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Key points A fundamental question in cardiovascular and muscle physiology is how the heart operates in synchrony with distinct muscles to regulate homeostasis, enable movement and adapt to exercise demands and fatigue. Here we investigate how autonomic regulation of cardiac function synchronizes and integrates as a network with the activity of distinct muscles during exercise. Further, we establish how the network of cardio‐muscular interactions reorganizes with fatigue. Thirty healthy young adults performed two body weight squat tests until exhaustion. Simultaneous recordings were taken of a 3‐lead electrocardiogram (EKG) along with electromyography (EMG) signals from the left and right vastus lateralis, and left and right erector spinae. We first obtained instantaneous heart rate (HR) derived from the EKG signal and decomposed the EMG recordings in 10 frequency bands (F1–F10). We next quantified pair‐wise coupling (cross‐correlation) between the time series for HR and all EMG spectral power frequency bands in each leg and back muscle. We uncovered the first profiles of cardio‐muscular network interactions, which depend on the role muscles play during exercise and muscle fibre histochemical characteristics. Additionally, we observed a significant decline in the degree of cardio‐muscular coupling with fatigue, characterized by complex transitions from synchronous to asynchronous behaviour across a range of timescales. The network approach we utilized introduces new avenues for the development of novel network‐based markers, with the potential to characterize multilevel cardio‐muscular interactions to assess global health, levels of fatigue, fitness status or the effectiveness of cardiovascular and muscle injury rehabilitation programmes. The heart operates in synchrony with muscles to regulate homeostasis, enable movement, and adapt to exercise demands and fatigue. However, the precise mechanisms regulating cardio‐muscular coupling remain unknown. This study introduces a pioneering approach to assess cardio‐muscular network interactions by examining the synchronization of cardiac function with muscle activity during exercise and fatigue. We uncover the first profiles of cardio‐muscular interactions characterized by specific hierarchical organization of link strength. We observe a significant decline in the degree of cardio‐muscular coupling with fatigue, marked by complex transitions from synchronous to asynchronous behaviour. This network approach offers new network‐based markers to characterize multilevel cardio‐muscular interactions to assess global health, levels of fatigue, fitness status or the effectiveness of cardiovascular and muscle injury rehabilitation programmes. [ABSTRACT FROM AUTHOR]

The atrioventricular node (AVN) is a crucial component of the cardiac conduction system. Despite its pivotal role in regulating the transmission of electrical signals between atria and ventricles, a comprehensive understanding of the cellular electrophysiological mechanisms governing AVN function has remained elusive. This paper presents a detailed computational model of mouse AVN cell action potential (AP). Our model builds upon previous work and introduces several key refinements, including accurate representation of membrane currents and exchangers, calcium handling, cellular compartmentalization, dynamic update of intracellular ion concentrations, and calcium buffering. We recalibrated and validated the model against existing and unpublished experimental data. In control conditions, our model reproduces the AVN AP experimental features, (e.g. rate = 175 bpm, experimental range [121, 191] bpm). Notably, our study sheds light on the contribution of L‐type calcium currents, through both Cav1.2 and Cav1.3 channels, in AVN cells. The model replicates several experimental observations, including the cessation of firing upon block of Cav1.3 or INa,r current. If block induces a reduction in beating rate of 11%. In summary, this work presents a comprehensive computational model of mouse AVN cell AP, offering a valuable tool for investigating pacemaking mechanisms and simulating the impact of ionic current blockades. By integrating calcium handling and refining formulation of ionic currents, our model advances understanding of this critical component of the cardiac conduction system, providing a platform for future developments in cardiac electrophysiology. Key points: This paper introduces a comprehensive computational model of mouse atrioventricular node (AVN) cell action potentials (APs).Our model is based on the electrophysiological data from isolated mouse AVN cells and exhibits an action potential and calcium transient that closely match the experimental records.By simulating the effects of blocking specific ionic currents, the model effectively predicts the roles of L‐type Cav1.2 and Cav1.3 channels, T‐type calcium channels, sodium currents (TTX‐sensitive and TTX‐resistant), and the funny current (If) in AVN pacemaking.The study also emphasizes the significance of other ionic currents, including IKr, Ito, IKur, in regulating AP characteristics and cycle length in AVN cells.The model faithfully reproduces the rate dependence of action potentials under pacing, opening the possibility of use in impulse propagation models.The population‐of‐models approach showed the robustness of this new AP model in simulating a wide spectrum of cellular pacemaking in AVN. [ABSTRACT FROM AUTHOR]

Electromechanical reciprocity – comprising electro‐mechanical (EMC) and mechano‐electric coupling (MEC) – provides cardiac adaptation to changing physiological demands. Understanding electromechanical reciprocity and its impact on function and heterogeneity in pathological conditions – such as (drug‐induced) acquired long QT syndrome (aLQTS) – might lead to novel insights in arrhythmogenesis. Our aim is to investigate how electrical changes impact on mechanical function (EMC) and vice versa (MEC) under physiological conditions and in aLQTS. To measure regional differences in EMC and MEC in vivo, we used tissue phase mapping cardiac MRI and a 24‐lead ECG vest in healthy (control) and IKr‐blocker E‐4031‐induced aLQTS rabbit hearts. MEC was studied in vivo by acutely increasing cardiac preload, and ex vivo by using voltage optical mapping (OM) in beating hearts at different preloads. In aLQTS, electrical repolarization (heart rate corrected RT‐interval, RTn370) was prolonged compared to control (P < 0.0001) with increased spatial and temporal RT heterogeneity (P < 0.01). Changing electrical function (in aLQTS) resulted in significantly reduced diastolic mechanical function and prolonged contraction duration (EMC), causing increased apico‐basal mechanical heterogeneity. Increased preload acutely prolonged RTn370 in both control and aLQTS hearts (MEC). This effect was more pronounced in aLQTS (P < 0.0001). Additionally, regional RT‐dispersion increased in aLQTS. Motion‐correction allowed us to determine APD‐prolongation in beating aLQTS hearts, but limited motion correction accuracy upon preload‐changes prevented a clear analysis of MEC ex vivo. Mechano‐induced RT‐prolongation and increased heterogeneity were more pronounced in aLQTS than in healthy hearts. Acute MEC effects may play an additional role in LQT‐related arrhythmogenesis, warranting further mechanistic investigations. Key points: Electromechanical reciprocity comprising excitation‐contraction coupling (EMC) and mechano‐electric feedback loops (MEC) is essential for physiological cardiac function.Alterations in electrical and/or mechanical heterogeneity are known to have potentially pro‐arrhythmic effects.In this study, we aimed to investigate how electrical changes impact on the mechanical function (EMC) and vice versa (MEC) both under physiological conditions (control) and in acquired long QT syndrome (aLQTS).We show that changing the electrical function (in aLQTS) results in significantly altered mechanical heterogeneity via EMC and, vice versa, that increasing the preload acutely prolongs repolarization duration and increases electrical heterogeneity, particularly in aLQTS as compared to control.Our results substantiate the hypothesis that LQTS is an ‛electro‐mechanical', rather than a 'purely electrical', disease and suggest that acute MEC effects may play an additional role in LQT‐related arrhythmogenesis. [ABSTRACT FROM AUTHOR]

