SGK1 inhibition attenuated the action potential duration in patient- and genotype-specific re-engineered heart cells with congenital long QT syndrome.

Background: Long QT syndrome (LQTS) stems from pathogenic variants in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3) and is characterized by action potential duration (APD) prolongation. Inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) is proposed as a novel therapeutic for LQTS. Objective: The study sought to test the efficacy of novel, selective SGK1 inhibitors in induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) models of LQTS. Methods: The mexiletine (MEX)-sensitive SCN5A-P1332L iPSC-CMs were tested initially compared with a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 SCN5A-P1332L variant-corrected isogenic control (IC). The SGK1-I1 therapeutic efficacy, compared with MEX, was tested for APD at 90% repolarization (APD90) shortening in SCN5A-P1332L, SCN5A-R1623Q, KCNH2-G604S, and KCNQ1-V254M iPSC-CMs using FluoVolt. Results: The APD90 was prolonged in SCN5A-P1332L iPSC-CMs compared with its IC (646 ± 7 ms vs 482 ± 23 ms; P < .0001). MEX shortened the APD90 to 560 ± 7 ms (52% attenuation, P < .0001). SGK1-I1 shortened the APD90 to 518 ± 5 ms (78% attenuation, P < .0001) but did not shorten the APD90 in the IC. SGK1-I1 shortened the APD90 of the SCN5A-R1623Q iPSC-CMs (753 ± 8 ms to 475 ± 19 ms compared with 558 ± 19 ms with MEX), the KCNH2-G604S iPSC-CMs (666 ± 10 ms to 574 ± 18 ms vs 538 ± 15 ms after MEX), and the KCNQ1-V254M iPSC-CMs (544 ± 10 ms to 475 ± 11ms; P = .0004). Conclusions: Therapeutically inhibiting SGK1 effectively shortens the APD in human iPSC-CM models of the 3 major LQTS genotypes. These preclinical data support development of SGK1 inhibitors as novel, first-in-class therapy for patients with congenital LQTS.

SGK1 inhibition attenuates the action potential duration in reengineered heart cell models of drug-induced QT prolongation.

BACKGROUND: Drug-induced QT prolongation (DI-QTP) is a clinical entity in which administration of a human ether-à-go-go-related gene/rapid delayed rectifier potassium current blocker such as dofetilide prolongs the cardiac action potential duration (APD) and the QT interval on the electrocardiogram. Inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) reduces the APD at 90% repolarization (APD90) in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from patients with congenital long QT syndrome. OBJECTIVE: Here, we test the efficacy of 2 novel SGK1 inhibitors-SGK1-I1 and SGK1-I2-in iPSC-CM models of dofetilide-induced APD prolongation. METHODS: Normal iPSC-CMs were treated with dofetilide to produce a DI-QTP iPSC-CM model. SGK1-I1's and SGK1-I2's therapeutic efficacy for shortening the dofetilide-induced APD90 prolongation was compared to mexiletine. The APD90 values were recorded 4 hours after treatment using a voltage-sensing dye. RESULTS: The APD90 was prolonged in normal iPSC-CMs treated with dofetilide (673 ± 8 ms vs 436 ± 4 ms; P < .0001). While 10 mM mexiletine shortened the APD90 of dofetilide-treated iPSC-CMs from 673 ± 4 to 563 ± 8 ms (46% attenuation; P < .0001), 30 nM of SGK1-I1 shortened the APD90 from 673 ± 8 to 502 ± 7 ms (72% attenuation; P < .0001). Additionally, 300 nM SGK1-I2 shortened the APD90 of dofetilide-treated iPSC-CMs from 673 ± 8 to 460 ± 7 ms (90% attenuation; P < .0001). CONCLUSION: These novel SGK1-Is substantially attenuated the pathological APD prolongation in a human heart cell model of DI-QTP. These preclinical data support the development of this therapeutic strategy to counter and neutralize DI-QTP, thereby increasing the safety profile for patients receiving drugs with torsadogenic potential.

Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I-IV, with the most N-terminal domain involving residues 77-466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants. Isogenic control iPSCs were generated using CRISPR-Cas9/PiggyBac. Fluo-4 Ca2+ imaging assessed Ca2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca2+ sparking and Ca2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca2+ amplitude and sparking in variant iPSC-CMs. Molecular dynamic simulations visualized the structural role of these variants. We provide the first functional evidence that these most proximal N-terminal localizing variants alter calcium handling similar to CPVT1. These variants are located at the N-terminal domain and the central domain interface and could destabilize the RYR2 channel promoting Ca2+ leak-triggered arrhythmias.

Acacetin, a Potent Transient Outward Current Blocker, May Be a Novel Therapeutic for KCND3-Encoded Kv4.3 Gain-of-Function-Associated J-Wave Syndromes.

