RESUMO
Long QT syndrome (LQTS) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. We functionally analyzed the KCNH2 (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the transcription factor Tbx20) variants found by next-generation sequencing in two siblings with LQTS in a Spanish family of African ancestry. Affected relatives harbor a heterozygous mutation in KCNH2 that encodes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to generate any current when transfected into Chinese hamster ovary (CHO) cells but, surprisingly, exerted "chaperone-like" effects over native hERG channels in both CHO cells and mouse atrial-derived HL-1 cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistinguishable from that generated by WT channels alone. Some affected relatives also harbor the p.R311C mutation in Tbx20. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 enhanced human KCNH2 gene expression and hERG currents (IhERG) and shortened action-potential duration (APD). However, Tbx20 did not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not increase KCNH2 expression in hiPSC-CMs, which led to decreased IhERG and increased APD. Our results suggest that Tbx20 controls the expression of hERG channels responsible for the rapid component of the delayed rectifier current. On the contrary, p.R311C Tbx20 specifically disables the Tbx20 protranscriptional activity over KCNH2 Therefore, TBX20 can be considered a KCNH2-modifying gene.
Assuntos
Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Potenciais de Ação/genética , Animais , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Células CHO , Linhagem Celular , Cricetulus , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Masculino , Camundongos , Mutação/genética , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Both increase and decrease of cardiac inward rectifier current (I(K1)) are associated with severe cardiac arrhythmias. Flecainide, a widely used antiarrhythmic drug, exhibits ventricular proarrhythmic effects while effectively controlling ventricular arrhythmias associated with mutations in the gene encoding Kir2.1 channels that decrease I(K1) (Andersen syndrome). Here we characterize the electrophysiological and molecular basis of the flecainide-induced increase of the current generated by Kir2.1 channels (I(Kir2.1)) and I(K1) recorded in ventricular myocytes. Flecainide increases outward I(Kir2.1) generated by homotetrameric Kir2.1 channels by decreasing their affinity for intracellular polyamines, which reduces the inward rectification of the current. Flecainide interacts with the HI loop of the cytoplasmic domain of the channel, Cys311 being critical for the effect. This explains why flecainide does not increase I(Kir2.2) and I(Kir2.3), because Kir2.2 and Kir2.3 channels do not exhibit a Cys residue at the equivalent position. We further show that incubation with flecainide increases expression of functional Kir2.1 channels in the membrane, an effect also determined by Cys311. Indeed, flecainide pharmacologically rescues R67W, but not R218W, channel mutations found in Andersen syndrome patients. Moreover, our findings provide noteworthy clues about the structural determinants of the C terminus cytoplasmic domain of Kir2.1 channels involved in the control of gating and rectification.
Assuntos
Cisteína/metabolismo , Flecainida/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Antiarrítmicos/metabolismo , Antiarrítmicos/farmacologia , Ligação Competitiva , Células Cultivadas , Cisteína/genética , Relação Dose-Resposta a Droga , Flecainida/metabolismo , Cobaias , Humanos , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Poliaminas/metabolismo , Poliaminas/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/química , Canais de Potássio Corretores do Fluxo de Internalização/genética , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
RATIONALE: The cardiac inwardly rectifying K(+) current (I(K1)) plays a critical role in modulating excitability by setting the resting membrane potential and shaping phase 3 of the cardiac action potential. OBJECTIVE: This study aims to analyze the effects of nitric oxide (NO) on human atrial I(K1) and on Kir2.1 channels, the major isoform of inwardly rectifying channels present in the human heart. METHODS AND RESULTS: Currents were recorded in enzymatically isolated myocytes and in transiently transfected CHO cells, respectively. NO at myocardial physiological concentrations (25 to 500 nmol/L) increased inward and outward I(K1) and I(Kir2.1). These effects were accompanied by hyperpolarization of the resting membrane potential and a shortening of the duration of phase 3 of the human atrial action potential. The I(Kir2.1) increase was attributable to an increase in the open probability of the channel. Site-directed mutagenesis analysis demonstrated that NO effects were mediated by the selective S-nitrosylation of Kir2.1 Cys76 residue. Single ion monitoring experiments performed by liquid chromatography/tandem mass spectrometry suggested that the primary sequence that surrounds Cys76 determines its selective S-nitrosylation. Chronic atrial fibrillation, which produces a decrease in NO bioavailability, decreased the S-nitrosylation of Kir2.1 channels in human atrial samples as demonstrated by a biotin-switch assay, followed by Western blot. CONCLUSIONS: The results demonstrated that, under physiological conditions, NO regulates human cardiac I(K1) through a redox-related process.
