Rectifier Potassium Current Research Articles

Sophoridine manifests as a leading compound for anti-arrhythmia with multiple ion-channel blocking effects

BackgroundSophoridine (SR) has shown the potential to be an antiarrhythmic agent. However, SR's electrophysiological properties and druggability research are relatively inadequate, which limits the development of SR as an antiarrhythmic candidate. PurposeTo facilitate the development process of SR as an antiarrhythmic candidate, we performed integrated studies on the electrophysiological properties of SR in vitro and ex vivo to gain more comprehensive insights into the multi-ion channel blocking effects of SR, which provided the foundation for the further drugability studies in antiarrhythmic and safety studies. Firstly, SR's electrophysiological properties and antiarrhythmic potentials were recorded and assessed at the cell and tissue levels by comprehensively integrating the patch clamp with the Electrical and Optical Mapping systems. Subsequently, the antiarrhythmic effects of SR were validated by aconitine and ouabain-induced arrhythmia in vivo. Finally, the safety of SR as an antiarrhythmic candidate compound was evaluated based on the guidelines of the Comprehensive in Vitro Proarrhythmia Assay (CiPA). Study designThe antiarrhythmic effect of SR was evaluated at the in vitro, ex vivo, and in vivo levels. MethodsIsolated primary cardiomyocytes and stable cell lines were prepared to explore the electrophysiologic properties of being a multiple ion-channel blocker in vitro by whole-cell patch clamp. Using electrical and optical mapping, the negative chronotropic effect of SR was determined in langendorff-perfused rat or guinea-pig hearts.The antiarrhythmic activity of SR was assessed by the ex vivo tachyarrhythmia models induced by left coronary artery ligation (LCAL) and isoproterenol (ISO). Canonical models of aconitine and ouabain-induced arrhythmia were used to verify the antiarrhythmic effects in vivo. Finally, the pro-arrhythmic risk of SR was detected in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hSCCMs) using a Microelectrode array (MEA). ResultsSingle-cell patch assay validated the multiple ion-channel blockers of SR in transient outward current potassium currents (Ito), l-type calcium currents (ICa-l), and rapid activation delayed rectifier potassium currents (IKr). SR ex vivo depressed heart rates (HR) and ventricular conduction velocity (CV) and prolonged Q-T intervals in a concentration-dependent manner. Consistent with the changes in HRs, SR extended the active time of hearts and increased the action potential duration measured at 90% repolarization (APD90). SR could also significantly lengthen the onset time and curtail the duration of spontaneous ventricular tachycardia (VT) in the ex vivo arrhythmic model induced by LCAL. Meanwhile, SR could also significantly upregulate the programmed electrical stimulation (PES) frequency after the ISO challenge in forming electrical alternans and re-entrant excitation. Furthermore, SR exerted antiarrhythmic effects in the tachyarrhythmia models induced by aconitine and ouabain in vivo. Notably, the pro-arrhythmic risk of SR was shallow for a moderate inhibition of the human ether-à-go-go-related gene (hERG) channel. Moreover, SR prolonged field potential duration (FPDc) of hSCCMs in a concentration-dependent manner without early after depolarization (EAD) and arrhythmia occurrence. ConclusionOur results indicated that SR manifested as a multiple ion-channel blocker in the electrophysiological properties and exerts antiarrhythmic effects ex vivo and in vivo. Meanwhile, due to the low pro-arrhythmic risk in the hERG inhibition assay and the induction of EAD, SR has great potential as a leading candidate in the treatment of ventricular tachyarrhythmia.

