Patient-specific, re-engineered cardiomyocyte model confirms the circumstance-dependent arrhythmia risk associated with the African-specific common SCN5A polymorphism p.S1103Y: Implications for the increased sudden deaths observed in black individuals during the COVID-19 pandemic.

BACKGROUND: During the early stages of the coronavirus disease 2019 (COVID-19) pandemic, a marked increase in sudden cardiac death (SCD) was observed. The p.S1103Y-SCN5A common variant, which is present in ∼8% of individuals of African descent, may be a circumstance-dependent, SCD-predisposing, proarrhythmic polymorphism in the setting of hypoxia-induced acidosis or QT-prolonging drug use. OBJECTIVE: The purpose of this study was to ascertain the effects of acidosis and hydroxychloroquine (HCQ) on the action potential duration (APD) in a patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model of p.S1103Y-SCN5A. METHODS: iPSC-CMs were generated from a 14-year-old p.S1103Y-SCN5A-positive African American male. The patient's variant-corrected iPSC-CMs (isogenic control [IC]) were generated using CRISPR/Cas9 technology. FluoVolt voltage-sensitive dye was used to assess APD90 values in p.S1103Y-SCN5A iPSC-CMs compared to IC before and after an acidotic state (pH 6.9) or 24 hours of treatment with 10 µM HCQ. RESULTS: Under baseline conditions (pH 7.4), there was no difference in APD90 values of p.S1103Y-SCN5A vs IC iPSC-CMs (P = NS). In the setting of acidosis (pH 6.9), there was a significant increase in fold-change of APD90 in p.S1103Y-SCN5A iPSC-CMs compared to IC iPSC-CMs (P <.0001). Similarly, with 24-hour 10 µM HCQ treatment, the fold-change of APD90 was significantly higher in p.S1103Y-SCN5A iPSC-CMs compared to IC iPSC-CMs (P <.0001). CONCLUSION: Although the African-specific p.S1103Y-SCN5A common variant had no effect on APD90 under baseline conditions, the physiological stress of either acidosis or HCQ treatment significantly prolonged APD90 in patient-specific, re-engineered heart cells.

Development of a Patient-Specific p.D85N-Potassium Voltage-Gated Channel Subfamily E Member 1-Induced Pluripotent Stem Cell-Derived Cardiomyocyte Model for Drug-Induced Long QT Syndrome.

BACKGROUND: Prior epidemiological studies demonstrated that the p.D85N-Potassium voltage-gated channel subfamily E member 1 (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 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 heart rate-corrected QT interval response to metoclopramide (ΔQTc of 160 ms). Clustered regularly interspaced short palindromic repeats-associated 9 technology was used to generate gene-corrected isogenic iPSCs. Field potential duration and action potential duration (APD) were measured from iPSC-CMs. RESULTS: At baseline, p.D85N-KCNE1 iPSC-CMs displayed significantly longer field potential duration (281±15 ms, n=13 versus 223±8.6 ms, n=14, P<0.01) and action potential duration at 90% repolarization (APD90; 579±22 ms, n=24 versus 465±33 ms, n=26, P<0.01) than isogenic-control iPSC-CMs. Dofetilide at a concentration of 2 nM increased significantly field potential duration (379±20 ms, n=13, P<0.01) and APD90 (666±11 ms, n=46, P<0.01) in p.D85N-KCNE1 iPSC-CMs but not in isogenic-control. The effect of dofetilide on APD90 (616±54 ms, n=7 versus 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 1 nM exaggerated further dofetilide-induced APD90 prolongation (696±9 ms, n=81, P<0.01) and caused more early afterdepolarizations (11.7%) compared with isogenic control (APD90: 618±8 ms, n=115 and early afterdepolarizations: 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 proarrhythmic.

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.

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.

MRAS Variants Cause Cardiomyocyte Hypertrophy in Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Additional Evidence for MRAS as a Definitive Noonan Syndrome-Susceptibility Gene.

BACKGROUND: MRAS was identified recently as a novel Noonan syndrome (NS)-susceptibility gene. Phenotypically, both patients with NS, harboring pathogenic MRAS variants, displayed severe cardiac hypertrophy. This study aimed to demonstrate both the necessity and sufficiency of a patient-specific variant (p.Gly23Val-MRAS) to cause NS through the generation and characterization of patient-specific, isogenic control, and disease modeled induced pluripotent stem cell (iPSC) lines. METHODS: iPSCs were derived from a patient with a p.Gly23Val-MRAS variant to assess the effect of MRAS variants on pathogenesis of NS-associated cardiac hypertrophy. CRISPR/Cas9 gene editing was used to correct the pathogenic p.Gly23Val-MRAS variant in patient cells (isogenic control) and to introduce the pathogenic variant into unrelated control cells (disease modeled) to determine the necessity and sufficiency of the p.Gly23Val-MRAS variant to elicit the disease phenotype in iPSC-derived cardiomyocytes (iPSC-CMs). iPSC-CMs were analyzed by microscopy and immunofluroesence, single-cell RNAseq, Western blot, room temperature-quantitative polymerase chain reaction, and live-cell calcium imaging to define an in vitro phenotype of MRAS-mediated cardiac hypertrophy. RESULTS: Compared with controls, both patient and disease modeled iPSC-CMs were significantly larger and demonstrated changes in gene expression and intracellular pathway signaling characteristic of cardiac hypertrophy. Additionally, patient and disease modeled iPSC-CMs displayed impaired Ca2+ handling, including increased frequency of irregular Ca2+ transients and changes in Ca2+ handling kinetics. CONCLUSIONS: p.Gly23Val-MRAS is both necessary and sufficient to elicit a cardiac hypertrophy phenotype in iPSC-CMs that includes increased cell size, changes in cardiac gene expression, and abnormal calcium handling-providing further evidence to establish the monogenetic pathogenicity of p.Gly23Val-MRAS in NS with cardiac hypertrophy.