scholarly journals Suppression-Replacement KCNQ1 Gene Therapy for Type 1 Long QT Syndrome

Circulation ◽  
2021 ◽  
Author(s):  
Steven M. Dotzler ◽  
C.S. John Kim ◽  
William A.C. Gendron ◽  
Wei Zhou ◽  
Dan Ye ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Long Qt Syndrome ◽  
Delayed Rectifier ◽  
Loss Of Function ◽  
Wild Type ◽  
Long Qt ◽  
Α Subunit ◽  
Cardiac Model ◽  
Qt Syndrome

Background: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1 -encoded K v 7.1 potassium channel α-subunit which is essential for cardiac repolarization, providing the slow delayed rectifier current (IKs). No current therapies target the molecular cause of LQT1. Methods: A dual-component "suppression-and-replacement" (SupRep) KCNQ1 gene therapy was created by cloning a KCNQ1 shRNA and a "shRNA-immune" (shIMM) KCNQ1 cDNA modified with silent variants in the shRNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated via heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from four patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for two LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep. Results: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type KCNQ1 and three LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of KCNQ1-shIMM as measured by allele-specific qRT-PCR and western blot. Using FluoVolt voltage dye to measure the cardiac APD in the four LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% (APD 90 ) and 50% (APD 50 ) repolarization resulting in APD values similar to those of the two isogenic controls. Conclusions: This study provides the first proof-of-principle gene therapy for complete correction of LQTS. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, co-transfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression-and-replacement of KCNQ1 . In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.

scholarly journals Molecular Mechanism of Autosomal Recessive Long QT-Syndrome 1 without Deafness

2021 ◽  
Vol 22 (3) ◽  
pp. 1112
Author(s):  
Annemarie Oertli ◽  
Susanne Rinné ◽  
Robin Moss ◽  
Stefan Kääb ◽  
Gunnar Seemann ◽  
...  
Keyword(s):  
Action Potential ◽  
Molecular Mechanism ◽  
Long Qt Syndrome ◽  
Autosomal Recessive ◽  
Delayed Rectifier ◽  
Loss Of Function ◽  
Wild Type ◽  
Long Qt ◽  
Qt Syndrome ◽  
Iks Channel

KCNQ1 encodes the voltage-gated potassium (Kv) channel KCNQ1, also known as KvLQT1 or Kv7.1. Together with its ß-subunit KCNE1, also denoted as minK, this channel generates the slowly activating cardiac delayed rectifier current IKs, which is a key regulator of the heart rate dependent adaptation of the cardiac action potential duration (APD). Loss-of-function mutations in KCNQ1 cause congenital long QT1 (LQT1) syndrome, characterized by a delayed cardiac repolarization and a prolonged QT interval in the surface electrocardiogram. Autosomal dominant loss-of-function mutations in KCNQ1 result in long QT syndrome, called Romano–Ward Syndrome (RWS), while autosomal recessive mutations lead to Jervell and Lange-Nielsen syndrome (JLNS), associated with deafness. Here, we identified a homozygous KCNQ1 mutation, c.1892_1893insC (p.P631fs*20), in a patient with an isolated LQT syndrome (LQTS) without hearing loss. Nevertheless, the inheritance trait is autosomal recessive, with heterozygous family members being asymptomatic. The results of the electrophysiological characterization of the mutant, using voltage-clamp recordings in Xenopus laevis oocytes, are in agreement with an autosomal recessive disorder, since the IKs reduction was only observed in homomeric mutants, but not in heteromeric IKs channel complexes containing wild-type channel subunits. We found that KCNE1 rescues the KCNQ1 loss-of-function in mutant IKs channel complexes when they contain wild-type KCNQ1 subunits, as found in the heterozygous state. Action potential modellings confirmed that the recessive c.1892_1893insC LQT1 mutation only affects the APD of homozygous mutation carriers. Thus, our study provides the molecular mechanism for an atypical autosomal recessive LQT trait that lacks hearing impairment.

