Citations

Introduction: Over 100 FDA-approved medications, electrolyte perturbations, and many disease states can prolong the QTc beyond its 99 th percentile value resulting in acquired QT prolongation. In contrast, approximately 1 in 2000 people have congenital long QT syndrome (LQTS) hallmarked by pathological QT prolongation secondary to genetic defects in the heart. Hypothesis: An artificial intelligence (AI) deep neural network (DNN) can distinguish patients with LQTS from those with acquired QT prolongation. Methods: The study cohort included all patients with LQTS evaluated in the Windland Smith Rice Genetic Heart Rhythm Clinic and controls from Mayo Clinic’s ECG data vault comprising over 2.5 million patients. For the AI-DNN model, every patient/control with ≥ 1 ECG above age- and sex- specific 99 th percentile values for QTc [> 460 ms for all patients (male/female) < 13 years of age, or > 470 ms for men and > 480 ms for women above this age] was included. An AI-DNN involving a multi-layer c onvolutional n eural n etwork (CNN) was developed to classify patients. LQTS patients were age- and sex- matched to controls at 1:5 ratio. Results: Among the 1,599 patients with genetically confirmed LQTS, 808 had ≥ 1 ECG with QTc above the threshold (2,987 ECGs) compared to 361,069/2.5M controls (14% of Mayo Clinic patients getting an ECG, ‘presumed negative’; 989,313 ECGs). Following age- and sex- matching and splitting, 3,309 (training), 411 (validation) and 887 (testing) control ECGs were used. This model distinguished LQTS from those with acquired QT prolongation with an AUC of 0.896 (accuracy 85%, sensitivity 77%, specificity 88%, PPV 0.58, NPV 0.94). After exclusion of patients with a wide QRS (>150 ms) or pacemaker, the model remained successful in distinguishing the two groups (AUC 0.85, accuracy 78%, sensitivity 78%, specificity 87%, PPV 59%, NPV 94%). Conclusions: For patients with a QTc exceeding its 99 th percentile values, this novel AI-DNN functions as a LQTS mutation detector being able to identify patients with abnormal QT prolongation secondary to a LQTS-causative mutation rather than acquired QT prolongation with a >50% positive predictive value. This algorithm may facilitate screening for this potentially lethal, yet highly treatable, genetic heart disease.

Introduction: Calmodulin (CaM)-mediated long QT syndrome (CaM-LQTS) is a genetic arrhythmia disorder characterized by a high prevalence of life-threatening ventricular arrhythmias occurring very early in life, frequent T-wave alternans, markedly prolonged QT intervals (QTc > 600 ms), and intermittent 2:1 AV block. Three distinct genes ( CALM1 , CALM2 , and CALM3 ) all encode for the identical CaM protein. Conventional LQTS pharmacotherapies fail to adequately protect against potentially lethal cardiac events in patients with CaM-LQTS. Here, we developed a CALM1 “ suppression-replacement (SupRep)” gene therapy with the goal of rescuing the prolonged cardiac action potential duration (APD) and increased Ca 2+ amplitude (ΔF/F0) in patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a patient with CALM1 -mediated LQTS. Methods: Custom designed CALM1 -targeting shRNAs were tested for knockdown efficiency using TSA201 cells and RT-qPCR. A dual-component SupRep CALM1 gene therapy was created by cloning into a single construct a custom-designed CALM1 shRNA that produces knockdown (suppression) and a “shRNA-immune” (shIMM) CALM1 cDNA (replacement). Patient-specific CALM1-F142L iPSC-CMs were generated from a patient with severe LQTS (QTc = 515 ms). FluoVolt voltage-sensing dye was used to measure the APD at 90% repolarization (APD 90 ). Calcium transient amplitude was measured using Fluo-4 indicator. Results: Overall, 6 unique candidate CALM1 -targeting shRNAs were tested for knockdown efficiency in a transient transfection CALM1 over-expression TSA201 cell system. One candidate shRNA had an 80% knockdown efficiency of CALM1. Following transfection with CALM1 -SupRep gene therapy, the APD 90 was significantly decreased in F142L iPSC-CMs compared to untreated cells (F142L: 665 ± 9 ms [n=40] vs F142L + SupRep: 353 ± 9 ms [n=20], p<0.0001). Ca 2+ amplitude was significantly decreased following SupRep transfection when compared to untreated cells (0.99 ± 0.04 vs 0.8 ± 0.06, p=0.02). Conclusions: Here, we provide the first proof-of-principle SupRep gene therapy for CALM1-mediated LQTS. CALM1 -SupRep gene therapy shortened significantly the pathologically prolonged APD 90 in CALM1 -F142L patient-derived iPSC-CMs.

