Action Potential Duration Research Articles

Electrophysiological tolerance - a new concept for understanding the electrical stability of the heart.

The coordinated electrical activity of ∼2 billion cardiac cells ensures stability of the heartbeat. Indeed, the remarkably low incidence (<1%) of ventricular arrhythmias in the healthy heart is only possible when the electrical event across this syncytium is closely controlled. In contrast, the diseased myocardium is associated with increased electrophysiological heterogeneity, unstable rhythm, and increased incidence of lethal arrhythmias. But what is the link between cellular and tissue level heterogeneity? Recent research has shown the existence of considerable cellular heterogeneity even in the healthy heart, suggesting that cell-to-cell variability in electrical (e.g. action potential duration) and mechanical performance (e.g. twitch amplitude) is a normal property. This observation has been previously unappreciated because the aggregated function in the form of QT-interval and cardiac output varies <1% on a beat-to-beat basis. This article describes the underlying cellular variability that is tolerated - and perhaps needed - by different regions of the heart for normal function and indicates why this variability is not apparent in function at the chamber and organ level. Thus, in contrast to the current dominant view, this article postulates that heterogeneity is normal and potentially endows various functional benefits. This new view of how the component parts of the heart come together to function also suggests novel mechanisms for cardiac pathologies, namely that dysfunction may emerge from changes in the extent and/or nature of heterogeneity. Once understood, restoring normal forms of heterogeneity could be a novel approach to treatment.

Cellular calcium handling and electrophysiology are modulated by chronic physiological pacing in human induced pluripotent stem cell-derived cardiomyocytes.

Electric pacing of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) has been increasingly used to simulate cardiac arrhythmias in vitro and to enhance cardiomyocyte maturity. However, the impact of electric pacing on cellular electrophysiology and Ca2+ handling in differentiated hiPSC-CM is less characterized. Here we studied the effects of electric pacing for 24 h or 7 days at a physiological rate of 60 beats/min on cellular electrophysiology and Ca2+ cycling in late-stage, differentiated hiPSC-CM (>90% troponin+, >60 days postdifferentiation). Electric culture pacing for 7 days did not influence cardiomyocyte cell size, apoptosis, or generation of reactive oxygen species in differentiated hiPSC-CM compared with 24-h pacing. However, epifluorescence measurements revealed that electric pacing for 7 days improved systolic Ca2+ transient amplitude and Ca2+ transient upstroke, which could be explained by elevated sarcoplasmic reticulum Ca2+ load and SERCA activity. Diastolic Ca2+ leak was not changed in line-scanning confocal microscopy, suggesting that the improvement in systolic Ca2+ release was not associated with a higher open probability of ryanodine receptor (RyR)2 during diastole. Whereas bulk cytosolic Na+ concentration and Na+/Ca2+ exchanger (NCX) activity were not changed, patch-clamp studies revealed that chronic pacing caused a slight abbreviation of the action potential duration (APD) in hiPSC-CM. We found in whole cell voltage-clamp measurements that chronic pacing for 7 days led to a decrease in late Na+ current, which might explain the changes in APD. In conclusion, our results show that chronic pacing improves systolic Ca2+ handling and modulates the electrophysiology of late-stage, differentiated hiPSC-CM. This study might help to understand the effects of electric pacing and its numerous applications in stem cell research including arrhythmia simulation.NEW & NOTEWORTHY Electric pacing is increasingly used in research with human induced pluripotent stem cell cardiomyocytes (hiPSC-CM), for example to simulate arrhythmias but also to enhance maturity. Therefore, it is mandatory to understand the effects of pacing itself on cellular electrophysiology in late-stage, matured hiPSC-CM. This study provides an electrophysiological characterization of the effects of chronic electric pacing at a physiological rate on differentiated hiPSC-CM.

Significance of J wave with inverted ST-T polarity induced by ill-timed atrial premature beats and its association with Torsade de Pointes in patients with acute acquired long QT syndrome

Abstract Background When ventricular cardiomyocytes become depolarized during their relative refractory period (RRP), the amplitude of the action potential (AP) becomes lower than that of preceding AP, since some sodium channels are in their refractory state. If the membrane voltage does not reach a level high enough to activate voltage-dependent calcium channels, the AP duration becomes markedly shortened due to loss of AP dome. In patients with long QT syndrome (LQTS), intrinsic differences in AP duration and/or AP restitution kinetics exist between ventricular endocardium and epicardium. Thus, we hypothesized that atrial premature beats (APBs) can capture the RRP and amplify a transmural voltage gradient that manifests as a J wave with inverted ST-T polarity (APB-induced J wave). Purpose To investigate the prevalence of APB-induced J wave and examine the contribution to the development of Torsade de Pointes (TdP) in patients with acute acquired LQTS. Methods A total of 34 consecutive patients with acute acquired LQTS (14 men; median age, 69 years; median QTc, 645.5 ms) and documented TdP were enrolled. APBs were observed before the onset of TdP in 18 (52.9%) patients. The 34 patients were divided into 2 groups based on the presence or absence of APB-induced J wave with inverted ST-T polarity: APB-J wave group (n=8) and non-APB-J wave group (n=26). Results APB-J wave group constituted 23.5% (8/34) of the studied patients. There were no significant differences in clinical characteristics (sex, age and QTc) between the 2 groups. Macroscopic T-wave alternans was more frequently observed before TdP onset in APB-J wave group than non-APB-J wave group (75% vs 15.4%, p=0.003). The ill-timed APBs with J wave directly initiated TdP in all patients in APB-J wave group. APB-J wave group experienced TdP even after initiating conventional therapy more frequently than non-APB-J wave group (3.5 vs 1 event, p=0.04). Conclusions APB-induced J wave is observed in approximately one-fourth of acute acquired LQTS patients with TdP and is associated with an increased risk of TdP. Our observations suggest that APBs can amplify the transmural dispersion of repolarization manifested as a J wave.Figure1Figure2

