Shortening Of Action Potential Duration Research Articles

Multiplatform modeling of atrial fibrillation identifies phospholamban as a central regulator of cardiac rhythm.

Atrial fibrillation (AF) is a common and genetically inheritable form of cardiac arrhythmia; however, it is currently not known how these genetic predispositions contribute to the initiation and/or maintenance of AF-associated phenotypes. One major barrier to progress is the lack of experimental systems to investigate the effects of gene function on rhythm parameters in models with human atrial and whole-organ relevance. Here, we assembled a multi-model platform enabling high-throughput characterization of the effects of gene function on action potential duration and rhythm parameters using human induced pluripotent stem cell-derived atrial-like cardiomyocytes and a Drosophila heart model, and validation of the findings using computational models of human adult atrial myocytes and tissue. As proof of concept, we screened 20 AF-associated genes and identified phospholamban loss of function as a top conserved hit that shortens action potential duration and increases the incidence of arrhythmia phenotypes upon stress. Mechanistically, our study reveals that phospholamban regulates rhythm homeostasis by functionally interacting with L-type Ca2+ channels and NCX. In summary, our study illustrates how a multi-model system approach paves the way for the discovery and molecular delineation of gene regulatory networks controlling atrial rhythm with application to AF.

Human leukocyte antigen F-associated transcript 10 regulates the IKs potassium channel by competing for Kv7.1 ubiquitination.

The protein expression and function changes from the slow-delayed rectifying K+ current, IKs, are tightly associated with ventricular cardiac arrhythmias. Human leukocyte antigen F-associated transcript 10 (FAT10), a member of the ubiquitin-like-modifier family, exerts a protective effect against myocardial ischaemia. However, whether or how FAT10 influences the function of IKs remains unclear. Here, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and Fat10 knockout HEK293 (Fat10-/-) cells through CRISPR-Cas9 technology were used to evaluate the novel modulation of FAT10 in IKs function. Patch-clamp studies showed that the overexpression of FAT10 significantly enhanced the current density of IKs both in hiPSC-CMs and HEK293-Fat10-/- cells. In addition, a shortened action potential duration (APD) was seen from hiPSC-CMs transfected with the ad-Fat10 virus. Then, a series of molecular approaches from neonatal rat cardiomyocytes, H9C2 cells and HEK293 cells were used to determine the regulatory mechanism of FAT10 in IKs. First, western blot assays indicated that the expression of Kv7.1, the alpha-subunit of IKs, was increased when FAT10 was overexpressed. Furthermore, immunofluorescence and co-immunoprecipitation assays demonstrated that FAT10 could interact with Kv7.1. Notably, FAT10 impedes Kv7.1 ubiquitination and degradation, thereby stabilizing its expression. Finally, a hypoxia model of hiPSC-CMs was established, and the overexpression of FAT10 showed a protective effect against hypoxia-induced decreases in the current density of IKs. Taken together, these findings revealed a novel role of FAT10 in the regulation of the IKs potassium channel by competing for Kv7.1 ubiquitination, which provides a new electrophysiological insight that FAT10 could modulate Kv7.1. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

PO-01-205 COMPUTATIONAL MODELING UNCOVERS A UNIFYING MECHANISM LINKING CONDUCTION BLOCK TO VT LOCALIZATION AND STABILITY IN ARRHYTHMOGENIC CARDIOMYOPATHY

