarily researched as a potential target for mitigating obesity. However, the physiological significance of BAT in relation to cachexia remains poorly understood. The objective of this study was to investigate the putative contribution of BAT on different components of energy metabolism in emphysematous chronic obstructive pulmonary disease (COPD) patients. METHODS: Twenty COPD patients (mean ± SD age 62 ± 6, 50% female, median [range] BMI 22.4 [15.1-32.5] kg/m2 and 85% low FFMI) were studied. Basal metabolic rate (BMR) was assessed by ventilated hood, total daily energy expenditure (TDEE) by doubly labelled water and physical activity by triaxial accelerometry. BMR was adjusted for fat-free mass (FFM) as assessed by deuterium dilution. Analysis of BAT and WAT was conducted in a subset of ten patients and six age-matched, gender-matched and BMI-matched healthy controls. BAT glucose uptake was assessed by means of cold-stimulated integrated [18F]FDG positron-emission tomography and magnetic resonance imaging. WAT was collected from subcutaneous abdominal biopsies to analyse metabolic and inflammatory gene expression levels. Lung function was assessed by spirometry and body plethysmography and systemic inflammation by high sensitivity C-reactive protein. RESULTS: Mean TDEE was 2209 ± 394 kcal/day, and mean BMR was 1449 ± 214 kcal/day corresponding to 120% of predicted. FFM-adjusted BMR did not correlate with lung function or C-reactive protein. Upon cooling, energy expenditure increased, resulting in a non-shivering thermogenesis of (median [range]) 20.1% [3.3-41.3] in patients and controls. Mean BAT glucose uptake was comparable between COPD and controls (1.5 [0.1-6.2] vs. 1.1 [0.7-3.9]). In addition, no correlation was found between BMR adjusted for FFM and BAT activity or between cold-induced non-shivering energy expenditure and BAT activity. Gene expression levels of the brown adipocyte or beige markers were also comparable between the groups. No (serious) adverse events were reported. CONCLUSIONS: Although COPD patients were hypermetabolic at rest, no correlation was found between BMR or TDEE and BAT activity. Furthermore, both BAT activity and gene expression levels of the brown adipocyte or beige markers were comparable between COPD patients and controls.
Assuntos
Tecido Adiposo Marrom , Doença Pulmonar Obstrutiva Crônica , Tecido Adiposo Marrom/metabolismo , Idoso , Metabolismo Energético , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons , Doença Pulmonar Obstrutiva Crônica/complicações , Doença Pulmonar Obstrutiva Crônica/metabolismo , Termogênese/genéticaRESUMO
One cluster of the extrapulmonary manifestations in chronic obstructive pulmonary disease (COPD) is related to the brain, which includes anxiety, depression and cognitive impairment. Brain-related comorbidities are related to worsening of symptoms and increased mortality in COPD patients. In this study, a murine model of COPD was used to examine the effects of emphysema and repetitive pulmonary inflammatory events on systemic inflammatory outcomes and brain function. In addition, the effect of a dietary intervention on brain-related parameters was assessed. Adult male C57Bl/6J mice were exposed to elastase or vehicle intratracheally (i.t.) once a week on three consecutive weeks. Two weeks after the final administration, mice were i.t. exposed to lipopolysaccharide (LPS) or vehicle for three times with a 10 day interval. A dietary intervention enriched with omega-3 PUFAs, prebiotic fibers, tryptophan and vitamin D was administered from the first LPS exposure onward. Behavior and cognitive function, the degree of emphysema and both pulmonary and systemic inflammation as well as blood-brain barrier (BBB) integrity and neuroinflammation in the brain were assessed. A lower score in the cognitive test was observed in elastase-exposed mice. Mice exposed to elastase plus LPS showed less locomotion in the behavior test. The enriched diet seemed to reduce anxiety-like behavior over time and cognitive impairments associated with the presented COPD model, without affecting locomotion. In addition, the enriched diet restored the disbalance in splenic T-helper 1 (Th1) and Th2 cells. There was a trend toward recovering elastase plus LPS-induced decreased expression of occludin in brain microvessels, a measure of BBB integrity, as well as improving expression levels of kynurenine pathway markers in the brain by the enriched diet. The findings of this study demonstrate brain-associated comorbidities - including cognitive and behavioral impairments - in this murine model for COPD. Although no changes in lung parameters were observed, exposure to the specific enriched diet in this model appeared to improve systemic immune disbalance, BBB integrity and derailed kynurenine pathway which may lead to reduction of anxiety-like behavior and improved cognition.
