scholarly journals Promise and Potential Peril With Lumacaftor for the Trafficking Defective Type 2 Long-QT Syndrome-Causative Variants, p.G604S, p.N633S, and p.R685P, Using Patient-Specific Re-Engineered Cardiomyocytes

2020 ◽  
Vol 13 (5) ◽  
pp. 466-475
Author(s):  
Bailey J. O’Hare ◽  
C.S. John Kim ◽  
Samantha K. Hamrick ◽  
Dan Ye ◽  
David J. Tester ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Delayed Rectifier ◽  
Long Qt ◽  
Novel Therapy ◽  
Channel Trafficking ◽  
K Current ◽  
Qt Syndrome

Background: The KCNH2 -encoded Kv11.1 hERG (human ether-a-go-go related gene) potassium channel is a critical regulator of cardiomyocyte action potential duration (APD). The majority of type 2 long-QT syndrome (LQT2) stems from trafficking defective KCNH2 mutations. Recently, Food and Drug Administration-approved cystic fibrosis protein trafficking chaperone, lumacaftor, has been proposed as novel therapy for LQT2. Here, we test the efficacy of lumacaftor treatment in patient-specific induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) derived from 2 patients with known LQT2 trafficking defective mutations and a patient with novel KCNH2 variant, p.R685P. Methods: Patient-specific iPSC-CM models of KCNH2-G604S, KCNH2-N633S, and KCNH2-R685P were generated from 3 unrelated patients diagnosed with severe LQT2 (rate-corrected QT>500 ms). Lumacaftor efficacy was also tested by ANEPPS, FluoVolt, and ArcLight voltage dye-based APD90 measurements. Results: All 3 mutations were hERG trafficking defective in iPSC-CMs. While lumacaftor treatment failed to rescue the hERG trafficking defect in TSA201 cells, lumacaftor rescued channel trafficking for all mutations in the iPSC-CM model. All 3 mutations conferred a prolonged APD90 compared with control. While lumacaftor treatment rescued the phenotype of KCNH2-N633S and KCNH2-R685P, lumacaftor paradoxically prolonged the APD90 in KCNH2-G604S iPSC-CMs. Lumacaftor-mediated APD90 rescue was affected by rapidly activating delayed rectifier K+ current blocker consistent with the increase of rapidly activating delayed rectifier K+ current by lumacaftor is the underlying mechanism of the LQT2 rescue. Conclusions: While lumacaftor is an effective hERG channel trafficking chaperone and may be therapeutic for LQT2, we urge caution. Without understanding the functionality of the mutant channel to be rescued, lumacaftor therapy could be harmful.

scholarly journals R534C mutation in hERG causes a trafficking defect in iPSC-derived cardiomyocytes from patients with type 2 long QT syndrome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Fernanda C. P. Mesquita ◽  
Paulo C. Arantes ◽  
Tais H. Kasai-Brunswick ◽  
Dayana S. Araujo ◽  
Fernanda Gubert ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Mononuclear Cells ◽  
Patient Specific ◽  
Control Line ◽  
Long Qt ◽  
Peripheral Blood Mononuclear ◽  
Channel Trafficking ◽  
Qt Syndrome ◽  
Induced Pluripotent

AbstractPatient-specific cardiomyocytes obtained from induced pluripotent stem cells (CM-iPSC) offer unprecedented mechanistic insights in the study of inherited cardiac diseases. The objective of this work was to study a type 2 long QT syndrome (LQTS2)-associated mutation (c.1600C > T in KCNH2, p.R534C in hERG) in CM-iPSC. Peripheral blood mononuclear cells were isolated from two patients with the R534C mutation and iPSCs were generated. In addition, the same mutation was inserted in a control iPSC line by genome editing using CRISPR/Cas9. Cells expressed pluripotency markers and showed spontaneous differentiation into the three embryonic germ layers. Electrophysiology demonstrated that action potential duration (APD) of LQTS2 CM-iPSC was significantly longer than that of the control line, as well as the triangulation of the action potentials (AP), implying a longer duration of phase 3. Treatment with the IKr inhibitor E4031 only caused APD prolongation in the control line. Patch clamp showed a reduction of IKr on LQTS2 CM-iPSC compared to control, but channel activation was not significantly affected. Immunofluorescence for hERG demonstrated perinuclear staining in LQTS2 CM-iPSC. In conclusion, CM-iPSC recapitulated the LQTS2 phenotype and our findings suggest that the R534C mutation in KCNH2 leads to a channel trafficking defect to the plasma membrane.

