Repolarizing Potassium Currents Research Articles

Characterization of atrial cardiomyopathy subtypes - A cellular electrophysiological comparison and evaluation of appropriate treatment options

Abstract Background Atrial fibrillation occurs in the context of atrial remodeling processes which are the result of an underlying atrial cardiomyopathy (AC). Insufficient understanding of the pathophysiological mechanisms behind AC contributes significantly to the fact that current clinical strategies for the treatment of atrial fibrillation are often ineffective. A systematic investigation of these cellular remodeling processes in the context of the various cardiac diseases underlying AC is still missing. Purpose The aim of this study was to characterize different subtypes of AC associated with common cardiac disease entities by systematic cellular electrophysiological characterization of the atria in combination with molecular and structural investigations. Methods For a comprehensive analysis of the molecular changes associated with AC, we characterized several mouse models of common heart diseases, including models for dilated cardiomyopathy (DCM), HFpEF, mitral regurgitation and immune checkpoint inhibitor (ICI-)mediated myocarditis. Patch clamp experiments were performed on isolated atrial cardiomyocytes to study atrial action potentials (aAP) and different ion currents contributing to the aAP. The cellular electrophysiological parameters of the different models were correlated to clinical parameters (ecg, echocardiography of the atria) of the mouse models and with data from RNA sequencing of the mice’ atria. Results Patch clamp measurements performed on isolated left and right atrial cardiomyocytes revealed distinct differences among the examined models. In all pathophysiological models, the aAP was significantly shorter than in the corresponding wild type mice. The different ion current components we measured various changes in potassium, sodium and calcium currents. The repolarizing TASK-1 potassium current was significantly increased in all models. The DCM model caused by an R636Q mutation in the RBM20 gene showed the most severe changes in cellular electrophysiology. Considering the detected atrial changes, we investigated the effects of SGLTi on atrial cardiomyocytes of the different models. When elevated concentrations of dapa-, empa- and sotagliflozin were administrated on atrial cardiomyocytes, a class-I-antiarrhythmic effect with reduced aAP inducibility and changes in the aAP duration could be observed. Conclusion Characterization of murine models of cardiac disease with respect to classification of AC subtypes revealed various electrical remodeling processes at the level of ion currents involved in aAP formation. A potent inductor of atrial proarrhythmogenity in the AC subtypes was an aAP shortening caused by an increase in repolarizing potassium currents, mediated especially by TASK-1. The most severe electrical remodeling in atria cardiomyocytes was observed in the RBM20-mutant model (DCM). SGLTi exhibit anti-arrhythmic effects in different murine disease models and may provide future treatment options for patients with AC.

Multichannel modulation of depolarizing and repolarizing ion currents increases the positive rate‐dependent action potential prolongation

Prolongation of the action potential duration (APD) could prevent reentrant arrhythmias if prolongation occurs at the fast excitation rates of tachycardia with minimal prolongation at slow excitation rates (i.e., if prolongation is positive rate-dependent). APD prolongation by current anti-arrhythmic agents is either reverse (larger APD prolongation at slow rates than at fast rates) or neutral (similar APD prolongation at slow and fast rates), which may not result in an effective anti-arrhythmic action. In this report we show that, in computer models of the human ventricular action potential, the combined modulation of both depolarizing and repolarizing ion currents results in a stronger positive rate-dependent APD prolongation than modulation of repolarizing potassium currents. A robust positive rate-dependent APD prolongation correlates with an acceleration of phase 2 repolarization and a deceleration of phase 3 repolarization, which leads to a triangulation of the action potential. A positive rate-dependent APD prolongation decreases the repolarization reserve with respect to control, which can be managed by interventions that prolong APD at fast excitation rates and shorten APD at slow excitation rates. For both computer models of the action potential, ICaL and IK1 are the most important ion currents to achieve a positive rate-dependent APD prolongation. In conclusion, multichannel modulation of depolarizing and repolarizing ion currents, with ion channel activators and blockers, results in a robust APD prolongation at fast excitation rates, which should be anti-arrhythmic, while minimizing APD prolongation at slow heart rates, which should reduce pro-arrhythmic risks.

RNA secondary structure modulates the co-translational association of hERG1a and 1b mRNAs.