KCNH2 encodes a nuclear-targeted polypeptide that mediates hERG1 channel gating and expression

KCNH2 encodes hERG1, the voltage-gated potassium channel that conducts the rapid delayed rectifier potassium current (IKr) in human cardiac tissue. hERG1 is one of the first channels expressed during early cardiac development, and its dysfunction is associated with intrauterine fetal death, sudden infant death syndrome, cardiac arrhythmia, and sudden cardiac death. Here, we identified a hERG1 polypeptide (hERG1NP) that is targeted to the nuclei of immature cardiac cells, including human stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat cardiomyocytes. The nuclear hERG1NP immunofluorescent signal is diminished in matured hiPSC-CMs and absent from adult rat cardiomyocytes. Antibodies targeting distinct hERG1 channel epitopes demonstrated that the hERG1NP signal maps to the hERG1 distal C-terminal domain. KCNH2 deletion using CRISPR simultaneously abolished IKr and the hERG1NP signal in hiPSC-CMs. We then identified a putative nuclear localization sequence (NLS) within the distal hERG1 C-terminus, 883-RQRKRKLSFR-892. Interestingly, the distal C-terminal domain was targeted almost exclusively to the nuclei when overexpressed HEK293 cells. Conversely, deleting the NLS from the distal peptide abolished nuclear targeting. Similarly, blocking α or β1 karyopherin activity diminished nuclear targeting. Finally, overexpressing the putative hERG1NP peptide in the nuclei of HEK cells significantly reduced hERG1a current density, compared to cells expressing the NLS-deficient hERG1NP or GFP. These data identify a developmentally regulated polypeptide encoded by KCNH2, hERG1NP, whose presence in the nucleus indirectly modulates hERG1 current magnitude and kinetics.

The Effect of the Tongyang Huoxue Recipe (TYHX) on the I to/I Kur in Ischemia/Reperfusion Sinoatrial Node Cells.

The transient outward potassium current (I to) and the ultrarapid delayed rectifier potassium current (I Kur) are major potassium currents involved in the repolarization process of sinoatrial node cells (SNCs). The SNCs of neonatal rats were divided into control, ischemia/reperfusion (I/R), I/R+blank serum, and Tongyang Huoxue recipe (TYHX) serum groups. I to and I Kur were recorded using the whole cell patch-clamp technique, and the current-voltage (I-V), steady-state activation (SSA), steady-state inactivation (SSI), and recovery from inactivation (RFI) curves were plotted, respectively. Compared to the control group, both the peak current density and the current density at the voltage of I to and I Kur decreased obviously in SNCs after simulated I/R, the SSA curves moved right, and the SSI curves moved left. After TYHX was added to the extracellular solution of SNCs, both the peak current density and the current density at the voltage of I to and I Kur increased significantly, the SSA curves moved left, and the SSI curves moved right with a significant difference of V 1/2. The recovery from the I Kur RFI curves was slightly restored, and the I to curves did not change. TYHX increases the peak current density, accelerates the activation, and decreases the inactivation of the I to and I Kur. This may be the mechanism of TYHX in shortening the action potential duration of repolarization, which accelerates spontaneous pulsation.

In silico mechanisms of arsenic trioxide-induced cardiotoxicity.

It has been found that arsenic trioxide (ATO) is effective in treating acute promyelocytic leukemia (APL). However, long QT syndrome was reported in patients receiving therapy using ATO, which even led to sudden cardiac death. The underlying mechanisms of ATO-induced cardiotoxicity have been investigated in some biological experiments, showing that ATO affects human ether-à-go-go-related gene (hERG) channels, coding rapid delayed rectifier potassium current (I Kr ), as well as L-type calcium (I CaL ) channels. Nevertheless, the mechanism by which these channel reconstitutions induced the arrhythmia in ventricular tissue remains unsolved. In this study, a mathematical model was developed to simulate the effect of ATO on ventricular electrical excitation at cellular and tissue levels by considering ATO's effects on I Kr and I CaL . The ATO-dose-dependent pore block model was incorporated into the I Kr model, and the enhanced degree of ATO to I CaL was based on experimental data. Simulation results indicated that ATO extended the action potential duration of three types of ventricular myocytes (VMs), including endocardial cells (ENDO), midmyocardial cells (MCELL), and epicardial cells (EPI), and exacerbated the heterogeneity among them. ATO could also induce alternans in all three kinds of VMs. In a cable model of the intramural ventricular strand, the effects of ATO are reflected in a prolonged QT interval of simulated pseudo-ECG and a wide vulnerable window, thus increasing the possibility of spiral wave formation in ventricular tissue. In addition to showing that ATO prolonged QT, we revealed that the heterogeneity caused by ATO is also an essential hazard factor. Based on this, a pharmacological intervention of ATO toxicity by resveratrol was undertaken. This study provides a further understanding of ATO-induced cardiotoxicity, which may help to improve the treatment for APL patients.

HERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (IKr) and is critical for human cardiac repolarization. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased IKr. Silencing hERG1b's impact on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of IKr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines IKr proton sensitivity in hiPSC-CMs.

Bioengineered peptibodies as blockers of ion channels

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

Effects of the gaseous signalling molecule nitroxyl (HNO) on myenteric neurons governing intestinal motility.

The main function of myenteric neurons is the control of gut motility. As we recently showed that nitroxyl (HNO) induces intestinal smooth muscle relaxation, it was of interest to evaluate the effects of this signalling molecule on myenteric neurons in order to distinguish its properties in regard to myocytes. Myenteric neurons isolated from the ileum of 4-10days old rats were used. HNO-induced changes in intracellular concentration of Ca2+ or membrane potential and ion currents were measured using the Ca2+-sensitive fluorescent dye fura-2 AM or by electrophysiological whole-cell recordings, respectively. Changes in intracellular thiol groups pool were evaluated using thiol tracker violet. Angeli's salt was used as HNO donor. The HNO donor Angeli's salt induced a significant increase in the cytosolic Ca2+ concentration at the concentration 50µM and a membrane hyperpolarization from a resting membrane potential of-56.1±8.0mV to-63.1±8.7mV (n=7). Although potassium channels primarily drive membrane potential changes in these cells, outwardly rectifying potassium currents were not significantly affected by 50µM Angeli's salt. Fast inward sodium currents were slightly but not significantly reduced by HNO. In more sensitive cells, HNO tended to reduce the pool of thiol groups. As in the case of smooth muscle cells, HNO causes hyperpolarization of myenteric neurons, an effect also associated with an increase in intracellular Ca2+ concentration. Pathways other than activation of potassium currents appear to drive the hyperpolarization evoked by HNO.

Verapamil inhibits Kir2.3 channels by binding to the pore and interfering with PIP2 binding

The inwardly rectifying potassium current of the cardiomyocyte (IK1) is the main determinant of the resting potential. Ion channels Kir2.1, Kir2.2, and Kir2.3 form tetramers and are the molecular correlate of macroscopic IK1 current. Verapamil is an antiarrhythmic drug used to suppress atrial and ventricular arrhythmias. Its primary mechanism of action is via blocking calcium channels. In addition, it has been demonstrated to block IK1 current and the Kir2.1 subunit. Its effect on other subunits that contribute to IK1 current has not been studied to date. We therefore analyzed the effect of verapamil on the Kir channels 2.1, 2.2, and 2.3 in the Xenopus oocyte expression system. Kir2.1, Kir2.2, and Kir2.3 channels were heterologously expressed in Xenopus oocytes. Respective currents were measured with the voltage clamp technique and the effect of verapamil on the current was measured. At a concentration of 300 µM, verapamil inhibited Kir2.1 channels by 41.36% ± 2.7 of the initial current, Kir2.2 channels by 16.51 ± 3.6%, and Kir2.3 by 69.98 ± 4.2%. As a verapamil effect on kir2.3 was a previously unknown finding, we analyzed this effect further. At wash in with 300 µM verapamil, the maximal effect was seen within 20 min of the infusion. After washing out with control solution, there was only a partial current recovery. The current reduction from verapamil was the same at − 120 mV (73.2 ± 3.7%), − 40 mV (85.5 ± 6.5%), and 0 mV (61.5 ± 10.6%) implying no voltage dependency of the block. Using site directed mutations in putative binding sites, we demonstrated a decrease of effect with pore mutant E291A and absence of verapamil effect for D251A. With mutant I214L, which shows a stronger affinity for PIP2 binding, we observed a normalized current reduction to 61.9 ± 0.06% of the control current, which was significantly less pronounced compared to wild type channels. Verapamil blocks Kir2.1, Kir2.2, and Kir2.3 subunits. In Kir2.3, blockade is dependent on sites E291 and D251 and interferes with activation of the channel via PIP2. Interference with these sites and with PIP2 binding has also been described for other Kir channels blocking drugs. As Kir2.3 is preferentially expressed in atrium, a selective Kir2.3 blocking agent would constitute an interesting antiarrhythmic concept.