scholarly journals Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes

2017 ◽  
Vol 113 (5) ◽  
pp. 531-541 ◽  
Author(s):  
Marcella Rocchetti ◽  
Luca Sala ◽  
Lisa Dreizehnter ◽  
Lia Crotti ◽  
Daniel Sinnecker ◽  
...  
Keyword(s):  
Stem Cell ◽  
Long Qt Syndrome ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Long Qt ◽  
Qt Syndrome ◽  
Induced Pluripotent

scholarly journals Patient-Specific Induced Pluripotent Stem-Cell Models for Long-QT Syndrome

2010 ◽  
Vol 363 (15) ◽  
pp. 1397-1409 ◽  
Author(s):  
Alessandra Moretti ◽  
Milena Bellin ◽  
Andrea Welling ◽  
Christian Billy Jung ◽  
Jason T. Lam ◽  
...  
Keyword(s):  
Stem Cell ◽  
Long Qt Syndrome ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Cell Models ◽  
Long Qt ◽  
Qt Syndrome ◽  
Induced Pluripotent ◽  
Stem Cell Models

scholarly journals From patient-specific induced pluripotent stem cells to clinical translation in long QT syndrome Type 2

2019 ◽  
Vol 40 (23) ◽  
pp. 1832-1836 ◽  
Author(s):  
Peter J Schwartz ◽  
Massimiliano Gnecchi ◽  
Federica Dagradi ◽  
Silvia Castelletti ◽  
Gianfranco Parati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Long Qt Syndrome ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Syndrome Type ◽  
Long Qt ◽  
Qt Syndrome ◽  
Induced Pluripotent

scholarly journals Generation of human induced pluripotent stem cell line from a patient with a long QT syndrome type 2

2016 ◽  
Vol 16 (2) ◽  
pp. 304-307 ◽  
Author(s):  
Azra Fatima ◽  
Dina Ivanyuk ◽  
Stefan Herms ◽  
Stefanie Heilmann-Heimbach ◽  
Orla O'Shea ◽  
...  
Keyword(s):  
Stem Cell ◽  
Long Qt Syndrome ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Syndrome Type ◽  
Stem Cell Line ◽  
Long Qt ◽  
Qt Syndrome ◽  
Induced Pluripotent

Long QT Syndrome 2 PAS Domain Variant Induces hERG1a/1b Subunit Imbalance in Patient-specific iPSC-cardiomyocytes

2021 ◽  
Author(s):  
Li Feng ◽  
Jianhua Zhang ◽  
ChangHwan Lee ◽  
Gina Kim ◽  
Fang Liu ◽  
...  
Keyword(s):  
Long Qt Syndrome ◽  
Protein Trafficking ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Pas Domain ◽  
Long Qt ◽  
Pathogenic Variants ◽  
Human Ipscs ◽  
Qt Syndrome ◽  
Domain Variant