Since their discovery nearly 30 years ago, fibroblast growth factor homologous factors (FHFs) are now known to control the functionality of excitable tissues through a range of mechanisms. Nervous and cardiac system dysfunctions are caused by loss‐ or gain‐of‐function mutations in FHF genes. The best understood 'canonical' targets for FHF action are voltage‐gated sodium channels, and recent studies have expanded the repertoire of ways that FHFs modulate sodium channel gating. Additional 'non‐canonical' functions of FHFs in excitable and non‐excitable cells, including cancer cells, have been reported over the past dozen years. This review summarizes and evaluates reported canonical and non‐canonical FHF functions. [ABSTRACT FROM AUTHOR]

T‐tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well‐co‐ordinated excitation–contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all‐optical approach to stimulate and record trans‐membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT‐generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho‐functional changes reduce the electrical connectivity between SS and TT membranes. Key points: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T‐tubular system) that propagate action potentials to the core of the cell, causing uniform excitation–contraction coupling across the cell.In the present study, we investigated whether the T‐tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma.For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains.Our findings demonstrate that T‐tubules are intrinsically excitable, revealing distinct characteristics of self‐generated T‐tubular action potentials.This active electrical capability would protect cells from voltage drops potentially occurring within the T‐tubular network. [ABSTRACT FROM AUTHOR]

Cardiac function is tightly regulated by the autonomic nervous system (ANS). Activation of the sympathetic nervous system increases cardiac output by increasing heart rate and stroke volume, while parasympathetic nerve stimulation instantly slows heart rate. Importantly, imbalance in autonomic control of the heart has been implicated in the development of arrhythmias and heart failure. Understanding of the mechanisms and effects of autonomic stimulation is a major challenge because synapses in different regions of the heart result in multiple changes to heart function. For example, nerve synapses on the sinoatrial node (SAN) impact pacemaking, while synapses on contractile cells alter contraction and arrhythmia vulnerability. Here, we present a multiscale neurocardiac modelling and simulator tool that predicts the effect of efferent stimulation of the sympathetic and parasympathetic branches of the ANS on the cardiac SAN and ventricular myocardium. The model includes a layered representation of the ANS and reproduces firing properties measured experimentally. Model parameters are derived from experiments and atomistic simulations. The model is a first prototype of a digital twin that is applied to make predictions across all system scales, from subcellular signalling to pacemaker frequency to tissue level responses. We predict conditions under which autonomic imbalance induces proarrhythmia and can be modified to prevent or inhibit arrhythmia. In summary, the multiscale model constitutes a predictive digital twin framework to test and guide high‐throughput prediction of novel neuromodulatory therapy. Key points: A multi‐layered model representation of the autonomic nervous system that includes sympathetic and parasympathetic branches, each with sparse random intralayer connectivity, synaptic dynamics and conductance based integrate‐and‐fire neurons generates firing patterns in close agreement with experiment.A key feature of the neurocardiac computational model is the connection between the autonomic nervous system and both pacemaker and contractile cells, where modification to pacemaker frequency drives initiation of electrical signals in the contractile cells.We utilized atomic‐scale molecular dynamics simulations to predict the association and dissociation rates of noradrenaline with the β‐adrenergic receptor.Multiscale predictions demonstrate how autonomic imbalance may increase proclivity to arrhythmias or be used to terminate arrhythmias.The model serves as a first step towards a digital twin for predicting neuromodulation to prevent or reduce disease. [ABSTRACT FROM AUTHOR]

Cardiac voltage‐gated sodium (Na+) channels (Nav1.5) are crucial for myocardial electrical excitation. Recent studies based on single‐channel recordings have suggested that Na+ channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de‐trend and idealize single‐channel currents, and assessed possible functional interactions in cell‐attached patch clamp experiments in HEK293 cells expressing human Nav1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de‐trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de‐trended sweeps. This reconstruction was achieved by non‐linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild‐type Nav1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na+ channel interactions. Key points: Nav1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating.Manual interventions when processing single‐channel recordings can lead to bias and inaccurate data interpretation.We developed an automated pipeline to de‐trend and idealize single‐channel currents and assessed possible functional interactions between Nav1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell‐attached patch clamp technique.In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild‐type Nav1.5 channels.Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins. [ABSTRACT FROM AUTHOR]

The article discusses the role of phosphorylation in the regulation of the cardiac ryanodine receptor (RyR2), a major calcium release channel in cardiomyocytes. The authors highlight the importance of three phosphorylation sites (Serine-2808, Serine-2030, and Serine-2814) in RyR2 and their association with different kinases. They also discuss the impact of phosphorylation on RyR2 activity and the potential implications for heart failure and arrhythmogenesis. The article presents recent research on the role of Serine-2030 and its relationship to RyR2 hyperactivity and arrhythmias. The authors emphasize the need for further research to fully understand the complex regulatory mechanisms of RyR2 and its potential as a therapeutic target for cardiac arrhythmias and failure. [Extracted from the article]

