RESUMO
Heart failure is one of the leading causes of death worldwide. RhoA, a small GTPase, governs actin dynamics in various tissue and cell types, including cardiomyocytes; however, its involvement in cardiac function has not been fully elucidated. Here, we generated cardiomyocyte-specific RhoA conditional knockout (cKO) mice, which demonstrated a significantly shorter lifespan with left ventricular dilation and severely impaired ejection fraction. We found that the cardiac tissues of the cKO mice exhibited structural disorganization with fibrosis and also exhibited enhanced senescence compared with control mice. In addition, we show that cardiomyocyte mitochondria were structurally abnormal in the aged cKO hearts. Clearance of damaged mitochondria by mitophagy was remarkably inhibited in both cKO cardiomyocytes and RhoA-knockdown HL-1 cultured cardiomyocytes. In RhoA-depleted cardiomyocytes, we reveal that the expression of Parkin, an E3 ubiquitin ligase that plays a crucial role in mitophagy, was reduced, and expression of N-Myc, a negative regulator of Parkin, was increased. We further reveal that the RhoA-Rho kinase axis induced N-Myc phosphorylation, which led to N-Myc degradation and Parkin upregulation. Re-expression of Parkin in RhoA-depleted cardiomyocytes restored mitophagy, reduced mitochondrial damage, attenuated cardiomyocyte senescence, and rescued cardiac function both in vitro and in vivo. Finally, we found that patients with idiopathic dilated cardiomyopathy without causal mutations for dilated cardiomyopathy showed reduced cardiac expression of RhoA and Parkin. These results suggest that RhoA promotes Parkin-mediated mitophagy as an indispensable mechanism contributing to cardioprotection in the aging heart.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Animais , Camundongos , Cardiomiopatia Dilatada/metabolismo , Insuficiência Cardíaca/metabolismo , Mitocôndrias/metabolismo , Mitofagia/genética , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.
Assuntos
Síndrome de Brugada/genética , Predisposição Genética para Doença , Proteínas de Membrana/genética , Mutação , Miócitos Cardíacos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Adulto , Animais , Síndrome de Brugada/patologia , Feminino , Humanos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Linhagem , Adulto JovemRESUMO
BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.
Assuntos
Fibrilação Atrial , Bloqueio Atrioventricular , Bradicardia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Doenças Genéticas Inatas , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/metabolismo , Bloqueio Atrioventricular/patologia , Bloqueio Atrioventricular/fisiopatologia , Benzopiranos/farmacologia , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/patologia , Bradicardia/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Doenças Genéticas Inatas/fisiopatologia , Humanos , Masculino , Xenopus laevis , Peixe-ZebraRESUMO
Vascular endothelial growth factors (VEGFs) include five molecules (VEGF-A, -B, -C, -D, and placental growth factor), and have various roles that crucially regulate cellular functions in many kinds of cells and tissues. Intracellular signal transduction induced by VEGFs has been extensively studied and is usually initiated by their binding to two classes of transmembrane receptors: receptor tyrosine kinase VEGF receptors (VEGF receptor-1, -2 and -3) and neuropilins (NRP1 and NRP2). In addition to many established results reported by other research groups, we have previously identified small G proteins, especially Ras homologue gene (Rho) and Ras-related protein (Rap), as important mediators of VEGF-A-stimulated signaling in cancer cells as well as endothelial cells. This review article describes the VEGF-A-induced signaling pathways underlying diverse cellular functions, including cell proliferation, migration, and angiogenesis, and the involvement of Rho, Rap, and their related molecules in these pathways.
Assuntos
Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Humanos , Modelos Biológicos , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: Pressure overload induces cardiac hypertrophy, which often ends in heart failure. Afadin is an adaptor protein that is ubiquitously expressed and, in the heart, it localizes at intercalated disks. The current study aimed to examine the afadin-mediated cardiac phenotype in mice exposed to different types of pressure overload: transverse aortic constriction (TAC) burden and angiotensin II (Ang II) stimulation.MethodsâandâResults:Conditional knockout mice with selective deletion of afadin (afadin cKO) in cardiomyocytes were generated. TAC-operated and Ang II-infused mice at 4 weeks had a similar degree of pressure overload and cardiac hypertrophy in the heart. In afadin cKO mice, TAC operation caused progressive left ventricular dysfunction and heart failure, while Ang II infusion did not deteriorate cardiac function. Furthermore, TAC operation produced more fibrosis and apoptosis in the heart than Ang II infusion, and the expression of growth differentiation factor 15, which can promote apoptosis, in the afadin cKO heart was higher in TAC-operated mice than Ang II-infused ones. CONCLUSIONS: In the 2 pressure overload models, myocardial afadin is involved in mechanical stress-induced, but not pharmacological Ang II-related, compensated cardiac hypertrophy.
