Ackerman, Michael J.

Introduction: Cardiac differentiation protocols enable derivation of cardiomyocytes (CM). from human induced pluripotent stem cell (iPS). However, limited tools are available to ensure enhanced iPS-CM maturation without the need for prolonged and complex culture. Research Question: This study assessed whether the mesodermal and cardiopoiesis inductor, Brachyury (T), complements standard cardiac differentiation and augments structural and functional maturation of iPS-CM. Aims: The impact of T mRNA introduction prior to initiation of cardiac differentiation was characterized on: i) cardiac transcription factor expression; ii) structural and functional hallmarks of maturity; and iii) cardiotoxicity. Methods: RT-qPCR was employed to monitor the expression of cardiac-specific transcription factors. Transmission electron microscopy enabled the assessment of cellular ultrastructure. Seahorse measurements were used to determine basal respiration. Fluo4 staining was used to measure calcium transients with external field stimulation. Cardiotoxicity was measured as the impact of doxorubicin on calcium handling and cell survival. Results: Transfection with T prompted early and sustained upregulation of the cardiac transcription factors Nkx2.5 (10-fold; FDR<0.001) and MEF2C (7.2-fold; FDR<0.001). Mitochondrial area was increased with T transfection (P<0.001) and T-transfected iPS-CM exhibited greater basal respiration (FDR<0.05), greater calcium handling resilience in response to field stimulation, and a more mature transient morphology. Following doxorubicin exposure T transfected iPS-CM exhibited a greater compromise in calcium handling (FDR<0.001), and exaggerated cell death. Conclusion: Induction of human iPS with T prior to initiation of cardiac differentiation enhanced signatures of structural and functional maturity, and provided more robust readouts of drug toxicity.

BACKGROUND: Long QT syndrome is a lethal arrhythmia syndrome, frequently caused by rare loss-of-function variants in the potassium channel encoded by KCNH2 . Variant classification is difficult, often because of lack of functional data. Moreover, variant-based risk stratification is also complicated by heterogenous clinical data and incomplete penetrance. Here we sought to test whether variant-specific information, primarily from high-throughput functional assays, could improve both classification and cardiac event risk stratification in a large, harmonized cohort of KCNH2 missense variant heterozygotes. METHODS: We quantified cell-surface trafficking of 18 796 variants in KCNH2 using a multiplexed assay of variant effect (MAVE). We recorded KCNH2 current density for 533 variants by automated patch clamping. We calibrated the strength of evidence of MAVE data according to ClinGen guidelines. We deeply phenotyped 1458 patients with KCNH2 missense variants, including QTc, cardiac event history, and mortality. We correlated variant functional data and Bayesian long QT syndrome penetrance estimates with cohort phenotypes and assessed hazard ratios for cardiac events. RESULTS: Variant MAVE trafficking scores and automated patch clamping peak tail currents were highly correlated (Spearman rank-order ρ=0.69; n=433). The MAVE data were found to provide up to pathogenic very strong evidence for severe loss-of-function variants. In the cohort, both functional assays and Bayesian long QT syndrome penetrance estimates were significantly predictive of cardiac events when independently modeled with patient sex and corrected QT interval (QTc); however, MAVE data became nonsignificant when peak tail current and penetrance estimates were also available. The area under the receiver operator characteristic curve for 20-year event outcomes based on patient-specific sex and QTc (area under the curve, 0.80 [0.76–0.83]) was improved with prospectively available penetrance scores conditioned on MAVE (area under the curve, 0.86 [0.83–0.89]) or attainable automated patch clamping peak tail current data (area under the curve, 0.84 [0.81–0.88]). CONCLUSIONS: High-throughput KCNH2 variant MAVE data meaningfully contribute to variant classification at scale, whereas long QT syndrome penetrance estimates and automated patch clamping peak tail current measurements meaningfully contribute to risk stratification of cardiac events in patients with heterozygous KCNH2 missense variants.