Background: Long QT syndrome (LQTS) is a genetic heart disease affecting about 1 in 2000 persons. Around 40% of patients with LQTS can have a nondiagnostic, normal QTc at rest. The treadmill exercise stress test (TEST) can help unmask patients with concealed LQTS with a characteristic maladaptive, paradoxical increase in QTc ( 460 ms) during the recovery phase. In fact, the recovery phase QTc value has been added to the latest diagnostic score for LQTS. Objective: To characterize the TEST-derived QTc profile of untreated patients with LQTS and determine its diagnostic and prognostic utility. Methods: Retrospective analysis was performed on TEST data derived from 249 patients with LQTS (129 LQT1, 64 LQT2 and 56 LQT3) untreated at the time of their TEST compared with 236 controls. The Bazett’s corrected QTc was calculated at rest (sitting, supine, and standing), during exercise at 1 min, 3 mins, and peak exercise, and during recovery phase at 1, 3 and 5 mins. Results: The mean resting QTc was significantly higher in LQTS patients (457 ± 30 ms) compared to controls (420 ± 24 ms; p<0.0001) as well as at every stage during the TEST (p<0.001). For LQT1 patients, highest QTc was observed at peak exercise (mean 496 ± 38 ms) and during the third minute of recovery phase (mean 500 ± 40 ms). In contrast, LQT2 patients showed the lowest QTc (mean 452 ± 48 ms) during peak exercise but the greatest QTc latency (QTc at 5 min - QTc at 1 min recovery; mean QTc latency 33 ± 77 ms; p=0.005) compared to controls. For LQT3, the mean QTc was greatest at rest compared to controls (p<0.001), and there was no difference during exercise and recovery phases. Among 162 LQT1 and LQT2 patients that presented with concealed LQTS, 119 (73%) showed a maladaptive QT response. ROC analysis showed that in patients with LQT1, a maladaptive QTc > 523 ms at 3 min of recovery was highly predictive of breakthrough cardiac events (AUC 0.84; sensitivity 100%, specificity 72%); no such association was identified for LQT2 or 3. Conclusions: This comprehensive characterization of the QTc response before, during, and after a TEST in the largest untreated cohort of patients with LQTS provides important genotype-specific characteristics and can be used as a predictor for potential LQT1-triggered cardiac events after diagnosis and treatment.

Introduction: Rare cardiac complications of COVID-19 and COVID-19 mRNA vaccination include pericarditis and myocarditis. Patients with recurrent pericarditis may be at increased risk of triggered recurrence. The known efficacy of the interleukin (IL)-1 antagonist rilonacept in treating and preventing recurrence could inform COVID-19 disease and vaccination management. Methods: The Phase 3 trial RHAPSODY was a global, double-blind, placebo-controlled, randomized-withdrawal study. A long-term extension (LTE) enabled up to 24 months further open-label treatment from May 2020 until June 2022. Concomitant vaccination data and adverse events, including COVID-19, were collected. Results: No COVID-19 cases were reported during the run-in and randomized withdrawal periods (n=86). As of the 6 May 2022 data cutoff, a COVID-19 AE was reported in 19% (14/74) of LTE subjects, of whom 12 were on rilonacept: 5 of 44 fully-vaccinated (≥14 days beyond second serial dose) subjects (11.4%) and 9 of 30 unvaccinated or partially-vaccinated subjects (30.0%). Of the fully-vaccinated subjects, 1 case occurred ~11 months post primary vaccination cycle (no booster). COVID-19 severity was mild (n= 11), moderate (n=2), or severe (n=1). All cases resolved except the severe AE complicated by comorbidities; the subject discontinued. Of 12 subjects receiving rilonacept at the time of COVID-19 diagnosis, 10 maintained dosing with rilonacept, 1 interrupted rilonacept for ~4 weeks, and 1 discontinued rilonacept permanently; none experienced a pericarditis recurrence (PR) after the AE. The remaining 2 subjects had previously interrupted rilonacept for off-treatment observation: one had a PR >4.5 months after suspending rilonacept and 18 days after COVID-19 resolution; no PR in the other (>6 months). There were no PR AEs associated with COVID-19 vaccination (all subjects were receiving rilonacept). Conclusions: In patients on rilonacept in this study, although numbers are small, vaccination reduced COVID-19 events, pericarditis recurrence was not increased by vaccination or disease, and no pericarditis recurrences occurred during rilonacept treatment. One pericarditis recurrence occurred remotely after rilonacept suspension in the peri-COVID-19 period.