Assuntos
Fatores Relaxantes Dependentes do Endotélio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Óxido Nítrico/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Cisteína/genética , Cisteína/metabolismo , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Feminino , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Masculino , Potenciais da Membrana/fisiologia , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/citologia , Óxido Nítrico/metabolismo , Oxirredução/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/genéticaRESUMO
Endocannabinoids are amides and esters of long chain fatty acids that can modulate ion channels through both receptor-dependent and receptor-independent effects. Nowadays, their effects on cardiac K(+) channels are unknown even when they can be synthesized within the heart. We have analyzed the direct effects of endocannabinoids, such as anandamide (AEA), 2-arachidonoylglycerol (2-AG), the endogenous lipid lysophosphatidylinositol, and cannabinoid analogues such as palmitoylethanolamide (PEA), and oleoylethanolamide, as well as the fatty acids from which they are endogenously synthesized, on human cardiac Kv4.3 channels, which generate the transient outward K(+) current (I(to1)). Currents were recorded in Chinese hamster ovary cells, which do not express cannabinoid receptors, by using the whole-cell patch-clamp. All these compounds inhibited I(Kv4.3) in a concentration-dependent manner, AEA and 2-AG being the most potent (IC(50) approximately 0.3-0.4 microM), while PEA was the least potent. The potency of block increased as the complexity and the number of C atoms in the fatty acyl chain increased. The effects were not mediated by modifications in the lipid order and microviscosity of the membrane and were independent of the presence of MiRP2 or DPP6 subunits in the channel complex. Indeed, effects produced by AEA were reproduced in human atrial I(to1) recorded in isolated myocytes. Moreover, AEA effects were exclusively apparent when it was applied to the external surface of the cell membrane. These results indicate that at low micromolar concentrations the endocannabinoids AEA and 2-AG directly block human cardiac Kv4.3 channels, which represent a novel molecular target for these compounds.
Assuntos
Moduladores de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Endocanabinoides , Canais de Potássio Shal/antagonistas & inibidores , Canais de Potássio Shal/metabolismo , Amidas , Animais , Ácido Araquidônico/farmacologia , Ácidos Araquidônicos/farmacologia , Células CHO , Cricetinae , Cricetulus , Etanolaminas , Ácidos Graxos/farmacologia , Glicerídeos/farmacologia , Coração/efeitos dos fármacos , Humanos , Ácidos Oleicos/farmacologia , Ácidos Palmíticos/farmacologia , Alcamidas Poli-Insaturadas/farmacologiaRESUMO
AIMS: Atrial fibrillation (AF) produces rapid changes in the electrical properties of the atria (electrical remodelling) that promote its own recurrence. In chronic AF (CAF) patients, up-regulation of the slow delayed rectifier K(+) current (IKs) and down-regulation of the voltage-gated Ca(2+) current (ICa,L) are hallmarks of electrical remodelling and critically contribute to the abbreviation of action potential duration and atrial refractory period. Recent evidences suggested that Pitx2c, a bicoid-related homeodomain transcription factor involved in directing cardiac asymmetric morphogenesis, could play a role in atrial remodelling. However, its effects on IKs and ICa,L are unknown. METHODS AND RESULTS: Real-time quantitative polymerase chain reaction analysis showed that Pitx2c mRNA expression was significantly higher in human atrial myocytes from CAF patients than those from sinus rhythm patients. The expression of Pitx2c was positively and negatively correlated with IKs and ICa,L densities, respectively. Expression of Pitx2c in HL-1 cells increased IKs density and reduced ICa,L density. Luciferase assays demonstrated that Pitx2c increased transcriptional activity of KCNQ1 and KCNE1 genes. Conversely, its effects on ICa,L could be mediated by the atrial natriuretic peptide. CONCLUSION: Our results demonstrated for the first time that CAF increases Pitx2c expression in isolated human atrial myocytes and suggested that this transcription factor could contribute to the CAF-induced IKs increase and ICa,L reduction observed in humans.
Assuntos
Fibrilação Atrial/metabolismo , Cálcio/metabolismo , Átrios do Coração/metabolismo , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/metabolismo , Potássio/metabolismo , Fatores de Transcrição/genética , Potenciais de Ação/efeitos dos fármacos , Animais , Fibrilação Atrial/fisiopatologia , Regulação para Baixo , Humanos , Camundongos , Técnicas de Patch-Clamp/métodos , Regulação para Cima , Proteína Homeobox PITX2RESUMO