Arrhythmogenic effects of DEHP exposure: insights into cardiac electrophysiology and myocardial cells

Abstract Introduction Hemodialysis patients, who are exposed to plasticizers through the hemodialysis circuit, face a high risk of cardiovascular disease (CVD). Plasticizers, such as the endocrine-disrupting compound Di(2-ethylhexyl) phthalate (DEHP), have emerged as potential contributors to CVD. This study explores the impact of DEHP on cardiac electrophysiology and myocardial cells, providing insights into its cardiotoxic effects. Methods We employed optical mapping techniques to assess cardiac electrophysiology in Langendorff-perfused rat hearts exposed to DEHP, comparing them to a control group. Additionally, we evaluated the effect of DEHP on cardiac muscle cells using H9C2 rat cardiomyoblasts. Results Prolonged DEHP exposure resulted in elevated heart rate, increased electrocardiographic variability, and higher low-frequency values, indicative of sympathetic nervous system overactivity. Action potential duration at APD80 increased, leading to greater action potential triangulation. Electrical alternans observed in the DEHP-treated group heightened the risk of arrhythmias. Single-cell analysis revealed severe mitochondrial damage, myocardial cell apoptosis, and disrupted calcium ion balance. Conclusion This study highlights DEHP's cardiotoxicity, suggesting its potential role in arrhythmogenesis and cardiac dysfunction. Environmental pollutants like DEHP warrant consideration as contributors to cardiovascular diseases, emphasizing the need for further research on preventive strategies.

Characterization of knock-in zebrafish models with a novel SLC4A3 variant identified in a short QT syndrome family

Abstract Background Short QT syndrome (SQTS) is one of the lethal inherited arrhythmia syndromes leading to ventricular fibrillation (VF) and cardiac death. It is mainly caused by pathogenic variants in ion channel related genes, especially KCNH2 which produces IKr. Recently, SLC4A3 encoding a membrane-localized anion exchanger 3 (AE3) that regulates intracellular pH (pHi), has been reported as a causative gene for SQTS. However, the molecular mechanism of SLC4A3 variants which cause SQTS remains unclear. Purpose Functional characterization of a novel SLC4A3 variant identified in a SQTS family. Methods We performed target gene and whole exome sequencing to detect pathogenic variants in a family with SQTS. Patch-clamp analyses were performed to determine the electrophysiological properties using HEK293 cells. We constructed stable cell lines expressing wild-type (WT) or the SLC4A3 variant based on HEK293 cells. To evaluate the intracellular pH (pHi) level, we measured pHi using BCECF-AM via time-lapse microscopy. To clarify the pathogenesis of the SLC4A3 variant in vivo, we injected CRISPR-Cas9 and a repair template into one-cell stage zebrafish embryos and established a SLC4A3 knock-in (KI) zebrafish model (hereafter referred to as slc4a3 zebrafish). Action potential durations (APD) were recorded in slc4a3 zebrafish at 3 days post-fertilization (dpf). Results We identified two novel variants in KCNH2 (c.280 c&amp;gt;t, p.H70Y) and SLC4A3 (c.1059 c&amp;gt;a, p.N353K) in a SQTS patient. Both variants were co-segregated in the patient’s family with short QT intervals (Figure A). By electrophysiological analysis, KCNH2-H70Y did not increase IKr. We over-expressed KCNH2 (WT and H70Y), KCNQ1, and KCNJ2 in the SLC4A3 stable cell lines (WT and N353K) though no increase of IKr, IKs, and IK1 was observed. In the analysis of pHi, cells expressing SLC4A3-N353K showed significantly slower pHi alternations than those with WT, indicating the loss-of-function effect of transport kinetics in the regulation of pHi. In the electrophysiological analysis of the slc4a3 zebrafish, which harbors the paralogous variant of SLC4A3-N353K, APD20, 50, and 70 were significantly shortened in the slc4a3 KI zebrafish (HOMO) compared to the control (WT), providing evidence for the shortened QT interval with the SLC4A3 variant in vivo (Figure B). Furthermore, slc4a3 zebrafish developed severe cardiac edema (HET and HOMO: Figure C, arrows) with no gross morphological defects in other organs, which may indicate the specific role of SLC4A3-N353K in cardiac function. Conclusion We identified a novel SLC4A3 variant, p.N353K, in a family with SQTS and revealed its contributions to the pHi control and the shortened APD. Further analysis will be necessary to clarify the mechanism by which SLC4A3-N353K causes QT shortening.

