RESUMO
Muscle may contribute to the systemic inflammatory environment during critical illness, but leukocyte interaction and cytokine influence on muscle and its response has not been fully explored in this context. Using an in vivo model of intratracheal lipopolysaccharide (IT LPS)-induced acute lung injury, we show that skeletal muscle rapidly responds with expression of proinflammatory genes, which may be explained by migration of LPS into the circulation. Treatment of mature C2C12 myotubes with LPS at a level achieved in the circulation following IT LPS elicited a proinflammatory cytokine expression profile similar to that of in vivo murine muscle following IT LPS. Stimulation with toll-like receptor (TLR) 2 and 3 agonists provoked comparable responses in C2C12 myotubes. Additionally, co-cultures of C2C12 myotubes and bone marrow-derived macrophages (BMDM) identified the capacity of macrophages to increase myotube proinflammatory gene expression, with tumor necrosis factor-α (TNFα) gene and protein expression largely attributable to BMDM. To investigate the contribution of TNFα in the synergy of the co-culture environment, C2C12 myotubes were treated with recombinant TNFα, co-cultures were established using TNF-deficient BMDM, and co-cultures were also depleted of TNFα using antibodies. To determine whether the in vitro observations were relevant in vivo, mice received intramuscular administration of LPS ± TNFα or TNFα-neutralizing antibodies and showed that TNFα is both sufficient and necessary to induce synergistic cytokine release from muscle. Taken together, these data demonstrate how skeletal muscle tissue may contribute proinflammatory cytokines following acute endotoxin injury and the potential of leukocytes to augment this response via TNFα secretion.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Macrófagos/metabolismo , Músculo Esquelético/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Citocinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/metabolismoRESUMO
Exercise has numerous benefits for patients with cancer, but implementation is challenging because of practical and logistical hurdles. This study examined whether neuromuscular electrical stimulation (NMES) can serve as a surrogate for classic exercise by eliciting an exercise training response in skeletal muscle of women diagnosed with breast cancer undergoing chemotherapy. Patients (n = 22) with histologically confirmed stage I, II, or III breast cancer scheduled to receive neoadjuvant or adjuvant chemotherapy were randomized to 8 wk of bilateral neuromuscular electrical stimulation (NMES; 5 days/wk) to their quadriceps muscles or control. Biopsy of the vastus lateralis was performed at baseline and after 8 wk of intervention to assess muscle fiber size, contractility, and mitochondrial content. Seventeen patients (8 control/9 NMES) completed the trial and were included in analyses. NMES promoted muscle fiber hypertrophy (P < 0.001), particularly in fast-twitch, myosin heavy chain (MHC) IIA fibers (P < 0.05) and tended to induce fiber type shifts in MHC II fibers. The effects of NMES on single-muscle fiber contractility were modest, and it was unable to prevent declines in the function in MHC IIA fibers. NMES did not alter intermyofibrillar mitochondrial content/structure but was associated with reductions in subsarcolemmal mitochondria. Our results demonstrate that NMES induces muscle fiber hypertrophy and fiber type shifts in MHC II fibers but had minimal effects on fiber contractility and promoted reductions in subsarcolemmal mitochondria. Further studies are warranted to evaluate the utility of NMES as an exercise surrogate in cancer patients and other conditions.NEW & NOTEWORTHY This is the first study to evaluate whether neuromuscular electrical stimulation (NMES) can be used as an exercise surrogate to improve skeletal muscle fiber size or function in cancer patients receiving treatment. We show that NMES promoted muscle fiber hypertrophy and fiber type shifts but had minimal effects on single-fiber contractility and reduced subsarcolemmal mitochondria.
