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1.
J Am Heart Assoc ; 4(7)2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26199228

RESUMO

BACKGROUND: Ubiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined. METHODS AND RESULTS: The tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated. CONCLUSIONS: These results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.


Assuntos
Metabolismo Energético , Insuficiência Cardíaca/enzimologia , Contração Miocárdica , Miócitos Cardíacos/enzimologia , Tiorredoxina Redutase 2/deficiência , Disfunção Ventricular Esquerda/enzimologia , Função Ventricular Esquerda , Fatores Etários , Animais , Autofagia , Pressão Sanguínea , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Predisposição Genética para Doença , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Membrana Lisossomal/genética , Proteínas de Membrana Lisossomal/metabolismo , Metabolômica/métodos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias Cardíacas/enzimologia , Mitocôndrias Cardíacas/ultraestrutura , Miócitos Cardíacos/ultraestrutura , Estresse Oxidativo , Fenótipo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Volume Sistólico , Tiorredoxina Redutase 2/genética , Fatores de Tempo , Disfunção Ventricular Esquerda/genética , Disfunção Ventricular Esquerda/fisiopatologia
2.
J Cell Sci ; 127(Pt 13): 2849-61, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24816559

RESUMO

The GTPase Ras can either promote or inhibit cell survival. Inactivating mutations in Drosophila RasGAP (encoded by vap), a Ras GTPase-activating protein, lead to age-related brain degeneration. Genetic interactions implicate the epidermal growth factor receptor (EGFR)-Ras pathway in promoting neurodegeneration but the mechanism is not known. Here, we show that the Src homology 2 (SH2) domains of RasGAP are essential for its neuroprotective function. By using affinity purification and mass spectrometry, we identify a complex containing RasGAP together with Sprint, which is a Ras effector and putative activator of the endocytic GTPase Rab5. Formation of the RasGAP-Sprint complex requires the SH2 domains of RasGAP and tyrosine phosphorylation of Sprint. RasGAP and Sprint colocalize with Rab5-positive early endosomes but not with Rab7-positive late endosomes. We demonstrate a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppresses neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila is crucially dependent on the activities of two GTPases, Ras and Rab5, regulated by the interplay of RasGAP and Sprint.


Assuntos
Drosophila/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Sobrevivência Celular/fisiologia , Drosophila/genética , Endocitose , Feminino , Masculino , Fosforilação , Transdução de Sinais
3.
Circulation ; 124(25): 2892-902, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22144571

RESUMO

BACKGROUND: Excessive formation of reactive oxygen species contributes to tissue injury and functional deterioration after myocardial ischemia/reperfusion. Especially, mitochondrial reactive oxygen species are capable of opening the mitochondrial permeability transition pore, a harmful event in cardiac ischemia/reperfusion. Thioredoxins are key players in the cardiac defense against oxidative stress. Mutations in the mitochondrial thioredoxin reductase (thioredoxin reductase-2, Txnrd2) gene have been recently identified to cause dilated cardiomyopathy in patients. Here, we investigated whether mitochondrial thioredoxin reductase is protective against myocardial ischemia/reperfusion injury. METHODS AND RESULTS: In mice, α-MHC-restricted Cre-mediated Txnrd2 deficiency, induced by tamoxifen (Txnrd2-/-ic), aggravated systolic dysfunction and cardiomyocyte cell death after ischemia (90 minutes) and reperfusion (24 hours). Txnrd2-/-ic was accompanied by a loss of mitochondrial integrity and function, which was resolved on pretreatment with the reactive oxygen species scavenger N-acetylcysteine and the mitochondrial permeability transition pore blocker cyclosporin A. Likewise, Txnrd2 deletion in embryonic endothelial precursor cells and embryonic stem cell-derived cardiomyocytes, as well as introduction of Txnrd2-shRNA into adult HL-1 cardiomyocytes, increased cell death on hypoxia and reoxygenation, unless N-acetylcysteine was coadministered. CONCLUSIONS: We report that Txnrd2 exerts a crucial function during postischemic reperfusion via thiol regeneration. The efficacy of cyclosporin A in cardiac Txnrd2 deficiency may indicate a role for Txnrd2 in reducing mitochondrial reactive oxygen species, thereby preventing opening of the mitochondrial permeability transition pore.