Low conduction velocity is a key factor for localisation of reentrant drivers for atrial fibrillation

Abstract Background Atrial Fibrillation (AF) is a prevalent cardiac arrhythmia globally, associated with heightened risks of stroke, dementia, and heart failure. Catheter ablation, the sole curative therapy for AF, yields suboptimal success rates for persistent AF cases. Understanding the mechanisms and dynamics of AF remains challenging, hindering treatment advancements. Novel ablation strategies aimed at targeting substrates harbouring AF reentrant drivers (RD), such as low-voltage ablation and rotor-based ablation, have yet to demonstrate superiority over the gold standard ablation strategy – pulmonary vein isolation. Purpose The purpose of the study is to integrate in-silico 3D left atrial (LA) simulations and explainable artificial intelligence (AI) to pinpoint key factors for RD localisation in persistent AF, aiming to improve the mechanistic understanding of AF and help develop a more effective ablation strategy. Methods 3D LA models were derived from magnetic resonance images of 41 patients and combined with atrial cell electrophysiology models. Persistent AF was initiated in each patient-specific LA model with fast pacing, and five functional features (action potential duration (APD), conduction velocity (CV), wavelength, APD spatial gradient, CV spatial gradient, and wavelength spatial gradient) and three structural features (fibrotic density, fibrotic entropy, and image intensity ratio) were evaluated. A random forest AI model was trained to classify phase singularities corresponding to the RDs (PS-RD) in five distinct tissue classes (total LA tissue, healthy tissue, fibrotic border zone, dense fibrotic tissue, and PS-RD tissue) based on the functional and structural features. Shapley additive explanations (SHAP) analysis was then used to find the most influential feature in the AI model’s decision process. Results Simulations of the patient-specific LA models revealed that RDs were typically localised in regions with the lowest atrial CV, which often coincided with patches of dense fibrotic tissue. In a five-fold cross-validation, the random forest AI model achieved high accuracy in classifying PS-RD regions: a mean ROC AUC of 0.99 ± 0.010, a mean F1 score of 0.86 ± 0.034, a mean recall of 0.95 ± 0.058, and a mean precision score of 0.79 ± 0.085. The further SHAP analysis revealed that low CV was the key determinant in classifying the PS-RD regions. Conclusion Our study combined AF computational modelling and explainable AI to identify the key factors in RD localisation. Both the computational and AI models concluded that low CV was the most important determinant in localising RDs. These findings provide mechanistic evidence for the efficacy of ablation strategies that target low CV atrial areas for persistent AF patients.

Computational modeling of cardiac electrophysiology applied to a swine model of Long QT Syndrome

Abstract Background Computational models are pivotal for understanding electrophysiological aspects of cardiac diseases. Despite pigs' increased use in research, limited in vitro porcine data impedes accurate mathematical modeling. In this context, we recently developed a novel knock-in porcine model of long QT syndrome type 8 (LQT8) [1]. Purpose To develop a novel computational model for electrophysiology and Ca2+ handing in wild-type and LQT8 swine ventricular cardiomyocytes. Methods We designed a mathematical model of wild-type swine action potential (AP) based on a wide range of cellular electrophysiological data. Using the ORd model [2] as a foundation, we re-parametrized main ionic currents using the Nelder-Mead algorithm. We then modified the model to simulate LQT8, evaluating its utility in exploring arrhythmogenic mechanisms and pharmacological approaches. For the L-type Ca Channel (LTCC), we replaced the original HH formulation with a Markov model based on [3], which was subsequently modified to account for a population of mutant p.G406R channels. Selected biomarkers for validation were AP duration at 20, 50, and 90% of repolarization, maximum upstroke velocity, AP amplitude and resting membrane potential. To assess the robustness of the model, we generated a random population of 500 models accounting for biological variability by sampling key parameters in the range [-20 to +20%] of their original values, using the Latin Hypercube Sampling. Simulations were executed using MATLAB R2023a. Numerical integration was performed utilizing the MATLAB function ode15s. Results The mathematical model accurately reproduced a wide range of experimental data, including steady-state curves of the main ionic currents, kinetic properties of LTCC, APD rate dependency, maximum upstroke velocity and Ca2+transient morphology. The model successfully simulated AP prolongation in LQT8. Also, it replicated key features of the cellular phenotype such as steeper APD rate adaptation, increased duration and amplitude of the Ca2+ transients, increased CaMKII-mediated late Na+ current, pathologically decreased upstroke velocity with increasing pacing frequency, due to a CaMKII-mediated mechanism. As a proof-of-concept, the model was utilized to investigate arrhythmogenic mechanisms and pharmacological approaches for LQT8. According to model’s prediction, early afterdepolarizations (EADs) in LQT8 can stem from three distinct mechanisms: improper kinetics of late Na+ current, improper kinetics of LTCC, and enhanced late-systolic SR Ca release. Notably, the model also predicts that full CaMKII inhibition effectively eliminates EADs for all three mechanisms, showcasing the potential efficacy of this pharmacological intervention. Conclusions A novel mathematical swine ventricular myocyte model explores arrhythmogenic mechanisms and therapeutic interventions, aiding LQT8 research and clinical translation in cardiac disorders.

