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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 <.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. [ABSTRACT FROM AUTHOR]

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 dualcomponent 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. [ABSTRACT FROM AUTHOR]

We investigate rare decays of tau leptons that occur via exchange of heavy on-shell neutrinos Nj (j = 1, 2). These neutrinos can be either Dirac or Majorana, and are considered to be almost degenerate in mass. The decays can thus be either lepton number conserving (LNC), τ∓ → π∓Nj → π∓μ∓π±, or lepton number violating (LNV), τ∓ → π∓Nj → π∓μ±π∓. If neutrinos are Dirac, only LNC decays are possible. If they are Majorana, both LNC and LNV are possible. We derive the corresponding expressions for the effective decay widths Γ eff , ∓ X (X=LNC, LNV) of these rare decays, where we account for N1-N2 overlap and oscillation effects and for the finite detector length effects. We then numerically evaluate these decay widths as well as the related CP violation asymmetry width ∆ Γ CP X = Γ eff , − X − Γ eff , + X . We conclude that for certain, presently allowed, ranges of the heavy-light neutrino mixing parameters, such decays and asymmetries could be observed in Belle II experiment. [ABSTRACT FROM AUTHOR]

We investigate rare decays of tau leptons that occur via exchange of heavy on-shell neutrinos Nj (j = 1, 2). These neutrinos can be either Dirac or Majorana, and are considered to be almost degenerate in mass. The decays can thus be either lepton number conserving (LNC), τ∓ → π∓Nj → π∓μ∓π±, or lepton number violating (LNV), τ∓ → π∓Nj → π∓μ±π∓. If neutrinos are Dirac, only LNC decays are possible. If they are Majorana, both LNC and LNV are possible. We derive the corresponding expressions for the effective decay widths Γ eff , ∓ X (X=LNC, LNV) of these rare decays, where we account for N1-N2 overlap and oscillation effects and for the finite detector length effects. We then numerically evaluate these decay widths as well as the related CP violation asymmetry width ∆ Γ CP X = Γ eff , − X − Γ eff , + X . We conclude that for certain, presently allowed, ranges of the heavy-light neutrino mixing parameters, such decays and asymmetries could be observed in Belle II experiment. [ABSTRACT FROM AUTHOR]

BACKGROUND Patients with prostate cancer who have high-risk biochemical recurrence have an increased risk of progression. The efficacy and safety of enzalutamide plus an- drogen-deprivation therapy and enzalutamide monotherapy, as compared with androgen-deprivation therapy alone, are unknown. METHODS In this phase 3 trial, we enrolled patients with prostate cancer who had high-risk biochemical recurrence with a prostate-specific antigen doubling time of 9 months or less. Patients were randomly assigned, in a 1:1:1 ratio, to receive enzalutamide (160 mg) daily plus leuprolide every 12 weeks (combination group), placebo plus leuprolide (leuprolide-alone group), or enzalutamide monotherapy (monotherapy group). The primary end point was metastasis-free survival, as assessed by blinded independent central review, in the combination group as compared with the leu- prolide-alone group. A key secondary end point was metastasis-free survival in the monotherapy group as compared with the leuprolide-alone group. Other secondary end points were patient-reported outcomes and safety. RESULTS A total of 1068 patients underwent randomization: 355 were assigned to the com- bination group, 358 to the leuprolide-alone group, and 355 to the monotherapy group. The patients were followed for a median of 60.7 months. At 5 years, metastasis-free survival was 87.3% (95% confidence interval [CI], 83.0 to 90.6) in the combination group, 71.4% (95% CI, 65.7 to 76.3) in the leuprolide-alone group, and 80.0% (95% CI, 75.0 to 84.1) in the monotherapy group. With respect to metastasis-free survival, enzalutamide plus leuprolide was superior to leuprolide alone (hazard ratio for metastasis or death, 0.42; 95% CI, 0.30 to 0.61; P<0.001); enzalutamide monotherapy was also superior to leuprolide alone (hazard ratio for metastasis or death, 0.63; 95% CI, 0.46 to 0.87; P=0.005). No new safety signals were observed, with no substantial between-group differences in quality-of-life measures. CONCLUSIONS In patients with prostate cancer with high-risk biochemical recurrence, enzalutamide plus leuprolide was superior to leuprolide alone with respect to metastasis- free survival; enzalutamide monotherapy was also superior to leuprolide alone. The safety profile of enzalutamide was consistent with that shown in previous clinical studies, with no apparent detrimental effect on quality of life. [ABSTRACT FROM AUTHOR]

