Shortening Of Action Potential Duration Research Articles

TLR4 pathway induces proarrhythmogenic slow conduction by impaired depolarisation

Abstract Background Cardiac inflammation driven by the Toll-Like-Receptor 4 (TLR4) is correlated to increased risk of arrhythmia. Cardiac arrhythmogenesis and the formation of re-entry tachycardia is highly dependent on conduction velocity (CV) and action potential (AP) duration (APD). As TLR4 induced APD shortening has been shown, we analyze in this study for the first time the TLR4 effect on conductance disturbances in a LPS induced septic mouse model. Methods Systemic activation of TLR4 in mice was achieved by intraperitoneal LPS injection 3.5 hours prior to experiments. In vivo electrophysiological investigation (EPI) was performed using an octapolar transvenous catheter placed to the right heart. Ex vivo experiments were performed on Langendorff-perfused hearts. AP propagation was measured by optical voltage mapping (OVM) with voltage sensitive dye Di-4-ANEPPS. For AP analysis including RMP, intracellular electrical recordings were performed using sharp microelectrodes. Wildtype mice after LPS injection were compared to wildtype mice after vehicle (NaCl) injection or ubiquitous TLR4 knockout (TLR4−/−) mice with LPS or vehicle application. Results In vivo EPI showed a tendency to more atrial fibrillation after LPS injection (+LPS 5/6, +NaCl 2/6, p=0.2). Ventricular stimulation evoked ventricular tachycardia in every LPS treated WT mouse but less in controls (+LPS 6/6, +NaCl 1/6, p=0.01). OVM measured decreased CV in both atria and ventricle after LPS treatment (atria: +LPS: 43.1±3.1cm/s, n=5; +NaCl: 72.6±9.8cm/s, n=10, p=0.04; ventricle: +LPS: 50.2±2.2cm/s, n=6; +NaCl: 67.7±5.0cm/s, n=10, p=0.02). In analysis of AP in atria upstroke velocity was slightly decreased (max.dV/dt: +LPS: 123.1±4.8V/s, n=22; +NaCl: 158.5±5.3V/s, n=39, p=0.04) but highly reduced in ventricle (max.dV/dt: +LPS: 91.8±3.6V/s, n=27; +NaCl: 140.7±6.3V/s, n=35, p<0.0001). RMP in atria was depolarised after LPS injection (+LPS: −70.1±1.9mV, n=22; +NaCl: −81.1±1.2mV, n=39, p=0.004) explaining decreased upstroke velocity and CV slowing in atria by voltage-dependent Na+ channel inactivation. Ventricular RMP was unaffected by LPS injection (+LPS: −75.1±1.1mV, n=44; +NaCl: −76.3±0.9mV, n=55, p=0.83). Therefore the Na+ currents were measured in isolated ventricular cardiomyocytes using whole cell patch clamp revealing the maximum Na+ current density lowered after LPS treatment (+LPS: −20.6±1.7 pA/pF, n=16, +NaCl 27.1±2.6 pA/pF, n=10, p=0.03). LPS did not affect EPI, CV, upstroke velocity or current density in TLR4−/− mice. Conclusion Herein we report for the first time impaired cardiac depolarisation and conduction after short term activation of TLR4 in vivo. Pro arrhythmogenic mechanisms differ in atria and ventricle: Increased atrial RMP inactivates Na+ current leading to reduction of CV. Ventricular slow CV is caused by reduced current density of Na+ channels. This different TLR4 effect might be important for novel antiarrhythmic and antiinflammatory applications. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): GEROK-grant, University Bonn

Coupling the Cardiac Voltage-Gated Sodium Channel to Channelrhodopsin-2 Generates Novel Optical Switches for Action Potential Studies.