We recently showed in a murine model of arrhythmogenic cardiomyopathy (ACM) that VT was driven by discrete rotors deep within the RV. Mechanisms underlying the evolution of these RV-pinned rotors, however, remained unknown considering that the initial wavebreaks that preceded VT were at the LV/RV interface. We hypothesized that the unique spatial action potential duration (APD) profile (RV>LV) in ACM during rapid pacing and adrenergic stimulation, independent of other factors such as structural remodeling, is sufficient to explain the rapid evolution of wavebreaks into discrete rotors that efficiently translocate and stabilize in the RV to maintain VT. We developed 2D virtual sheets of mouse ventricular myocardium using isotropic cellular automaton models with realistic restitution properties that mimic experimentally measured LV to RV APD gradients in wildtype (WT) and ACM hearts (Fig A). VT rotors were induced by burst stimulus trains at increasing frequencies. VT threshold was defined as the duration of the burst stimulus train required to initiate stable VT. Rotors were localized in space and time using phase singularity (PS) analysis. To determine the relationship between spatial APD profile and rotor dynamics, we varied the steepness of the LV-to-RV APD gradient. Once induced, we examined rotor dynamics, speed of rotor drift, meander, stability and dominant frequency (DF). VT threshold was markedly decreased in ACM (2.9s) vs WT (14.5s) reflecting increased vulnerability. VT rotors were more readily induced when stimuli were applied from the zone of short versus long APD. While VT rotors were randomly distributed across homogeneous tissues, PS density analysis revealed exclusive localization of VT rotors within areas of increased APD in heterogeneous tissues (Fig B). Varying the slope of the APD gradient revealed divergent effects on forward drift and DF of VT rotors (Fig C). As the slope of the APD gradient steepened, the velocity of rotor forward drift to the RV increased exponentially while rotor DF decreased in a linear manner consistent with increased stability. Simulation of experimentally measured LV-to-RV APD gradient in ACM caused forward drift of multiple rotors at 2.18cm/sec before pinning within narrow RV loci. The unique spatial APD profile at early stages of ACM is sufficient to explain not only the initiation but also the translocation, dynamics and stability of VT rotors within the RV.

Gene- and variant-specific efficacy of serum/glucocorticoid-regulated kinase 1 inhibition in long QT syndrome types 1 and 2.

Current long QT syndrome (LQTS) therapy, largely based on beta-blockade, does not prevent arrhythmias in all patients; therefore, novel therapies are warranted. Pharmacological inhibition of the serum/glucocorticoid-regulated kinase 1 (SGK1-Inh) has been shown to shorten action potential duration (APD) in LQTS type 3. We aimed to investigate whether SGK1-Inh could similarly shorten APD in LQTS types 1 and 2. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hiPSC-cardiac cell sheets (CCS) were obtained from LQT1 and LQT2 patients; CMs were isolated from transgenic LQT1, LQT2, and wild-type (WT) rabbits. Serum/glucocorticoid-regulated kinase 1 inhibition effects (300 nM-10 µM) on field potential durations (FPD) were investigated in hiPSC-CMs with multielectrode arrays; optical mapping was performed in LQT2 CCS. Whole-cell and perforated patch clamp recordings were performed in isolated LQT1, LQT2, and WT rabbit CMs to investigate SGK1-Inh (3 µM) effects on APD. In all LQT2 models across different species (hiPSC-CMs, hiPSC-CCS, and rabbit CMs) and independent of the disease-causing variant (KCNH2-p.A561V/p.A614V/p.G628S/IVS9-28A/G), SGK1-Inh dose-dependently shortened FPD/APD at 0.3-10 µM (by 20-32%/25-30%/44-45%). Importantly, in LQT2 rabbit CMs, 3 µM SGK1-Inh normalized APD to its WT value. A significant FPD shortening was observed in KCNQ1-p.R594Q hiPSC-CMs at 1/3/10 µM (by 19/26/35%) and in KCNQ1-p.A341V hiPSC-CMs at 10 µM (by 29%). No SGK1-Inh-induced FPD/APD shortening effect was observed in LQT1 KCNQ1-p.A341V hiPSC-CMs or KCNQ1-p.Y315S rabbit CMs at 0.3-3 µM. A robust SGK1-Inh-induced APD shortening was observed across different LQT2 models, species, and genetic variants but less consistently in LQT1 models. This suggests a genotype- and variant-specific beneficial effect of this novel therapeutic approach in LQTS.

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes

BackgroundAlthough human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.MethodsiPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).ResultsWe showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.ConclusionsOverexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Enhanced Ca2+-Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation.

