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Congenital muscular dystrophy with laminin α2 chain-deficiency (LAMA2-CMD) is a severe neuromuscular disorder without a cure. Using transcriptome and proteome profiling as well as functional assays, we previously demonstrated significant metabolic impairment in skeletal muscle from LAMA2-CMD patients and mouse models. Reactive oxygen species (ROS) increase when oxygen homeostasis is not maintained and, here, we investigate whether oxidative stress indeed is involved in the pathogenesis of LAMA2-CMD. We also analyze the effects of two antioxidant molecules, N-acetyl-L-cysteine (NAC) and vitamin E, on disease progression in the dy2J/dy2J mouse model of LAMA2-CMD. We demonstrate increased ROS levels in LAMA2-CMD mouse and patient skeletal muscle. Furthermore, NAC treatment (150 mg/kg IP for 6 days/week for 3 weeks) led to muscle force loss prevention, reduced central nucleation and decreased the occurrence of apoptosis, inflammation, fibrosis and oxidative stress in LAMA2-CMD muscle. In addition, vitamin E (40 mg/kg oral gavage for 6 days/week for 2 weeks) improved morphological features and reduced inflammation and ROS levels in dy2J/dy2J skeletal muscle. We suggest that NAC and to some extent vitamin E might be potential future supportive treatments for LAMA2-CMD as they improve numerous pathological hallmarks of LAMA2-CMD.
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Congenital muscular dystrophy with laminin α2 chain-deficiency (LAMA2-CMD) is a severe muscle disorder with complex underlying pathogenesis. We have previously employed profiling techniques to elucidate molecular patterns and demonstrated significant metabolic impairment in skeletal muscle from LAMA2-CMD patients and mouse models. Thus, we hypothesize that skeletal muscle metabolism may be a promising pharmacological target to improve muscle function in LAMA2-CMD. Here, we have investigated whether the multifunctional medication metformin could be used to reduce disease in the dy2J/dy2J mouse model of LAMA2-CMD. First, we show gender disparity for several pathological hallmarks of LAMA2-CMD. Second, we demonstrate that metformin treatment significantly increases weight gain and energy efficiency, enhances muscle function and improves skeletal muscle histology in female dy2J/dy2J mice (and to a lesser extent in dy2J/dy2J males). Thus, our current data suggest that metformin may be a potential future supportive treatment that improves many of the pathological characteristics of LAMA2-CMD.
Assuntos
Disparidades nos Níveis de Saúde , Lamina Tipo A/deficiência , Metformina/administração & dosagem , Músculo Esquelético/efeitos dos fármacos , Distrofias Musculares/tratamento farmacológico , Distrofia Muscular Animal/tratamento farmacológico , Administração Oral , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Lamina Tipo A/genética , Laminina/genética , Masculino , Camundongos , Músculo Esquelético/patologia , Distrofias Musculares/genética , Distrofias Musculares/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Fatores Sexuais , Resultado do TratamentoRESUMO
Skeletal muscle has high energy requirement and alterations in metabolism are associated with pathological conditions causing muscle wasting and impaired regeneration. Congenital muscular dystrophy type 1A (MDC1A) is a severe muscle disorder caused by mutations in the LAMA2 gene. Leigh syndrome (LS) is a neurometabolic disease caused by mutations in genes related to mitochondrial function. Skeletal muscle is severely affected in both diseases and a common feature is muscle weakness that leads to hypotonia and respiratory problems. Here, we have investigated the bioenergetic profile in myogenic cells from MDC1A and LS patients. We found dysregulated expression of genes related to energy production, apoptosis and proteasome in myoblasts and myotubes. Moreover, impaired mitochondrial function and a compensatory upregulation of glycolysis were observed when monitored in real-time. Also, alterations in cell cycle populations in myoblasts and enhanced caspase-3 activity in myotubes were observed. Thus, we have for the first time demonstrated an impairment of the bioenergetic status in human MDC1A and LS muscle cells, which could contribute to cell cycle disturbance and increased apoptosis. Our findings suggest that skeletal muscle metabolism might be a promising pharmacological target in order to improve muscle function, energy efficiency and tissue maintenance of MDC1A and LS patients.
