RESUMO
Potassium voltage-gated channel subfamily Q member 1 opposite strand 1 (KCNQ1OT1), a long non-coding RNA found in the KCNQ1 locus, has been evidenced to play important roles in the aggravation of inflammatory and oxidative stresses under hypoxia, but whether and how KCNQ1OT1 contributes to neuronal damages in the cerebral ischemic stroke remains unknown. In the present study, we found a dominant upregulation of KCNQ1OT1 both in the plasma of cerebral ischemia patients and in an oxygen-glucose deprivation and reperfusion (OGD/R) model in PC12 cells. KCNQ1OT1 knocking-down significantly ameliorated the inflammation, oxidative stress, and cell apoptosis induced by OGD/R. We further demonstrated that KCNQ1OT1 directly bound to and suppressed the expression of miR-140-3p. Overexpressing miR-140-3p significantly alleviated both the inflammation, oxidative stress, and cell apoptosis in OGD/R, while all those cytoprotective effects of miR-140-3p-overexpression were hindered by the co-overexpression of KCNQ1OT1. Furthermore, we found a direct interaction between miR-140-3p and the hypoxia-inducible factor-1α (HIF-1α), which was suppressed by the upregulation of KCNQ1OT1 in OGD/R. Our results indicate that KCNQ1OT1 exacerbates cerebral ischemia-reperfusion injury by targeted binding to miR-140-3p, thus interfering its direct interaction with HIF-1α. These data provide novel therapeutic targets in the cerebral ischemic stroke.
Assuntos
Isquemia Encefálica/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Isquemia Encefálica/patologia , Técnicas de Silenciamento de Genes/métodos , Humanos , Células PC12 , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ligação Proteica , RNA Interferente Pequeno/farmacologia , Ratos , Traumatismo por Reperfusão/patologiaRESUMO
Sudden cardiac death (SCD) is the leading global cause of mortality. SCD often arises from cardiac ischemia reperfusion (IR) injury, pathologic sequence variants within ion channel genes, or a combination of the two. Alternative approaches are needed to prevent or ameliorate ventricular arrhythmias linked to SCD. Here, we investigated the efficacy of remote ischemic preconditioning (RIPC) of the limb versus the liver in reducing ventricular arrhythmias in a mouse model of SCD. Mice lacking the Kcne2 gene, which encodes a potassium channel ß subunit associated with acquired Long QT syndrome were exposed to IR injury via coronary ligation. This resulted in ventricular arrhythmias in all mice (15/15) and SCD in 5/15 mice during reperfusion. Strikingly, prior RIPC (limb or liver) greatly reduced the incidence and severity of all ventricular arrhythmias and completely prevented SCD. Biochemical and pharmacological analysis demonstrated that RIPC cardioprotection required ERK1/2 and/or AKT phosphorylation. A lack of alteration in GSK-3ß phosphorylation suggested against conventional reperfusion injury salvage kinase (RISK) signaling pathway protection. If replicated in human studies, limb RIPC could represent a noninvasive, nonpharmacological approach to limit dangerous ventricular arrhythmias associated with ischemia and/or channelopathy-linked SCD.
Assuntos
Arritmias Cardíacas/prevenção & controle , Morte Súbita Cardíaca/prevenção & controle , Extremidades/irrigação sanguínea , Precondicionamento Isquêmico/métodos , Fígado/irrigação sanguínea , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Circulação Hepática , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Fosforilação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Fluxo Sanguíneo Regional , Transdução de SinaisRESUMO
Nonalcoholic fatty liver disease (NAFLD) is an increasing health problem worldwide, with genetic, epigenetic, and environmental components. Here, we describe the first example of NAFLD caused by genetic disruption of a mammalian potassium channel subunit. Mice with germline deletion of the KCNE2 potassium channel ß subunit exhibited NAFLD as early as postnatal day 7. Using mouse genetics, histology, liver damage assays and transcriptomics we discovered that iron deficiency arising from KCNE2-dependent achlorhydria is a major factor in early-onset NAFLD in Kcne2(â/â) mice, while two other KCNE2-dependent defects did not initiate NAFLD. The findings uncover a novel genetic basis for NAFLD and an unexpected potential factor in human KCNE2-associated cardiovascular pathologies, including atherosclerosis.
