Horváth, András

Abstract Background and Aims Type 1 short QT syndrome (SQT1) is a genetic channelopathy caused by gain-of-function variants in KCNH2, resulting in shortened cardiac repolarization and QT intervals, which predispose patients to ventricular arrhythmias and sudden cardiac death. This study aimed to investigate the therapeutic efficacy of KCNH2-specific suppression-and-replacement (KCNH2-SupRep) gene therapy in a transgenic rabbit model of SQT1. Methods KCNH2-SupRep was developed by combining a KCNH2-shRNA with its corresponding shRNA-immune KCNH2-cDNA into an AAV9 vector, delivered directly into the aortic root (1 × 1010 vg/kg). Therapeutic efficacy was evaluated in vivo by electrocardiogram, ex vivo by optical mapping, and at cellular levels by patch-clamp, calcium imaging, and qPCR in ventricular cardiomyocytes (VCMs). Results In vivo, KCNH2-SupRep normalized the heart rate-corrected QT interval (QTc) in SQT1 rabbits, without affecting repolarization heterogeneity. Ex vivo, KCNH2-SupRep corrected the action potential duration (APD90) and resolved the increased apicobasal APD90 heterogeneity observed in untreated (UT)-SQT1 hearts, supporting an antiarrhythmic effect, which was further validated by reduced re-entry formation in silico. At cellular levels, KCNH2-SupRep prolonged APD90 in VCMs from SupRep-SQT1 rabbits closer to wildtype levels compared with UT- and sham-SQT1. Additionally, KCNH2-SupRep restored the cellular surrogate of the electro-mechanical window and normalized IKr in nearly 50% of VCMs, in line with a 50%–60% suppression of the mutant KCNH2 transcript. Conclusions This proof-of-concept study is the first to demonstrate the efficacy of gene therapy for SQT1 in a medium-sized animal model. KCNH2-SupRep gene therapy successfully corrected the pathologic phenotype in vivo, ex vivo, and at cellular levels in transgenic SQT1 rabbits.

Abstract Aims Pronounced sex-differences are known in the incidence of atrial fibrillation (AF). In this study, we aimed to investigate the atrial electrophysiological properties that may underlie sex-differences in AF incidence in the younger population, focusing on IK1, a cardiac ion current important for action potential (AP) stability and triggered activity. Methods and results We assessed sex-differences in P-wave morphology in 12-lead ECG in healthy young New Zealand White rabbits. Males presented longer PWD and larger P-wave area compared to females. Patch-clamp experiments were performed in isolated rabbit atrial cardiomyocytes (CMs). Male atrial CMs presented higher delayed after depolarizations (DAD) incidence, amplitude, and area under the curve (AUC) than females, potentially facilitating the presence of atrial triggered activity in males. Male atrial CMs showed a less hyperpolarized resting membrane potential (RMP), a 50% smaller IK1, and a 26% reduction in Kir2.1 protein expression, a pore forming subunit of IK1, than females. Dihydrotestosterone (DHT) effects were investigated acutely and semi-chronically ex vivo. Experiments showed that the sex-difference in IK1 could be mimicked by DHT. In female atrial CMs, acute and semi-chronic (24 h) DHT administration reduced IK1. In the presence of a PKC-inhibitor, DHT-mediated IK1 reduction was not observed in atrial female CMs, suggesting it to be PKC-mediated. Chronic DHT-effects were investigated in vivo in female rabbits after hormone-releasing pellet implantation. After 2 weeks, animals showed a significantly prolonged and larger P-wave, a smaller atrial IK1 and a trend towards an increased DAD amplitude and AUC. Conclusion Sex impacts on atrial electrophysiology, leading to sex-differences in P-wave morphology, triggered activity, RMP, and IK1. These sex-differences can be mimicked by sex hormone-treatment, suggesting that sex hormones—particularly DHT—play a pivotal role in mediating sex-differences in atrial electrophysiology. Such sex-differences might impact on the propensity to develop AF, particularly in the younger population.

Abstract Background and Aims Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. Methods KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. Results KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P < .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. Conclusions This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

Abstract Background Short QT syndrome type 1 (SQT1) is a genetic channelopathy characterized by gain-of-function variants in the KCNH2-encoded potassium channel underlying the IKr current, leading to a pathologically shortened cardiac action potential duration (APD). Patients with SQT1 manifest shortening of the QT duration on 12-lead ECG and are prone to cardiac arrhythmias that can culminate in sudden cardiac death. Purpose To investigate the effect of a KCNH2-specific suppression-replacement (KCNH2-SupRep) gene therapy on the pathogenic phenotype in transgenic SQT1 rabbits. Methods A novel dual-component KCNH2-SupRep gene therapy was developed by combining a custom-designed KCNH2 shRNA (suppression) and an shRNA-immune (shIMM) KCNH2 cDNA (replacement) into AAV9. In vivo AAV9-mediated KCNH2-SupRep / sham treatment was performed by intra-aortic root injection of the KCNH2-SupRep AAV9 (1E10 vg/kg) / NaCl 0.9% during balloon occlusion using a Swan Ganz catheter. Three weeks after administration, 12-lead ECGs were assessed in adult transgenic SQT1 (KCNH2-N588K, gain of IKr due to impaired inactivation) and wild-type (WT) rabbits to determine the effect of KCNH2-SupRep / sham treatment on the rabbit’s QTc duration and QT index. Patch-clamp recordings were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of KCNH2-SupRep / sham treatment on the CM’s action potential duration at 90% repolarization (APD90). Results After in vivo AAV9-mediated KCNH2-SupRep gene therapy injection (1E10 vg/kg) in 7 SQT1 rabbits, QTc duration and QT index significantly increased in all SQT1 rabbits (QTc: untreated(UT)-SQT1: 153.2±3.8ms vs treated-SQT1: 166.5±4.6ms, p=0.0002, N=7 each; QTi: UT-SQT1: 92.5±1.9% vs treated-SQT1: 99.7±2.7%, p=0.0007, N=7 each), reaching a QT index of ~100% typically observed in WT rabbits. No such changes were observed in the sham-SQT1 rabbits. Furthermore, cardiomyocytes isolated from 7 SQT1 rabbits that received in vivo SupRep gene therapy demonstrated APD90 prolongation closer to WT rabbits compared to UT-SQT1 and sham-SQT1 rabbits (APD90: UT-SQT1: 314.2±11.9ms, WT: 404.5±13.8ms, treated-SQT1: 362.9±10.6ms; UT-SQT1 vs WT: p<0.0001; UT-SQT1 vs treated-SQT1: p=0.019; WT vs Treated-SQT1: p=0.031). In line with these observations, first IKr measurements demonstrate smaller IKr currents in SupRep-SQT1 than in sham-SQT1 CMs. Conclusion In vivo KCNH2-SupRep gene therapy normalizes the pathologically shortened QTc duration and QT index in transgenic SQT1 rabbits. Moreover, KCNH2-SupRep treatment prolongs APD90 in SQT1 compared to UT-SQT1 and sham-SQT1 reaching levels closer to WT.