Voltage-gated sodium (Na+) channels respond to short membrane depolarization with conformational changes leading to pore opening, Na+ influx, and action potential (AP) upstroke. In the present study, we coupled channelrhodopsin-2 (ChR2), the key ion channel in optogenetics, directly to the cardiac voltage-gated Na+ channel (Nav1.5). Fusion constructs were expressed in Xenopus laevis oocytes, and electrophysiological recordings were performed by the two-microelectrode technique. Heteromeric channels retained both typical Nav1.5 kinetics and light-sensitive ChR2 properties. Switching to the current-clamp mode and applying short blue-light pulses resulted either in subthreshold depolarization or in a rapid change of membrane polarity typically seen in APs of excitable cells. To study the effect of individual K+ channels on the AP shape, we co-expressed either Kv1.2 or hERG with one of the Nav1.5-ChR2 fusions. As expected, both delayed rectifier K+ channels shortened AP duration significantly. Kv1.2 currents remarkably accelerated initial repolarization, whereas hERG channel activity efficiently restored the resting membrane potential. Finally, we investigated the effect of the LQT3 deletion mutant ΔKPQ on the AP shape and noticed an extremely prolonged AP duration that was directly correlated to the size of the non-inactivating Na+ current fraction. In conclusion, coupling of ChR2 to a voltage-gated Na+ channel generates optical switches that are useful for studying the effect of individual ion channels on the AP shape. Moreover, our novel optogenetic approach provides the potential for an application in pharmacology and optogenetic tissue-engineering.

Use of nicorandil is associated with increased risk of incident atrial fibrillation.

Background: Nicorandil will activate ATP-sensitive potassium channel (KATP). However, activation of potassium channels plays an important role in the mechanism of atrial fibrillation (AF) or atrial flutter (AFL). Whether use of nicorandil might contribute to initiation and/or perpetuation of AF/AFL remained unknown. We determined the relationship between use of nicorandil and risk of atrial fibrillation and determined its molecular mechanism.Methods: We performed a nested case-control study using a cohort from the National Health Insurance Research Database (NHIRD) of Taiwan. The association between nicorandil use and risk of atrial fibrillation/flutter was estimated by logistic regression model. We also performed molecular, cellular and animal studies to explain the association.Results: A total of 715 individuals who experienced AF/atrial flutter were matched to 72,215 controls. New use of nicorandil was found to be associated with increased risk for AF/AFL (odds ratio [OR], 2.34; 95% CI 1.07–5.13) compared to nitrate use. We found the expression of KATP subunits Kir6.2 and SUR2A in human and rat left atrial tissues. Furthermore, nicorandil directly shortened action potential duration (APD) in rat left atrium and shortened the QT interval of cultured human induced pluripotent stem cell (iPSC) derived cardiomyocytes (iPSC-CMs).Conclusions: Use of nicorandil was found to be associated with increased risk of AF/AFL. We also showed the expression of KATP subunits in human atria, and a possible mechanism that use of nicorandil increases the risk of AF through activation of KATP and shortening of atrial APD.

Abstract P2096: Long Qt Syndrome Type 3 Phenotype Dependence On Extracellular Sodium And The Perinexus In A Genetic Mouse Model

Background: Long QT Syndrome Type 3 (LQT3) is caused by cardiac voltage-gated sodium channel (Nav1.5) gain of function and characterized by action potential duration (APD) prolongation. A guinea pig model of drug-induced LQT3 demonstrated that elevating sodium and widening the perinexus, an intercalated disc cleft adjacent to gap junctions, individually and synergistically exacerbate APD prolongation. The synergistic effect suggests that high sodium with cardiac edema is a risk factor for LQT3 patients. However, it remains unclear if this synergistic effect occurs in a genetic model of LQT3. Purpose: To investigate individual and combined effects of sodium concentration and perinexal widening in a genetic mouse model of LQT3. Methods: Ventricular action potential duration at 30 (APD30) and 90 (APD90) percent of repolarization was measured from optically mapped, Langendorff-perfused wild type (WT) and ΔKPQ mouse hearts. Hearts were perfused with sodium concentrations of 145 or 160 mM, in the absence or presence of an intercalated disc adhesion inhibitor (βadp1) inducing perinexal widening. Results: At baseline with 145 mM sodium, APD30 and APD90 were significantly greater in ΔKPQ than WT hearts. Surprisingly, increased sodium (160 mM), significantly decreased APD90 in WT but not ΔKPQ hearts, which is inconsistent with results from the drug-induced LQT3 guinea pig model. In contrast, 160mM sodium significantly decreased APD30 in both genotypes suggesting elevated sodium increases the transient outward potassium current (Ito). Interestingly, with 145 mM sodium, βadp1 treatment did not significantly change APD30 or APD90 in either genotype. However, increasing sodium from 145 to 160 mM with βadp1 significantly increased APD90 in ΔKPQ hearts but not WT. Conclusions: Synergistic APD prolongation was modest in a genetic LQT3 mouse when elevating sodium and widening the perinexus relative to drug-induced LQT3 in guinea pig. A significant difference in action potential morphology between mice and guinea pig is the over-expression of Ito in mice and its absence in guinea pig. Future studies will investigate whether transient outward potassium channels contribute to the effect of high sodium on APD shortening during early repolarization.

