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1.
Proc Natl Acad Sci U S A ; 117(6): 2795-2804, 2020 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-31980532

RESUMO

The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.


Assuntos
Canal de Potássio ERG1/química , Canal de Potássio ERG1/metabolismo , Motivos de Aminoácidos , Canal de Potássio ERG1/genética , Mutação com Ganho de Função , Humanos , Cinética , Síndrome do QT Longo/genética , Síndrome do QT Longo/metabolismo , Mutação de Sentido Incorreto , Potássio/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
2.
J Biochem Mol Toxicol ; 34(2): e22423, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31729781

RESUMO

MicroRNAs are endogenous small noncoding RNAs that posttranscriptionally regulate the expression of target genes and play crucial roles in diverse physiopathologic processes. In the current study, we examined the microRNA (miRNA) expression profile of high-glucose-treated neonatal rat cardiomyocytes and the potential mechanisms. Differentially expressed miRNAs were analyzed by a miRNA microarray and validated by a quantitative real-time polymerase chain reaction in high-glucose-treated rat cardiomyocytes. Based on the results of our previous study and the bioinformatics prediction, we identified miR-195-5p/SGK1/Nedd4-2/hERG as the top-ranked signal pathway in diabetes cell model in vitro. In summary, our present study provides novel insights into the regulatory mechanism of miR-195-5p/SGK1/Nedd4-2/hERG in rat cardiomyocytes under high-glucose stress, which may provide a novel idea for the development of diagnostic and therapeutic strategies for diabetic cardiomyopathy in the future.


Assuntos
Cardiomiopatias Diabéticas/metabolismo , Glucose/farmacologia , MicroRNAs/genética , Miócitos Cardíacos/efeitos dos fármacos , Transcriptoma , Regiões 3' não Traduzidas/genética , Animais , Sítios de Ligação , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Transfecção
3.
Toxicol Lett ; 319: 40-48, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31706004

RESUMO

Two synthetic tryptamines, namely [3-[2-(diethylamino)ethyl]-1H-indol-4-yl] acetate (4-AcO-DET) and 3-[2-[ethyl(methyl)amino]ethyl]-1H-indol-4-ol (4-HO-MET), are abused by individuals seeking recreational hallucinogens. These new psychoactive substances (NPSs) can cause serious health problems because their adverse effects are mostly unknown. In the present study, we evaluated the cardiotoxicity of 4-AcO-DET and 4-HO-MET using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, electrocardiography (ECG), and the human ether-a-go-go-related gene (hERG) assay. In addition, we analyzed the expression level of p21 (CDC42/RAC)-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, 4-AcO-DET- and 4-HO-MET-treated H9c2 cells proliferated in a concentration-dependent manner. Moreover, both substances increased QT intervals (as determined using ECG) in Sprague-Dawley rats and inhibited potassium channels (as verified by the hERG assay) in Chinese hamster ovary cells. However, there was no change in PAK1 expression. Collectively, the results indicated that 4-AcO-DET and 4-HO-MET might cause adverse effects on the cardiovascular system. Further studies are required to confirm the relationship between PAK1 expression and cardiotoxicity. The findings of the present study would provide science-based evidence for scheduling the two NPSs.


Assuntos
Cardiotoxinas/toxicidade , Alucinógenos/toxicidade , Triptaminas/toxicidade , Animais , Células CHO , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Canal de Potássio ERG1/metabolismo , Eletrocardiografia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/toxicidade , Ratos , Ratos Sprague-Dawley , Quinases Ativadas por p21/biossíntese , Quinases Ativadas por p21/genética
4.
Am J Pathol ; 190(1): 48-56, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31839145

