Cardiac Cell Excitation Research Articles

Control of spiral waves in optogenetically modified cardiac tissue by periodic optical stimulation

Resonant drift of nonlinear spiral waves occurs in various types of excitable media under periodic stimulation. Recently a novel methodology of optogenetics has emerged, which allows to affect excitability of cardiac cells and tissues by optical stimuli. In this paper we study if resonant drift of spiral waves in the heart can be induced by a homogeneous weak periodic optical stimulation of cardiac tissue. We use a two-variable and a detailed model of cardiac tissue and add description of depolarizing and hyperpolarizing optogenetic ionic currents. We show that weak periodic optical stimulation at a frequency equal to the natural rotation frequency of the spiral wave induces resonant drift for both depolarizing and hyperpolarizing optogenetic currents. We quantify these effects and study how the speed of the drift and its direction depend on the initial conditions. We also derive analytical formulas based on the response function theory which correctly predict the drift velocity and its trajectory. We conclude that optogenetic methodology can be used for control of spiral waves in cardiac tissue and discuss its possible applications.

Three-Week-Old Rabbit Ventricular Cardiomyocytes as a Novel System to Study Cardiac Excitation and EC Coupling.

Cardiac arrhythmias significantly contribute to cardiovascular morbidity and mortality. The rabbit heart serves as an accepted model system for studying cardiac cell excitation and arrhythmogenicity. Accordingly, primary cultures of adult rabbit ventricular cardiomyocytes serve as a preferable model to study molecular mechanisms of human cardiac excitation. However, the use of adult rabbit cardiomyocytes is often regarded as excessively costly. Therefore, we developed and characterized a novel low-cost rabbit cardiomyocyte model, namely, 3-week-old ventricular cardiomyocytes (3wRbCMs). Ventricular myocytes were isolated from whole ventricles of 3-week-old New Zealand White rabbits of both sexes by standard enzymatic techniques. Using wheat germ agglutinin, we found a clear T-tubule structure in acutely isolated 3wRbCMs. Cells were adenovirally infected (multiplicity of infection of 10) to express Green Fluorescent Protein (GFP) and cultured for 48 h. The cells showed action potential duration (APD90 = 253 ± 24 ms) and calcium transients similar to adult rabbit cardiomyocytes. Freshly isolated and 48-h-old-cultured cells expressed critical ion channel proteins: calcium voltage-gated channel subunit alpha1 C (Cavα1c), sodium voltage-gated channel alpha subunit 5 (Nav1.5), potassium voltage-gated channel subfamily D member 3 (Kv4.3), and subfamily A member 4 (Kv1.4), and also subfamily H member 2 (RERG. Kv11.1), KvLQT1 (K7.1) protein and inward-rectifier potassium channel (Kir2.1). The cells displayed an appropriate electrophysiological phenotype, including fast sodium current (INa), transient outward potassium current (Ito), L-type calcium channel peak current (ICa,L), rapid and slow components of the delayed rectifier potassium current (IKr and IKs), and inward rectifier (IK1). Although expression of the channel proteins and some currents decreased during the 48 h of culturing, we conclude that 3wRbCMs are a new, low-cost alternative to the adult-rabbit-cardiomyocytes system, which allows the investigation of molecular mechanisms of cardiac excitation on morphological, biochemical, genetic, physiological, and biophysical levels.

Characterization of a novel LQT3 variant with a selective efficacy of mexiletine treatment