AIMS: Cardiac excitability and refractoriness are largely determined by the function and number of inward rectifier K⺠channels (Kir2.1-2.3), which are differentially expressed in the atria and ventricles, and Nav1.5 channels. We have focused on how Nav1.5 and Kir2.x function within a macromolecular complex by elucidating the molecular determinants that govern Nav1.5/Kir2.x reciprocal modulation. METHODS AND RESULTS: The results demonstrate that there is an unexpected 'internal' PDZ-like binding domain located at the N-terminus of the Nav1.5 channel that mediates its binding to α1-syntrophin. Nav1.5 N-terminal domain, by itself (the 132 aa peptide) (Nter), exerts a 'chaperone-like' effect that increases sodium (I(Na)) and inward rectifier potassium (I(K1)) currents by enhancing the expression of Nav1.5, Kir2.1, and Kir2.2 channels as demonstrated in Chinese hamster ovary (CHO) cells and in rat cardiomyocytes. Site-directed mutagenesis analysis demonstrates that the Nter chaperone-like effect is determined by Serine 20. Nav1.5-Kir2.x reciprocal positive interactions depend on a specific C-terminal PDZ-binding domain sequence (SEI), which is present in Kir2.1 and Kir2.2 channels but not in Kir2.3. Therefore, in human atrial myocytes, the presence of Kir2.3 isoforms precludes reciprocal I(K1)-INa density modulation. Moreover, results in rat and human atrial myocytes demonstrate that binding to α1-syntrophin is necessary for the Nav1.5-Kir2.x-positive reciprocal modulation. CONCLUSIONS: The results demonstrate the critical role of the N-terminal domain of Nav1.5 channels in Nav1.5-Kir2.x-reciprocal interactions and suggest that the molecular mechanisms controlling atrial and ventricular cellular excitability may be different.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Cricetinae , Ventrículos do Coração/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Miócitos Cardíacos/metabolismo , RatosRESUMO
BACKGROUND: The human cardiac action potential in atrial and ventricular cells is initiated by a fast-activating, fast-inactivating sodium current generated by the SCN5A/Nav1.5 channel in association with its ß1/SCN1B subunit. The role of Nav1.5 in the etiology of many cardiac diseases strongly suggests that proper regulation of cell biology and function of the channel is critical for normal cardiac function. Hence, numerous recent studies have focused on the regulatory mechanisms of Nav1.5 biosynthetic and degradation processes as well as its subcellular localization. OBJECTIVE: The purpose of this study was to investigate the role of microRNAs in the Scn5a/Nav1.5 posttranscriptional regulation. METHODS: Quantitative polymerase chain reaction, immunohistochemical and electrophysiological measurements of distinct microRNA gain-of-function experiments in cardiomyocytes for the assessment of Scn5a expression. RESULTS: Functional studies of HL-1 cardiomyocytes and luciferase assays in fibroblasts demonstrate that Scn5a is directly (miR-98, miR-106, miR-200, and miR-219) and indirectly (miR-125 and miR-153) regulated by multiple microRNAs displaying distinct time-dependent profiles. Cotransfection experiments demonstrated that miR-219 and miR-200 have independent opposite effects on Scn5a expression modulation. Of all the microRNAs studied, only miR-219 increases Scn5a expression levels, leading to altered contraction rhythm of HL-1 cardiomyocytes. Electrophysiological analyses in HL-1 cells revealed that miR-219 increases the sodium current. In vivo administration of miR-219 does not alter normal cardiac rhythm, but abolishes some of the effects of flecainide intoxication in mice, particularly QRS prolongation. CONCLUSION: This study demonstrates the involvement of multiple microRNAs in the regulation of Scn5a. Particularly, miR-219 increases Scn5a/Nav1.5 transcript and protein expression. Our data suggest that microRNAs, such as miR-219, constitute a promising therapeutical tool to treat sodium cardiac arrhythmias.
Assuntos
Flecainida/intoxicação , MicroRNAs/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/análise , Animais , Células Cultivadas , Eletrocardiografia , Eletrofisiologia , Imuno-Histoquímica , Camundongos , Miócitos Cardíacos , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA/fisiologia , TransfecçãoRESUMO
AIMS: We hypothesize that some drugs, besides flecainide, increase the inward rectifier current (IK1) generated by Kir2.1 homotetramers (IKir2.1) and thus, exhibit pro- and/or antiarrhythmic effects particularly at the ventricular level. To test this hypothesis, we analysed the effects of propafenone, atenolol, dronedarone, and timolol on Kir2.x channels. METHODS AND RESULTS: Currents were recorded with the patch-clamp technique using whole-cell, inside-out, and cell-attached configurations. Propafenone (0.1 nM-1 µM) did not modify either IK1 recorded in human right atrial myocytes or the current generated by homo- or heterotetramers of Kir2.2 and 2.3 channels recorded in transiently transfected Chinese hamster ovary cells. On the other hand, propafenone increased IKir2.1 (EC50 = 12.0 ± 3.0 nM) as a consequence of its interaction with Cys311, an effect which decreased inward rectification of the current. Propafenone significantly increased mean open time and opening frequency at all the voltages tested, resulting in a significant increase of the mean open probability of the channel. Timolol, which interacted with Cys311, was also able to increase IKir2.1. On the contrary, neither atenolol nor dronedarone modified IKir2.1. Molecular modelling of the Kir2.1-drugs interaction allowed identification of the pharmacophore of drugs that increase IKir2.1. CONCLUSIONS: Kir2.1 channels exhibit a binding site determined by Cys311 that is responsible for drug-induced IKir2.1 increase. Drug binding decreases channel affinity for polyamines and current rectification, and can be a mechanism of drug-induced pro- and antiarrhythmic effects not considered until now.