Assuntos
Doença de Leigh/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Distrofias Musculares/metabolismo , Mioblastos/metabolismo , Fosforilação Oxidativa , Apoptose , Células Cultivadas , Glicólise , Humanos , Laminina/genética , Distrofias Musculares/genética , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Adult skeletal tissue is composed of heterogeneous population of cells that constantly self-renew by means of a controlled process of activation and proliferation of tissue-resident stem cells named satellite cells. Many growth factors, cytokines and myokines produced by skeletal muscle cells play critical roles in local regulation of the inflammatory process and skeletal muscle regeneration during different pathological conditions. IL-6 is a pleiotropic cytokine released in large amount during infection, autoimmunity and cancer. Low levels of IL-6 can promote activation of satellite cells and myotube regeneration while chronically elevated production promote skeletal muscle wasting. These distinct effects may be explained by a crosstalk of the IL-6/IL-6 receptor and gp130 trans-signaling pathway that oppose to regenerative and anti-inflammatory of the classical IL-6 receptor signaling pathway. Here we discuss on potential therapeutic strategies using monoclonal antibodies to IL-6R for the treatment of skeletal muscle wasting and cachexia. We also highlight on the IL-6/JAK/STAT and FGF/p38αß MAPK signaling pathways in satellite cell activation and the use of protein kinase inhibitors for tailoring and optimizing satellite cell proliferation during the skeletal muscle renewal. Future investigations on the roles of the IL-6 classical and trans-signaling pathways in both immune and non-immune cells in skeletal muscle tissue will provide new basis for therapeutic approaches to reverse atrophy and degeneration of skeletal muscles in cancer and inflammatory diseases.
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BACKGROUND: We previously identified dermicidin (DCD), which encodes a growth and survival factor, as a gene amplified and overexpressed in a subset of breast tumors. Patients with DCD-positive breast cancer have worse prognostic features. We therefore searched for specific molecular signatures in DCD-positive breast carcinomas from patients and representative cell lines. METHODS: DCD expression was evaluated by qRT-PCR, immunohistochemical and immunoblot assays in normal and neoplastic tissues and cell lines. To investigate the role of DCD in breast tumorigenesis, we analyzed the consequences of its downregulation in human breast cancer cell lines using three specific shRNA lentiviral vectors. Genes up- and down-regulated by DCD were identified using Affymetrix microarray and analyzed by MetaCore Platform. RESULTS: We identified DCD splice variant (DCD-SV) that is co-expressed with DCD in primary invasive breast carcinomas and in other tissue types and cell lines. DCD expression in breast tumors from patients with clinical follow up data correlated with high histological grade, HER2 amplification and luminal subtype. We found that loss of DCD expression led to reduced cell proliferation, resistance to apoptosis, and suppressed tumorigenesis in immunodeficient mice. Network analysis of gene expression data revealed perturbed ERBB signaling following DCD shRNA expression including changes in the expression of ERBB receptors and their ligands. CONCLUSIONS: These findings imply that DCD promotes breast tumorigenesis via modulation of ERBB signaling pathways. As ERBB signaling is also important for neural survival, HER2+ breast tumors may highjack DCD's neural survival-promoting functions to promote tumorigenesis.
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Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Dermocidinas/genética , Dermocidinas/metabolismo , Receptor ErbB-2/metabolismo , Transdução de Sinais , Processamento Alternativo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Biomarcadores , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Imuno-Histoquímica , Receptor ErbB-2/antagonistas & inibidores , Trastuzumab/administração & dosagem , Trastuzumab/farmacologia , Carga Tumoral/efeitos dos fármacosRESUMO
An alteration of energy balance is the immediate cause of the so-called cachexia. Although alterations of energy intake are often associated with cachexia, it has lately became clear that an increased energy expenditure is the main cause of wasting associated with different types of pathological conditions, such as cancer, infections or chronic heart failure among others. Different types of molecular mechanisms contribute to energy expenditure and, therefore, involuntary body weight loss; among them, adenosine triphosphate (ATP) consumption by sarcoplasmic reticulum Ca(2+) pumps could represent a key mechanism. In other cases, an increase in energy inefficiency will further contribute to energy imbalance.
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Congenital muscular dystrophy with laminin α2 chain deficiency (MDC1A) is one of the most severe forms of muscular disease and is characterized by severe muscle weakness and delayed motor milestones. The genetic basis of MDC1A is well known, yet the secondary mechanisms ultimately leading to muscle degeneration and subsequent connective tissue infiltration are not fully understood. In order to obtain new insights into the molecular mechanisms underlying MDC1A, we performed a comparative proteomic analysis of affected muscles (diaphragm and gastrocnemius) from laminin α2 chain-deficient dy(3K)/dy(3K) mice, using multidimensional protein identification technology combined with tandem mass tags. Out of the approximately 700 identified proteins, 113 and 101 proteins, respectively, were differentially expressed in the diseased gastrocnemius and diaphragm muscles compared with normal muscles. A large portion of these proteins are involved in different metabolic processes, bind calcium, or are expressed in the extracellular matrix. Our findings suggest that metabolic alterations and calcium dysregulation could be novel mechanisms that underlie MDC1A and might be targets that should be explored for therapy. Also, detailed knowledge of the composition of fibrotic tissue, rich in extracellular matrix proteins, in laminin α2 chain-deficient muscle might help in the design of future anti-fibrotic treatments. All MS data have been deposited in the ProteomeXchange with identifier PXD000978 (http://proteomecentral.proteomexchange.org/dataset/PXD000978).
