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Background Mutations in the HERG potassium channel are a major cause of long QT syndrome type 2 (LQT2), which can lead to sudden cardiac death. The HERG channel plays a critical role in the repolarization of the myocardial action potential, and loss-of-function mutations prolong cardiac repolarization. Methods In this study, we investigated the efficacy and underlying molecular mechanism of ICA-105574, an HERG activator, in shortening the duration of cardiac repolarization in severe LQT2 variants. We characterized the efficacy of ICA-105574 in vivo, using an animal model to assess its ability to shorten the QT interval and in vitro, in cellular models mimicking severe HERG channel mutations (A561V, G628S, and L779P) to evaluate its impact in enhancing I Kr current. Additionally, molecular dynamics simulations were used to investigate the molecular mechanism of ICA-105574 action. Results In vivo, ICA-105574 significantly shortened the QT interval. LQT2 mutations drastically reduced I Kr amplitude and suppressed tail currents in cellular models. ICA-105574 restored I Kr in A561V and G628S. Finally, in silico data showed that ICA-105574 stabilizes a pattern of interactions similar to gain-of-function SQT1 mutations and can reverse the G628S modifications, through an allosteric network linking the binding site to the selectivity filter and the S5P turret helix, thereby restoring its K + ion permeability. Conclusions Our results support the development of HERG activators like ICA-105574 as promising pharmacological molecules against some severe LQT2 mutations and suggest that molecular dynamics simulations can be used to test the ability of molecules to modulate HERG function in silico, paving the way for the rational design of new HERG activators.

The voltage‐gated hERG (human‐Ether‐à‐go‐go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss‐of‐function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain‐of‐function mutations lead to short QT syndrome. A recent open channel cryo‐EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure‐function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full‐length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole‐cell measurements of hERG current (IhERG) were made at 37°C from HEK 293 cells expressing wild‐type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end‐pulse nor tail IhERG density significantly differed between WT and hERGT. However, the IhERG deactivation rate was significantly slower for hERGT. Half‐maximal activation voltage (V0.5) was positively shifted for hERGT by ~+8 mV (p < .05 versus WT), without significant change to the activation relation slope factor. Neither the voltage dependence of inactivation, nor time course of development of inactivation significantly differed between WT and hERGT, but recovery of IhERG from inactivation was accelerated for hERGT (p < .05 versus WT). Steady‐state “window” current was positively shifted for hERGT with a modest increase in the window current peak. Under action potential (AP) voltage clamp, hERGT IhERG showed modestly increased current throughout the AP plateau phase with a significant increase in current integral during the AP. The observed consequences for hERGT IhERG of deletion of the two cytoplasmic regions may reflect changes to electrostatic interactions influencing the voltage sensor domain. The hERGTtruncation mutant was made to derive the cryo‐EM structure that is employed to study hERG K+channel structure function. This paper shows that the hERGT mutant exhibits modest differences in hERG current kinetics from the wild‐type channel. Voltage‐dependent activation and “window current” were positively voltage‐shifted, deactivation was slowed and recovery from inactivation was faster for hERGT compared to the wild‐type channel.

Background Cyclic Nucleotide-Binding Domain (CNBD)-family channels display distinct voltage-sensing properties despite sharing sequence and structural similarity. For example, the human Ether-a-go-go Related Gene (hERG) channel and the Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) channel share high amino acid sequence similarity and identical domain structures. hERG conducts outward current and is activated by positive membrane potentials (depolarization), whereas HCN conducts inward current and is activated by negative membrane potentials (hyperpolarization). The structural basis for the “opposite” voltage-sensing properties of hERG and HCN remains unknown. Results We found the voltage-sensing domain (VSD) involves in modulating the gating polarity of hERG. We identified that a long-QT syndrome type 2-related mutation within the VSD, K525N, mediated an inwardly rectifying non-deactivating current, perturbing the channel closure, but sparing the open state and inactivated state. K525N rescued the current of a non-functional mutation in the pore helix region (F627Y) of hERG. K525N&F627Y switched hERG into a hyperpolarization-activated channel. The reactivated inward current induced by hyperpolarization mediated by K525N&F627Y can be inhibited by E-4031 and dofetilide quite well. Moreover, we report an extracellular interaction between the S1 helix and the S5-P region is crucial for modulating the gating polarity. The alanine substitution of several residues in this region (F431A, C566A, I607A, and Y611A) impaired the inward current of K525N&F627Y. Conclusions Our data provide evidence that a potential cooperation mechanism in the extracellular vestibule of the VSD and the PD would determine the gating polarity in hERG.

Background: Congenital long QT syndrome (LQTS) is a rare cardiac disorder caused by repolarization abnormalities in the myocardium that predisposes to ventricular arrhythmias and sudden cardiac death. Potassium channel-mediated LQT1 and LQT2 are the most common types of channelopathy. Recently, LQTS has been acknowledged as an electromechanical disease. Methods: A total of 87 genotyped LQT1/LQT2 patients underwent cardiac evaluation. A comparison between LQT1 and LQT2 electrical and mechanical parameters was performed. Results: LQT2 patients had worse electrical parameters at rest: a longer QTc interval (p = 0.007), a longer Tpe in lead V2 (p = 0.028) and in lead V5 (p < 0.001), and a higher Tpe/QT ratio in lead V2 (p = 0.011) and in lead V5 (p = 0.005). Tpe and Tpe/QT remained significantly higher in the LQT2 group after brisk standing. Tpe was longer in LQT2 patients compared with LQT1 patients during peak exercise (p = 0.007) and almost all recovery periods in lead V2 during EST. The mid-cavity myocardium mean radial contraction duration (CD) was longer in LQT2 patients (p = 0.02). LQT2 patients had a longer mean radial CD in mid-septal (p = 0.015), mid-inferior (p = 0.034), and mid-posterior (p = 0.044) segments. Conclusions: Potassium channel-mediated LQTS has different effects on cardiac electromechanics with a more pronounced impact on LQT2 patients. Tpe was more prominent in the LQT2 cohort, not only at rest and brisk standing but also during EST exercise and at recovery phases. The altered mean radial CD in the mid-cavity myocardium was also specific for LQT2 patients.

Mutations in the HERG gene encoding the potassium ion channel HERG, represent one of the most frequent causes of long QT syndrome type-2 (LQT2). The same genetic mutation frequently presents different clinical phenotypes in the family. Our study aimed to model LQT2 and study functional differences between the mutation carriers of variable clinical phenotypes. We derived human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) from asymptomatic and symptomatic HERG mutation carriers from the same family. When comparing asymptomatic and symptomatic single LQT2 hiPSC-CMs, results from allelic imbalance, potassium current density, and arrhythmicity on adrenaline exposure were similar, but a difference in Ca2+ transients was observed. The major differences were, however, observed at aggregate level with increased susceptibility to arrhythmias on exposure to adrenaline or potassium channel blockers on CM aggregates derived from the symptomatic individual. The effect of this mutation was modeled in-silico which indicated the reactivation of an inward calcium current as one of the main causes of arrhythmia. Our in-vitro hiPSC-CM model recapitulated major phenotype characteristics observed in LQT2 mutation carriers and strong phenotype differences between LQT2 asymptomatic vs. symptomatic were revealed at CM-aggregate level.

Background: Methadone is prescribed to heroin addicts to decrease illicit opioid use. Prolongation of the QT interval in the ECG of patients with torsade de pointes (TdP) has been reported in methadone users. As heroin addicts sometimes faint while using illicit drugs, doctors might attribute too many episodes of syncope to illicit drug use and thereby underestimate the incidence of TdP in this special population, and the high mortality in this population may, in part, be caused by the proarrhythmic effect of methadone. Methods: In this cross-sectional study interview, ECGs and blood samples were collected in a population of adult heroin addicts treated with methadone or buprenorphine on a daily basis. Of the patients at the Drug Addiction Service in the municipal of Copenhagen, 450 (∼52%) were included. The QT interval was estimated from 12 lead ECGs. All participants were interviewed about any experience of syncope. The association between opioid dose and QT, and methadone dose and reporting of syncope was assessed using multivariate linear regression and logistic regression, respectively. Results: Methadone dose was associated with longer QT interval of 0.140 ms/mg (p = 0.002). No association between buprenorphine and QTc was found. Among the subjects treated with methadone, 28% men and 32% women had prolonged QTc interval. None of the subjects treated with buprenorphine had QTc interval >0.440s½. A 50 mg higher methadone dose was associated with a 1.2 (95% CI 1.1 to 1.4) times higher odds for syncope. Conclusions: Methadone is associated with QT prolongation and higher reporting of syncope in a population of heroin addicts.

Background The protective effect of beta-blockers in patients with inherited Long-QT syndrome is well established. Recent reports have suggested that beta-blockers are not equally effective in Long-QT (LQT). Bisoprolol is an attractive candidate for use in LQT because of its cardioselective properties and favorable side-effect profile. Methods We performed a retrospective cohort study of 114 consecutive patients with gene-positive Long-QT syndrome type 1 (LQT1) or Long-QT syndrome type 2 (LQT2) treated with bisoprolol, nadolol or atenolol with a total of 580 person-years of follow-up. Electrocardiogram (ECG) parameters and cardiac events during follow-up were compared. In addition, exercise treadmill testing was performed in bisoprolol-treated patients. Results Fifty-nine patients were treated with bisoprolol, 39 with atenolol and 16 with nadolol. Overall, 59 % were females and 62 % had LQT1. Baseline heart rate and corrected QT (QTc) interval were similar between the groups. QTc shortening was observed in individuals on bisoprolol (ΔQTc −5 ± 31 ms; p  = 0.049) and nadolol (ΔQTc −13 ± 16 ms; p  = 0.02) but not on atenolol (ΔQTc +9 ± 24 ms; p  = 0.16). Median follow-up was similar for bisoprolol and nadolol (3 years), but longer for atenolol (6 years; p  = 0.03); one cardiac event occurred in the bisoprolol group (1.7 %) and two events occurred in the atenolol group (5.1 %; p  = 0.45), whereas none occurred in nadolol-treated patients. Beta-blocker efficacy was not affected by the underlying genotype. The antiadrenergic effect of bisoprolol correlated with the reduction of peak heart rates at exercise testing. Conclusions Bisoprolol treatment results in QTc shortening in gene-positive LQT1 and LQT2 patients and is well tolerated during long-term administration. The equivalence of bisoprolol for protection from ventricular arrhythmia in LQT patients compared to established beta-blockers remains unknown. Further large-scale studies are required.

Background Due to its availability, atenolol is the primary beta‐blocker used in Australia for children with long QT syndrome. There is limited data on long‐term follow‐up of its use. Methods A single‐tertiary‐center, retrospective, observational study investigating all children and adolescents who had genetically proven long QT syndrome type 1 (LQT1) and type 2 (LQT2) was conducted. Their pretreatment exercise tests were evaluated for QTc intervals into the recovery phase of exercise. Results Eighty six patients were identified (LQT1, 67, and LQT2, 19) from 2004 to 2014. The majority (86%) of patients were initially referred for family screening. Atenolol was administered at a mean dose of 1.58 ± 0.51 mg/kg/day. During the median follow‐up period of 4.29 years, only one proband developed ventricular arrhythmia whilst taking atenolol, No patient had cardiac arrest or aborted cardiac arrest. With respect to side effects of atenolol, only two patients had intolerable side effects necessitating changes of medication. Evaluation of exercise tests (pretreatment) demonstrated that corrected QT (QTc) intervals at 2–3 min into the recovery phase of exercise were significantly prolonged for LQT1 patients. LQT1 patients with transmembrane mutation had longer QTc intervals than their C‐terminus mutation counterparts, reaching statistical significance at 3 min into the recovery phase of exercise. Conclusions Atenolol is an effective treatment for genetically proven LQT1 and LQT2 children and adolescents, with good tolerability. In LQT1 patients, QTc intervals at 2–3 min into the recovery phase of exercise were significantly prolonged, particularly in patients with transmembrane mutations.

Genetic diagnosis of an inherited disease or cancer often involves analysis for unknown point mutations in several genes; therefore, rapid and automated techniques that can process a large number of samples are needed. We describe a method for high‐throughput single‐strand conformation polymorphism (SSCP) analysis using automated capillary electrophoresis. The operating temperature of a commercially available capillary electrophoresis instrument (ABI PRISM 310) was expanded by installation of a cheap in‐house designed cooling system, thereby allowing us to perform automated SSCP analysis at 14–45°C. We have used the method for detection of point mutations associated with the inherited cardiac disorders long QT syndrome (LQTS) and hypertrophic cardiomyopathy (HCM). The sensitivity of the method was 100% when 34 different point mutations were analyzed, including two previously unpublished LQTS‐associated mutations (F157C in KVLQT1 and G572R in HERG), as well as eight novel normal variants in HERG and MYH7. The analyzed polymerase chain reaction (PCR) fragments ranged in size from 166 to 1,223 bp. Seventeen different sequence contexts were analyzed. Three different electrophoresis temperatures were used to obtain 100% sensitivity. Two mutants could not be detected at temperatures greater than 20°C. The method has a high resolution and good reproducibility and is very robust, making multiplex SSCP analysis and pattern‐based identification of known allelic variants as single nucleotide polymorphisms (SNPs) possible. These possibilities, combined with automation and short analysis time, make the method suitable for high‐throughput tasks, such as genetic screening. Hum Mutat 13:318–327, 1999. © 1999 Wiley‐Liss, Inc.

The hERG channel has an unusually long (39 amino acids) extracellular loop between the transmembrane S5 segment and the pore region that may play a role in channel function. We explored this possibility by mutating two histidine residues in this region (H578 and H587, referred to as H1 and H2) to various residues and examined the resulting changes in channel function. Both positions could tolerate drastic changes in side-chain properties (proline, cysteine, glutamate and lysine), indicating that they are solvent exposed. None of the H1 mutations affected hERG channel function. On the other hand, although replacing H2 with glutamate had little or no effect on hERG properties, putting a proline or lysine at this position disrupted the C-type inactivation process and the pore's K selectivity. There was also a hyperpolarizing shift in the voltage dependence of activation. The phenotype of the H2C mutant was similar to that of H2P or H2K. However, dithiothreitol (DTT, a thiol-reducing agent) treatment converted the H2C phenotype to that of the wild-type channel. These observations suggest that the peptide backbone conformation around position 587 in the extracellular S5-P loop of hERG channel can affect the channel's gating and ion selectivity functions.