Abstract P1104: Lower Troponin Expression In The Right Ventricle Of Rats Explains Interventricular Differences In E-c Coupling

Despite distinctive functional and anatomical differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, IonOptix system and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single cell contractility and cytosolic Ca 2+ ([Ca 2+ ] i ), respectively. Myofilament proteins were analyzed by immunoblotting. RVCMs showed significantly shorter action potential duration (APD) and higher density of transient outward K + current ( I to ). However, the triggered [Ca 2+ ] i change (Ca 2+ transient) was not different, while the decay rate of the Ca 2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was approximately 60% lower in RVCM, partly responsible for the slower decay of the Ca 2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca 2+ buffering capacity ( κ S ), which was proved by in situ analysis. The introduction of these new levels of cTn, I to and SERCA into a mathematical model of rat LVCM reproduced the similar Ca 2+ transient, slower Ca 2+ decay, shorter APD and smaller ΔSL of RVCM. Taken together, we show reduced expression of cTn proteins in the RVCMs, which provides an explanation for the inter-ventricular difference in the E-C coupling kinetics.

Abstract P1090: Polycystin-1 Regulates Calcium Handling And Contractility In Human IPSC-derived Ventricular Cardiomyocytes

Mutations in polycystin-1 (PC1) cause 85% of autosomal dominant polycystic kidney disease cases. This disease is characterized by progressive renal disease and multiple cardiovascular alterations. Evidence suggests that cardiovascular disease precedes the onset of renal failure. We recently demonstrated that PC1 ablation shortens cardiomyocyte action potential, decreases Ca 2+ transient amplitude, and reduces contractility in adult cardiomyocytes isolated from cardiomyocyte-specific PC1 knock-out mice compared to control mice. Alterations in Ca 2+ cycling in PC1-deficient cardiomyocytes were demonstrated to be due to action potential duration shortening and impaired sarcoendoplasmic reticulum ATPase activity. However, mouse and human cardiomyocytes have considerable differences in their action potential shapes and the ion channels involved. It remains unknown whether PC1 regulates these events in human cardiomyocytes. Here, we sought to investigate whether PC1 regulates excitation-contraction coupling in human ventricular cardiomyocytes. We differentiated commercially available human-induced pluripotent stem cell lines (hiPSC) into ventricular cardiomyocytes (hiPSC-CM) using published protocols (measurements were performed 60-90 days after differentiation). We developed a protocol to simultaneously measure action potentials (FluoVolt), intracellular Ca 2+ (Calbryte-590) and contractility using the IonOptix algorithm to detect changes in contrast as contractility measurements. PC1 ablation using siRNA decreased action potential duration and impaired Ca 2+ transients and contractility in hiPSC-CMs. Because hiPSC-CMs have several limitations due to their immature state, we employed a well-validated method to improve their maturation that utilizes a Matrigel mattress and hormone supplementation (dexamethasone; Dexa / thyroid hormone; T3; PMID: 28974554; developed by Dr. Bjorn Knollmann’s lab). Using the Matrigel mattress/Dexa/T3 technique, we increased hiPSC-CM maturation measured as follows: cardiomyocytes were oriented and contracted in the longitudinal axis, ryanodine receptor type 2 and alpha-actinin localized with a striated pattern, exhibited robust inward K + rectifier currents (I K1 ) and fired action potentials that resemble human adult cardiomyocytes. In this model, PC1 knockdown also decreased action potential duration and impaired Ca 2+ transients and decreased contractility measured as fractional shortening. Altogether, this evidence shows for the first time that PC1 regulates human cardiomyocyte physiology and suggests that cardiac alterations in patients with autosomal dominant polycystic kidney disease may emerge as a result of impaired cardiomyocyte contractile function.

Tetramisole is a new IK1 channel agonist and exerts IK1 -dependent cardioprotective effects in rats.