Pathogenic variants in the human SCN5A gene encoding the a-subunit of the principle Na+ channel (Nav1.5) are associated with long QT syndrome (LQTS) 3. LQT3 patients display variable responses to Na+ channel blockers demanding for the development of variant-specific therapeutic strategies. Here we performed a combined electrophysiological analysis with in silico simulation of variant channel to elucidate mechanisms of therapeutic responsiveness. We identified a novel SCN5A variant (A1656D) in a LQTS patient with a distinct response to mexiletine resulting in suppression of non-sustained ventricular tachycardia and manifestation of premature atrial contraction. Patch clamp analysis revealed that A1656D variant exerted gain-of-function effects including hyperpolarizing shift of the voltage-dependence of activation, depolarizing shift in the voltage-dependence of inactivation, and slowing of fast inactivation. Among ranolazine, flecainide, and mexiletine, only mexiletine restored inactivation kinetics of A1656D currents. In silico simulation to assess the effect of A1656D variant on ventricular cardiac cell excitation predicted a prolonged action potential which is consistent with the prolonged QT and non-sustained ventricular tachycardia of the patient. It also predicted that only mexiletine suppressed the prolonged action potential of human ventricular myocytes expressing A1656D. These data elucidate the underlying mechanism of the distinct response to mexiletine in this patient.

Stilbene derivative as a photosensitive compound to control the excitability of neonatal rat cardiomyocytes.

Substances that can be used as photosensitizers for cardiac tissue are very helpful in modeling various excitation patterns in a cardiac tissue culture and may have prospective use in the temporary and permanent ablation of unwanted excitation sources in the heart.The aim of the present work is to study the effect of stilbene derivative c-TAB (2- {4- [(E) -2- (4-ethoxyphenyl) vinyl] phenoxy} ethyl) trimethylammonium bromide) on the cardiomyocyte layers and voltage-gated ion channels in cardiac cells. C-TAB is a structural analog to AzoTAB, reported previously as a photoswitch for cardiac and neural cells, in which the azobenzene moiety is replaced by a stilbene grouping. Such a replacement makes c-TAB less toxic to living cells. c-TAB has been shown to successfully inhibit excitation in cardiac cells in both trans- and cis- forms. The excitation inhibition of cardiac cells under c-TAB is reversible and can be overturned easily by washing out the c-TAB; however, not by light illumination. The irradiation of cardiac cells with near-UV, when the trans- form of c-TAB is applied, changes reversible inhibition to a permanent one that cannot be overturned by a washout.

Formation of virtual isthmus: A new scenario of spiral wave death after a decrease in excitability

Termination of rotating (spiral) waves or reentry is crucial when fighting with the most dangerous cardiac tachyarrhythmia. To increase the efficiency of the antiarrhythmic drugs as well as finding new prospective ones it is decisive to know the mechanisms how they act and influence the reentry dynamics. The most popular view on the mode of action of the contemporary antiarrhythmic drugs is that they increase the core of the rotating wave (reentry) to that extent that it is not enough space in the real heart for the reentry to exist. Since the excitation in cardiac cells is essentially change of the membrane potential, it relies on the functioning of the membrane ion channels. Thus, membrane ion channels serve as primary targets for the substances, which may serve as antiarrhythmics. At least, the entire group of antiarrhythmics class I (modulating activity of sodium channels) and partially class IV (modulating activity of calcium channels) are believed to destabilize and terminate reentry by decreasing the excitability of cardiac tissue. We developed an experimental model employing cardiac tissue culture and photosensitizer (AzoTAB) to study the process of the rotating wave termination while decreasing the excitability of the tissue. A new scenario of spiral wave cessation was observed: an asymmetric growth of the rotating wave core and subsequent formation of a virtual isthmus, which eventually caused a conduction block and the termination of the reentry.

