Cardiac Action Potential Propagation Research Articles

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.

Nonmyocytes as electrophysiological contributors to cardiac excitation and conduction.

Although cardiac action potential (AP) generation and propagation have traditionally been attributed exclusively to cardiomyocytes (CM), other cell types in the heart are also capable of forming electrically conducting junctions. Interactions between CM and nonmyocytes (NM) enable and modulate each other's activity. This review provides an overview of the current understanding of heterocellular electrical communication in the heart. Although cardiac fibroblasts were initially thought to be electrical insulators, recent studies have demonstrated that they form functional electrical connections with CM in situ. Other NM, such as macrophages, have also been recognized as contributing to cardiac electrophysiology and arrhythmogenesis. Novel experimental tools have enabled the investigation of cell-specific activity patterns in native cardiac tissue, which is expected to yield exciting new insights into the development of novel or improved diagnostic and therapeutic strategies.

A Simulation Study of the Effects of His Bundle Pacing in Left Bundle Branch Block.

His bundle pacing (HBP) has emerged as a feasible alternative to right (RVP) and biventricular pacing (BVP) for Cardiac Resynchronization Therapy (CRT). This study sought to assess, in ex-vivo experimental models, the optimal setup for HBP in terms of electrode placement and pacing protocol to achieve superior electrical synchrony in the case of complete His-Purkinje block and left bundle branch block (LBBB). We developed a 3D model of His bundle and bundle branches, embedded in a patient-specific biventricular heart model reconstructed from CT images. A monodomain reaction-diffusion model was adopted to describe the propagation of cardiac action potential, and a custom procedure was developed to compute pseudo-ECGs. Experimental measurements of tip electrode potential waveforms have been performed on ex-vivo swine myocardium to determine the appropriate boundary condition for delivering the electrical stimulus in the numerical model. An extended parametric analysis, investigating the effect of the electrode orientation and helix length, pacing protocol, and atrioventricular delay, allowed us to determine the optimal setup for HBP therapy. Both selective (S-HBP) and non-selective (NS-HBP) His bundle pacing were tested, as the variable anatomical location of the His bundle can result in the activation of the surrounding myocardium. Our study indicates a perpendicular placement of the electrode as the most advantageous for restoring the physiological function of the His-Purkinje system. We found that higher-energy protocols can compensate for the effects of an angled placement though concurring to potential tip fibrosis. Promisingly, we also revealed that an increased electrode helix length can provide optimal resynchronization even with low-energy pacing protocols. Our results provide informative guidance for implant procedure and therapy optimization, which will hopefully have clinical implications further improving the procedural success rates and patients' quality of life, due to reduced incidence of lead revision and onset of complications.

Optogenetic manipulation of cardiac electrical dynamics using sub-threshold illumination: dissecting the role of cardiac alternans in terminating rapid rhythms

Abstract Funding Acknowledgements Type of funding sources: None. Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ions channel recovery and intracellular Ca2+-cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose a novel approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination) but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsins-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We use an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role that cardiac alternans plays in spontaneous termination of ventricular tachycardias (VTs). We find that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT.

Optogenetic manipulation of cardiac electrical dynamics using sub-threshold illumination: dissecting the role of cardiac alternans in terminating rapid rhythms

Cardiac action potential (AP) shape and propagation are regulated by several key dynamic factors such as ion channel recovery and intracellular Ca2+ cycling. Experimental methods for manipulating AP electrical dynamics commonly use ion channel inhibitors that lack spatial and temporal specificity. In this work, we propose an approach based on optogenetics to manipulate cardiac electrical activity employing a light-modulated depolarizing current with intensities that are too low to elicit APs (sub-threshold illumination), but are sufficient to fine-tune AP electrical dynamics. We investigated the effects of sub-threshold illumination in isolated cardiomyocytes and whole hearts by using transgenic mice constitutively expressing a light-gated ion channel (channelrhodopsin-2, ChR2). We find that ChR2-mediated depolarizing current prolongs APs and reduces conduction velocity (CV) in a space-selective and reversible manner. Sub-threshold manipulation also affects the dynamics of cardiac electrical activity, increasing the magnitude of cardiac alternans. We used an optical system that uses real-time feedback control to generate re-entrant circuits with user-defined cycle lengths to explore the role of cardiac alternans in spontaneous termination of ventricular tachycardias (VTs). We demonstrate that VT stability significantly decreases during sub-threshold illumination primarily due to an increase in the amplitude of electrical oscillations, which implies that cardiac alternans may be beneficial in the context of self-termination of VT.