BACKGROUND: Genetic testing for cardiac channelopathies is the standard of care. However, many rare genetic variants remain classified as variants of uncertain significance (VUS) due to lack of epidemiological and functional data. Whether deep protein language models may aid in VUS resolution remains unknown. Here, we set out to compare how 2 deep protein language models perform at VUS resolution in the 3 most common long-QT syndrome–causative genes compared with the gold-standard patch clamp. METHODS: A total of 72 rare nonsynonymous VUS (9 KCNQ1, 19 KCNH2 , and 50 SCN5A ) were engineered by site-directed mutagenesis and expressed in either HEK293 cells or TSA201 cells. Whole-cell patch-clamp technique was used to functionally characterize these variants. The protein language models, evolutionary scale modeling, version 1b and AlphaMissense, were used to predict the variant effect of missense variants and compared with patch clamp. RESULTS: Considering variants in all 3 genes, the evolutionary scale modeling, version 1b model had a receiver operating characteristic curve–area under the curve of 0.75 ( P =0.0003). It had a sensitivity of 88% and a specificity of 50%. AlphaMissense performed well compared with patch-clamp with an receiver operating characteristic curve-area under the curve of 0.85 ( P <0.0001), sensitivity of 80%, and specificity of 76%. CONCLUSIONS: Deep protein language models aid in VUS resolution with high sensitivity but lower specificity. Thus, these tools cannot fully replace functional characterization but can aid in reducing the number of variants that may require functional analysis.

Abstract Background and Aims Type 1 long QT syndrome (LQT1) is caused by pathogenic variants in the KCNQ1-encoded Kv7.1 potassium channels, which pathologically prolong ventricular action potential duration (APD). Herein, the pathologic phenotype in transgenic LQT1 rabbits is rescued using a novel KCNQ1 suppression-replacement (SupRep) gene therapy. Methods KCNQ1-SupRep gene therapy was developed by combining into a single construct a KCNQ1 shRNA (suppression) and an shRNA-immune KCNQ1 cDNA (replacement), packaged into adeno-associated virus serotype 9, and delivered in vivo via an intra-aortic root injection (1E10 vg/kg). To ascertain the efficacy of SupRep, 12-lead electrocardiograms were assessed in adult LQT1 and wild-type (WT) rabbits and patch-clamp experiments were performed on isolated ventricular cardiomyocytes. Results KCNQ1-SupRep treatment of LQT1 rabbits resulted in significant shortening of the pathologically prolonged QT index (QTi) towards WT levels. Ventricular cardiomyocytes isolated from treated LQT1 rabbits demonstrated pronounced shortening of APD compared to LQT1 controls, leading to levels similar to WT (LQT1-UT vs. LQT1-SupRep, P &lt; .0001, LQT1-SupRep vs. WT, P = ns). Under β-adrenergic stimulation with isoproterenol, SupRep-treated rabbits demonstrated a WT-like physiological QTi and APD90 behaviour. Conclusions This study provides the first animal-model, proof-of-concept gene therapy for correction of LQT1. In LQT1 rabbits, treatment with KCNQ1-SupRep gene therapy normalized the clinical QTi and cellular APD90 to near WT levels both at baseline and after isoproterenol. If similar QT/APD correction can be achieved with intravenous administration of KCNQ1-SupRep gene therapy in LQT1 rabbits, these encouraging data should compel continued development of this gene therapy for patients with LQT1.