RESUMO

Kv11.1 potassium channels are essential for heart repolarization. Prescription medication that blocks Kv11.1 channels lengthens the ventricular action potential and causes cardiac arrhythmias. Surprisingly little is known about the Kv11.1 channel expression and function in the lung tissue. Here we report that Kv11.1 channels were abundantly expressed in the large pulmonary arteries (PAs) of healthy lung tissues from humans and rats. Kv11.1 channel expression was increased in the lungs of humans affected by chronic obstructive pulmonary disease-associated pulmonary hypertension and in the lungs of rats with pulmonary arterial hypertension (PAH). In healthy lung tissues from humans and rats, Kv11.1 channels were confined to the large PAs. In humans with chronic obstructive pulmonary disease-associated pulmonary hypertension and in rats with PAH, Kv11.1 channels were expressed in both the large and small PAs. The increase in Kv11.1 channel expression closely followed the time-course of the development of pulmonary vascular remodeling in PAH rats. Treatment of PAH rats with dofetilide, an Kv11.1 channel blocker approved by the US Food and Drug Administration for use in the treatment of arrythmia, inhibited PAH-associated pulmonary vascular remodeling. Taken together, the findings from this study uncovered a novel role of Kv11.1 channels in lung function and their potential as new drug targets in the treatment of pulmonary hypertension. The protective effect of dofetilide raises the possibility of repurposing this antiarrhythmic drug for the treatment of patients with pulmonary hypertension.


Assuntos
Arritmias Cardíacas/prevenção & controle , Canal de Potássio ERG1/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Fenetilaminas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Hipertensão Arterial Pulmonar/complicações , Sulfonamidas/farmacologia , Remodelação Vascular/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/patologia , Estudos de Casos e Controles , Canal de Potássio ERG1/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Prognóstico , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/patologia , Ratos Sprague-Dawley
5.
Cardiovasc Ther ; 2019: 6032631, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31772612

RESUMO

Background: LCZ696 (valsartan/sacubitril) therapy significantly reduced mortality in patients with heart failure (HF). Although a clinical trial (PARADISE-MI Trial) has been ongoing to examine the effects of LCZ696 in myocardial infarction (MI) patients, the effects of LCZ696 on remodeling of cardiac electrophysiology in animal models remain largely unclear. Methods: We performed coronary artery ligation to create MI in Sprague-Dawley rats. Echocardiography was performed one week after MI to confirm the development of HF with left ventricular ejection fraction ≤ 40%. MI rats were randomly assigned to receive medical therapy for 4 weeks: LCZ696, enalapril, or vehicle. The sham-operation rats received sham operation without MI creation. In vivo electrophysiological exams were performed under general anesthesia. Western blot analyses were conducted to quantify ion channel proteins. Results: The HF-vehicle group did not show significant changes in LVEF. Both enalapril and LCZ696 therapy significantly improved LVEF. The HF-vehicle group had higher ventricular arrhythmia (VA) inducibility than the sham group. As compared with the HF-vehicle group, LCZ696 therapy significantly reduced VA inducibility, but enalapril therapy did not. Western blot analyses showed significant downregulation of NaV1.5, ERG, KCNE1, and KCNE2 channel proteins in the HF vehicle group compared with the sham group. LCZ696 therapy upregulated protein expression of ERG, KCNE1, and KCNE2. Conclusion: As compared with enalapril therapy, LCZ696 therapy led to improvement of LVEF, reduced VA inducibility, and upregulated expression of K+ channel proteins.


Assuntos
Aminobutiratos/farmacologia , Antiarrítmicos/farmacologia , Arritmias Cardíacas/prevenção & controle , Insuficiência Cardíaca/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Infarto do Miocárdio/complicações , Tetrazóis/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Modelos Animais de Doenças , Canal de Potássio ERG1/metabolismo , Feminino , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Ventrículos do Coração/metabolismo , Ventrículos do Coração/fisiopatologia , Masculino , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Ratos Sprague-Dawley , Volume Sistólico/efeitos dos fármacos
6.
Elife ; 82019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31670657

RESUMO

Catastrophic arrhythmias and sudden cardiac death can occur with even a small imbalance between inward sodium currents and outward potassium currents, but mechanisms establishing this critical balance are not understood. Here, we show that mRNA transcripts encoding INa and IKr channels (SCN5A and hERG, respectively) are associated in defined complexes during protein translation. Using biochemical, electrophysiological and single-molecule fluorescence localization approaches, we find that roughly half the hERG translational complexes contain SCN5A transcripts. Moreover, the transcripts are regulated in a way that alters functional expression of both channels at the membrane. Association and coordinate regulation of transcripts in discrete 'microtranslatomes' represents a new paradigm controlling electrical activity in heart and other excitable tissues.