Small-conductance Ca2+-activated K+ (SK)-channel inhibitors have antiarrhythmic effects in animal models of atrial fibrillation (AF), presenting a potential novel antiarrhythmic option. However, the regulation of SK-channels in human atrial cardiomyocytes and its modification in patients with AF are poorly understood and were the object of this study. Apamin-sensitive SK-channel current (ISK) and action potentials were recorded in human right-atrial cardiomyocytes from sinus rhythm control (Ctl) patients or patients with (long-standing persistent) chronic AF (cAF). ISK was significantly higher, and apamin caused larger action potential prolongation in cAF- versus Ctl-cardiomyocytes. Sensitivity analyses in an in silico human atrial cardiomyocyte model identified IK1 and ISK as major regulators of repolarization. Increased ISK in cAF was not associated with increases in mRNA/protein levels of SK-channel subunits in either right- or left-atrial tissue homogenates or right-atrial cardiomyocytes, but the abundance of SK2 at the sarcolemma was larger in cAF versus Ctl in both tissue-slices and cardiomyocytes. Latrunculin-A and primaquine (anterograde and retrograde protein-trafficking inhibitors) eliminated the differences in SK2 membrane levels and ISK between Ctl- and cAF-cardiomyocytes. In addition, the phosphatase-inhibitor okadaic acid reduced ISK amplitude and abolished the difference between Ctl- and cAF-cardiomyocytes, indicating that reduced calmodulin-Thr80 phosphorylation due to increased protein phosphatase-2A levels in the SK-channel complex likely contribute to the greater ISK in cAF-cardiomyocytes. Finally, rapid electrical activation (5 Hz, 10 minutes) of Ctl-cardiomyocytes promoted SK2 membrane-localization, increased ISK and reduced action potential duration, effects greatly attenuated by apamin. Latrunculin-A or primaquine prevented the 5-Hz-induced ISK-upregulation. ISK is upregulated in patients with cAF due to enhanced channel function, mediated by phosphatase-2A-dependent calmodulin-Thr80 dephosphorylation and tachycardia-dependent enhanced trafficking and targeting of SK-channel subunits to the sarcolemma. The observed AF-associated increases in ISK, which promote reentry-stabilizing action potential duration shortening, suggest an important role for SK-channels in AF auto-promotion and provide a rationale for pursuing the antiarrhythmic effects of SK-channel inhibition in humans.

Levosimendan attenuates electrical alternans and prevents ventricular arrhythmia during therapeutic hypothermia in isolated rabbit hearts

Therapeutic hypothermia (TH) increases the susceptibility to ventricular arrhythmias (VAs) by prolonging action potential duration (APD) and facilitating arrhythmogenic spatially discordant alternans (SDA). Levosimendan, a calcium sensitizer, has been reported to shorten APD by enhancing the adenosine triphosphate (ATP)-sensitive K current. The purpose of this study was to test the hypothesis that, during TH, levosimendan shortens the already prolonged APD, attenuates SDA, and prevents VA. Langendorff-perfused isolated rabbit hearts were subjected to TH (30°C) for 15 minutes, followed by treatment with either levosimendan 0.5 μM (n = 9) or vehicle (n = 8) for an additional 30 minutes under TH. Using an optical mapping system, epicardial APD was evaluated by S1 pacing. SDA threshold was defined as the longest pacing cycle length (PCL) that induces the phenomenon of SDA. Ventricular fibrillation (VF) inducibility was evaluated by burst pacing for 30 seconds at the shortest PCL that achieved 1:1 ventricular capture. During TH, levosimendan shortened ventricular APD (PCL 400 ms; from 259 ± 8 ms to 241 ± 18 ms; P = .036) and decreased SDA threshold (from 327 ± 88 ms to 311 ± 68 ms; P = .011). VF inducibility was lowered from 39% ± 30% to 14% ± 12% with levosimendan (P = .018), whereas APD at PCL 400 ms (P = .161), SDA threshold (P = 1), and VF inducibility (P = .173) were not changed by vehicle. During TH, levosimendan could protect hearts against VA by shortening APD and decreasing SDA threshold. Enhancing ATP-sensitive K current with levosimendan might be a novel approach to preventing VA during TH.

