hERG Current Research Articles

Intracellular Binding of Terfenadine Competes with Its Access to Pancreatic ß-cell ATP-Sensitive K+ Channels and Human ether-à-go-go-Related Gene Channels

Most blockers of both hERG (human ether-à-go-go-related gene) channels and pancreatic ß-cell ATP-sensitive K+ (KATP) channels access their binding sites from the cytoplasmic side of the plasma membrane. It is unknown whether binding to intracellular components competes with binding of these substances to K+ channels. The whole-cell configuration of the patch-clamp technique, a laser-scanning confocal microscope, and fluorescence correlation spectroscopy (FCS) were used to study hERG channels expressed in HEK (human embryonic kidney) 293 cells and KATP channels from the clonal insulinoma cell line RINm5F. When applied via the pipette solution in the whole-cell configuration, terfenadine blocked both hERG and KATP currents with much lower potency than after application via the bath solution, which was not due to P-glycoprotein-mediated efflux of terfenadine. Such a difference was not observed with dofetilide and tolbutamide. 37–68% of hERG/EGFP (enhanced green-fluorescent protein) fusion proteins expressed in HEK 293 cells were slowly diffusible as determined by laser-scanning microscopy in the whole-cell configuration and by FCS in intact cells. Bath application of a green-fluorescent sulphonylurea derivative (Bodipy-glibenclamide) induced a diffuse fluorescence in the cytosol of RINm5F cells under whole-cell patch-clamp conditions. These observations demonstrate the presence of intracellular binding sites for hERG and KATP channel blockers not dialyzable by the patch-pipette solution. Intracellular binding of terfenadine was not influenced by a mutated hERG (Y652A) channel. In conclusion, substances with high lipophilicity are not freely diffusible inside the cell but steep concentration gradients might exist within the cell and in the sub-membrane space.Graphical

A Comprehensive Evaluation of Sdox, a Promising H2S-Releasing Doxorubicin for the Treatment of Chemoresistant Tumors.

Sdox is a hydrogen sulfide (H2S)-releasing doxorubicin effective in P-glycoprotein-overexpressing/doxorubicin-resistant tumor models and not cytotoxic, as the parental drug, in H9c2 cardiomyocytes. The aim of this study was the assessment of Sdox drug-like features and its absorption, distribution, metabolism, and excretion (ADME)/toxicity properties, by a multi- and transdisciplinary in silico, in vitro, and in vivo approach. Doxorubicin was used as the reference compound. The in silico profiling suggested that Sdox possesses higher lipophilicity and lower solubility compared to doxorubicin, and the off-targets prediction revealed relevant differences between Dox and Sdox towards several cancer targets, suggesting different toxicological profiles. In vitro data showed that Sdox is a substrate with lower affinity for P-glycoprotein, less hepatotoxic, and causes less oxidative damage than doxorubicin. Both anthracyclines inhibited CYP3A4, but not hERG currents. Unlike doxorubicin, the percentage of zebrafish live embryos at 72 hpf was not affected by Sdox treatment. In conclusion, these findings demonstrate that Sdox displays a more favorable drug-like ADME/toxicity profile than doxorubicin, different selectivity towards cancer targets, along with a greater preclinical efficacy in resistant tumors. Therefore, Sdox represents a prototype of innovative anthracyclines, worthy of further investigations in clinical settings.

Combining in-silico cardiac action potentials with HERG currents expressed in HEK cells using dynamic clamp in synthetic cell mode

We used real-time dynamic clamp to allow heterologously-expressed cloned ion channel currents expressed in cells to be input into an in silico cardiac action potential (AP) model (Synthetic Cell Mode). The effect of dofetilide, a HERG blocker, on AP duration was measured. HERG currents were recorded from a stable cell line (HERG_A:HEK, BPS Bioscience) in whole cell voltage clamp. HERG currents were monitored until stable. In Synthetic Cell Mode, we input the HERG current into an in silico AP model consisting of electronically expressed INa, IK1, IKS, ICa_L and Ito.