Abstract 13309: Specific Deletion of Microrna1’S Biophysical Action Causes a Functional Deterioration of the Heart and Increases the Vulnerability to Arrhythmias

MicroRNAs (miRs) are involved in most biological events via a broadly-recognized RNA interference (RNAi) mechanism. miR1, encoded by miR1-1 and miR1-2 genes, is the predominant miRs of the heart and plays a critical role in heart development and cardiac diseases. Previous studies of miRs have focused on the canonical RNAi mechanism. Recently, we discovered a novel biophysical action of miR and found that miR1 physically binds to Kir2.1 and directly suppresses the inward rectifier potassium current (I K1 ). However, the physiological significance of this newly-discovered biophysical action of miR remains unknown. Importantly, we found that a human single nucleotide polymorphism (hSNP)-hSNP14A/G (rs776480338), in which the 14th nucleotide of miR1 is mutated from A to G, specifically disrupts this biophysical action while maintaining its RNAi function. We then created a single nucleotide mutation of hSNP14A/G on mouse miR1-1 and miR1-2 genes by CRISPR/Cas9 technology and have successfully developed 14G-mutated homozygous transgenic mice (14G-Homo), in which the biophysical function of miR1 is specifically removed while the RNAi function is maintained. We found that 14G-Homo mice had higher mortality than wild-type (WT) mice. Echocardiogram showed that, compared to WT hearts, adult 14G-homo heart (2-month age) had significantly lower ejection fraction and fractional shortening with a thinner wall of the left ventricle, which became more severe at the age of 6 months; some mice developed severe heart failure. Surface ECG showed that 14G-Homo hearts had a slower heart rate and the prolonged QRS interval and QT interval. Optical mapping revealed a slower conduction velocity in the 14G-Homo heart with high inducibility of ventricular arrhythmias. Patch clamping of isolated ventricular cardiomyocytes showed that, compared to WT cells, 14G-homo cardiomyocytes had an increased amplitude and prolonged duration of action potential with bigger I K1 and L-type calcium currents. In conclusion, our discoveries demonstrate that the biophysical action of miR1 is deeply involved in maintaining the homeostasis of the heart. Specific deletion of the biophysical function causes a functional deterioration of the heart and increases the vulnerability to arrhythmias.

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

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

IKACh is constitutively active via PKC epsilon in aging mediated atrial fibrillation

Atrial fibrillation (AF), the most common abnormal heart rhythm, is a major cause for stroke. Aging is a significant risk factor for AF; however, specific ionic pathways that can elucidate how aging leads to AF remain elusive. We used young and old wild-type and PKC epsilon- (PKCϵ) knockout mice, whole animal, and cellular electrophysiology, as well as whole heart, and cellular imaging to investigate how aging leads to the aberrant functioning of a potassium current, and consequently to AF facilitation. Our experiments showed that knocking out PKCϵ abrogates the effects of aging on AF by preventing the development of a constitutively active acetylcholine sensitive inward rectifier potassium current (IKACh). Moreover, blocking this abnormal current in the old heart reduces AF inducibility. Our studies demonstrate that in the aging heart, IKACh is constitutively active in a PKCϵ-dependent manner, contributing to the perpetuation of AF.

A novel de novo KCNB1 variant altering channel characteristics in a patient with periventricular heterotopia, abnormal corpus callosum, and mild seizure outcome.