Assuntos
Antiarrítmicos/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/agonistas , Propafenona/farmacologia , Animais , Antiarrítmicos/química , Antiarrítmicos/metabolismo , Antiarrítmicos/toxicidade , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Sítios de Ligação , Células CHO , Cricetulus , Cisteína , Relação Dose-Resposta a Droga , Cobaias , Humanos , Potenciais da Membrana , Moduladores de Transporte de Membrana/química , Moduladores de Transporte de Membrana/metabolismo , Moduladores de Transporte de Membrana/toxicidade , Simulação de Acoplamento Molecular , Estrutura Molecular , Miócitos Cardíacos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Propafenona/química , Propafenona/metabolismo , Propafenona/toxicidade , Ligação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Fatores de Tempo , TransfecçãoRESUMO
BACKGROUND: Atrial fibrillation is characterized by progressive atrial structural and electrical changes (atrial remodeling) that favor arrhythmia recurrence and maintenance. Reduction of L-type Ca(2+) current (I(Ca,L)) density is a hallmark of the electrical remodeling. Alterations in atrial microRNAs could contribute to the protein changes underlying atrial fibrillation-induced atrial electrical remodeling. This study was undertaken to compare miR-21 levels in isolated myocytes from atrial appendages obtained from patients in sinus rhythm and with chronic atrial fibrillation (CAF) and to determine whether L-type Ca(2+) channel subunits are targets for miR-21. METHODS AND RESULTS: Quantitative polymerase chain reaction analysis showed that miR-21 was expressed in human atrial myocytes from patients in sinus rhythm and that its expression was significantly greater in CAF myocytes. There was an inverse correlation between miR-21 and the mRNA of the α1c subunit of the calcium channel (CACNA1C) expression and I(Ca,L) density. Computational analyses predicted that CACNA1C and the mRNA of the ß2 subunit of the calcium channel (CACNB2) could be potential targets for miR-21. Luciferase reporter assays demonstrated that miR-21 produced a concentration-dependent decrease in the luciferase activity in Chinese Hamster Ovary cells transfected with CACNA1C and CACNB2 3' untranslated region regions. miR-21 transfection in HL-1 cells produced changes in I(Ca,L) properties qualitatively similar to those produced by CAF (ie, a marked reduction of I(Ca,L) density and shift of the inactivation curves to more depolarized potentials). CONCLUSIONS: Our results demonstrated that CAF increases miR-21 expression in enzymatically isolated human atrial myocytes. Moreover, it decreases I(Ca,L) density by downregulating Ca(2+) channel subunits expression. These results suggested that this microRNA could participate in the CAF-induced I(Ca,L) downregulation and in the action potential duration shortening that maintains the arrhythmia.
Assuntos
Apêndice Atrial/metabolismo , Fibrilação Atrial/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Regiões 3' não Traduzidas , Potenciais de Ação , Idoso , Apêndice Atrial/fisiopatologia , Fibrilação Atrial/genética , Fibrilação Atrial/fisiopatologia , Sítios de Ligação , Canais de Cálcio Tipo L/genética , Doença Crônica , Regulação para Baixo , Feminino , Genes Reporter , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Fatores de Tempo , Transfecção , Regulação para CimaRESUMO
AIMS: ß-adrenergic stimulation has profound influence in the genesis and maintenance of atrial fibrillation (AF). However, the effects of ß-Adrenoceptor stimulation on repolarizing currents and action potential (AP) characteristics in human atrial myocytes from left (LAA) and right atrial appendages (RAA) obtained from sinus rhythm (SR) and chronic atrial fibrillation (CAF) patients have not been compared yet. METHODS AND RESULTS: Currents and APs were recorded using whole-cell patch-clamp in RAA and LAA myocytes from SR and CAF patients. Isoproterenol concentration-dependently decreased the Ca(2+)-independent 4-aminopyridine-sensitive component of the transient outward current (I(to1)) and the inward rectifying current (I(K1)). CAF significantly enhanced this inhibition, this effect being more marked in the left than in the right atria. CAF dramatically enhanced ß-Adrenoceptor-mediated increase in the slow component of the delayed rectifier current (I(Ks)), whose density was already markedly increased by CAF. Conversely, the ultrarapid component of the delayed rectifier current (I(Kur)) of both SR and CAF myocytes was insensitive to low isoproterenol concentrations. As a consequence, stimulation of ß1-Adrenoceptors in SR myocytes lengthened, whereas in CAF myocytes shortened, the AP duration. Quantitative PCR revealed that CAF up-regulated ß1-Adrenoceptor expression, preferentially in the left atria. CONCLUSION: The present results demonstrate that CAF increases the effects of ß1-Adrenoceptor stimulation on repolarizing currents by means of a chamber-specific up-regulation of the receptors. This, together with the ion channel derangements produced by CAF, could contribute to the long-term stabilization of the arrhythmia by shortening the AP duration.