A Simple Regulation of Cardiomyocyte Excitability

Introduction: Recently, genome wide association studies (GWAS) identified LITAF (lipopolysaccharide-induced TNF factor) as one of the novel loci associated with prolongation of QT interval. Our goal is to investigate the role of LITAF in regulating cardiomyocyte excitability and action potential duration (APD). Methods: Optical mapping was performed in zebrafish hearts to determine Ca2+ transients and transmembrane (TM) potentials. Propagation velocities of depolarizing waves were determined. Action potential (AP) was digitally imaged in adenoviral infected neonatal rabbit cardiomyocytes (NRbCM). Live cell confocal calcium imaging was performed on adult rabbit ventricular myocytes (ARbCM) to determine intracellular Ca2+ handling. A current-voltage (I-V) relationship for the L-type calcium current (ICa,L ) was determined using whole cell patch clamping. To study the effect of LITAF on Cav1.2 channel expression, surface biotinylation and western blot assays were performed using tSA201 cells. Results: Our studies demonstrate that loss-of-function of LITAF in vivo (zebrafish) increases the Ca2+ transient amplitude (which primarily depends on calcium influx), slows atrial-ventricular conduction, and prolongs the APD. In contrast, in vitro gain-of-function experiments in rabbit CM established that overexpression of LITAF downregulates the total and surface pools of Cav1.2 channel polypeptides, decreases Ca2+ transients and L-type Ca2+ conductance, and shorten the APD. Studies in heterologous expression systems revealed that one of the auxiliary subunits of Cav1.2 channels, Cavβ2b, is likely involved in LITAF-mediated inhibition of recombinant Cav1.2 channels. Conclusion: LITAF regulates the expression and function of Cav1.2 channels, thereby determining APD and cardiac cell excitation.

How do sex hormones modify arrhythmogenesis in long QT syndrome? Sex hormone effects on arrhythmogenic substrate and triggered activity.

Gender differences in cardiac repolarization and the arrhythmogenic risk of patients with inherited and acquired long QT syndromes are well appreciated clinically. Enhancing our knowledge of the mechanisms underlying these differences is critical to improve our therapeutic strategies for preventing sudden cardiac death in such patients. This review summarizes the effects of sex hormones on the expression and function of ion channels that control cardiac cell excitation and repolarization as well as key proteins that regulate Ca(2+) dynamics at the cellular level. Moreover, it examines the role of sex hormones in modifying the dynamic spatiotemporal (regional and transmural) heterogeneities in action potential duration (eg, the arrhythmogenic substrate) and the susceptibility to (sympathetic) triggered activity at the tissue, organ, and whole animal levels. Finally, it explores the implications of these effects on the management of patients with LQTS.

Kinetic Model of Nav1.5 Channel Provides a Subtle Insight into Slow Inactivation Associated Excitability in Cardiac Cells

Voltage-gated sodium channel Nav1.5 has been linked to the cardiac cell excitability and a variety of arrhythmic syndromes including long QT, Brugada, and conduction abnormalities. Nav1.5 exhibits a slow inactivation, corresponding to a duration-dependent bi-exponential recovery, which is often associated with various arrhythmia syndromes. However, the gating mechanism of Nav1.5 and the physiological role of slow inactivation in cardiac cells remain elusive. Here a 12-state two-step inactivation Markov model was successfully developed to depict the gating kinetics of Nav1.5. This model can simulate the Nav1.5 channel in not only steady state processes, but also various transient processes. Compared with the simpler 8-state model, this 12-state model is well-behaved in simulating and explaining the processes of slow inactivation and slow recovery. This model provides a good framework for further studying the gating mechanism and physiological role of sodium channel in excitable cells.

Greater Cardiac Cell Excitation Efficiency With Rapidly Switching Multidirectional Electrical Stimulation

Electric field (E) stimulation is widely used in experiments with myocardial preparations and in the clinical setting (e.g., defibrillation). As a rule, stimuli are applied in a single direction, which limits excitatory cell recruitment because myocytes are disposed in different directions and their sensitivity to E depends on the stimulus orientation with respect to the cell major axis. Here, we propose a stimulatory approach, namely rapidly switching multidirectional stimulation (RSMS), in which stimuli are delivered in three directions within the electric refractory period. In populations of randomly oriented isolated rat cardiomyocytes, RSMS doubled the percentage of cells excited by near-threshold E (P < 0.001), which was more than the increase in recruitment in a single direction achieved by doubling E intensity. This effect was similar for monophasic and biphasic pulses, but for the latter, a given percent recruitment was obtained with 20-30% lower E intensity ( P < 0.01), so that RSMS with biphasic pulses allowed at least 60% reduction of E intensity for recruitment of >70% of the cells. RSMS can be applied to improve stimulation efficiency in experiments with isolated cardiac myocytes, and may be a promising alternative for decreasing shock intensity requirements for cardioversion and defibrillation.