Assuntos
Cardiomegalia/etiologia , Proteínas dos Microfilamentos/fisiologia , Pressão/efeitos adversos , Estresse Mecânico , Angiotensina II/farmacologia , Animais , Apoptose , Fibrose , Humanos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Miocárdio/químicaRESUMO
Our goals are to simultaneously determine the three-dimensional distribution patterns of KCNQ1 and KCNE1 in cardiac myocytes and to study the mechanism and functional implications for variations in KCNQ1/KCNE1 colocalization in myocytes. We monitored the distribution patterns of KCNQ1, KCNE1, and markers for subcellular compartments/organelles using immunofluorescence/confocal microscopy and confirmed the findings in ventricular myocytes by directly observing fluorescently tagged KCNQ1-GFP and KCNE1-dsRed expressed in these cells. We also monitored the effects of stress on KCNQ1-GFP and endoplasmic reticulum (ER) remodeling during live cell imaging. The data showed that 1) KCNE1 maintained a stable cell surface localization, whereas KCNQ1 exhibited variations in the cytosolic compartment (striations versus vesicles) and the degree of presence on the cell surface; 2) the degree of cell surface KCNQ1/KCNE1 colocalization was positively correlated with slow delayed rectifier (IKs) current density; 3) KCNQ1 and calnexin (an ER marker) shared a cytosolic compartment; and 4) in response to stress ([Ca(2+)]i elevation, oxidative overload, or AT1R stimulation), KCNQ1 exited the cytosolic compartment and trafficked to the cell periphery in vesicles. This was accompanied by partial ER fragmentation. We conclude that the cellular milieu regulates KCNQ1 distribution in cardiac myocytes and that stressful conditions can increase IKs by inducing KCNQ1 movement to the cell surface. This represents a hitherto unrecognized mechanism by which IKs fulfills its function as a repolarization reserve in ventricular myocytes.
Assuntos
Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Canal de Potássio KCNQ1/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Células COS , Calnexina/metabolismo , Membrana Celular/metabolismo , Chlorocebus aethiops , Citosol/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cobaias , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Canal de Potássio KCNQ1/genética , Estresse Oxidativo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Transporte Proteico , Ratos , Receptor Tipo 1 de Angiotensina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.
Assuntos
Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteólise , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cães , Feminino , Cobaias , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Humanos , Masculino , Modelos Animais , Dados de Sequência Molecular , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Ratos , Ratos Endogâmicos SHR , Canais de Potássio Shal , Regulador Transcricional ERGRESUMO
HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Antiarrítmicos/farmacologia , Western Blotting , Linhagem Celular , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/genética , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Nisoldipino/farmacologia , Técnicas de Patch-Clamp , Piperidinas/farmacologia , Potássio/metabolismo , Piridinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Long QT syndrome (LQTS) is an inherited disease involving mutations in the genes encoding a number of cardiac ion channels and a membrane adaptor protein. Among the genes that are responsible for LQTS, KCNE1 and KCNE2 are members of the KCNE family of genes, and function as ancillary subunits of Kv channels. The third KCNE gene, KCNE3, is expressed in cardiac myocytes and interacts with KCNQ1 to change the channel properties. However, KCNE3 has never been linked to LQTS. To investigate the association between KCNE3 and LQTS, we conducted a genetic screening of KCNE3 mutations and single nucleotide polymorphisms (SNPs) in 485 Japanese LQTS probands using DHPLC-WAVE system and direct sequencing. Consequently, we identified two KCNE3 missense mutations, located in the N- and C-terminal domains. The functional effects of these mutations were examined by heterologous expression systems using CHO cells stably expressing KCNQ1. One mutation, p.R99lambdaH was identified in a 76-year-old woman who suffered torsades de pointes (TdP) after administration of disopyramide. Another mutation, p.T4A was identified in a 16-year-old boy and 67-year-old woman. Although the boy carried another KCNH2 mutation, he was asymptomatic. On the other hand, the woman suffered from hypokalemia-induced TdP. In a series of electrophysiological analyses, the KCNQ1(Q1)+KCNE3(E3)-R99lambdaH channel significantly reduced outward current compared to Q1+E3-WT, though the current density of the Q1+E3-T4A channel displayed no statistical significance. This is the first report of KCNE3 mutations associated with LQTS. Screening for variants in the KCNE3 gene is of clinical importance for LQTS patients.