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Cálcio/metabolismo , Diafragma/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Laminina/deficiência , Músculo Esquelético/metabolismo , Animais , Diafragma/patologia , Modelos Animais de Doenças , Fibrose/genética , Fibrose/patologia , Expressão Gênica/genética , Perfilação da Expressão Gênica , Laminina/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/patologia , Distrofia Muscular Animal , Espectrometria de Massas em TandemRESUMO
Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted.
Assuntos
Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Laminina/deficiência , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular Animal/patologia , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Peso Corporal/efeitos dos fármacos , Bortezomib , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Laminina/metabolismo , Locomoção/efeitos dos fármacos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Células Musculares/patologia , Músculos/efeitos dos fármacos , Músculos/metabolismo , Músculos/patologia , Distrofia Muscular Animal/genética , Especificidade de Órgãos/efeitos dos fármacos , Especificidade de Órgãos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Análise de SobrevidaRESUMO
INTRODUCTION: Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. METHODS: By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts. RESULTS: The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals. CONCLUSIONS: The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.
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Carcinoma Hepatocelular/fisiopatologia , Acoplamento Excitação-Contração/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias Hepáticas/fisiopatologia , Músculo Esquelético/fisiopatologia , Animais , Caquexia/etiologia , Caquexia/genética , Caquexia/fisiopatologia , Cálcio/metabolismo , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/genética , Modelos Animais de Doenças , Metabolismo Energético/fisiologia , Acoplamento Excitação-Contração/genética , Homeostase/fisiologia , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/genética , Masculino , Ratos , Ratos WistarRESUMO
Approximately half of all cancer patients present with cachexia, a condition in which disease-associated metabolic changes lead to a severe loss of skeletal muscle mass. Working toward an integrated and mechanistic view of cancer cachexia, we investigated the hypothesis that cancer promotes mitochondrial uncoupling in skeletal muscle. We subjected mice to in vivo phosphorous-31 nuclear magnetic resonance (31P NMR) spectroscopy and subjected murine skeletal muscle samples to gas chromatography/mass spectrometry (GC/MS). The mice used in both experiments were Lewis lung carcinoma models of cancer cachexia. A novel 'fragmented mass isotopomer' approach was used in our dynamic analysis of 13C mass isotopomer data. Our 31P NMR and GC/MS results indicated that the adenosine triphosphate (ATP) synthesis rate and tricarboxylic acid (TCA) cycle flux were reduced by 49% and 22%, respectively, in the cancer-bearing mice (p<0.008; t-test vs. controls). The ratio of ATP synthesis rate to the TCA cycle flux (an index of mitochondrial coupling) was reduced by 32% in the cancer-bearing mice (p=0.036; t-test vs. controls). Genomic analysis revealed aberrant expression levels for key regulatory genes and transmission electron microscopy (TEM) revealed ultrastructural abnormalities in the muscle fiber, consistent with the presence of abnormal, giant mitochondria. Taken together, these data suggest that mitochondrial uncoupling occurs in cancer cachexia and thus point to the mitochondria as a potential pharmaceutical target for the treatment of cachexia. These findings may prove relevant to elucidating the mechanisms underlying skeletal muscle wasting observed in other chronic diseases, as well as in aging.
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Trifosfato de Adenosina/biossíntese , Ciclo do Ácido Cítrico , Músculo Esquelético/metabolismo , Neoplasias/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Caquexia/complicações , Caquexia/metabolismo , Caquexia/patologia , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Neoplasias/complicações , Neoplasias/patologiaRESUMO
BACKGROUND: Cachexia is a wasting condition that manifests in several types of cancer, and the main characteristic is the profound loss of muscle mass. METHODS: The Yoshida AH-130 tumor model has been used and the samples have been analyzed using transmission electronic microscopy, real-time PCR and Western blot techniques. RESULTS: Using in vivo cancer cachectic model in rats, here we show that skeletal muscle loss is accompanied by fiber morphologic alterations such as mitochondrial disruption, dilatation of sarcoplasmic reticulum and apoptotic nuclei. Analyzing the expression of some factors related to proteolytic and thermogenic processes, we observed in tumor-bearing animals an increased expression of genes involved in proteolysis such as ubiquitin ligases Muscle Ring Finger 1 (MuRF-1) and Muscle Atrophy F-box protein (MAFBx). Moreover, an overexpression of both sarco/endoplasmic Ca(2+)-ATPase (SERCA1) and adenine nucleotide translocator (ANT1), both factors related to cellular energetic efficiency, was observed. Tumor burden also leads to a marked decreased in muscle ATP content. CONCLUSIONS: In addition to muscle proteolysis, other ATP-related pathways may have a key role in muscle wasting, both directly by increasing energetic inefficiency, and indirectly, by affecting the sarcoplasmic reticulum-mitochondrial assembly that is essential for muscle function and homeostasis. GENERAL SIGNIFICANCE: The present study reports profound morphological changes in cancer cachectic muscle, which are visualized mainly in alterations in sarcoplasmic reticulum and mitochondria. These alterations are linked to pathways that can account for energy inefficiency associated with cancer cachexia.
