Cardiac Excitability Research Articles

The many faces of SCN5A pathogenic variants: from channelopathy to cardiomyopathy.

The SCN5A gene encodes the alpha subunit of the cardiac sodium channel, which plays a fundamental role in the generation and propagation of the action potential in the heart muscle. During the past years our knowledge concerning the function of the cardiac sodium channel and the diseases caused by mutations of the SCN5A gene has grown. Although initially SCN5A pathogenic variants were mainly associated with channelopathies, increasing recent evidence suggests an association with structural heart disease in the form of heart muscle disease. The pathways leading to a cardiomyopathic phenotype remain unclear and require further elucidation. The aim of the present review is to provide a concise summary regarding the mechanisms through which SCN5A pathogenic variants result in heart disease, focusing in cardiomyopathy, highlighting along the way the complex role of the SCN5A gene at the intersection of cardiac excitability and contraction networks.

A Non-Invasive and DNA-free Approach to Upregulate Mammalian Voltage-Gated Calcium Channels and Neuronal Calcium Signaling via Terahertz Stimulation.

Mammalian voltage-gated calcium channels (CaV) play critical roles in cardiac excitability, synaptic transmission, and gene transcription. Dysfunctions in CaV are implicated in a variety of cardiac and neurodevelopmental disorders. Current pharmacological approaches to enhance CaV activity are limited by off-target effects, drug metabolism issues, cytotoxicity, and imprecise modulation. Additionally, genetically-encoded channel activators and optogenetic tools are restricted by gene delivery challenges and biosafety concerns. Here a novel terahertz (THz) wave-based method to upregulate CaV1.2, a key subtype of CaV, and boost CaV1-mediated Ca2+ signaling in neurons without introducing exogenous DNA is presented. Using molecular dynamics simulations, it is shown that 42.5 THz (7.05µm, 1418cm-1) waves enhance Ca2+ conductance in CaV1.2 by resonating with the stretching mode of the -COO- group in the selectivity filter. Electrophysiological recordings and Ca2+ imaging confirm that these waves rapidly, reversibly, and non-thermally increase calcium influx of CaV1.2 in HEK293 cells and induce acute Ca2+ signals in neurons. Furthermore, this irradiation upregulates critical CaV1 signals, including CREB phosphorylation and c-Fos expression, in vitro and in vivo, without raising significant biosafety risks. This DNA-free, non-invasive approach offers a promising approach for modulating CaV gating and Ca2+ signaling and treating diseases characterized by deficits in CaV functions.

The evolution of patch-clamp electrophysiology: robotic, multiplex, and dynamic.

The patch-clamp technique has been the gold standard for analysis of excitable cells. Since its development in the 1980s it has contributed immensely to our understanding of neurons, muscle cells, and cardiomyocytes, and the ion channels and receptors that reside within them. This technique, predicated on Ohm's law, enables precise measurements of macroscopic excitability patterns, and ionic and gating conductances that can be assessed even down to the single channel level. Over the years, patch-clamp electrophysiology has undergone extensive modifications, with the introduction of new applications that have enhanced its power and reach. The most recent evolution of this technique occurred with the introduction of robotic high throughput automated platforms that enable high quality simultaneous recordings, in both voltage- and current-clamp modes, from 10s to 100s of cells, including cells freshly isolated from their native tissues. Combined with new dynamic-clamp applications, these new methods provide increasingly powerful tools for studying the contributions of ion channels and receptors to electrogenesis. In this brief review, we provide an overview of these enhanced patch-clamp techniques, followed by some of the applications presently being pursued, and a perspective into the potential future of the patch-clamp method. Significance Statement The patch-clamp technique, introduced in the 1980s, has revolutionized understanding of electrogenesis. Predicated on Ohm's law, this approach facilitates exploration of ionic conductances, gating mechanisms of ion channels and receptors, and their roles in neuronal, muscular, and cardiac excitability. Robotic platforms for high-throughput patch-clamp, and dynamic-clamp, have recently expanded its reach. Here, we outline new advances in patch-clamp including high throughput analysis of freshly-isolated neurons, and discuss the increasingly powerful trajectory of new patch-clamp techniques.