KCNB1 encodes the α-subunit of Kv2.1, the main contributor to neuronal delayed rectifier potassium currents. The subunit consists of six transmembrane α helices (S1-S6), comprising the voltage-sensing domain (S1-S4) and the pore domain (S5-P-S6). Heterozygous KCNB1 pathogenic variants are associated with developmental and epileptic encephalopathy. Here we report an individual who shows the milder phenotype compared to the previously reported cases, including delayed language development, mild intellectual disability, attention deficit hyperactivity disorder, late-onset epilepsy responsive to an antiepileptic drug, elevation of serum creatine kinase, and peripheral axonal neuropathy. On the other hand, his brain MRI showed characteristic findings including periventricular heterotopia, polymicrogyria, and abnormal corpus callosum. Exome sequencing identified a novel de novo KCNB1 variant c.574G>A, p.(Ala192Thr) located in the S1 segment of the voltage-sensing domain. Functional analysis using the whole-cell patch-clamp technique in Neuro2a cells showed that the Ala192Thr mutant reduces both activation and inactivation of the channel at membrane voltages in the range of -50 to -30 mV. Our case could expand the phenotypic spectrum of patients with KCNB1 variants, and suggested that variants located in the S1 segment might be associated with a milder outcome of seizures.

Spiral- and scroll-wave dynamics in mathematical models for canine and human ventricular tissue with varying Potassium and Calcium currents

We conduct a systematic, direct-numerical-simulation (DNS) study, in mathematical models for ventricular tissue, of the dependence of spiral- and scroll-wave dynamics on GKr, the maximal conductance of the delayed rectifier Potassium current (IKr) channel, and the parameter γCao, which determines the magnitude and shape of the current ICaL for the L-type calcium-current channel, in both square and anatomically realistic, whole-ventricle simulation domains. We study canine and human models. In the former, we use a canine-ventricular geometry, with fiber-orientation details, obtained from diffusion-tensor-magnetic-resonance-imaging (DTMRI) data; and we employ the physiologically realistic Hund-Rudy-Dynamic (HRD) model for a canine ventricular myocyte. To focus on the dependence of spiral- and scroll-wave dynamics on GKr and γCao, we restrict ourselves to an HRD-model parameter regime, which does not produce spiral- and scroll-wave instabilities because of other, well-studied causes like a very sharp action-potential-duration-restitution (APDR) curve or early after depolarizations (EADs) at the single-cell level. We find that spiral- or scroll-wave dynamics are affected predominantly by a simultaneous change in ICaL and IKr from their original values in the model, rather than by a change in any one of these currents; other currents do not have such a large effect on these wave dynamics in this parameter regime of the HRD model. In particular, we examine spiral-wave dynamics for ten different values of GKr and ten different values of γCao in our 2D DNSs. For our 3D DNSs in an anatomically realistic domain, we chose 16 parameter sets. In the parameter regime we begin with, the system displays broken spiral or scroll states with S1–S2 initial conditions (see below). We show that, by simultaneously increasing GKr and reducing γCao, we can get to a parameter regime in which the system displays single, stable rotating spirals or scroll waves. We obtain stability diagrams (or phase diagrams) in the GKr−γCao plane; and we find that these diagrams are significantly different in our 2D and 3D studies. In the 3D case, the geometry of the domain itself supports the confinement of the scroll waves and makes them more stable compared to their spiral-wave counterparts in our flat, 2D simulation domain. Thus, a combination of functional and geometrical mechanisms produce different dynamics for 3D scroll waves and their 2D spiral-wave counterparts. In particular, the former do not break easily because, in an anatomically realistic ventricular geometry, they are not easily absorbed at boundaries, nor do they break near boundaries. We have also carried out a comparison of our HRD results with their counterparts for the human-ventricular TP06 model; and we have found important differences between wave dynamics in these two models. The region in parameter space, where we obtain broken spiral or scroll waves in the HRD model is the region of stable rotating waves in the TP06 model; the default parameter values produce broken waves in the HRD model, but stable scrolls in the TP06 model. In both these models, to make a transition, (most simply, from broken-wave to stable-scroll states) we must simultaneously increase IKr and decrease ICaL; a modification of only one of these currents is not enough to effect this transition. Furthermore, the converse, i.e., an increase in ICaL along with a decrease in IKr does not yield any interesting dynamical transitions in the HRD model, for, in this range of currents, this model does not sustain spiral or scroll waves or broken waves.