Identification through action potential clamp of proarrhythmic consequences of the short QT syndrome T618I hERG ‘hotspot’ mutation

The T618I KCNH2-encoded hERG mutation is the most frequently observed mutation in genotyped cases of the congenital short QT syndrome (SQTS), a cardiac condition associated with ventricular fibrillation and sudden death. Most T618I hERG carriers exhibit a pronounced U wave on the electrocardiogram and appear vulnerable to ventricular, but not atrial fibrillation (AF). The basis for these effects is unclear. This study used the action potential (AP) voltage clamp technique to determine effects of the T618I mutation on hERG current (IhERG) elicited by APs from different cardiac regions. Whole-cell patch-clamp recordings were made at 37 °C of IhERG from hERG-transfected HEK-293 cells. Maximal IhERG during a ventricular AP command was increased ∼4-fold for T618I IhERG and occurred much earlier during AP repolarization. The mutation also increased peak repolarizing currents elicited by Purkinje fibre (PF) APs. Maximal wild-type (WT) IhERG current during the PF waveform was 87.2 ± 4.5% of maximal ventricular repolarizing current whilst for the T618I mutant, the comparable value was 47.7 ± 2.7%. Thus, the T618I mutation exacerbated differences in repolarizing IhERG between PF and ventricular APs; this could contribute to heterogeneity of ventricular-PF repolarization and consequently to the U waves seen in T618I carriers. The comparatively shorter duration and lack of pronounced plateau of the atrial AP led to a smaller effect of the T618I mutation during the atrial AP, which may help account for the lack of reported AF in T618I carriers. Use of a paired ventricular AP protocol revealed an alteration to protective IhERG transients that affect susceptibility to premature excitation late in AP repolarization/early in diastole. These observations may help explain altered arrhythmia susceptibility in this form of the SQTS.

Investigation of PAS and CNBH domain interactions in hERG channels and effects of long-QT syndrome-causing mutations with surface plasmon resonance

Human ether-á-go-go-related gene (hERG) channels are key regulators of cardiac repolarization, neuronal excitability, and tumorigenesis. hERG channels contain N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBH) domains with many long-QT syndrome (LQTS)-causing mutations located at the interface between these domains. Despite the importance of PAS/CNBH domain interactions, little is known about their affinity. Here, we used the surface plasmon resonance (SPR) technique to investigate interactions between isolated PAS and CNBH domains and the effects of LQTS-causing mutations R20G, N33T, and E58D, located at the PAS/CNBH domain interface, on these interactions. We determined that the affinity of the PAS/CNBH domain interactions was ∼1.4 μM. R20G and E58D mutations had little effect on the domain interaction affinity, while N33T abolished the domain interactions. Interestingly, mutations in the intrinsic ligand, a conserved stretch of amino acids occupying the beta-roll cavity in the CNBH domain, had little effect on the affinity of PAS/CNBH domain interactions. Additionally, we determined that the isolated PAS domains formed oligomers with an interaction affinity of ∼1.6 μM. Coexpression of the isolated PAS domains with the full-length hERG channels or addition of the purified PAS protein inhibited hERG currents. These PAS/PAS interactions can have important implications for hERG function in normal and pathological conditions associated with increased surface density of channels or interaction with other PAS-domain-containing proteins. Taken together, our study provides the first account of the binding affinities for wild-type and mutant hERG PAS and CNBH domains and highlights the potential functional significance of PAS/PAS domain interactions.

Assessment of proarrhythmogenic risk for chloroquine and hydroxychloroquine using the CiPA concept

Chloroquine and hydroxychloroquine have been proposed recently as therapy for SARS-CoV-2-infected patients, but during 3 months of extensive use concerns were raised related to their clinical effectiveness and arrhythmogenic risk. Therefore, we estimated for these compounds several proarrhythmogenic risk predictors according to the Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm. Experiments were performed with either CytoPatch™2 automated or manual patch-clamp setups on HEK293T cells stably or transiently transfected with hERG1, hNav1.5, hKir2.1, hKv7.1+hMinK, and on Pluricyte® cardiomyocytes (Ncardia), using physiological solutions. Dose-response plots of hERG1 inhibition fitted with Hill functions yielded IC50 values in the low micromolar range for both compounds. We found hyperpolarizing shifts of tens of mV, larger for chloroquine, in the voltage-dependent activation but not inactivation, as well as a voltage-dependent block of hERG current, larger at positive potentials. We also found inhibitory effects on peak and late INa and on IK1, with IC50 of tens of μM and larger for chloroquine. The two compounds, tested on Pluricyte® cardiomyocytes using the β-escin-perforated method, inhibited IKr, ICaL, INa peak, but had no effect on If. In current-clamp they caused action potential prolongation. Our data and those from literature for Ito were used to compute proarrhythmogenic risk predictors Bnet (Mistry HB, 2018) and Qnet (Dutta S et al., 2017), with hERG1 blocking/unblocking rates estimated from time constants of fractional block. Although the two antimalarials are successfully used in autoimmune diseases, and chloroquine may be effective in atrial fibrillation, assays place these drugs in the intermediate proarrhythmogenic risk group.

