RESUMEN
There is clinical evidence that patients with heart failure and concomitant iron deficiency have increased skeletal muscle fatigability and impaired exercise tolerance. It was expected that a skeletal muscle cell line subjected to different degrees of iron availability and/or concomitant hypoxia would demonstrate changes in cell morphology and in the expression of atrophy markers. L6G8C5 rat skeletal myocytes were cultured in normoxia or hypoxia at optimal, reduced or increased iron concentrations. Experiments were performed to evaluate the iron content in cells, cell morphology, and the expression of muscle specific atrophy markers [Atrogin1 and musclespecific RINGfinger 1 (MuRF1)], a gene associated with the atrophy/hypertrophy balance [mothers against decapentaplegic homolog 4 (SMAD4)] and a muscle classIII intermediate filament protein (Desmin) at the mRNA and protein level. Hypoxic treatment caused, as compared to normoxic conditions, an increase in the expression of Atrogin1 (P<0.001). Irondeficient cells exhibited morphological abnormalities and demonstrated a significant increase in the expression of Atrogin1 (P<0.05) and MuRF1 (P<0.05) both in normoxia and hypoxia, which indicated activation of the ubiquitin proteasome pathway associated with protein degradation during muscle atrophy. Depleted iron in cell culture combined with hypoxia also induced a decrease in SMAD4 expression (P<0.001) suggesting modifications leading to atrophy. In contrast, cells cultured in a medium enriched with iron during hypoxia exhibited inverse changes in the expression of atrophy markers (both P<0.05). Desmin was upregulated in cells subjected to both iron depletion and iron excess in normoxia and hypoxia (all P<0.05), but the greatest augmentation of mRNA expression occurred when iron depletion was combined with hypoxia. Notably, in hypoxia, an increased expression of Atrogin1 and MuRF1 was associated with an increased expression of transferrin receptor 1, reflecting intracellular iron demand (R=0.76, P<0.01; R=0.86, P<0.01). Hypoxia and iron deficiency when combined exhibited the most detrimental impact on skeletal myocytes, especially in the context of muscle atrophy markers. Conversely, iron supplementation in in vitro conditions acted in a protective manner on these cells.
Asunto(s)
Deferoxamina/farmacología , Compuestos Férricos/farmacología , Hierro/metabolismo , Fibras Musculares Esqueléticas/efectos de los fármacos , Atrofia Muscular/metabolismo , Atrofia Muscular/prevención & control , Compuestos de Amonio Cuaternario/farmacología , Animales , Hipoxia de la Célula/efectos de los fármacos , Línea Celular , Desmina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/etiología , Atrofia Muscular/patología , Ratas , Proteína Smad4/genéticaRESUMEN
Diagnosis of fatal hypothermia is considered to be difficult in forensic practice because of the lack of any specific pathological findings. The mechanism that induces abnormal behavior such as undressing or hiding during the state of hypothermia has not been clarified. In order to solve these problems, we made a rat model of fatal hypothermia and investigated the expression of some mRNA within the hypothalamus and the frontal cortex. The expression of aldehyde dehydrogenase 6 family, member A1 (ALDH6A1), cocaine- and amphetamine-regulated transcript peptide (CARTPT), desmin (DES), heat shock 70kDa protein 4 (HSPA4), serotonin receptor 2A (HTR2A), opioid receptor, delta 1 (OPRD1) and transthyretin (TTR) supposedly related to fatal hypothermia was determined using quantitative real-time PCR. The expression of OPRD1 in the hypothalamus of fatal hypothermia was significantly increased, while the expression of TTR within the frontal cortex was significantly decreased compared to that in the control. These findings suggest that OPRD1 and TTR may be involved in thermoregulation at a low ambient temperature.