BACKGROUND: CaM (calmodulin)-mediated long-QT syndrome is a genetic arrhythmia disorder (calmodulinopathies) characterized by a high prevalence of life-threatening ventricular arrhythmias occurring early in life. Three distinct genes ( CALM1 , CALM2 , and CALM3 ) encode for the identical CaM protein. Conventional pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with calmodulinopathy. METHODS: Five custom-designed CALM1 -, CALM2 -, and CALM3 -targeting short hairpin RNAs (shRNAs) were tested for knockdown (KD) efficiency using TSA201 cells and reverse transcription-quantitative polymerase chain reaction. A dual-component suppression and replacement (SupRep) CALM gene therapy (CALM-SupRep) was created by cloning into a single construct CALM1 -, CALM2 -, and CALM3-specific shRNAs that produce KD (suppression) of each respective gene and a shRNA-immune CALM1 cDNA (replacement). CALM1-F142L, CALM2-D130G, and CALM3-D130G induced pluripotent stem cell-derived CMs were generated from patients with CaM-mediated long-QT syndrome. A voltage-sensing dye was used to measure action potential duration at 90% repolarization (APD90). RESULTS: Following shRNA KD efficiency testing, a candidate shRNA was identified for CALM1 (86% KD), CALM2 (71% KD), and CALM3 (94% KD). The APD90 was significantly prolonged in CALM2-D130G (647±9 ms) compared with CALM2-WT (359±12 ms; P <0.0001). Transfection with CALM -SupRep shortened the average APD90 of CALM2-D130G to 457±19 ms (66% attenuation; P <0.0001). Additionally, transfection with CALM -SupRep shortened the APD90 of CALM1-F142L (665±9 to 410±15 ms; P <0.0001) and CALM3-D130G (978±81 to 446±6 ms; P <0.001). CONCLUSIONS: We provide the first proof-of-principle suppression-replacement gene therapy for CaM-mediated long-QT syndrome. The CALM-SupRep gene therapy shortened the pathologically prolonged APD90 in CALM1 -, CALM2 -, and CALM3-variant CaM-mediated long-QT syndrome induced pluripotent stem cell-derived CM lines. The single CALM-SupRep construct may be able to treat all calmodulinopathies, regardless of which of the 3 CaM-encoding genes are affected.

BACKGROUND: Whether vigorous exercise increases risk of ventricular arrhythmias for individuals diagnosed and treated for congenital long QT syndrome (LQTS) remains unknown. METHODS: The National Institutes of Health–funded LIVE-LQTS study (Lifestyle and Exercise in the Long QT Syndrome) prospectively enrolled individuals 8 to 60 years of age with phenotypic and/or genotypic LQTS from 37 sites in 5 countries from May 2015 to February 2019. Participants (or parents) answered physical activity and clinical events surveys every 6 months for 3 years with follow-up completed in February 2022. Vigorous exercise was defined as ≥6 metabolic equivalents for >60 hours per year. A blinded Clinical Events Committee adjudicated the composite end point of sudden death, sudden cardiac arrest, ventricular arrhythmia treated by an implantable cardioverter defibrillator, and likely arrhythmic syncope. A National Death Index search ascertained vital status for those with incomplete follow-up. A noninferiority hypothesis (boundary of 1.5) between vigorous exercisers and others was tested with multivariable Cox regression analysis. RESULTS: Among the 1413 participants (13% <18 years of age, 35% 18–25 years of age, 67% female, 25% with implantable cardioverter defibrillators, 90% genotype positive, 49% with LQT1, 91% were treated with beta-blockers, left cardiac sympathetic denervation, and/or implantable cardioverter defibrillator), 52% participated in vigorous exercise (55% of these competitively). Thirty-seven individuals experienced the composite end point (including one sudden cardiac arrest and one sudden death in the nonvigorous group, one sudden cardiac arrest in the vigorous group) with overall event rates at 3 years of 2.6% in the vigorous and 2.7% in the nonvigorous exercise groups. The unadjusted hazard ratio for experience of events for the vigorous group compared with the nonvigorous group was 0.97 (90% CI, 0.57–1.67), with an adjusted hazard ratio of 1.17 (90% CI, 0.67–2.04). The upper 95% one-sided confidence level extended beyond the 1.5 boundary. Neither vigorous or nonvigorous exercise was found to be superior in any group or subgroup. CONCLUSIONS: Among individuals diagnosed with phenotypic and/or genotypic LQTS who were risk assessed and treated in experienced centers, LQTS-associated cardiac event rates were low and similar between those exercising vigorously and those not exercising vigorously. Consistent with the low event rate, CIs are wide, and noninferiority was not demonstrated. These data further inform shared decision-making discussions between patient and physician about exercise and competitive sports participation. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02549664.

