Cardiac Channel Research Articles

Rapid changes of mRNA expressions of cardiac ion channels affected by Torsadogenic drugs influence susceptibility of rat hearts to arrhythmias induced by Beta-Adrenergic stimulation.

Drug-induced long QT syndrome (LQTS) and Torsades de Pointes (TdP) are serious concerns in drug development. Although rats are a useful scientific tool, their hearts, unlike larger species, usually do not respond to torsadogenic drugs. Consequently, their resistance to drug-induced arrhythmias is poorly understood. Here, we challenged rats with rapid delayed rectifier current (Ikr)-inhibiting antibiotic clarithromycin (CLA), loop diuretic furosemide (FUR) or their combination (CLA + FUR), and examined functional and molecular abnormalities after stimulation with isoproterenol. Clarithromycin and furosemide were administered orally at 12-h intervals for 7 days. To evaluate electrical instability, electrocardiography (ECG) was recorded either in vivo or ex vivo using the Langendorff-perfused heart method under basal conditions and subsequently under beta-adrenergic stimulation. Gene expression was measured using real-time quantitative PCR in left ventricular tissue. Indeed, FUR and CLA + FUR rats exhibited hypokalemia. CLA and CLA + FUR treatment resulted in drug-induced LQTS and even an episode of TdP in one CLA + FUR rat. The combined treatment dysregulated gene expression of several ion channels subunits, including KCNQ1, calcium channels and Na+/K + -ATPase subunits, while both monotherapies had no impact. The rat with recorded TdP exhibited differences in the expression of ion channel genes compared to the rest of rats within the CLA + FUR group. The ECG changes were not detected in isolated perfused hearts. Hence, we report rapid orchestration of ion channel reprogramming of hearts with QT prolongation induced by simultaneous administration of clarithromycin and furosemide in rats, which may account for their ability to avoid arrhythmias triggered by beta-adrenergic stimulation.

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKs currents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

Investigation of cardiac pacemaker channel HCN4 leads to new therapeutic approaches in the treatment of viral diseases associated with bradycardia or cardiac arrest

Investigation of cardiac pacemaker channel HCN4 leads to new therapeutic approaches in the treatment of viral diseases associated with bradycardia or cardiac arrest

Assessment of the proarrhythmic effects of repurposed antimalarials for COVID-19 treatment using a comprehensive in vitro proarrhythmia assay (CiPA).

Due to the outbreak of the SARS-CoV-2 virus, drug repurposing and Emergency Use Authorization have been proposed to treat the coronavirus disease 2019 (COVID-19) during the pandemic. While the efficiency of the drugs has been discussed, it was identified that certain compounds, such as chloroquine and hydroxychloroquine, cause QT interval prolongation and potential cardiotoxic effects. Drug-induced cardiotoxicity and QT prolongation may lead to life-threatening arrhythmias such as torsades de pointes (TdP), a potentially fatal arrhythmic symptom. Here, we evaluated the risk of repurposed pyronaridine or artesunate-mediated cardiac arrhythmias alone and in combination for COVID-19 treatment through in vitro and in silico investigations using the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative. The potential effects of each drug or in combinations on cardiac action potential (AP) and ion channels were explored using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and Chinese hamster ovary (CHO) cells transiently expressing cardiac ion channels (Nav1.5, Cav1.2, and hERG). We also performed in silico computer simulation using the optimized O'Hara-Rudy human ventricular myocyte model (ORd model) to classify TdP risk. Artesunate and dihydroartemisinin (DHA), the active metabolite of artesunate, are classified as a low risk of inducing TdP based on the torsade metric score (TMS). Moreover, artesunate does not significantly affect the cardiac APs of hiPSC-CMs even at concentrations up to 100 times the maximum serum concentration (Cmax). DHA modestly prolonged at APD90 (10.16%) at 100 times the Cmax. When considering Cmax, pyronaridine, and the combination of both drugs (pyronaridine and artesunate) are classified as having an intermediate risk of inducing TdP. However, when considering the unbound concentration (the free fraction not bound to carrier proteins or other tissues inducing pharmacological activity), both drugs are classified as having a low risk of inducing TdP. In summary, pyronaridine, artesunate, and a combination of both drugs have been confirmed to pose a low proarrhythmogenic risk at therapeutic and supratherapeutic (up to 4 times) free Cmax. Additionally, the CiPA initiative may be suitable for regulatory use and provide novel insights for evaluating drug-induced cardiotoxicity.