Asunto(s)
Lóbulo Frontal/metabolismo , Hipotálamo/metabolismo , Hipotermia/metabolismo , ARN Mensajero/metabolismo , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Animales , Desmina/genética , Desmina/metabolismo , Patologia Forense , Proteínas del Choque Térmico HSP110/genética , Proteínas del Choque Térmico HSP110/metabolismo , Masculino , Modelos Animales , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Reacción en Cadena de la Polimerasa , Prealbúmina/genética , Prealbúmina/metabolismo , Ratas , Ratas Wistar , Receptores Opioides delta/genética , Receptores Opioides delta/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismoRESUMEN
This case report describes a pregnant female patient who presented with new-onset congestive heart failure symptoms and prolonged QTc, with strong family history of sudden death. Endomyocardial biopsy and genetic testing revealed myocardial desmin accumulation and a previously described mutation in the DES (desmin) gene, as well as variants in two LQT genes, SCN5A and KCNH2. The case highlights the phenotypic variability for a particular desmin genotype, and the possible interaction of desminopathy with LQT variants not independently associated with large differences in current properties or QT prolongation from wild type.
Asunto(s)
Desmina/genética , Insuficiencia Cardíaca/genética , Síndrome de QT Prolongado/genética , Mutación , Miositis por Cuerpos de Inclusión/genética , Complicaciones Cardiovasculares del Embarazo/genética , Adulto , Muerte Súbita Cardíaca/etiología , Muerte Súbita Cardíaca/prevención & control , Desfibriladores Implantables , Canal de Potasio ERG1 , Electrocardiografía , Técnicas Electrofisiológicas Cardíacas , Canales de Potasio Éter-A-Go-Go/genética , Exones , Femenino , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/terapia , Humanos , Síndrome de QT Prolongado/terapia , Miositis por Cuerpos de Inclusión/patología , Miositis por Cuerpos de Inclusión/terapia , Canal de Sodio Activado por Voltaje NAV1.5 , Embarazo , Canales de Sodio/genética , Resultado del TratamientoRESUMEN
BACKGROUND: Hepatic oval cells, progenitor cells in the liver, can differentiate into hepatocytes and bile duct cells both in vitro and in vivo. Although hepatic stellate cells are another important cell component in the liver, less attention has been focused on the relationship between hepatic oval cells and hepatic stellate cells. METHODS: Hepatic oval cells were isolated from rats fed a choline-deficient diet supplemented with 0.1% ethionine for 6 weeks and characterized by electron microscopy, flow cytometry, reverse transcription polymerase chain reaction, Western blot and bi-direction differentiation. After treatment with transforming growth factor-beta1 (TGF-beta1), changes in cell viability, morphology, extracellular matrix (ECM) expression and immune phenotype were analysed in these cultured and adherent hepatic oval cells. RESULTS: The primary cultured hepatic oval cells were positive for the oval cell-specific markers OV-6, BD-1/BD-2 and M2PK as well as the hepatocyte markers albumin and alpha-foetoprotein. These hepatic oval cells differentiated bipotentially into hepatocytes or bile duct-like cells under appropriate conditions. It is noteworthy that these bipotential hepatic oval cells expressed ECM genes stably, including collagens, matrix metalloproteinases and tissue inhibitor of mellatoproteinase. Furthermore, except for growth inhibition and morphological changes in the hepatic oval cells after exposure to TGF-beta1, there was an increased expression of ECM genes, the onset expression of snail and loss expression of E-cadherin. During this process, TGF-beta1 treatment induced an upregulation of marker genes for hepatic stellate cells in hepatic oval cells, such as desmin and GFAP. CONCLUSION: Except for the expression of ECM, the cultured hepatic oval cells could induce an increased expression of hepatic stellate cell markers by TGF-beta1 through an epithelial-mesenchymal transition process, which might indicate the contribution of hepatic oval cells to liver fibrosis.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Células Madre/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Albúminas/metabolismo , Animales , Antimetabolitos/administración & dosificación , Antimetabolitos/efectos adversos , Conductos Biliares/efectos de los fármacos , Conductos Biliares/metabolismo , Conductos Biliares/patología , Biomarcadores/metabolismo , Cadherinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Deficiencia de Colina/etiología , Deficiencia de Colina/metabolismo , Deficiencia de Colina/patología , Desmina/genética , Desmina/metabolismo , Modelos Animales de Enfermedad , Etionina/administración & dosificación , Etionina/efectos adversos , Proteínas de la Matriz Extracelular/efectos de los fármacos , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/farmacología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/patología , Masculino , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Células Madre/efectos de los fármacos , Células Madre/ultraestructura , Factor de Crecimiento Transformador beta/farmacología , alfa-Fetoproteínas/metabolismoAsunto(s)