BACKGROUND: Despite major advances in the clinical management of long QT syndrome, some patients are not fully protected by beta-blocker therapy. Mexiletine is a well-known sodium channel blocker, with proven efficacy in patients with sodium channel–mediated long QT syndrome type 3. Our aim was to evaluate the efficacy of mexiletine in long QT syndrome type 2 (LQT2) using cardiomyocytes derived from patient-specific human induced pluripotent stem cells, a transgenic LQT2 rabbit model, and patients with LQT2. METHODS: Heart rate–corrected field potential duration, a surrogate for QTc, was measured in human induced pluripotent stem cells from 2 patients with LQT2 (KCNH2-p.A561V, KCNH2-p.R366X) before and after mexiletine using a multiwell multi-electrode array system. Action potential duration at 90% repolarization (APD 90 ) was evaluated in cardiomyocytes isolated from transgenic LQT2 rabbits (KCNH2-p.G628S) at baseline and after mexiletine application. Mexiletine was given to 96 patients with LQT2. Patients were defined as responders in the presence of a QTc shortening ≥40 ms. Antiarrhythmic efficacy of mexiletine was evaluated by a Poisson regression model. RESULTS: After acute treatment with mexiletine, human induced pluripotent stem cells from both patients with LQT2 showed a significant shortening of heart rate–corrected field potential duration compared with dimethyl sulfoxide control. In cardiomyocytes isolated from LQT2 rabbits, acute mexiletine significantly shortened APD 90 by 113 ms, indicating a strong mexiletine-mediated shortening across different LQT2 model systems. Mexiletine was given to 96 patients with LQT2 either chronically (n=60) or after the acute oral drug test (n=36): 65% of the patients taking mexiletine only chronically and 75% of the patients who performed the acute oral test were responders. There was a significant correlation between basal QTc and ∆QTc during the test ( r = −0.8; P <0.001). The oral drug test correctly predicted long-term effect in 93% of the patients. Mexiletine reduced the mean yearly event rate from 0.10 (95% CI, 0.07–0.14) to 0.04 (95% CI, 0.02–0.08), with an incidence rate ratio of 0.40 (95% CI, 0.16–0.84), reflecting a 60% reduction in the event rate ( P =0.01). CONCLUSIONS: Mexiletine significantly shortens cardiac repolarization in LQT2 human induced pluripotent stem cells, in the LQT2 rabbit model, and in the majority of patients with LQT2. Furthermore, mexiletine showed antiarrhythmic efficacy. Mexiletine should therefore be considered a valid therapeutic option to be added to conventional therapies in higher-risk patients with LQT2.

Importance Sudden death and cardiac arrest frequently occur without explanation, even after a thorough clinical evaluation. Calcium release deficiency syndrome (CRDS), a life-threatening genetic arrhythmia syndrome, is undetectable with standard testing and leads to unexplained cardiac arrest. Objective To explore the cardiac repolarization response on an electrocardiogram after brief tachycardia and a pause as a clinical diagnostic test for CRDS. Design, Setting, and Participants An international, multicenter, case-control study including individual cases of CRDS, 3 patient control groups (individuals with suspected supraventricular tachycardia; survivors of unexplained cardiac arrest [UCA]; and individuals with genotype-positive catecholaminergic polymorphic ventricular tachycardia [CPVT]), and genetic mouse models (CRDS, wild type, and CPVT were used to define the cellular mechanism) conducted at 10 centers in 7 countries. Patient tracings were recorded between June 2005 and December 2023, and the analyses were performed from April 2023 to December 2023. Intervention Brief tachycardia and a subsequent pause (either spontaneous or mediated through cardiac pacing). Main Outcomes and Measures Change in QT interval and change in T-wave amplitude (defined as the difference between their absolute values on the postpause sinus beat and the last beat prior to tachycardia). Results Among 10 case patients with CRDS, 45 control patients with suspected supraventricular tachycardia, 10 control patients who experienced UCA, and 3 control patients with genotype-positive CPVT, the median change in T-wave amplitude on the postpause sinus beat (after brief ventricular tachycardia at ≥150 beats/min) was higher in patients with CRDS ( P &amp;lt; .001). The smallest change in T-wave amplitude was 0.250 mV for a CRDS case patient compared with the largest change in T-wave amplitude of 0.160 mV for a control patient, indicating 100% discrimination. Although the median change in QT interval was longer in CRDS cases ( P = .002), an overlap between the cases and controls was present. The genetic mouse models recapitulated the findings observed in humans and suggested the repolarization response was secondary to a pathologically large systolic release of calcium from the sarcoplasmic reticulum. Conclusions and Relevance There is a unique repolarization response on an electrocardiogram after provocation with brief tachycardia and a subsequent pause in CRDS cases and mouse models, which is absent from the controls. If these findings are confirmed in larger studies, this easy to perform maneuver may serve as an effective clinical diagnostic test for CRDS and become an important part of the evaluation of cardiac arrest.