The Arf6/PIP5K pathway activates IKACh in cigarette smoke mediated atrial fibrillation

Cigarette smoking (CS) is a major cause of cardiovascular diseases. Smokers are at a significantly higher risk for developing atrial fibrillation (AF), a dangerous and abnormal heart rhythm. In the US, 15.5% of adults are current smokers, and it is becoming clear that CS is an independent risk factor for AF, but a detailed mechanistic understanding of how CS contributes to the molecular patho-electrophysiology of AF remains elusive. We investigated if CS related AF is in part mediated through a mechanism that depends on the cardiac acetylcholine activated inward rectifier potassium current (IKACh). We tested the hypothesis that CS increases IKACh via phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5K) and ADP ribosylation factor 6 (Arf6) signaling, leading to AF perpetuation. In vivo inducibility of AF was assessed in mice exposed to CS for 8 weeks. AF duration was increased in CS exposed mice, and TertiapinQ, an IKACh blocker prevented AF development in CS exposed mice. In HEK293 cells stably transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, CS exposure increased the expression of the Kir3.1 and Kir3.4 proteins at the cell surface, activated Arf6 and increased the IKACh current. Inhibition of PIP5K, or of Kir3.1/Kir3.4 trafficking via Arf6 abrogated the CS effects on IKACh. Cigarette smoke modifies the atrial electrophysiological substrate, leading to arrhythmogenesis, in part, through IKACh activation via an Arf6/PIP5K dependent pathway.

Open channel block of Kv1.5 channels by HMQ1611.

Kv1.5 channels conduct the ultra-rapid delayed rectifier potassium current (I Kur). Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as an effective treatment of re-entrant based atrial fibrillation, because Kv1.5 is highly expressed in human cardiac atria but scarcely in ventricles. The Kv1.5 blockade is also expected to be used in cancer therapeutics since Kv1.5 is overexpressed in some types of human tumors. Here, we investigated the blockade of hKv1.5 channels by HMQ1611, a symmetrical biphenyl derivative. hKv1.5 channels were heterologously expressed in Chinese hamster ovary cells. The effects of HMQ1611 on wild-type and 13 hKv1.5 mutant channels were examined using the whole-cell patch-clamp method, and molecular docking simulation was conducted to predict the docking position of HMQ1611 within Kv1.5 channels. We showed that HMQ1611 reversibly inhibited the hKv1.5 current in a concentration-dependent manner (IC50 = 2.07 μM). HMQ1611 blockade of hKv1.5 current developed with time during depolarizing voltage-clamp steps, and this blockade was also voltage-dependent with a steep increase over the voltage range for channel openings. HMQ1611 inhibition was significantly reduced in the T479A, T480A, V505A, I508A, L510A, V512A, and V516A hKv1.5 mutant channels. Molecular docking analysis predicted that V505, V512, and T480 were involved in the blocking action of HMQ1611 on hKv1.5 channels. These results suggest that HMQ1611 inhibits hKv1.5 currents as an open channel blocker. Amino acid residues located at the base of the selectivity filter (T479 and T480) and in the S6 segment (V505, I508, L510, V512, and V516) of hKv1.5 appear to constitute potential binding sites for HMQ1611.

Aldehyde dehydrogenase 2 and arrhythmogenesis.

Cardiac arrhythmia is a common cardiovascular disease that leads to considerable economic burdens and significant global public health challenges. Despite the remarkable progress made in recent decades, antiarrhythmic therapy remains suboptimal. Aldehyde dehydrogenase 2 (ALDH2), a critical detoxifying enzyme, catalyzes toxic aldehydes and protects individuals from damage caused by oxidative stress. Accumulating evidence has demonstrated that ALDH2 activation has potential antiarrhythmic benefits. The correlation between ALDH2 deficiency and arrhythmogenesis has been widely recognized. In this review, we summarize recent research on the potential role of ALDH2 activation and antiarrhythmic protection, as well as the role played by the ALDH2∗2 polymorphism (rs671) in promoting arrhythmic risk. Additionally, we discuss important new findings illustrating the use of ALDH2 activators, which may prove to be promising antiarrhythmic therapy agents.