Assuntos
Anemia Ferropriva/complicações , Hepatopatia Gordurosa não Alcoólica/etiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Animais , Proteína C-Reativa/análise , Dieta Hiperlipídica , Feminino , Redes Reguladoras de Genes , Mutação em Linhagem Germinativa , Homocisteína/sangue , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Deleção de Sequência , Transcriptoma , Triglicerídeos/sangueRESUMO
Iron homeostasis is a dynamic process that is tightly controlled to balance iron uptake, storage, and export. Reduction of dietary iron from the ferric to the ferrous form is required for uptake by solute carrier family 11 (proton-coupled divalent metal ion transporters), member 2 (Slc11a2) into the enterocytes. Both processes are proton dependent and have led to the suggestion of the importance of acidic gastric pH for the absorption of dietary iron. Potassium voltage-gated channel subfamily E, member 2 (KCNE2), in combination with potassium voltage-gated channel, KQT-like subfamily, member 1 (KCNQ1), form a gastric potassium channel essential for gastric acidification. Deficiency of either Kcne2 or Kcnq1 results in achlorhydia, gastric hyperplasia, and neoplasia, but the impact on iron absorption has not, to our knowledge, been investigated. Here we report that Kcne2-deficient mice, in addition to the previously reported phenotypes, also present with iron-deficient anemia. Interestingly, impaired function of KCNQ1 results in iron-deficient anemia in Jervell and Lange-Nielsen syndrome patients. We speculate that impaired function of KCNE2 could result in the same clinical phenotype.
Assuntos
Anemia Ferropriva/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Anemia Ferropriva/sangue , Animais , Dieta Ocidental , Índices de Eritrócitos , Eritropoetina/sangue , Feminino , Ferritinas/sangue , Heterogeneidade Genética , Hematócrito , Humanos , Canal de Potássio KCNQ1/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Caracteres Sexuais , Especificidade da Espécie , Organismos Livres de Patógenos Específicos , Transferrina/análiseRESUMO
The mechanotransduction of vestibular sensory cells depends on the high endolymphatic potassium concentration ([K+]) maintained by a fine balance between K+ secretion and absorption by epithelial cells. Despite the crucial role of endolymph as an electrochemical motor for mechanotransduction, little is known about the processes that govern endolymph formation. To address these, we took advantage of an organotypic rodent model, which regenerates a genuine neonatal vestibular endolymphatic compartment, facilitating the determination of endolymphatic [K+] and transepithelial potential (Vt) during endolymph formation. While mature Vt levels are almost immediately achieved, K+ accumulates to reach a steady [K+] by day 5 in culture. Inhibition of sensory cell K+ efflux enhances [K+] regardless of the blocker used (FM1.43, amikacin, gentamicin, or gadolinium). Targeting K+ secretion with bumetanide partially and transiently reduces [K+], while ouabain application and Kcne1 deletion almost abolishes it. Immunofluorescence studies demonstrate that dark cells do not express Na-K-2Cl cotransporter 1 (the target of bumetanide) in cultured and young mouse utricles, while Na/K-ATPase (the target of ouabain) is found in dark cells and transitional cells. This global analysis of the involvement of endolymphatic homeostasis actors in the immature organ (1) confirms that KCNE1 channels are necessary for K+ secretion, (2) highlights Na/K-ATPase as the key endolymphatic K+ provider and shows that Na-K-2Cl cotransporter 1 has a limited impact on K+ influx, and (3) demonstrates that transitional cells are involved in K+ secretion in the early endolymphatic compartment.
Assuntos
Endolinfa/metabolismo , Células Epiteliais/fisiologia , Sáculo e Utrículo/crescimento & desenvolvimento , Sáculo e Utrículo/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Aminoglicosídeos/farmacologia , Animais , Animais Recém-Nascidos , Bumetanida/farmacologia , Endocitose/genética , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Feminino , Gadolínio/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Ouabaína/farmacologia , Potássio/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Ratos , Ratos Wistar , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Fatores de TempoRESUMO
BACKGROUND: Voltage-gated potassium channels are thought to be the target of antibodies associated with limbic encephalitis. However, antibody testing using cells expressing voltage-gated potassium channels is negative; hence, we aimed to identify the real autoantigen associated with limbic encephalitis. METHODS: We analysed sera and CSF of 57 patients with limbic encephalitis and antibodies attributed to voltage-gated potassium channels and 148 control individuals who had other disorders with or without antibodies against voltage-gated potassium channels. Immunohistochemistry, immunoprecipitation, and mass spectrometry were used to characterise the antigen. An assay with HEK293 cells transfected with leucine-rich, glioma-inactivated 1 (LGI1) and disintegrin and metalloproteinase domain-containing protein 22 (ADAM22) or ADAM23 was used as a serological test. The identity of the autoantigen was confirmed by immunoabsorption studies and immunostaining of Lgi1-null mice. FINDINGS: Immunoprecipitation and mass spectrometry analyses showed that antibodies from patients with limbic encephalitis previously attributed to voltage-gated potassium channels recognise LGI1, a neuronal secreted protein that interacts with presynaptic ADAM23 and postsynaptic ADAM22. Immunostaining of HEK293 cells transfected with LGI1 showed that sera or CSF from patients, but not those from control individuals, recognised LGI1. Co-transfection of LGI1 with its receptors, ADAM22 or ADAM23, changed the pattern of reactivity and improved detection. LGI1 was confirmed as the autoantigen by specific abrogation of reactivity of sera and CSF from patients after immunoabsorption with LGI1-expressing cells and by comparative immunostaining of wild-type and Lgi1-null mice, which showed selective lack of reactivity in brains of Lgi1-null mice. One patient with limbic encephalitis and antibodies against LGI1 also had antibodies against CASPR2, an autoantigen we identified in some patients with encephalitis and seizures, Morvan's syndrome, and neuromyotonia. INTERPRETATION: LGI1 is the autoantigen associated with limbic encephalitis previously attributed to voltage-gated potassium channels. The term limbic encephalitis associated with antibodies against voltage-gated potassium channels should be changed to limbic encephalitis associated with LGI1 antibodies, and this disorder should be classed as an autoimmune synaptic encephalopathy. FUNDING: National Institutes of Health, National Cancer Institute, and Euroimmun.