Inhibition of the hERG potassium channel by phenanthrene: a polycyclic aromatic hydrocarbon pollutant

The lipophilic polycyclic aromatic hydrocarbon (PAH) phenanthrene is relatively abundant in polluted air and water and can access and accumulate in human tissue. Phenanthrene has been reported to interact with cardiac ion channels in several fish species. This study was undertaken to investigate the ability of phenanthrene to interact with hERG (human Ether-à-go-go-Related Gene) encoded Kv11.1 K+ channels, which play a central role in human ventricular repolarization. Pharmacological inhibition of hERG can be proarrhythmic. Whole-cell patch clamp recordings of hERG current (IhERG) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT IhERG1a was inhibited by phenanthrene with an IC50 of 17.6 ± 1.7 µM, whilst IhERG1a/1b exhibited an IC50 of 1.8 ± 0.3 µM. WT IhERG block showed marked voltage and time dependence, indicative of dependence of inhibition on channel gating. The inhibitory effect of phenanthrene was markedly impaired by the attenuated inactivation N588K mutation. Remarkably, mutations of S6 domain aromatic amino acids (Y652, F656) in the canonical drug binding site did not impair the inhibitory action of phenanthrene; the Y652A mutation augmented IhERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit IhERG, as did the S624A mutation below the selectivity filter region. Computational docking using a cryo-EM derived hERG structure supported the mutagenesis data. Thus, phenanthrene acts as an inhibitor of the hERG K+ channel by directly interacting with the channel, binding to a distinct site in the channel pore domain.

The Effect of a Synthetic Estrogen, Ethinylestradiol, on the hERG Block by E-4031.

Inhibition of K+-conductance through the human ether-a-go-go related gene (hERG) channel leads to QT prolongation and is associated with cardiac arrhythmias. We previously reported that physiological concentrations of some estrogens partially suppress the hERG channel currents by interacting with the S6 residue F656 and increase the sensitivity of hERG blockade by E-4031. Although these studies suggested that clinically used synthetic estrogens with similar structures have the marked potential to alter hERG functions, the hERG interactions with synthetic estrogens have not been assessed. We therefore examined whether ethinylestradiol (EE2), a synthetic estrogen used in oral contraceptives, affects hERG function and blockade by drugs. Supratherapeutic concentrations of EE2 did not alter amplitudes or kinetics of the hERG currents elicited by train pulses at 20 mV (0.1 Hz). On the other hand, EE2 at therapeutic concentrations reduced the degree of hERG current suppression by E-4031. The administration of EE2 followed by E-4031 blockade reversed the current suppression, suggesting that the interaction of EE2 and E-4031 alters hERG at the drug-binding site. The effects of EE2 on hERG blockade raised the possibility that other estrogens, including synthetic estrogens, can alter hERG blockade by drugs that cause QT prolongation and ventricular arrhythmias.

Abstract P402: Caveolin-1 Upregulation Leads To Decreased Erg And Stress-induced Ventricular Tachycardia In Cystic Fibrosis Hearts