The postnatal mammalian heart undergoes remarkable developmental changes, which are stimulated by the transition from the intrauterine to extrauterine environment. With birth, increased oxygen levels promote metabolic, structural and biophysical maturation of cardiomyocytes, resulting in mature muscle with increased efficiency, contractility and electrical conduction. In this Topical Review article, we highlight key studies that inform our current understanding of human cardiomyocyte maturation. Collectively, these studies suggest that human atrial and ventricular myocytes evolve quickly within the first year but might not reach a fully mature adult phenotype until nearly the first decade of life. However, it is important to note that fetal, neonatal and paediatric cardiac physiology studies are hindered by a number of limitations, including the scarcity of human tissue, small sample size and a heavy reliance on diseased tissue samples, often without age‐matched healthy controls. Future developmental studies are warranted to expand our understanding of normal cardiac physiology/pathophysiology and inform age‐appropriate treatment strategies for cardiac disease. [ABSTRACT FROM AUTHOR]

Daily variations in cardiac electrophysiology and the incidence for different types of arrhythmias reflect ≈24 h changes in the environment, behaviour and internal circadian rhythms. This article focuses on studies that use animal models to separate the impact that circadian rhythms, as well as changes in the environment and behaviour, have on 24 h rhythms in heart rate and ventricular repolarization. Circadian rhythms are initiated at the cellular level by circadian clocks, transcription–translation feedback loops that cycle with a periodicity of 24 h. Several studies now show that the circadian clock in cardiomyocytes regulates the expression of cardiac ion channels by multiple mechanisms; underlies time‐of‐day changes in sinoatrial node excitability/intrinsic heart rate; and limits the duration of the ventricular action potential waveform. However, the 24 h rhythms in heart rate and ventricular repolarization are primarily driven by autonomic signalling. A functional role for the cardiomyocyte circadian clock appears to buffer the heart against perturbations. For example, the cardiomyocyte circadian clock limits QT‐interval prolongation (especially at slower heart rates), and it may facilitate the realignment of the 24 h rhythm in heart rate to abrupt changes in the light cycle. Additional studies show that modifying rhythmic behaviours (including feeding behaviour) can dramatically impact the 24 h rhythms in heart rate and ventricular repolarization. If these mechanisms are conserved, these studies suggest that targeting endogenous circadian mechanisms in the heart, as well as modifying the timing of certain rhythmic behaviours, could emerge as therapeutic strategies to support heart function against perturbations and regulate 24 h rhythms in cardiac electrophysiology. [ABSTRACT FROM AUTHOR]

It has been proposed that when gap junctional coupling is reduced in cardiac tissue, action potential propagation can be supported via ephaptic coupling, a mechanism mediated by negative electric potentials occurring in narrow intercellular clefts of intercalated discs (IDs). Recent studies showed that sodium (Na+) channels form clusters near gap junction plaques in nanodomains called perinexi, where the ID cleft is even narrower. To examine the electrophysiological relevance of Na+ channel clusters being located in perinexi, we developed a 3D finite element model of two longitudinally abutting cardiomyocytes, with a central Na+ channel cluster on the ID membranes. When this cluster was located in the perinexus of a closely positioned gap junction plaque, varying perinexal width greatly modulated impulse transmission from one cell to the other, with narrow perinexi potentiating ephaptic coupling. This modulation occurred via the interplay of Na+ currents, extracellular potentials in the cleft and patterns of current flow within the cleft. In contrast, when the Na+ channel cluster was located remotely from the gap junction plaque, this modulation by perinexus width largely disappeared. Interestingly, the Na+ current in the ID membrane of the pre‐junctional cell switched from inward to outward during excitation, thus contributing ions to the activating channels on the post‐junctional ID membrane. In conclusion, these results indicate that the localization of Na+ channel clusters in the perinexi of gap junction plaques is crucial for ephaptic coupling, which is furthermore greatly modulated by perinexal width. These findings are relevant for a comprehensive understanding of cardiac excitation. Key points: Ephaptic coupling is a cardiac conduction mechanism involving nanoscale‐level interactions between the sodium (Na+) current and the extracellular potential in narrow intercalated disc clefts.When gap junctional coupling is reduced, ephaptic coupling acts in conjunction with the classical cardiac conduction mechanism based on gap junctional current flow.In intercalated discs, Na+ channels form clusters that are preferentially located in the periphery of gap junction plaques, in nanodomains known as perinexi, but the electrophysiological role of these perinexi has never been examined.In our new 3D finite element model of two cardiac cells abutting each other with their intercalated discs, a Na+ channel cluster located inside a narrowed perinexus facilitated impulse transmission via ephaptic coupling.Our simulations demonstrate the role of narrowed perinexi as privileged sites for ephaptic coupling in pathological situations when gap junctional coupling is decreased. [ABSTRACT FROM AUTHOR]

Key points: In adult ventricular myocytes, the slow delayed rectifier (IKs) channels are distributed on the surface sarcolemma, not t‐tubules.In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools.KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface.Liquid chromatography/tandem mass spectrometry applied to affinity‐purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly.Microtubule plus‐end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface.An LQT1‐associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. Slow delayed rectifier (IKs) channels consist of KCNQ1 and KCNE1. IKs functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β‐adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form IKs channels, and the distribution pattern of IKs channels relative to β‐adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or IKs, to provide new insights into these issues. 'Retention‐using‐selected‐hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide‐bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected IKs channels in surface sarcolemma but not t‐tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein‐tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into IKs channels. This mode of delayed channel assembly helps IKs fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus‐end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer. Key points: In adult ventricular myocytes, the slow delayed rectifier (IKs) channels are distributed on the surface sarcolemma, not t‐tubules.In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools.KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into IKs channels on the cell surface.Liquid chromatography/tandem mass spectrometry applied to affinity‐purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly.Microtubule plus‐end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface.An LQT1‐associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer. [ABSTRACT FROM AUTHOR]