Assuntos
Autoantígenos/imunologia , Encefalite Límbica/imunologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/imunologia , Proteínas/imunologia , Proteínas ADAM/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Autoanticorpos , Autoantígenos/genética , Linhagem Celular , Epilepsia/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Ligantes , Encefalite Límbica/genética , Encefalite Límbica/metabolismo , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteínas/genética , Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
Egr2 is a transcription factor required for peripheral nerve myelination in rodents, and mutations in Egr2 are associated with congenital hypomyelinating neuropathy (CHN) in humans. To further study its role in myelination, we generated mice harboring a hypomorphic Egr2 allele (Egr2Lo) that survive for up to 3 weeks postnatally, a period of active myelination in rodents. These Egr2Lo/Lo mice provided the opportunity to study the molecular effects of Egr2 deficiency on Schwann cell biology, an analysis that was not possible previously, because of the perinatal lethality of Egr2-null mice. Egr2Lo/Lo mice phenocopy CHN, as evidenced by the severe hypomyelination and increased numbers of proliferating Schwann cells of the peripheral nerves. Comparison of sciatic nerve gene expression profiles during development and after crush injury with those of Egr2Lo/Lo Schwann cells revealed that they are developmentally arrested, with down-regulation of myelination-related genes and up-regulation of genes associated with immature and promyelinating Schwann cells. One of the abnormally elevated genes in Egr2Lo/Lo Schwann cells, Sox2, encodes a transcription factor that is crucial for maintenance of neural stem cell pluripotency. Wild-type Schwann cells infected with Sox2 adenovirus or lentivirus inhibited expression of myelination-associated genes (e.g., myelin protein zero; Mpz), and failed to myelinate axons in vitro, but had an enhanced proliferative response to beta-neuregulin. The characterization of a mouse model of CHN has provided insight into Schwann cell differentiation and allowed the identification of Sox2 as a negative regulator of myelination.
Assuntos
Proteínas de Ligação a DNA/genética , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Proteínas Nucleares/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/deficiência , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Células de Schwann/patologia , Animais , Diferenciação Celular , Doenças Desmielinizantes/metabolismo , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Perfilação da Expressão Gênica , Proteínas HMGB , Camundongos , Camundongos Mutantes Neurológicos , Bainha de Mielina/metabolismo , Nervos Periféricos/metabolismo , Nervos Periféricos/patologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/patologia , Fatores de Transcrição SOXB1 , Células de Schwann/metabolismo , Fatores de TranscriçãoRESUMO
BACKGROUND: Oxygen (O2)-sensitive K+ channels mediate acute O2 sensing in many tissues. At birth, initial functional closure of the ductus arteriosus (DA) results from O2-induced vasoconstriction. This mechanism often fails in premature infants, resulting in persistent DA, a common form of congenital heart disease. We hypothesized that the basis for impaired O2 constriction in preterm DA is reduced expression and function of O2-sensitive, voltage-gated (Kv) channels. METHODS AND RESULTS: Preterm rabbit DA rings have reduced O2 constriction (even after inhibition of prostaglandin and nitric oxide synthases), and preterm DA smooth muscle cells (DASMCs) display reduced O2-sensitive K+ current. This is associated with decreased mRNA and protein expression of certain O2-sensitive Kv channels (Kv1.5 and Kv2.1) but equivalent expression of the L-type calcium channel. Transmural Kv1.5 or Kv2.1 gene transfer "rescues" the developmental deficiency, conferring O2 responsiveness to preterm rabbit DAs. Targeted SMC Kv1.5 gene transfer also enhances O2 constriction in human DAs. CONCLUSIONS: These data demonstrate a central role for developmentally regulated DASMC O2-sensitive Kv channels in the functional closure of the DA. Modulation of Kv channels may have therapeutic potential in diseases associated with impaired O2 responsiveness, including persistent DA.