Assuntos
Síndrome do QT Longo/genética , Mutação de Sentido Incorreto , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Feminino , Humanos , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Síndrome do QT Longo/fisiopatologia , Masculino , Potenciais da Membrana , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Ligação Proteica , TransfecçãoRESUMO
A repolarizing conduction in the heart augmented by hyposmotic or mechanically induced membrane stretch is the slow component of delayed rectifier K(+) current (I (Ks)). I (Ks) upregulation is recognized as a factor promoting appearance of atrial fibrillation (AF) since gain-of-function mutations of the channel genes have been detected in congenital AF. Mechanical stretch activates angiotensin II type 1 (AT(1)) receptor in the absence of its physiological ligand angiotensin II. We investigated the functional role of AT(1) receptor in I (Ks) enhancement in hyposmotically challenged guinea pig atrial myocytes using the whole-cell patch-clamp method. In atrial myocytes exposed to hyposmotic solution with osmolality decreased to 70% of the physiological level, I (Ks) was enhanced by 84.1%, the duration of action potential at 90% repolarization (APD(90)) was decreased by 16.8%, and resting membrane potential was depolarized (+4.9 mV). The hyposmotic-induced effects on I (Ks) and APD(90) were significantly attenuated by specific AT(1) receptor antagonist candesartan (1 and 5 muM). Pretreatment of atrial myocytes with protein tyrosine kinase inhibitors tyrphostin A23 and A25 suppressed but the presence of tyrosine phosphatase inhibitor orthovanadate augmented hyposmotic stimulation of I (Ks). The above results implicate AT(1) receptor and tyrosine kinases in the hyposmotic modulation of atrial I (Ks) and suggest acute antiarrhythmic properties of AT(1) antagonists in the settings of stretch-related atrial tachyarrhythmias.
Assuntos
Fenômenos Eletrofisiológicos/fisiologia , Átrios do Coração/metabolismo , Potássio/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Angiotensina II/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Benzimidazóis/farmacologia , Compostos de Bifenilo , Forma Celular/efeitos dos fármacos , Forma Celular/fisiologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Cobaias , Átrios do Coração/citologia , Soluções Hipotônicas/farmacologia , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Concentração Osmolar , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Receptor Tipo 1 de Angiotensina/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tetrazóis/farmacologiaRESUMO
BACKGROUND: In the heart, slow delayed rectifier channels provide outward currents (IKs) for action potential (AP) repolarization in a region- and context-dependent manner. In diseased hearts, chronic elevation of angiotensin II (Ang II) may remodel IKs in a region-dependent manner, contributing to atrial and ventricular arrhythmias of different mechanisms. OBJECTIVE: The purpose of this study was to study whether/how chronic in vivo Ang II administration remodels IKs in atrial and ventricular myocytes. METHODS: We used the guinea pig (GP) model whose myocytes express robust IKs. GPs were implanted with minipumps containing Ang II or vehicle. Treatment continued for 4-6 weeks. We used patch clamp, immunofluorescence/confocal microscopy, and immunoblots to evaluate changes in IKs function and to explore the underlying mechanisms. RESULTS: We confirmed the pathologic state of the heart after chronic Ang II treatment. IKs density was increased in atrial myocytes but decreased in ventricular myocytes in Ang II- vs vehicle-treated animals. The former was correlated with an increase in KCNQ1/KCNE1 colocalization in myocyte periphery, whereas the latter was correlated with a decrease in KCNQ1 protein level. Interestingly, these changes in IKs were not translated into expected alterations in AP duration or plateau voltage, indicating that other currents were involved. In atrial myocytes from Ang II-treated animals, the L-type Ca channel current was increased, contributing to AP plateau elevation and AP duration prolongation. CONCLUSION: IKs is differentially modulated by chronic in vivo Ang II administration between atrial and ventricular myocytes. Other currents remodeled by Ang II treatment also contribute to changes in action potentials.