Background: Type 2 long QT syndrome (LQT2) is an inherited arrhythmia syndrome caused by pathogenic variants in the KCNH2 gene which result in QT prolongation and confers susceptibility to arrhythmic syncope/seizures and sudden cardiac arrest/death. Oral progestin-based contraceptives increase the risk of cardiac events in women with LQT2 and we previously reported the case of a LQT2 woman with recurrent cardiac events attributed to the use of the progestin-based contraceptive, Medroxyprogesterone Acetate (“Depo-Provera”, Depo). Objective: To evaluate the arrhythmic-risk of Depo in a LQT2-specific, induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) model. Methods: An iPSC-CM line was generated from the previously reported woman (KCNH2-G1006A*fs) as well as a control line. Voltage-sensing dye was used to assess the optical action potential duration (APD) with 10 μM Depo. 3-D erratic beating patterns were then assessed using multiple electrode array (MEA) with Depo and isoproterenol (ISO) to mimic an adrenergic surge. Results: Addition of Depo to the G1006A*fs iPSC-CMs decreased the optical APD from baseline 438±14 ms (n=6) to 363±35 ms (n=12; p=0.0001). Despite the APD shortening at baseline, in the setting of ISO, Depo administration induced erratic beating patterns in MEA, characterized as alternating spike amplitudes, alternans, or early after depolarization-like phenomena. Combined Depo and ISO treatment increased the percent of electrodes displaying erratic beating in G1006A*fs [baseline 8%±13 vs. Depo + ISO 38%±40 (p=0.0097)] but not in controls [baseline 2%±5 vs. Depo + ISO 7%±11 (p=0.14)]. Neither Depo nor ISO alone changed beating patterns in G1006A*fs [baseline 8%±13, Depo 30%±41 (p=0.08), ISO 21%±30, (p=0.17)] and controls [baseline 2%±5, Depo 3%±7 (p=0.61), ISO 0%±0 (p=0.4)]. Conclusion: This patient-specific, re-engineered heart cell study provides a potential mechanism for the patient’s clinically documented, Depo-associated episodes of recurrent ventricular fibrillation. This in-vitro data should prompt a large-scale clinical assessment of Depo’s potential pro-arrhythmic effect in women with LQT2.

Introduction: Rilonacept reduced pericarditis recurrence in the phase 3 trial RHAPSODY. The long-term extension (LTE) enabled further insights into efficacy, safety, and clinical decision making. Methods: The RHAPSODY run-in (RI) and randomized-withdrawal periods were followed by an LTE that enabled open-label rilonacept treatment up to 24 additional months. A decision at 18 months after the most recent recurrence was based on clinical status at investigator discretion: 1) continue rilonacept on-study, 2) suspend rilonacept for observation or 3) discontinue from the LTE without observation. Endpoints included recurrence and quality of life. Results: In May 2020, 74 of 75 eligible subjects continued to LTE; US subjects (n=45) switched to commercial therapy or discontinued in April 2021 (treatment duration from RI baseline [median; maximum] 18; 27 months). Non-US subjects (n=29) completed the LTE in June 2022 (treatment duration from RI baseline [median; maximum] 27; 33 months). Concomitant oral medications during LTE: 22% NSAIDS, 20% colchicine, 0% corticosteroids, 43% none. Inflammation signs (CRP), RP symptoms (PGIPS), and recurrences were low on-treatment. Before the 18-month decision (n=74), there were 3 investigator-assessed recurrences (0.04 per patient-year). At the 18-month decision (n=52), 64% (n=33) continued rilonacept on-study, 15% (n=8) suspended rilonacept for observation, and 21% (n=11) discontinued the study without observation, of whom 7 switched to commercial rilonacept. There was a 75% recurrence rate (n=6/8) in the off-treatment observation group (median time to event 11.8 [3.7, Not-Estimable (NE)] weeks) but only 1 recurrence in subjects on rilonacept (time to event NE [too few events]) associated with a 4-week interruption; HR 0.018, p<0.0001; Fig 1. Conclusions: Continued rilonacept treatment resulted in continued treatment response, whereas treatment suspension at 18 months may be associated with pericarditis recurrence.