Background - Inherited long QT syndrome type 2 (LQT2) results from variants in the KCNH2 gene encoding the hERG1 potassium channel. Two main isoforms, hERG1a and hERG1b, assemble to form tetrameric channel. The N-terminal Per-Arnt-Sim (PAS) domain, present only on hERG1a subunits, is a hotspot for pathogenic variants, but it is unknown whether PAS domain variants impact hERG1b expression to contribute to the LQT2 phenotype. We aimed to use patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate the pathogenesis of the hERG1a PAS domain variant hERG1-H70R. Methods - Human iPSCs were derived from a LQT2 patient carrying the PAS domain variant hERG1-H70R. CRISPR/Cas9 gene editing produced isogenic control iPSC lines. Differentiated iPSC-CMs were evaluated for their electrophysiology, hERG1a/1b mRNA expression, and hERG1a/1b protein expression. Results - Action potentials from single hERG1-H70R iPSC-CMs were prolonged relative to controls, and voltage clamp studies showed an underlying decrease in I Kr with accelerated deactivation. In hERG1-H70R iPSC-CMs, transcription of hERG1a and hERG1b mRNA was unchanged compared to controls based on nascent nuclear transcript analysis, but hERG1b mRNA was significantly increased as was the ratio of hERG1b/hERG1a in mRNA complexes, suggesting post-transcriptional changes. Expression of complex glycosylated hERG1a in hERG1-H70R iPSC-CMs was reduced due to impaired protein trafficking, whereas the expression of the complex glycosylated form of hERG1b was unchanged. Conclusions - Patient-specific hERG1-H70R iPSC-CMs reveal a newly appreciated mechanism of pathogenesis of the LQT2 phenotype due to both impaired trafficking of hERG1a and maintained expression of hERG1b that produces subunit imbalance and reduced I Kr with accelerated deactivation.

Development of a Patient-Specific p.D85N-KCNE1 Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for Drug-Induced Long QT Syndrome

2021 ◽  
Author(s):  
Maengjo Kim ◽  
Dan Ye ◽  
CS John Kim ◽  
Wei Zhou ◽  
David J. Tester ◽  
...  
Keyword(s):  
Stem Cell ◽  
Long Qt Syndrome ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Common Variant ◽  
Patient Specific ◽  
Long Qt ◽  
Drug Induced ◽  
Qt Syndrome ◽  
Induced Pluripotent

Background - Prior epidemiological studies demonstrated that the p.D85N-KCNE1 common variant reduces repolarization reserve and predisposes to drug-induced QT prolongation/torsades de pointes. We sought to develop a cellular model for drug-induced long QT syndrome (DI-LQTS) using a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM). Methods - p.D85N-KCNE1 iPSCs were generated from a 23-year-old female with an exaggerated QTc response to metoclopramide (ΔQTc of 160 ms). CRISPR/Cas9 technology was used to generate "gene-corrected" isogenic iPSCs. Field potential duration (FPD) and action potential duration (APD) were measured from iPSC-CMs. Results - At baseline, p.D85N-KCNE1 iPSC-CMs displayed significantly longer FPD (281 ± 15 ms, n=13 vs. 223 ± 8.6 ms, n=14, p<0.01) and APD 90 (579 ± 22 ms, n=24 vs. 465 ± 33 ms, n=26, p<0.01) than isogenic-control iPSC-CMs. Dofetilide at a concentration of 2nM increased significantly FPD (379 ± 20 ms, n=13, p<0.01) and APD 90 (666 ± 11 ms, n=46, p<0.01) in p.D85N-KCNE1 iPSC-CMs, but not in isogenic-control. The effect of dofetilide on APD 90 (616 ± 54 ms, n=7 vs. 526 ± 54 ms, n=10, p<0.05) was confirmed by Patch-clamp. Interestingly, treatment of p.D85N-KCNE1 iPSC-CMs with estrogen at a concentration of 1nM exaggerated further dofetilide-induced APD 90 prolongation (696 ± 9 ms, n=81, p<0.01) and caused more early afterdepolarizations (EADs) (11.7%) compared to isogenic control (APD 90 : 618 ± 8 ms, n=115 and EADs: 2.6%, p<0.05) Conclusions - This iPSC-CM study provides further evidence that the p.D85N-KCNE1 common variant in combination with environmental factors such as QT prolonging drugs and female sex is pro-arrhythmic.

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.

scholarly journals Inhibition of Cardiac Delayed Rectifier K + Current by Overexpression of the Long-QT Syndrome HERG G628S Mutation in Transgenic Mice

1998 ◽  
Vol 83 (6) ◽  
pp. 668-678 ◽  
Author(s):  
Philip Babij ◽  
G. Roger Askew ◽  
Bart Nieuwenhuijsen ◽  
Chien-Min Su ◽  
Terry R. Bridal ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Long Qt Syndrome ◽  
Delayed Rectifier ◽  
Long Qt ◽  
K Current ◽  
Cardiac Delayed Rectifier ◽  
Qt Syndrome

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.

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