Assuntos
Potenciais de Ação , Fibrilação Atrial/fisiopatologia , Receptores Adrenérgicos beta 1/fisiologia , Potenciais de Ação/efeitos dos fármacos , Idoso , Doença Crônica , Feminino , Átrios do Coração/fisiopatologia , Humanos , Isoproterenol/farmacologia , Masculino , Modelos Biológicos , Fatores de TempoRESUMO
INTRODUCTION: We functionally analyzed a frameshift mutation in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) found in a proband with repeated episodes of ventricular fibrillation who presented bradycardia and paroxysmal atrial fibrillation. Seven relatives also carry the mutation and showed a Brugada syndrome with an incomplete and variable expression. The mutation (p.D1816VfsX7) resulted in a severe truncation (201 residues) of the Nav1.5 C-terminus. METHODS AND RESULTS: Wild-type (WT) and mutated Nav1.5 channels together with hNavß1 were expressed in CHO cells and currents were recorded at room temperature using the whole-cell patch-clamp. Expression of p.D1816VfsX7 alone resulted in a marked reduction (≈90%) in peak Na(+) current density compared with WT channels. Peak current density generated by p.D1816VfsX7+WT was ≈50% of that generated by WT channels. p.D1816VfsX7 positively shifted activation and inactivation curves, leading to a significant reduction of the window current. The mutation accelerated current activation and reactivation kinetics and increased the fraction of channels developing slow inactivation with prolonged depolarizations. However, late INa was not modified by the mutation. p.D1816VfsX7 produced a marked reduction of channel trafficking toward the membrane that was not restored by decreasing incubation temperature during cell culture or by incubation with 300 µM mexiletine and 5 mM 4-phenylbutirate. CONCLUSION: Despite a severe truncation of the C-terminus, the resulting mutated channels generate currents, albeit with reduced amplitude and altered biophysical properties, confirming the key role of the C-terminal domain in the expression and function of the cardiac Na(+) channel.
Assuntos
Síndrome de Brugada/genética , Mutação da Fase de Leitura/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Transporte Biológico/efeitos dos fármacos , Feminino , Humanos , Mexiletina/farmacologia , Pessoa de Meia-Idade , Fenilbutiratos/farmacologiaRESUMO
BACKGROUND: We identified 2 compound heterozygous mutations (p.D1690N and p.G1748D) in the SCN5A gene encoding cardiac Na(+) channels (Nav1.5) in a proband diagnosed with Brugada syndrome type 1. Furthermore, in the allele encoding the p.D1690N mutation, the p.H558R polymorphism was also detected. OBJECTIVE: The purpose of this study was to analyze the functional properties of the mutated channels as well as the putative modulator effects produced by the presence of the polymorphism. METHODS: Wild-type and mutated human Nav1.5 channels were expressed in Chinese hamster ovary cells and recorded using whole-cell patch-clamp technique. RESULTS: Separately, both p.D1690N and p.G1748D mutations produced a marked reduction in peak Na(+) current density (>80%), mainly due to their limited trafficking toward the membrane. Furthermore, p.G1748D mutation profoundly affected channel gating. Both p.D1690N and p.G1748D produced a marked dominant negative effect when cotransfected with either wild-type or p.H558R channels. Conversely, p.H558R was able to rescue defective trafficking of p.D1690N channels toward the membrane when both polymorphism and mutation were in the same construct. Surprisingly, cotransfection with p.D1690N, either alone or together with the polymorphism (p.H558R-D1690N), completely restored the profound gating defects exhibited by p.G1748D channels but only slightly rescued their trafficking. CONCLUSIONS: Our results add further support to the hypothesis that Nav1.5 subunits interact among them before trafficking toward the membrane and suggest that a missense mutation can "rescue" the defective gating produced by another missense mutation.
Assuntos
Síndrome de Brugada/genética , Ativação do Canal Iônico/genética , Mutação de Sentido Incorreto/genética , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Animais , Síndrome de Brugada/fisiopatologia , Células Cultivadas , Cricetinae , DNA Complementar/metabolismo , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Células HEK293 , Heterozigoto , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Sensibilidade e EspecificidadeRESUMO
Human cardiac inward rectifier current (IK1) is generated by Kir2.x channels. Inhibition of IK1 could offer a useful antiarrhythmic strategy against fibrillatory arrhythmias. Therefore, elucidation of Kir2.x channels pharmacology, which still remains elusive, is mandatory. We characterized the electrophysiological and molecular basis of the inhibition produced by the antiarrhythmic propafenone of the current generated by Kir2.x channels (IKir2.x) and the IK1 recorded in human atrial myocytes. Wild type and mutated human Kir2.x channels were transiently transfected in CHO and HEK-293 cells. Macroscopic and single-channel currents were recorded using the patch-clamp technique. At concentrations >1µM propafenone inhibited IKir2.x the order of potency being Kir2.3â¼IK1>Kir2.2>Kir2.1 channels. Blockade was irrespective of the extracellular K(+) concentration whereas markedly increased when the intracellular K(+) concentration was decreased. Propafenone decreased inward rectification since at potentials positive to the K(+) equilibrium potential propafenone-induced block decreased in a voltage-dependent manner. Importantly, propafenone favored the occurrence of subconductance levels in Kir2.x channels and decreased phosphatidylinositol 4,5-bisphosphate (PIP2)-channel affinity. Blind docking and site-directed mutagenesis experiments demonstrated that propafenone bound Kir2.x channels at the cytoplasmic domain, close to, but not in the pore itself, the binding site involving two conserved Arg residues (residues 228 and 260 in Kir2.1). Our results suggested that propafenone incorporated into the cytoplasmic domain of the channel in such a way that it decreased the net negative charge sensed by K(+) ions and polyamines which, in turn, promotes the appearance of subconductance levels and the decrease of PIP2 affinity of the channels.