Cardiac ischemia, hypoxia, arrhythmias, and heart failure share the common electrophysiological changes featured by the elevation of intracellular Ca2+ (Ca2+ overload) and inhibition of the inward rectifier potassium (IK1) channel. IK1 channel agonists have been considered a new type of anti‐arrhythmia and cardioprotective agents. We predicted using a drug repurposing strategy that tetramisole (Tet), a known anthelminthic agent, was a new IK1 channel agonist. The present study aimed to experimentally identify the above prediction and further demonstrate that Tet has cardioprotective effects. Results of the whole‐cell patch clamp technique showed that Tet at 1–100 μmol/L enhanced IK1 current, hyperpolarized resting potential (RP), and shortened action potential duration (APD) in isolated rat cardiomyocytes, while without effects on other ion channels or transporters. In adult Sprague–Dawley (SD) rats in vivo, Tet showed anti‐arrhythmia and anticardiac remodeling effects, respectively, in the coronary ligation‐induced myocardial infarction model and isoproterenol (Iso, i.p., 3 mg/kg/day, 10 days) infusion‐induced cardiac remodeling model. Tet also showed anticardiomyocyte remodeling effect in Iso (1 μmol/L) infused adult rat ventricular myocytes or cultured H9c2 (2‐1) cardiomyocytes. Tet at 0.54 mg/kg in vivo or 30 μmol/L in vitro showed promising protections on acute ischemic arrhythmias, myocardial hypertrophy, and fibrosis. Molecular docking was performed and identified the selective binding of Tet with Kir2.1. The cardioprotection of Tet was associated with the facilitation of IK1 channel forward trafficking, deactivation of PKA signaling, and inhibition of intracellular calcium overload. Enhancing IK1 may play dual roles in anti‐arrhythmia and antiventricular remodeling mediated by restoration of Ca2+ homeostasis.

Chronic Administration of COVID-19 Drugs Fluvoxamine and Lopinavir Shortens Action Potential Duration by Inhibiting the Human Ether-à-go-go-Related Gene and Cav1.2.

Background: Old drugs for new indications in the novel coronavirus disease of 2019 (COVID-19) pandemic have raised concerns regarding cardiotoxicity, especially the development of drug-induced QT prolongation. The acute blocking of the cardiac hERG potassium channel is conventionally thought to be the primary mechanism of QT prolongation induced by COVID-19 drugs fluvoxamine (FLV) and lopinavir (LPV). The chronic impact of these medications on the hERG expression has yet to be determined. Methods: To investigate the effect of long-term incubation of FLV and LPV on the hERG channel, we used electrophysiological assays and molecular experiments, such as Western blot, RT-qPCR, and immunofluorescence, in HEK-293 cells stably expressing hERG and human-induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). Results: Compared to the acute effects, chronic incubation for FLV and LPV generated much lower half-maximal inhibitory concentration (IC50) values, along with a left-shifted activation curve and retarded channel activation. Inconsistent with the reduction in current, we unexpectedly found that the chronic effects of drugs promoted the maturation of hERG proteins, accompanied by the high expression of Hsp70 and low expression of Hsp90. Targeting Hsp70 using siRNA was able to reverse the effects of these drugs on hERG proteins. In addition, FLV and LPV resulted in a significant reduction of APD90 and triggered the early after-depolarizations (EADs), as well as inhibited the protein level of the L-type voltage–operated calcium channel (L-VOCC) in hiPSC-CMs. Conclusion: Chronic incubation with FLV and LPV produced more severe channel-blocking effects and contributed to altered channel gating and shortened action potential duration by inhibiting hERG and Cav1.2.

Impact of Adenosine on Wavefront Propagation in Persistent Atrial Fibrillation: Insights From Global Noncontact Charge Density Mapping of the Left Atrium.