Abstract Mo134: Restoring NaV1.5 Mutant Functionality in Arrhythmia with ManNAc Supplementation

The SCN5A gene encodes the human cardiac sodium channel NaV1.5, essential for cardiac excitability and action potential propagation. Mutations in SCN5A are linked to various cardiac disorders, including Brugada Syndrome (BrS), typically leading to channel loss-of-function. Understanding alterations in NaV1.5 expression and sodium current density is vital for comprehending arrhythmogenesis and formulating therapeutic strategies. Post-translational modifications, especially sialylation, significantly impact channel functionality. We previously demonstrated that BrS patients show decreased protein sialylation, correlating with reduced sodium channel activity. This study aimed to investigate sialic acid's role in regulating NaV1.5 activity, testing if sialylation induction via mannosamine (ManNAc) supplementation could restore channel activity in-vitro. Preliminary tests with exogenous sialidases and ManNAc supplementation affirmed our hypothesis. We examined ManNAc's effect on NaV1.5 mutants common in BrS, which typically exhibit partial functional loss. We overexpressed mutations p.A344S, p.K1479A, and p.G1406R in HEK293A cells and measured sodium currents before and after ManNAc supplementation to stimulate sialic acid synthesis. ManNAc effectively restored sodium currents in mutants with intracellular and extracellular loop mutations. However, the p.1406G>A mutant, affecting the pore structure, showed no improvement, suggesting that sialic acid mainly influences channel membrane trafficking rather than direct glycosylation. This underscores the complexity of sialic acid's role and points to trafficking pathways as potential therapeutic targets. Notably, the treatment neither increased nor decreased sodium currents in wild-type NaV1.5 channels or in the CaV3.2 calcium channel, underscoring its specificity. In summary, our findings demonstrate that ManNAc supplementation can selectively restore the function of certain NaV1.5 mutants without affecting wild-type channels, highlighting the potential of targeting sialylation pathways for SCN5A-related arrhythmia treatments. Future research should aim to unravel these mechanisms further, providing insights into novel treatments for SCN5A-related arrhythmias.

Acupuncture modulation of the ACE/Ang II/AT1R and ACE2/Ang(1-7)/MasR pathways in the rostral ventrolateral medulla reduces sympathetic output and prevents cardiac injury caused by SHR hypertension.

Acupuncture can reduce blood pressure, heart rate (HR), and ameliorate cardiac damage by modulating the excitability of the sympathetic nervous system, but the exact mechanism of this effect remains unclear. This study investigated the potential mechanisms of acupuncture in the treatment of cardiac damage in hypertension. Spontaneously hypertensive rats (SHR) were used as the hypertension model with Wistar-Kyoto rats as the control. Manual acupuncture, electroacupuncture, and metoprolol were used as interventions. Systolic and diastolic blood pressure (SBP, DBP) plus HR were monitored with cardiac structure determined using Masson staining. Angiotensin II (Ang II) and norepinephrine in myocardium were detected with ELISA as was Ang(1-7) and gamma aminobutyric acid (GABA) in the rostral ventrolateral medulla (RVLM). Expression of mRNA for collagen type I (Col-I), Col-III, actin α1 (ACTA1), and thrombospondin 4 (THBS4) in myocardium was detected using real-time PCR. Expression of angiotensin converting enzyme (ACE), Ang II, angiotensin II type 1 receptor (AT1R), ACE2, and Mas receptor (MasR) proteins in RVLM was monitored using western blot. After manual acupuncture and electroacupuncture treatment, SHRs showed decreased SBP, DBP and HR, reduced myocardial damage. There was decreased expression of the ACE/Ang II/AT1R axis, and increased expression of the ACE2/Ang(1-7)/MasR axis within the RVLM. GABA levels were increased within the RVLM and norepinephrine levels were decreased in myocardial tissue. Metoprolol was more effective than either manual acupuncture or electroacupuncture. Acupuncture directed against hypertensive cardiac damage may be associated with regulation of ACE/Ang II/AT1R and the ACE2/Ang(1-7)/MasR pathway within the RLVM to reduce cardiac sympathetic excitability.