Rate-dependent effects of state-specific sodium channel blockers in cardiac tissue: Insights from idealized models

A common side effect of pharmaceutical drugs is an increased propensity for cardiac arrhythmias. Many drugs bind to cardiac ion-channels in a state-specific manner, which alters the ionic conductances in complicated ways, making it difficult to identify the mechanisms underlying pro-arrhythmic drug effects. To better understand the fundamental mechanisms underlying the diverse effects of state-dependent sodium (Na+) channel blockers on cellular excitability, we consider two canonical motifs of drug-ion-channel interactions and compare the effects of Na+ channel blockers on the rate-dependence of peak upstroke velocity, conduction velocity, and vulnerable window size. In the literature, both motifs are referred to as “guarded receptor,” but here we distinguish between state-specific binding that does not alter channel gating (referred to here as “guarded receptor”) and state-specific binding that blocks certain gating transitions (“gate immobilization”). For each drug binding motif, we consider drugs that bind to the inactivated state and drugs that bind to the non-inactivated state of the Na+ channel. Exploiting the idealized nature of the canonical binding motifs, we identify the fundamental mechanisms underlying the effects on excitability of the various binding interactions. Specifically, we derive the voltage-dependence of the drug binding time constants and the equilibrium fractions of channels bound to drug, and we then derive a formula that incorporates these time constants and equilibrium fractions to elucidate the fundamental mechanisms. In the case of charged drug, we find that drugs that bind to inactivated channels exhibit greater rate-dependence than drugs that bind to non-inactivated channels. For neutral drugs, the effects of guarded receptor interactions are rate-independent, and we describe a novel mechanism for reverse rate-dependence resulting from neutral drug binding to non-inactivated channels via the gate immobilization motif.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

Abstract P1037: Dysfunction Of N-terminal Acetylation Causes Multiple Electrical Abnormalities Leading To Long QT Syndrome And Dilated Cardiomyopathy

Introduction: NAA10-related syndrome is characterized by long QT syndrome (LQTS), cardiomyopathy, hypotonia, and neurodevelopmental delay. NAA10 complexes with the regulatory subunit NAA15 to make the post-translationally modifying N-terminal acetyltransferase, NatA. Despite the association of LQTS and NAA10 mutations, the mechanism is unknown. Hypothesis: NAA10 dysfunction affects the function of cardiac ion channels or related proteins causing LQTS, and sarcomere abnormalities causing cardiomyopathy. Methods: Target sequencing of large kindred with multiple family members who had LQTS and sudden death revealed a segregating mutation in NAA10 (c.10C>A; p.R4S). Somatic cell reprogramming was used to create an induced pluripotent stem cell (iPSC) line from a gene-positive male patient with LQTS and dilated cardiomyopathy (pNAA10 R4S ). Genome-editing was used to create the control iPSC line (eNAA10 R4S ). iPSC lines were differentiated into cardiomyocytes (iPSC-CMs) for further analysis. In addition, we performed biochemical assays to investigate the enzymatic activities of the NAA10-R4S mutation. Results: The NAA10-R4S mutation had reduced enzymatic activity, decreased expression levels of NAA10/NAA15 and destabilized the NatA complex. Action potential duration was prolonged in pNAA10 R4S - and eNAA10 R4S -iPSC-CMs as compared to WT iPSC-CMs (498.9 ± 38.8 ms and 514.1 ± 52.9 ms, vs 316.7 ± 48.2 ms; p<0.05). Investigation of the underlying ion channel currents revealed increased late I Na (Late I Na / Peak I Na : pNAA10 R4S : 0.062 ± 0.010 % and eNAA10 R4S : 0.074 ± 0.020 % vs. WT: 0.026 ± 0.005 %; p<0.05) and reduced I Ks (pNAA10 R4S : 0.18 ± 0.05 pA/pF and eNAA10 R4S : 0.26 ± 0.04 pA/pF vs. WT: 0.61 ± 0.15 pA/pF; p<0.05). There were no significant differences in I Kr and I CaL . Diastolic Ca 2+ levels were increased in mutant iPSC-CMs (F340/F380: 0.18 ± 0.02 vs 0.04 ± 0.01; p<0.05) consistent with impaired Ca 2+ handling. Plating iPSC-CMs on micro-contact printed islands revealed alterations in sarcomeric structure. Conclusion: We demonstrate that the NAA10 mutation cause abnormalities in ion channels and sarcomere development. Our data indicated a novel role for N-terminal acetylation in cardiac ion channel regulation leading to cardiovascular disorders.