Background: Long QT syndrome (LQTS) stems from cardiac action potential duration (APD) prolonging pathogenic variants in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3). Sodium/glucose co-transporter 2 inhibitor (SGLT2i), a class of drugs used to treat type II diabetes and all forms of heart failure, reduces cardiac late sodium channel current. Objective: To test the ability of three commercially available SGLT2i to reduce APD in patient-specific induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) models of LQT1, LQT2, and LQT3. Method: Patient-specific iPSC-CMs and a CRISPR/Cas9 gene-edited variant corrected, isogenic control (IC) iPSC-CMs were generated. The variant-corrected IC was used to determine the curative APD in order to establish the degree of APD attenuation. The SCN5A-P1332L (LQT3) and IC iPSC-CMs were tested with 10 μM of either canagliflozin (CANA), dapagliflozin (DAPA), or empagliflozin (EMPA). An ATXII (5μM)-induced APD prolongation iPSC-CM model, mimicking drug-induced LQT3, was also treated. The APD was measured 30 minutes, 1 hour, and 2 hours after treatment using local extracellular action potential recordings on multielectrode array. The efficacy of these SGLT2i were also tested in KCNQ1-V254M (LQT1) and KCNH2-G604S (LQT2) iPSC-CMs. Results: The APD90 was prolonged in SCN5A-P1332L iPSC-CMs compared to its IC (469 ± 4 ms vs 305 ± 2 ms, p<0.0001). While 10 μM of SGLT2i had no APD shortening effect on the KCNQ1-V254M or KCNH2-G604S iPSC-CMs, all three SGLT2i compounds shortened the APD90 of the SCN5A-P1332L iPSC-CMs after 1 hour of treatment from 463±5 ms to 432±6 ms (EMPA, 18.8% attenuation, p=0.0002), 459±8 ms to 430±5 ms (CANA, 17.5%, p=0.004), and 453±6 ms to 434±4 ms (DAPA, 11.4%, p=0.01). Additionally, IC iPSC-CMs treated with 5 μM of ATXII induced significant APD prolongation from 318±9 ms to 379±12 ms (p=0.0003) after 30 min incubation. However, co-incubation of ATXII and SGLT2i abolished the APD prolonging effect of ATXII. Conclusions: Therapeutically inhibiting SGLT2 effectively shortens the cardiomyocyte APD in human heart cell models of the LQT3, but not LQT1 or LQT2. This pre-clinical data compels further assessment of SGLT2i as a novel therapy for patients with congenital LQT3.

Background: Long QT syndrome (LQTS) often stems from pathogenic variants in KCNQ1 (LQT1), KCNH2 (LQT2), or SCN5A (LQT3) and is characterized cellularly by prolongation of the cardiac action potential duration (APD). We have shown that inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) reduces I NaL and is proposed as a novel therapeutic strategy for LQT3. Objective: To test the efficacy of a potent and selective SGK1 inhibitor (SGK1-I) in human cardiomyocyte models of LQT1, LQT2, and LQT3. Methods: Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) were generated from patients with either LQT1, LQT2, or LQT3. The mexiletine (MEX)-sensitive SCN5A-P1332L iPSC-CMs were tested initially. A CRISPR/Cas9 P1332L variant-corrected isogenic control (IC) was used as a control. The SGK1-I’s therapeutic efficacy was compared to MEX and to a commercially available SGK1 inhibitor (EMD638683). The novel SGK-I was then tested for APD shortening in SCN5A-R1623Q, KCNH2-G604S and KCNQ1-V254M iPSC-CMs. The APD90 values were recorded 4 hours after treatment using FluoVolt. Results: The APD90 was significantly prolonged in SCN5A-P1332L iPSC-CMs compared to its IC (646 ± 7 ms vs 482 ± 23 ms, p<0.0001). MEX shortened the average APD90 to 560 ± 7 ms (52% attenuation). Interestingly, SGK1-I significantly shortened the APD to 518 ± 5 ms (78% attenuation) but did not further shorten the APD in the IC. SGK1-I also shortened the APD90 of the SCN5A-R1623Q iPSC-CMs from 753 ± 8 ms to 475 ± 19 ms compared to 558 ± 19 ms with MEX. The SGK1-I shortened the APD90 in KCNH2-G604S (666 ± 10ms to 574 ± 18ms for SGK1-I versus 538 ± 15 ms after MEX). EMD had limited APD shortening effects in SCN5A and KCNH2 iPSC-CMs. Interestingly, while neither MEX nor EMD reduced the APD90 in the KCNQ1-V254M iPSC-CMs, the novel SGK1-I reduced the APD90 from 544 ± 10 ms to 475 ± 11ms (p=0.0004). Conclusions: Therapeutically inhibiting SGK1 effectively shortens the cardiomyocyte APD in human heart cell models of the 3 major LQTS genotypes. The novel SGK1-I normalized the pathological APD prolongation almost fully (> 70%) in the patient-derived SCN5A-P1332L iPSC-CM model. These pre-clinical data support further development of SGK1-I as a novel, first-in-class therapy for patients with congenital LQTS.