Background: Cardiac manifestations of cystic fibrosis are classically accepted as cor pulmonale, yet emerging evidence supports that cardiac abnormalities can develop early when respiratory function is preserved. Myocardial cystic fibrosis transmembrane regulator (CFTR) channels, inactive in normal conditions, are activated in response to catecholamine stress. Methods and Results: In multiple mouse models of homozygous CFTR mutations (S489X or F508d), we demonstrate increased susceptibility to ventricular tachycardia (VT) in response to epinephrine, and this is associated with prolongation of QTc intervals (repolarization). Despite having normal left ventricular ejection fraction and histology, CF S489X mut/mut hearts showed 460 proteins significantly altered compared with WT hearts by tandem mass tag (TMT) proteomics. The Ingenuity Pathway Analysis of proteomics data revealed mitochondrial dysfunction and increased caveolin-1 signaling among the top pathways affected in CF S489X mut/mut hearts. In mouse hearts and human induced pluripotent stem cells derived cardiomyocytes (iPS-CM) with CFTR mutations, we observed increased ER stress and caveolin-1 (cav-1). These changes are concomitant with reduced levels of the ERG channel, a K+ channel contributing to phase 3 repolarization, mutations of which cause long QT syndrome II and VT. Patch clamp analysis showed decreased phase 2-3 repolarization current (including hERG current) in CF F508d iPS-CM compared with control iPS-CM, consistent with the findings of decreased membranous hERG and elevated Cav-1. Suppression of Cav-1 successfully restores membranous hERG in two separate lines of mutant iPS-CM generated from CF patients with F508d mutation. In F508d mut/mut mice, suppression of cardiac Cav-1 upregulation by reducing ER stress using TUDCA or crossing with heterozygous Cav-1 +/- abolished the stress-induced VT. Our analysis of a large-scale real-world clinical database from TriNetX, including 4520 CF men, 3510 CF women and the same number of well-matched controls demonstrated novel findings of increased risk for QT prolongation and VT in CF patients. Retrospective analysis of 126 CF patients with available EKG in our hospital revealed significant QTc prolongation in CF patients compared with matched controls, for both males (CF 447.8±4.5 vs cont. 429.2±2.5) and females (CF 454.0±3.5 vs cont.437.5±2.4). The QTc interval is not affected by lung function (%predicted FEV1) and is an independent predictor of survival in CF patients. Conclusions: In mice and iPS-CM models of CF, we demonstrated impaired repolarization and increased susceptibility to stress-induced VT. This is mediated by ER-stress-induced upregulation of Cav-1, which leads to internalization of ERG and subsequent decrease in repolarization current.

Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243.

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.

Dual Mechanisms of Cardiac Action Potential Prolongation by 4-Oxo-Nonenal Increasing the Risk of Arrhythmia; Late Na+ Current Induction and hERG K+ Channel Inhibition.

4-Oxo-nonenal (4-ONE) is an endogenous lipid peroxidation product that is more reactive than 4-hydroxy-nonenal (4-HNE). We previously reported the arrhythmic potential of 4-HNE by suppression of cardiac human Ether-a-go-go Related Gene (hERG) K+ channels with prolonged action potential duration (APD) in cardiomyocytes. Here, we illustrate the higher arrhythmic risk of 4-ONE by modulating the cardiac hNaV1.5 channel currents (INaV). Although the peak amplitude of INaV was not significantly changed by 4-ONE up to 10 μM, the rate of INaV inactivation was slowed, and the late Na+ current (INaL) became larger by 10 μM 4-ONE. The chemical modification of specific residues in hNaV1.5 by 4-ONE was identified using MS-fingerprinting analysis. In addition to the changes in INaV, 4-ONE decreased the delayed rectifier K+ channel currents including the hERG current. The L-type Ca2+ channel current was decreased, whereas its inactivation was slowed by 4-ONE. The APD prolongation by 10 μM of 4-ONE was more prominent than that by 100 μM of 4-HNE. In the computational in silico cardiomyocyte simulation analysis, the changes of INaL by 4-ONE significantly exacerbated the risk of arrhythmia exhibited by the TdP marker, qNet. Our study suggests an arrhythmogenic effect of 4-ONE on cardiac ion channels, especially hNaV1.5.

Proton Pump Inhibitors Directly Block hERG-Potassium Channel and Independently Increase the Risk of QTc Prolongation in a Large Cohort of US Veterans

[Figure: see text].