PITX2 Knockout Induces Key Findings of Electrical Remodeling as Seen in Persistent Atrial Fibrillation.

Electrical remodeling in human persistent atrial fibrillation is believed to result from rapid electrical activation of the atria, but underlying genetic causes may contribute. Indeed, common gene variants in an enhancer region close to PITX2 (paired-like homeodomain transcription factor 2) are strongly associated with atrial fibrillation, but the mechanism behind this association remains unknown. This study evaluated the consequences of PITX2 deletion (PITX2-/-) in human induced pluripotent stem cell-derived atrial cardiomyocytes. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) was used to delete PITX2 in a healthy human iPSC line that served as isogenic control. Human induced pluripotent stem cell-derived atrial cardiomyocytes were differentiated with unfiltered retinoic acid and cultured in atrial engineered heart tissue. Force and action potential were measured in atrial engineered heart tissues. Single human induced pluripotent stem cell-derived atrial cardiomyocytes were isolated from atrial engineered heart tissue for ion current measurements. PITX2-/- atrial engineered heart tissue beats slightly slower than isogenic control without irregularity. Force was lower in PITX2-/- than in isogenic control (0.053±0.015 versus 0.131±0.017 mN, n=28/3 versus n=28/4, PITX2-/- versus isogenic control; P<0.0001), accompanied by lower expression of CACNA1C and lower L-type Ca2+ current density. Early repolarization was weaker (action potential duration at 20% repolarization; 45.5±13.2 versus 8.6±5.3 ms, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P<0.0001), and maximum diastolic potential was more negative (-78.3±3.1 versus -69.7±0.6 mV, n=18/3 versus n=12/4, PITX2-/- versus isogenic control; P=0.001), despite normal inward rectifier currents (both IK1 and IK,ACh) and carbachol-induced shortening of action potential duration. Complete PITX2 deficiency in human induced pluripotent stem cell-derived atrial cardiomyocytes recapitulates some findings of electrical remodeling of atrial fibrillation in the absence of fast beating, indicating that these abnormalities could be primary consequences of lower PITX2 levels.

Engineered cardiac tissue as a novel in vitro human myocardium model for studying cardiac stretch.

Heart disease is the largest contributor to death worldwide and research is limited by a lack of adequate human tissue in vitro models. To address this, we developed a protocol for generating 3D, atrial-like engineered cardiac tissue (ECT) comprised of cardiomyocytes (CM) and cardiac fibroblasts, derived from human-induced pluripotent stem cells (hiPSC). Control and atrial-like hiPSC-CM were produced using GiWi differentiation supplemented without and with 0.75 µM retinoic acid, respectively. ECTs were generated using hiPSC-cardiac fibroblasts and Day 30 hiPSC-CM combined in a fibrin matrix and molded via a FlexCell Tissue TrainTM system. ECTs were cultured for 30 days and evaluated for action potentials (AP) as well as mRNA and protein expression profiles. A subset of control ECTs were cyclically and incrementally stretched for 7 days up to 18% elongation. Stretch-induced changes were evaluated via transmission electron microscopy (TEM) and cell culture medium (LC-MS/MS). Atrial-like ECTs recapitulated various atrial phenotypes. They exhibit an atrial-like electrophysiology with a shorter AP duration and increased repolarization fraction (p<0.05). Atrial-like ECT show atrial-specific mRNA/protein expression, such as lower ventricular-like MYL2 and higher atrial-like MYL7 levels (p<0.05). They also demonstrate distinct contraction mechanics with faster kinetics (p<0.05) and decreased normalized contraction force, compared to control ECT. Cyclic stretch reduced cardiomyocyte membrane convolution index (estimating membrane tension) and caveolae membrane structure density (p<0.05). The latter was rescued via neutral sphingomyelinase inhibitor GW4869 (20 µM, p<0.05). LC-MS/MS of stretch-conditioned culture medium revealed decreases in relative sphingomyelins and phosphatidylcholines (p<0.05), ostensibly indicating membrane damage which was reversed by GW4869. Overall, our findings demonstrate retinoic acid-induced atrial-like phenotypes in hiPSC-CM are sustained in ECT form. We also introduce a novel ECT stretch protocol that recapitulates changes observed in animal models of heart pressure overload.