scholarly journals Electrophysiological studies of transgenic long QT type 1 and type 2 rabbits reveal genotype-specific differences in ventricular refractoriness and His conduction

2010 ◽  
Vol 299 (3) ◽  
pp. H643-H655 ◽  
Author(s):  
Katja E. Odening ◽  
Malcolm Kirk ◽  
Michael Brunner ◽  
Ohad Ziv ◽  
Peem Lorvidhaya ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Optical Mapping ◽  
Delayed Rectifier ◽  
Syndrome Type ◽  
Long Qt ◽  
Av Conduction ◽  
Electrophysiological Studies ◽  
Qt Syndrome

We have generated transgenic rabbits lacking cardiac slow delayed-rectifier K+ current [ IKs; long QT syndrome type 1 (LQT1)] or rapidly activating delayed-rectifier K+ current [ IKr; long QT syndrome type 2 (LQT2)]. Rabbits with either genotype have prolonged action potential duration and QT intervals; however, only LQT2 rabbits develop atrioventricular (AV) blocks and polymorphic ventricular tachycardia. We therefore sought to characterize the genotype-specific differences in AV conduction and ventricular refractoriness in LQT1 and LQT2 rabbits. We carried out in vivo electrophysiological studies in LQT1, LQT2, and littermate control (LMC) rabbits at baseline, during isoproterenol infusion, and after a bolus of dofetilide and ex vivo optical mapping studies of the AV node/His-region at baseline and during dofetilide perfusion. Under isoflurane anesthesia, LQT2 rabbits developed infra-His blocks, decremental His conduction, and prolongation of the Wenckebach cycle length. In LQT1 rabbits, dofetilide altered the His morphology and slowed His conduction, resulting in intra-His block, and additionally prolonged the ventricular refractoriness, leading to pseudo-AV block . The ventricular effective refractory period (VERP) in right ventricular apex and base was significantly longer in LQT2 than LQT1 ( P < 0.05) or LMC ( P < 0.01), with a greater VERP dispersion in LQT2 than LQT1 rabbits. Isoproterenol reduced the VERP dispersion in LQT2 rabbits by shortening the VERP in the base more than in the apex but had no effect on VERP in LQT1. EPS and optical mapping experiments demonstrated genotype-specific differences in AV conduction and ventricular refractoriness. The occurrence of infra-His blocks in LQT2 rabbits under isoflurane and intra-His block in LQT1 rabbits after dofetilide suggest differential regional sensitivities of the rabbit His-Purkinje system to drugs blocking IKr and IKs.

Abstract 17137: Unmasking Pathological Mechanisms of Long QT Syndrome KCNH2 H70R Variant Using Human iPSC-Cardiomyocytes

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_1) ◽  
Author(s):  
Li Feng ◽  
Gina Kim ◽  
Catherine A Eichel ◽  
Fang Liu ◽  
Evi Lim ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Herg Channel ◽  
Patient Specific ◽  
Delayed Rectifier ◽  
Pas Domain ◽  
Loss Of Function ◽  
Long Qt ◽  
Unrelated Control ◽  
Qt Syndrome ◽  
Human Ipsc