Cardiac IKr is an important repolarizing potassium current of the human ventricular action potential. It is conducted by heteromeric assemblies of the human ether-à-go-go-related gene (hERG1) 1a and 1b subunits. These subunits are encoded by alternate transcripts of the hERG1/KCNH2 gene and differ only in their amino-terminal regions. hERG1a/1b heteromerization is vital for normal CM function, as the imbalance of the subunit's expression and/or function results in cellular pro-arrhythmic behaviors. hERG1a/1b assembly is mediated by the co-translational association of the encoding mRNAs in HEK293 cells, cardiomyocytes derived from human induced pluripotent stem cells, and human myocardium. New evidence suggests that neither 1a nor 1b proteins are required for the co-translational complex assembly. Thus, we hypothesize that hERG1a and 1b mRNAs’ physico-chemical properties, especially their structure, contribute to their co-translational association. In an in vitro reconstitution assay, hERG1a and 1b mRNA transcribed and labeled with Cy3 and Cy5, respectively, undergo phase separation in isolation. When mixed, the two transcripts modulate each other's morphology as represented by size, shape, and area covered, suggesting they interact. Synonymous mutations predicted to alter hERG1a secondary structure, without affecting channel function, reduce hERG1a and 1b interaction in vitro. Confocal live-cell imaging in HeLa cells transfected with labeled hERG1a and 1b mRNA show condensate morphologies consistent with those seen in vitro. Co-transfection of the in-vitro labeled mRNAs results in spatial overlap of the respective signals as well as translation of hERG1b protein apparently within the same macromolecular complexes, indicating they are translational. In addition, the findings indicate that co-transcription of the mRNAs is not required for their co-translational association. These results suggest that 1a and 1b mRNA physico-chemical properties modulate their association and subsequent co-translation in live cells.

Arrhythmogenic Effects of Cardiac Memory.

Cardiac memory is the term used to describe an interesting electrocardiographic phenomenon. Whenever a QRS complex is wide and abnormal, such as during ventricular pacing, the T waves will also be abnormal and will point to the opposite direction of the wide QRS. If the QRS then normalizes, such as after cessation of ventricular pacing, the T waves will normalize as well, but at a later stage. The period of cardiac memory is the phase between the sudden normalization of the QRS and the eventual and gradual return of the T waves to their baseline morphology. Cardiac memory is assumed to be an innocent electrocardiographic curiosity. However, during cardiac memory, reduction of repolarizing potassium currents increases left ventricular repolarization gradients. Therefore, when cardiac memory occurs in patients who already have a prolonged QT interval (for whatever reason), it can lead to a frank long QT syndrome with QT-related ventricular arrhythmias (torsades de pointes). These arrhythmogenic effects of cardiac memory are not generally appreciated and are reviewed here for the first time.

Common Ancestry-Specific Ion Channel Variants Predispose to Drug-Induced Arrhythmias.

Multiple reports associate the cardiac sodium channel gene (SCN5A) variants S1103Y and R1193Q with type 3 congenital long QT syndrome and drug-induced long QT syndrome. These variants are too common in ancestral populations to be highly arrhythmogenic at baseline, however: S1103Y allele frequency is 8.1% in African Americans and R1193Q 6.1% in East Asians. R1193Q is known to increase late sodium current (INa-L) in cardiomyocytes derived from induced pluripotent stem cells but the role of these variants in modulating repolarization remains poorly understood. We determined the effect of S1103Y on QT intervals among African-American participants in a large electronic health record. Using cardiomyocytes derived from induced pluripotent stem cells carrying naturally occurring or genome-edited variants, we studied action potential durations (APDs) at baseline and after challenge with the repolarizing potassium current (IKr) blocker dofetilide and INa-L and IKr at baseline. In 1479 African-American participants with no confounding medications or diagnoses of heart disease, QT intervals in S1103Y carriers was no different from that in noncarriers. Baseline APD was no different in cells expressing the Y allele (SY, YY cells) compared with isogenic cells with the reference allele (SS cells). However, INa-L was increased in SY and YY cells and the INa-L blocker GS967 shortened APD in SY/YY but not SS cells (P<0.001). IKr was increased almost 2-fold in SY/YY cells compared with SS cells (tail current: 0.66±0.1 versus 1.2±0.1 pA/pF; P<0.001). Dofetilide challenge prolonged APD at much lower concentrations in SY (4.1 nmol/L [interquartile range, 1.5-9.3]; n=11) and YY (4.2 nmol/L [1.7-5.0]; n=5) than in SS cells (249 nmol/L [22.3-2905]; n=14; P<0.001 and P<0.01, respectively) and elicited afterdepolarizations in 8/16 SY/YY cells but only in 1/14 SS cells. R1193Q cells similarly displayed no difference in baseline APD but increased IKr and increased dofetilide sensitivity. These common ancestry-specific variants do not affect baseline repolarization, despite generating increased INa-L. We propose that increased IKr serves to maintain normal repolarization but increases the risk of manifest QT prolongation with IKr block in variant carriers. Our findings emphasize the need for inclusion of diverse populations in the study of adverse drug reactions.