High Performance Flexible Organic Electrochemical Transistors for Monitoring Cardiac Action Potential.

Flexible and transparent electronic devices possess crucial advantages over conventional silicon based systems for bioelectronic applications since they are able to adapt to nonplanar surfaces, cause less chronic immunoreactivity, and facilitate easy optical inspection. Here, organic electrochemical transistors (OECTs) are embedded in a flexible matrix of polyimide to record cardiac action potentials. The wafer-scale fabricated devices exhibit transconductances (12 mS V-1 ) and drain-source on-to-off current ratios (≈105 ) comparable to state of the art nonflexible and superior to other reported flexible OECTs. The transfer characteristics of the devices are preserved even after experiencing extremely high bending strain and harsh crumpling. A sub-micrometer poly(3,4-ethylenedioxythiophene) doped with poly(styrenesulfonate) layer results in a fast transport of ions between the electrolyte and the polymer channel characterized by a cut-off frequency of 1200 Hz. Excellent device performance is proved by mapping the propagation of cardiac action potentials with high signal-to-noise ratio. These results demonstrate that the electrical performance of flexible OECTs can compete with hard-material-based OECTs and thus potentially be used for in vivo applications.

Protein⁻Protein Interactions with Connexin 43: Regulation and Function.

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.

Directed fusion of cardiac spheroids into larger heterocellular microtissues enables investigation of cardiac action potential propagation via cardiac fibroblasts.

Multicellular spheroids generated through cellular self-assembly provide cytoarchitectural complexities of native tissue including three-dimensionality, extensive cell-cell contacts, and appropriate cell-extracellular matrix interactions. They are increasingly suggested as building blocks for larger engineered tissues to achieve shapes, organization, heterogeneity, and other biomimetic complexities. Application of these tissue culture platforms is of particular importance in cardiac research as the myocardium is comprised of distinct but intermingled cell types. Here, we generated scaffold-free 3D cardiac microtissue spheroids comprised of cardiac myocytes (CMs) and/or cardiac fibroblasts (CFs) and used them as building blocks to form larger microtissues with different spatial distributions of CMs and CFs. Characterization of fusing homotypic and heterotypic spheroid pairs revealed an important influence of CFs on fusion kinetics, but most strikingly showed rapid fusion kinetics between heterotypic pairs consisting of one CF and one CM spheroid, indicating that CMs and CFs self-sort in vitro into the intermixed morphology found in the healthy myocardium. We then examined electrophysiological integration of fused homotypic and heterotypic microtissues by mapping action potential propagation. Heterocellular elongated microtissues which recapitulate the disproportionate CF spatial distribution seen in the infarcted myocardium showed that action potentials propagate through CF volumes albeit with significant delay. Complementary computational modeling revealed an important role of CF sodium currents and the spatial distribution of the CM-CF boundary in action potential conduction through CF volumes. Taken together, this study provides useful insights for the development of complex, heterocellular engineered 3D tissue constructs and their engraftment via tissue fusion and has implications for arrhythmogenesis in cardiac disease and repair.