Prior studies modelled how the cardiac autonomic nervous system (ANS) influences high-level cardiac physiology (Kember et al., [[8], [7]]) but models directly linking the cardiac ANS to cardiac electrophysiology are lacking. Keywords: autonomic nervous system; cardiac electrophysiology; computational modelling; modelling; neurocardiology EN autonomic nervous system cardiac electrophysiology computational modelling modelling neurocardiology 3693 3694 2 09/05/23 20230901 NES 230901 Neurocardiology is a rapidly progressing scientific and clinical field that shows significant promise in the prevention and treatment of arrhythmias (Shivkumar et al., [10]). The true advantage of simultaneously simulating both ANS electrophysiology and cardiac myocyte electrophysiology as opposed to simply pre-programing autonomic input on rails is the opportunity for feedback loops and emergent behaviours to develop. [Extracted from the article]

Key points: Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.PKA phosphorylation attenuates ISK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i.This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+/voltage‐dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465. Key points: Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.PKA phosphorylation attenuates ISK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i.This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. [ABSTRACT FROM AUTHOR]

Key points: Mutations in the caveolae scaffolding protein, caveolin‐3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood.In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation‐specific gain of function effects on Cav1.2‐encoded L‐type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv4.2 and Kv4.3 channels responsible for Ito.A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3‐F97C is the slowly inactivating ICa,L but, for Cav3‐S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation.Overall, the LQT9 Cav3‐F97C and Cav3‐S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation‐specific therapeutic approaches. Mutations in the CAV3 gene encoding caveolin‐3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long‐QT syndrome (LQT9). Initial studies demonstrated that LQT9‐associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole‐cell, patch clamp technique to characterize the effect of Cav3‐F97C and Cav3‐S141R mutations on heterologously expressed Cav1.2+Cavβ2cN4 channels, as well as Kv4.2 and Kv4.3 channels, in HEK 293 cells. Expression of Cav3‐S141R increased ICa,L density without changes in gating properties, whereas expression of Cav3‐F97C reduced Ca2+‐dependent inactivation of ICa,L without changing current density. The Cav3‐F97C mutation reduced current density and altered the kinetics of IKv4.2 and IKv4.3 and also slowed recovery from inactivation. Cav3‐S141R decreased current density and also slowed activation kinetics and recovery from inactivation of IKv4.2 but had no effect on IKv4.3. Using the O'Hara–Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation‐induced changes in Ito are predicted to have negligible effect on APD, whereas blunted Ca2+‐dependent inactivation of ICa,L by Cav3‐F97C is predicted to be primarily responsible for APD prolongation, although increased ICa,L and late INa by Cav3‐S141R contribute equally to APD prolongation. Thus, LQT9 Cav3‐associated mutations, F97C and S141R, produce mutation‐specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation‐specific therapeutic approaches in the future. Key points: Mutations in the caveolae scaffolding protein, caveolin‐3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood.In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation‐specific gain of function effects on Cav1.2‐encoded L‐type Ca2+ channels responsible for ICa,L and also cause loss of function effects on heterologously expressed Kv4.2 and Kv4.3 channels responsible for Ito.A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3‐F97C is the slowly inactivating ICa,L but, for Cav3‐S141R, both increased ICa,L and increased late Na+ current contribute equally to action potential duration prolongation.Overall, the LQT9 Cav3‐F97C and Cav3‐S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation‐specific therapeutic approaches. [ABSTRACT FROM AUTHOR]

Keywords: cardiac electrophysiology; cardiac function; excitation-contraction coupling; mechano-chemo transduction; mechano-electrical transduction; mitochondria EN cardiac electrophysiology cardiac function excitation-contraction coupling mechano-chemo transduction mechano-electrical transduction mitochondria 1275 1277 3 04/06/20 20200401 NES 200401 GRAPH Remembering Jeremy Rice, a wonderful colleague gone too soon Before discussing the 2018 meeting content, we wish to commemorate Dr J. Jeremy Rice, who died tragically during the conference. The 2018 symposium focused on cardiac mechanics and energetics in eight sessions: (1) Mechanics and energetics in heart diseases and arrhythmias; (2) Genetic mutations illuminate mechanics-energetics interplay; (3) Mechano-chemo-transduction; (4) Mechano-electro-transduction; (5) Mitochondria energetics in heart diseases and arrhythmias; (6) Mitochondrial Ca SP 2+ sp and reactive oxygen species (ROS); (7) Feedbacks between energetics and excitation-contraction coupling; (8) Mechanics-energetics feedbacks and therapeutic targets. ([10]) summarizes conference presentations and discussions on the cardiac mechanics and mechanotransduction topics (mechano-electro-transduction or coupling (MEC) and mechano-chemo-transduction (MCT)). Cardiac electrophysiology, cardiac function, excitation-contraction coupling, mechano-chemo transduction, mechano-electrical transduction, mitochondria. [Extracted from the article]