Abstract P3142: Engineering A Genetically Encoded Enhancer Of L-type Ca 2+ Currents

L-type calcium channels (LTCC) play a crucial role as they serve as vital portals for Ca 2+ entry into cardiac myocytes, neurons, and other physiological systems. Altered LTCC function is linked to various pathologies including cardiac arrhythmias and neurodevelopmental disorders. In particular, genetic studies have identified loss-of-function mutations in Ca V 1.2 channels in patients with shortened QT interval and Brugada Syndrome, as well as neuropsychiatric disorders. Given the broad physiological importance of LTCC in various tissues, conventional pharmacological agonists of LTCC may yield off-target effects and toxicity. Therefore, new approaches to upregulate LTCC function in a cell-type dependent manner is sought after. To do so, we here develop a genetically encoded enhancer of calcium channels (GeeCC, “geek”) by leveraging both a cardiac specific L-type channel modulator, leucine rich repeat containing protein 10 (Lrrc10), and selective nanobodies that target the LTCC complex. Specifically, Lrrc10 co-expression markedly upregulates LTCC currents. Furthermore, mutations in Lrrc10 are linked to cardiac diseases, including cardiomyopathies. Through extensive structure-function analysis, we identified a minimal action domain within Lrrc10 that is critical for functional modulation of LTCC. However, as this segment alone has a low affinity for the channel complex, its expression alone yielded minimal changes in Ca 2+ current amplitude. However, fusion of this Lrrc10 domain to a nanobody (nb.F3) that targets the Ca 2+ channel beta subunit resulted in a significant increase in the LTCC current amplitude. In depth analysis revealed exquisite selectivity of GeeCC for Ca V 1.2/Ca V 1.3 channels. In mouse cardiomyocytes, deployment of GeeCC via adenovirus enhances endogenous L-type currents when compared to uninfected controls or cardiomyocytes infected with nanobodies alone. Additionally, in neurons, co-expression of GeeCC enhanced excitation-transcription coupling. In all, we have developed a genetically-encoded approach to selectively enhance L-type currents. This strategy may enable new insights into cardiovascular and neuronal physiology and pathophysiology, and may have potential therapeutic applications.

Model-guided concurrent data assimilation for calibrating cardiac ion-channel kinetics

Potassium channels (Kv) are responsible for repolarizing the action potential in cardiomyocytes. There is a variety of Kv isoforms and corresponding currents (e.g. IKto, IKslow1, IKslow2) that contribute to different phases of repolarization. Because only the sum of their activities can be measured in the form of currents (IKsum), there is a need to delineate individual K+ currents. Most existing studies make inference of Kv activities via curve-fitting procedures but encounter certain limitations as follows: (1) curve-fitting decomposition only relies on the shape of K+ current traces, which does not discern the underlying kinetics; (2) IKsum traces can only be fitted for one clamp voltage at each time, and then analyzed in a population-averaged way later. This paper presents a novel concurrent data assimilation method to calibrate biophysics-based models and delineate kinetics of Kv isoforms with multiple voltage-clamp responses simultaneously. The proposed method is evaluated and validated with whole-cell IKsum recordings from wild-type and chronically glycosylation-deficient cardiomyocytes. Experimental results show that the proposed method effectively handles multiple-response data and describes glycosylation-conferred perturbations to Kv isoforms. Further, we develop a graphical-user-interface (GUI) application that provides an enabling tool to biomedical scientists for data-driven modeling and analysis of Kv kinetics in various heart diseases.

Mechanistic understanding of KCNQ1 activating polyunsaturated fatty acid analogs.