Assuntos
Citoplasma/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Propafenona/farmacologia , Eletricidade Estática , Idoso , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Patch-ClampRESUMO
BACKGROUND: Long QT syndrome (LQTS) is characterized by a prolonged QT interval that can lead to severe ventricular arrhythmias (torsades de pointes) and sudden death. Congenital LQTS type 2 (LQT2) is due to loss-of-function mutations in the KCNH2 gene encoding Kv11.1 channels responsible for the rapid component of the delayed rectifier current. OBJECTIVE: The purpose of this study was to determine the functional properties of the LQT2-associated mutation p.E637G found in a Spanish family. METHODS: Wild-type (WT) and p.E637G Kv11.1 channels were transiently transfected in Chinese hamster ovary cells, and currents were recorded using the patch-clamp technique. RESULTS: The p.E637G channels lost inward rectification and K(+) selectivity, generating small but measurable slowly activating, noninactivating currents. These important alterations were corrected neither by cotransfection with WT channels nor by incubation at low temperatures or with pharmacological chaperones. As a consequence of its effects on channel gating, the mutation significantly reduced the outward repolarizing current during the action potential (AP), resulting in a marked lengthening of the duration of a simulated human ventricular AP. CONCLUSION: We have identified and characterized an LQT2-associated mutation that through removal of C-type inactivation and reduction of K(+) selectivity causes the QT prolongation observed in the patients carrying the mutation. Moreover, the results obtained demonstrate the importance of the glutamic acid at position 637 for the inactivation process and K(+) selectivity of Kv11.1 channels.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Síndrome do QT Longo/genética , Mutação de Sentido Incorreto/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Animais , Cricetinae , Cricetulus , Canais de Potássio de Retificação Tardia/genética , Canal de Potássio ERG1 , Ácido Glutâmico/genética , Humanos , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Transporte ProteicoRESUMO
AIMS: Endocannabinoids are synthesized from lipid precursors at the plasma membranes of virtually all cell types, including cardiac myocytes. Endocannabinoids can modulate neuronal and vascular ion channels through receptor-independent actions; however, their effects on cardiac K(+) channels are unknown. This study was undertaken to determine the receptor-independent effects of endocannabinoids such as anandamide (N-arachidonoylethanolamine, AEA), 2-arachidonoylglycerol (2-AG), and endocannabinoid-related compounds such as N-palmitoylethanolamine (PEA), N-oleoylethanolamine (OEA), the endogenous lipid lysophosphatidylinositol (LPI), and the fatty acids from which some of these compounds are endogenously synthesized, on human cardiac Kv1.5 channels, which generate the ultrarapid delayed rectifier current (I(Kur)). METHODS AND RESULTS: hKv1.5 currents (I(hKv1.5)) were recorded in mouse fibroblasts (Ltk(-) cells) by using the whole-cell patch-clamp technique. Most of these compounds inhibited I(hKv1.5) in a concentration-dependent manner, the potency being determined by the number of C atoms in the fatty acyl chain. Indeed, AEA and 2-AG, which are arachidonic acid (20:4) derivatives, exhibited the highest potency (IC(50) approximately 0.9-2.5 microM), whereas PEA, a palmitic acid (PA-16:0) derivative, exhibited the lowest potency. The inhibition was independent of cannabinoid receptor engagement and of changes in the order and microviscosity of the membrane. Furthermore, blockade induced by AEA and 2-AG was abolished upon mutation of the R487 residue, which determines the external tetraethylammonium sensitivity and is located in the external entryway of the pore. AEA significantly prolonged the duration of action potentials (APs) recorded in mouse left atria. CONCLUSION: These results indicate that endocannabinoids block human cardiac Kv1.5 channels by interacting with an extracellular binding site, a mechanism by which these compounds regulate atrial AP shape.
Assuntos
Moduladores de Receptores de Canabinoides/farmacologia , Canabinoides/farmacologia , Endocanabinoides , Coração/efeitos dos fármacos , Canal de Potássio Kv1.5/antagonistas & inibidores , Receptores de Canabinoides/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Ácidos Araquidônicos/farmacologia , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Ácidos Graxos/farmacologia , Polarização de Fluorescência , Humanos , Camundongos , Alcamidas Poli-Insaturadas/farmacologia , Proteína Quinase C/fisiologiaRESUMO
OBJECTIVES: The purpose of this study was to compare the voltage-dependent K(+) currents of human cells of the right and left atria and determine whether electrical remodeling produced by chronic atrial fibrillation (CAF) is chamber-specific. BACKGROUND: Several data point to the existence of interatrial differences in the repolarizing currents. Therefore, it could be possible that CAF-induced electrical remodeling differentially affects voltage-dependent K(+) currents in each atrium. METHODS: Currents were recorded using the whole-cell patch-clamp in myocytes from left (LAA) and right atrial appendages (RAA) obtained from sinus rhythm (SR) and CAF patients. RESULTS: In SR, LAA and RAA myocytes were divided in 3 types, according to their main voltage-dependent repolarizing K(+) current. CAF differentially modified the proportion of these 3 types of cells on each atrium. CAF reduced the Ca(2+)-independent 4-aminopyridine-sensitive component of the transient outward current (I(to1)) more markedly in the LAA than in the RAA. Therefore, an atrial right-to-left I(to1) gradient was created by CAF. In contrast, the ultrarapid component of the delayed rectifier current (I(Kur)) was more markedly reduced in the RAA than in the LAA, thus abolishing the atrial right-to-left I(Kur) gradient observed in SR. Importantly, in both atria, CAF increased the slow component of the delayed rectifier current (I(Ks)). CONCLUSIONS: Our results demonstrated that in SR there are intra-atrial heterogeneities in the repolarizing currents. CAF decreases I(to1) and I(Kur) differentially in each atrium and increases I(Ks) in both atria, an effect that further promotes re-entry.