Introduction: patients exposed to chest, breast or mediastinal RT, may present with accelerated atherosclerosis and CAD due to diffuse micro and macrovascular fibrosis. However, CAD risk has not been well established in a community-based study. Aims: Describe CAD risk in patients treated with chemotherapy with and without RT and evaluate the cumulative incidence of CAD (coronary revascularization, MI and abnormal stress test and coronary angiogram). Methods: From 1985-2010, we identified 744 patients with a new diagnosis of breast cancer or lymphoma, using the Rochester Epidemiology Project (REP) who received chemotherapy with or without RT. CAD risk was assessed by a proportional hazards model for time to event. RT was treated as a time dependent co-variable, with index date being chemotherapy start date, significance was considered as a p<.05. Characteristics were evaluated by a multivariable Cox model to consider RT and time censoring. Results: 744 patients treated with chemotherapy were identified, of these 488 (65.6%) had breast cancer, 256 (34.4%) lymphoma, and 367 (49%) also received RT. The mean total RT dose was 5.5 ± 1.2 (Gy) for those that had CAD and 5.4 (Gy) ± 1.1 for non-CAD patients, p=0.4. The cumulative incidence of CAD events (accounting for death as a competing risk) at 5 and 10 years of follow-up was 1.9% (CI 1.14-3.2) and 3.06% (CI 2.01-4.7), respectively p<.05 figure1. Baseline differences between those receiving RT or not, were not significant for DM and HTN; however, males and ages 70-79 were less likely to receive RT, table1. In a multivariable model, there was no difference in the CAD outcomes between those with and without RT, HR= 1.21 [0.48-3.06] p=.69, after adjusting for age, gender, DM, and HTN and those <49 y/o had a lower risk of CAD. Table2 Conclusion: In this REP retrospective cohort study, the risk of developing a CAD event was similar between chemotherapy plus RT and chemotherapy only regimens, even after considering gender, age, HTN and DM.

Introduction: Gain-of-function mutations in SCN5A- encoded cardiac sodium channel (Na V 1.5) cause type 3 long QT syndrome (LQT3). Patients with SCN5A variants localizing to the local anesthetic binding domain might not be sensitive to lidocaine or mexiletine treatment. Hypothesis: Phenytoin, a sodium channel blocker used to treat epilepsy, may be a novel LQT3-directed therapy for a variant involving a critical residue of the Nav1.5’s local anesthetic binding domain. Methods: SCN5A-F1760C was identified in a now deceased infant with severe LQT3 (QTc of 680-1200 ms) who was refractory to left cardiac sympathetic denervation and pharmacological treatments with either lidocaine or mexiletine. SCN5A-F1760C was engineered by site directed mutagenesis and expressed in TSA201 cells. Patient-specific pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from the SCN5A-F1760C-positive infant and gene-edited/variant-corrected isogenic control (IC) iPSC-CMs were generated. Na V 1.5 currents were recorded in TSA201 cells transfected with SCN5A-WT/ F1760C and action potentials (APs) from iPSC-CMs were recorded before and after treatment with 5μM Phenytoin using the whole cell patch-clamp technique. Results: SCN5A-F1760C did not change the NaV1.5 current density. However, the V1/2 of inactivation was right-shifted significantly by +14.1 mV from -86.3±0.9 mV (WT) to -72.2±0.7 mV (F1760C, p<0.0001) resulting in a marked increase in window current. F1760C increased sodium late current 2-fold from 0.18±0.04% in WT to 0.49±0.07% in F1760C (p=0.0005). Baseline APD to 90% repolarization (APD90) was prolonged significantly in F1760C-derived iPSC-CMs (641±40 ms) compared to IC iPSC-CMs (412±22 ms, p<0.0001). Treatment with 5μM phenytoin normalized the APD90 in F1760C-derived iPSC-CMs to 423+34 ms (p<0.0001), values approximating the ‘gene/variant-edited’ cure. Conclusions: Na V 1.5 gain-of-function caused by a novel SCN5A-F1760C variant results in marked APD90 prolongation which were rescued by phenytoin indicating the antiepileptic drug, phenytoin, may be a mutation-specific, LQT3-directed therapy for those LQT3-causative variants that disrupt the local anesthetic/lidocaine-binding domain.