The KCNQ1 channel is important for the repolarization phase of the cardiac action potential. Loss of function mutations in KCNQ1 can cause long QT syndrome (LQTS), which can lead to cardiac arrythmia and even sudden cardiac death. We have previously shown that polyunsaturated fatty acids (PUFAs) and PUFA analogs can activate the cardiac KCNQ1 channel, making them potential therapeutics for the treatment of LQTS. PUFAs bind to KCNQ1 at two different binding sites: one at the voltage sensor (Site I) and one at the pore (Site II). PUFA interaction at Site I shifts the voltage dependence of the channel to the left, while interaction at Site II increases maximal conductance. The PUFA analogs, linoleic-glycine and linoleic-tyrosine, are more effective than linoleic acid at Site I, but less effective at Site II. Using both simulations and experiments, we find that the larger head groups of linoleic-glycine and linoleic-tyrosine interact with more residues than the smaller linoleic acid at Site I. We propose that this will stabilize the negatively charged PUFA head group in a position to better interact electrostatically with the positively charges in the voltage sensor. In contrast, the larger head groups of linoleic-glycine and linoleic-tyrosine compared with linoleic acid prevent a close fit of these PUFA analogs in Site II, which is more confined. In addition, we identify several KCNQ1 residues as critical PUFA-analog binding residues, thereby providing molecular models of specific interactions between PUFA analogs and KCNQ1. These interactions will aid in future drug development based on PUFA-KCNQ1 channel interactions.

Interpretation and management of premature ventricular beats in athletes: An expert opinion document of the Italian Society of Sports Cardiology (SICSPORT)

Premature ventricular beats (PVBs) are recorded in a sizeable proportion of athletes during pre-participation screening, especially if the evaluation includes both resting and exercise ECG. While in the majority of cases no underlying heart disease is present, in others PVBs may be the sign of a condition at risk of sudden cardiac death, including cardiomyopathies, congenital, coronary artery, heart valves and ion channels diseases. In this expert opinion document of the Italian Society of Sports Cardiology, we propose a multiparametric interpretation approach to PVBs in athletes and a stepwise diagnostic algorithm. The clinical work-up should include the assessment of the probable site of origin based on the ECG pattern of the ectopic QRS and of the arrhythmia behavior (including the number of different PVB morphologies, complexity, response to exercise and reproducibility), as well as first-line tests such as echocardiography. Based on this initial evaluation, most athletes can be reassured of the benign nature of PVBs and cleared for competition under periodical follow-up. However, when the clinical suspicion is high, further investigations with non-invasive (e.g. cardiac magnetic resonance, cardiac computed tomography, genetic testing) and, in very selected cases, invasive (e.g. endocardial voltage mapping and endomyocardial biopsy) tests should be carried out to rule out a high-risk condition. Importantly, such advanced tests should be performed in centers with a consolidated experience not only in the technique, but also in evaluation of athletes.

LncRNA GAS5 Attenuates Cardiac Electrical Remodeling Induced by Rapid Pacing via the miR-27a-3p/HOXa10 Pathway.

Previous studies indicated long non-coding RNAs (lncRNAs) participated in the pathogenesis of atrial fibrillation (AF). However, little is known about the role of lncRNAs in AF-induced electrical remodeling. This study aimed to investigate the regulatory effect of lncRNA GAS5 (GAS5) on the electrical remodeling of neonatal rat cardiomyocytes (NRCMs) induced by rapid pacing (RP). RNA microarray analysis yielded reduced GAS5 level in NRCMs after RP. RT-qPCR, western blot, and immunofluorescence yielded downregulated levels of Nav1.5, Kv4.2, and Cav1.2 after RP, and whole-cell patch-clamp yielded decreased sodium, potassium, and calcium current. Overexpression of GAS5 attenuated electrical remodeling. Bioinformatics tool prediction analysis and dual luciferase reporter assay confirmed a direct negative regulatory effect for miR-27a-3p on lncRNA-GAS5 and HOXa10. Further analysis demonstrated that either miR-27a-3p overexpression or the knockdown of HOXa10 further downregulated Nav1.5, Kv4.2, and Cav1.2 expression. GAS5 overexpression antagonized such effects in Nav1.5 and Kv4.2 but not in Cav1.2. These results indicate that, in RP-treated NRCMs, GAS5 could restore Nav1.5 and Kv4.2 expression via the miR-27a-3p/HOXa10 pathway. However, the mechanism of GAS5 restoring Cav1.2 level remains unclear. Our study suggested that GAS5 regulated cardiac ion channels via the GAS5/miR-27a-3p/HOXa10 pathway and might be a potential therapeutic target for AF.

CardioMotion: identification of functional and structural cardiotoxic liabilities in small molecules through brightfield kinetic imaging.