Assuntos
Caquexia/metabolismo , Núcleo Celular/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Atrofia Muscular/metabolismo , Sarcoma de Yoshida/metabolismo , Retículo Sarcoplasmático/metabolismo , Translocador 1 do Nucleotídeo Adenina/genética , Translocador 1 do Nucleotídeo Adenina/metabolismo , Trifosfato de Adenosina/deficiência , Animais , Apoptose/genética , Caquexia/complicações , Caquexia/patologia , Núcleo Celular/ultraestrutura , Metabolismo Energético/genética , Expressão Gênica , Masculino , Mitocôndrias/ultraestrutura , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/complicações , Atrofia Muscular/patologia , Proteólise , Ratos , Ratos Wistar , Proteínas Ligases SKP Culina F-Box/genética , Proteínas Ligases SKP Culina F-Box/metabolismo , Sarcoma de Yoshida/complicações , Sarcoma de Yoshida/patologia , Retículo Sarcoplasmático/ultraestrutura , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND AND AIMS: The aim of the present investigation was to examine the anti-wasting effects of theophylline (a methylxantine present in tea leaves) on a rat model of cancer cachexia. METHODS: The in vitro effects of the nutraceuticals on proteolysis were examined on muscle cell cultures submitted to hyperthermia. Individual muscle weights, muscle gene expression, body composition and cardiac function were measured in rats bearing the Yoshida AH-130 ascites hepatoma, following theophylline treatment. RESULTS: Theophylline treatment inhibited proteolysis in C2C12 cell line and resulted in an anti-proteolytic effect on muscle tissue (soleus and heart), which was associated with a decrease in circulating TNF-alpha levels and with a decreased proteolytic systems gene expression. Treatment with the nutraceutical also resulted in an improvement in body composition and cardiac function. CONCLUSION: Theophylline - alone or in combination with drugs - may be a candidate molecule for the treatment of cancer cachexia.
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Interleukin-15 (IL-15) is an anabolic factor for skeletal muscle and several reports have described its important role as a regulator of energy homeostasis. In this study, we analyzed the effects of IL-15 on adipocyte differentiation using the 3T3-L1 preadipose cell line. The data show that IL-15 tends to reduce the rate of adipocyte proliferation, induces apoptosis, and partially stops differentiation. The signaling molecules behind these actions of the cytokine on adipose cells are: p42/p44 MAPK (which seem to be associated with the reduced rate of proliferation induced by the cytokine), STAT5 (which is related to the actions of IL-15 on differentiation), and SAPK/JNK (which are related to the increased apoptosis induced by IL-15). In conclusion, using the 3T3-L1 adipocyte cell line, the results presented here show that IL-15 exerts important effects on differentiation, proliferation and apoptosis. Altogether, the results presented here reinforce the idea that IL-15 is an important mediator that regulates adipose size and, therefore, the role of the cytokine in affecting body weight and obesity deserves additional studies.
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Adipócitos/fisiologia , Diferenciação Celular , Interleucina-15/fisiologia , Obesidade/patologia , Células 3T3-L1 , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Compostos Azo , Proliferação de Células , Expressão Gênica , Interleucina-15/farmacologia , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
The hypothesis we tested was that administering corticotropin-releasing factor receptor agonists preserves muscle mass during cancer that is related to changes in tissue gene expression. cDNA microarrays were used to compare mRNAs from muscle and adipose tissues of non-treated and agonist-treated tumor-bearing rats. In muscle of non-tumor-bearing agonist-treated animals we observed decreased expression of genes associated with fatty acid uptake and esterification. In tumor-bearing animals, CRF2R agonist administration produced decreased mRNA content of the atrogene lipin-1. In white adipose tissue, agonist treatment of non-tumor-bearing animals induced genes typically related to muscle structure and function. The fact that this treatment decreased expression of atrogenes could have clinical application. In addition, agonist treatment changed the gene pattern of adipose tissue to render it similar to that of skeletal muscle; thus, treatment with this agonist alters the gene pattern to what could be called "muscularization of white adipose tissue."