APD Prolongation Research Articles

Abstract 10669: Phenytoin, an Anti-Epileptic Sodium Channel Blocker, May Be a Novel Therapy for Type 3 Long Qt Syndrome Mediated by the Lidocaine-Insensitive SCN5A-F1760C Variant

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.

Abstract 11027: SGK1 Inhibition and Attenuation of the Action Potential Duration in Re-Engineered Heart Cell Models of Drug-Induced QT Prolongation

Background: Drug-induced QT prolongation (DI-QTP) is a clinical entity in which administration of a HERG/I Kr blocker such as dofetilide prolongs the cardiac action potential duration (APD) at the cellular level and the QT interval clinically, and increases the risk for a potentially lethal, QT-triggered ventricular arrhythmia. There may be a role for inhibition of I NaL to counter DI-QTP. Recently, we have shown that inhibition of serum and glucocorticoid regulated kinase-1 (SGK1) reduces the APD90 in induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) from patients with LQT3 and ameliorates increases in I NaL . Objective: To test the efficacy of novel SGK1 inhibitors (SGK-I) in re-engineered cardiomyocyte models of dofetilide-induced APD prolongation. Methods: Normal iPSC-CMs were treated with dofetilide [5 nM] to produce a DI-QTP iPSC-CM model. The SGK1-I’s therapeutic efficacy for shortening the dofetilide-induced APD90 prolongation was compared to mexiletine. The APD90 values were recorded 4 hours after treatment using FluoVolt, a voltage-sensitive fluorescent dye. Results: The APD90 was significantly prolonged in normal iPSC-CMs treated with dofetilide (673 ± 8 ms with dofetilide vs 436 ± 4 ms with DMSO, p<0.0001). While 10 μM mexiletine shortened the average APD90 of dofetilide-treated normal iPSC-CMs from 673 ± 4 ms to 563 ± 8 ms (46% attenuation, p<0.0001), 30 nM of SGK-I shortened the APD90 from 673 ± 4 ms to 478 ± 10 ms (82% attenuation, p<0.0001). Conclusions: Therapeutically inhibiting SGK1 effectively shortens the cardiomyocyte APD in a human heart cell model of DI-QTP. The novel SGK1 inhibitor normalized the pathological APD prolongation almost fully (> 80%) in the iPSC-CM model treated with dofetilide. This pre-clinical data supports further development of this therapeutic strategy to counter and neutralize the threat of DI-QTP.

Radiation-induced electrophysiological remodelling in neonatal rat ventricular cardiomyocytes

Abstract Background Ventricular tachycardia (VT) carries a relatively high risk of recurrence and significantly impacts patients' quality of life, despite state of the art treatment including antiarrhythmic drug therapy and catheter ablation. Cardiac stereotactic body radiotherapy (SBRT) has emerged as a promising non-invasive treatment option for refractory VT. A marked reduction in VT episodes is observed within days of treating high-risk patients with a single fraction SBRT. The cellular mechanisms underlying the clinically demonstrated antiarrhythmic effect are still poorly understood. Radiation-induced fibrosis is observed several months after irradiation, suggesting other mechanisms that explain the immediate benefit of radiotherapy on the VT substrate. Purpose We hypothesized that early antiarrhythmic effects of single-fraction radiation are produced by direct molecular and functional electrophysiological changes, independent of fibrosis. Our aim was to investigate the effects of 20 Gy ionizing photon radiation on the electrophysiology of ventricular neonatal rat cardiomyocytes at time periods from 24 hours to 96 hours after irradiation. Methods Ventricular neonatal rat cardiomyocytes were isolated, cultivated for 24 to 96 hours and then exposed to photon beam with a single dose of 20 Gy. Expression changes of ion channels, calcium handling proteins and structural proteins affecting cellular electrophysiology were assessed via RT-qPCR and Western blotting. Beat frequency was evaluated by video analysis and action potentials (APD) were recorded using the whole-cell voltage clamp technique in current-clamp mode. Intracellular calcium transients in Fura-2 AM loaded cardiomyocytes were recorded with an IonOptix recording system. Results Irradiation lead to significant changes in the mRNA and protein level of ion channels, with most distinct changes being observed 96h after radiation. Increased mRNA and protein expression of (Cav1.2) and of the potassium channels Kcnd3 (Kv4.3), Kcnh2 (Kv11.1), Kcnq1 (Kv7.1), Kcnk2 (K2P2.1) and Kcnj2 (Kir2.1) was observed. Additionally, Connexin 43 (Cx43) was upregulated. Irradiated cardiomyocytes showed a significant increase in beat rate over time. APD was significantly shortened, with a significantly increased upstroke velocity. Regarding calcium handling, significant upregulations of Ryr2 (RYR2) and Slc8a1 (NCX) transcript and protein level was detected. Additionally, calcium handling properties were altered. Conclusion Our results suggest that acute irradiation effects are associated with electrophysiological remodelling in cardiomyocytes. Changes of cellular ion channel expression and calcium homeostasis lead to normalization and shortening of heart-failure associated APD prolongation. These results reveal new insights into acute antiarrhythmic effects on cardiomyocytes after SBRT-therapy. Funding Acknowledgement Type of funding sources: None.