Update on Antiarrhythmic Therapy: Dofetilide

Dofetilide is one of the several novel methanesulfonamide compounds that selectively inhibits the rapid component of the delayed rectifier potassium current, IKr, thus prolonging the effective refractory period (ERP) and the action potential duration (APD) in both the atria and the ventricles. It has no effects on sodium and calcium channels, and therefore, has minimal effects on myocardial conduction velocity. Dofetilide can be administered either orally or intravenously, and has a rapid onset of action. It was recently approved in the United States for the maintenance and conversion of normal sinus rhythm in patients with atrial fibrillation (AF).

Purification and membrane interactions of human KCNQ1100–370 potassium ion channel

KCNQ1 (Kv7.1 or KvLQT1) is a voltage-gated potassium ion channel that is involved in the ventricular repolarization following an action potential in the heart. It forms a complex with KCNE1 in the heart and is the pore forming subunit of slow delayed rectifier potassium current (Iks). Mutations in KCNQ1, leading to a dysfunctional channel or loss of activity have been implicated in a cardiac disorder, long QT syndrome. In this study, we report the overexpression, purification, biochemical characterization of human KCNQ1100–370, and lipid bilayer dynamics upon interaction with KCNQ1100–370. The recombinant human KCNQ1 was expressed in Escherichia coli and purified into n-dodecylphosphocholine (DPC) micelles. The purified KCNQ1100–370 was biochemically characterized by SDS-PAGE electrophoresis, western blot and nano-LC-MS/MS to confirm the identity of the protein. Circular dichroism (CD) spectroscopy was utilized to confirm the secondary structure of purified protein in vesicles. Furthermore, 31P and 2H solid-state NMR spectroscopy in DPPC/POPC/POPG vesicles (MLVs) indicated a direct interaction between KCNQ100–370 and the phospholipid head groups. Finally, a visual inspection of KCNQ1100–370 incorporated into MLVs was confirmed by transmission electron microscopy (TEM). The findings of this study provide avenues for future structural studies of the human KCNQ1 ion channel to have an in depth understanding of its structure-function relationship.

Repetitive transcranial magnetic stimulation enhances the neuronal excitability of mice by regulating dynamic characteristics of Granule cells' Ion channels.

This study aims to explore the effects of acute high-frequency repetitive transcranial magnetic stimulation (hf-rTMS) on neuronal excitability of granule cells in the hippocampal dentate gyrus, as well as the underlying intrinsic mediating mechanisms by which rTMS regulates neuronal excitability. First, high-frequency single TMS was used to measure the motor threshold (MT) of mice. Then, rTMS with different intensities of 0 MT (control), 0.8 MT, and 1.2 MT were applied to acute mice brain slices. Next, patch-clamp technique was used to record the resting membrane potential and evoked nerve discharge of granule cells, as well as the voltage-gated sodium current (I Na) of voltage-gated sodium channels (VGSCs), transient outward potassium current (I A) and delayed rectifier potassium current (I K) of voltage-gated potassium channels (Kv). Results showed that acute hf-rTMS in both 0.8 MT and 1.2 MT groups significantly activated I Na and inhibited I A and I K compared with control group, due to the changes of dynamic characteristics of VGSCs and Kv. Acute hf-rTMS in both 0.8 MT and 1.2 MT groups significantly increased membrane potential and nerve discharge frequency. Therefore, changing dynamic characteristics of VGSCs and Kv, activating I Na and inhibiting I A and I K might be one of the intrinsic mediating mechanisms by which rTMS enhanced the neuronal excitability of granular cells, and this regulatory effect increased with the increase of stimulus intensity.

SNTA1 gene rescues ion channel function and is antiarrhythmic in cardiomyocytes derived from induced pluripotent stem cells from muscular dystrophy patients.

Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients. To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays. Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers. We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias. Supported by National Institutes of Health R01 HL122352 grant; 'la Caixa' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.