RESUMEN
KCNQ1 and hERG encode the voltage-gated potassium channel α-subunits of the cardiac repolarizing currents I(Ks) and I(Kr), respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs), and loss-of-function mutations in these channels manifest clinically as long QT syndrome, characterized by the prolongation of the QT interval, polymorphic ventricular tachycardia, and sudden cardiac death. Previous cellular electrophysiology experiments in transgenic rabbit cardiomyocytes and heterologous cell lines demonstrated functional downregulation of complementary repolarizing currents. Biochemical assays indicated direct, protein-protein interactions between KCNQ1 and hERG may underlie the interplay between I(Ks) and I(Kr). Our objective was to investigate hERG-KCNQ1 interactions in the intact cellular environment primarily through acceptor photobleach FRET (apFRET) experiments. We quantitatively assessed the extent of interactions based on fluorophore location and the potential regulation of interactions by physiologically relevant signals. apFRET experiments established specific hERG-KCNQ1 associations in both heterologous and primary cardiomyocytes. The largest FRET efficiency (E(f); 12.0 ± 5.2%) was seen between ion channels with GFP variants fused to the COOH termini. Acute treatment with forskolin + IBMX or a membrane-permeable cAMP analog significantly and specifically reduced the extent of hERG-KCNQ1 interactions (by 41 and 38%, respectively). Our results demonstrate direct interactions between KCNQ1 and hERG occur in both intact heterologous cells and primary cardiomyocytes and are mediated by their COOH termini. Furthermore, this interplay between channel proteins is regulated by intracellular cAMP.
Asunto(s)
AMP Cíclico/química , Canales de Potasio Éter-A-Go-Go/química , Canal de Potasio KCNQ1/química , 1-Metil-3-Isobutilxantina/administración & dosificación , Potenciales de Acción/fisiología , Animales , Células CHO , Células Cultivadas , Colforsina/administración & dosificación , Cricetinae , Cricetulus , AMP Cíclico/agonistas , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/fisiología , Femenino , Células HEK293 , Corazón/efectos de los fármacos , Corazón/fisiología , Humanos , Canal de Potasio KCNQ1/fisiología , Masculino , Inhibidores de Fosfodiesterasa/administración & dosificación , ConejosRESUMEN
BACKGROUND: Anemia is a hematologic disorder with decreased number of erythrocytes. Erythropoiesis, the process by which red blood cells differentiate, are conserved in humans, mice and zebrafish. The only known agents available to treat pathological anemia are erythropoietin and its biologic derivatives. However, erythropoietin therapy elicits unwanted side-effects, high cost and intravenous or subcutaneous injection, warranting the development of a more cost effective and non-peptide alternative. Ginger (Zingiber officinale) has been widely used in traditional medicine; however, to date there is no scientific research documenting the potential of ginger to stimulate hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Here, we utilized gata1:dsRed transgenic zebrafish embryos to investigate the effect of ginger extract on hematopoiesis in vivo and we identified its bioactive component, 10-gingerol. We confirmed that ginger and 10-gingerol promote the expression of gata1 in erythroid cells and increase the expression of hematopoietic progenitor markers cmyb and scl. We also demonstrated that ginger and 10-gingerol can promote the hematopoietic recovery from acute hemolytic anemia in zebrafish, by quantifying the number of circulating erythroid cells in the dorsal aorta using video microscopy. We found that ginger and 10-gingerol treatment during gastrulation results in an increase of bmp2b and bmp7a expression, and their downstream effectors, gata2 and eve1. At later stages ginger and 10-gingerol can induce bmp2b/7a, cmyb, scl and lmo2 expression in the caudal hematopoietic tissue area. We further confirmed that Bmp/Smad pathway mediates this hematopoiesis promoting effect of ginger by using the Bmp-activated Bmp type I receptor kinase inhibitors dorsomorphin, LND193189 and DMH1. CONCLUSIONS/SIGNIFICANCE: Our study provides a strong foundation to further evaluate the molecular mechanism of ginger and its bioactive components during hematopoiesis and to investigate their effects in adults. Our results will provide the basis for future research into the effect of ginger during mammalian hematopoiesis to develop novel erythropoiesis promoting agents.