Interleukin-6 levels in atrium and onset of spontaneous atrial fibrillation: is there a causal relationship

Abstract Background Cumulative evidence suggests that atrial interleukin-6 (IL-6) may play a significant role in the development of spontaneous postoperative atrial fibrillation (sPOAF) in coronary artery bypass grafting (CABG) patients. Our previous studies found that the IL-6 produced by the atrium is positively related to the development of sPOAF. However, the causal relationship between IL-6 and sPOAF is not established. Purpose This study aims to explore the potential causality between atrial IL-6 and spontaneous AF(sAF), alongside assessing the impact of IL-6 on the electrophysiological properties of atrial myocytes (AM). Methods To determine the causal link between IL-6 and sAF, mice were randomly divided into eight groups: seven receiving injections of varying doses of recombinant IL-6 protein groups (15ng/kg to180ng/kg) into the left atrial wall, and one control group (vehicle). Mice were monitored for sAF for 7 days using implanted telemetry device. At 48 hrs post-injection, the left atrial appendages of another set of mice were collected for analyzing levels of IL-6, Myeloperoxidase (MPO), Transforming Growth Factor-β1 (TGF-β1), and Tumor Necrosis Factor-α (TNF-α). To assess the effect of IL-6 on the electrophysiological characteristics of AM, AMs were isolated from the hearts of 12-week-old mice and divided into experimental and control groups for electrophysiological testing. The experimental group was perfused with extracellular solutions containing 100 ng/mL of IL-6 while the control group was perfused with standard extracellular solutions. The measurements focused on action potential (AP), transient outward potassium current (Ito), and inward rectifier potassium current (Ik1) after a 30-minute perfusion. Results As depicted in Fig. 1A, a dose-response relationship between IL-6 levels and sAF occurrence was observed, with incidence increasing from 0% at 15 ng/kg to 75% at 180 ng/kg (χ²= 16.691, P = 0.005). The injection of IL-6 also induced the production of MPO, TGF-β1, and TNF-α with a dose-dependent effects (Fig. 2). In comparison to control, the AP duration at 90% (APD90) and APD50 of repolarization of atrial myocytes were significantly shortened in IL-6 group (Fig. 1B). For voltages from -10mV to +70mV, atrial myocytes in the IL-6 group showed significantly lower Ito current density than those in the control (Fig. 1C). No significant alterations were observed in the impact of IL-6 on Ik1 current density in atrial myocytes (Fig.1D). Conclusion To the best of our knowledge, this is the first study that experimentally tested the causal relation between IL-6 and sAF. The injection of IL-6 could cause the onset of sAF and produce MPO, TGF-β1, and TNF-α with a significant dose-dependent effect. The effect of IL-6 on inducing sAF might be through its effect on reducing APD and Ito current density in mouse atrial myocytes. This suggests that inhibition of atrial IL-6 might be a potential novel sPOAF prevention strategy.

Eleclazine Suppresses Ventricular Fibrillation in Failing Rabbit Hearts with Ischemia- Reperfusion Injury Undergoing Therapeutic Hypothermia.

Eleclazine is a highly selective late sodium current inhibitor, possibly effective in reducing ventricular fibrillation (VF) in heart failure (HF) with ischemia-reperfusion (IR) injury. The electrophysiological effects of eleclazine at therapeutic hypothermia (TH) are unknown. We investigated the effects of eleclazine in suppressing VF in failing rabbit hearts with IR injury undergoing TH. HF was induced by right ventricular pacing. An IR model was created using coronary artery ligation for 60 min, followed by reperfusion for 30 min. Hearts were excised and Langendorff-perfused for optical mapping and electrophysiological studies. Electrophysiological studies were repeated after TH (33 oC) for 30 min or eleclazine (1 μM) infusion for 20 min. In failing IR-injured hearts, eleclazine reduced action potential duration (APD) dispersion and accelerated intracellular Ca2+ uptake to suppress arrhythmogenic alternans, but also exacerbated rate-dependent conduction slowing, resulting in neutral effects on VF inducibility at normothermia. TH increased VF severity. Eleclazine after TH ameliorated TH-induced APD dispersion and further depressed conduction to reduce VF inducibility and severity. TH after eleclazine also slowed conduction to a greater extent to reduce VF inducibility and severity by extrastimulus pacing. In control IR-injured hearts, eleclazine increased VF severity by dynamic pacing at normothermia, which was counteracted by TH. Eleclazine does not prevent VF at normothermia, but reduces VF inducibility and severity by extrastimulus pacing at TH in isolated failing hearts with regional IR injury.