Introduction: Inherited long QT syndrome type 2 (LQT2) results from loss-of-function mutations in the KCNH2 gene encoding the hERG channel, which conducts I Kr , the rapid component of the delayed rectifier K + current. The N-terminal Per-Arnt-Sim (PAS) domain present on the hERG1a subunit, but not the hERG1b subunit, is the site of multiple LQT2-linked missense variants. The mechanism of loss of function by many of these missense PAS variants is unclear given conflicting results from different heterologous expression systems expressing hERG1a. Hypothesis: Patient-specific LQT2 human iPSC-cardiomyocytes (hiPSC-CMs) which naturally express hERG1a/1b carrying the KCNH2 H70R variant in the PAS domain will exhibit loss of I Kr associated with APD prolongation due to impaired channel protein trafficking. Methods and Results: Human iPSCs were derived from a patient carrying the LQT2-associated PAS domain mutation KCNH2 H70R, which has been reported to cause impaired hERG channel trafficking without effects on channel gating when expressed in HEK 293 cells but accelerated deactivation kinetics of I hERG when expressed in Xenopus laevis oocytes. Two clones of KCNH2 H70R and unrelated control hiPSCs (DF19-9-11T) were differentiated using monolayer-base, small molecule protocol to CMs, evaluated with whole-cell patch clamp. Action potentials from single hiPSC-CMs paced at 1Hz were prolonged in the hERG-H70R group compared to control (APD 90 439.9 ± 15.3 ms vs. 363.7 ± 29.0ms, p =0.003, H70R: n=11, control: n=9, Temp 36 ± 1°C). Voltage clamp studies showed hERG-H70R hiPSC-CMs had a significantly smaller peak tail I Kr current density (1.1 ± 0.3 vs. 2.9 ± 0.5 pA/pF, p <0.001, H70R: n=11, control: n=7, Temp 36 ± 1°C). The voltage dependence of I Kr activation (V½ and k) were not affected by the mutation; however, the fast (τf) and slow (τs) deactivation time constants were significantly decreased in hERG-H70R hiPSC-CMs. Further, Western blot characterization revealed impaired trafficking of hERG-H70R channels relative to control. Conclusions: The LQT2 PAS domain variant hERG-H70R results in loss of function of I Kr by both reduced membrane trafficking and accelerated deactivation of hERG in a hiPSC-CMs model which informs therapeutic approaches.

scholarly journals Enhanced Effects of Isoflurane on the Long QT Syndrome 1–associated A341V Mutant

2015 ◽  
Vol 122 (4) ◽  
pp. 806-820 ◽  
Author(s):  
Ikuomi Mikuni ◽  
Carlos G. Torres ◽  
Tania Bakshi ◽  
Akihito Tampo ◽  
Brian E. Carlson ◽  
...  
Keyword(s):  
Action Potential ◽  
Long Qt Syndrome ◽  
Volatile Anesthetics ◽  
Hek293 Cells ◽  
Dominant Negative Effect ◽  
Wild Type ◽  
Long Qt ◽  
Α Subunit ◽  
Qt Syndrome ◽  
The Impact

Abstract Background: The impact of volatile anesthetics on patients with inherited long QT syndrome (LQTS) is not well understood. This is further complicated by the different genotypes underlying LQTS. No studies have reported on the direct effects of volatile anesthetics on specific LQTS-associated mutations. We investigated the effects of isoflurane on a common LQTS type 1 mutation, A341V, with an unusually severe phenotype. Methods: Whole cell potassium currents (IKs) were recorded from HEK293 and HL-1 cells transiently expressing/coexpressing wild-type KCNQ1 (α-subunit), mutant KCNQ1, wild-type KCNE1 (β-subunit), and fusion KCNQ1 + KCNE1. Current was monitored in the absence and presence of clinically relevant concentration of isoflurane (0.54 ± 0.05 mM, 1.14 vol %). Computer simulations determined the resulting impact on the cardiac action potential. Results: Isoflurane had significantly greater inhibitory effect on A341V + KCNE1 (62.2 ± 3.4%, n = 8) than on wild-type KCNQ1 + KCNE1 (40.7 ± 4.5%; n = 9) in transfected HEK293 cells. Under heterozygous conditions, isoflurane inhibited A341V + KCNQ1 + KCNE1 by 65.2 ± 3.0% (n = 13) and wild-type KCNQ1 + KCNE1 (2:1 ratio) by 32.0 ± 4.5% (n = 11). A341V exerted a dominant negative effect on IKs. Similar differential effects of isoflurane were also observed in experiments using the cardiac HL-1 cells. Mutations of the neighboring F340 residue significantly attenuated the effects of isoflurane, and fusion proteins revealed the modulatory effect of KCNE1. Action potential simulations revealed a stimulation frequency–dependent effect of A341V. Conclusions: The LQTS-associated A341V mutation rendered the IKs channel more sensitive to the inhibitory effects of isoflurane compared to wild-type IKs in transfected cell lines; F340 is a key residue for anesthetic action.