Assuntos
Canal de Potássio ERG1/metabolismo , Regulação da Expressão Gênica , Coração/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Canal de Potássio ERG1/genética , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Transfecção
7.
Int J Mol Sci ; 20(13)2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31261773

RESUMO

Human ether-a-gogo related gene (hERG) product is the membrane potassium channel Kv11.1, which is involved in the electrical activity of the heart. As such, it is a key player in the toxicity of many drug candidates. Therefore, having this protein at hand during earlier stages of drug discovery is important for preventing later toxicity. Furthermore, having a fair quantity of functional channels may help in the development of the necessary techniques for gaining insight in this channel structure. Thus, we performed a comparative study of methods for over-expressing a mutated but functional, hERG in different orthologous hosts, such as yeast, bacteria, insect and human cell lines. We also engineered the protein to test various constructs of a functional channel. We obtained a significant amount of a functional mutant channel from HEK cells that we thoroughly characterized. The present work paves the way for the expression of large amounts of this protein, with which protein crystallization or cryo-electronic microscopy will be attempted. This will be a way to gain information on the structure of the hERG active site and its modelization to obtain data on the pauses of various reference compounds from the pharmacopeia, as well as to gain information about the thermodynamics of the hERG/ligand relationship.


Assuntos
Canal de Potássio ERG1/genética , Engenharia de Proteínas/métodos , Animais , Fracionamento Químico/métodos , Cristalografia por Raios X/métodos , Canal de Potássio ERG1/química , Canal de Potássio ERG1/metabolismo , Células HEK293 , Humanos , Pichia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Xenopus
8.
Mol Pharmacol ; 96(3): 330-344, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31253645

RESUMO

hERG K+ channel is important for controlling the duration of cardiac action potentials. Amiodarone (AMD), a widely prescribed class III antiarrhythmic, could inhibit hERG currents with relatively few tachyarrhythmic adverse events. We use injected Xenopus oocyte with two-electrode voltage clamp techniques to characterize the action of AMD on hERG channels. We found that AMD binds to the resting hERG channel with an apparent dissociation constant of ∼1.4 µM, and inhibits hERG currents at mild and strong depolarization pulses by slowing activation and enhancing inactivation, respectively, at 22°C. The activation kinetics of hERG channel activation are much faster, but inactivation kinetics are slower at 37°C. AMD accordingly has a 15% to 20% weaker and stronger inhibitory effect at mild and strong depolarization (e.g., -60 and +30 mV, 0.3-second pulse), respectively. In the meanwhile, the resurgent hERG tail currents are dose-dependently inhibited by AMD without altering the kinetics of current decay at both 22°C and 37°C, indicating facilitation of recovery from inactivation via the silent route. Most importantly, AMD no longer inhibits but enhances hERG currents at a mild pulse shortly after a prepulse at 37°C, but not so much at 22°C. We conclude that AMD is an effective hERG channel-gating modifier capable of lengthening the plateau phase of cardiac action potential (without increasing the chance of afterdepolarization). AMD, however, should be used with caution in hypothermia or the other scenarios that slow hERG channel activation. SIGNIFICANCE STATEMENT: It is known that amiodarone (AMD) acts on hERG K+ channels to treat cardiac arrhythmias with relatively little arrhythmogenicity. We found that AMD enhances hERG channel inactivation but slows activation as well as recovery from inactivation, and thus has a differential inhibition and enhancement effect on hERG currents at different phases of membrane voltage changes, especially at 37°C, but not so much at 22°C. AMD is therefore a relatively ideal agent against tachyarrhythmia at 37°C, but should be more cautiously used at lower temperatures or relevant pathophysiological/pharmacological scenarios associated with slower hERG channel activation because of the increased chances of adverse events.