Antiviral and anti-inflammatory drugs to combat COVID-19: Effects on cardiac ion channels and risk of ventricular arrhythmias.

Introduction: Drugs with no indication for the treatment of cardiovascular diseases (e.g., drugs employed to treat COVID-19) can increase the risk of arrhythmias. Of interest, a six-fold increase in the number of arrhythmic events was reported in patients with severe COVID-19. In this study, we reviewed (i) the pro-arrhythmic action of drugs given to patients with COVID-19 infection, and (ii) the effects of inflammatory cytokines on cardiac ion channels and possible generation of arrhythmias. Methods: We conducted a literature search on the drugs with purported or demonstrated efficacy against COVID-19 disease, emphasizing the mechanisms by which anti-COVID-19 drugs and inflammatory cytokines interfere with cardiac ion channels. Results: Antibiotics (azithromycin), antimalarials (hydroxychloroquine, chloroquine), antivirals (ritonavir/lopinavir, atazanavir), and some of the tyrosine kinase inhibitors (vandetanib) could induce long QT and increase risk for ventricular arrhythmias. The pro-arrhythmic action results from drug-induced inhibition of Kv11.1 (hERG) channels interfering with the repolarizing potassium IKr currents, leading to long QT and increased risk of triggered arrhythmias. At higher concentrations, these drugs may interfere with IKs, IK1, and/or Ito potassium currents, and even inhibit sodium (INa) and calcium (ICa) currents, inducing additional cardiac toxicity. Ibrutinib, an inhibitor of Bruton’s TK, increased the incidence of atrial fibrillation and ventricular tachycardia associated with a short QT interval. Inflammatory cytokines IL-6 and TNF-α inhibit IKr and Ito repolarizing potassium currents. High levels of inflammatory cytokines could contribute to the arrhythmic events. For remdesivir, favipiravir, dexamethasone, tocilizumab, anakinra, baricitinib, and monoclonal antibodies (bamlanivimab, etesevimab, and casirivimab), no evidence supports significant effects on cardiac ion channels, changes in the QT interval, and increased risk for ventricular arrhythmias. Conclusion: This study supports the concept of hERG channel promiscuity. Different drug classes given to COVID-19 patients might delay repolarization, and increase the risk of ventricular arrhythmias. The presence of comorbid pro-arrhythmic disease states, and elevated levels of pro-arrhythmic cytokines, could increase the risk of ventricular arrhythmias. Discontinuation of nonessential drugs and correction of electrolyte abnormalities could prevent severe ventricular arrhythmias. Altogether, the most effective therapies against COVID-19 (remdesivir, dexamethasone, monoclonal antibodies) lack pro-arrhythmic activity.

Repolarizing potassium currents in working myocardium of Japanese quail: a novel translational model for cardiac electrophysiology