644-P: Discovery of Nonpeptide, Orally Available Small Molecule GLP-1 Receptor Agonists

GLP-1R agonists comprise a growing class of agents that deliver unprecedented efficacy in the treatment of diabetes. We discovered promising compounds for the development of a novel small molecule GLP-1R agonist and examined their efficacy in cynomolgus monkey. The in vitro activities of the compounds were evaluated by measuring cAMP accumulation in cells expressing human GLP-1R. All compounds demonstrated potent cAMP accumulation of EC50 value below 20 nM, with one compound showing 1.5 nM. In addition, the compounds showed excellent selectivity against related class B GPCR, with no stimulatory activity on human GIP, GCG, and GLP-2 receptors. In the intravenous glucose tolerance test, according to the present disclosure, the compounds increased insulin secretion in cynomolgus monkey (dosed 50 mg/kg, p.o.), demonstrating their efficacy as orally available GLP-1 receptor agonists. Moreover, inhibitory effect on the hERG current was investigated and the compounds were shown to have favorable safety profile with IC50 values ranging from 34.6 to more than 100 μM. In conclusion, we discovered promising compounds which have potential to be developed as an orally active, non-peptide GLP-1R agonist for the treatment of type 2 diabetes with favorable safety profile regarding the hERG current. Disclosure A. Im: None. Y. Jun: None. E. Jang: None. Y. Yoo: None. W. Jeon: None. K. An: None. J. Park: None. D. Hong: None. I. Je: None. J. Kim: None. M. Chang: None. C. Hong: None. J. Shin: None. T. Sohn: None.

Post myocardial infarction ionic remodelling promotes repolarisation dispersions and abnormalities in acute and chronic stages

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Wellcome Trust Background Sudden cardiac death after a myocardial infarction (MI) was reported in both the acute stage and the chronic stage with the development of heart failure (HF). Around 50% of these deaths were due to ventricular tachyarrhythmia which is related to the temporal dispersion of repolarisation caused by the post-MI remodelling of ionic currents. While variable extent of ionic current changes was observed in multiple animal species in different post-MI phases, the proarrhythmic effects of such remodelling in post infarction human hearts is not fully explored. Purpose The goal of this study is to explore the proarrhythmic effects of post-MI ionic remodelling in human ventricular myocyte models of electrophysiology for both the acute and the chronic stages. Methods The ToR-ORd human ventricular cellular model was used as the baseline model, and a new formulation of the calcium activated potassium current was implemented. We constructed and calibrated a population of 253 human ventricular cell models (POM) to represent electrophysiological variability of normal zone (NZ) myocytes. For the acute phase, three types of border zone (BZ) remodelling (Acute BZ1-3) were introduced based on dog experimental data collected within one week after infarction. For the chronic phase with the development of HF, BZ (Chronic BZ) and one type of remote zone (RZ) remodelling (Chronic RZ1) were introduced based on minipig data collected 5 months post-MI. Another type of RZ remodelling (Chronic RZ2) was introduced based on experimental data from failing human hearts. The cellular models were paced at cycle lengths of 1000ms, 500ms, 400ms and 300ms for 500 beats before the analysis of repolarization abnormalities. Results For both the acute and the chronic stages post-MI, the extent of repolarisation dispersion between BZ and the corresponding NZ and RZ varied from 2ms up to 120ms, and largely depended on the level of potassium current remodelling. A weaker calcium transient was always observed in the BZ. Early after depolarisations (EADs) and repolarisation failure (RF) were observed in the chronic stages, caused by the reactivation of L-type calcium current due to the augmentation of late sodium current and the inhibition of hERG current. Beat-to-beat repolarization alternans was observed in both the acute and the chronic stages, with the key ionic mechanisms being the inhibition of SERCA activity and the slower calcium release due to enhanced CaMKII. Conclusions Post-MI ionic remodelling of the acute and chronic stages can cause a substantial dispersion of repolarisation in human hearts by inducing the prolongation of action potential durations, EADs, RFs, and alternans, providing substrates for the generation and maintenance of lethal reentrant waves. Abstract Figure. BZ and RZ ionic current remodelling

Rutaecarpine targets hERG channels and participates in regulating electrophysiological properties leading to ventricular arrhythmia.

Drug‐mediated or medical condition‐mediated disruption of hERG function accounts for the main cause of acquired long‐QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG‐HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (IhERG) was measured by patch‐clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the IhERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell‐based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD90 and APD50 and the increase of DF, numbers of PSs, incidence of early after‐depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the IhERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long‐term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.