Assuntos
Amiodarona/farmacologia , Canal de Potássio ERG1/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Xenopus laevis/genética , Animais , Animais Geneticamente Modificados , Fenômenos Biofísicos , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/genética , Humanos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Temperatura , Xenopus laevis/crescimento & desenvolvimento
9.
Cell Physiol Biochem ; 53(1): 36-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31169990

RESUMO

BACKGROUND/AIMS: Ivabradine lowers the heart rate by inhibition of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels mediating the 'funny' pacemaker current If in the sinoatrial node. It is clinically approved for the treatment of heart failure and angina pectoris. Due to its proposed high selectivity for If administration of ivabradine is not associated with the side effects commonly observed following the application of other heart rate lowering agents. Recent evidence, however, has shown significant affinity of ivabradine towards Kv11.1 (ether-a-go-go related gene, ERG) potassium channels. Despite the inhibition of Kv11.1 channels by ivabradine, cardiac action potential (AP) duration and heart rate corrected QT interval (QTc) of the human electrocardiogram (ECG) were not prolonged. We thus surmised that compensatory mechanisms might counteract the drug's inhibitory action on Kv11.1. METHODS: The effects of ivabradine on human Kv11.1 and Kv7.1 potassium, Cav1.2 calcium, and Nav1.5 sodium channels, heterologously expressed in tsA-201 cells, were studied in the voltage-clamp mode of the whole cell patch clamp technique. In addition, changes in action potential parameters of human induced pluripotent stem cell (iPSC) derived cardiomyocytes upon application of ivabradine were studied with current-clamp experiments. RESULTS: Here we show that ivabradine exhibits significant affinity towards cardiac ion channels other than HCN. We demonstrate for the first time inhibition of human voltage-gated Nav1.5 sodium channels at therapeutically relevant concentrations. Within this study we also confirm recent findings of human Kv11.1 inhibition by low µM concentrations of ivabradine and observed no prolongation of ventricular-like APs in cardiomyocytes derived from iPSCs. CONCLUSION: Our results provide an explanation why ivabradine, despite its affinity for Kv11.1 channels, does not prolong the cardiac AP and QTc interval. Furthermore, our results suggest the inhibition of voltage-gated Nav1.5 sodium channels to underlie the recent observations of slowed atrioventricular conduction by increased atrial-His bundle intervals upon administration of ivabradine.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Fármacos Cardiovasculares/farmacologia , Canais Iônicos/metabolismo , Ivabradina/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Canais Iônicos/antagonistas & inibidores , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/química , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp
10.
Biochem Biophys Res Commun ; 512(4): 845-851, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-30929919

RESUMO

Pathogenic long QT mutations often comprise high phenotypic variability and particularly variants in ANK2 (long QT syndrome 4) frequently lack QT prolongation. We sought to elucidate the genetic and functional background underlying the clinical diversity in a 3-generation family with different cardiac arrhythmias. Next-generation sequencing-based screening of patients with QT prolongation identified the index patient of the family carrying an ANK2-E1813K variant and a previously uncharacterized KCNH2-H562R mutation in a double heterozygous conformation. The patient presented with a severe clinical phenotype including a markedly prolonged QTc interval (544 ms), recurrent syncope due to Torsade de Pointes tachycardias, survived cardiopulmonary resuscitation, progressive cardiac conduction defect, and atrial fibrillation. Evaluation of other family members identified a sister and a niece solely carrying the ANK2-E1813K variant, who showed age-related conduction disease. An asymptomatic second sister solely carried the KCNH2-H562R mutation. Voltage-clamp recordings in Xenopus oocytes revealed that KCNH2-H562R subunits were non-functional but did not exert dominant-negative effects on wild-type subunits. Expression of KCNH2-H562R in HEK293 cells showed a trafficking deficiency. Co-expression of the C-terminal regulatory domain of ANK2 in Xenopus oocytes revealed that ANK2-E1813K diminished currents mediated by the combination of wild-type and H562R KCNH2 subunits. Our data suggest that ANK2 functionally interacts with KCNH2 leading to a stronger current suppression and marked aggravation of long QT syndrome in the patient carrying variants in both proteins.


Assuntos
Anquirinas/genética , Canal de Potássio ERG1/genética , Síndrome do QT Longo/genética , Mutação , Adulto , Idoso , Animais , Anquirinas/metabolismo , Canal de Potássio ERG1/metabolismo , Feminino , Células HEK293 , Humanos , Síndrome do QT Longo/etiologia , Masculino , Pessoa de Meia-Idade , Oócitos/metabolismo , Linhagem , Xenopus laevis
11.
Mol Pharmacol ; 96(1): 1-12, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31015282