The Formula 1 Heart of the Etruscan Shrew Suncus etruscus: Using the Extreme to Exemplify Basic Principals in Cardiovascular Physiology

Modern teaching in physiology is greatly enhanced by strategies such as case‐based instruction. A variant of this employs a comparative approach to illustrate basic physiological principles and promote mechanistic thinking. As with clinical cases, examples from comparative physiology can illustrate dramatic responses of physiological systems under extreme stresses, or specializations, optimizations and integration of physiological processes underlying extreme performance. A typical course in physiology considers the structure and function of muscles, and specializations particular to cardiac muscle. Characteristics of cardiac myocytes are illustrated which underlie myogenic pacemakers and syncytial propagation of cardiac action potential between adjacent cells. Considered are patterns of excitation distribution from cardiac pacemakers through the conducting system leading to precise patterns of muscle contraction ensuring synchronized blood flow through the chambers of the heart. Mechanisms coupling changes in membrane polarization to myofibril cross‐bridge formation and ion fluxes and channel properties integral to muscle contraction, relaxation and refractory periods are all described. Integral to these concepts are the temporal durations of basic physiological processes resulting in a coordinated pattern of autorhythmic systole and diastole, and the capacities for autonomic modulation of the heart to match cardiac output to metabolic demands. These basic principles are commonly only described in relation to humans, and resulting in a healthy heart beating at 72 beats per minute. The Etruscan Shrew, Suncus etruscus, presents as a compelling comparative example, having a heart rate as high as 1500 bpm. We consider the shrew to illustrate basic physiological mechanisms, what is known, what can be predicted and what mysteries remain as to the capacities necessary to accommodate extreme cardiac cycle frequencies approaching 25 Hz.Support or Funding InformationWork reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards 1R15HL126105 and 1SC2GM112570. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cardiac Action Potential Propagation through Compact Fibroblasts in 3D Cardiac Microtissues Engineered from Self-Assembled Spheroids as Building Blocks

Cardiac Action Potential Propagation through Compact Fibroblasts in 3D Cardiac Microtissues Engineered from Self-Assembled Spheroids as Building Blocks

Simulation study on compressive laminar optical tomography for cardiac action potential propagation.

To measure the activity of tissue at the microscopic level, laminar optical tomography (LOT), which is a microscopic form of diffuse optical tomography, has been developed. However, obtaining sufficient recording speed to determine rapidly changing dynamic activity remains major challenges. For a high frame rate of the reconstructed data, we here propose a new LOT method using compressed sensing theory, called compressive laminar optical tomography (CLOT), in which novel digital micromirror device-based illumination and data reduction in a single reconstruction are applied. In the simulation experiments, the reconstructed volumetric images of the action potentials that were acquired from 5 measured images with random pattern featured a wave border at least to a depth of 2.5 mm. Consequently, it was shown that CLOT has potential for over 200 fps required for the cardiac electrophysiological phenomena.

Brugada syndrome genetic testing: A reliable sanger sequencing protocol

The Brugada syndrome (BrS) is a potentially lethal, inheritable arrhythmia syndrome affecting about 5 in 10,000 people worldwide. This cardiac disorder, triggered by mutations in SCN5A gene (encoding for cardiac sodium channels), results in changes in the onset or propagation of cardiac action potential that can generate arrhythmias, potentially fatal. In the last few years, the genetic studies are performed with next generation sequencing (NGS), obtaining global genomic information. However, this strategy requires capillary electrophoresis data validations. In this study, we developed and optimized a Sanger sequencing workflow for SCN5A gene exons.

High-order finite element methods for cardiac monodomain simulations.

Computational modeling of tissue-scale cardiac electrophysiology requires numerically converged solutions to avoid spurious artifacts. The steep gradients inherent to cardiac action potential propagation necessitate fine spatial scales and therefore a substantial computational burden. The use of high-order interpolation methods has previously been proposed for these simulations due to their theoretical convergence advantage. In this study, we compare the convergence behavior of linear Lagrange, cubic Hermite, and the newly proposed cubic Hermite-style serendipity interpolation methods for finite element simulations of the cardiac monodomain equation. The high-order methods reach converged solutions with fewer degrees of freedom and longer element edge lengths than traditional linear elements. Additionally, we propose a dimensionless number, the cell Thiele modulus, as a more useful metric for determining solution convergence than element size alone. Finally, we use the cell Thiele modulus to examine convergence criteria for obtaining clinically useful activation patterns for applications such as patient-specific modeling where the total activation time is known a priori.