Birds developed endothermy and four-chambered high-performance heart independently from mammals. Though avian embryos are extensively studied and widely used as various models for heart research, little is known about cardiac physiology of adult birds. Meanwhile, cardiac electrophysiology is in search for easily accessible and relevant model objects which resemble human myocardium in the pattern of repolarizing currents (IKr, IKs, IKur and Ito). This study focuses on the configuration of electrical activity and electrophysiological phenotype of working myocardium in adult Japanese quails (Coturnix japonica). The resting membrane potential and action potential (AP) waveform in quail atrial myocardium were similar to that in working myocardium of rodents. Using whole-cell patch clamp and sharp glass microelectrodes, we demonstrated that the repolarization of quail atrial and ventricular myocardium is determined by voltage-dependent potassium currents IKr, IKs and Ito – the latter was previously considered as an exclusive evolutionary feature of mammals. The specific blockers of these currents, dofetilide (3 μmol l−1), HMR 1556 (30 μmol l−1) and 4-aminopyridine (3 mmol l−1), prolonged AP in both ventricular and atrial myocardial preparations. The expression of the corresponding channels responsible for these currents in quail myocardium was investigated with quantitative RT-PCR and western blotting. In conclusion, the described pattern of repolarizing ionic currents and channels in quail myocardium makes this species a novel and suitable experimental model for translational cardiac research and reveals new information related to the evolution of cardiac electrophysiology in vertebrates.

Thyroid and Dexamethasone Treatment Enhances the Expression of Repolarizing Potassium Currents and Improve Electrophysiological Maturation of Human IPSC-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising tool for disease modeling. However, their utility as an adult human cardiomyocyte model is limited by the high amount of funny currents but low or near-complete lack of repolarizing potassium currents. In this study, our goal is to increase repolarizing potassium currents and reduce funny current in hiPSC-CMs without prolonging culture time, resulting in a more matured electrophysiological phenotype. Day 16th hiPSC-CMs were treated with 100 nM triiodothyronine (T3) and 1 μM Dexamethasone (Dex) or vehicle for 14 days. At day 30th, T3+Dex- and vehicle-treated hiPSC-CMs were dissociated and plated on the flexible Matrigel Mattress at the single-cell level. The results showed that T3+Dex-treated hiPSC-CMs had slower spontaneous beating rate than vehicle (T3+Dex 11.96 ± 0.82 vs. vehicle 17.48 ± 0.84 beats per minute, P< 0.05). This was due to a 3-fold decreased funny current (If) in T3+Dex-treated hiPSC-CMs. Furthermore, T3+Dex-treated hiPSC-CMs exhibited more hyperpolarized resting membrane potential (T3+Dex −72.00 ± 1.10 vs. vehicle −65.04 ± 1.20 mV, P< 0.05). This was associated with a 5-fold elevated inward rectifier potassium current (IK1) in T3+Dex-treated hiPSC-CMs. In addition, T3+Dex-treated hiPSC-CMs showed shorter action potential duration at single cells and in 2D monolayers. This was caused by a 1.9- and 6.5-fold increased rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium currents, respectively. This study showed that 14-day T3+Dex treatment elevates IK1, IKr, and IKs and reduces If currents, which increases the application of hiPSC-CMs in disease modeling.

Differential hERG Subunit Transcription Drives Changes in IKr During Cardiac Maturation

hERG conducts the cardiac repolarizing potassium current, IKr, which is one of the first currents to appear in the developing heart. Disrupting IKr is linked with sudden infant death syndrome and intrauterine fetal death. Despite this, the mechanisms regulating hERG during cardiac maturation and its impact on excitability in the immature heart are unknown. At least two subunits, hERG 1a and hERG 1b, comprise native hERG channels. Shifts in the relative abundance of hERG 1a and hERG 1b alter IKr gating kinetics and cardiac repolarization. Decreasing the relative abundance of hERG 1b slows IKr gating kinetics, reduces repolarizing IKr, and prolongs the action potential (AP) in cardiomyocytes derived from human induced pluripotent stem cells (hIPSC-CMs). A previous study demonstrated that hERG 1a mRNA was upregulated and hERG 1b mRNA downregulated in adult ventricular tissue compared to fetal ventricular tissue. We hypothesized that hERG subunits are selectively regulated to meet the physiological needs of the maturing myocardium. To define the mechanisms regulating hERG subunit abundance during cardiac maturation, we measured hERG 1a and hERG 1b mRNA and protein, as well as IKr and AP morphology in immature and matured hIPSC-CMs. We found that matured hIPSC-CMs had significantly slower IKr kinetics, increased peak tail IKr density, and reduced repolarizing charge conducted by IKr during a ventricular AP voltage command. In agreement with the observed shift in IKr kinetics, hERG 1a immunofluorescence significantly increased whereas hERG 1b immunofluorescence significantly decreased in matured hiPSC-CMs. Furthermore, qRT-PCR showed that upregulated Tbx20 levels selectively increased hERG 1a mRNA levels in matured hiPSC-CMs compared to immature hiPSC-CMs. These data demonstrate that cardiac maturation drives transcriptional subunit-specific regulation of hERG and modifies IKr. These data will help clinicians predict the pathogenicity of subunit-specific hERG variants during development.