RESUMO

The human ether-à-go-go-related gene (hERG) encodes the channel that conducts the rapidly activating delayed rectifier potassium current (IKr) in the heart. Reduction in IKr causes long QT syndrome, which can lead to fatal arrhythmias triggered by stress. One potential link between stress and hERG function is protein kinase C (PKC) activation; however, seemingly conflicting results regarding PKC regulation of hERG have been reported. We investigated the effects of PKC activation using phorbol 12-myristate 13-acetate (PMA) on hERG channels expressed in human embryonic kidney cell line 293 (HEK293) cells and IKr in isolated neonatal rat ventricular myocytes. Acute activation of PKC by PMA (30 nM, 30 minutes) reduced both hERG current (IhERG) and IKr Chronic activation of PKC by PMA (30 nM, 16 hours) increased IKr in cardiomyocytes and the expression level of hERG proteins; however, chronic (30 nM, 16 hours) PMA treatment decreased IhERG, which became larger than untreated control IhERG after PMA removal for 4 hours. Deletion of amino acid residues 2-354 (Δ2-354 hERG) or 1-136 of the N terminus (ΔN 136 hERG) abolished acute PMA (30 nM, 30 minutes)-mediated IhERG reduction. In contrast to wild-type hERG channels, chronic activation of PKC by PMA (30 nM, 16 hours) increased both Δ2-354 hERG and ΔN136 hERG expression levels and currents. The increase in hERG protein was associated with PKC-induced phosphorylation (inhibition) of Nedd4-2, an E3 ubiquitin ligase that mediates hERG degradation. We conclude that PKC regulates hERG in a balanced manner, increasing expression through inhibiting Nedd4-2 while decreasing current through targeting a site(s) within the N terminus.


Assuntos
Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Canal de Potássio ERG1/química , Ativação Enzimática/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Fosforilação , Proteólise , Deleção de Sequência
12.
Clin Pharmacol Ther ; 106(3): 642-651, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30947366

RESUMO

Short QT syndrome (SQTS) predisposes afflicted patients to sudden cardiac death. Until now, only one drug-quinidine-has been shown to be effective in patients with SQTS type 1(SQTS1). The objective of this study was to use human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with SQTS1 to search for potentially effective drugs for the treatment of SQTS1 patients. Patch clamp and single-cell contraction measurements were employed to assess drug effects. Ivabradine, mexiletine, and ajmaline but not flecainide, ranolazine, or amiodarone prolonged the action potential duration (APD) in hiPSC-CMs from an SQTS1 patient. Ivabradine, ajmaline, and mexiletine inhibited KCNH2 channel currents significantly, which may underlie their APD-prolonging effects. Under proarrhythmic epinephrine stimulation in spontaneously beating SQTS1 hiPSC-CMs, ivabradine, mexiletine, and ajmaline but not flecainide reduced the epinephrine-induced arrhythmic events. The results demonstrate that ivabradine, ajmaline, and mexiletine may be candidate drugs for preventing tachyarrhythmias in SQTS1 patients.


Assuntos
Fármacos Cardiovasculares/farmacologia , Canal de Potássio ERG1/metabolismo , Sistema de Condução Cardíaco/anormalidades , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Arritmias Cardíacas , Relação Dose-Resposta a Droga , Epinefrina/farmacologia , Sistema de Condução Cardíaco/efeitos dos fármacos , Cardiopatias Congênitas , Humanos
13.
Prog Biophys Mol Biol ; 149: 86-98, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30826123

RESUMO

The human Ether-à-go-go Related Gene (hERG) encodes the pore forming subunit of the channel that conducts the rapid delayed rectifier potassium current IKr. IKr drives repolarization in the heart and when IKr is dysfunctional, cardiac repolarization delays, the QT interval on the electrocardiogram (ECG) prolongs and the risk of developing lethal arrhythmias such as Torsade de Pointes (TdP) increases. TdP risk is incorporated in drug safety screening for cardiotoxicity where hERG is the main target since the IKr channels appear highly sensitive to blockage. hERG block is also included as an important read-out in the Comprehensive in Vitro Proarrhythmia Assay (CiPA) initiative which aims to combine in vitro and in silico experiments on induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to screen for cardiotoxicity. However, the hERG channel has some unique features to consider for drug safety screening, which we will discuss in this study. The hERG channel consists of different isoforms, hERG1a and hERG1b, which individually influence the kinetics of the channel and the drug response in the human heart and in iPSC-CMs. hERG1b is often underappreciated in iPSC-CM studies, drug screening assays and in silico models, and the fact that its contribution might substantially differ between iPSC-CM and healthy but also diseased human heart, adds to this problem. In this study we show that the activation kinetics in iPSC-CMs resemble hERG1b kinetics using Cs+ as a charge carrier. Not including hERG1b in drug safety testing might underestimate the actual role of hERG1b in repolarization and drug response, and might lead to inappropriate conclusions. We stress to focus more on including hERG1b in drug safety testing concerning IKr.