Changes of myocardial calcium currents in rats with myocardial injury induced by running exercise during acute hypoxia

To investigate the changes in myocardial calcium currents in rats subjected to forced running exercise during acute hypoxia and their association with myocardial injury. Forty SD rats were randomized into quiescent group and running group either in normal oxygen (NQ and NR groups, respectively) or in acute hypoxia (HQ and HR groups, respectively). Hypoxia was induced by keeping the rats in a hypobaric oxygen chamber (PaO2=61.6kpa) for 4 h a day; the rats in the two running groups were forced to run on running wheels for 4 h each day. Rat ventricular myocytes was isolated by enzymatic digestion for recording action potentials and currents using patch clamp technique, and confocal Ca2+ imaging was used to monitor intracellular Ca2+ levels. The expressions of Cav1.2 channel and the cardiac ryanodine receptor (RyR2) were determined using Western blotting. Compared with those in NQ group, the rats in HR group showed significantly decreased SOD activity (P < 0.01), increased h-FABP, hs-CRP and IMA levels (P < 0.05 or 0.01), obvious myocardial pathology, and prolonged APD50 and APD90 (P < 0.05). Of the different stress conditions, forced running in acute hypoxia resulted in the most prominent increase of the densities of ICa, L currents, causing also a significant left shift of the steady state activation curve and a significant right shift of the steady state inactivation curve. Compared with those in NQ group, the rats in NR, HQ and HR groups all exhibited higher rates of spontaneous calcium wave events in the cardiac myocytes, increased frequency of calcium sparks with lowered amplitude, enhanced calcium release amplitude in the ventricular myocytes, and delayed calcium ion reabsorption; in particular, these changes were the most conspicuous in HR group (P < 0.05 or 0.01). There was also a significant increase in the protein levels of Cav1.2 channel and RyR2 receptor in HR group (P < 0.05 or 0.01). The mechanism of myocardial injury in rats subjected to forced running in acute hypoxia may involve the increase of oxidative stress and calcium current and intracellular calcium overload.

Aging-associated susceptibility to stress-induced ventricular arrhythmogenesis is attenuated by tetrodotoxin

Aging is associated with increased prevalence of life-threatening ventricular arrhythmias, but mechanisms underlying higher susceptibility to arrhythmogenesis and means to prevent such arrhythmias under stress are not fully defined. We aimed to define differences in aging-associated susceptibility to ventricular fibrillation (VF) induction between young and aged hearts. VF induction was attempted in isolated perfused hearts of young (6-month) and aged (24-month-old) male Fischer-344 rats by rapid pacing before and following isoproterenol (1μM) or global ischemia and reperfusion (I/R) injury with or without pretreatment with low-dose tetrodotoxin, a late sodium current blocker. At baseline, VF could not be induced; however, the susceptibility to inducible VF after isoproterenol and spontaneous VF following I/R was 6-fold and 3-fold higher, respectively, in old hearts (P<0.05). Old animals had longer epicardial monophasic action potential at 90% repolarization (APD90; P<0.05) and displayed a loss of isoproterenol-induced shortening of APD90 present in the young. In isolated ventricular cardiomyocytes from older but not younger animals, 4-aminopyridine prolonged APD and induced early afterdepolarizations (EADs) and triggered activity with isoproterenol. Low-dose tetrodotoxin (0.5μM) significantly shortened APD without altering action potential upstroke and prevented 4-aminopyridine-mediated APD prolongation, EADs, and triggered activity. Tetrodotoxin pretreatment prevented VF induction by pacing in isoproterenol-challenged hearts. Vulnerability to VF following I/R or catecholamine challenge is significantly increased in old hearts that display reduced repolarization reserve and increased propensity to EADs, triggered activity, and ventricular arrhythmogenesis that can be suppressed by low-dose tetrodotoxin, suggesting a role of slow sodium current in promoting arrhythmogenesis with aging.