Cardiac voltage-gated sodium (Na + ) channels (Na v 1.5) are crucial for myocardial electrical excitation. Recent studies based on single-channel recordings have suggested that Na + channels interact functionally and exhibit coupled gating. However, the analysis of such recordings frequently relies on manual interventions, which can lead to bias. Here, we developed an automated pipeline to de-trend and idealize single-channel currents, and assessed possible functional interactions in cell-attached patch clamp experiments in HEK293 cells expressing human Na v 1.5 channels as well as in adult mouse and rabbit ventricular cardiomyocytes. Our pipeline involved de-trending individual sweeps by linear optimization using a library of predefined functions, followed by digital filtering and baseline offset. Subsequently, the processed sweeps were idealized based on the idea that the ensemble average of the idealized current identified by thresholds between current levels reconstructs at best the ensemble average current from the de-trended sweeps. This reconstruction was achieved by non-linear optimization. To ascertain functional interactions, we examined the distribution of the numbers of open channels at every time point during the activation protocol and compared it to the distribution expected for independent channels. We also examined whether the channels tended to synchronize their openings and closings. However, we did not uncover any solid evidence of such interactions in our recordings. Rather, our results indicate that wild-type Na v 1.5 channels are independent entities or exhibit only very weak functional interactions that are probably irrelevant under physiological conditions. Nevertheless, our unbiased analysis will be important for further studies examining whether auxiliary proteins potentiate functional Na + channel interactions. KEY POINTS: Na v 1.5 channels are critical for cardiac excitation. They are part of macromolecular interacting complexes, and it was previously suggested that two neighbouring channels may functionally interact and exhibit coupled gating. Manual interventions when processing single-channel recordings can lead to bias and inaccurate data interpretation. We developed an automated pipeline to de-trend and idealize single-channel currents and assessed possible functional interactions between Na v 1.5 channels in HEK293 cells and cardiomyocytes during activation protocols using the cell-attached patch clamp technique. In recordings consisting of up to 1000 sweeps from the same patch, our analysis did not reveal any evidence of functional interactions or coupled gating between wild-type Na v 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na + channel interactions are affected by mutations and auxiliary proteins., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key points: Although optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all‐optical platform to monitor and control electrical activity in real‐time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed‐loop approach was also applied to simulate a re‐entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof‐of‐concept that a real‐time all‐optical stimulation can control cardiac rhythm in normal and abnormal conditions. Abstract: Optogenetics has provided new insights in cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all‐optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide‐field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free‐run mode with submillisecond temporal resolution or in a closed‐loop fashion: a tailored hardware and software platform allowed real‐time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real‐time intraventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real‐time resynchronization therapy and cardiac defibrillation. Furthermore, the closed‐loop approach was applied to simulate a re‐entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof‐of‐concept that a real‐time optically based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart. [ABSTRACT FROM AUTHOR]

Key points: It has been proposed that ephaptic conduction, relying on interactions between the sodium (Na+) current and the extracellular potential in intercalated discs, might contribute to cardiac conduction when gap junctional coupling is reduced, but this mechanism is still controversial. In intercalated discs, Na+ channels form clusters near gap junction plaques, but the functional significance of these clusters has never been evaluated. In HEK cells expressing cardiac Na+ channels, we show that restricting the extracellular space modulates the Na+ current, as predicted by corresponding simulations accounting for ephaptic effects. In a high‐resolution model of the intercalated disc, clusters of Na+ channels that face each other across the intercellular cleft facilitate ephaptic impulse transmission when gap junctional coupling is reduced. Thus, our simulations reveal a functional role for the clustering of Na+ channels in intercalated discs, and suggest that rearrangement of these clusters in disease may influence cardiac conduction. Abstract: It has been proposed that ephaptic interactions in intercalated discs, mediated by extracellular potentials, contribute to cardiac impulse propagation when gap junctional coupling is reduced. However, experiments demonstrating ephaptic effects on the cardiac Na+ current (INa) are scarce. Furthermore, Na+ channels form clusters around gap junction plaques, but the electrophysiological significance of these clusters has never been investigated. In patch clamp experiments with HEK cells stably expressing human Nav1.5 channels, we examined how restricting the extracellular space modulates INa elicited by an activation protocol. In parallel, we developed a high‐resolution computer model of the intercalated disc to investigate how the distribution of Na+ channels influences ephaptic interactions. Approaching the HEK cells to a non‐conducting obstacle always increased peak INa at step potentials near the threshold of INa activation and decreased peak INa at step potentials far above threshold (7 cells, P = 0.0156, Wilcoxon signed rank test). These effects were consistent with corresponding control simulations with a uniform Na+ channel distribution. In the intercalated disc computer model, redistributing the Na+ channels into a central cluster of the disc potentiated ephaptic effects. Moreover, ephaptic impulse transmission from one cell to another was facilitated by clusters of Na+ channels facing each other across the intercellular cleft when gap junctional coupling was reduced. In conclusion, our proof‐of‐principle experiments demonstrate that confining the extracellular space modulates cardiac INa, and our simulations reveal the functional role of the aggregation of Na+ channels in the perinexus. These findings highlight novel concepts in the physiology of cardiac excitation. [ABSTRACT FROM AUTHOR]

Key points At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics., Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality., Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic-banded rats exhibiting diastolic dysfunction., The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L-type Ca2+ channel and sarco/endoplasmic reticulum Ca2+ATPase as the principal regulators of systolic and diastolic Ca2+, respectively., Results show that the ability to dynamically change systolic Ca2+, through changes in expression of key Ca2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca2+ homeostasis in an efficient way to minimize systolic dysfunction., Abstract Elevated left ventricular afterload leads to myocardial hypertrophy, diastolic dysfunction, cellular remodelling and compromised calcium dynamics. At the cellular scale this remodelling of the ionic channels, pumps and exchangers gives rise to changes in the Ca2+ transient. However, the relative roles of the underlying subcellular processes and the positive or negative impact of each remodelling mechanism are not fully understood. Biophysical cardiac cell models were created to simulate electrophysiology and calcium dynamics in myocytes from control rats (SHAM) and aortic-banded rats exhibiting diastolic dysfunction. The model parameters and framework were validated and the fitted parameters demonstrated to be unique for explaining our experimental data. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L-type Ca2+ channel (LCC) and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) as the principal regulators of systolic and diastolic Ca2+, respectively. In the aortic banding model, the sensitivity of systolic Ca2+ to LCC density and diastolic Ca2+ to SERCA density decreased by 16-fold and increased by 23%, respectively, relative to the SHAM model. The energy cost of ionic homeostasis is maintained across the two models. The models predict that changes in ionic pathway densities in compensated aortic banding rats maintain Ca2+ function and efficiency. The ability to dynamically alter systolic function is significantly diminished, while the capacity to maintain diastolic Ca2+ is moderately increased. [ABSTRACT FROM AUTHOR]