A simulation study on the antiarrhythmic mechanisms of established agents in myocardial ischemia and infarction.

Patients with myocardial ischemia and infarction are at increased risk of arrhythmias, which in turn, can exacerbate the overall risk of mortality. Despite the observed reduction in recurrent arrhythmias through antiarrhythmic drug therapy, the precise mechanisms underlying their effectiveness in treating ischemic heart disease remain unclear. Moreover, there is a lack of specialized drugs designed explicitly for the treatment of myocardial ischemic arrhythmia. This study employs an electrophysiological simulation approach to investigate the potential antiarrhythmic effects and underlying mechanisms of various pharmacological agents in the context of ischemia and myocardial infarction (MI). Based on physiological experimental data, computational models are developed to simulate the effects of a series of pharmacological agents (amiodarone, telmisartan, E-4031, chromanol 293B, and glibenclamide) on cellular electrophysiology and utilized to further evaluate their antiarrhythmic effectiveness during ischemia. On 2D and 3D tissues with multiple pathological conditions, the simulation results indicate that the antiarrhythmic effect of glibenclamide is primarily attributed to the suppression of efflux of potassium ion to facilitate the restitution of [K+]o, as opposed to recovery of IKATP during myocardial ischemia. This discovery implies that, during acute cardiac ischemia, pro-arrhythmogenic alterations in cardiac tissue's excitability and conduction properties are more significantly influenced by electrophysiological changes in the depolarization rate, as opposed to variations in the action potential duration (APD). These findings offer specific insights into potentially effective targets for investigating ischemic arrhythmias, providing significant guidance for clinical interventions in acute coronary syndrome.

Photoinhibition of the hERG potassium channel PAS domain by ultraviolet light speeds channel closing

hERG potassium channels are critical for cardiac excitability. hERG channels have a Per-Arnt-Sim (PAS) domain at their N-terminus, and here, we examined the mechanism for PAS domain regulation of channel opening and closing (gating). We used TAG codon suppression to incorporate the noncanonical amino acid 4-benzoyl-L-phenylalanine (BZF), which is capable of forming covalent cross-links after photoactivation by ultraviolet (UV) light, at three locations (G47, F48, and E50) in the PAS domain. We found that hERG-G47BZF channels had faster closing (deactivation) when irradiated in the open state (at 0 mV) but showed no measurable changes when irradiated in the closed state (at −100 mV). hERG-F48BZF channels had slower activation, faster deactivation, and a marked rightward shift in the voltage dependence of activation when irradiated in the open (at 0 mV) or closed (at −100 mV) state. hERG-E50BZF channels had no measurable changes when irradiated in the open state (at 0 mV) but had slower activation, faster deactivation, and a rightward shift in the voltage dependence of activation when irradiated in the closed state (at −100mV), indicating that hERG-E50BZF had a state-dependent difference in UV photoactivation, which we interpret to mean that PAS underwent molecular motions between the open and closed states. Moreover, we propose that UV-dependent biophysical changes in hERG-G47BZF, F48BZF, and E50BZF were the direct result of photochemical cross-linking that reduced dynamic motions in the PAS domain and broadly stabilized the closed state relative to the open state of the channel.

Cardiac function is regulated by the sodium-dependent inhibition of the sodium-calcium exchanger NCX1

The Na+-Ca2+ exchanger (NCX1) is the dominant Ca2+ extrusion mechanism in cardiac myocytes. NCX1 activity is inhibited by intracellular Na+ via a process known as Na+-dependent inactivation. A central question is whether this inactivation plays a physiological role in heart function. Using CRISPR/Cas9, we inserted the K229Q mutation in the gene (Slc8a1) encoding for NCX1. This mutation removes the Na+-dependent inactivation while preserving transport properties and other allosteric regulations. NCX1 mRNA levels, protein expression, and protein localization are unchanged in K229Q male mice. However, they exhibit reduced left ventricular ejection fraction and fractional shortening, while displaying a prolonged QT interval. K229Q ventricular myocytes show enhanced NCX1 activity, resulting in action potential prolongation, higher incidence of aberrant action potentials, a faster decline of Ca2+ transients, and depressed cell shortening. The results demonstrate that NCX1 Na+-dependent inactivation plays an essential role in heart function by affecting both cardiac excitability and contractility.