Abstract Background Short QT syndrome type 1 (SQT1) is a genetic channelopathy characterized by gain-of-function variants in the KCNH2-encoded potassium channel underlying the IKr current, leading to a pathologically shortened cardiac action potential duration (APD). Patients with SQT1 manifest shortening of the QT duration on 12-lead ECG and are prone to cardiac arrhythmias that can culminate in sudden cardiac death. Purpose To investigate the effect of a KCNH2-specific suppression-replacement (KCNH2-SupRep) gene therapy on the pathogenic phenotype in transgenic SQT1 rabbits. Methods A novel dual-component KCNH2-SupRep gene therapy was developed by combining a custom-designed KCNH2 shRNA (suppression) and an shRNA-immune (shIMM) KCNH2 cDNA (replacement) into AAV9. In vivo AAV9-mediated KCNH2-SupRep / sham treatment was performed by intra-aortic root injection of the KCNH2-SupRep AAV9 (1E10 vg/kg) / NaCl 0.9% during balloon occlusion using a Swan Ganz catheter. Three weeks after administration, 12-lead ECGs were assessed in adult transgenic SQT1 (KCNH2-N588K, gain of IKr due to impaired inactivation) and wild-type (WT) rabbits to determine the effect of KCNH2-SupRep / sham treatment on the rabbit’s QTc duration and QT index. Patch-clamp recordings were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of KCNH2-SupRep / sham treatment on the CM’s action potential duration at 90% repolarization (APD90). Results After in vivo AAV9-mediated KCNH2-SupRep gene therapy injection (1E10 vg/kg) in 7 SQT1 rabbits, QTc duration and QT index significantly increased in all SQT1 rabbits (QTc: untreated(UT)-SQT1: 153.2±3.8ms vs treated-SQT1: 166.5±4.6ms, p=0.0002, N=7 each; QTi: UT-SQT1: 92.5±1.9% vs treated-SQT1: 99.7±2.7%, p=0.0007, N=7 each), reaching a QT index of ~100% typically observed in WT rabbits. No such changes were observed in the sham-SQT1 rabbits. Furthermore, cardiomyocytes isolated from 7 SQT1 rabbits that received in vivo SupRep gene therapy demonstrated APD90 prolongation closer to WT rabbits compared to UT-SQT1 and sham-SQT1 rabbits (APD90: UT-SQT1: 314.2±11.9ms, WT: 404.5±13.8ms, treated-SQT1: 362.9±10.6ms; UT-SQT1 vs WT: p&lt;0.0001; UT-SQT1 vs treated-SQT1: p=0.019; WT vs Treated-SQT1: p=0.031). In line with these observations, first IKr measurements demonstrate smaller IKr currents in SupRep-SQT1 than in sham-SQT1 CMs. Conclusion In vivo KCNH2-SupRep gene therapy normalizes the pathologically shortened QTc duration and QT index in transgenic SQT1 rabbits. Moreover, KCNH2-SupRep treatment prolongs APD90 in SQT1 compared to UT-SQT1 and sham-SQT1 reaching levels closer to WT.