Assuntos
Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Estimulação Elétrica , Feminino , Humanos , Contração Muscular , Fibras Musculares Esqueléticas , Músculo Esquelético , Músculo QuadrícepsRESUMO
Factors secreted from tumors/tumor cells are hypothesized to cause skeletal muscle wasting in cancer patients. We examined whether cancer cells secrete factors to promote atrophy by evaluating the effects of conditioned media (CM) from murine lung cancer cells and primary cultures of human lung tumor cells on cultured myotubes. We evaluated murine Lewis lung carcinoma (LLC) and KRASG12D cells, and primary cell lines derived from tumor biopsies from patients with lung cancer (hTCM; n = 6). In all experiments, serum content was matched across treatment groups. We hypothesized that CM from murine and human tumor cells would reduce myotube myosin content, decrease mitochondrial content, and increase mitochondrial reactive oxygen species (ROS) production. Treatment of myotubes differentiated for 7 days with CM from LLC and KRASG12D cells did not alter any of these variables. Effects of murine tumor cell CM were observed when myotubes differentiated for 4 days were treated with tumor cell CM and compared with undiluted differentiation media. However, these effects were not apparent if tumor cell CM treatments were compared with control cell CM or dilution controls. Finally, CM from human lung tumor primary cell lines did not modify myosin content or mitochondrial content or ROS production compared with either undiluted differentiated media, control cell CM, or dilution controls. Our results do not support the hypothesis that factors released from cultured lung cancer/tumor cells promote myotube wasting or mitochondrial abnormalities, but we cannot dismiss the possibility that these cells could secrete such factors in vivo within the native tumor microenvironment.
Assuntos
Caquexia/metabolismo , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Meios de Cultivo Condicionados/farmacologia , Neoplasias Pulmonares/metabolismo , Mitocôndrias Musculares/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Miosinas/metabolismo , Adenocarcinoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Caquexia/etiologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos , Neoplasias/complicações , Neoplasias/metabolismo , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Células Tumorais CultivadasRESUMO
Muscle contraction may protect against the effects of chemotherapy to cause skeletal muscle atrophy, but the mechanisms underlying these benefits are unclear. To address this question, we utilized in vitro modeling of contraction and mechanotransduction in C2C12 myotubes treated with doxorubicin (DOX; 0.2 µM for 3 days). Myotubes expressed contractile proteins and organized these into functional myofilaments, as electrical field stimulation (STIM) induced intracellular calcium (Ca2+) transients and contractions, both of which were prevented by inhibition of membrane depolarization. DOX treatment reduced myotube myosin content, protein synthesis, and Akt (S308) and forkhead box O3a (FoxO3a; S253) phosphorylation and increased muscle RING finger 1 (MuRF1) expression. STIM (1 h/day) prevented DOX-induced reductions in myotube myosin content and Akt and FoxO3a phosphorylation, as well as increases in MuRF1 expression, but did not prevent DOX-induced reductions in protein synthesis. Inhibition of myosin-actin interaction during STIM prevented contraction and the antiatrophic effects of STIM without affecting Ca2+ cycling, suggesting that the beneficial effect of STIM derives from mechanotransductive pathways. Further supporting this conclusion, mechanical stretch of myotubes recapitulated the effects of STIM to prevent DOX suppression of FoxO3a phosphorylation and upregulation of MuRF1. DOX also increased reactive oxygen species (ROS) production, which led to a decrease in mitochondrial content. Although STIM did not alter DOX-induced ROS production, peroxisome proliferator-activated receptor-γ coactivator-1α and antioxidant enzyme expression were upregulated, and mitochondrial loss was prevented. Our results suggest that the activation of mechanotransductive pathways that downregulate proteolysis and preserve mitochondrial content protects against the atrophic effects of chemotherapeutics.
Assuntos
Doxorrubicina/efeitos adversos , Regulação da Expressão Gênica , Mecanotransdução Celular , Mitocôndrias/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Animais , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Transformada , Doxorrubicina/antagonistas & inibidores , Estimulação Elétrica , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Contração Muscular/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Atrofia Muscular/prevenção & controle , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Miosinas/genética , Miosinas/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
How breast cancer and its treatments affect skeletal muscle is not well defined. To address this question, we assessed skeletal muscle structure and protein expression in 13 women who were diagnosed with breast cancer and receiving adjuvant chemotherapy following tumor resection and 12 nondiseased controls. Breast cancer patients showed reduced single-muscle fiber cross-sectional area and fractional content of subsarcolemmal and intermyofibrillar mitochondria. Drugs commonly used in breast cancer patients (doxorubicin and paclitaxel) caused reductions in myosin expression, mitochondrial loss, and increased reactive oxygen species (ROS) production in C2C12 murine myotube cell cultures, supporting a role for chemotherapeutics in the atrophic and mitochondrial phenotypes. Additionally, concurrent treatment of myotubes with the mitochondrial-targeted antioxidant MitoQ prevented chemotherapy-induced myosin depletion, mitochondrial loss, and ROS production. In patients, reduced mitochondrial content and size and increased expression and oxidation of peroxiredoxin 3, a mitochondrial peroxidase, were associated with reduced muscle fiber cross-sectional area. Our results suggest that chemotherapeutics may adversely affect skeletal muscle in patients and that these effects may be driven through effects of these drugs on mitochondrial content and/or ROS production.