Activation of small conductance Ca2+ -activated K+ channels suppresses Ca2+ transient and action potential alternans in ventricular myocytes.

At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and Ca2+ transient (CaT) amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We tested the hypothesis that in single rabbit ventricular myocytes pharmacological modulation of SK channels plays a causative role for the development of pacing-induced CaT and AP duration (APD) alternans. SK channel blockers (apamin, UCL1684) had only a minor effect on AP repolarization. However, SK channel activation by NS309 resulted in significant APD shortening, demonstrating that functional SK channels are well expressed in ventricular myocytes. The effects of NS309 were prevented or reversed by apamin and UCL1684, indicating that NS309 acted on SK channels. SK channel activation abolished or reduced the degree of pacing-induced CaT and APD alternans. Inhibition of KV 7.1 (with HMR1556) and KV 11.1 (with E4031) channels was used to mimic conditions of long QT syndromes type-1 and type-2, respectively. Both HMR1556 and E4031 enhanced CaT alternans that was prevented by SK channel activation. In AP voltage-clamped cells the SK channel activator had no effect on CaT alternans, confirming that suppression of CaT alternans was caused by APD shortening. APD shortening contributed to protection from alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest that SK activation could be a potential intervention to avert development of alternans with important ramifications for arrhythmia prevention and therapy for patients with long QT syndrome. KEY POINTS: At the cellular level, cardiac alternans is observed as beat-to-beat alternations in contraction strength, action potential (AP) morphology and intracellular Ca2+ release amplitude, and is a risk factor for cardiac arrhythmia. The (patho)physiological roles of small conductance Ca2+ -activated K+ (SK) channels in ventricles are poorly understood. We investigated whether pharmacological modulation of SK channels affects the development of cardiac alternans in normal ventricular cells and in cells with drug-induced long QT syndrome (LQTS). While SK channel blockers have only a minor effect on AP morphology, their activation leads to AP shortening and abolishes or reduces the degree of pacing-induced Ca2+ and AP alternans. AP shortening contributed to protection against alternans by lowering sarcoplasmic reticulum Ca2+ content and curtailing Ca2+ release. The data suggest SK activation as a potential intervention to avert the development of alternans with important ramifications for arrhythmia prevention for patients with LQTS.

Scenarios for Increasing, Decreasing and Stability of Tpe/QT Ratio (Simulation Study)

The objective of this simulation was to study the physiological meaning of the novel index for arrhythmic risk stratification – Tpe/QT ratio (the interval from the peak to the end of the T-wave (Tpe) on electrocardiogram divided by QT interval). Methods and Results: The role of two factors determining Tpe/QT ratio – action potential duration (APD) and dispersion of repolarization (DOR) – was studied in silico at two levels: ventricular wall segment and the whole heart ventricles. The simulations performed in the framework of both the segment and the entire ventricles’ models showed that Tpe/QT magnitude reflects the dynamic relationship between the longest and the shortest APD rather than the magnitude of DOR, as interval Tpe does. Tpe/QT ratio remained unchanged even at large DOR, provided that the longer the ventricular APD, the greater the gap between the longest and the shortest of them. The imbalance between the longest and the shortest APD values led to the increased or decreased Tpe/QT. Conclusion: Simulations showed that Tpe/QT is not just a corrected Tpe interval, but it reflects the balance between the longest and the shortest APD in the heart ventricles.