Assuntos
Fibrilação Atrial/diagnóstico , Miócitos Cardíacos/fisiologia , Canais de Potássio/metabolismo , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/fisiopatologia , Doença Crônica , Eletrocardiografia , Eletrofisiologia , Feminino , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Estudos de AmostragemRESUMO
Acute heart failure represents a major public health problem due to its high prevalence, high rates of mortality and readmissions and significant healthcare costs. Patients with AHF and low cardiac output represent a small subgroup of patients with very high mortality rates that require inotropic support to improve cardiac systolic function. Classical inotropic agents, such as beta1-adrenergic agonists (dobutamine, dopamine) and phosphodiesterase III inhibitors (milrinone, enoximone) improve symptoms and hemodynamics by increasing free intracellular Ca(2+) levels, but also increase myocardial O(2) demands and exert arrhythmogenic effects. These actions explain why these drugs increase both short- and long-term mortality, particularly in patients with AHF and coronary artery disease. Thus, we need new inotropic agents that do not increase cytosolic Ca(2+) or myocardial oxygen demands or produce arrhythmogenesis for the treatment of high-risk patients with acute heart failure and low cardiac output. This review describes three new classes of investigational agents: levosimendan, a calcium sensitizer and potassium channel opener, istaroxime, the first new luso-inotropic agent and cardiac myosin activators.
Assuntos
Cardiotônicos/uso terapêutico , Drogas em Investigação/uso terapêutico , Insuficiência Cardíaca/tratamento farmacológico , Doença Aguda , Animais , Cardiotônicos/farmacologia , Ensaios Clínicos como Assunto/métodos , Drogas em Investigação/farmacologia , Insuficiência Cardíaca/metabolismo , HumanosRESUMO
AIMS: Chronic atrial fibrillation (CAF) is characterized by a shortening of the plateau phase of the action potentials (AP) and a decrease in the bioavailability of nitric oxide (NO). In this study, we analysed the effects of NO on Kv4.3 (I(Kv4.3)) and on human transient outward K(+) (I(to1)) currents as well as the signalling pathways responsible for them. We also analysed the expression of NO synthase 3 (NOS3) in patients with CAF. METHODS AND RESULTS: I(Kv4.3) and I(to1) currents were recorded in Chinese hamster ovary cells and in human atrial and mouse ventricular dissociated myocytes using the whole-cell patch clamp. The expression of NOS3 was analysed by western blotting. AP were recorded using conventional microelectrode techniques in mouse atrial preparations. NO and NO donors inhibited I(Kv4.3) and human I(to1) in a concentration- and voltage-dependent manner (IC(50) for NO: 375.0 +/- 48 nM) as a consequence of the activation of adenylate cyclase and the subsequent activation of the cAMP-dependent protein kinase and the serine-threonine phosphatase 2A. The density of the I(to1) recorded in ventricular myocytes from wild-type (WT) and NOS3-deficient mice (NOS3(-/-)) was not significantly different. Furthermore, the duration of atrial AP repolarization in WT and NOS3(-/-) mice was not different. The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atrial AP and lengthened the AP duration measured at 20 and 50% of repolarization of the human atrial CAF-remodelled AP as determined using a mathematical model. However, the expression of NOS3 was not modified in left atrial appendages from CAF patients. CONCLUSION: Our results suggested that the increase in the atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the I(to1) as a result of the activation of non-canonical enzymatic pathways.