Using a Failing Human Ventricular Cardiomyocyte Model to Re-Evaluate Ca2+ Cycling, Voltage Dependence, and Spark Characteristics

Previous studies have observed alterations in excitation–contraction (EC) coupling during end-stage heart failure that include action potential and calcium (Ca2+) transient prolongation and a reduction of the Ca2+ transient amplitude. Underlying these phenomena are the downregulation of potassium (K+) currents, downregulation of the sarcoplasmic reticulum Ca2+ ATPase (SERCA), increase Ca2+ sensitivity of the ryanodine receptor, and the upregulation of the sodium–calcium (Na=-Ca2+) exchanger. However, in human heart failure (HF), debate continues about the relative contributions of the changes in calcium handling vs. the changes in the membrane currents. To understand the consequences of the above changes, they are incorporated into a computational human ventricular myocyte HF model that can explore the contributions of the spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). The reduction of transient outward K+ current (Ito) is the main membrane current contributor to the decrease in RyR2 open probability and L-type calcium channel (LCC) density which emphasizes its importance to phase 1 of the action potential (AP) shape and duration (APD). During current-clamp conditions, RyR2 hyperphosphorylation exhibits the least amount of Ca2+ release from the SR into the cytosol and SR Ca2+ fractional release during a dynamic slow–rapid–slow (0.5–2.5–0.5 Hz) pacing, but it displays the most abundant and more lasting Ca2+ sparks two-fold longer than a normal cell. On the other hand, under voltage-clamp conditions, HF by decreased SERCA and upregulated INCX show the least SR Ca2+ uptake and EC coupling gain, as compared to HF by hyperphosphorylated RyR2s. Overall, this study demonstrates that the (a) combined effect of SERCA and NCX, and the (b) RyR2 dysfunction, along with the downregulation of the cardiomyocyte’s potassium currents, could substantially contribute to Ca2+ mishandling at the spark level that leads to heart failure.

Advancing cardiac 3D organoid-on-chip: integrating iPSC-derived MELAS syndrome models to study electromechanical synchronicity and cardiac memory using contactless technology

Abstract Background Mitochondrial DNA mutations affect 1:5000 newborns, posing life-threatening risks, especially to high-energy organs such as the brain, heart, and muscles. Mortality rates can reach 71% when cardiomyopathy is present. MELAS syndrome (Mitochondrial Encephalopathy with Lactic Acidosis and Stroke-like Episodes), primarily affecting protein synthesis (MTTL1 gene, tRNALeu(UUR)), disrupts the electron transport chain complex I and is associated with cardiac conduction defects and hypertrophy. Manifesting symptoms typically appear between ages 3 and adulthood, presenting a complex clinical scenario. Human induced pluripotent stem cells (hiPSCs) offer a unique platform for studying cardiac manifestations of this disease when differentiated into cardiomyocytes. Purpose We adopted a highly adaptable in-vitro model, organized in 3D cardioids, to explore potential impairments in electromechanical coupling of cells. Methods We utilized hiPSCs carrying the MELAS mutation and their respective isogenic controls differentiated into cardiomyocytes, cultured in 3D cardioids. Electromechanical coupling was assessed using laser-poration technology combined with video kinematics evaluation under various stimulation patterns. Results Morphological analysis revealed a statistically significant reduction in the diameter of MELAS cardioids compared to the isogenic 3D structures (368±17.15 μm vs. 527±36.76 μm). In a spontaneous regimen, MELAS samples exhibited higher beating frequency (0.88±0.28 Hz vs. 0.17±0.05 Hz) and contractile speed (99.35±39.11 μm/s vs. 33.46±5.57 μm/s). Additionally, the response to different electric field stimulation protocols varied significantly, with the isogenic cell line showing better adaptation to sinusoidal stimuli than the pathological one, characterized by rectangular monophasic stimuli. Laser-poration technology, combined with MEA chips and video kinematic evaluation, revealed modulation of electromechanical coupling, affecting action potential upstroke and duration, reducing contraction velocity, and altering electromechanical delay. Furthermore, MELAS hiPSCs and primary cardiomyocytes exhibited reduced protein levels of various OXPHOS subunits (SDHB, ATPB, and COX IV) compared to isogenic controls. The mutated cell line also showed increased ROS production in non-stimulated samples compared to stimulated ones (1.3-fold vs. 1.2-fold), with this difference magnifying after 24 hours from stimulation (1.8-fold vs. 1.3-fold). Conclusions This study characterizes MELAS hiPSCs differentiated into cardiac organoids, offering insight into the cardiac aspect of this complex syndrome. Our findings deepen understanding of the syndrome's cardiac impact and response to extracellular stimuli, potentially benefiting affected tissues. This research defines critical physiological and kinematic parameters for MELAS, paving the way for future investigations.