BackgroundAdenosine shortens action potential duration and refractoriness and provokes atrial fibrillation. This study aimed to evaluate the effect of adenosine on mechanisms of wavefront propagation during atrial fibrillation.Methods and ResultsThe study included 22 patients undergoing catheter ablation for persistent atrial fibrillation. Left atrial mapping was performed using the AcQMap charge density system before and after administration of intravenous adenosine at 1 or more of 3 time points during the procedure (before pulmonary vein isolation, after pulmonary vein isolation, and after nonpulmonary vein isolation ablation). Wave‐front propagation patterns were evaluated allowing identification and quantification of localized rotational activation (LRA), localized irregular activation, and focal firing. Additional signal processing was performed to identify phase singularities and calculate global atrial fibrillation cycle length and dominant frequency. A total of 35 paired maps were analyzed. Adenosine shortened mean atrial fibrillation cycle length from 181.7±14.3 to 165.1±16.3, (mean difference 16.6 ms; 95% CI, 11.3–21.9, P<0.0005) and increased dominant frequency from 6.0±0.7 Hz to 6.6±0.8 Hz (95% CI, 0.4–0.9, P<0.0005). This was associated with a 50% increase in the number of LRA occurrences (16.1±7.6–24.2±8.1; mean difference 8.1, 95% CI, 4.1–12, P<0.0005) as well as a 20% increase in the number of phase singularities detected (30.1±7.8–36.6±9.3; mean difference 6.5; 95% CI, 2.6–10.0, P=0.002). The percentage of left atrial surface area with LRA increased with adenosine and 42 of 70 zones (60%) with highest density of LRA coincided with high density LRA zones at baseline with only 28% stable across multiple maps.ConclusionsAdenosine accelerates atrial fibrillation and promotes rotational activation patterns with no impact on focal activation. There is little evidence that rotational activation seen with adenosine represents promising targets for ablation aimed at sites of stable arrhythmogenic sources in the left atrium.

Understanding the contribution of stretch-activated ion channels to cardiac arrhythmogenesis using computational modelling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch heart foundation, ERA-CVD Introduction Cardiac electrophysiology and mechanics are strongly interconnected. Among other things, their interaction is mediated by cardiac mechano-electric feedback through stretch-activated ion-channels (SACs). These channels are also thought to contribute to the development of arrhythmias, but their precise role remains unclear. Purpose To elucidate the contribution of SACs to arrhythmias using a novel computational model of cardiac electromechanics. Methods We implemented two types of SACs in the O’Hara-Rudy model (ORd) of human ventricular electrophysiology: potassium-selective SACs and non-selective SACs (conducting sodium and potassium). The model was calibrated based on experimental human and rodent data of cardiomyocytes undergoing stretch. The calibration also considered inter-species differences, age, and upregulation of SACs under disease conditions. Subsequently, we varied the amplitude, duration, and timing of the simulated stretch to investigate their effects on action potential (AP). Results The model reproduced APs measured experimentally. Early afterdepolarizations, delayed afterdepolarizations, and ectopic beats were observed when applying stretch with short duration (e.g. 20ms) and high amplitude (e.g. 40%). When varying the time of application, stretch applied closer to the subsequent beat (cycle length: 1000ms) also shortened the following AP duration (APD) (shortening of 30ms with stretch at t=600ms, 130ms at t=800ms). Higher sensitivity to stretch was observed when simulating disease-related remodelling of SACs. Milder effects (no APD shortening with stretch at t=600ms and t=800ms) were seen with a lower stretch amplitude (e.g. 10% with SACs disease-related remodelling, 15% without). Failure of repolarization only occurred when sustained stretch (1000ms duration) of more than 10% (with SACs disease-related remodelling) or 15% (without) was applied. Conclusions Using a novel human electromechanical computational model, we quantified the contribution of SACs to cardiac AP changes. We showed that both disease-related SAC remodelling and variations of amplitude, timing, and duration of cardiomyocyte stretch modulated the effects on cardiac electrophysiology. We also showed that SACs may lead to afterdepolarizations and shorten the subsequent AP duration, factors which may potentially contribute to the generation of arrhythmias.

Transmural voltage gradient dispersion and heterogeneity in brugada syndrome phenotype - a novel workflow for advanced mapping using endocardial unipolar electrograms J-point elevation