Modelling the Structure and Function of Cardiac Cell Transverse-Axial-Tubules

Modelling the Structure and Function of Transverse-Axial-TubulesThe past decades have seen significant strides being made in understanding the structure and function of the heart using experimental and computational approaches. Mathematical models of the heart can now represent the anatomical structure of the whole-heart and simulate the heart beat using biophysically based methods on the computer. At the cellular-level, however, mathematical models typically simulate the function using black-box mass kinetics approaches and make several simplifying assumptions regarding the spatial scale and organization of the key cellular components in excitation-contraction coupling. While these studies have been pivotal in gaining quantitative insights on cardiac cell activity, a deeper understanding of structure function relationships at the cellular scale can only be gained by incorporating more realistic models of the geometry. With this aim, we present an anatomically realistic finite element model of the cardiac cell sarcolemma, and its transverse-axial tubule system. Confocal microscopy images of the sarcolemma and t-tubules of an isolated wistar rat myocyte were acquired, segmented and skeletonized. A two-dimensional surface mesh was fitted to the sarcolemma data, and one-dimensional finite elements were used to represent the t-tubules from the skeletonized image in three-dimensional space. This representation will form the basis to simulate calcium diffusion into the cell cytosol during the excitation phase of excitation-contraction coupling. We present preliminary results from these simulations, and provide first quantitative insights into cardiac cell excitation in 3D.

Elimination of Pulses and Spirals by External Forces in Luo–Rudy Model

The Luo–Rudy model is one of the standard models for the excitation of cardiac cells. Traveling pulses and spiral waves appear in one-dimensional or two-dimensional Luo–Rudy models. Spiral waves in two-dimensional systems are interpreted as a possible origin of ventricular fibrillation, which is the most serious type of cardiac arrhythmia. Defibrillation is a process to eliminate spiral waves and stop ventricular fibrillation. We perform numerical simulations to eliminate traveling pulses and spiral waves by applying direct current or alternating current to the Luo–Rudy model.

Non-Tikhonov Asymptotic Properties of Cardiac Excitability

Models of electric excitability of cardiac cells can be studied by singular perturbation techniques. To do this one should take into account parameters appearing in equations in nonstandard ways. The physical reason for this is near-perfect switch behavior of ionic current gates. This leads to a definition of excitability different from the currently accepted one. The asymptotic structure revealed by our analysis can be used to devise simplified caricature models, to obtain approximate analytical solutions, and to facilitate numerical simulations.

Class III antiarrhythmics and phenytoin: teratogenicity due to embryonic cardiac dysrhythmia and reoxygenation damage.

Class III antiarrhythmic drugs, like almokalant, dofetilide and ibutilide, cause a spectrum of malformations in experimental teratology studies. The pattern of developmental toxic effects is very similar to those reported for phenytoin, which is an established human and animal teratogen. The toxic effects are characterised by embryonic death, decreased fetal weights, and stage specific malformations, such as distal digital reductions, orofacial clefts and cardiovascular defects. Class III antiarrhythmics decrease the excitability of cardiac cells by selectively blocking the rapid component of the delayed rectified potassium channel (IKr), resulting in prolongation of the repolarisation phase of the action potential. Phenytoin, which decrease the excitability of neurones, has recently also been shown to block IKr, in addition to its known blockade of sodium channels. Animal studies indicate that IKr is expressed in the embryo and that the embryonic heart is extremely susceptible to IKr-blockers during a restricted period in early development. At concentrations not affecting the maternal heart, the embryonic heart reacts with bradycardia, arrhythmia and cardiac arrest when exposed to such drugs. Available studies strongly support the idea that birth defects after in utero exposure to both selective and non-selective IKr-blockers (like phenytoin) are initiated by concentration dependent embryonic bradycardia/arrhythmia resulting in 1) hypoxia; explaining embryonic death and growth retardation, 2) episodes of severe hypoxia, followed by generation of reactive oxygen species within the embryo during reoxygenation, causing orofacial clefts and distal digital reductions, and 3) alterations in embryonic blood flow and blood pressure, inducing cardiovascular defects.