Assuntos
Mitocôndrias/enzimologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Estresse Oxidativo/fisiologia , Compostos de Sulfidrila/metabolismo , Tiorredoxina Redutase 2/metabolismo , Acetilcisteína/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Células Cultivadas , Ciclosporina/farmacologia , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Inibidores Enzimáticos/farmacologia , Sequestradores de Radicais Livres/farmacologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Células-Tronco Hematopoéticas/citologia , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/citologia , Estresse Oxidativo/efeitos dos fármacos , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 1/metabolismo , Tiorredoxina Redutase 2/genética
4.
J Am Assoc Lab Anim Sci ; 50(5): 665-74, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22330713

RESUMO

Regular work tasks in the washroom of laboratory animal facilities include cleaning of cages and bottles and handling of chow and bedding. These operations largely are carried out by hand. We quantitatively determined the musculoskeletal load on the trunk and upper limbs of washroom employees in an animal facility with a holding capacity of 35,000 rodent cages by using a computer-assisted, quantitative, recording, and long-term analysis (CUELA) system, which volunteers wore during routine work. Parallel video recording allowed exact assignment of each movement of body and limbs to the data recorded by the sensors. For the most part, trunk movements were unassociated with risk of injury. Evaluation of upper limb movements by CUELA indicated elevated burden on shoulder, elbows, and wrists due to the high repetitiveness and range of movements and postures. However, after additional work factors like low effort and the presence of micropauses were taken into account, workers were not at risk for the development of musculoskeletal disorders of the upper limbs. Handling bottles, chow, and bedding and maneuvering trolleys that entailed greater musculoskeletal loads did not yield evidence of overstraining, because the actions typically were executed alternately and were of short duration during daily shifts. The results represent quantitative information on the musculoskeletal load of regular washroom operations in a laboratory animal facility. These data provide the basis for ergonomic redesign of operations and implementation of automation for highly repetitive movements.


Assuntos
Experimentação Animal , Transtornos Traumáticos Cumulativos/patologia , Laboratórios , Remoção , Esforço Físico/fisiologia , Postura/fisiologia , Adulto , Fenômenos Biomecânicos , Humanos , Masculino , Pessoa de Meia-Idade , Saneamento/métodos , Gravação em Vídeo , Suporte de Carga
5.
Biol Chem ; 388(10): 1083-90, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17937622

RESUMO

Thioredoxin reductases (Txnrds) are a group of selenoenzymes participating in cellular redox regulation. Three Txnrd isoforms are known, each of which exhibits distinct cellular localisation and tissue-specific expression pattern. Txnrd1 is found in the cytoplasm, expression of Txnrd2 is restricted to mitochondria and Txnrd3 shows testis-specific expression. Recently, it was shown that Txnrd2 strongly affects the development of blood cells, since mouse embryos deficient for Txnrd2 are severely anaemic, show increased apoptosis in foetal liver and possess haematopoietic liver stem cells of reduced capacity to proliferate in vitro. However, because Txnrd2-deficient mice die at embryonic day 13.5, it was not known how this enzyme affects blood cell function in the adult animal. In the present study we show that conditional Txnrd2 knockouts generated using CD4- and CD19Cre transgenic mice lack Txnrd2 expression in CD4-- and CD19-positive T- and B-lymphocytes, respectively. However, the development and differentiation of both cell types in thymus and bone marrow was not significantly impaired. In addition, B-cell proliferation and activation in response to CD40 and IL-4 was unaltered in Txnrd2-deficient B-cells.