Indoxyl sulfate deteriorates cardiac electrophysiological function and increases the likelihood of arrhythmias in human ventricular myocardial cells

Abstract Background Approximately 10% of the world population is suffering from chronic kidney disease (CKD). In these patients, an increased mortality and morbidity, predominantly derived from cardiovascular diseases, can be observed. Purpose The impact of CKD on myocardial cell mechanisms is only partially understood. This study aims to investigate the impact of the uremic toxin indoxyl sulfate (IS) on human ventricular myocardial cells. Methods Human left ventricular myocardium was obtained from patients with severe aortic stenosis who underwent surgical valve replacement. In addition, human ventricular induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) obtained from donors without any known cardiovascular disease were used. Before measurements were conducted, isolated single primary human cardiomyocytes were incubated for 15 minutes with an IS concentration of 120 mg/l (highest measured IS-level in dialysis patients). Further, iPSC-CM were incubated for one week with IS 45 mg/l and IS 120 mg/l, with IS 45 mg/l representing the average dialysis patients IS-level. Epifluorescence and confocal microscopy were performed to investigate Ca2+hemostasis and patch clamp measurements assessed cardiac action potentials and the occurrence of arrythmias. Results Experiments were conducted using human ventricular myocardium from 10 patients with a mean left ventricular ejection fraction of 51.5±12% and a mean glomerular filtration rate of 66.3±22.7 ml/min. Compared to the control group, higher systolic Ca2+ transient amplitudes were observed after acute incubation with IS 120 mg/l in single primary human cardiomyocytes. Measurements after 7 days of IS incubation (IS 45 mg/l and 120 mg/l) in iPSC-CM confirmed these previous results by showing higher Ca2+ transient amplitudes (Figure 1). Additionally, cells showed a diminished diastolic Ca2+ level compared to controls. An increased Ca2+ spark frequency in IS-treated iPSC-CM versus the control group was unveiled by confocal microscopy measurements. While an increased number of delayed afterdepolarizations could be observed, IS seemed to have no effect on cardiac action potential duration in iPSC-CM. Conclusion For the first time, we could reveal the impact of IS on cellular electrophysiology of primary human myocardium cells and iPSC-CM and identify IS as a proarrhythmogenic trigger. Therefore, IS may be of relevance in explaining the high cardiovascular mortality in end-stage CKD patients. To improve the outcome of these patients, new therapeutic approaches will be necessary since IS, a protein-bound uremic toxin, is only insufficiently removed by dialysis.

Combined class I and class III antiarrhythmic effects of sotagliflozin - insights from heterologous expressions systems and atrial cardiomyocytes

Abstract Background The demand for more effective treatment options for atrial fibrillation (AF), the most common sustained arrhythmia, is driven by its direct impact on morbidity and mortality. Current therapies, such as antiarrhythmic drugs and catheter ablation, are limited by suboptimal efficacy and adverse effects or periinterventional complications. Therefore, safe and effective treatment of AF remains a major unmet clinical need. Sodium-glucose cotransporter (SGLT) inhibitors have shown promise in reducing the incidence of AF in large cardiovascular endpoint trials. However, the exact mechanisms of action remain unclear. While only two SGLT2 inhibitors are approved and clinically used in Europe, sotagliflozin, which inhibits both SGLT1 and SGLT2, might also have potential for the treatment of AF. Purpose This study aims to elucidate the effects of sotagliflozin on cardiac action potential formation. This understanding could optimise its clinical use, identify new targets for the treatment of atrial fibrillation and ultimately improve the quality of life of patients suffering from AF. Methods The effects of sotagliflozin on cardiac ionic currents were assessed using two-electrode voltage clamp measurements of several sodium and potassium channels heterologously expressed in Xenopus laevis oocytes. Subsequently, ascending concentrations of sotagliflozin were administered to isolated human atrial cardiomyocytes during patch-clamp measurements to investigate its effect on atrial action potential morphology. Results At a concentration of 100 µM, sotagliflozin showed significant inhibitory effects on three out of ten ion channels tested: NaV1.5 sodium channel current was reduced by 22,3 % %, hERG and KV4.3 potassium currents were reduced by 30,4 % and 22,5 % respectively. Patch-clamp experiments, performed on isolated human atrial cardiomyocytes showed a dose-dependent reduction in upstroke velocity and action potential amplitude by 20,8 % and 13,8 % at a concentration of 20 µM, reflecting class-I-antiarrhythmic effects. Furthermore, a prolongation of AP duration measured at 50 % (APD50) and 90 % (APD90) of repolarization by 57,7 % and 27,9 % at a sotagliflozin concentration of 20 µM was noted, implying additional class-III-antiarrhythmic action. Conclusions Sotagliflozin exerts direct inhibitory effects on heterologously expressed NaV1.5, hERG and KV4.3 channels, resulting in a reduction in upstroke velocity and action potential amplitude, as well as a prolongation of APD50 and APD90 levels in isolated human atrial cardiomyocytes. These results extend the pharmacological profile of sotagliflozin and suggest a potential role for the clinical use of combined SGLT1/SGLT2 inhibitors in the treatment of AF.