Assuntos
Miócitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Canais de Potássio Shal/antagonistas & inibidores , Transdução de Sinais/fisiologia , Potenciais de Ação/fisiologia , Idoso , Animais , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Células CHO , Células Cultivadas , Doença Crônica , Cricetinae , Cricetulus , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Teóricos , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Shal/metabolismoRESUMO
La ranolazina es un derivado piperazínico con un nuevo mecanismo de acción que ha sido aprobado para su uso en combinación en pacientes con angina crónica estable. La isquemia miocárdica aumenta la corriente tardía de entrada de Na+ en las células cardiacas (INaL) e incrementa la concentración de sodio intracelular ([Na+ ] i ), lo que, a su vez, activa el modo inverso del intercambiador Na+ -Ca2+ y aumenta la concentración de calcio intracelular ([Ca2+] i ). Este aumento en Na+ i y Ca2+ i conduce a una disfunción mecánica (aumenta la presión diastólica y reduce la contractilidad y el aporte coronario de O2 ), eléctrica (produce arritmias) y mitocondrial (aumenta las demandas miocárdicas de O2 y reducción de la formación de adenosintrifosfato). La ranolazina inhibe selectivamente la INaL, reduce la acumulación intracelular de Na+ y la posterior de Ca2+ inducido por el Na+ , así como las anomalías mecánicas, eléctricas y metabólicas en el miocardio isquémico o insuficiente. En ensayos clínicos controlados realizados en pacientes con angina crónica estable, la ranolazina ejerce acciones antianginosas y antiisquémicas y, en pacientes con síndrome coronario agudo, ejerce acciones antiarrítmicas. Además, reduce la glucohemoglobina en pacientes coronarios diabéticos y mejora la función ventricular en pacientes con cardiopatía isquémica o insuficiencia cardiaca crónica. La ranolazina es un fármaco bien tolerado cuyos efectos adversos más comunes son náuseas, mareos, astenia y estreñimiento. Por todo lo anterior, la ranolazina representa una alternativa segura y eficaz en pacientes con angina crónica estable con síntomas no controlados o que no toleran los fármacos antianginosos convencionales (AU)
Ranolazine is a piperazine derivative that has a novel mechanism of action and which has been approved as add-on therapy for patients with chronic stable angina. Myocardial ischemia increases the late inward sodium ion (Na+ ) current in cardiac cells (INaL) and raises the intracellular sodium concentration (Na+ i ), which in turn activates the reverse mode of the sodium-calcium (Na+ - Ca2+) exchanger and increases the intracellular calcium concentration (Ca2+ i ). This increase in Na+ i and Ca2+ i leads to mechanical dysfunction (i.e. diastolic pressure increases, and contractility and myocardial oxygen supply decrease), electrical dysfunction (i.e. the induction of arrhythmias) and mitochondrial dysfunction (i.e. myocardial oxygen demand increases and the rate of ATP formation decreases). Ranolazine selectively inhibits the increase in INaL, reduces intracellular Na+ accumulation and the subsequent Ca2+ accumulation induced by Na+ , and decreases mechanical, electrical and metabolic dysfunction in the ischemic or failing myocardium. Controlled clinical trials have shown that ranolazine has both antianginal and anti-ischemic effects in patients with chronic stable angina, and an antiarrhythmic effect in patients with acute coronary syndrome. Moreover, ranolazine reduces the glycosylated hemoglobin level in diabetic patients with coronary heart disease and improves ventricular function in patients with ischemic heart disease or chronic heart failure. Ranolazine is well tolerated, with the most common adverse effects being nausea, dizziness, asthenia and constipation. For these reasons, ranolazine is a safe and effective option for patients with chronic stable angina whose symptoms are not under control or who can not tolerate conventional anti-anginal drugs (AU)
Assuntos
Humanos , Angina Estável/tratamento farmacológico , Ranolazina/farmacologia , Isquemia Miocárdica/tratamento farmacológico , Síndrome do QT Longo/fisiopatologia , Diabetes Mellitus/fisiopatologia , Segurança do PacienteRESUMO
La renina es la enzima que limita la síntesis de angiotensina II y la activación del sistema renina-angiotensina (SRA), por lo que desde hace más de 50 años se ha intentado desarrollar fármacos activos por vía oral capaces de inhibir directamente la renina. Recientemente se ha demostrado que prorrenina y renina son moléculas activas que interactúan con un receptor específico, (P)RR, y que su estimulación activa diversas vías de señalización independientes de las activadas por la angiotensina II. Aliskiren, el primer inhibidor directo de la renina (IDR) no peptídico, activo por vía oral, ha mostrado eficacia y seguridad en el tratamiento de la hipertensión arterial. Este artículo revisa el desarrollo de los IDR, el papel fisiopatológico de la prorrenina y la renina, la estructura y las vías de señalización acopladas a la activación del (P)RR, el mecanismo de acción y las posibles diferencias, ventajas y/o desventajas de los IDR con respecto a otros fármacos que inhiben el SRA, como los inhibidores de la enzima de conversión y los antagonistas de los receptores AT1 (AU)
Renin is an enzyme that limits angiotensin-II synthesis and activates the renin-angiotensin system (RAS). Therefore, for almost 50 years, multiple attempts were made to develop orally active direct renin inhibitors (DRIs). Recently, it has been demonstrated that both prorenin and renin are active molecules that interact with a specific receptor, (P)RR, and that stimulation of this receptor leads to the activation of signal transduction pathways independent of angiotensin-II activity. Aliskiren, the first non-peptide orally active DRI, has been found to be safe and effective in the treatment of hypertension. The present article reviews the development of DRIs, the pathophysiological roles of prorenin and renin, the structure of and the signal transduction pathways involved in activation of the (P)RR, and the mechanisms of action of, the possible differences between, and the comparative advantages and disadvantages of DRIs and other RAS inhibitors, mainly angiotensin-converting enzyme inhibitors and angiotensin AT1 receptor blockers (AU)