Toward in vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Cannabidiol inhibits multiple cardiac ion channels and shortens ventricular action potential duration in vitro

Cannabidiol (CBD) is a non-psychoactive component of Cannabis which has recently received regulatory consideration for the treatment of intractable forms of epilepsy such as the Dravet and the Lennox-Gastaut syndromes. The mechanisms of the antiepileptic effects of CBD are unclear, but several pre-clinical studies suggest the involvement of ion channels. Therefore, we have evaluated the effects of CBD on seven major cardiac currents shaping the human ventricular action potential and on Purkinje fibers isolated from rabbit hearts to assess the in vitro cardiac safety profile of CBD. We found that CBD inhibits with comparable micromolar potencies the peak and late components of the NaV1.5 sodium current, the CaV1.2 mediated L-type calcium current, as well as all the repolarizing potassium currents examined except Kir2.1. The most sensitive channels were KV7.1 and the least sensitive were KV11.1 (hERG), which underly the slow (IKs) and rapid (IKr) components, respectively, of the cardiac delayed-rectifier current. In the Purkinje fibers, CBD decreased the action potential (AP) duration more potently at half-maximal than at near complete repolarization, and slightly decreased the AP amplitude and its maximal upstroke velocity. CBD had no significant effects on the membrane resting potential except at the highest concentration tested under fast pacing rate. These data show that CBD impacts cardiac electrophysiology and suggest that caution should be exercised when prescribing CBD to carriers of cardiac channelopathies or in conjunction with other drugs known to affect heart rhythm or contractility.

COMBINATION THERAPY WITH DOFETILIDE AND MEXILETINE FOR ATRIAL FIBRILLATION: INCREASED EFFICACY AND DECREASED QT INTERVAL PROLONGATION

Dofetilide, a class III antiarrhythmic is indicated for conversion of atrial fibrillation (AF) to sinus rhythm and maintenance of sinus rhythm. Dofetilide works by blocking the repolarizing potassium currents and hence prolongs QT interval. QT prolongation can predispose to serious ventricular

Contribution of potassium channels to action potential repolarization of human embryonic stem cell-derived cardiomyocytes.

The electrophysiological properties of human pluripotent stem cell-derived cardiomyocytes (CMs) have not yet been characterized in a syncytial context. This study systematically characterized the contributions of different repolarizing potassium currents in human embryonic stem cell-derived CMs (hESC-CMs) during long-term culture as cell monolayers. The H9 hESC line was differentiated to CMs and plated to form confluent cell monolayers. Optical mapping was used to record the action potentials (APs) and conduction velocity (CV) during electrophysiological and pharmacological experiments. RT-PCR and Western blot were used to detect the presence and expression levels of ion channel subunits. Long-term culture of hESC-CMs led to shortened AP duration (APD), faster repolarization rate, and increased CV. Selective block of IKr , IKs , IK1 , and IKur significantly affected AP repolarization and APD in a concentration- and culture time-dependent manner. Baseline variations in APD led to either positive or negative APD dependence of drug response. Chromanol 293B produced greater relative AP prolongation in mid- and late-stage cultures, while DPO-1 had more effect in early-stage cultures. CV in cell monolayers in early- and late-stage cultures was most susceptible to slowing by E-4031 and BaCl2 respectively. IKr , IKs , IK1 , and IKur all play an essential role in the regulation of APD and CV in hESC-CMs. During time in culture, increased expression of IKr and IK1 helps to accelerate repolarization, shorten APD, and increase CV. We identified a new pro-arrhythmic parameter, positive APD dependence of ion channel block, which can increase APD and repolarization gradients.

A current understanding of drug-induced QT prolongation and its implications for anticancer therapy.