RESUMO
Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3ß (GSK-3ß), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3ß using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3ß suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3ß inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3ß dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3ß enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3ß.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Músculo Esquelético/enzimologia , Atrofia Muscular/enzimologia , Mioblastos/enzimologia , Animais , Linhagem Celular , Cromonas/farmacologia , Dexametasona/farmacologia , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Cloreto de Lítio/farmacologia , Camundongos , Morfolinas/farmacologia , Proteínas Musculares/biossíntese , Músculo Esquelético/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Mioblastos/efeitos dos fármacos , Mioblastos/fisiologia , Cadeias Pesadas de Miosina/biossíntese , Cadeias Leves de Miosina/biossíntese , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/metabolismo , Proteínas Ligases SKP Culina F-Box/biossíntese , Transdução de Sinais/efeitos dos fármacos , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/biossínteseRESUMO
BACKGROUND: Patients with COPD are often characterized by disturbed metabolic health which is reflected in altered body composition. Current studies in healthy subjects suggest that resveratrol improves metabolic health by enhancing muscle mitochondrial function and adipose tissue morphology. The primary objective was to investigate the effect of four weeks resveratrol supplementation on muscle mitochondrial function in patients with COPD. Secondary objectives were to investigate the effect of resveratrol on adipose tissue inflammatory and metabolic gene expression, systemic inflammation and body composition in patients with COPD. METHODS: In a double-blind randomized placebo-controlled proof-of-concept study, 21 COPD patients (FEV1: 53 ± 15% predicted; age: 67 ± 9 years and BMI: 24.5 ± 3.3 kg/m2) received resveratrol (150 mg/day) or placebo for four weeks. Before and after intervention, blood samples, quadriceps muscle and subcutaneous abdominal fat biopsies were obtained for metabolic and inflammatory profiling. Body composition was assessed by dual energy X-ray absorptiometry. RESULTS: Muscle mitochondrial biogenesis regulators AMPK, SIRT1 and PGC-1α as well as mitochondrial respiration, Oxphos complexes, oxidative enzyme activities and kynurenine aminotransferases were not improved by resveratrol. Plasma high-sensitive C-reactive protein and kynurenine did not change after resveratrol supplementation. Adipose tissue inflammatory markers were unaffected by resveratrol, while markers of glycolysis and lipolysis were significantly increased compared to placebo supplementation. Body weight decreased after resveratrol supplementation (resveratrol -0.95 ± 1.01 kg vs placebo -0.16 ± 0.66 kg, p = 0.049) due to a reduction in lean mass (resveratrol -1.79 ± 1.67 kg vs 0.37 ± 0.86 kg, p = 0.026). CONCLUSION: We do not confirm previously reported positive effects of resveratrol on skeletal muscle mitochondrial function in patients with COPD, but show an unexpected decline in lean mass. CLINICAL TRIAL REGISTRY: Clinicaltrials.gov NCT02245932.