Assuntos
Canal de Potássio ERG1/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Arritmias Cardíacas/metabolismo , Linhagem Celular , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1/genética , Humanos , Cinética , Potássio/metabolismo , Isoformas de Proteínas , Segurança , Torsades de Pointes/metabolismo
14.
J Biol Chem ; 294(16): 6506-6521, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30808709

RESUMO

Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, KV10.1 and human ether-a-go-go-related gene (hERG or KV11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5L) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6T) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5L to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in KV10.2 channels. Using cysteine bridges and S4-S5L-mimicking peptides, we show that the ligand/receptor model is conserved in KV10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in KV10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.


Assuntos
Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go , Ativação do Canal Iônico , Modelos Moleculares , Animais , Células COS , Chlorocebus aethiops , Canal de Potássio ERG1/química , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Peptídeos/química , Domínios Proteicos
15.
Cell Death Dis ; 10(3): 180, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792401

RESUMO

Potassium ion channels are critical in the regulation of cell motility. The acquisition of cell motility is an essential parameter of cancer metastasis. However, the role of K+ channels in cancer metastasis has been poorly studied. High expression of the hG1 gene, which encodes for Kv11.1 channel associates with good prognosis in estrogen receptor-negative breast cancer (BC). We evaluated the efficacy of the Kv11.1 activator NS1643 in arresting metastasis in a triple negative breast cancer (TNBC) mouse model. NS1643 significantly reduces the metastatic spread of breast tumors in vivo by inhibiting cell motility, reprogramming epithelial-mesenchymal transition via attenuation of Wnt/ß-catenin signaling and suppressing cancer cell stemness. Our findings provide important information regarding the clinical relevance of potassium ion channel expression in breast tumors and the mechanisms by which potassium channel activity can modulate tumor biology. Findings suggest that Kv11.1 activators may represent a novel therapeutic approach for the treatment of metastatic estrogen receptor-negative BC. Ion channels are critical factor for cell motility but little is known about their role in metastasis. Stimulation of the Kv11.1 channel suppress the metastatic phenotype in TNBC. This work could represent a paradigm-shifting approach to reducing mortality by targeting a pathway that is central to the development of metastases.


Assuntos
Canal de Potássio ERG1/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias de Mama Triplo Negativas/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cresóis/farmacologia , Cresóis/uso terapêutico , Canal de Potássio ERG1/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/mortalidade , Neoplasias de Mama Triplo Negativas/patologia , beta Catenina/antagonistas & inibidores , beta Catenina/genética
16.
Mol Pharmacol ; 95(5): 537-550, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30770456

RESUMO

Current guidelines around preclinical screening for drug-induced arrhythmias require the measurement of the potency of block of voltage-gated potassium channel subtype 11.1 (Kv11.1) as a surrogate for risk. A shortcoming of this approach is that the measured IC50 of Kv11.1 block varies widely depending on the voltage protocol used in electrophysiological assays. In this study, we aimed to investigate the factors that contribute to these differences and to identify whether it is possible to make predictions about protocol-dependent block that might facilitate the comparison of potencies measured using different assays. Our data demonstrate that state preferential binding, together with drug-binding kinetics and trapping, is an important determinant of the protocol dependence of Kv11.1 block. We show for the first time that differences in IC50 measured between protocols occurs in a predictable way, such that machine-learning algorithms trained using a selection of simple voltage protocols can indeed predict protocol-dependent potency. Furthermore, we also show that the preference of a drug for binding to the open versus the inactivated state of Kv11.1 can also be inferred from differences in IC50 values measured between protocols. Our work therefore identifies how state preferential drug binding is a major determinant of the protocol dependence of IC50 values measured in preclinical Kv11.1 assays. It also provides a novel method for quantifying the state dependence of Kv11.1 drug binding that will facilitate the development of more complete models of drug binding to Kv11.1 and improve our understanding of proarrhythmic risk associated with compounds that block Kv11.1.