Assuntos
Angiotensina II/administração & dosagem , Átrios do Coração/fisiopatologia , Ventrículos do Coração/fisiopatologia , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Taquicardia Ventricular/tratamento farmacológico , Animais , Células Cultivadas , Modelos Animais de Doenças , Cobaias , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Técnicas de Patch-Clamp , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologia , Vasoconstritores/administração & dosagemRESUMO
Tumor metastasis is the most common cause of cancer death. Elucidation of the mechanism of tumor metastasis is therefore important in the development of novel, effective anti-cancer therapies to reduce cancer mortality. Interaction between cancer cells and surrounding stromal cells in the tumor microenvironment is a key factor in tumor metastasis. Using a co-culture assay system with human prostate cancer LNCaP cells and primary human prostate stromal cells, we identified epithelial membrane protein 1 (EMP1) as a gene with elevated expression in the cancer cells. The orthotopic injection of LNCaP cells overexpressing EMP1 (EMP1-LNCaP cells) into the prostate of nude mice induced lymph node and lung metastases, while that of control LNCaP cells did not. EMP1-LNCaP cells had higher cell motility and Rac1 activity than control LNCaP cells. These results were also observed in other lines of cancer cells. We newly identified copine-III as an intracellular binding partner of EMP1. Knockdown of copine-III attenuated the increased cell motility and Rac1 activity in EMP1-LNCaP cells. Reduced cell motility and Rac1 activity following knockdown of copine-III in EMP1-LNCaP cells were recovered by re-expression of wild-type copine-III, but not of a copine-III mutant incapable of interacting with EMP1, suggesting the importance of the EMP1-copine-III interaction. Phosphorylated and activated Src and a Rac guanine nucleotide exchange factor Vav2 were found to be involved in the EMP1-induced enhancement of cell motility and Rac1 activation. Moreover, EMP1 was highly expressed in prostate cancer samples obtained from patients with higher Gleason score. These results demonstrate that upregulation of EMP1 significantly increases cancer cell migration that leads to tumor metastasis, suggesting that EMP1 may play an essential role as a positive regulator of tumor metastasis.
Assuntos
Movimento Celular , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Transdução de Sinais , Células Estromais/patologia , Regulação para CimaRESUMO
BACKGROUND: Angiotensin II (Ang II) has diverse actions on cardiac electrical activity. Little information is available, however, regarding immediate electrophysiological effects of Ang II on cardiac repolarization. METHODS AND RESULTS: The present study investigated the immediate effects of Ang II on the slow component of delayed rectifier K+ current (IKs) and action potentials in guinea pig atrial myocytes using the whole-cell patch-clamp technique. Bath application of Ang II increased the amplitude of IKs (EC50, 6.16 nmol/L) concentration dependently. The stable analogue Sar1-Ang II was also effective at increasing IKs. The voltage dependence of IKs activation and the kinetics of deactivation were not significantly affected by these agonists. The enhancement of IKs was blocked by the Ang II type 1 (AT1) receptor antagonist valsartan (1 micromol/L) and was markedly attenuated by inclusion of GDPbetaS (2 mmol/L) in the pipette, indicating an involvement of G protein-coupled AT(1) receptor. The stimulatory effect was also significantly reduced by the phospholipase C inhibitor compound 48/80 (100 micromol/L) and the protein kinase C inhibitors bisindolylmaleimide I (200 nmol/L) and H-7 (10 micromol/L), suggesting that AT1 receptor acts through phospholipase C-protein kinase C signaling cascade to potentiate I(Ks). As expected from its stimulatory action on IKs, Sar1-Ang II markedly shortened the action potential duration, which could be reversed by valsartan. CONCLUSIONS: The potentiation of IKs via AT1 stimulation in atrial myocytes, accompanied by a shortening of the action potential duration, suggests a potential mechanism by which elevated levels of Ang II may promote atrial fibrillation in heart failure and warrants further investigation.