Endurance training induced cellular electrophysiological remodeling in a small and a large animal athlete’s heart model

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Funding: It was supported by NKFIH grants (K-19992, K- 135464, GINOP-2.3.2-15-2016-00047). Background The positive impact of regular exercise on a healthy and fulfilling lifestyle. Therefore, active athletes are considered the healthiest members of our society. However, there is an increasing amount of evidence that long-term high-intensity sporting activity could also have adverse effects on the heart, such as impaired electrophysiological properties. Heavy long-term training can lead to structural and functional remodelling of the heart, which in turn, can evoke malignant cardiac arrhythmias. Purpose To develop animal models with a significant translational value of the human athlete’s heart and to investigate underlying malignant drivers of cardiac arrhythmias due to the long-term endurance training in in vitro studies. Methods 24 dogs from both sexes and 26 male guinea pigs were randomly assigned to sedentary (’Sed’) and trained (’Tr’) groups (n = 12-12; n=13-13). The latter group underwent a long-term endurance interval-training program on the treadmill 5 days a week for 4 months. ECG recordings and echocardiography validated the characteristic of athlete’s heart. After heart removal, the degree of interstitial fibrosis was quantified and ventricular myocytes were enzymatically dissociated via retrograde perfusion. The transmembrane ionic currents were recorded using the whole-cell configuration of the patch-clamp technique. The action potentials were measured by the perforated patch-clamp technique. Immunocytochemistry measurements were performed to determine the density of transmembrane ion channels. Results Based on the ECG and ECHO results, the vigorous training program resulted in significant cardiac adaptation in both species. In addition, it caused mild ventricular fibrosis. The repolarization is reflected as the 90 percent of action potential duration (APD90). It was significantly lengthened in the left ventricular myocytes of ‘Tr’ dogs. (‘Tr’ vs. ‘Sed’ 472.8±29.6 ms; =29 vs. 369.3±31.4 ms; n=24, p=0.023) and there was no difference in the case of guinea pigs. The amplitude of the transient outward current (Ito), which is not expressed in the guinea pig heart, was significantly smaller in the ‘Tr’ dogs (‘Tr’ vs. ‘Sed’ 7.6±0.6 pA/pF, n=54 vs. 10.2±1.0 pA/pF, n=42, p&amp;lt;0.05). Under the currently used protocols, no differences were detected in the magnitude of other ionic currents. The HCN4 gene expression was significantly higher in isolated myocytes in ’Tr’ dogs. Conclusion Increased ectopic activity is not rare among top athletes. Our results suggest an association between increased arrhythmia susceptibility and impaired repolarisation reserve related to down-regulation of Ito and prolonged APD90 and enhanced fibrotic changes. The overexpression of HCN4 gene in hypertrophic hearts, similar to heart failure, may evoke malignant ventricular arrhythmias. Further studies are warranted to clarify this hypothesis in more detail.

PO-615-03 MUTANT ATRIAL NATRIURETIC PEPTIDE INDUCES MITOCHONDRIAL DEFECTS AND ION CHANNEL REMODELING IN A HUMAN INDUCED PLURIPOTENT STEM CELL-DERIVED ATRIAL FIBRILLATION MODEL

We identified a mutation in NPPA, encoding atrial natriuretic peptide (ANP), as the first non-ion channel gene causing familial atrial fibrillation (AF). However, lack of a mature and comprehensive model prevents a deeper understanding of the underlying cellular mechanisms. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) are ideally suited for modeling heritable AF and personalized pharmacological therapy, but there is limited data on the maturation of iPSC-atrial (a) CMs. We hypothesized that an electro-metabolic maturation (EMM) approach using biochemical cues (T3, IGF-1, dexamethasone), bioenergetic supplement (fatty acids), and electrical stimulation, synergistically promotes metabolic, structural, and electrophysiological maturity of iPSC-aCMs to a level comparable to human atrial cardiomyocytes (haCMs) and will reveal the novel underlying cellular mechanisms of the NPPA mutation. We applied our EMM approach to iPSC-aCMs and compared the metabolic (Seahorse Analyzer, western blots [WB], RT-PCR), structural (immunofluorescence, WB), electrophysiological (patch clamping, optical voltage mapping), and transcriptomic (RNA-seq) maturity with immature iPSC-aCMs and adult haCMs from the same patient. EMM matured iPSC-aCMs from a family carrying the NPPA-S64R mutation, and an isogenic control using CRISPR-Cas9, elucidated the novel underlying mechanism by which the mutation causes AF. EMM iPSC-aCMs displayed improved sarcomeric organization (Fig. 1a) and oxidative capacity (Fig. 1b), as well as more hyperpolarized RMP and increased APA (Fig. 1c,d). Only mature NPPA-S64R iPSC-aCMs demonstrated that the NPPA mutation caused mitochondrial defects resulting in reduced oxidative capacity and electron transport chain dysfunction (Fig. 2a), as well as electrophysiological remodeling causing prolonged APD (Fig. 2b,c) via increased IKs density (Fig. 2d-g). We established a combinatorial EMM approach that promoted comprehensive maturation of iPSC-aCMs. Using this maturation approach, we unmasked the novel electrophysiological remodeling and mitochondrial dysfunction underlying an AF-causing NPPA mutation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

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.