Cardioprotective effect of abscisic acid in a rat model of type 3 cardio-renal syndrome: Role of NOX-4, P-53, and HSP-70

Cardiorenal syndrome (CRS) is a complex heart and kidney pathophysiologic disorder that leads to a bidirectional interrelationship between them. Abscisic acid (ABA) is a phytohormone that is present in plants, and is known to regulate fundamental physiological functions. This study aimed to explore the efficacy of ABA in surgically induced-CRS type 3 rats. Rats were randomly and equally divided into four groups. Rats in Group 1 received saline (Sham group), Group 2 included control induced-CRS rats, Group 3 rats (CRS+ABA) included CRS rats treated with ABA and Group 4 (CRS + ABA + Verapamil + propofol) were CRS rats treated with Verapamil, propofol and ABA. The rats were treated with the drugs daily for four weeks. At the end of the study, relative heart weight corrected QT interval (QTc), mean blood pressure (MBP), kidney functions, oxidative stress, NADPH oxidase 4 (NOX4), protein 53 (P53), and heat shock proteins-70 (HSP-70) expression was assessed and recorded.ABA led to a significant shortening of the ventricular action potential duration indicated by QTc. Furthermore, it significantly lowered heart weight, MBP, serum creatinine, NOX-4, and P-53 expression and augmented HSP-70 expression. In contrast, adding calcium channel blockers (CCBs) to ABA mitigated this effect. The results suggested that ABA has a potential protective role in CRS-induced cardiac hypertrophy and arrhythmia that could be mediated through inhibition of P-53, NOX-4, and an increase in HSP-70 expression.

GPER limits adverse changes to Ca2+ signalling and arrhythmogenic activity in ovariectomised guinea pig cardiomyocytes.

Background: The increased risk of post-menopausal women developing abnormalities of heart function emphasises the requirement to understand the effect of declining oestrogen levels on cardiac electrophysiology and structure, and investigate possible therapeutic targets, namely the G protein-coupled oestrogen receptor 1 (GPER). Methods: Female guinea pigs underwent sham or ovariectomy (OVx) surgeries. Cardiomyocytes were isolated 150-days post-operatively. Membrane structure was assessed using di-8-ANEPPs staining and scanning ion conductance microscopy. Imunnohistochemistry (IHC) determined the localisation of oestrogen receptors. The effect of GPER activation on excitation-contraction coupling mechanisms were assessed using electrophysiological and fluorescence techniques. Downstream signalling proteins were investigated by western blot. Results: IHC staining confirmed the presence of nuclear oestrogen receptors and GPER, the latter prominently localised to the peri-nuclear region and having a clear striated pattern elsewhere in the cells. Following OVx, GPER expression increased and its activation reduced Ca2+ transient amplitude (by 40%) and sarcomere shortening (by 32%). In these cells, GPER activation reduced abnormal spontaneous Ca2+ activity, shortened action potential duration and limited drug-induced early after-depolarisation formation. Conclusion: In an animal species with comparable steroidogenesis and cardiac physiology to humans, we show the expression and localisation of all three oestrogen receptors in cardiac myocytes. We found that following oestrogen withdrawal, GPER expression increased and its activation limited arrhythmogenic behaviours in this low oestrogen state, indicating a potential cardioprotective role of this receptor in post-menopausal women.

Abstract 10026: Augmentation of Slow Repolarizing Potassium Channel (I Ks ) Links Truncated Titin Mutations to Atrial Fibrillation

Introduction: Rare titin-truncating variants ( TTN tv) are associated with early-onset atrial fibrillation (AF) but the pathogenic mechanisms and therapeutic implications remain unclear. To determine the function of a highly conserved immunoglobulin-like domain of the A-band of TTN , a region enriched in AF-associated variants, we employed CRISPR-Cas9-mediated deletion of 9 amino acids (Δ9) in human induced pluripotent stem cells and then derived atrial cardiomyocytes (hiPSC-aCMs) for electrophysiological (EP) analysis. Hypothesis: We hypothesized that the TTN -Δ9 mutation results in augmentation of the slow delayed rectifier potassium current (I Ks ) resulting in shortened action potential duration (APD). Methods: TTN -WT hiPSC-aCMs (healthy control) and TTN -Δ9 hiPSC-aCMs (isogenic mutant) were matured with metabolic conditioning, electrical stimulation, co-culture with atrial fibroblasts, and micropatterning. We used transmission electron microscopy (TEM) to assess sarcomeric defects, RNA-sequencing and RT-qPCR to assess molecular ion channel remodeling, and high throughput automated patch clamp and optical voltage mapping to assess the EP properties of TTN -Δ9 hiPSC-aCMs. We also performed targeted pharmacological inhibition of I Ks . Results: We showed that TTN -Δ9 hiPSC-aCMs displayed disrupted organization and myofibril assembly ( Fig. 1A-B ), upregulated voltage-gated potassium channel complexes and increased KCNQ1 expression ( Fig 1C-D ), and shortened APD ( Fig. 1E-F ). I Ks was significantly augmented in TTN -Δ9 hiPSC-aCMs ( Fig. 1G-H ). Infusion of the I Ks -specific blocker HMR-1556, and not I Kr blocker dofetilide, fully restored the shortened APD 90 and partially recovered defects in contractility in TTN -Δ9 hiPSC-aCMs compared to TTN -WT hiPSC-aCMs ( Fig. 1I-J ). Conclusion: Our findings suggest that augmentation of I Ks contributes to the pathogenic mechanism of TTN -mediated AF, pointing to I Ks as a potential therapeutic target.