Asunto(s)
Anemia/tratamiento farmacológico , Proteínas Morfogenéticas Óseas/metabolismo , Hematopoyesis/efectos de los fármacos , Extractos Vegetales/farmacología , Transducción de Señal/efectos de los fármacos , Zingiber officinale , Anemia/genética , Anemia/metabolismo , Animales , Animales Modificados Genéticamente , Catecoles/farmacología , Catecoles/uso terapéutico , Alcoholes Grasos/farmacología , Alcoholes Grasos/uso terapéutico , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Hematopoyesis/fisiología , Extractos Vegetales/uso terapéutico , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND/AIMS: Sodium thiosulfate (STS) has been shown to be an antioxidant and calcium solubilizer, but the possible role of STS in dysfunctional ventricles remains unknown. Here, we assessed the effects of STS in the failing heart. METHODS: Heart failure was created by an arteriovenous fistula (AVF). Mice were divided into 4 groups: sham, AVF, sham + STS, and AVF + STS. STS (3 mg/ml) was supplemented with drinking water for 6 weeks in the appropriate surgery groups after surgery. RESULTS: M-mode echocardiograms showed ventricular contractile dysfunction with reduced aortic blood flow in AVF mice, whereas STS treatment prevented the decline in cardiac function. Ventricular collagen, MMP-2 and -9, and TIMP-1 were robustly increased with a decreasing trend in adenylate cyclase VI expression; however, STS supplementation reversed these effects in AVF mice. Among 2 enzymes that produce endogenous hydrogen sulfide (H(2)S), cystathionine-gamma-lyase (CSE) expression was attenuated in AVF mice with no changes in cystathionine-beta-synthase (CBS) expression. In addition, reduced production of H(2)S in AVF ventricular tissue was normalized with STS supplementation. Moreover, cardiac tissues were more responsive to H(2)S when AVF mice were supplemented with STS compared to AVF alone. CONCLUSIONS: These results suggested that STS modulated cardiac dysfunction and the extracellular matrix, in part, by increasing ventricular H(2)S generation.
Asunto(s)
Cardiotónicos/farmacología , Insuficiencia Cardíaca/tratamiento farmacológico , Sulfuro de Hidrógeno/metabolismo , Tiosulfatos/farmacología , Adenilil Ciclasas/genética , Adenilil Ciclasas/metabolismo , Animales , Aorta/fisiopatología , Fístula Arteriovenosa , Enfermedad Crónica , Colágeno/efectos de los fármacos , Colágeno/metabolismo , Cistationina gamma-Liasa/efectos de los fármacos , Cistationina gamma-Liasa/metabolismo , Ecocardiografía , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Insuficiencia Cardíaca/fisiopatología , Masculino , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/efectos de los fármacos , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Contracción Miocárdica/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/efectos de los fármacos , Inhibidor Tisular de Metaloproteinasa-1/metabolismoRESUMEN
Elevated levels of homocysteine (Hcy) (known as hyperhomocysteinemia HHcy) are involved in dilated cardiomyopathy. Hcy chelates copper and impairs copper-dependent enzymes. Copper deficiency has been linked to cardiovascular disease. We tested the hypothesis that copper supplement regresses left ventricular hypertrophy (LVH), fibrosis and endothelial dysfunction in pressure overload DCM mice hearts. The mice were grouped as sham, sham + Cu, aortic constriction (AC), and AC + Cu. Aortic constriction was performed by transverse aortic constriction. The mice were treated with or without 20 mg/kg copper supplement in the diet for 12 weeks. The cardiac function was assessed by echocardiography and electrocardiography. The matrix remodeling was assessed by measuring matrix metalloproteinase (MMP), tissue inhibitor of metalloproteinases (TIMPs), and lysyl oxidase (LOX) by Western blot analyses. The results suggest that in AC mice, cardiac function was improved with copper supplement. TIMP-1 levels decreased in AC and were normalized in AC + Cu. Although MMP-9, TIMP-3, and LOX activity increased in AC and returned to baseline value in AC + Cu, copper supplement showed no significant effect on TIMP-4 activity after pressure overload. In conclusion, our data suggest that copper supplement helps improve cardiac function in a pressure overload dilated cardiomyopathic heart.
Asunto(s)
Cardiomiopatía Dilatada/tratamiento farmacológico , Cobre/administración & dosificación , Suplementos Dietéticos , Insuficiencia Cardíaca/tratamiento farmacológico , Hemodinámica/efectos de los fármacos , Homocisteína/metabolismo , Hipertrofia Ventricular Izquierda/tratamiento farmacológico , Miocardio/metabolismo , Animales , Aorta/cirugía , Presión Sanguínea , Western Blotting , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/fisiopatología , Constricción , Cobre/metabolismo , Modelos Animales de Enfermedad , Ecocardiografía , Electrocardiografía , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiopatología , Femenino , Fibrosis , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Homocisteína/sangre , Hipertrofia Ventricular Izquierda/metabolismo , Hipertrofia Ventricular Izquierda/fisiopatología , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocardio/enzimología , Miocardio/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Remodelación Ventricular/efectos de los fármacosRESUMEN
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.