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. 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. 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. 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). 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.

EN-580-03 SUPPRESSION AND REPLACEMENT GENE THERAPY FOR KCNH2-MEDIATED ARRHYTHMIAS

KCNH2-mediated arrhythmias are caused by either loss-of-function (type 2 long QT syndrome, LQT2) or gain-of-function (type 1 short QT syndrome, SQT1) pathogenic variants in the KCNH2-encoded Kv11.1 potassium channel which is essential for the rapid delayed rectifier current (IKr) that contributes to the cardiac action potential (AP). No current therapies target the molecular cause of either LQT2 or SQT1. To rescue the pathologic phenotype in cell models of LQT2 and SQT1 using our novel gene therapy. A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ∼80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA (replacement). Patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and their CRISPR-Cas9 variant-corrected isogenic control (IC) iPSC-CMs were made for 3 LQT2- (G604S, G628S, N633S) and 1 SQT1- (N588K) causative variants. All 4 variant lines were treated with KCNH2-SupRep or non-targeting control shRNA (sham). FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90). KCNH2-SupRep achieved mutation-independent rescue of the pathologic phenotype in both LQT2 and SQT1. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD90 to near curative (IC-like) APD90 levels (G604S IC, 471±25ms; G628S IC, 429±16ms; N633S IC, 405±55ms) compared to treatment with sham (G604S: SupRep-treated, 452±76ms vs. sham-treated, 550±41ms, p<0.0001; G628S: SupRep-treated, 491±38ms vs. sham-treated, 674±20ms, p<0.0001; N633S: SupRep-treated, 399±105ms vs. sham-treated, 577±39ms, p<0.0001). Conversely, for the SQT1-causative N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD90 (IC: 429±16ms; SupRep-treated: 396±61ms; sham-treated: 274±12ms). We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. Akin to our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep gene therapy successfully corrected/normalized the pathologic APD90, thereby eliminating the pathognomonic feature of both LQT2 and SQT1.

EN-510-02 SUPPRESSION AND REPLACEMENT GENE THERAPY FOR KCNH2-MEDIATED ARRHYTHMIAS

KCNH2-mediated arrhythmias are caused by either loss-of-function (type 2 long QT syndrome, LQT2) or gain-of-function (type 1 short QT syndrome, SQT1) pathogenic variants in the KCNH2-encoded Kv11.1 potassium channel which is essential for the rapid delayed rectifier current (IKr) that contributes to the cardiac action potential (AP). No current therapies target the molecular cause of either LQT2 or SQT1. To rescue the pathologic phenotype in cell models of LQT2 and SQT1 using our novel gene therapy. A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ∼80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA (replacement). Patient-derived induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and their CRISPR-Cas9 variant-corrected isogenic control (IC) iPSC-CMs were made for 3 LQT2- (G604S, G628S, N633S) and 1 SQT1- (N588K) causative variants. All 4 variant lines were treated with KCNH2-SupRep or non-targeting control shRNA (sham). FluoVolt voltage dye was used to measure the APD at 90% repolarization (APD90). KCNH2-SupRep achieved mutation-independent rescue of the pathologic phenotype in both LQT2 and SQT1. For LQT2-causative variants, treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD90 to near curative (IC-like) APD90 levels (G604S IC, 471±25ms; G628S IC, 429±16ms; N633S IC, 405±55ms) compared to treatment with sham (G604S: SupRep-treated, 452±76ms vs. sham-treated, 550±41ms, p<0.0001; G628S: SupRep-treated, 491±38ms vs. sham-treated, 674±20ms, p<0.0001; N633S: SupRep-treated, 399±105ms vs. sham-treated, 577±39ms, p<0.0001). Conversely, for the SQT1-causative N588K, treatment with KCNH2-SupRep resulted in therapeutic prolongation of the pathologically shortened APD90 (IC: 429±16ms; SupRep-treated: 396±61ms; sham-treated: 274±12ms). We provide the first proof-of-principle gene therapy for correction of both LQT2 and SQT1. Akin to our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep gene therapy successfully corrected/normalized the pathologic APD90, thereby eliminating the pathognomonic feature of both LQT2 and SQT1.