BACKGROUND: The transient outward current (Ito) that mediates early (phase 1) repolarization is conducted by the KCND3-encoded Kv4.3 pore-forming α-subunit. KCND3 gain-of-function mutations have been reported previously as a pathogenic substrate for J wave syndromes (JWS), including the Brugada syndrome and early repolarization syndrome, as well as autopsy-negative sudden unexplained death (SUD). Acacetin, a natural flavone, is a potent Ito current blocker. Acacetin may be a novel therapeutic for KCND3-mediated J wave syndrome. METHODS: KCND3-V392I was identified in an 18-year-old male with J wave syndrome/early repolarization syndrome, and a history of cardiac arrest including ventricular tachycardia/ventricular fibrillation and atrial fibrillation/atrial flutter. Pathogenic KCND3 mutation was engineered by site-directed mutagenesis and co-expressed with wild-type KChIP2 in TSA201 cells. Gene-edited/variant-corrected isogenic control and patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the p. Val392Ile-KCND3-positive patient were generated. Ito currents and action potentials were recorded before and after treatment with Acacetin using the whole cell patch-clamp and multielectrode array technique. Western blot and immunocytochemistry were performed to investigate KCND3 expression. RESULTS: KCND3-V392I demonstrated a marked gain-of-function phenotype, increasing peak Ito current density by 92.2% (P<0.05 versus KCND3-WT). KCND3 expression was significantly increased in KCND3-V392I-derived iPSC-CMs (P<0.05 versus isogenic control). While KCND3-WT revealed an IC50 of 7.2±1.0 µmol/L for acacetin effect, 30 µmol/L acacetin dramatically inhibited KCND3-V392I peak Ito current density by 96.2% (P<0.05 versus before Acacetin). Ito was also increased by 60.9% in Kv4.3-V392I iPSC-CM (P<0.05 versus isogenic control iPSC-CM). Ten micromoles per liter acacetin, a concentration approaching its IC50 value, inhibited Ito by ≈50% in patient-derived iPSC-CMs and reduced the accentuated action potential notch displayed in KCND3-V392I-derived iPSC-CMs. CONCLUSIONS: This preclinical study provides pharmacological and functional evidence to suggest that Acacetin may be a novel therapeutic for patients with KCND3 gain-of-function-associated J wave syndrome by inhibiting Ito and abolishing the accentuated action potential notch in patient-derived iPSC-CMs.

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.

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.

Molecular characterization of the calcium release channel deficiency syndrome.

We identified a potentially novel homozygous duplication involving the promoter region and exons 1-4 of the gene encoding type 2 cardiac ryanodine receptor (RYR2) that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2 duplication (RYR2-DUP) induced pluripotent stem cell cardiomyocytes (iPSC-CMs) were generated from 2 unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT-iPSC-CM and RYR2-DUP-iPSC-CM lines. However, compared with WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine-stimulated and isoproterenol-stimulated (ISO-stimulated) Ca2+ transient amplitude, suggesting RyR2 loss of function. There was a greater than 50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared with WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared with WT-iPSC-CMs, there was significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5-fold, 6.8-fold, and 2.4-fold, respectively, from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multiexon duplication precipitated a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss of function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.

Identification of a Novel Homozygous Multi-Exon Duplication in RYR2 Among Children With Exertion-Related Unexplained Sudden Deaths in the Amish Community.

Importance: The exome molecular autopsy may elucidate a pathogenic substrate for sudden unexplained death. Objective: To investigate the underlying cause of multiple sudden deaths in young individuals and sudden cardiac arrests that occurred in 2 large Amish families. Design, Setting, and Participants: Two large extended Amish families with multiple sudden deaths in young individuals and sudden cardiac arrests were included in the study. A recessive inheritance pattern was suggested based on an extended family history of sudden deaths in young individuals and sudden cardiac arrests, despite unaffected parents. A family with exercise-associated sudden deaths in young individuals occurring in 4 siblings was referred for postmortem genetic testing using an exome molecular autopsy. Copy number variant (CNV) analysis was performed on exome data using PatternCNV. Chromosomal microarray validated the CNV identified. The nucleotide break points of the CNV were determined by mate-pair sequencing. Samples were collected for this study between November 2004 and June 2019. Main Outcomes and Measures: The identification of an underlying genetic cause for sudden deaths in young individuals and sudden cardiac arrests consistent with the recessive inheritance pattern observed in the families. Results: A homozygous duplication, involving approximately 26 000 base pairs of intergenic sequence, RYR2's 5'UTR/promoter region, and exons 1 through 4 of RYR2, was identified in all 4 siblings of a family. Multiple distantly related relatives experiencing exertion-related sudden cardiac arrest also had the identical RYR2 homozygous duplication. A second, unrelated family with multiple exertion-related sudden deaths and sudden cardiac arrests in young individuals, with the same homozygous duplication, was identified. Several living, homozygous duplication-positive symptomatic patients from both families had nondiagnostic cardiologic testing, with only occasional ventricular ectopy occurring during exercise stress tests. Conclusions and Relevance: In this analysis, we identified a novel, highly penetrant, homozygous multiexon duplication in RYR2 among Amish youths with exertion-related sudden death and sudden cardiac arrest but without an overt phenotype that is distinct from RYR2-mediated catecholaminergic polymorphic ventricular tachycardia. Considering that no cardiac tests reliably identify at-risk individuals and given the high rate of consanguinity in Amish families, identification of unaffected heterozygous carriers may provide potentially lifesaving premarital counseling and reproductive planning.