Key points We constructed a comprehensive mathematical model of the spontaneous electrical activity of a human sinoatrial node (SAN) pacemaker cell, starting from the recent Severi-DiFrancesco model of rabbit SAN cells., Our model is based on electrophysiological data from isolated human SAN pacemaker cells and closely matches the action potentials and calcium transient that were recorded experimentally., Simulated ion channelopathies explain the clinically observed changes in heart rate in corresponding mutation carriers, providing an independent qualitative validation of the model., The model shows that the modulatory role of the 'funny current' ( If) in the pacing rate of human SAN pacemaker cells is highly similar to that of rabbit SAN cells, despite its considerably lower amplitude., The model may prove useful in the design of experiments and the development of heart-rate modulating drugs., Abstract The sinoatrial node (SAN) is the normal pacemaker of the mammalian heart. Over several decades, a large amount of data on the ionic mechanisms underlying the spontaneous electrical activity of SAN pacemaker cells has been obtained, mostly in experiments on single cells isolated from rabbit SAN. This wealth of data has allowed the development of mathematical models of the electrical activity of rabbit SAN pacemaker cells. The present study aimed to construct a comprehensive model of the electrical activity of a human SAN pacemaker cell using recently obtained electrophysiological data from human SAN pacemaker cells. We based our model on the recent Severi-DiFrancesco model of a rabbit SAN pacemaker cell. The action potential and calcium transient of the resulting model are close to the experimentally recorded values. The model has a much smaller 'funny current' ( If) than do rabbit cells, although its modulatory role is highly similar. Changes in pacing rate upon the implementation of mutations associated with sinus node dysfunction agree with the clinical observations. This agreement holds for both loss-of-function and gain-of-function mutations in the HCN4, SCN5A and KCNQ1 genes, underlying ion channelopathies in If, fast sodium current and slow delayed rectifier potassium current, respectively. We conclude that our human SAN cell model can be a useful tool in the design of experiments and the development of drugs that aim to modulate heart rate. [ABSTRACT FROM AUTHOR]

Key points A mathematical model of a small conductance Ca2+-activated potassium (SK) channel was developed and incorporated into a physiologically detailed ventricular myocyte model., Ca2+-sensitive K+ currents promote negative intracellular Ca2+ to membrane voltage (CAi2+→ Vm) coupling., Increase of Ca2+-sensitive K+ currents can be responsible for electromechanically discordant alternans and quasiperiodic oscillations at the cellular level., At the tissue level, Turing-type instability can occur when Ca2+-sensitive K+ currents are increased., Abstract Cardiac alternans is a precursor to life-threatening arrhythmias. Alternans can be caused by instability of the membrane voltage ( Vm), instability of the intracellular Ca2+ ( [ABSTRACT FROM AUTHOR]

Key points [Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current ( IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol., Rabbit ventricular rapidly activating delayed rectifier K+ current ( IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i., When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 n m., Abstract The slowly activating delayed rectifier K+ current ( IKs) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca2+ ([Ca2+]i) and β-adrenergic receptor (β-AR) stimulation modulate IKs amplitude and kinetics, but details of these important IKs regulators and their interaction are limited. We assessed the [Ca2+]i dependence of IKs in steady-state conditions and with dynamically changing membrane potential and [Ca2+]i during an AP. IKs was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca2+]i, we found that raising [Ca2+]i from 100 to 600 n m produced similar increases in IKs as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca2+]i increased maximally activated tail IKs, negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, IKs recorded during a normal physiological Ca2+ transient was similar to IKs measured with [Ca2+]i clamped at 500-600 n m. Thus, our study provides novel quantitative data as to how physiological [Ca2+]i regulates IKs amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca2+]i, in the submembrane or junctional cleft space, is not required to maximize [Ca2+]i-dependent IKs activation during normal Ca2+ transients. [ABSTRACT FROM AUTHOR]

Pacemaker activity of the sino-atrial node generates the heart rate. Disease of the sinus node and impairment of atrioventricular conduction induce an excessively low ventricular rate (bradycardia), which cannot meet the needs of the organism. Bradycardia accounts for about half of the total workload of clinical cardiologists. The 'sick sinus' syndrome (SSS) is characterized by sinus bradycardia and periods of intermittent atrial fibrillation. Several genetic or acquired risk factors or pathologies can lead to SSS. Implantation of an electronic pacemaker constitutes the only available therapy for SSS. The incidence of SSS is forecast to double over the next 50 years, with ageing of the general population thus urging the development of complementary or alternative therapeutic strategies. In recent years an increasing number of mutations affecting ion channels involved in sino-atrial automaticity have been reported to underlie inheritable SSS. L-type Cav1.3 channels play a major role in the generation and regulation of sino-atrial pacemaker activity and atrioventricular conduction. Mutation in the CACNA1D gene encoding Cav1.3 channels induces loss-of-function in channel activity and underlies the sino-atrial node dysfunction and deafness syndrome (SANDD). Mice lacking Cav1.3 channels ( Cav1.3−/−) fairly recapitulate SSS and constitute a precious model to test new therapeutic approaches to handle this disease. Work in our laboratory shows that targeting G protein-gated K+ ( IKACh) channels effectively rescues SSS of Cav1.3−/− mice. This new concept of 'compensatory' ion channel targeting shines new light on the principles underlying the pacemaker mechanism and may open the way to new therapies for SSS. [ABSTRACT FROM AUTHOR]

The article presents an editorial on the medical topic of Cardian K+ channels and cardiac electrophysiology techniques in the treatment of high-risk heart diseases including arrhythmia and heart failure. Details related to the release of white papers in the medical study of cardiac K+ channels is presented.