Oxytocin exerts harmful cardiac repolarization prolonging effects in drug-induced LQTS.

BackgroundOxytocin is used therapeutically in psychiatric patients. Many of these also receive anti-depressant or anti-psychotic drugs causing acquired long-QT-syndrome (LQTS) by blocking HERG/IKr. We previously identified an oxytocin-induced QT-prolongation in LQT2 rabbits, indicating potential harmful effects of combined therapy. We thus aimed to analyze the effects of dual therapy with oxytocin and fluoxetine/risperidone on cardiac repolarization.MethodsEffects of risperidone, fluoxetine and oxytocin on QT/QTc, short-term variability (STV) of QT, and APD were assessed in rabbits using in vivo ECG and ex vivo monophasic AP recordings in Langendorff-perfused hearts. Underlying mechanisms were assessed using patch clamp in isolated cardiomyocytes.ResultsOxytocin, fluoxetine and risperidone prolonged QTc and APD in whole hearts. The combination of fluoxetine + oxytocin resulted in further QTc- and APD-prolongation, risperidone + oxytocin tended to increase QTc and APD compared to monotherapy. Temporal QT instability, STVQTc was increased by oxytocin, fluoxetine / fluoxetine + oxytocin and risperidone / risperidone + oxytocin. Similar APD-prolonging effects were confirmed in isolated cardiomyocytes due to differential effects of the compounds on repolarizing ion currents: Oxytocin reduced IKs, fluoxetine and risperidone reduced IKr, resulting in additive effects on IKtotal-tail. In addition, oxytocin reduced IK1, further reducing the repolarization reserve.ConclusionOxytocin, risperidone and fluoxetine prolong QTc / APD. Combined treatment further prolongs QTc/APD due to differential effects on IKs and IK1 (block by oxytocin) and IKr (block by risperidone and fluoxetine), leading to pronounced impairment of repolarization reserve. Oxytocin should be used with caution in patients in the context of acquired LQTS.

Deciphering the pathogenic role of a variant with uncertain significance for short QT and Brugada syndromes using gene‐edited human‐induced pluripotent stem cell‐derived cardiomyocytes and preclinical drug screening

Deciphering the pathogenic role of a variant with uncertain significance for short QT and Brugada syndromes using gene‐edited human‐induced pluripotent stem cell‐derived cardiomyocytes and preclinical drug screening

Abstract 12269: Investigating Arrhythmogenic Potential of a Novel KCNH2 Mutation Leading to Long QT Syndrome

Background: Mutations in the gene KCNH2 have been associated with both Short and Long QT syndrome. In this study, we identified a 1-day old infant that exhibited T-wave alternans coupled with Long QT Syndrome leading to aborted sudden infant death. Methods: Genetic analysis of the patient revealed a mutation in KCNH2 resulting in a proline to leucine substitution at position 632 (P632L). Functional electrophysiological studies were performed with the hERG mutation transiently transfected into either Human Embryonic Kidney (HEK) cells or human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The functional effects of P632L were incorporated into biophysical computational models of hiPSC-CM as well as adult human cardiomyocytes to further assess its arrhythmogenic potential. Results: The male infant displayed multiple repolarization disorders including T-wave alternans and a QTc =510 ms that appeared 1 day after birth. Genetic screening revealed a heterozygous missense mutation (P632L) in KCNH2 in the infant. Patch clamp analysis of HEK cells transiently transfected with P632L mutation showed a complete loss of function of HERG current compared to WT HERG. Transient transfection of P632L into WT hiPSC myocytes resulted in prolongation of the hiPSC action potential compared to untransfected hiPSC myocytes. Co-transfection of WT and non-functional P632L channels into HEK cells resulted in a dramatic loss of current suggesting a dominant negative effect. Implementing the effects of a complete block of HERG current in the hiPSC-CM computer model prolonged the action potential by 45% and depolarized the resting membrane potentials. In adult human myocyte models, the effects of HERG blockade were more severe in Purkinje cells than that in ventricular myocytes (71% vs. 16% APD prolongation, respectively). Conclusions: The mutation P632L causes a complete loss of HERG current and results in QT prolongation. Numerical simulations suggest a more severe phenotype in Purkinje cells than in ventricular myocytes which could be proarrhythmic.