Abstract 12044: Injectable Contraceptive, Medroxyprogesterone Acetate, Produces Erratic Beating Patterns in Patient-Specific Re-Engineered Heart Cells With Type 2 Long QT Syndrome

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.

Abstract 10986: SGK1 Inhibition Attenuated the Action Potential Duration In-Patient and Genotype-Specific Re-Engineered Heart Cells With Congenital Long QT Syndrome

Background: Long QT syndrome (LQTS) 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&lt;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 (&gt; 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.

Abstract 12449: Inhibition of the Sodium Potassium ATPase Alpha 3 (ATP1A3) Pump Function Alters Repolarization Time and is Proarrhythmic in Cardiac Myocytes

Introduction: Inhibition of Na + /K + -ATPase (NKA) is arrhythmogenic, but the specific effect of alpha 3 NKA isoform (ATP1A3) inhibition is unknown. Variants in human ATP1A3 can lead to ventricular arrhythmias, yet the mechanism remains unknown. Hypothesis: Loss of NKA function results in shortening of QT interval and increased arrhythmia susceptibility due to intracellular Na+ and Ca2+ overload. Pharmacological inhibition of sodium-calcium exchanger (NCX) can rescue ECG parameter changes and arrhythmic phenotypes by reducing Ca2+ overload in the cell. Methods: Whole-cell patch clamp action potential recordings were conducted on induced pluripotent stem cell cardiomyocytes (iPSC-CMs) from an ostensibly healthy individual to measure action potential duration (APD) and presence of delayed after depolarizations (DADs). Cells were exposed to 10 -7 M ouabain and 10 -5 M ORM-10103 (a selective inhibitor of NCX). ECGs and in vivo intracardiac electrophysiology studies were performed in mice at 12-14 weeks of age after intraperitoneal drug administration of 5 μg/g ouabain and 10 μg/g ORM-10103. Results: APD recordings of iPSC-CMs demonstrated both shortening and lengthening of APD with ouabain exposure. In both models, DADs were found in 6 of 18 cells with ouabain treatment. These findings were rescued with pretreatment using ORM-10103, as 0 of 8 cells developed DADs. On mouse ECG recordings, ouabain led to QRS and QT prolongation. During programmed electrical stimulation, no arrhythmia was noted with vehicle-treated mice. Following ouabain administration, 2 in 12 mice developed induced monomorphic ventricular tachycardia. Pretreatment of ORM-10103 in mice did not rescue QRS and QT prolongation with ouabain administration. Conclusions: Pharmacologic inhibition of ATP1A3 variably altered cardiac repolarization time in a species-specific manner, yet was associated with a substrate for triggered arrhythmias. The arrhythmogenic phenotype was rescued with inhibition of NCX in human cardiac myocytes, suggesting that NCX plays a role in the arrhythmogenic mechanism of ATP1A3. However, these effects were not seen in murine cardiac myocytes. Exploration of specifies-specific differences in NKA isoform expression and cardiac repolarization is needed.