It has been proposed that when gap junctional coupling is reduced in cardiac tissue, action potential propagation can be supported via ephaptic coupling, a mechanism mediated by negative electric potentials occurring in narrow intercellular clefts of intercalated discs (IDs). Recent studies showed that sodium (Na + ) channels form clusters near gap junction plaques in nanodomains called perinexi, where the ID cleft is even narrower. To examine the electrophysiological relevance of Na + channel clusters being located in perinexi, we developed a 3D finite element model of two longitudinally abutting cardiomyocytes, with a central Na + channel cluster on the ID membranes. When this cluster was located in the perinexus of a closely positioned gap junction plaque, varying perinexal width greatly modulated impulse transmission from one cell to the other, with narrow perinexi potentiating ephaptic coupling. This modulation occurred via the interplay of Na + currents, extracellular potentials in the cleft and patterns of current flow within the cleft. In contrast, when the Na + channel cluster was located remotely from the gap junction plaque, this modulation by perinexus width largely disappeared. Interestingly, the Na + current in the ID membrane of the pre-junctional cell switched from inward to outward during excitation, thus contributing ions to the activating channels on the post-junctional ID membrane. In conclusion, these results indicate that the localization of Na + channel clusters in the perinexi of gap junction plaques is crucial for ephaptic coupling, which is furthermore greatly modulated by perinexal width. These findings are relevant for a comprehensive understanding of cardiac excitation. KEY POINTS: Ephaptic coupling is a cardiac conduction mechanism involving nanoscale-level interactions between the sodium (Na + ) current and the extracellular potential in narrow intercalated disc clefts. When gap junctional coupling is reduced, ephaptic coupling acts in conjunction with the classical cardiac conduction mechanism based on gap junctional current flow. In intercalated discs, Na + channels form clusters that are preferentially located in the periphery of gap junction plaques, in nanodomains known as perinexi, but the electrophysiological role of these perinexi has never been examined. In our new 3D finite element model of two cardiac cells abutting each other with their intercalated discs, a Na + channel cluster located inside a narrowed perinexus facilitated impulse transmission via ephaptic coupling. Our simulations demonstrate the role of narrowed perinexi as privileged sites for ephaptic coupling in pathological situations when gap junctional coupling is decreased., (© 2021 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: In adult ventricular myocytes, the slow delayed rectifier (I Ks ) channels are distributed on the surface sarcolemma, not t-tubules. In adult ventricular myocytes, KCNQ1 and KCNE1 have distinct cell surface and cytoplasmic pools. KCNQ1 and KCNE1 traffic from the endoplasmic reticulum to the plasma membrane by separate routes, and assemble into I Ks channels on the cell surface. Liquid chromatography/tandem mass spectrometry applied to affinity-purified KCNQ1 and KCNE1 interacting proteins reveals novel interactors involved in protein trafficking and assembly. Microtubule plus-end binding protein 1 (EB1) binds KCNQ1 preferentially in its dimer form, and promotes KCNQ1 to reach the cell surface. An LQT1-associated mutation, Y111C, reduces KCNQ1 binding to EB1 dimer., Abstract: Slow delayed rectifier (I Ks ) channels consist of KCNQ1 and KCNE1. I Ks functions as a 'repolarization reserve' in the heart by providing extra current for ventricular action potential shortening during β-adrenergic stimulation. There has been much debate about how KCNQ1 and KCNE1 traffic in cells, where they associate to form I Ks channels, and the distribution pattern of I Ks channels relative to β-adrenergic signalling complex. We used experimental strategies not previously applied to KCNQ1, KCNE1 or I Ks , to provide new insights into these issues. 'Retention-using-selected-hook' experiments showed that newly translated KCNE1 constitutively trafficked through the conventional secretory path to the cell surface. KCNQ1 largely stayed in the endoplasmic reticulum, although dynamic KCNQ1 vesicles were observed in the submembrane region. Disulphide-bonded KCNQ1/KCNE1 constructs reported preferential association after they had reached cell surface. An in situ proximity ligation assay detected I Ks channels in surface sarcolemma but not t-tubules of ventricular myocytes, similar to the reported location of adenylate cyclase 9/yotiao. Fluorescent protein-tagged KCNQ1 and KCNE1, in conjunction with antibodies targeting their extracellular epitopes, detected distinct cell surface and cytoplasmic pools of both proteins in myocytes. We conclude that, in cardiomyocytes, KCNQ1 and KCNE1 traffic by different routes to surface sarcolemma where they assemble into I Ks channels. This mode of delayed channel assembly helps I Ks fulfil its function of repolarization reserve. Proteomic experiments revealed a novel KCNQ1 interactor, microtubule plus-end binding protein 1 (EB1). EB1 dimer (active form) bound KCNQ1 and increased its surface level. An LQT1 mutation, Y111C, reduced KCNQ1 binding to EB1 dimer., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