Abstract 10689: C-src Contributes to Mitochondrial Ca 2+ Uniporter Increase and Arrhythmic Risk in Ischemic Cardiomyopathy

Introduction: We have described previously an arrhythmic mechanism in nonischemic heart failure (HF) involving an increased mitochondrial Ca 2+ uptake that results in QT prolongation and lethal arrhythmias during cardiomyopathy. This arrhythmic mechanism is associated with mitochondrial Ca 2+ uniporter (MCU) tyrosine phosphorylation (p-Tyr). Hypothesis: Here, we determined which kinase was responsible for MCU phosphorylation in ischemic cardiomyopathy. Methods: Ischemic HF was induced in wild type FVB/NJ mice for three weeks by ligated left anterior descending coronary artery. Western blot, immunoprecipitation, fluorescence resonance energy transfer (FRET) and patch-clamp techniques were employed in isolated mouse cardiomyocytes. Results: When compared to sham hearts, the protein level of p-Src increased significantly in ischemic HF mouse hearts. In a heterologous expression system, c-Src could bind MCU and tyrosine phosphorylate MCU. This phosphorylation was blocked by a c-Src inhibitor, PP1. Overexpression of constitutive active c-Src (Src-Y527F) significantly increased MCU inward current at -160 mV. Downregulation of C-terminal Src kinase (CSK), a regulatory kinase inversely related to c-Src activity, increased p-Src, enhanced mitochondrial and SR contact, and facilitated mitochondrial Ca 2+ uptake in ischemic HF mouse cardiomyocytes. Protein expression of CaMKII, PyK2, and p-PyK2 did not change during ischemic HF. MI activated c-Src by S-nitrosylation of caveolin-1 with subsequent release of the kinase. c-Src activation was associated with APD prolongation and arrhythmia following myocardial infarction in mice. Human heart tissue showed that ischemic HF patients had significantly increased p-Src associated with increased QTc prolongation and arrhythmia. Conclusions: During ischemic HF, c-Src activation directly increased MCU current that contributed to QT prolongation and arrhythmic risk in ischemic cardiomyopathy.

Abstract 13939: Triiodothyronine and Dexamethasone Enhances Electrophysiological Maturation of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) technology opens new avenues for disease modeling and drug development. However, hiPSC-CMs remain functionally immature, which hinders their utility as a model of human cardiomyocytes. Objective: To improve the electrophysiological maturation of hiPSC-CMs. Methods and Results: Day 16 th hiPSC-CMs were treated with vehicle or 100 nmol/L triiodothyronine (T3) and 1 μmol/L Dexamethasone (Dex) for 14 days. On day 30 th , hiPSC-CMs were dissociated and replated as cell sheets or single cells. Optical mapping and patch-clamp technique were used to examine the electrophysiological properties of vehicle- and T3+Dex-treated hiPSC-CMs. T3+Dex-treated hiPSC-CMs had a slower spontaneous beating rate (vehicle 17 ± 0.8 vs . T3+Dex 12 ± 0.8 beats/min, P &lt; 0.01), more hyperpolarized resting membrane potential (vehicle -65.04 ± 1.20 vs. T3+Dex -72.00 ± 1.10 mV, P &lt; 0.01), faster maximal upstroke velocity (vehicle 86.08 ± 8.12 vs. T3+Dex 131.78 ± 8.49 mV/ms, P &lt; 0.01), and shorter action potential duration at 90% repolarization (APD 90 , vehicle 550.44 ± 39.54 vs. T3+Dex 418.59 ± 21.58 ms, P &lt; 0.01) compared to the vehicle. Changes in spontaneous activity and AP were mediated by decreasing the funny current (I f , vehicle -12.20 ± 2.15 vs. T3+Dex -6.98 ± 0.89 pA/pF at -120 mV, P &lt; 0.05) and increasing the inward rectifier potassium current (I K1 , vehicle -4.62 ± 0.59 pA/pF vs . T3+Dex -18.79 ± 1.98 pA/pF at -120 mV, P &lt; 0.01), the sodium current (I Na , vehicle -9.8 ± 1.86 vs. T3+Dex -44.9 ± 7.10 pA/pF at -30 mV, P &lt; 0.01), and the rapidly and slowly activating delayed rectifier potassium currents (I Kr , 0.81 ± 0.07 vs. T3+Dex: 1.35 ± 0.19 pA/pF at 20 mV, P &lt; 0.01; I Ks , vehicle 0.38 ± 0.06 vs. T3+Dex 0.72 ± 0.17 pA/pF at 40 mV, P &lt; 0.05). Furthermore, T3+Dex-treated hiPSC-CM cell sheets (hiPSC-CCSs) exhibited a faster conduction velocity and shorter APD than the vehicle. Inhibition of I K1 by 100 μM BaCl 2 significantly slowed conduction velocity and prolonged APD in T3+Dex-treated hiPSC-CCSs but had no effect in the vehicle group, demonstrating the important role of I K1 for conduction velocity and APD. Conclusion: T3+Dex treatment is an effective approach to rapidly enhance electrophysiological maturation of hiPSC-CMs.