Assuntos
Bioensaio/métodos , Canal de Potássio ERG1/genética , Canal de Potássio ERG1/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Arritmias Cardíacas/induzido quimicamente , Células CHO , Linhagem Celular , Cricetulus , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Cinética , Bloqueadores dos Canais de Potássio/efeitos adversos
17.
Molecules ; 24(3)2019 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759820

RESUMO

Natural and synthetic small molecules from the NCI Developmental Therapeutics Program (DTP) were employed in molecular dynamics-based docking with DNA repair proteins whose RNA-Seq based expression was associated with overall cancer survival (OS) after adjustment for the PCNA metagene. The compounds employed were required to elicit a sensitive response (vs. resistance) in more than half of the cell lines tested for each cancer. Methodological approaches included peptide sequence alignments and homology modeling for 3D protein structure determination, ligand preparation, docking, toxicity and ADME prediction. Docking was performed for unique lists of DNA repair proteins which predict OS for AML, cancers of the breast, lung, colon, and ovaries, GBM, melanoma, and renal papillary cancer. Results indicate hundreds of drug-like and lead-like ligands with best-pose binding energies less than -6 kcal/mol. Ligand solubility for the top 20 drug-like hits approached lower bounds, while lipophilicity was acceptable. Most ligands were also blood-brain barrier permeable with high intestinal absorption rates. While the majority of ligands lacked positive prediction for HERG channel blockage and Ames carcinogenicity, there was a considerable variation for predicted fathead minnow, honey bee, and Tetrahymena pyriformis toxicity. The computational results suggest the potential for new targets and mechanisms of repair inhibition and can be directly employed for in vitro and in vivo confirmatory laboratory experiments to identify new targets of therapy for cancer survival.


Assuntos
Reparo do DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/mortalidade , Antígeno Nuclear de Célula em Proliferação/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Barreira Hematoencefálica/metabolismo , Desenho de Fármacos , Canal de Potássio ERG1/metabolismo , Humanos , Absorção Intestinal/efeitos dos fármacos , Ligantes , Simulação de Acoplamento Molecular/métodos , Simulação de Dinâmica Molecular , Neoplasias/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas/metabolismo , Relação Estrutura-Atividade
18.
Toxicol Lett ; 304: 50-57, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30658151

RESUMO

Two emerging psychoactive substances, 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine (25D-NBOMe) and N-(2-methoxybenzyl)-2,5-dimethoxy-4-chlorophenethylamine (25C-NBOMe), are being abused, leading to fatal and non-fatal intoxications. However, most of their adverse effects have been reported anecdotally. In the present study, cardiotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), and human ether-a-go-go-related gene (hERG) assay. Expression levels of p21 (CDC42/RAC)-activated kinase 1 (PAK1), one of known biomarkers for cardiotoxicity, were also analyzed. Both 25D-NBOMe and 25C-NBOMe at 100 µM reduced cell viability in MTT assay. At 2.0 mg/kg and 0.75 mg/kg, they prolonged QT intervals in rat ECG. PAK1 was down-regulated by treatment with these two test compounds. Furthermore, potassium channels were inhibited by 25D-NBOMe treatment in hERG assay. Taken together, these results suggest that both 25D-NBOMe and 25C-NBOMe have potential cardiotoxicity, especially regarding cardiac rhythm. Further studies are needed to confirm the relationship between PAK1 down-regulation and cardiotoxicity.