We demonstrate the sensitive detection of dimethyl methylphosphonate (DMMP, a hydrogen-bond (HB) basic phosphonate ester) using additional optical loss induced in an interband cascade laser with top optical cladding layer replaced by an exposed sensing window coated by a HB acidic sorbent layer. Thin coatings of the sorbents HCSFA2 and oapBPAF were deposited on the sensing window to allow reversible capture and concentration of DMMP for optical interrogation. Analyte levels down to 0.1 mg/m 3 (∼20 ppb) were tested and successfully detected by monitoring the laser's threshold or its output power at a fixed bias as a function of DMMP delivery concentration.

Triadin knockout syndrome (TKOS) is a malignant arrhythmia disorder caused by recessive null variants in TRDN-encoded cardiac triadin. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two unrelated TKOS patients and an unrelated control. CRISPR-Cas9 gene editing was used to insert homozygous TRDN-p.D18fs ∗ 13 into a control line to generate a TKOS model (TRDN -/- ). Western blot confirmed total knockout of triadin in patient-specific and TRDN -/- iPSC-CMs. iPSC-CMs from both patients revealed a prolonged action potential duration (APD) at 90% repolarization, and this was normalized by protein replacement of triadin. APD prolongation was confirmed in TRDN -/- iPSC-CMs. TRDN -/- iPSC-CMs revealed that loss of triadin underlies decreased expression and co-localization of key calcium handling proteins, slow and decreased calcium release from the sarcoplasmic reticulum, and slow inactivation of the L-type calcium channel leading to frequent cellular arrhythmias, including early and delayed afterdepolarizations and APD alternans., Competing Interests: Conflict of interests M.J.A. is a consultant for Abbott, ARMGO Pharma, Boston Scientific, Daiichi Sankyo, Invitae, LQT Therapeutics, Medtronic, and UpToDate. M.J.A. and Mayo Clinic are involved in equity/royalty relationships with AliveCor and Anumana. These relationships are all modest, and none of these entities have contributed to this study in any manner., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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. I to 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 I to 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 I to and abolishing the accentuated action potential notch in patient-derived iPSC-CMs.

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 Ca 2+ imaging assessed Ca 2+ handling with/without isoproterenol (ISO), nadolol (Nad), and flecainide (Flec) treatment. CPVT1 iPSC-CMs displayed increased Ca 2+ sparking and Ca 2+ transient amplitude following ISO compared with control. Combined Nad treatment/ISO stimulation reduced Ca 2+ 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 Ca 2+ leak-triggered arrhythmias., Competing Interests: Conflict of interests M.J.A. is a consultant for Abbott, ARMGO Pharma, Boston Scientific, Daiichi Sankyo, Invitae, LQT Therapeutics, and Medtronic. M.J.A. and/or Mayo Clinic are involved in an equity/royalty relationship with AliveCor, Anumana, Pfizer, and UpToDate. However, none of these entities were involved in this study in any manner. A.R.M. and Columbia University own shares in ARMGO Pharma, a biotech company developing RyR-targeted therapeutics., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 APD 90 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 APD 90 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 APD 90 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 APD 90 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 APD 90 under baseline conditions, the physiological stress of either acidosis or HCQ treatment significantly prolonged APD 90 in patient-specific, re-engineered heart cells., (Copyright © 2021 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)