Abstract 14260: Suppression and Replacement Gene Therapy for Type 2 Long QT Syndrome

Introduction: Type 2 long QT syndrome (LQT2) is caused by loss-of-function variants in the KCNH2 -encoded K v 11.1 potassium channel which is essential for the rapid delayed rectifier channel (I Kr ) that governs phase 3 of cardiac action potential (AP). No current therapies target the molecular cause of LQT2. Methods: A dual-component “suppression-and-replacement” (SupRep) KCNH2 gene therapy was created by cloning into a single construct a custom-designed KCNH2 shRNA that produces ~80% knockdown (suppression) and a “shRNA-immune” (shIMM) KCNH2 cDNA modified by the introduction of synonymous single nucleotide substitutions within the shRNA target site (replacement). The ability of KCNH2-SupRep gene therapy to suppress and replace LQT2-causative variants in KCNH2 was evaluated via heterologous expression in TSA201 cells. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from two patients with LQT2 (KCNH2-G604S and -N633S). CRISPR-Cas9 corrected isogenic control iPSC-CMs were made. Both lines were treated with KCNH2-SupRep or non-targeting control shRNA (shCT). FluoVolt voltage dye was used to measure the APD. Results: KCNH2-SupRep achieved mutation-independent suppression of wild-type KCNH2 and both LQT2-causative variants with simultaneous replacement of KCNH2-shIMM as measured by allele-specific qRT-PCR. Treatment with KCNH2-SupRep resulted in shortening of the pathologically prolonged APD at 90% repolarization (APD 90 ) to near isogenic control APD 90 levels (G604S: isogenic controls, 424 + 39 ms and N633S: isogenic controls, 433 + 16 ms) compared to treatment with non-targeting shCT (G604S: SupRep treated, 399±105 ms vs. shCT treated, 577±39 ms, p&lt;0.0001; N633S: SupRep treated, 432±85 ms vs. shCT treated, 483±65 ms, p&lt;0.005). Conclusions: We provide the first proof-of-principle gene therapy for correction of LQT2. Like our sentinel discovery of SupRep gene therapy for LQT1, KCNH2-SupRep successfully suppressed and replaced KCNH2 to normal wild-type levels. In TSA201 cells, co-transfection of LQT2 variants and KCNH2-SupRep caused mutation-independent suppression-and-replacement of KCNH2 . In LQT2 iPSC-CMs, KCNH2-SupRep shortened the APD, thereby eliminating the pathognomonic feature of LQT2.

Abstract 10691: Single Cell Florescence Imaging in Hypertrophic Cardiomyopathy Reveals a Proarrhythmic Substrate: Implications for Arrhythmogenesis and Antiarrhythmic Therapy

Introduction: Hypertrophic cardiomyopathy (HCM) is associated with high morbidity and mortality and remains the leading cause of sudden death in young people. Despite 5 decades of research, there is no disease modifying therapy for HCM. The cellular mechanisms leading to a proarrhythmic substrate are not yet fully elucidated but may be patient-specific. Here, we present single cell electrophysiology results of adult cardiomyocytes isolated from a cohort of HCM patients who underwent septal reduction therapy with surgical myectomy. Methods: Freshly isolated surgical specimens from HCM patients were enzymatically digested into single ventricular myocytes and compared to discarded wild-type (WT) donor hearts as controls. Myocytes displayed characteristic rod-shaped morphology, clear sarcolemmal ultrastructure, and intact plasma membranes without significant inclusions. After Ca 2+ readaptation and labeling with both a voltage and Ca 2+ sensitive fluorescent dye, cells were field stimulated at various pacing frequencies to obtain voltage and Ca 2+ tracings. APD restitution, response to drug therapy, and baseline data was compared to patient’s clinical data including electrocardiography, pathology, and multimodality imaging. Results: As compared to WT controls, HCM myocytes displayed significantly prolonged APD and Ca 2+ transients, as well as steeper APD restitution at all pacing frequencies tested. These results correlated with clinical QTc prolongation which was evident in HCM patients’ baseline ECGs that was not seen in controls. Despite genetic diversity in HCM samples, the HCM specimens appeared to share a common proarrhythmic phenotype. Finally, pathologic analysis of the myectomy specimens revealed evidence of myocyte hypertrophy, fibrosis, and cellular disarray, consistent with the clinical diagnosis of HCM. Conclusions: The single cell electrophysiology of HCM is markedly different than WT controls and displays a proarrhythmic phenotype. Single cell fluorescence imaging of a patient’s myocytes is a promising approach to elucidate patient specific electrophysiologic signatures and predict precision therapies for these patients.