Assuntos
Composição Corporal/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Músculo Quadríceps/efeitos dos fármacos , Resveratrol/uso terapêutico , Absorciometria de Fóton , Adiposidade , Idoso , Método Duplo-Cego , Feminino , Nível de Saúde , Humanos , Irlanda , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Estudo de Prova de Conceito , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Músculo Quadríceps/metabolismo , Músculo Quadríceps/fisiopatologia , Resveratrol/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Redução de Peso/efeitos dos fármacosRESUMO
BACKGROUND: Cachexia, highly prevalent in patients with non-small cell lung cancer (NSCLC), impairs quality of life and is associated with reduced tolerance and responsiveness to cancer therapy and decreased survival. MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in post-transcriptional gene regulation. Changes in intramuscular levels of miRNAs have been implicated in muscle wasting conditions. Here, we aimed to identify miRNAs that are differentially expressed in skeletal muscle of cachectic lung cancer patients to increase our understanding of cachexia and to allow us to probe their potential as therapeutic targets. METHODS: A total of 754 unique miRNAs were profiled and analysed in vastus lateralis muscle biopsies of newly diagnosed treatment-naïve NSCLC patients with cachexia (n = 8) and age-matched and sex-matched healthy controls (n = 8). miRNA expression analysis was performed using a TaqMan MicroRNA Array. In silico network analysis was performed on all significant differentially expressed miRNAs. Differential expression of the top-ranked miRNAs was confirmed using reverse transcription-quantitative real-time PCR in an extended group (n = 48) consisting of NSCLC patients with (n = 15) and without cachexia (n = 11) and healthy controls (n = 22). Finally, these miRNAs were subjected to univariate and multivariate Cox proportional hazard analysis using overall survival and treatment-induced toxicity data obtained during the follow-up of this group of patients. RESULTS: We identified 28 significant differentially expressed miRNAs, of which five miRNAs were up-regulated and 23 were down-regulated. In silico miRNA-target prediction analysis showed 158 functional gene targets, and pathway analysis identified 22 pathways related to the degenerative or regenerative processes of muscle tissue. Subsequently, the expression of six top-ranked miRNAs was measured in muscle biopsies of the entire patient group. Five miRNAs were detectable with reverse transcription-quantitative real-time PCR analysis, and their altered expression (expressed as fold change, FC) was confirmed in muscle of cachectic NSCLC patients compared with healthy control subjects: miR-424-5p (FC = 4.5), miR-424-3p (FC = 12), miR-450a-5p (FC = 8.6), miR-144-5p (FC = 0.59), and miR-451a (FC = 0.57). In non-cachectic NSCLC patients, only miR-424-3p was significantly increased (FC = 5.6) compared with control. Although the statistical support was not sufficient to imply these miRNAs as individual predictors of overall survival or treatment-induced toxicity, when combined in multivariate analysis, miR-450-5p and miR-451a resulted in a significant stratification between short-term and long-term survival. CONCLUSIONS: We identified differentially expressed miRNAs putatively involved in lung cancer cachexia. These findings call for further studies to investigate the causality of these miRNAs in muscle atrophy and the mechanisms underlying their differential expression in lung cancer cachexia.
Assuntos
Caquexia/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Músculo Esquelético/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Physical inactivity contributes to muscle wasting and reductions in mitochondrial oxidative phenotype (OXPHEN), reducing physical performance and quality of life during aging and in chronic disease. Previously, it was shown that inactivation of glycogen synthase kinase (GSK)-3ß stimulates muscle protein accretion, myogenesis, and mitochondrial biogenesis. Additionally, GSK-3ß is inactivated during recovery of disuse-induced muscle atrophy. AIM: Therefore, we hypothesize that GSK-3 inhibition is required for reloading-induced recovery of skeletal muscle mass and OXPHEN. METHODS: Wild-type (WT) and whole-body constitutively active (C.A.) Ser21/9 GSK-3α/ß knock-in mice were subjected to a 14-day hind-limb suspension/14-day reloading protocol. Soleus muscle mass, fiber cross-sectional area (CSA), OXPHEN (abundance of sub-units of oxidative phosphorylation (OXPHOS) complexes and fiber-type composition), as well as expression levels of their main regulators (respectively protein synthesis/degradation, myogenesis and peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) signaling) were monitored. RESULTS: Subtle but consistent differences suggesting suppression of protein turnover signaling and decreased expression of several OXPHOS sub-units and PGC-1α signaling constituents were observed at baseline in C.A. GSK-3 versus WT mice. Although soleus mass recovery during reloading occurred more rapidly in C.A. GSK-3 mice, this was not accompanied by a parallel increased CSA. The OXPHEN response to reloading was not distinct between C.A. GSK-3 and WT mice. No consistent or significant differences in reloading-induced changes in the regulatory steps of protein turnover, myogenesis or muscle OXPHEN were observed in C.A. GSK-3 compared to WT muscle. CONCLUSION: This study indicates that GSK-3 inactivation is dispensable for reloading-induced recovery of muscle mass and OXPHEN.