Abstract 13496: Sequencing in Over 50,000 Cases Identifies Coding and Structural Variation Underlying Atrial Fibrillation Risk

Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia and is associated with substantial morbidity and mortality. AF is known to have a heritable component, with &gt;100 associated common variant loci. Rare variant studies have yielded limited robust associations for AF. We aimed to utilize large genome and exome sequencing data to discover rare genetic variants conferring large effects on AF risk. Methods: We meta-analyzed genome and exome sequencing data from 36 studies, including TOPMed, CCDG, UK Biobank and FOURIER. We performed exome-wide gene burden testing of rare (MAF&lt;0.1%) loss-of-function and deleterious missense variants, and single variant testing of low-frequency and rare (MAF&lt;1%) coding variants. Within genome sequenced samples, we performed gene burden testing of rare structural variants. Novel signals were replicated in MyCode. Finally, we functionally validated a novel gene by siRNA knockdown in pluripotent-induced atrial cardiomyocytes. Results: We included 52,416 AF cases and 277,762 controls, of which 49.6% were female, 83.4% were of European ancestry, and the mean baseline age was 56 years. In analysis of rare coding variation, we identified 4 novel genes associated with AF, including MYBPC3 (OR 3.5, P =2.1x10 -15 ), LMNA (OR 5.7, P =8.8x10 -11 ), PKP2 (OR 1.9, P =5.2x10 -8 ) and KDM5B (OR 2.3, P =3.0x10 -6 ). These signals were robust to removal of heart failure and cardiomyopathy cases and were replicated in independent datasets. Single variant analysis identified 2 novel signals in FAM189A2 (OR 3.9, P =7.86x10 -8 ) and ZFC3H1 (OR 5.9, P =9.7x10 -8 ). Rare deletions in CTNNA3 (OR 4.5, P =7.0x10 -9 ) were associated with increased AF risk and were supported by independent coding variant analyses, while duplications of GATA4 (OR 0.24, P =2.1x10 -5 ) were associated with reduced AF risk. In functional studies, knockdown of KDM5B resulted in shortening of the atrial action potential duration. Conclusions: Our analyses show the contribution of rare coding and structural variants to AF risk, highlight the shared genetic pathways underlying cardiomyopathy and AF, and implicate the histone demethylase gene KDM5B in AF susceptibility. In sum, we expanded our understanding of the rare variant architecture of this common arrhythmia.

Abstract 11556: Sodium/Glucose Co-Transporter 2 Inhibition and Attenuation of the Action Potential Duration in Patient-Specific Re-Engineered Heart Cells With Congenital Long QT Syndrome

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&lt;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.

Testosterone does not shorten action potential duration in Langendorff-perfused rabbit ventricles.

Women have longer baseline QT intervals than men. Because previous studies showed that testosterone and 5α-dihydrotestosterone shorten the ventricular action potential duration (APD) in animal models, differential testosterone concentrations may account for the sex differences in QT interval. The purpose of this study was to test the hypothesis that testosterone shortens the APD in Langendorff-perfused rabbit ventricles. We performed optical mapping studies in hearts with or without testosterone administration. Acute studies included 26 hearts using 2 different protocols, including 17 without and 9 with atrioventricular (AV) block. For chronic studies, we implanted testosterone pellets subcutaneously in 7 female rabbits for 2-3 weeks before optical mapping studies during complete AV block. Six rabbits without pellet implantation served as controls. The hearts in the acute studies were paced with a pacing cycle length (PCL) of 200-300 ms and mapped at baseline and after administration of 1 nM, 10 nM, 100 nM, and 3 μM of testosterone. There was no shortening of APD80 at any PCL. Instead, a lengthening of APD80 was noted at higher concentrations. There were no sex differences in testosterone responses. In chronic studies, heart rates were 136 ± 5 bpm before and 148 ± 9 bpm after (P = .10) while QTc intervals were 314 ± 9 ms before and 317 ± 99 ms after (P= .69) testosterone pellet implantation, respectively. Overall, ventricular APD80 in the pellet group was longer than in the control group at 300- to 700-ms PCL. Testosterone does not shorten ventricular repolarization in rabbit hearts.