The endocannabinoid ARA-S facilitates the activation of cardiac Kv7.1/KCNE1 channels from different species

ABSTRACT The endogenous endocannabinoid-like compound N-arachidonoyl-L-serine (ARA-S) facilitates activation of the human Kv7.1/KCNE1 channel and shortens a prolonged action potential duration and QT interval in guinea pig hearts. Hence, ARA-S is interesting to study further in cardiac models to explore the functional impact of such Kv7.1/KCNE1-mediated effects. To guide which animal models would be suitable for assessing ARA-S effects, and to aid interpretation of findings in different experimental models, it is useful to know whether Kv7.1/KCNE1 channels from relevant species respond similarly to ARA-S. To this end, we used the two-electrode voltage clamp technique to compare the effects of ARA-S on Kv7.1/KCNE1 channels from guinea pig, rabbit, and human Kv7.1/KCNE1, when expressed in Xenopus laevis oocytes. We found that the activation of Kv7.1/KCNE1 channels from all tested species was facilitated by ARA-S, seen as a concentration-dependent shift in the voltage-dependence of channel opening and increase in current amplitude and conductance over a broad voltage range. The rabbit channel displayed quantitatively similar effects as the human channel, whereas the guinea pig channel responded with more prominent increase in current amplitude and maximal conductance. This study suggests that rabbit and guinea pig models are both suitable for studying ARA-S effects mediated via Kv7.1/KCNE1.

Identification of the optimal reference genes for atrial fibrillation model established by iPSC-derived atrial myocytes

BackgroundAtrial fibrillation (AF) stands as a prevalent and detrimental arrhythmic disorder, characterized by intricate pathophysiological mechanisms. The availability of reliable and reproducible AF models is pivotal in unraveling the underlying mechanisms of this complex condition. Unfortunately, the researchers are still confronted with the absence of consistent in vitro AF models, hindering progress in this crucial area of research.MethodsHuman induced pluripotent stem cells derived atrial myocytes (hiPSC-AMs) were generated based on the GiWi methods and were verified by whole-cell patch clamp, immunofluorescent staining, and flow cytometry. Then hiPSC-AMs were employed to establish the AF model by HS. Whole-cell patch clamp technique and calcium imaging were used to identify the AF model. The stability of 29 reference genes was evaluated using delta-Ct, GeNorm, NormFinder, and BestKeeper algorithms;ResultsHiPSC-AMs displayed atrial myocyte action potentials and expressed the atrial-specific protein MLC-2 A and NR2F2, about 70% of the cardiomyocytes were MLC-2 A positive. After HS, hiPSC-AMs showed a significant increase in beating frequency, a shortened action potential duration, and increased calcium transient frequency. Of the 29 candidate genes, the top five most stably ranked genes were ABL1, RPL37A, POP4, RPL30, and EIF2B1. After normalization using ABL1, KCNJ2 was significantly upregulated in the AF model; Conclusions: In the hiPSC-AMs AF model established by HS, ABL1 provides greater normalization efficiency than commonly used GAPDH.

Clinical usefulness of digital twin guided virtual amiodarone test in patients with atrial fibrillation ablation

It would be clinically valuable if the efficacy of antiarrhythmic drugs could be simulated in advance. We developed a digital twin to predict amiodarone efficacy in high-risk atrial fibrillation (AF) patients post-ablation. Virtual left atrium models were created from computed tomography and electroanatomical maps to simulate AF and evaluate its response to varying amiodarone concentrations. As the amiodarone concentration increased in the virtual setting, action potential duration lengthened, peak upstroke velocities decreased, and virtual AF termination became more frequent. Patients were classified into effective (those with virtually terminated AF at therapeutic doses) and ineffective groups. The one-year clinical outcomes after AF ablation showed significantly better results in the effective group compared to the ineffective group, with AF recurrence rates of 20.8% vs. 45.1% (log-rank p = 0.031, adjusted hazard ratio, 0.37 [0.14-0.98]; p = 0.046). This study highlights the potential of a digital twin-guided approach in predicting amiodarone’s effectiveness and improving personalized AF management. Clinical Trial Registration Name: The Evaluation for Prognostic Factors After Catheter Ablation of Atrial Fibrillation: Cohort Study, Registration number: NCT02138695. The date of registration: 2014-05. URL: https://www.clinicaltrials.gov; Unique identifier: NCT02138695.

Development of new Kir2.1 channel openers from propafenone analogues.

Reduced inward rectifier potassium channel (Kir2.1) functioning is associated with heart failure and may cause Andersen-Tawil Syndrome, among others characterized by ventricular arrhythmias. Most heart failure or Andersen-Tawil Syndrome patients are treated with β-adrenoceptor antagonists (β-blockers) or sodium channel blockers; however, these do not specifically address the inward rectifier current (IK1) nor aim to improve resting membrane potential stability. Consequently, additional pharmacotherapy for heart failure and Andersen-Tawil Syndrome treatment would be highly desirable. Acute propafenone treatment at low concentrations enhances IK1 current, but it also exerts many off-target effects. Therefore, discovering and exploring new IK1-channel openers is necessary. Effects of propafenone and 10 additional propafenone analogues were analysed. Currents were measured by single-cell patch-clamp electrophysiology. Kir2.1 protein expression levels were determined by western blot analysis and action potential characteristics were further validated in human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMCs). Molecular docking was performed to obtain detailed information on drug-channel interactions. Analogues GPV0019, GPV0057 and GPV0576 strongly increased the outward component of IK1 while not affecting the Kir2.1 channel expression levels. GPV0057 did not block IKr at concentrations below 0.5μmol L-1 nor NaV1.5 current below 1μmol L-1. Moreover, hiPSC-CMC action potential duration was also not affected by GPV0057 at 0.5 and 1μmol L-1. Structure analysis indicates a mechanism by which GPV0057 might enhance Kir2.1 channel activation. GPV0057 has a strong efficiency towards increasing IK1, which makes it a good candidate to address IK1 deficiency-associated diseases.