Abstract Funding Acknowledgements Type of funding sources: None. Background Differential action potential duration shortening across the right ventricular (RV) myocardial wall is primarily responsible for the Brugada Syndrome (BrS) phenotype [1]. To date, data on electrical substrate characterization in humans with BrS phenotype is limited and risk evaluation is still controversial. Purpose We hypothesized that Uni-JEl mapping could be used as a marker of transmural voltage gradient dispersion resulting. Our aim was to evaluate Uni-JEl mapping in defining arrhythmogenic substrates in patients with BrS phenotype. Methods 12 patients were included in our analysis. 2 normal patients provided control data and 10 asymptomatic subjects with spontaneous type-1 BrS underwent 3D RV mapping (CARTO3 System, Biosense Webster). Among BrS patients we had 3 patients with arrhythmic events (aborted sudden death or appropriate ICD therapies) during follow-up (median 56, interquartile range: 46-74 months) and 7 patients without arrhythmic events. In the former group we had 1 patient with inducibility of VT/VF during EPS (EPS+) and 2 patients non-inducibles during EPS (EPS-), in the latter group we had 3 patients with EPS+ and 4 patients with EPS-. Electrophysiological data and signals were exported and OpenEP [2] was used to convert Carto proprietary data formats into Matlab format (Fig.1). Uni-JEl was calculated for each point map as the unipolar value at J point on surface electrocardiogram. Uni-JEl values were then interpolated in Paraview to create Uni-JEl maps, interpolating data points on the mesh cell (Fig.1). Finally, a region of interest (ROI) was selected and the calculation of mean Uni-JEI (MUni-JEI, as a measure of voltage gradient dispersion), interquartile range and range (intrqUni-JEI and ∆Uni-JEI, as markers of heterogeneity of dispersion) was performed. Results are shown as mean ± standard deviation for the group of BrS patients and the actual values for the two controls. Results BrS patients showed Muni-JEl, intrqUni-JEl and ∆Uni-JEI higher than controls (2.03 mV ± 0.31 mV vs 0,82 mV and 1,1 mV, 1.90 mV ± 0.82 mV vs 1,04 mV and 1,18 mV 6.26 mV ± 1.98 mV vs 3,54 mV and 4,01 mV, respectively). BrS patients with arrhythmic events during the follow-up showed higher intrqUni-JEl and the ∆Uni-JEI respect to BrS with EPS+ and without arrhythmic events during follow-up (2.31 mV ± 0.44 mV vs 0.78 mV ± 0.11 mV and 6.69 mV ± 2.27 mV vs 3.98 mV ± 0.31 mV). Figure 2 shows some examples of calculated Uni-JEl maps for each group under study. Conclusions In this work we introduced a novel workflow for the electrical substrate characterization of subjects with BrS phenotype. The results from our preliminary analysis indicate that a higher transmural voltage gradient dispersion and heterogeneity can be found in type-1 BrS with respect to normal subjects. Voltage gradient dispersion heterogeneity could be used to better recognize high risk BrS patients regardless of VT/VF inducibility during EPS.

Paradoxical prolongation of QT interval during exercise in patients with hypertrophic cardiomyopathy: cellular mechanisms and implications for diastolic function.

AimsVentricular cardiomyocytes from hypertrophic cardiomyopathy (HCM) patient hearts show prolonged action potential duration (APD), impaired intracellular Ca2+ homeostasis and abnormal electrical response to beta -adrenergic stimulation. We sought to determine whether this behaviour is associated with abnormal changes of repolarization during exercise and worsening of diastolic function, ultimately explaining the intolerance to exercise experienced by some patients without obstruction.Methods and resultsNon-obstructive HCM patients (178) and control subjects (81) underwent standard exercise testing, including exercise echocardiography. Ventricular myocytes were isolated from myocardial samples of 23 HCM and eight non-failing non-hypertrophic surgical patients. The APD shortening in response to high frequencies was maintained in HCM myocytes, while β-adrenergic stimulation unexpectedly prolonged APDs, ultimately leading to a lesser shortening of APDs in response to exercise. In HCM vs. control subjects, we observed a lesser shortening of QT interval at peak exercise (QTc: +27 ± 52 ms in HCM, −4 ± 50 ms in controls, P < 0.0001). In patients showing a marked QTc prolongation (>30 ms), the excessive shortening of the electrical diastolic period was linked with a limited increase of heart-rate and deterioration of diastolic function at peak effort.ConclusionsAbnormal balance of Ca2+- and K+-currents in HCM cardiomyocytes determines insufficient APD and Ca2+-transient shortening with exercise. In HCM patients, exercise-induced QTc prolongation was associated with impaired diastolic reserve, contributing to the reduced exercise tolerance. Our results support the idea that severe electrical cardiomyocyte abnormalities underlie exercise intolerance in a subgroup of HCM patients without obstruction.