The Chronotropic Function of Propofol and the Underlying Mechanism in Rabbits.

The report of bradycardia caused by propofol is increasing. In the experiment, we investigated the chronotropic function of propofol and the underlying mechanism. Rabbits of both sexes were randomly divided into 4 groups: propofol 5 mg/kg group, 10 mg/kg group, 15 mg/kg group, and sham group. Heart rate and frequency of vagal efferent discharge were recorded before the injection and 0, 0.5, 1, 2, and 10 min after the injection through intravenous mode. Then, their hearts were removed, and sinoatrial nodes were dissected. The action potentials of the sinus node pacemaker cells were recorded by the intracellular glass microelectrode technique, and the sinoatrial (SA) node was exposed to propofol 1, 3, 5, and 10 µM respectively. The action potentials were recorded after the sinoatrial nodes were exposed to each concentration of propofol for 15 min. Our results show that the heart rate significantly decreased, and the vagal efferent discharge was significantly increased at 0, 0.5, 1, and 2 min after the injection, respectively. Besides, as the dose increases, the magnitude of change shows a dose-dependent manner. Propofol exerts a negative chronotropic action on sinoatrial node pacemaker cells. The drug significantly decreased APA, VDD, RPF, and prolonged APD90 in a concentration-dependent manner. These effects may be the main mechanism of propofol-induced bradycardia in clinical study.

Species dependent cardiac electrophysiological effects elicited by various potassium channel blocking drugs

Rodents are commonly used as models in electrophysiology. However, distinct differences exist between large animals and rodents in terms of their ion channel expression and action potential shapes, possibly limiting the translational value of findings obtained in rodents. We aimed for a direct comparison of the possible impact of selective inhibition of ion channels on the cardiac repolarization in preparations from human hearts and from model species. We applied the standard microelectrode technique at 37°C on cardiac ventricular preparations (papillary muscles and trabecules) from human (n = 63), dog (n = 47), guinea pig (n = 53), rat (n = 43), and rabbit (n = 16) hearts, paced at 1 Hz. To selectively block the IKur current, 1 µM XEN-D101; IK1 current, 10 µM barium chloride; IKr current, 50 nM dofetilide; IKs current, 500 nM HMR-1556; and Ito current, 100 µM chromanol-293B were applied directly to the tissue bath. The block of IKur and IK1 elicited significantly more prominent prolongation of APD in rats (35.6% and 67.9%, respectively) when compared with the other species, including that of human (1.0% and 2.6%, respectively). On the other hand, IKr block did not affect APD in rat preparations (1.6%), whereas it elicited marked prolongation in other species (9.0-47.7%), especially being pronounced in human preparations (60.3%). IKs inhibition elicited similar but minor APD prolongation (0.3-11.4%) in all species. Inhibition of Ito moderately lengthened APD in dog (22.3%) and rabbit (17.5%) preparations but elicited no change of APD in human preparations. In contrast, block of Ito caused marked APD prolongation in rat preparations (33.2%). Our findings suggest that the specific inhibition of various ion channels elicits fundamentally different effects in rodent ventricular action potential when compared with those of other species, including human. Therefore, from a translational standpoint, rodent models in cardiac electrophysiological and arrhythmia research should be used with great caution.

Impact of Ghrelin on Ventricular Arrhythmia and Related Mechanism After Myocardial Infarction

Introduction: Ghrelin is an endogenous peptide with potential protective effects on ischemic heart. Methods: Synthetic ghrelin was administered (100 μg·kg^-1 subcutaneous injection, twice daily) for 4 weeks in a rat model of myocardial infarction (MI) with coronary artery occlusion. At the 5th week, electrocardiogram, monophasic action potentials and autonomic nerve function were evaluated. Cardiac tyrosine hydroxylase (TH) was determined by immunofluorescence staining. Results: MI significantly increased sympathetic nerve activity (SNA) and ventricular arrhythmias, and prolonged APD dispersion and APD alternans (p < 0.01). Ghrelin treatment significantly increased ventricular fibrillation threshold (VFT), shortened APD dispersion and APD alternans, inhibited SNA and promoted vagus nerve activities (p < 0.01). Ghrelin also markedly reversed abnormal expression of TH in the peri-infarcted area of the heart (p < 0.01). Discussion/Conclusion: Ghrelin provides a sustained electrophysiological protection by the increase of VFT and improvement of APD dispersion and APD alternans. The mechanism may be related to the regulation of autonomic nerve and sympathetic nerve remodeling. Thus, ghrelin represents a novel drug to prevent ventricular arrhythmia in ischemic heart disease.