Assuntos
Glicogênio Sintase Quinase 3 beta/genética , Desenvolvimento Muscular/genética , Atrofia Muscular/tratamento farmacológico , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Animais , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Elevação dos Membros Posteriores , Humanos , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Fenótipo , Qualidade de Vida , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
Exacerbations in Chronic obstructive pulmonary disease (COPD) are often accompanied by pulmonary and systemic inflammation, and are associated with an increased susceptibility to weight loss and muscle wasting. As the emphysematous phenotype in COPD appears prone to skeletal muscle wasting, the aims of this study were to evaluate in emphysematous compared to control mice following repetitive exacerbations (1) changes in muscle mass and strength and, (2) whether muscle mass recovery and its underlying processes are impaired. Emphysema was induced by intra-tracheal (IT) elastase instillations, followed by three weekly IT-LPS instillations to mimic repetitive exacerbations. Loss of muscle mass and strength were measured, and related to analyses of muscle protein turnover and myogenesis signaling in tissue collected during and following recovery. Emphysematous mice showed impaired muscle mass recovery in response to pulmonary inflammation-induced muscle atrophy. Proteolysis and protein synthesis signaling remained significantly higher in emphysematous mice during recovery from LPS. Myogenic signaling in skeletal muscle was altered, and fusion capacity of cultured muscle cells treated with plasma derived from LPS-treated emphysematous mice was significantly decreased. In conclusion, repetitive cycles of pulmonary inflammation elicit sustained muscle wasting in emphysematous mice due to impaired muscle mass recovery, which is accompanied by aberrant myogenesis.
Assuntos
Desenvolvimento Muscular , Atrofia Muscular/fisiopatologia , Enfisema Pulmonar/fisiopatologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiopatologia , Atrofia Muscular/metabolismo , Enfisema Pulmonar/metabolismo , Recuperação de Função Fisiológica , Transdução de SinaisRESUMO
BACKGROUND: Sarcopenia was recently recognized as an independent condition by an International Classification of Diseases, Tenth Revision, Clinical Modification code, and is a frequently observed comorbidity in chronic obstructive pulmonary disease (COPD). Muscle mass is primarily dictated by the balance between protein degradation and synthesis, but their relative contribution to sarcopenia is unclear. OBJECTIVE: We aimed to assess potential differential molecular regulation of protein degradation and synthesis, as well as myogenesis, in the skeletal muscle of COPD patients with and without sarcopenia. METHODS: Muscle biopsies were obtained from the vastus lateralis muscle. Patients with COPD were clustered based on sarcopenia defined by low appendicular skeletal muscle mass index (nonsarcopenic COPD, n = 53; sarcopenic COPD, n = 39), and compared with healthy nonsarcopenic controls (n = 13). The mRNA and protein expression of regulators and mediators of ubiquitin-proteasome system (UPS), autophagy-lysosome system (autophagy), and protein synthesis were analyzed. Furthermore, mRNA expression of myogenesis markers was assessed. RESULTS: UPS signaling was unaltered, whereas indices of UPS regulation (eg, FOXO1 protein; p-FOXO3/FOXO3), autophagy signaling (eg, LC3BII/I; p-ULK1[Ser757]/ULK1), and protein synthesis signaling (eg, AKT1; p-GSK3B/GSK3B; p-4E-BP1/4E-BP1) were increased in COPD. These alterations were even more pronounced in COPD patients with sarcopenia (eg, FOXO1 protein; p-FOXO1/FOXO1; LC3BII/I; p-ULK(Ser555); p-AKT1/AKT1; AKT1; p-4E-BP1). Furthermore, myogenic signaling (eg, MYOG) was increased in COPD despite a concomitant increase of myostatin (MSTN) mRNA expression, with no difference between sarcopenic and nonsarcopenic COPD patients. CONCLUSION: Together with elevated myogenic signaling, the increase in muscle protein turnover signaling in COPD, which is even more prominent in COPD patients with sarcopenia, reflects molecular alterations associated with muscle repair and remodeling.