The network of cardiac KIR2.1: its function, cellular regulation, electrical signaling, diseases and new drug avenues.

The functioning of the human heart relies on complex electrical and communication systems that coordinate cardiac contractions and sustain rhythmicity. One of the key players contributing to this intricate system is the KIR2.1 potassium ion channel, which is encoded by the KCNJ2 gene. KIR2.1 channels exhibit abundant expression in both ventricular myocytes and Purkinje fibers, exerting an important role in maintaining the balance of intracellular potassium ion levels within the heart. And by stabilizing the resting membrane potential and contributing to action potential repolarization, these channels have an important role in cardiac excitability also. Either gain- or loss-of-function mutations, but also acquired impairments of their function, are implicated in the pathogenesis of diverse types of cardiac arrhythmias. In this review, we aim to elucidate the system functions of KIR2.1 channels related to cellular electrical signaling, communication, and their contributions to cardiovascular disease. Based on this knowledge, we will discuss existing and new pharmacological avenues to modulate their function.

Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCβ. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCβ do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

Effects of aconitine on membrane currents and action potentials in neonatal rat ventricular myocytes and its impact on electrocardiographic changes

This study presents an analysis of the electrophysiological effects of aconitine, a toxic diterpenoid alkaloid derived from Aconitum plants, on neonatal rat ventricular myocytes (NRVMs). The research investigates the potential impact of aconitine on various ion currents and cardiac action potentials, shedding light on its arrhythmogenic properties. Whole-cell patch-clamp experiments were conducted to assess the effects of aconitine on delayed-rectifier K+ currents (IK(DR)) and inwardly rectifying K+ currents (IK(IR) in NRVMs. The findings indicate that aconitine exposure led to the inhibition of (IK(DR)) and (IK(IR)), suggesting its potential influence on cardiac repolarization and excitability. Notably, aconitine induced transient inward current (ITI) and early after-depolarizations (EADs) in a concentration- dependent manner, both of which have implications for cardiac arrhythmias. Moreover, the study examined the electrocardiogram changes in Sprague-Dawley rats upon aconitine injection, revealing a prolonged QT interval and the emergence of polymorphic ventricular tachycardia (VTs), indicative of arrhythmic effects. The study emphasizes the importance of understanding the electrophysiological impact of aconitine and similar compounds, considering their potential therapeutic applications and associated toxicities.

Two-pore channels (TPCs) acts as a hub for excitation-contraction coupling, metabolism and cardiac hypertrophy signalling

Ca2+ signaling is essential for cardiac contractility and excitability in heart function and remodeling. Intriguingly, little is known about the role of a new family of ion channels, the endo-lysosomal non-selective cation “two-pore channel” (TPCs) in heart function. Here we have used double TPC knock-out mice for the 1 and 2 isoforms of TPCs (Tpcn1/2−/−) and evaluated their cardiac function. Doppler-echocardiography unveils altered left ventricular (LV) systolic function associated with a LV relaxation impairment. In cardiomyocytes isolated from Tpcn1/2−/- mice, we observed a reduction in the contractile function with a decrease in the sarcoplasmic reticulum Ca2+ content and a reduced expression of various key proteins regulating Ca2+ stores, such as calsequestrin. We also found that two main regulators of the energy metabolism, AMP-activated protein kinase and mTOR, were down regulated. We found an increase in the expression of TPC1 and TPC2 in a model of transverse aortic constriction (TAC) mice and in chronically isoproterenol infused WT mice. In this last model, adaptive cardiac hypertrophy was reduced by Tpcn1/2 deletion. Here, we propose a central role for TPCs and lysosomes that could act as a hub integrating information from the excitation-contraction coupling mechanisms, cellular energy metabolism and hypertrophy signaling.