Cardiovascular toxicity is an important cause of drug failures in the later stages of drug development, early clinical safety assessment, and even postmarket withdrawals. Early-stage in vitro assessment of potential cardiovascular liabilities in the pharmaceutical industry involves assessment of interactions with cardiac ion channels, as well as induced pluripotent stem cell-derived cardiomyocyte-based functional assays, such as calcium flux and multielectrode-array assays. These methods are appropriate for the identification of acute functional cardiotoxicity but structural cardiotoxicity, which manifests effects after chronic exposure, is often only captured in vivo. CardioMotion is a novel, label-free, high throughput, in vitro assay and analysis pipeline which records and assesses the spontaneous beating of cardiomyocytes and identifies compounds which impact beating. This is achieved through the acquisition of brightfield images at a high framerate, combined with an optical flow-based python analysis pipeline which transforms the images into waveform data which are then parameterized. Validation of this assay with a large dataset showed that cardioactive compounds with diverse known direct functional and structural mechanisms-of-action on cardiomyocytes are identified (sensitivity = 72.9%), importantly, known structural cardiotoxins also disrupt cardiomyocyte beating (sensitivity = 86%) in this method. Furthermore, the CardioMotion method presents a high specificity of 82.5%.

Protein networks for cardiac ion channels share protein components.

Protein networks for cardiac ion channels share protein components.

Protein-Mediated Electroporation in a Cardiac Voltage-Sensing Domain Due to an nsPEF Stimulus.

This study takes a step in understanding the physiological implications of the nanosecond pulsed electric field (nsPEF) by integrating molecular dynamics simulations and machine learning techniques. nsPEF, a state-of-the-art technology, uses high-voltage electric field pulses with a nanosecond duration to modulate cellular activity. This investigation reveals a relatively new and underexplored phenomenon: protein-mediated electroporation. Our research focused on the voltage-sensing domain (VSD) of the NaV1.5 sodium cardiac channel in response to nsPEF stimulation. We scrutinized the VSD structures that form pores and thereby contribute to the physical chemistry that governs the defibrillation effect of nsPEF. To do so, we conducted a comprehensive analysis involving the clustering of 142 replicas simulated for 50 ns under nsPEF stimuli. We subsequently pinpointed the representative structures of each cluster and computed the free energy between them. We find that the selected VSD of NaV1.5 forms pores under nsPEF stimulation, but in a way that significant differs from the traditional VSD opening. This study not only extends our understanding of nsPEF and its interaction with protein channels but also adds a new effect to further study.

Long QT syndrome-associated calmodulin variants disrupt the activity of the slowly activating delayed rectifier potassium channel.

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+ )-dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT interval), increasing their risk of incurring life-threatening arrhythmic events. Loss-of-function mutations to Kv7.1 (which drives the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50% of cases). CaM modulates Kv7.1 to produce a Ca2+ -sensitive IKs, but little is known about the consequences of LQTS-associated CaM mutations on Kv7.1 function. Here, we present novel data characterising the biophysical and modulatory properties of three LQTS-associated CaM variants (D95V, N97I and D131H). We showed that mutations induced structural alterations in CaM and reduced affinity for Kv7.1, when compared with wild-type (WT). Using HEK293T cells expressing Kv7.1 channel subunits (KCNQ1/KCNE1) and patch-clamp electrophysiology, we demonstrated that LQTS-associated CaM variants reduced current density at systolic Ca2+ concentrations (1μm), revealing a direct QT-prolonging modulatory effect. Our data highlight for the first time that LQTS-associated perturbations to CaM's structure impede complex formation with Kv7.1 and subsequently result in reduced IKs. This provides a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype. KEY POINTS: Calmodulin (CaM) is a ubiquitous, highly conserved calcium (Ca2+ ) sensor playing a key role in cardiac muscle contraction. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS), a life-threatening cardiac arrhythmia syndrome. LQTS-associated CaM variants (D95V, N97I and D131H) induced structural alterations, altered binding to Kv7.1 and reduced IKs. Our data provide a novel mechanistic insight into how the perturbed structure-function relationship of CaM variants contributes to the LQTS phenotype.

Autoimmune Atrial Fibrillation.