scholarly journals Clinical and electrophysiological characterization of a novel mutation (F193L) in the KCNQ1 gene associated with long QT syndrome

2003 ◽  
Vol 104 (4) ◽  
pp. 377-382 ◽  
Author(s):  
Masato YAMAGUCHI ◽  
Masami SHIMIZU ◽  
Hidekazu INO ◽  
Hidenobu TERAI ◽  
Kenshi HAYASHI ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Novel Mutation ◽  
Expression System ◽  
Clinical Manifestations ◽  
Delayed Rectifier ◽  
Polymorphism Analysis ◽  
Long Qt ◽  
Α Subunit ◽  
Kcnq1 Gene ◽  
Qt Syndrome

KCNQ1 is a gene encoding an α subunit of voltage-gated cardiac K+ channels, with properties similar to the slowly activating delayed rectifier K+ current, and one of the genes causing long QT syndrome (LQTS). However, genotype–phenotype correlations of the KCNQ1 gene mutations are not fully understood. The aims of this study were to identify a mutation in the KCNQ1 gene in patients with LQTS, and to characterize the clinical manifestations and electrophysiological properties of the mutation. We screened and identified mutations by PCR, single-strand conformational polymorphism analysis and DNA sequencing. We identified a novel mutation [Phe193Leu (F193L)] in the KCNQ1 gene in one family with LQTS. The patients with this mutation showed a mildly affected phenotype. The proband was a 17-year-old girl who had a prolonged QT interval. Her elder brother, father and paternal grandmother also had the mutation. None of them had any history of syncope. Sudden death was not found in this family. Next, we studied the electrophysiological characteristics of the F193L mutation in the KCNQ1 gene using the expression system in Xenopus oocytes and the two-microelectrode voltage-clamp technique. Co-expression of F193L KCNQ1 with the K+ channel minK suppressed peak (by 23.3%) and tail (by 38.2%) currents compared with those obtained by the combination of wild-type (WT) KCNQ1 and minK. Time constants of current activation in F193L KCNQ1 and F193L KCNQ1+minK were significantly slower than those of WT KCNQ1 and WT KCNQ1+minK. This electrophysiological study indicates that F193L causes less severe KCNQ1 current suppression, and thereby this mutation may result in a mildly affected phenotype.

Readthrough of long-QT syndrome type 1 nonsense mutations rescues function but alters the biophysical properties of the channel

2012 ◽  
Vol 443 (3) ◽  
pp. 635-642 ◽  
Author(s):  
Stephen C. Harmer ◽  
Jagdeep S. Mohal ◽  
Duncan Kemp ◽  
Andrew Tinker
Keyword(s):  
Current Density ◽  
Long Qt Syndrome ◽  
Syndrome Type ◽  
Wild Type ◽  
Biophysical Properties ◽  
Long Qt ◽  
Nonsense Mutations ◽  
Channel Function ◽  
Qt Syndrome

The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause LQT1 (long-QT syndrome type 1) and result in a complete loss of IKs channel function. In the present study we attempted to rescue the function of these mutants, in HEK (human embryonic kidney)-293 cells, by promoting readthrough of their PTCs (premature termination codons) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418 acted to promote full-length channel protein expression from R518X at 100 μM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced from R518X, while similar, were not identical with wild-type as the voltage-dependence of activation was significantly (P<0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible, it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.

hERG channel trafficking: novel targets in drug-induced long QT syndrome

2007 ◽  
Vol 35 (5) ◽  
pp. 1060-1063 ◽  
Author(s):  
A. Dennis ◽  
L. Wang ◽  
X. Wan ◽  
E. Ficker
Keyword(s):  
Long Qt Syndrome ◽  
Heat Shock Protein 90 ◽  
Delayed Rectifier ◽  
Cardiac Events ◽  
Long Qt ◽  
Drug Induced ◽  
Α Subunit ◽  
Human Cardiomyocytes ◽  
Qt Syndrome ◽  
Therapeutic Compounds