Trapped re-entry challenges the binary view on cardiac arrhythmias

Abstract Background There is currently a binary view on cardiac arrhythmias: (1) arrhythmias are present and thereby affect the atria or ventricles as a whole, or (2) they are not present and therefore the heart is in sinus rhythm. However, complex fractionated atrial electrograms (CFAEs) under sinus rhythm (SR) are observed in patients with atrial fibrillation (AF), raising the question whether both cardiac states (arrhythmia and SR) could coexist. Although CFAEs in AF patients have been associated with functional and structural heterogeneities (e.g. dense fibrotic regions), the underlying mechanisms of fractionation under SR remain incompletely understood. Purpose To test the hypothesis that an arrhythmia can exist locally with the majority of cardiac tissue in sinus rhythm, made possible through dense local fibrotic regions forming a small electrically isolated re-entrant circuit that can "trap" excitation waves, which can only be "released" under dynamic tissue changes at an isthmus connected to the bulk of the tissue. Methods By harnessing the unique possibilities of light-gated depolarizing ion channels (CatCh) to precisely control in vitro cardiac excitability in time and space, complemented with advanced computational modelling of whole human atria, we explored the geometry of such an electrically isolated circuit and assessed its clinical relevance. Results Optical mapping studies, in monolayers of CatCh-activated neonatal rat atrial cardiomyocytes (n=8), revealed that re-entry can be established and trapped by creating an electrically isolated pathway with a bulk-connecting isthmus causing source-sink mismatch. A tachyarrhythmia was shown to exist locally with SR prevailing in the bulk of the monolayer. Next, conditions were found under which re-entry could escape this pathway, thereby converting a local dormant arrhythmic source into an active driver with global impact. Escape could be established by overcoming the source-sink mismatch through widening of the isthmus or a reduction of the gap junctional coupling. In a digital twin of the human atria, it was revealed that the conditions for "trapped re-entry" and its release can be realized as well. Unipolar pseudo-electrograms derived from these complementary computational 3D studies showed CFAEs at the site of "trapped re-entry" in coexistence with normal electrograms of SR in the bulk of the atria. Upon release of the re-entry, acute arrhythmia onset occurred, affecting the complete atria as evidenced by wave front and electrogram visualization. Conclusion Through the concept of "trapped re-entry", we not only present a new mechanism of acute manifestation of focal tachyarrhythmias, but also provide novel insight into the origin of fractionated electrograms. This insight may provide new rationales for treatment of cardiac arrhythmias, especially for ablation by site-directed targeting.Trapped re-entry in vitro and in silico

Abstract 16957: PRKAG2 Directly Interacts With Myosin Heavy Chains to Modulate Myocyte Electrical and Morphological Changes in Cardiac Hypertrophy

Introduction: Mutations in the PRKAG2 gene cause a complex myocardial disorder characterized by hypertrophic cardiomyopathy (HCM) and conduction disturbances. While prior studies associated PRKAG2 -linked hypertrophy with increased glycogen storage, many HCM phenotypes in the disorder remain unexplained by this effect alone. We aimed to uncover the molecular mechanisms by which PRKAG2 mutations induce myocyte hypertrophy and electrical changes during cardiac development. Methods: We created transgenic zebrafish expressing WT (Tg WT ) and pathological mutant (Tg R299Q ) murine Prkag2 cDNA under a myocardium-specific promoter. Using these models, we studied the impact of Prkag2 variant on myocyte electrical activity, metabolism, and cytoarchitecture across cardiogenesis and in mature hearts. Results: Tg R299Q adult fish showed hypertrophic cardiomyocytes, swollen mitochondria, and shortened sarcomere length compared to Tg WT and WT, mirroring human HCM phenotypes. The glycogen content was elevated in adult hearts but not during early cardiogenesis. Despite the absence of glycogen accumulation at 6-day post fertilization, Tg R299Q hearts showed electrical abnormalities, including a reduced conduction velocity, decreased Ca 2+ amplitude, and prolonged action potential durations in both atria and ventricles, compared to Tg WT and WT. Co-immunoprecipitation and proximity ligation assay revealed physical interactions between Prkag2 and myosin heavy chain (MYH), with the R299Q variant enhancing this interaction and altering the subcellular localization of Prkag2 from mitochondria to sarcomeres. Knockdown of the fish homolog of MYH7 ( vmhcl ) restored the early electrical disturbances caused by Tg R299Q . This abnormal MYH-Prkag2 interaction disturbed mitochondria and sarcomere organization contributing to altered myocyte cytoarchitecture observed in Tg R299Q . Conclusions: Mutant PRKAG2 altered cardiac excitability and Ca 2+ handling during embryogenesis, preceding glycogen accumulation. Enhanced binding between Prkag2 and MYHs contributed to these early changes. Our study revealed a novel link between sarcomere proteins and metabolic regulators in cardiac hypertrophy, suggesting a new therapeutic avenue for treating HCM.