Simulating Cardiac Electrophysiology Using Unstructured All-Hexahedra Spectral Elements.

We discuss the application of the spectral element method to the monodomain and bidomain equations describing propagation of cardiac action potential. Models of cardiac electrophysiology consist of a system of partial differential equations coupled with a system of ordinary differential equations representing cell membrane dynamics. The solution of these equations requires solving multiple length scales due to the ratio of advection to diffusion that varies among the different equations. High order approximation of spectral elements provides greater flexibility in resolving multiple length scales. Furthermore, spectral elements are extremely efficient to model propagation phenomena on complex shapes using fewer degrees of freedom than its finite element equivalent (for the same level of accuracy). We illustrate a fully unstructured all-hexahedra approach implementation of the method and we apply it to the solution of full 3D monodomain and bidomain test cases. We discuss some key elements of the proposed approach on some selected benchmarks and on an anatomically based whole heart human computational model.

Automatic cycle averaging for denoising approximately periodic spatiotemporal signals.

Optical mapping has become a common tool for cardiovascular research, providing high resolution spatiotemporal data of cardiac action potential propagation. However, noise in cardiac optical mapping (COM) data hampers quantitative measurements and analyses. Spatial and temporal filters have been used to increase the signal-to-noise ratio (SNR) of COM data. Although these help reduce noise and increase SNR, they also lower the spatial and temporal resolution of the data, which is not desirable. This paper utilizes the approximate periodicity of COM data to perform denoising by averaging cycles. We consider the entire approximately periodic spatiotemporal (APST) COM data as a concatenation of random samples generated from a deterministic single cycle spatiotemporal signal. The image difference signal (IDS) was defined and calculated to provide "global" periodicity information. Parameters for cycle segmentation, scaling and alignment were estimated based on the IDS. Finally, these parameters were used to segment, align and average cycles from each individual signal, which produces a clean and denoised single cycle spatiotemporal signal. The novel, fully automated pipeline was validated both qualitatively and quantitatively on zebrafish heart optical mapping data.

0106: Influence of the ratio of co-expressed cardiac connexins Cx43 and Cx45 in the formation of gap junction channels and their electrical properties

The cardiac action potential (AP) propagation is regulated to permit the coordinated and rhythmic atrial and ventricular contractions. This regulation requires several factors, especially gap junctions, which ensure a direct pathway for electrical and biochemical signaling. They are clusters of few to hundred intercellular gap junction channels (GJC) made of two hemichannels docked in the membrane of adjacent cells, which are composed of six connexins (Cxs). Their distinct electrical properties are a key factor regulating the propagation of the AP. Four cardiac Cxs, Cx40, Cx43, Cx45 and Cx30.2, exhibit specific patterns of expression that change in the healthy and diseased heart, which leads to different possible configurations of GJC. The aim of this study is to investigate the function of the distinct ratio of co-expressed Cxs in regulating the formation and function of GJC. Electrical properties of GJC (junctional coupling, voltage dependence, unitary conductance) are determined by performing electrical recordings on cell pair by applying the dual voltage-clamp method. Rat Liver Epithelial cells stably transfected to induce accurate Cx43:Cx45 ratios of 0 (single Cx43 expression), 0.5, 1 and 2, are used. The ongoing recordings show distinct electrical properties before and after the induction of Cx45: induction of Cx45 decreases the cell-to-cell coupling and rectifies the voltage dependence of GJC. Preliminary unitary recordings suggest a distinct formation of GJC of mixed Cx43/Cx45 composition in function of the Cx43:Cx45 ratio. Further investigations will provide better understanding on the distinct contributions of Cx43 and Cx45 in the GJC make-up, electrical properties and function of the Cx43/Cx45 expression pattern in regulating the cardiac impulse propagation in the healthy heart, and the pro-arrhythmic behavior in the diseased heart.

Mechanistic insights into hypothermic ventricular fibrillation: the role of temperature and tissue size.