The QT interval, a global index of ventricular repolarization, varies among individuals and is influenced by diverse physiologic and pathophysiologic stimuli such as gender, age, heart rate, electrolyte concentrations, concomitant cardiac disease, and other diseases such as diabetes. Many drugs produce a small but reproducible effect on QT interval but in rare instances this is exaggerated and marked QT prolongation can provoke the polymorphic ventricular tachycardia 'torsades de pointes', which can cause syncope or sudden cardiac death. The generally accepted common mechanism whereby drugs prolong QT is block of a key repolarizing potassium current in heart, IKr, generated by expression of KCNH2, also known as HERG. Thus, evaluation of the potential that a new drug entity may cause torsades de pointes has relied on exposure of normal volunteers or patients to drug at usual and high concentrations, and on assessment of IKr block in vitro. More recent work, focusing on anticancer drugs with QT prolonging liability, is defining new pathways whereby drugs can prolong QT. Notably, the in vitro effects of some tyrosine kinase inhibitors to prolong cardiac action potentials (the cellular correlate of QT) can be rescued by intracellular phosphatidylinositol 3,4,5-trisphosphate, the downstream effector of phosphoinositide 3-kinase. This finding supports a role for inhibition of this enzyme, either directly or by inhibition of upstream kinases, to prolong QT through mechanisms that are being worked out, but include enhanced inward 'late' sodium current during the plateau of the action potential. The definition of non-IKr-dependent pathways to QT prolongation will be important for assessing risk, not only with anticancer therapies but also with other QT prolonging drugs and for generating a refined understanding how variable activity of intracellular signalling systems can modulate QT and associated arrhythmia risk.

Description of the Human Atrial Action Potential Derived From a Single, Congruent Data Source: Novel Computational Models for Integrated Experimental-Numerical Study of Atrial Arrhythmia Mechanisms.

Introduction: The development of improved diagnosis, management, and treatment strategies for human atrial fibrillation (AF) is a significant and important challenge in order to improve quality of life for millions and reduce the substantial social-economic costs of the condition. As a complex condition demonstrating high variability and relation to other cardiac conditions, the study of AF requires approaches from multiple disciplines including single-cell experimental electrophysiology and computational modeling. Models of human atrial cells are less well parameterized than those of the human ventricle or other mammal species, largely due to the inherent challenges in patch clamping human atrial cells. Such challenges include, frequently, unphysiologically depolarized resting potentials and thus injection of a compensatory hyperpolarizing current, as well as detecting certain ion currents which may be disrupted by the cell isolation process. The aim of this study was to develop a laboratory specific model of human atrial electrophysiology which reproduces exactly the conditions of isolated-cell experiments, including testing of multiple experimental interventions.Methods: Formulations for the primary ion currents characterized by isolated-cell experiments in the Workman laboratory were fit directly to voltage-clamp data; the fast sodium-current was parameterized based on experiments relating resting membrane potential to maximal action potential upstroke velocity; compensatory hyperpolarizing current was included as a constant applied current. These formulations were integrated with three independent human atrial cell models to provide a family of novel models. Extrapolated intact-cell models were developed through removal of the hyperpolarizing current and introduction of terminal repolarization potassium currents.Results: The isolated-cell models quantitatively reproduced experimentally measured properties of excitation in both control and pharmacological and dynamic-clamp interventions. Comparison of isolated and intact-cell models highlighted the importance of reproducing this cellular environment when comparing experimental and simulation data.Conclusion: We have developed a laboratory specific model of the human atrial cell which directly reproduces the experimental isolated-cell conditions and captures human atrial excitation properties. The model may be particularly useful for directly relating model to experiment, and offers a complementary tool to the available set of human atrial cell models with specific advantages resulting from the congruent input data source.