Key Points: Mutations in the caveolae scaffolding protein, caveolin-3 (Cav3), have been linked to the long QT type 9 inherited arrhythmia syndrome (LQT9) and the cause of underlying action potential duration prolongation is incompletely understood. In the present study, we show that LQT9 Cav3 mutations, F97C and S141R, cause mutation-specific gain of function effects on Ca v 1.2-encoded L-type Ca 2+ channels responsible for I Ca,L and also cause loss of function effects on heterologously expressed K v 4.2 and K v 4.3 channels responsible for I to . A computational model of the human ventricular myocyte action potential suggests that the major ionic current change causing action potential duration prolongation in the presence of Cav3-F97C is the slowly inactivating I Ca,L but, for Cav3-S141R, both increased I Ca,L and increased late Na + current contribute equally to action potential duration prolongation. Overall, the LQT9 Cav3-F97C and Cav3-S141R mutations differentially impact multiple ionic currents, highlighting the complexity of Cav3 regulation of cardiac excitability and suggesting mutation-specific therapeutic approaches., Abstract: Mutations in the CAV3 gene encoding caveolin-3 (Cav3), a scaffolding protein integral to caveolae in cardiomyocytes, have been associated with the congenital long-QT syndrome (LQT9). Initial studies demonstrated that LQT9-associated Cav3 mutations, F97C and S141R, increase late sodium current as a potential mechanism to prolong action potential duration (APD) and cause LQT9. Whether these Cav3 LQT9 mutations impact other caveolae related ion channels remains unknown. We used the whole-cell, patch clamp technique to characterize the effect of Cav3-F97C and Cav3-S141R mutations on heterologously expressed Ca v 1.2+Ca v β 2cN4 channels, as well as K v 4.2 and K v 4.3 channels, in HEK 293 cells. Expression of Cav3-S141R increased I Ca,L density without changes in gating properties, whereas expression of Cav3-F97C reduced Ca 2+ -dependent inactivation of I Ca,L without changing current density. The Cav3-F97C mutation reduced current density and altered the kinetics of I Kv4.2 and I Kv4.3 and also slowed recovery from inactivation. Cav3-S141R decreased current density and also slowed activation kinetics and recovery from inactivation of I Kv4.2 but had no effect on I Kv4.3 . Using the O'Hara-Rudy computational model of the human ventricular myocyte action potential, the Cav3 mutation-induced changes in I to are predicted to have negligible effect on APD, whereas blunted Ca 2+ -dependent inactivation of I Ca,L by Cav3-F97C is predicted to be primarily responsible for APD prolongation, although increased I Ca,L and late I Na by Cav3-S141R contribute equally to APD prolongation. Thus, LQT9 Cav3-associated mutations, F97C and S141R, produce mutation-specific changes in multiple ionic currents leading to different primary causes of APD prolongation, which suggests the use of mutation-specific therapeutic approaches in the future., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Key Points: At the cellular level cardiac hypertrophy causes remodelling, leading to changes in ionic channel, pump and exchanger densities and kinetics. Previous studies have focused on quantifying changes in channels, pumps and exchangers without quantitatively linking these changes with emergent cellular scale functionality. Two biophysical cardiac cell models were created, parameterized and validated and are able to simulate electrophysiology and calcium dynamics in myocytes from control sham operated rats and aortic-banded rats exhibiting diastolic dysfunction. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L-type Ca 2+ channel and sarco/endoplasmic reticulum Ca 2+ ATPase as the principal regulators of systolic and diastolic Ca 2+ , respectively. Results show that the ability to dynamically change systolic Ca 2+ , through changes in expression of key Ca 2+ modelling protein densities, is drastically reduced following the aortic banding procedure; however the cells are able to compensate Ca 2+ homeostasis in an efficient way to minimize systolic dysfunction., Abstract: Elevated left ventricular afterload leads to myocardial hypertrophy, diastolic dysfunction, cellular remodelling and compromised calcium dynamics. At the cellular scale this remodelling of the ionic channels, pumps and exchangers gives rise to changes in the Ca 2+ transient. However, the relative roles of the underlying subcellular processes and the positive or negative impact of each remodelling mechanism are not fully understood. Biophysical cardiac cell models were created to simulate electrophysiology and calcium dynamics in myocytes from control rats (SHAM) and aortic-banded rats exhibiting diastolic dysfunction. The model parameters and framework were validated and the fitted parameters demonstrated to be unique for explaining our experimental data. The contribution of each ionic pathway to the calcium kinetics was calculated, identifying the L-type Ca 2+ channel (LCC) and the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) as the principal regulators of systolic and diastolic Ca 2+ , respectively. In the aortic banding model, the sensitivity of systolic Ca 2+ to LCC density and diastolic Ca 2+ to SERCA density decreased by 16-fold and increased by 23%, respectively, relative to the SHAM model. The energy cost of ionic homeostasis is maintained across the two models. The models predict that changes in ionic pathway densities in compensated aortic banding rats maintain Ca 2+ function and efficiency. The ability to dynamically alter systolic function is significantly diminished, while the capacity to maintain diastolic Ca 2+ is moderately increased., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: [Ca 2+ ] i enhanced rabbit ventricular slowly activating delayed rectifier K + current (I Ks ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K + current (I Kr ) amplitude and voltage dependence were unaffected by high [Ca 2+ ] i . When measuring or simulating I Ks during an action potential, I Ks was not different during a physiological Ca 2+ transient or when [Ca 2+ ] i was buffered to 500 nm., Abstract: The slowly activating delayed rectifier K + current (I Ks ) contributes to repolarization of the cardiac action potential (AP). Intracellular Ca 2+ ([Ca 2+ ] i ) and β-adrenergic receptor (β-AR) stimulation modulate I Ks amplitude and kinetics, but details of these important I Ks regulators and their interaction are limited. We assessed the [Ca 2+ ] i dependence of I Ks in steady-state conditions and with dynamically changing membrane potential and [Ca 2+ ] i during an AP. I Ks was recorded from freshly isolated rabbit ventricular myocytes using whole-cell patch clamp. With intracellular pipette solutions that controlled free [Ca 2+ ] i , we found that raising [Ca 2+ ] i from 100 to 600 nm produced similar increases in I Ks as did β-AR activation, and the effects appeared additive. Both β-AR activation and high [Ca 2+ ] i increased maximally activated tail I Ks , negatively shifted the voltage dependence of activation, and slowed deactivation kinetics. These data informed changes in our well-established mathematical model of the rabbit myocyte. In both AP-clamp experiments and simulations, I Ks recorded during a normal physiological Ca 2+ transient was similar to I Ks measured with [Ca 2+ ] i clamped at 500-600 nm. Thus, our study provides novel quantitative data as to how physiological [Ca 2+ ] i regulates I Ks amplitude and kinetics during the normal rabbit AP. Our results suggest that micromolar [Ca 2+ ] i , in the submembrane or junctional cleft space, is not required to maximize [Ca 2+ ] i -dependent I Ks activation during normal Ca 2+ transients., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

The hyperpolarization-activated cation current (I(f)), and the hyperpolarization-activated cyclic nucleotide-modulated 'HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. I(f) and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing I(f), and the density of I(f) in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The I(f) blocker ZD7288 (0.3 microm) reduced I(f) and the beating rate of embryoid bodies. Taken together, the activation kinetics of I(f) and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of I(f) to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation.