Dataset Information

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene.

ABSTRACT:

Background and purpose

The reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel-in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve.

Experimental approach

We have developed a model for the long QT syndrome type-5 in rabbits (LQT5 ) with cardiac-specific overexpression of a mutant (G52R) KCNE1 β-subunit of the channel that carries the slow delayed-rectifier K(+) -current (IKs ). ECG parameters, including short-term variability of the QT interval (STVQT ), a biomarker for proarrhythmic risk, and arrhythmia development were recorded. In vivo, arrhythmia susceptibility was evaluated by i.v. administration of the IKr blocker dofetilide. K(+) currents were measured with the patch-clamp technique.

Key results

Patch-clamp studies in ventricular myocytes isolated from LQT5 rabbits revealed accelerated IKs and IKr deactivation kinetics. At baseline, LQT5 animals exhibited slightly but significantly prolonged heart-rate corrected QT index (QTi) and increased STVQT . Dofetilide provoked Torsade-de-Pointes arrhythmia in a greater proportion of LQT5 rabbits, paralleled by a further increase in STVQT .

Conclusion and implications

We have created a novel transgenic LQT5 rabbit model with increased susceptibility to drug-induced arrhythmias that may represent a useful model for testing proarrhythmic potential and for investigations of the mechanisms underlying arrhythmias and sudden cardiac death due to repolarization disturbances.

SUBMITTER: Major P

PROVIDER: S-EPMC4882500 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Publications

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene.

Major Péter P Baczkó István I Hiripi László L Odening Katja E KE Juhász Viktor V Kohajda Zsófia Z Horváth András A Seprényi György G Kovács Mária M Virág László L Jost Norbert N Prorok János J Ördög Balázs B Doleschall Zoltán Z Nattel Stanley S Varró András A Bősze Zsuzsanna Z

British journal of pharmacology 20160519 12

<h4>Background and purpose</h4>The reliable assessment of proarrhythmic risk of compounds under development remains an elusive goal. Current safety guidelines focus on the effects of blocking the KCNH2/HERG ion channel-in tissues and animals with intact repolarization. Novel models with better predictive value are needed that more closely reflect the conditions in patients with cardiac remodelling and reduced repolarization reserve.<h4>Experimental approach</h4>We have developed a model for the ...[more]

PMID: 27076034

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Project description:AimsShort QT syndrome type 1 (SQT1) is a genetic channelopathy caused by gain-of-function variants in human-ether-a-go-go (HERG) underlying the rapid delayed-rectifier K+ current (IKr), leading to QT-shortening, ventricular arrhythmias, and sudden cardiac death. Data on efficient pharmacotherapy for SQT1 are scarce. In patients with primary carnitine-deficiency, acquired-short QT syndrome (SQTS) has been observed and rescued by carnitine supplementation. Here, we assessed whether carnitine exerts direct beneficial (prolonging) effects on cardiac repolarization in genetic SQTS.Methods and resultsAdult wild-type (WT) and transgenic SQT1 rabbits (HERG-N588K, gain of IKr) were used. In vivo electrocardiograms (ECGs), ex vivo monophasic action potentials (APs) in Langendorff-perfused hearts, and cellular ventricular APs and ion currents were assessed at baseline and during L-Carnitine/C16-Carnitine-perfusion. Two-dimensional computer simulations were performed to assess re-entry-based ventricular tachycardia-inducibility. L-Carnitine/C16-Carnitine prolonged QT-intervals in WT and SQT1, leading to QT-normalization in SQT1. Similarly, monophasic and cellular AP duration (APD) was prolonged by L-Carnitine/C16-Carnitine in WT and SQT1. As underlying mechanisms, we identified acute effects on the main repolarizing ion currents: IKr-steady, which is pathologically increased in SQT1, was reduced by L-Carnitine/C16-Carnitine and deactivation kinetics were accelerated. Moreover, L-Carnitine/C16-Carnitine decreased IKs-steady and IK1. In silico modelling identified IKr changes as the main factor for L-Carnitine/C16-Carnitine-induced APD-prolongation. 2D simulations revealed increased sustained re-entry-based arrhythmia formation in SQT1 compared to WT, which was decreased to the WT-level when adding carnitine-induced ion current changes.ConclusionL-Carnitine/C16-Carnitine prolong/normalize QT and whole-heart/cellular APD in SQT1 rabbits. These beneficial effects are mediated by acute effects on IKr. L-Carnitine may serve as a potential future QT-normalizing, anti-arrhythmic therapy in SQT1.

Project description:Key pointsIn adult ventricular myocytes, the slow delayed rectifier (IKs ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer.AbstractSlow delayed rectifier (IKs ) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to β-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer.

Dataset Information

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene.

Background and purpose

Experimental approach

Key results

Conclusion and implications

Publications

A novel transgenic rabbit model with reduced repolarization reserve: long QT syndrome caused by a dominant-negative mutation of the KCNE1 gene.

Similar Datasets

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