Cardiomiopatía Dilatada/veterinaria , Enfermedades de los Perros/genética , Perros/genética , Actinas/genética , Animales , Proteínas de Unión al Calcio/genética , Cardiomiopatía Dilatada/genética , ADN Complementario/genética , Desmina/genética , Perros/clasificación , Escala de Lod , Proteínas Musculares/genética , Sarcoglicanos/genética , Especificidad de la Especie , Tropomodulina/genéticaRESUMEN
Several studies have already demonstrated that micro- and milli-calpains (CAPN 1-CAPN 2), calcium-dependent intracellular cysteine-proteases are involved in many biological phenomenon including muscle growth and development. More particularly, recent studies have demonstrated that milli-calpain is implicated in myoblast fusion. Moreover, in primary muscle cells, these proteases do not appear simultaneously throughout muscle cell differentiation. Because micro- and milli-calpains do not have the same intracellular localization, it appears likely that these two calcium-dependent proteases have different biological roles during muscle cell differentiation. The goal of this study is to determine the role of micro-calpain. We therefore, have developed a muscle cell line in which micro-calpain is over-expressed, using the inducible Tet Regulated Expression System. The outcome is observed by following the behavior of different proteins, considered to be potential substrates of the protease. The present study shows important decreases in the expression level of ezrin (68%), vimentin (64%) and caveolin 3 (76%) whereas many other cytoskeletal proteins remain remarkably stable. Concerning the myogenic transcription factors, only the level of myogenin decreased (59%) after the over-expression of micro-calpain. Ultra structural studies have shown that the myofibrils formed near the cell periphery are normally oriented, lying along the longitudinal axis. This regularity is lost progressively towards the cell center where the cytoskeleton presented an increasing disorganization. All these results indicate that micro-calpain is involved in regulation pathway of myogenesis via at least its action on ezrin, vimentin, caveolin 3 and myogenin, a muscle transcription factor.
Asunto(s)
Calpaína/fisiología , Células Musculares/metabolismo , Animales , Calpaína/genética , Caveolina 3 , Caveolinas/genética , Caveolinas/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular , Clonación Molecular , Proteínas del Citoesqueleto , ADN Complementario/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desmina/genética , Desmina/metabolismo , Doxiciclina/farmacología , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Células Musculares/fisiología , Células Musculares/ultraestructura , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Factor 5 Regulador Miogénico , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Vimentina/genética , Vimentina/metabolismoRESUMEN
Recent studies in desmin (-/-) mice have shown that the targeted ablation of desmin leads to pathological changes of the extrasarcomeric intermediate filament cytoskeleton, as well as structural and functional abnormalities of mitochondria in striated muscle. Here, we report on a novel heterozygous single adenine insertion mutation (c.5141_5143insA) in a 40-year-old patient with a distal myopathy. The insertion mutation leads to a frameshift and a truncated desmin (K239fs242). Using transfection studies in SW13 and BHK21 cells, we show that the K239fsX242 desmin mutant is incapable of forming a desmin intermediate filament network. Furthermore, it induces the collapse of a pre-existing desmin cytoskeleton, alters the subcellular distribution of mitochondria and leads to abnormal cytoplasmic protein aggregates reminiscent of desmin-immunoreactive granulofilamentous material seen in the ultrastructural analysis of the patient's muscle. Analysis of mitochondrial function in isolated saponin-permeablized skeletal muscle fibres from our patient showed decreased maximal rates of respiration with the NAD-dependent substrate combination glutamate and malate, as well as a higher amytal sensitivity of respiration, indicating an in vivo inhibition of complex I activity. Our findings suggest that the heterozygous K239fsX242 desmin insertion mutation has a dominant negative effect on the polymerization process of desmin intermediate filaments and affects not only the subcellular distribution, but also biochemical properties of mitochondria in diseased human skeletal muscle. As a consequence, the intermediate filament pathology-induced mitochondrial dysfunction may contribute to the degeneration/regeneration process leading to progressive muscle dysfunction in human desminopathies.