Effects of Estrogens on the Actions of hERG

Regulation of cardiac ion channels by sex hormones accounts for gender-differences in susceptibility of Torsades de Pointes (TdP) arrhythmias associated with QT prolongation, that is: women are more prone to develop drug-induced arrhythmias which can lead cardiac sudden death. We previously found that β-estradiol (E2) directly interacts with a drug-binding site, F656 of the hERG channel, and alters the effect of a selective hERG blocker. Although these actions were observed for other estrogens, the effect of estrogen on other drugs has been unknown. In this study, to reveal impacts of the actions of estrogens on hERG currents, we compared the hERG screen data in the absence and the presence of estrogens using manual and automated patch-clamp experiments. HEK293 cells stably-expressing the hERG channels were seeded into 384-well plates, and were cultured in a steroid-free condition. Whole-cell hERG currents were recorded by the patch-clamp technique with SyncroPatch 384i (Nanion). Effects of 22 compounds were assessed by percentage changes of hERG peak tail currents with or without estrogens (E2, estrone sulfate; E1S, and ethynylestradiol; EE2) and by molecular docking simulations. Our hERG screening depicted that some estrogens showed distinct effects on respective blockers. Estrogens did not change the degree of hERG block by most of compounds. However, we have newly found that inhibitory action of one drug was increased by E2, and that of another drug was decreased by EE2. This means that specific combinations of an estrogen and a hERG blocker can alter the hERG blockade significantly. Indeed, some combinations resulted in larger hERG currents. This study suggests potential risks using estrogen therapy/medication and the necessity of taking sex hormonal differences into consideration for more accurate prediction of QT prolongation proclivity of drug candidates.

A mechanism underlying hERG current increase by a blocker

Several hERG blockers with good clinical safety profiles share a common attribute: They all paradoxically increase hERG current at potentials near the threshold for its activation. We refer to this phenomenon as "facilitation." The molecular mechanism underlying facilitation remains unknown. Here, we propose a mechanism through which facilitation arises from drug effects on hERG channel gating kinetics. We found that 1) nifekalant, a hERG facilitator, accesses the receptor site within the pore of the open or inactivated channels at depolarized potentials, 2) upon return to the resting potentials, channels close trapping nifekalant inside, 3) trapped nifekalant biases the open-closed equilibrium towards the open state, and 4) the kinetics of drug escape from the channel are faster than channel closing rates at potentials where facilitation of hERG current is observed, thereby drug unbinding reveals channels that have been biased towards the open state. Simulations with a Markov model of such nifekalant-hERG interaction successfully reproduce key characteristics of hERG facilitation. Therefore, hERG facilitation arises from the coupling of the features of the channel gating, slow opening and closing, with a drug action on the gating, similar to a classic "foot-in-the-door" mechanism.

Berberine on the Prevention and Management of Cardiometabolic Disease: Clinical Applications and Mechanisms of Action.

Berberine is an alkaloid from several medicinal plants originally used to treat diarrhea and dysentery as a traditional Chinese herbal medicine. In recent years, berberine has been discovered to exhibit a wide spectrum of biological activities in the treatment of diverse diseases ranging from cancer and neurological dysfunctions to metabolic disorders and heart diseases. This review article summarizes the clinical practice and laboratory exploration of berberine for the treatment of cardiometabolic and heart diseases, with a focus on the novel insights and recent advances of the underlying mechanisms recognized in the past decade. Berberine was found to display pleiotropic therapeutic effects against dyslipidemia, hyperglycemia, hypertension, arrhythmia, and heart failure. The mechanisms of berberine for the treatment of cardiometabolic disease involve combating inflammation and oxidative stress such as inhibiting proprotein convertase subtilisin/kexin 9 (PCSK9) activation, regulating electrical signals and ionic channels such as targeting human ether-a-go-go related gene (hERG) currents, promoting energy metabolism such as activating adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, modifying gut microbiota to promote transforming of berberine into its intestine-absorbable form, and interacting with non-coding RNAs via targeting multiple signaling pathways such as AMPK, mechanistic target of rapamycin (mTOR), etc. Collectively, berberine appears to be safe and well-tolerated in clinical practice, especially for those who are intolerant to statins. Knowledge from this field may pave the way for future development of more effective pharmaceutical approaches for managing cardiometabolic risk factors and preventing heart diseases.