Caution for Multidrug Therapy: Significant Baroreflex Afferent Neuroexcitation Coordinated by Multi-Channels/Pumps Under the Threshold Concentration of Yoda1 and Dobutamine Combination

Multi-drug therapies are common in cardiovascular disease intervention; however, io channel/pump coordination has not been tested electrophysiologically. Apparently, inward currents were not elicited by Yoda1/10 nM or Dobutamine/100 nM alone in Ah-type baroreceptor neurons, but were by their combination. To verify this, electroneurography and the whole-cell patch-clamp technique were performed. The results showed that Ah- and C-volley were dramatically increased by the combination at 0.5 V and 5 V, in contrast to A-volley, as consistent with repetitive discharge elicited by step and ramp with markedly reduced current injection/stimulus intensity. Notably, a frequency-dependent action potential (AP) duration was increased with Iberiotoxin-sensitive K+ component. Furthermore, an increased peak in AP measured in phase plots suggested enhanced Na+ influx, cytoplasmic Ca2+ accumulation through reverse mode of Na+/Ca2+ exchanger, and, consequently, functional KCa1.1 up-regulation. Strikingly, the Yoda1- or Dbtm-mediated small/transient Na+/K+-pump currents were robustly increased by their combination, implying a quick ion equilibration that may also be synchronized by hyperpolarization-induced voltage-sag, enabling faster repetitive firing. These novel findings demonstrate multi-channel/pump collaboration together to integrate neurotransmission at the cellular level for baroreflex, providing an afferent explanation in sexual dimorphic blood pressure regulation, and raising the caution regarding the individual drug concentration in multi-drug therapies to optimize efficacy and minimize toxicity.

The mechanisms of effects of oil-derived polyaromatic hydrocarbons on cardiac electrical activity in navaga cod (Eleginus nawaga)

The intensive development of oil and gas industries in the Arctic threatens Arctic aquatic ecosystems. The toxic and primarily lethal cardiotoxic effects of oil in living organisms are believed to be associated with polyaromatic hydrocarbons (PAHs), and previous works revealed the electrophysiological mechanisms of action of individual oil-derived PAHs. However, the physiological effects of a complex PAHs mixture in oil water-soluble fraction (WSF) have not been previously studied. This study is focused on the effects of oil WSF on electrical activity and major ionic currents in the working myocardium of navaga (Eleginus nawaga), which is one of the most important commercial fish species in the Arctic. We found that 1% and 10% solutions of oil WSF cause a marked increase in the duration of action potentials (APs) in navaga cardiomyocytes. This effect appears to be due to the suppression of rapid delayed rectifying current IKr (IC50 about 3% in ventricular and atrial myocardium). At higher concentrations, oil WSF also suppressed calcium current ICaL (IC50 = 10.6%), which led to a decrease in the contractile activity in isolated myocardial preparations. Unlike individual tricyclic PAHs, oil WSF did not affect fast sodium current INa and AP upstroke velocity. An assessment of the content of tricyclic PAHs in 10% solution of oil WSF showed that their total concentration is relatively low and does not exceed 100 nM. Thus, oil WSF also has a powerful cardiotoxic effect in fish myocardium, but its effects differ from the previously studied effects of tricyclic PAHs and suggest the presence of yet unexplored oil compounds that have a more powerful toxic potential against ERG channels.

Reverse-engineered models reveal differential membrane properties of autonomic and cutaneous unmyelinated fibers.

Unmyelinated C-fibers constitute the vast majority of axons in peripheral nerves and play key roles in homeostasis and signaling pain. However, little is known about their ion channel expression, which controls their firing properties. Also, because of their small diameters (~ 1 μm), it has not been possible to characterize their membrane properties using voltage clamp. We developed a novel library of isoform-specific ion channel models to serve as the basis functions of our C-fiber models. We then developed a particle swarm optimization (PSO) framework that used the isoform-specific ion channel models to reverse engineer C-fiber membrane properties from measured autonomic and cutaneous C-fiber conduction responses. Our C-fiber models reproduced experimental conduction velocity, chronaxie, action potential duration, intracellular threshold, and paired pulse recovery cycle. The models also matched experimental activity-dependent slowing, a property not included in model optimization. We found that simple conduction responses, characterizing the action potential, were controlled by similar membrane properties in both the autonomic and cutaneous C-fiber models, but complicated conduction response, characterizing the afterpotenials, were controlled by differential membrane properties. The unmyelinated C-fiber models constitute important tools to study autonomic signaling, assess the mechanisms of pain, and design bioelectronic devices. Additionally, the novel reverse engineering approach can be applied to generate models of other neurons where voltage clamp data are not available.