Scar fibroblasts affect cardiac excitability.

Scar fibroblasts affect cardiac excitability.

Activation of neurokinin-III receptors modulates human atrial TASK-1 currents

RationaleThe neurokinin-III receptor was recently shown to regulate atrial cardiomyocyte excitability by inhibiting atrial background potassium currents. TASK-1 (hK2P3.1) two-pore-domain potassium channels, which are expressed atrial-specifically in the human heart, contribute significantly to atrial background potassium currents. As TASK-1 channels are regulated by a variety of intracellular signalling cascades, they represent a promising candidate for mediating the electrophysiological effects of the Gq-coupled neurokinin-III receptor. ObjectiveTo investigate whether TASK-1 channels mediate the neurokinin-III receptor activation induced effects on atrial electrophysiology. Methods and resultsIn Xenopus laevis oocytes, heterologously expressing neurokinin-III receptor and TASK-1, administration of the endogenous neurokinin-III receptor ligands substance P or neurokinin B resulted in a strong TASK-1 current inhibition. This could be reproduced by application of the high affinity neurokinin-III receptor agonist senktide. Moreover, preincubation with the neurokinin-III receptor antagonist osanetant blunted the effect of senktide. Mutagenesis studies employing TASK-1 channel constructs which lack either protein kinase C (PKC) phosphorylation sites or the domain which is regulating the diacyl glycerol (DAG) sensitivity domain of TASK-1 revealed a protein kinase C independent mechanism of TASK-1 current inhibition: upon neurokinin-III receptor activation TASK-1 channels are blocked in a DAG-dependent fashion. Finally, effects of senktide on atrial TASK-1 currents could be reproduced in patch-clamp measurements, performed on isolated human atrial cardiomyocytes. ConclusionsHeterologously expressed human TASK-1 channels are inhibited by neurokinin-III receptor activation in a DAG dependent fashion. Patch-clamp measurements, performed on human atrial cardiomyocytes suggest that the atrial-specific effects of neurokinin-III receptor activation on cardiac excitability are predominantly mediated via TASK-1 currents.

The fungicide tebuconazole modulates the sodium current of human NaV1.5 channels expressed in HEK293cells.

The fungicide Tebuconazole is a widely used pesticide in agriculture and may cause cardiotoxicity. In our present investigation the effect of Tebuconazole on the sodium current (INa) of human cardiac sodium channels (NaV1.5) was studied using a heterologous expression system and whole-cell patch-clamp techniques. Tebuconazole reduced the amplitude of the peak INa in a concentration- and voltage-dependent manner. At the holding potential of -120 mV the IC50 was estimated at 204.1±34.3μM, while at -80 mV the IC50 was 0.3±0.1μM. The effect of the fungicide is more pronounced at more depolarized potentials, indicating a state-dependent interaction. Tebuconazole caused a negative shift in the half-maximal inactivation voltage and delayed recovery from fast inactivation of INa. Also, it enhanced closed-state inactivation, exhibited use-dependent block in a voltage-dependent manner. Furthermore, Tebuconazole reduced the increase in late sodium current induced by the pyrethroid insecticide β-Cyfluthrin. These results suggest that Tebuconazole can interact with NaV1.5 channels and modulate INa. The observed effects may lead to decreased cardiac excitability through reduced INa availability, which could be a new mechanism of cardiotoxicity to be attributed to the fungicide.