Atrial fibrillation (AF) is by far the most common cardiac arrhythmia. In about 3% of individuals, AF develops as a primary disorder without any identifiable trigger (idiopathic or historically termed lone AF). In line with the emerging field of autoantibody-related cardiac arrhythmias, the objective of this study was to explore whether autoantibodies targeting cardiac ion channels can underlie unexplained AF. Peptide microarray was used to screen patient samples for autoantibodies. We compared patients with unexplained AF (n=37 pre-existent AF; n=14 incident AF on follow-up) to age- and sex-matched controls (n=37). Electrophysiological properties of the identified autoantibody were then tested in vitro with the patch clamp technique and in vivo with an experimental mouse model of immunization. A common autoantibody response against Kir3.4 protein was detected in patients with AF and even before the development of clinically apparent AF. Kir3.4 protein forms a heterotetramer that underlies the cardiac acetylcholine-activated inwardly rectifying K+ current, IKACh. Functional studies on human induced pluripotent stem cell-derived atrial cardiomyocytes showed that anti-Kir3.4 IgG purified from patients with AF shortened action potentials and enhanced the constitutive form of IKACh, both key mediators of AF. To establish a causal relationship, we developed a mouse model of Kir3.4 autoimmunity. Electrophysiological study in Kir3.4-immunized mice showed that Kir3.4 autoantibodies significantly reduced atrial effective refractory period and predisposed animals to a 2.8-fold increased susceptibility to AF. To our knowledge, this is the first report of an autoimmune pathogenesis of AF with direct evidence of Kir3.4 autoantibody-mediated AF.

The Strength of hERG Inhibition by Erythromycin at Different Temperatures Might Be Due to Its Interacting Features with the Channels.

Erythromycin is one of the few compounds that remarkably increase ether-a-go-go-related gene (hERG) inhibition from room temperature (RT) to physiological temperature (PT). Understanding how erythromycin inhibits the hERG could help us to decide which compounds are needed for further studies. The whole-cell patch clamp technique was used to investigate the effects of erythromycin on hERG channels at different temperatures. While erythromycin caused a concentration-dependent inhibition of cardiac hERG channels, it also shifted the steady-state activation and steady-state inactivation of the channel to the left and significantly accelerated the onset of inactivation at both temperatures, although temperature itself caused a profound change in the dynamics of hERG channels. Our data also suggest that the binding pattern to S6 of the channels changes at PT. In contrast, cisapride, a well-known hERG blocker whose inhibition is not affected by temperature, does not change its critical binding sites after the temperature is raised to PT. Our data suggest that erythromycin is unique and that the shift in hERG inhibition may not apply to other compounds.

Outlining cardiac ion channel protein interactors and their signature in the human electrocardiogram

Protein–protein interactions are essential for normal cellular processes and signaling events. Defining these interaction networks is therefore crucial for understanding complex cellular functions and interpretation of disease-associated gene variants. We need to build a comprehensive picture of the interactions, their affinities and interdependencies in the specific organ to decipher hitherto poorly understood signaling mechanisms through ion channels. Here we report the experimental identification of the ensemble of protein interactors for 13 types of ion channels in murine cardiac tissue. Of these, we validated the functional importance of ten interactors on cardiac electrophysiology through genetic knockouts in zebrafish, gene silencing in mice, super-resolution microscopy and patch clamp experiments. Furthermore, we establish a computational framework to reconstruct human cardiomyocyte ion channel networks from deep proteome mapping of human heart tissue and human heart single-cell gene expression data. Finally, we integrate the ion channel interactome with human population genetics data to identify proteins that influence the electrocardiogram (ECG). We demonstrate that the combined channel network is enriched for proteins influencing the ECG, with 44% of the network proteins significantly associated with an ECG phenotype. Altogether, we define interactomes of 13 major cardiac ion channels, contextualize their relevance to human electrophysiology and validate functional roles of ten interactors, including two regulators of the sodium current (epsin-2 and gelsolin). Overall, our data provide a roadmap for our understanding of the molecular machinery that regulates cardiac electrophysiology.

New Drug Targets and Preclinical Modelling Recommendations for Treating Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is the leading cause of morbidity and mortality worldwide and the primary underlying risk factor for heart failure. Despite decades of research and clinical trials, there are no drugs currently available to prevent organ damage from acute ischaemic injuries of the heart. In order to address the increasing global burden of heart failure, drug, gene, and cell-based regeneration technologies are advancing into clinical testing. In this review we highlight the burden of disease associated with AMI and the therapeutic landscape based on market analyses. New studies revealing the role of acid-sensitive cardiac ion channels and other proton-gated ion channels in cardiac ischaemia are providing renewed interest in pre- and post-conditioning agents with novel mechanisms of action that may also have implications for gene- and cell-based therapeutics. Furthermore, we present guidelines that couple new cell technologies and data resources with traditional animal modelling pipelines to help de-risk drug candidates aimed at treating AMI. We propose that improved preclinical pipelines and increased investment in drug target identification for AMI is critical to stem the increasing global health burden of heart failure.