Drug-Mediated Shortening of Action Potentials in LQTS2 Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are now a well-established modality for modeling genetic disorders of the heart. This is especially so for long QT syndrome (LQTS), which is caused by perturbation of ion channel function, and can lead to fainting, malignant arrhythmias and sudden cardiac death. LQTS2 is caused by mutations in KCNH2, a gene whose protein product contributes to IKr (also known as HERG), which is the predominant repolarizing potassium current in CMs. β-blockers are the mainstay treatment for patients with LQTS, functioning by reducing heart rate and arrhythmogenesis. However, they are not effective in around a quarter of LQTS2 patients, in part, because they do not correct the defining feature of the condition, which is excessively prolonged QT interval. Since new therapeutics are needed, in this report, we biopsied skin fibroblasts from a patient who was both genetically and clinically diagnosed with LQTS2. By producing LQTS-hiPSC-CMs, we assessed the impact of different drugs on action potential duration (APD), which is used as an in vitro surrogate for QT interval. Not surprisingly, the patient's own β-blocker medication, propranolol, had a marginal effect on APD in the LQTS-hiPSC-CMs. However, APD could be significantly reduced by up to 19% with compounds that enhanced the IKr current by direct channel binding or by indirect mediation through the PPARδ/protein 14-3-3 epsilon/HERG pathway. Drug-induced enhancement of an alternative potassium current, IKATP, also reduced APD by up to 21%. This study demonstrates the utility of LQTS-hiPSC-CMs in evaluating whether drugs can shorten APD and, importantly, shows that PPARδ agonists may form a new class of therapeutics for this condition.

Abstract 84: Remodeling of Repolarization and Arrhythmia Susceptibility in a Myosin Binding Protein C Knockout Mouse Model

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease (1:500 in the general population) and amongst the leading causes of sudden cardiac death (SCD) in the young. There is lack of an in-depth understanding of cellular mechanisms leading to SCD in HCM. Prior studies that explored the cellular electrophysiological alterations in HCM, focused on the role of calcium and sodium currents in the development of cardiac hypertrophy and arrhythmia. Repolarizing potassium currents in HCM have not been investigated in detail. Better definition of the cellular substrates for arrhythmias could improve risk stratification and lead to potential novel therapeutic targets for prevention of SCD. Objective: To characterize the arrhythmogenic phenotype, cellular electrophysiological properties and repolarizing potassium currents in Myosin binding Protein C (MyBPC) knockout (KO) mice. Methods and results: MyBPC KO mice demonstrate overt phenotype with severe hypertrophy and cardiac dysfunction compared to wild type (WT) control mice, as evidenced by echocardiography and heart weight/body weight analysis. Telemetric electrocardiographic recordings reveal prolongation of the QTc interval in the KO compared to WT control mice (53.2 ± 7.4 vs 30.5 ± 5 msec, n=4 KO, 4 WT, p&lt; 0.05) with frequent arrhythmias including preexcitation, ventricular and supraventricular tachycardia. Whole cell current and voltage clamp studies, comparing KO to WT mice, demonstrate myocyte hypertrophy (469 ± 44 vs 234 ± 15 pF, n=18 KO,15 WT, p&lt;0.01), action potential prolongation (Action potential duration (APD)50 of 30.4± vs 17.7 msec, APD90 of 125.7± vs 74.7 msec, n=11 KO, 12 WT, p &lt;0.05) and decreased repolarizing potassium currents (n=15 KO, 14 WT): I k Peak (-120 to 60 mV) decrease of 33-39%, I k 1 (-120 to -60 mV) decrease of 16-30%, I ks (20 to 60 mV) decrease 21-23% and I to (0 to 60mV) decrease of 46-61%. Conclusion: MyBPC KO mice demonstrate remodeling of repolarizing potassium currents, action potential and QTc intervals prolongation that could account for the arrhythmia susceptibility.

The investigation of the cellular electrophysiological and antiarrhythmic effects of a novel selective sodium-calcium exchanger inhibitor, GYKB-6635, in canine and guinea-pig hearts.

The sodium-calcium exchanger (NCX) is considered as the major transmembrane transport mechanism that controls Ca2+ homeostasis. Its contribution to the cardiac repolarization has not yet been directly studied due to lack of specific inhibitors, so that an urgent need for more selective compounds. In this study, the electrophysiological effects of GYKB-6635, a novel NCX inhibitor, on the NCX, L-type calcium, and main repolarizing potassium currents as well as action potential (AP) parameters were investigated. Ion currents and AP recordings were investigated by applying the whole-cell patch clamp and standard microelectrode techniques in canine heart at 37 °C. Effects of GYKB-6635 were studied in ouabain-induced arrhythmias in isolated guinea-pig hearts. At a concentration of 1 μmol/L, GYKB significantly reduced both the inward and outward NCX currents (57% and 58%, respectively). Even at a high concentration (10 μmol/L), GYKB-6635 did not change the ICaL, the maximum rate of depolarization (dV/dtmax), the main repolarizing K+ currents, and the main AP parameters. GYKB-6635 pre-treatment significantly delayed the time to the development of ventricular fibrillation (by about 18%). It is concluded that GYKB-6635 is a potent and highly selective inhibitor of the cardiac NCX and, in addition, it is suggested to also contribute to the prevention of DAD-based arrhythmias.