Assuntos
Angiotensina II/farmacologia , Canais de Potássio de Retificação Tardia/metabolismo , Átrios do Coração/citologia , Células Musculares/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Potenciais de Ação , Animais , Fibrilação Atrial/etiologia , Eletrofisiologia , Cobaias , Cinética , Técnicas de Patch-Clamp , Fosfolipases Tipo CRESUMO
BACKGROUND: Long QT syndromes (LQTS) are inherited diseases involving mutations to genes encoding a number of cardiac ion channels and a membrane adaptor protein. The MinK protein is a cardiac K-channel accessory subunit encoded by the KCNE1 gene, mutations of which are associated with the LQT5 form of LQTS. OBJECTIVE: The purpose of this study was to search for the KCNE1 mutations and clarify the function of those mutations. METHODS: We conducted a genetic screen of KCNE1 mutations in 151 Japanese LQTS patients using the denaturing high-performance liquid chromatography-WAVE system and direct sequencing. In two LQTS patients, we identified two KCNE1 missense mutations, located in the MinK N- and C-terminal domains. The functional effects of these mutations were examined by heterologous coexpression with KCNQ1 and KCNH2. RESULTS: One mutation, which was identified in a 67-year-old woman, A8V, was novel. Her electrocardiogram (ECG) revealed marked bradycardia and QT interval prolongation. Another mutation, R98W, was identified in a 19-year-old woman. She experienced syncope followed by palpitation in exercise. At rest, her ECG showed bradycardia with mild QT prolongation, which became more prominent during exercise. In electrophysiological analyses, R98W produced reduced I(Ks) currents with a positive shift in the half activation voltages. In addition, when the A8V mutation was coexpressed with KCNH2, this reduced current magnitude, which is suggestive of a modifier effect by the A8V KCNE1 mutation on I(Kr). CONCLUSION: KCNE1 mutations may be associated with mild LQTS phenotypes, and KCNE1 gene screening is of clinical importance for asymptomatic and mild LQTS patients.
Assuntos
Povo Asiático/genética , Síndrome do QT Longo/genética , Mutação de Sentido Incorreto , Fenótipo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Adulto , Idoso , Cromatografia Líquida de Alta Pressão , Canal de Potássio ERG1 , Eletrocardiografia , Técnicas Eletrofisiológicas Cardíacas , Canais de Potássio Éter-A-Go-Go/genética , Feminino , Expressão Gênica/genética , Testes Genéticos , Genótipo , Humanos , Japão , Canal de Potássio KCNQ1/genética , Polimorfismo de Nucleotídeo Único , Projetos de PesquisaRESUMO
Adhesive intercellular connections at cardiomyocyte intercalated disks (IDs) support contractile force and maintain structural integrity of the heart muscle. Disturbances of the proteins at IDs deteriorate cardiac function and morphology. An adaptor protein afadin, one of the components of adherens junctions, is expressed ubiquitously including IDs. At present, the precise role of afadin in cardiac physiology or disease is unknown. To explore this, we generated conditional knockout (cKO) mice with cardiomyocyte-targeted deletion of afadin. Afadin cKO mice were born according to the expected Mendelian ratio and have no detectable changes in cardiac phenotype. On the other hand, chronic pressure overload induced by transverse aortic constriction (TAC) caused systolic dysfunction, enhanced fibrogenesis and apoptosis in afadin cKO mice. Afadin deletion increased macrophage infiltration and monocyte chemoattractant protein-1 expression, and suppressed transforming growth factor (TGF) ß receptor signaling early after TAC procedure. Afadin also associated with TGFß receptor I at IDs. Pharmacological antagonist of TGFß receptor I (SB431542) augmented mononuclear infiltration and fibrosis in the hearts of TAC-operated control mice. In conclusion, afadin is a critical molecule for cardiac protection against chronic pressure overload. The beneficial effects are likely to be a result from modulation of TGFß receptor signaling pathways by afadin.