PO-629-02 NEW EVIDENCE TO CHALLENGE CLINGEN'S “DISPUTED EVIDENCE” DESIGNATION FOR AKAP9 AS A BONA FIDE SUSCEPTIBILITY GENE FOR CONGENITAL LONG QT SYNDROME

A-kinase-anchoring protein-9 (AKAP9) mediates the phosphorylation state of the KCNQ1-encoded Kv7.1 potassium channel. Loss-of-function (LOF) variants in KCNQ1 cause type 1 long QT syndrome (LQT1). In 2007, we published data suggesting AKAP9-S1570L as a novel LQTS-causative variant and AKAP9 as a novel LQTS-susceptibility gene. However, in a recent re-appraisal of LQTS genes, the Clinical Genome Resource (ClinGen) demoted AKAP9 to a “disputed evidence” gene due to apparent lack of sufficient evidence to support causation of LQTS. To characterize the electrophysiology of a LQTS patient-derived induced pluripotent stem cell-cardiomyocyte (iPSC-CM) AKAP9-S1570L model and its corresponding CRISPR/Cas9 gene-edited/variant-corrected isogenic control (IC). iPSC-CMs were generated from an AKAP9-S1570L positive female diagnosed with LQTS at 14 years of age with a QTc > 480 ms, history of syncope, a LQT1-suggestive treadmill stress test, and a positive family history of QT-prolongation. Standard whole-cell patch clamp technique was performed to measure the action potential duration (APD) of both the AKAP9-S1570L and IC iPSC-CMs. To determine if KCNQ1 contributes to the APD prolongation of the variant iPSC-CMs, the KCNQ1 channel specific activator, ML277 was used in additional APD measurements. APD50 (457 ± 21) and APD90 (549 ± 22 ms) were significantly prolonged in the AKAP9-S1570L iPSC-CMs compared to the IC iPSC-CMs (APD50, 335 ± 15 ms; APD90, 413 ± 16 ms; p<0.05). Addition of ML277 resulted in a greater degree of APD shortening in AKAP9-S1570L iPSC-CMs (APD50 shortening, 349 ± 40 ms (p<0.05); APD90 shortening, 397 ± 42 ms (p<0.05) than in IC iPSC-CMs (APD50 shortening, 219 ± 31 ms; APD90 shortening, 242 ± 33 ms). This indicates KCNQ1 LOF may contribute to the mechanism underlying APD prolongation in the patient-specific AKAP9-S1570L iPSC-CMs, consistent with our previously published data. Using patient specific iPSC-CMs and isogenic controls, we provide new evidence bolstering AKAP9-S1570L as the pathogenic LQTS-causative variant responsible for this patient’s LQTS. Whether ClinGen deems this sufficient evidence to rescue AKAP9 from genetic purgatory and re-classify it as a sufficient evidence LQTS-susceptibility gene, remains to be seen.

Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes

BackgroundGout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1.Methods and ResultsMacrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1β secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1β. MSU reduced intracellular K+ concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1β, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1β and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5.ConclusionsKv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF.

A benzodiazepine activator locks Kv7.1 channels open by electro-mechanical uncoupling

Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.

Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

Electrophysiology and Arrhythmogenesis in the Human Right Ventricular Outflow Tract.

Right ventricular outflow tract (RVOT) is a common source of ventricular tachycardia, which often requires ablation. However, the mechanisms underlying the RVOT's unique arrhythmia susceptibility remain poorly understood due to lack of detailed electrophysiological and molecular studies of the human RVOT. We conducted optical mapping studies in 16 nondiseased donor human RVOT preparations subjected to pharmacologically induced adrenergic and cholinergic stimulation to evaluate susceptibility to arrhythmias and characterize arrhythmia dynamics. We found that under control conditions, RVOT has shorter action potential duration at 80% repolarization relative to the right ventricular apical region. Treatment with isoproterenol (100 nM) shortened action potential duration at 80% repolarization and increased incidence of premature ventricular contractions (P=0.003), whereas acetylcholine (100 μM) stimulation alone had no effect on action potential duration at 80% repolarization or premature ventricular contractions. However, acetylcholine treatment after isoproterenol stimulation reduced the incidence of premature ventricular contractions (P=0.034) and partially reversed action potential duration at 80% repolarization shortening (P=0.029). Immunolabeling of RVOT (n=4) confirmed the presence of cholinergic marker VAChT (vesicular acetylcholine transporter) in the region. Rapid pacing revealed RVOT susceptibility to both concordant and discordant alternans. Investigation into transmural arrhythmia dynamics showed that arrhythmia wave fronts and phase singularities (rotors) were relatively more organized in the endocardium than in the epicardium (P=0.006). Moreover, there was a weak but positive spatiotemporal autocorrelation between epicardial and endocardial arrhythmic wave fronts and rotors. Transcriptome analysis (n=10 hearts) suggests a trend that MAPK (mitogen-activated protein kinase) signaling, calcium signaling, and cGMP-PKG (protein kinase G) signaling are among the pathways that may be enriched in the male RVOT, whereas pathways of neurodegeneration may be enriched in the female RVOT. Human RVOT electrophysiology is characterized by shorter action potential duration relative to the right ventricular apical region. Cholinergic right ventricular stimulation attenuates the arrhythmogenic effects of adrenergic stimulation, including increase in frequency of premature ventricular contractions and shortening of wavelength. Right ventricular arrhythmia is characterized by positive spatial-temporal autocorrelation between epicardial-endocardial arrhythmic wave fronts and rotors that are relatively more organized in the endocardium.

Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via. CaM/Src/Pitx2 Activation in Atrial Myocytes.

Hypertension is a major cardiovascular risk factor for atrial fibrillation (AF) worldwide. However, the role of mechanical stress caused by hypertension on downregulating the L-type calcium current (ICa,L), which is vital for AF occurrence, remains unclear. Therefore, the aim of the present study was to investigate the role of Piezo1, a mechanically activated ion channel, in the decrease of ICa,L in response to high hydrostatic pressure (HHP, one of the principal mechanical stresses) at 40 mmHg, and to elucidate the underlying pathways. Experiments were conducted using left atrial appendages from patients with AF, spontaneously hypertensive rats (SHRs) treated with valsartan (Val) at 30 mg/kg/day and atrium-derived HL-1 cells exposed to HHP. The protein expression levels of Piezo1, Calmodulin (CaM), and Src increased, while that of the L-type calcium channel a1c subunit protein (Cav1.2) decreased in the left atrial tissue of AF patients and SHRs. SHRs were more vulnerable to AF, with decreased ICa,L and shortened action potential duration, which were ameliorated by Val treatment. Validation of these results in HL-1 cells in the context of HHP also demonstrated that Piezo1 is required for the decrease of ICa,L by regulating Ca2+ transient and activating CaM/Src pathway to increase the expression of paired like homeodomain-2 (Pitx2) in atrial myocytes. Together, these data demonstrate that HHP stimulation increases AF susceptibility through Piezo1 activation, which is required for the decrease of ICa,L via. the CaM/Src/Pitx2 pathway in atrial myocytes.

Lower troponin expression in the right ventricle of rats explains interventricular differences in E-C coupling.

Despite distinctive functional and anatomic differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, the IonOptix system, and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single-cell contractility, and cytosolic Ca2+ ([Ca2+]i), respectively. Myofilament proteins were analyzed by immunoblotting. RVCM showed significantly shorter action potential duration (APD) and higher density of transient outward K+ current (Ito). However, the triggered [Ca2+]i change (Ca2+ transient) was not different, while the decay rate of the Ca2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was ∼60% lower in RVCM, which is partly responsible for the slower decay of the Ca2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca2+ buffering capacity (κS), which was proved by in situ analysis. The introduction of these new levels of cTn, Ito, and SERCA into a mathematical model of rat LVCM reproduced the similar Ca2+ transient, slower Ca2+ decay, shorter APD, and smaller ΔSL of RVCM. Taken together, these data show reduced expression of cTn proteins in the RVCM, which provides an explanation for the interventricular difference in the E-C coupling kinetics.

Enhanced β-adrenergic response in mice with dominant-negative expression of the PKD2L1 channel.

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.e., lacking the pore-forming domain). The resulting PKD2L1del-Tg mice exhibited supraventricular premature contraction, as well as enhanced sensitivity to β-adrenergic stimulation and unstable R-R intervals in electrocardiography. During spontaneous atrial contraction, PKD2L1del-Tg atria showed enhanced sensitivity to isoproterenol, norepinephrine, and epinephrine. Action potential recording revealed a shortened action potential duration in PKD2L1del-Tg atria in response to isoproterenol. These findings indicated increased adrenergic sensitivity in PKD2L1del-Tg mice, suggesting that PKD2L1 is involved in sympathetic regulation.