Assuntos
Antineoplásicos/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Caquexia/genética , Atrofia Muscular/genética , Peroxirredoxina III/genética , Idoso , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caquexia/induzido quimicamente , Caquexia/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/patologia , Miosinas/genética , Miosinas/metabolismo , Compostos Organofosforados/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
Estrogen replacement therapy reduces the incidence of type 2 diabetes in postmenopausal women; however, the mechanism is unknown. Therefore, the aim of this study was to evaluate the metabolic effects of estrogen replacement therapy in an experimental model of menopause. At 8 weeks of age, female mice were ovariectomized (OVX) or sham (SHAM) operated, and OVX mice were treated with vehicle (OVX) or estradiol (E2) (OVX+E2). After 4 weeks of high-fat diet feeding, OVX mice had increased body weight and fat mass compared with SHAM and OVX+E2 mice. OVX mice displayed reduced whole-body energy expenditure, as well as impaired glucose tolerance and whole-body insulin resistance. Differences in whole-body insulin sensitivity in OVX compared with SHAM mice were accounted for by impaired muscle insulin sensitivity, whereas both hepatic and muscle insulin sensitivity were impaired in OVX compared with OVX+E2 mice. Muscle diacylglycerol (DAG), content in OVX mice was increased relative to SHAM and OVX+E2 mice. In contrast, E2 treatment prevented the increase in hepatic DAG content observed in both SHAM and OVX mice. Increases in tissue DAG content were associated with increased protein kinase Cε activation in liver of SHAM and OVX mice compared with OVX+E2 and protein kinase Cθ activation in skeletal muscle of OVX mice compared with SHAM and OVX+E2. Taken together, these data demonstrate that E2 plays a pivotal role in the regulation of whole-body energy homeostasis, increasing O(2) consumption and energy expenditure in OVX mice, and in turn preventing diet-induced ectopic lipid (DAG) deposition and hepatic and muscle insulin resistance.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Estradiol/metabolismo , Estradiol/farmacologia , Resistência à Insulina/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Animais , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/prevenção & controle , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Estradiol/deficiência , Terapia de Reposição de Estrogênios , Feminino , Glucose/metabolismo , Homeostase/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Menopausa/metabolismo , Camundongos , Modelos Animais , Ovariectomia , Proteína Quinase C/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is the most frequent chronic liver disease in the United States and is strongly associated with hepatic insulin resistance. We examined whether the thyroid hormone receptor-α (Thra) would be a potential therapeutic target to prevent diet-induced NAFLD and insulin resistance. For that purpose, we assessed insulin action in high-fat diet-fed Thra gene knockout (Thra-0/0) and wild-type mice using hyperinsulinemic-euglycemic clamps combined with (3)H/(14)C-labeled glucose to assess basal and insulin-stimulated rates of glucose and fat metabolism. Body composition was assessed by (1)H magnetic resonance spectroscopy and energy expenditure by indirect calorimetry. Relative rates of hepatic glucose and fat oxidation were assessed in vivo using a novel proton-observed carbon-edited nuclear magnetic resonance technique. Thra-0/0 were lighter, leaner, and manifested greater whole-body insulin sensitivity than wild-type mice during the clamp, which could be attributed to increased insulin sensitivity both in liver and peripheral tissues. Increased hepatic insulin sensitivity could be attributed to decreased hepatic diacylglycerol content, resulting in decreased activation of protein kinase Cε and increased insulin signaling. In conclusion, loss of Thra protects mice from high-fat diet-induced hepatic steatosis and hepatic and peripheral insulin resistance. Therefore, thyroid receptor-α inhibition represents a novel pharmacologic target for the treatment of NAFLD, obesity, and type 2 diabetes.