Assuntos
Benzilaminas/efeitos adversos , Etilaminas/toxicidade , Cardiopatias/induzido quimicamente , Frequência Cardíaca/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Fenetilaminas/farmacologia , Psicotrópicos/efeitos adversos , Potenciais de Ação , Animais , Benzilaminas/farmacologia , Células CHO , Cardiotoxicidade , Sobrevivência Celular , Cricetulus , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fenetilaminas/efeitos adversos , Psicotrópicos/farmacologia , Ratos Sprague-Dawley , Quinases Ativadas por p21/metabolismo
19.
J Gen Physiol ; 151(2): 214-230, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30674563

RESUMO

Drug-induced block of the cardiac rapid delayed rectifying potassium current (I Kr), carried by the human ether-a-go-go-related gene (hERG) channel, is the most common cause of acquired long QT syndrome. Indeed, some, but not all, drugs that block hERG channels cause fatal cardiac arrhythmias. However, there is no clear method to distinguish between drugs that cause deadly arrhythmias and those that are clinically safe. Here we propose a mechanism that could explain why certain clinically used hERG blockers are less proarrhythmic than others. We demonstrate that several drugs that block hERG channels, but have favorable cardiac safety profiles, also evoke another effect; they facilitate the hERG current amplitude in response to low-voltage depolarization. To investigate how hERG facilitation impacts cardiac safety, we develop computational models of I Kr block with and without this facilitation. We constrain the models using data from voltage clamp recordings of hERG block and facilitation by nifekalant, a safe class III antiarrhythmic agent. Human ventricular action potential simulations demonstrate the ability of nifekalant to suppress ectopic excitations, with or without facilitation. Without facilitation, excessive I Kr block evokes early afterdepolarizations, which cause lethal arrhythmias. When facilitation is introduced, early afterdepolarizations are prevented at the same degree of block. Facilitation appears to prevent early afterdepolarizations by increasing I Kr during the repolarization phase of action potentials. We empirically test this prediction in isolated rabbit ventricular myocytes and find that action potential prolongation with nifekalant is less likely to induce early afterdepolarization than action potential prolongation with dofetilide, a hERG channel blocker that does not induce facilitation. Our data suggest that hERG channel blockers that induce facilitation increase the repolarization reserve of cardiac myocytes, rendering them less likely to trigger lethal ventricular arrhythmias.


Assuntos
Potenciais de Ação , Antiarrítmicos/farmacologia , Canal de Potássio ERG1/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Células Cultivadas , Canal de Potássio ERG1/antagonistas & inibidores , Células HEK293 , Humanos , Miócitos Cardíacos/fisiologia , Fenetilaminas/farmacologia , Pirimidinonas/farmacologia , Coelhos , Sulfonamidas/farmacologia , Xenopus
20.
J Biol Chem ; 294(1): 351-360, 2019 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-30401747

RESUMO

The QT interval is an important diagnostic feature on surface electrocardiograms because it reflects the duration of the ventricular action potential. A previous genome-wide association study has reported a significant linkage between a single-nucleotide polymorphism ∼11.7 kb downstream of the gene encoding the RING finger ubiquitin ligase rififylin (RFFL) and variability in the QT interval. This, along with results in animal studies, suggests that RFFL may have effects on cardiac repolarization. Here, we sought to determine the role of RFFL in cardiac electrophysiology. Adult rabbit cardiomyocytes with adenovirus-expressed RFFL exhibited reduced rapid delayed rectifier current (I Kr). Neonatal rabbit cardiomyocytes transduced with RFFL-expressing adenovirus exhibited reduced total expression of the potassium channel ether-a-go-go-related gene (rbERG). Using transfections of 293A cells and Western blotting experiments, we observed that RFFL and the core-glycosylated form of the human ether-a-go-go-related gene (hERG) potassium channel interact. Furthermore, RFFL overexpression led to increased polyubiquitination and proteasomal degradation of hERG protein and to an almost complete disappearance of I Kr, which depended on the intact RING domain of RFFL. Blocking the ER-associated degradation (ERAD) pathway with a dominant-negative form of the ERAD core component, valosin-containing protein (VCP), in 293A cells partially abolished RFFL-mediated hERG degradation. We further substantiated the link between RFFL and ERAD by showing an interaction between RFFL and VCP in vitro We conclude that RFFL is an important regulator of voltage-gated hERG potassium channel activity and therefore cardiac repolarization and that this ubiquitination-mediated regulation requires parts of the ERAD pathway.


Assuntos
Canal de Potássio ERG1/metabolismo , Degradação Associada com o Retículo Endoplasmático , Miócitos Cardíacos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Canal de Potássio ERG1/genética , Células HEK293 , Humanos , Transporte Proteico , Coelhos , Ubiquitina-Proteína Ligases/genética
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