The cardiac potassium channel hERG (human ether-a-go-go-related gene) encodes the α-subunit of the rapid delayed rectifier current IKr in the heart, which contributes to terminal repolarization in human cardiomyocytes. Direct block of hERG/IKr channels by a large number of therapeutic compounds produces acLQTS [acquired LQTS (long QT syndrome)] characterized by drug-induced QT prolongation and torsades de pointes arrhythmias. The cardiotoxicity associated with unintended hERG block has prompted pharmaceutical companies to screen developmental compounds for hERG blockade and made hERG a major target in drug safety programmes. More recently, a novel form of acLQTS has been discovered that may go undetected in most conventional safety assays. Several therapeutic compounds have been identified that reduce hERG/IKr currents not by direct block but by inhibition of hERG/IKr trafficking to the cell surface. Important examples are antineoplastic Hsp90 (heat-shock protein 90) inhibitors such as (i) geldanamycin, (ii) the leukaemia drug arsenic trioxide, (iii) the antiprotozoical pentamidine, (iv) probucol, a cholesterol-lowering drug, and (v) fluoxetine, a widely used antidepressant. Increased awareness of drug-induced hERG trafficking defects will help to further reduce the potentially lethal adverse cardiac events associated with acLQTS.

Functional Hyperactivity in Long QT Syndrome Type 1 Pluripotent Stem Cell-Derived Sympathetic Neurons

2021 ◽  
Author(s):  
Annika Winbo ◽  
Suganeya Ramanan ◽  
Emily Eugster ◽  
Annika Rydberg ◽  
Stefan Jovinge ◽  
...  
Keyword(s):  
Pluripotent Stem Cell ◽  
Sympathetic Neurons ◽  
Compound Heterozygous ◽  
Syndrome Type ◽  
Loss Of Function ◽  
Long Qt ◽  
Action Potential Frequency ◽  
Qt Syndrome ◽  
Potential Frequency

Sympathetic activation is an established trigger of life-threatening cardiac events in long QT syndrome type 1 (LQT1). KCNQ1 loss-of-function variants, which underlie LQT1, have been associated with both cardiac arrhythmia and neuronal hyperactivity pathologies. However, the LQT1 sympathetic neuronal phenotype is unknown. Here we aimed to study human induced pluripotent stem cell (hiPSC)-derived sympathetic neurons (SNs) to evaluate neuronal functional phenotype in LQT1. We generated hiPSC-SNs from two LQT1 patients with a history of sympathetically triggered arrhythmia and KCNQ1 loss-of-function genotypes (c.781_782delinsTC and p.S349W/p.R518X). Characterisation of hiPSC-SNs was performed using immunohistochemistry, enzyme-linked immunosorbent assay and whole-cell patch clamp electrophysiology, and functional LQT1 hiPSC-SN phenotypes compared to healthy control (WT) hiPSC-SNs. hiPSC-SNs stained positive for tyrosine hydroxylase, peripherin, KCNQ1, and secreted noradrenaline. hiPSC-SNs at 60±2.2 days in vitro had healthy resting membrane potentials (-60±1.3 mV), and fired rapid action potentials with mature kinetics in response to stimulation. Significant hyperactivity in LQT1 hiPSC-SNs was evident via increased noradrenaline release, increased spontaneous action potential frequency, increased total inward current density, and reduced afterhyperpolarisation, compared to age-matched WT hiPSC-SNs. A significantly higher action potential frequency upon current injection and larger synaptic current amplitudes in compound heterozygous p.S349W/p.R518X hiPSC-SNs compared to heterozygous c.781_782delinsTC hiPSC-SNs was also observed, suggesting a potential genotype-phenotype correlation. Together our data reveal increased neurotransmission and excitability in heterozygous and compound heterozygous patient-derived LQT1 sympathetic neurons, suggesting that the cellular arrhythmogenic potential in LQT1 is not restricted to cardiomyocytes.

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