Cardiovascular Action of Insulin in Health and Disease: Endothelial L-Arginine Transport and Cardiac Voltage-Dependent Potassium Channels.

Impairment of insulin signaling on diabetes mellitus has been related to cardiovascular dysfunction, heart failure, and sudden death. In human endothelium, cationic amino acid transporter 1 (hCAT-1) is related to the synthesis of nitric oxide (NO) and insulin has a vascular effect in endothelial cells through a signaling pathway that involves increases in hCAT-1 expression and L-arginine transport. This mechanism is disrupted in diabetes, a phenomenon potentiated by excessive accumulation of reactive oxygen species (ROS), which contribute to lower availability of NO and endothelial dysfunction. On the other hand, electrical remodeling in cardiomyocytes is considered a key factor in heart failure progression associated to diabetes mellitus. This generates a challenge to understand the specific role of insulin and the pathways involved in cardiac function. Studies on isolated mammalian cardiomyocytes have shown prolongated action potential in ventricular repolarization phase that produces a long QT interval, which is well explained by attenuation in the repolarizing potassium currents in cardiac ventricles. Impaired insulin signaling causes specific changes in these currents, such a decrease amplitude of the transient outward K+ (Ito) and the ultra-rapid delayed rectifier (IKur) currents where, together, a reduction of mRNA and protein expression levels of α-subunits (Ito, fast; Kv 4.2 and IKs; Kv 1.5) or β-subunits (KChIP2 and MiRP) of K+ channels involved in these currents in a MAPK mediated pathway process have been described. These results support the hypothesis that lack of insulin signaling can produce an abnormal repolarization in cardiomyocytes. Furthermore, the arrhythmogenic potential due to reduced Ito current can contribute to an increase in the incidence of sudden death in heart failure. This review aims to show, based on pathophysiological models, the regulatory function that would have insulin in vascular system and in cardiac electrophysiology.

Mechanisms of acquired long QT syndrome in patients with propionic academia

Propionic acidemia (PROP) is a rare metabolic disorder caused by deficiency of propionyl-CoA carboxylase. PROP patients demonstrate QT prolongations associated with ventricular tachycardia and syncopes. Mechanisms responsible for this acquired long QT syndrome (acqLQTS) are unknown. The aim of the study was to investigate acute and chronic effects of metabolites accumulating in PROP patients on major repolarizing potassium currents (IKs and IKr) and their channel subunits. Voltage clamp studies were performed in CHO-KCNQ1/KCNE1 or HEK-KCNH2 cells to determine effects of propionic acid (PA; 1-10 mM), propionylcarnitine (PC; 25 µM-10 mM), methylcitrate (MC; 25 µM-10 mM), 0.2 M phosphate buffer (PB), or patient serum on IKs and IKr currents. Metabolite effects on action potentials were recorded in current clamp mode in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Protein expression of α- and β-subunits of IKs (KCNQ1/KCNE1) and IKr (KCNH2) was evaluated with Western blots. Acute application of PA, PC, MC, and patient serum had no direct effect on net IKr densities (and KCNH2 expression), although it changed IKr gating kinetics. In contrast, PA, PC, MC, and patient serum all reduced IKs-tail (-67% ± 4.2%, -27% ± 6.7%, -16% ± 6.3%, -42.8% ± 5.15; P < .001) and IKs-end pulse currents. PA significantly prolonged action potential duration (APD) in hiPSC-CM and QT interval in wild-type but not in LQT1 rabbits lacking IKs. Moreover, PC and MC (1 mM) decreased KCNQ1 protein expression (relative density: 0.58 ± 0.08 and 0.16 ± 0.05; P < .01). Chronic exposure to 10 mM PA, in contrast, increased KCNQ1 5.4-fold (P < .001) owing to decreased protein degradation. Acute reduction of IKs by PROP metabolites may be responsible for APD prolongation and acqLQTS observed in PROP patients.