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

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.

Download Full-text

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

Circulation Genomic and Precision Medicine ◽

10.1161/circgen.120.002950 ◽

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.

Download Full-text

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

Circulation Arrhythmia and Electrophysiology ◽

10.1161/circep.120.009343 ◽

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.

Download Full-text

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

Circulation ◽

10.1161/circulationaha.120.051836 ◽

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.

Download Full-text

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

International Journal of Molecular Sciences ◽

10.3390/ijms22031112 ◽

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.

Download Full-text

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

Cardiovascular Research ◽

10.1093/cvr/cvx006 ◽

2017 ◽

Vol 113 (5) ◽

pp. 531-541 ◽

Cited By ~ 64

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

Download Full-text

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

New England Journal of Medicine ◽

10.1056/nejmoa0908679 ◽

2010 ◽

Vol 363 (15) ◽

pp. 1397-1409 ◽

Cited By ~ 813

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

Download Full-text

Fatty acid analogue N-arachidonoyl taurine restores function of IKs channels with diverse long QT mutations

eLife ◽

10.7554/elife.20272 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 13

Author(s):

Sara I Liin ◽

Johan E Larsson ◽

Rene Barro-Soria ◽

Bo Hjorth Bentzen ◽

H Peter Larsson

Keyword(s):

Fatty Acid ◽

Long Qt Syndrome ◽

Molecular Mechanisms ◽

Loss Of Function ◽

Long Qt ◽

Acid Analogue ◽

Fatty Acid Analogue ◽

Qt Syndrome ◽

Channel Currents ◽

Iks Channel

About 300 loss-of-function mutations in the IKs channel have been identified in patients with Long QT syndrome and cardiac arrhythmia. How specific mutations cause arrhythmia is largely unknown and there are no approved IKs channel activators for treatment of these arrhythmias. We find that several Long QT syndrome-associated IKs channel mutations shift channel voltage dependence and accelerate channel closing. Voltage-clamp fluorometry experiments and kinetic modeling suggest that similar mutation-induced alterations in IKs channel currents may be caused by different molecular mechanisms. Finally, we find that the fatty acid analogue N-arachidonoyl taurine restores channel gating of many different mutant channels, even though the mutations are in different domains of the IKs channel and affect the channel by different molecular mechanisms. N-arachidonoyl taurine is therefore an interesting prototype compound that may inspire development of future IKs channel activators to treat Long QT syndrome caused by diverse IKs channel mutations.

Download Full-text

Trafficking-deficient hERG K+ channels linked to long QT syndrome are regulated by a microtubule-dependent quality control compartment in the ER

AJP Cell Physiology ◽

10.1152/ajpcell.00494.2010 ◽

2011 ◽

Vol 301 (1) ◽

pp. C75-C85 ◽

Cited By ~ 24

Author(s):

Jennifer L. Smith ◽

Christie M. McBride ◽

Parvathi S. Nataraj ◽

Daniel C. Bartos ◽

Craig T. January ◽

...

Keyword(s):

Golgi Apparatus ◽

Long Qt Syndrome ◽

Functional Expression ◽

Delayed Rectifier ◽

Temperature Sensitive ◽

Missense Mutations ◽

Long Qt ◽

Delayed Rectifier Current ◽

Golgi Compartment ◽

Qt Syndrome

The human ether-a-go-go related gene ( hERG) encodes the voltage-gated K+ channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an “immature” N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.

Download Full-text