Ginsenoside Rg3 Alleviates Antithyroid Cancer Drug Vandetanib-Induced QT Interval Prolongation.

Inhibition of human ether-a-go-go-related gene (hERG) potassium channel is responsible for acquired long QT syndromes, which leads to life-threatening cardiac arrhythmia. A multikinase inhibitor, vandetanib, prolongs the progression-free survival time in advanced medullary thyroid cancer. However, vandetanib has been reported to induce significant QT interval prolongation, which limits its clinical application. Some studies have showed that ginsenoside Rg3 decelerated hERG K(+) channel tail current deactivation. Therefore, in this study, we aim to confirm whether ginsenoside Rg3 targeting hERG K(+) channel could be used to reverse the vandetanib-induced QT interval prolongation. Electrocardiogram (ECG) and monophasic action potential (MAP) were recorded using electrophysiology signal sampling and analysis system in Langendorff-perfused rabbit hearts. The current clamp mode of the patch-clamp technique was used to record transmembrane action potential. The whole-cell patch-clamp technique was used to record the hERG K+ current. In Langendorff-perfused hearts, vandetanib prolonged the QT interval in a concentration-dependent manner with an IC50 of 1.96 μmol/L. In human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), vandetanib significantly prolonged the action potential duration at 50%, 70%, and 90% repolarization (APD50, APD70, and APD90). In stable transfected human hERG gene HEK293 cells, vandetanib caused concentrate-dependent inhibition in the step and tail currents of hERG. As expected, ginsenoside Rg3 relieved vandetanib-induced QT interval prolongation in Langendorff-perfused heart and reversed vandetanib-induced APD prolongation in hiPSC-CMs. Furthermore, ginsenoside Rg3 alleviated vandetanib-induced hERG current inhibition and accelerated the process of the channel activation. Ginsenoside Rg3 may be a promising cardioprotective agent against vandetanib-induced QT interval prolongation through targeting hERG channel. These novel findings highlight the therapeutic potential of ginsenoside to prevent vandetanib-induced cardiac arrhythmia.

Abstract 17074: Methadone Potently Blocks Cardiac IK1 Leading to Membrane Instability

Introduction: Methadone is the second most frequently reported cause of drug-induced cardiac arrest in pharmacovigilance databases, yet the mechanism of its pro-arrhythmia is unclear. Methadone-induced QTU wave prolongation has been repeatedly observed and attributed to inhibition of the delayed rectifier hERG current, but QTU fusion suggests the inwardly rectifying K + current (IK1) might also be affected by methadone. Hypothesis: Methadone pro-arrhythmia is associated with potent block of the IK1 current responsible for rapid terminal repolarization of the cardiac action potential (AP). Methods: Human Kir2.2, encoding the IK1 current, was transiently expressed in COS cells. hERG1a was stably expressed in CHO cells. Cardiac myocytes from swine were obtained by ventricular enzymatic dissociation. Ionic current and APs were measured using patch-clamp methods. Methadone HCl (R+S racemates) was dissolved in Tyrode solution. Results: Methadone suppressed IK1 current with an IC 50 of 1.47 uM (Fig 1A). Methadone also suppressed outward IK1 (-60 mV) measured in swine myocytes (Ba 2+ -sensitive current) with an IC 50 of 1.52 μM. Methadone suppressed hERG currents with an IC 50 of 2.1 μM. APs measured in swine myocytes exhibited significant prolongation (13 ± 4 % increase of APD 90 , p<0.029, n=7) as well as slowing of the rate of terminal repolarization (a specific marker of IK1 blockade) in the presence of 1 μM methadone (Fig. 1B). Fluctuations of diastolic voltage increased by 30 ± 12 and 151 ± 27 % (n=3; p<0.04) in 0.1 and 1 μM methadone, respectively, consistent with a reduction in membrane stability. Conclusions: Methadone is an equipotent blocker of IK1 and hERG. The effect of IK1 block coupled with modest hERG block has a synergistic effect on terminal repolarization that may partially explain the pro-arrhythmic impact of methadone. Moreover, this observation may be generalized to other drugs where unsuspected IK1 blockade may contribute to pro-arrhythmia and torsade de pointes.