Hypothermia is well known to be pro-arrhythmic, yet it has beneficial effects as a resuscitation therapy and valuable during intracardiac surgeries. Therefore, we aim to study the mechanisms that induce fibrillation during hypothermia. A better understanding of the complex spatiotemporal dynamics of heart tissue as a function of temperature will be useful in managing the benefits and risks of hypothermia. We perform two-dimensional numerical simulations by using a minimal model of cardiac action potential propagation fine-tuned on experimental measurements. The model includes thermal factors acting on the ionic currents and the gating variables to correctly reproduce experimentally recorded restitution curves at different temperatures. Simulations are implemented using WebGL, which allows long simulations to be performed as they run close to real time. We describe (i) why fibrillation is easier to induce at low temperatures, (ii) that there is a minimum size required for fibrillation that depends on temperature, (iii) why the frequency of fibrillation decreases with decreasing temperature, and (iv) that regional cooling may be an anti-arrhythmic therapy for small tissue sizes however it may be pro-arrhythmic for large tissue sizes. Using a mathematical cardiac cell model, we are able to reproduce experimental observations, quantitative experimental results, and discuss possible mechanisms and implications of electrophysiological changes during hypothermia.

Trafficking Defective Mutations Modulate NaV1.5 N—Glycosylation States

Voltage gated sodium channels are membrane proteins that play a critical role in electrical signaling of excitable cells. Amongst this family, the isoform Nav1.5 is responsible for the initiation and propagation of cardiac action potentials. As most of membrane proteins, Nav1.5 is well known to be a glycoprotein with ∼5% of its total weight corresponding to carbohydrates. To date, it has been shown that Nav1.5 glycosylations such as sialylations influence biophysical properties of this channel protein.

Post-translational modifications of the cardiac Na channel: contribution of CaMKII-dependent phosphorylation to acquired arrhythmias

The voltage-gated Na channel isoform 1.5 (NaV1.5) is the pore forming α-subunit of the voltage-gated cardiac Na channel, which is responsible for the initiation and propagation of cardiac action potentials. Mutations in the SCN5A gene encoding NaV1.5 have been linked to changes in the Na current leading to a variety of arrhythmogenic phenotypes, and alterations in the NaV1.5 expression level, Na current density, and/or gating have been observed in acquired cardiac disorders, including heart failure. The precise mechanisms underlying these abnormalities have not been fully elucidated. However, several recent studies have made it clear that NaV1.5 forms a macromolecular complex with a number of proteins that modulate its expression levels, localization, and gating and is the target of extensive post-translational modifications, which may also influence all these properties. We review here the molecular aspects of cardiac Na channel regulation and their functional consequences. In particular, we focus on the molecular and functional aspects of Na channel phosphorylation by the Ca/calmodulin-dependent protein kinase II, which is hyperactive in heart failure and has been causally linked to cardiac arrhythmia. Understanding the mechanisms of altered NaV1.5 expression and function is crucial for gaining insight into arrhythmogenesis and developing novel therapeutic strategies.

A predictive method allowing the use of a single ionic model in numerical cardiac electrophysiology

One of the current debate about simulating the electrical activity in the heart is the following: Using a realistic anatomical setting, i.e. realistic geometries, fibres orientations, etc., is it enough to use a simplified 2-variable phenomenological model to reproduce the main characteristics of the cardiac action potential propagation, and in what sense is it sufficient? Using a combination of dimensional and asymptotic analysis, together with the well-known Mitchell − Schaeffer model, it is shown that it is possible to accurately control (at least locally) the solution while spatial propagation is involved. In particular, we reduce the set of parameters by writing the bidomain model in a new nondimensional form. The parameters of the bidomain model with Mitchell − Schaeffer ion kinetics are then set and shown to be in one-to-one relation with the main characteristics of the four phases of a propagated action potential. Explicit relations are derived using a combination of asymptotic methods and ansatz. These relations are tested against numerical results. We illustrate how these relations can be used to recover the time/space scales and speed of the action potential in various regions of the heart.