Assuntos
Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Sarcopenia/metabolismo , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/fisiopatologia , Atrofia Muscular/fisiopatologia , Sarcopenia/complicações , Transdução de Sinais/fisiologiaRESUMO
Tumor necrosis factor alpha (TNFalpha) has been implicated as a mediator of muscle wasting through nuclear factor kappa B (NF-kappaB) -dependent inhibition of myogenic differentiation. The aim of the present study was to identify the regulatory molecule(s) of myogenesis targeted by TNFalpha/NF-kappaB signaling. TNFalpha interfered with cell cycle exit and repressed the accumulation of transcripts encoding muscle-specific genes in differentiating C2C12 myoblasts. Overexpression of a p65 (RelA) mutant lacking the transcriptional activation domain attenuated the TNFalpha-mediated inhibition of muscle-specific gene transcription. The ability of muscle regulatory factor MyoD to induce muscle-specific transcription in 10T1/2 fibroblasts was also disrupted by wild-type p65, demonstrating that NF-kappaB transcriptional activity interferes with the function of MyoD. Inhibition of muscle-specific gene expression by TNFalpha was restored by overexpression of MyoD, whereas endogenous MyoD protein abundance and stability were reduced by TNFalpha through increased proteolysis of MyoD by the ubiquitin proteasome pathway. Last, the inhibitory effects of TNFalpha on myogenic differentiation were demonstrated in a mouse model of skeletal muscle regeneration, in which TNFalpha caused a delay in myoblast cell cycle exit. These results implicate that TNFalpha inhibits myogenic differentiation through destabilizing MyoD protein in a NF-kappaB-dependent manner, which interferes with skeletal muscle regeneration and may contribute to muscle wasting.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Proteína MyoD/metabolismo , Mioblastos/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Meia-Vida , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Proteína MyoD/química , Mioblastos/citologia , Mioblastos/metabolismo , NF-kappa B/metabolismo , Complexo de Endopeptidases do Proteassoma , Regeneração , Fator de Transcrição RelA , Ativação Transcricional , Ubiquitina/metabolismoRESUMO
Exercise training (ExTr) is largely used to improve functional capacity in patients with chronic obstructive pulmonary disease (COPD). However, ExTr only partially restores muscle function in patients with COPD, suggesting that confounding factors may limit the efficiency of ExTr. In the present study, we hypothesized that skeletal muscle adaptations triggered by ExTr could be compromised in hypoxemic patients with COPD. Vastus lateralis muscle biopsies were obtained from patients with COPD who were either normoxemic (n = 15, resting arterial Po2 = 68.5 ± 1.5 mmHg) or hypoxemic (n = 8, resting arterial Po2 = 57.0 ± 1.0 mmHg) before and after a 2-mo ExTr program. ExTr induced a significant increase in exercise capacity both in normoxemic and hypoxemic patients with COPD. However, ExTr increased citrate synthase and lactate dehydrogenase enzyme activities only in skeletal muscle of normoxemic patients. Similarly, muscle fiber cross-sectional area and capillary-to-fiber ratio were increased only in patients who were normoxemic. Expression of atrogenes (MuRF1, MAFbx/Atrogin-1) and autophagy-related genes (Beclin, LC3, Bnip, Gabarapl) remained unchanged in both groups. Phosphorylation of Akt (Ser473), GSK-3ß (Ser9), and p70S6k (Thr389) was nonsignificantly increased in normoxemic patients in response to ExTr, but it was significantly decreased in hypoxemic patients. We further showed on C2C12 myotubes that hypoxia completely prevented insulin-like growth factor-1-induced phosphorylation of Akt, GSK-3ß, and p70S6K. Together, our observations suggest a role for hypoxemia in the adaptive response of skeletal muscle of patients with COPD in an ExTr program.
Assuntos
Hipóxia/enzimologia , Condicionamento Físico Humano/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença Pulmonar Obstrutiva Crônica/complicações , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Citrato (si)-Sintase/metabolismo , Feminino , Humanos , Hipertrofia , Hipóxia/patologia , Hipóxia/fisiopatologia , Fator de Crescimento Insulin-Like I/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
Myogenic cell lines have been used extensively in the study of skeletal muscle development, regeneration, and homeostasis. To induce myogenic differentiation, culture media composed of a wide variety of growth factors and other additives have been used. Because the diversity in these components may modulate the differentiation process differentially, we describe a differentiation protocol that does not require the introduction of any factors to the differentiation media (DM) other than those present in the growth media. By culturing C2C12 skeletal myocytes on a coating of diluted Matrigel, a soluble basement membrane, consisting of collagen IV, laminin, heparan sulfate proteoglycans, and entactin, myogenic differentiation was accomplished by mere serum reduction. Assessment of myotube formation, creatine kinase activity, myosin heavy chain-fast, and myogenin demonstrated that the kinetics and extent of myogenic differentiation were superior using this protocol, compared with a commonly used differentiation protocol, in which an extracellular matrix is not provided and the DM contains horse serum. In addition, the elevated transactivation of a troponin-I promoter reporter construct suggested that myogenesis was enhanced at the transcriptional level. Finally, assessment of genomic deoxyribonucleic acid content revealed that the Matrigel differentiation protocol resulted in lowered proliferation. This protocol may aid studies aimed at elucidating mechanisms of myogenic differentiation, where a homogeneous population of myotubes is preferred.
Assuntos
Matriz Extracelular/fisiologia , Músculo Esquelético/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Fusão Celular , Linhagem Celular , Creatina Quinase/genética , DNA/genética , Regulação da Expressão Gênica/genética , Cinética , Camundongos , Músculo Esquelético/fisiologia , Miogenina/genética , Cadeias Pesadas de Miosina/genética , Plasmídeos , TransfecçãoRESUMO
Loss of skeletal muscle oxidative fiber types and mitochondrial capacity is a hallmark of chronic obstructive pulmonary disease and chronic heart failure. Based on in vivo human and animal studies, tissue hypoxia has been hypothesized as determinant, but the direct effect of hypoxia on muscle oxidative phenotype remains to be established. Hence, we determined the effect of hypoxia on in vitro cultured muscle cells, including gene and protein expression levels of mitochondrial components, myosin isoforms (reflecting slow-oxidative versus fast-glycolytic fibers), and the involvement of the regulatory PPAR/PGC-1α pathway. We found that hypoxia inhibits the PPAR/PGC-1α pathway and the expression of mitochondrial components through HIF-1α. However, in contrast to our hypothesis, hypoxia stimulated the expression of slow-oxidative type I myosin via HIF-1α. Collectively, this study shows that hypoxia differentially regulates contractile and metabolic components of muscle oxidative phenotype in a HIF-1α-dependent manner.
Assuntos
Hipóxia Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Animais , Linhagem Celular , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Fosforilação Oxidativa , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Being well-established in advanced chronic obstructive pulmonary disease (COPD), skeletal muscle dysfunction and its underlying pathology have been scarcely investigated in patients with mild-to-moderate airflow obstruction. We hypothesized that a loss of oxidative phenotype (oxphen) associated with decreased endurance is present in the skeletal muscle of patients with mild-to-moderate COPD. In quadriceps muscle biopsies from 29 patients with COPD (forced expiratory volume in 1 s [FEV1] 58 ± 16%pred, body mass index [BMI] 26 ± 4 kg/m(2)) and 15 controls (BMI 25 ± 3 kg/m(2)) we assessed fiber type distribution, fiber cross-sectional areas (CSA), oxidative and glycolytic gene expression, OXPHOS protein levels, metabolic enzyme activity, and levels of oxidative stress markers. Quadriceps function was assessed by isokinetic dynamometry, body composition by dual-energy X-ray absorptiometry, exercise capacity by an incremental load test, and physical activity level by accelerometry. Compared with controls, patients had comparable fat-free mass index, quadriceps strength, and fiber CSA, but quadriceps endurance was decreased by 29% (P = 0.002). Patients with COPD had a clear loss of muscle oxphen: a fiber type I-to-II shift, decreased levels of OXPHOS complexes IV and V subunits (47% and 31%, respectively; P < 0.05), a decreased ratio of 3-hydroxyacyl-CoA dehydrogenase/phosphofructokinase (PFK) enzyme activities (38%, P < 0.05), and decreased peroxisome proliferator-activated receptor-γ coactivator-1α (40%; P < 0.001) vs. increased PFK (67%; P < 0.001) gene expression levels. Within the patient group, markers of oxphen were significantly positively correlated with quadriceps endurance and inversely with the increase in plasma lactate relative to work rate during the incremental test. Levels of protein carbonylation, tyrosine nitration, and malondialdehyde protein adducts were comparable between patients and controls. However, among patients, oxidative stress levels were significantly inversely correlated with markers of oxphen and quadriceps endurance. Reduced muscle endurance associated with underlying loss of muscle oxphen is already present in patients with mild-to-moderate COPD without muscle wasting.