Assuntos
Aorta/fisiologia , Insuficiência Cardíaca Sistólica/genética , Junções Intercelulares/metabolismo , Macrófagos/imunologia , Proteínas dos Microfilamentos/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Animais , Apoptose/genética , Movimento Celular , Quimiocina CCL2/metabolismo , Constrição Patológica , Fibrose , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Contração Miocárdica , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVES: We examined the cellular and ionic mechanism for QT prolongation and subsequent Torsade de Pointes (TdP) and the effect of verapamil under conditions mimicking KCNQ1 (I(Ks) gene) defect linked to acquired long QT syndrome (LQTS). BACKGROUND: Agents with an I(Kr)-blocking effect often induce marked QT prolongation in patients with acquired LQTS. Previous reports demonstrated a relationship between subclinical mutations in cardiac K+ channel genes and a risk of drug-induced TdP. METHODS: Transmembrane action potentials from epicardial (EPI), midmyocardial (M), and endocardial (ENDO) cells were simultaneously recorded, together with a transmural electrocardiogram, at a basic cycle length of 2,000 ms in arterially perfused feline left ventricular preparations. RESULTS: The I(Kr) block (E-4031: 1 micromol/l) under control conditions (n = 5) prolonged the QT interval but neither increased transmural dispersion of repolarization (TDR) nor induced arrhythmias. However, the I(Kr) blocker under conditions with I(Ks) suppression by chromanol 293B 10 micromol/l mimicking the KCNQ1 defect (n = 10) preferentially prolonged action potential duration (APD) in EPI rather than M or ENDO, thereby dramatically increasing the QT interval and TDR. Spontaneous or epinephrine-induced early afterdepolarizations (EADs) were observed in EPI, and subsequent TdP occurred only under both I(Ks) and I(Kr) suppression. Verapamil (0.1 to 5.0 micromol/l) dose-dependently abbreviated APD in EPI more than in M and ENDO, thereby significantly decreasing the QT interval, TDR, and suppressing EADs and TdP. CONCLUSIONS: Subclinical I(Ks) dysfunction could be a risk of drug-induced TdP. Verapamil is effective in decreasing the QT interval and TDR and in suppressing EADs, thus preventing TdP in the model of acquired LQTS.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Síndrome do QT Longo/fisiopatologia , Canais de Potássio/fisiologia , Torsades de Pointes/fisiopatologia , Verapamil/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Antiarrítmicos/farmacologia , Gatos , Cromanos/farmacologia , Síndrome do QT Longo/induzido quimicamente , Piperidinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Piridinas/farmacologia , Sulfonamidas/farmacologia , Técnicas de Cultura de Tecidos , Torsades de Pointes/etiologia , Torsades de Pointes/prevenção & controleRESUMO
Dipeptidyl peptidase III (DPP III) cleaves dipeptide residues from the N terminus of polypeptides ranging from 3 to 10 amino acids in length and is implicated in pathophysiological processes through the breakdown of certain oligopeptides or their fragments. In this study, we newly identified the biochemical properties of DPP III for angiotensin II (Ang II), which consists of 8 amino acids. DPP III quickly and effectively digested Ang II with Km = 3.7×10(-6) mol/L. In the in vivo experiments, DPP III remarkably reduced blood pressure in Ang II-infused hypertensive mice without alteration of heart rate. DPP III did not affect hemodynamics in noradrenalin-induced hypertensive mice or normotensive mice, suggesting specificity for Ang II. When DPP III was intravenously injected every other day for 4 weeks after Ang II osmotic minipump implantation in mice, Ang II-induced cardiac fibrosis and hypertrophy were significantly attenuated. This DPP III effect was at least similar to that caused by an angiotensin receptor blocker candesartan. Furthermore, administration of DPP III dramatically reduced the increase in urine albumin excretion and kidney injury and inflammation markers caused by Ang II infusion. Both DPP III and candesartan administration showed slight additive inhibition in the albumin excretion. These results reveal a novel potential use of DPP III in the treatment of hypertension and its protective effects on hypertension-sensitive organs, such as the heart and kidneys.