Assuntos
Linfócitos B/enzimologia , Linfócitos T/enzimologia , Tiorredoxina Redutase 2/fisiologia , Animais , Linfócitos B/imunologia , Diferenciação Celular , Feminino , Citometria de Fluxo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Transgênicos , Células Precursoras de Linfócitos B/enzimologia , Células Precursoras de Linfócitos B/imunologia , Células Precursoras de Linfócitos T/enzimologia , Células Precursoras de Linfócitos T/imunologia , Linfócitos T/imunologia , Tiorredoxina Redutase 2/genética
6.
Biol Chem ; 388(10): 1091-7, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17937623

RESUMO

Thioredoxin reductase 1 (Txnrd1) and thioredoxin reductase 2 (Txnrd2) are selenoproteins whose expression and function depends on adequate supply of the trace element selenium (Se). As homozygous (-/-) knockout of both Txnrd1 and Txnrd2 is embryonically lethal, we investigated the effect of their hemizygosity (+/-) alone and in combination with dietary Se on enzymatic activity in various tissues. To assess the overall health of the corresponding mice, the growth, viability and fertility of the different experimental groups were also compared. Se depletion led to a marked decrease in Se organ contents. Se depletion was most prominent in lung, followed by liver, kidney, heart, muscle and brain. We found no major effect of Txnrd1 or Txnrd2 hemizygosity and/or Se on male fertility and the viability of offspring. A gene dose effect under Se-adequate conditions for Txnrd1 and Txnrd2 in all organs was observed. Haploid insufficiency decreased Txnrd activity to an extent that can be further decreased by Se deficiency, but not to levels below those observed for Se depletion alone. The only exception was Txnrd2 activity in kidney, heart and muscle, where we found an additive effect.


Assuntos
Selênio/farmacologia , Tiorredoxina Redutase 1/metabolismo , Tiorredoxina Redutase 2/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Knockout , Selênio/administração & dosagem , Tiorredoxina Redutase 1/genética , Tiorredoxina Redutase 2/genética
7.
Genesis ; 45(1): 11-6, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17216603

RESUMO

Inducible and tissue-specific gene inactivation in mice has become a powerful tool to bypass embryonic and postnatal lethality of knockout mice. The most frequently used inducible system is based on Cre recombinase fused to either one or two mutated estrogen receptor ligand binding domains, thus rendering Cre function tamoxifen-dependent. To achieve Cre-mediated inactivation of a given gene, 4-OH tamoxifen (4-OHT) dissolved either in alcohol and/or oil is usually administered by repeated intraperitoneal (i.p.) injections. Since this procedure imposes considerable stress on mice, we compared the effect of tamoxifen citrate, mixed into a standard mouse diet at different concentrations, with that of i.p. administration of 4-OHT on Cre-mediated, heart-specific inactivation of thioredoxin reductase 2. Here we show that tamoxifen citrate in the chow was equally effective as 4-OHT given i.p. Oral tamoxifen administration is thus a convenient and cost-saving way for gene induction, and, most importantly, it reduces stress and avoids adverse effects in mice.


Assuntos
Miocárdio/enzimologia , Tamoxifeno/farmacologia , Tiorredoxina Dissulfeto Redutase/biossíntese , Animais , Integrases/genética , Camundongos , Camundongos Transgênicos , Tamoxifeno/administração & dosagem , Tamoxifeno/efeitos adversos , Tiorredoxina Redutase 2 , Tiorredoxina Dissulfeto Redutase/genética
8.
Mol Biol Cell ; 14(1): 241-50, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12529440

RESUMO

Ras signaling has been shown to play an important role in promoting cell survival in many different tissues. Here we show that upregulation of Ras activity in adult Drosophila neurons induces neuronal cell death, as evident from the phenotype of vacuolar peduncle (vap) mutants defective in the Drosophila RasGAP gene, which encodes a Ras GTPase-activating protein. These mutants show age-related brain degeneration that is dependent on activation of the EGF receptor signaling pathway in adult neurons, leading to autophagic cell death (cell death type 2). These results provide the first evidence for a requirement of Egf receptor activity in differentiated adult Drosophila neurons and show that a delicate balance of Ras activity is essential for the survival of adult neurons.


Assuntos
Envelhecimento/fisiologia , Encéfalo/fisiologia , Drosophila/fisiologia , Receptores ErbB/metabolismo , Transdução de Sinais/fisiologia , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Morte Celular/fisiologia , Drosophila/genética , Mutação , Regulação para Cima , Vacúolos/genética , Vacúolos/metabolismo
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