Fibroblasts in heart scar tissue directly regulate cardiac excitability and arrhythmogenesis.

After heart injury, dead heart muscle is replaced by scar tissue. Fibroblasts can electrically couple with myocytes, and changes in fibroblast membrane potential can lead to myocyte excitability, which suggests that fibroblast-myocyte coupling in scar tissue may be responsible for arrhythmogenesis. However, the physiologic relevance of electrical coupling of myocytes and fibroblasts and its impact on cardiac excitability in vivo have never been demonstrated. We genetically engineered a mouse that expresses the optogenetic cationic channel ChR2 (H134R) exclusively in cardiac fibroblasts. After myocardial infarction, optical stimulation of scar tissue elicited organ-wide cardiac excitation and induced arrhythmias in these animals. Complementing computational modeling with experimental approaches, we showed that gap junctional and ephaptic coupling, in a synergistic yet functionally redundant manner, excited myocytes coupled to fibroblasts.

A bioelectrical phase transition patterns the first vertebrate heartbeats.

A regular heartbeat is essential to vertebrate life. In the mature heart, this function is driven by an anatomically localized pacemaker. By contrast, pacemaking capability is broadly distributed in the early embryonic heart1-3, raising the question of how tissue-scale activity is first established and then maintained during embryonic development. The initial transition of the heart from silent to beating has never been characterized at the timescale of individual electrical events, and the structure in space and time of the early heartbeats remains poorly understood. Using all-optical electrophysiology, we captured the very first heartbeat of a zebrafish and analysed the development of cardiac excitability and conduction around this singular event. The first few beats appeared suddenly, had irregular interbeat intervals, propagated coherently across the primordial heart and emanated from loci that varied between animals and over time. The bioelectrical dynamics were well described by a noisy saddle-node on invariant circle bifurcation with action potential upstroke driven by CaV1.2. Our work shows how gradual and largely asynchronous development of single-cell bioelectrical properties produces a stereotyped and robust tissue-scale transition from quiescence to coordinated beating.

SUMOylation of the cardiac sodium channel NaV1.5 modifies inward current and cardiac excitability

Decreased peak sodium current (INa) and increased late sodium current (INa,L), through the cardiac sodium channel NaV1.5 encoded by SCN5A, cause arrhythmias. Many NaV1.5 post-translational modifications have been reported. A recent report concluded that acute hypoxia increases INa,L by increasing a Small Ubiquitin-like MOdifier (SUMOylation) at K442-NaV1.5. To determine whether and by what mechanisms SUMOylation alters INa, INa,L and cardiac electrophysiology. SUMOylation of NaV1.5 was detected by immunoprecipitation and immunoblotting. INa was measured by patch clamp with/without SUMO1 overexpression in HEK293 cells expressing wild type (WT) or K442R-NaV1.5 and in neonatal rat cardiac myocytes (NRCMs). SUMOylation effects were studied in vivo by electrocardiograms and ambulatory telemetry using Scn5a heterozygous knockout (SCN5A+/-) mice and the de-SUMOylating protein SENP2 (AAV9-SENP2), AAV9-SUMO1, or the SUMOylation inhibitor anacardic acid. NaV1.5 trafficking was detected by immunofluorescence. NaV1.5 was SUMOylated in HEK293 cells, NRCMs and human heart tissue. HyperSUMOylation at NaV1.5-K442 increased INa in NRCMs and in HEK cells overexpressing WT but not K442R-Nav1.5. SUMOylation did not alter other channel properties including INa,L. AAV9-SENP2 or anacardic acid decreased INa, prolonged QRS duration, and produced heart block and arrhythmias in SCN5A+/- mice, while AAV9-SUMO1 increased INa and shortened QRS duration . SUMO1 overexpression enhanced membrane localization of NaV1.5. SUMOylation of K442-Nav1.5 increases peak INa without changing INa,L, at least in part by altering membrane abundance. Our findings do not support SUMOylation as a mechanism for changes in INa,L. Nav1.5 SUMOylation may modify arrhythmic risk in disease states and represents a potential target for pharmacological manipulation.