Assuntos
Angiotensina II/farmacologia , Dipeptidil Peptidases e Tripeptidil Peptidases/farmacologia , Hemodinâmica/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Análise de Variância , Animais , Pressão Sanguínea/efeitos dos fármacos , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Modelos Animais de Doenças , Taxa de Filtração Glomerular/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/enzimologia , Camundongos , Camundongos Endogâmicos ICR , Distribuição Aleatória , Papel (figurativo) , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
Vasculature is present in all tissues and therefore is indispensable for development, biology, and pathology of multicellular organisms. Endothelial cells guarantee proper function of the vessels and are the original component in angiogenesis. Morphogenesis of the vascular system utilizes processes like cell adhesion, motility, proliferation, and survival that are closely related to the dynamics of actin filaments and actin-tethered adhesion complexes. Here we review involvement of actin cytoskeleton-associated junctional molecules of endothelial cells in angiogenesis and lymphangiogenesis. Particularly, we focus on F-actin binding protein afadin, an adaptor protein involved in broad range of signaling mechanisms. Afadin mediates the pathways of vascular endothelial growth factor- (VEGF-) and sphingosine 1-phosphate-triggered angiogenesis and is essential for embryonic development of lymph vessels in mice. We propose that targeting actin-tethered junctional molecules, including afadin, may present a new approach to angiogenic therapy that in combination with today used medications like VEGF inhibitors will benefit against development of pathological angiogenesis.
Assuntos
Actinas/fisiologia , Regulação da Expressão Gênica , Linfangiogênese , Proteínas dos Microfilamentos/fisiologia , Neovascularização Patológica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Cateninas/metabolismo , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Humanos , Integrinas/metabolismo , Vasos Linfáticos/patologia , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Knockout , Neovascularização Fisiológica , Esfingosina/análogos & derivados , Esfingosina/metabolismo , beta Catenina/metabolismo , delta CateninaRESUMO
Zn-α2-glycoprotein (ZAG) (molecular weight=41 kDa) is one component in the α2 fraction of human plasma, and is reported to be associated with several diseases, such as cancers and metabolic syndromes. ZAG is also considered to be an important modulator of lipid metabolism. However, little is known about the correlation of serum ZAG levels with indicators of metabolic syndrome. Serum ZAG concentrations analyzed by enzyme-linked immunoassay were positively correlated with systolic and diastolic blood pressure in 326 subjects (236 males and 90 females) aged 17-79 years who had an annual health examination. By luciferase reporter and electrophoretic mobility shift assays, the core promoter region to regulate the ZAG gene expression was found to exist between -110 and -101. The transcription factor Sp1 interacted with this region, and Sp1 knockdown experiments showed that Sp1 critically regulated ZAG expression. Furthermore, ZAG increased the active form of RhoA, which was determined by pull-down assay. Increased serum ZAG concentrations induced, at least partly, by Sp1 may cause an increase in vascular tone through the activation of RhoA and contribute to elevated blood pressure.
Assuntos
Pressão Sanguínea/fisiologia , Proteínas de Plasma Seminal/sangue , Adolescente , Adulto , Idoso , Animais , Pressão Sanguínea/genética , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Síndrome Metabólica/sangue , Pessoa de Meia-Idade , Tono Muscular/genética , Regiões Promotoras Genéticas/genética , Ratos , Proteínas de Plasma Seminal/biossíntese , Proteínas de Plasma Seminal/genética , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/fisiologia , Adulto Jovem , Glicoproteína Zn-alfa-2 , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Afadin is an intracellular binding partner of nectins, cell-cell adhesion molecules, and plays important roles in the formation of cell-cell junctions. Afadin-knockout mice show early embryonic lethality, therefore little is known about the function of afadin during organ development. In this study, we generated mice lacking afadin expression in endothelial cells, and found that the majority of these mice were embryonically lethal as a result of severe subcutaneous edema. Defects in the lymphatic vessels of the skin were observed, although the morphology in the blood vessels was almost normal. Severe disruption of VE-cadherin-mediated cell-cell junctions occurred only in lymphatic endothelial cells, but not in blood endothelial cells. Knockout of afadin did not affect the differentiation and proliferation of lymphatic endothelial cells. Using in vitro assays with blood and lymphatic microvascular endothelial cells (BMVECs and LMVECs, respectively), knockdown of afadin caused elongated cell shapes and disruption of cell-cell junctions among LMVECs, but not BMVECs. In afadin-knockdown LMVECs, enhanced F-actin bundles at the cell periphery and reduced VE-cadherin immunostaining were found, and activation of RhoA was strongly increased compared with that in afadin-knockdown BMVECs. Conversely, inhibition of RhoA activation in afadin-knockdown LMVECs restored the cell morphology. These results indicate that afadin has different effects on blood and lymphatic endothelial cells by controlling the levels of RhoA activation, which may critically regulate the lymphangiogenesis of mouse embryos.