The old heart: operating on the edge.

Excitation of cardiac cells is accompanied by Ca2+ influx which triggers a transient increase in cytosolic [Ca2+], (Cai), and contraction. While the amplitudes of the Cai transient and contraction increase with the extent of cell Ca2+ loading, excess Ca2+ loading leads to dysregulation of Ca2+ homeostasis, impaired contraction, arrhythmia and cell death. The cell Ca2+ load is determined by membrane structure and permeability characteristics, the intensity of stimuli that modulate Ca2+ influx or efflux via regulatory function of proteins within membranes, and reactive oxygen species (ROS), which affect both membrane structure and function. Cardiocytes of senescent hearts exhibit a reduced threshold for pathologic manifestations of excess Ca2+ loading during stimulation (physiologic or pharmacologic) that increases Ca2+ influx, e.g. in response to neurotransmitters, post-ischaemic reperfusion, or oxidative stress. Cell 'remodelling' is one cause of the relative Ca2+ intolerance of cardiocytes in the senescent heart; cells increase in size and changes occur in the amounts of proteins that regulate Ca2+ handling due, in part, to altered gene expression; another cause is a change in the composition of membranes in which Ca2+ regulatory proteins reside, e.g. an increase in membrane omega 6:omega 3 polyunsaturated fatty acids (PUFA); a third cause is an enhanced likelihood for intracellular generation of ROS. Each class of these determinants changes with ageing and reduces the threshold for Ca2+ overload to occur with the older heart. The risk of excess Ca2+ loading within the senescent heart can potentially be reduced by gene therapy to restore Ca2+ regulatory proteins, by diet to reverse the membrane omega 6:omega 3 PUFA imbalance, or by antioxidants.

Mechanisms of cardiac cell excitation with premature monophasic and biphasic field stimuli: a model study

The mechanisms by which extracellular electric field stimuli induce the (re)excitation of cardiac cells in various stages of refractoriness are still not well understood. We modeled the interactions between an isolated cardiac cell and imposed extracellular electric fields to determine the mechanisms by which relatively low-strength uniform monophasic and biphasic field stimuli induce premature reexcitations. An idealized ventricular cell was simulated with 11 subcellular membrane patches, each of which obeyed Luo-Rudy (phase 1) kinetics. Implementing a standard S1-S2 pulse protocol, strength-interval maps of the cellular excitatory responses were generated for rectangular monophasic and symmetric biphasic field stimuli of 2, 5, 10, and 20 ms total duration. In contrast to previously documented current injection studies, our results demonstrate that a cardiac cell exhibits a significantly nonmonotonic excitatory response to premature monophasic and, to a much lesser degree, biphasic field stimuli. Furthermore, for monophasic stimuli at low field strengths, the cell is exquisitely sensitive to the timing of the shock, demonstrating a classic all-or-none depolarizing response. However, at higher field strengths this all-or-none sensitivity reverts to a more gradual transition of excitatory responses with respect to stimulus prematurity. In contrast, biphasic stimuli produce such graded responses at all suprathreshold stimulus strengths. Similar behaviors are demonstrated at all S2 stimulus durations tested. The generation of depolarizing (sodium) currents is triggered by one or more of the sharp field gradient changes produced at the stimulus edges-i.e., make, break, and transphasic (for biphasic stimuli)-with the magnitude of these edge-induced current contributions dependent on both the prematurity and the strength of the applied field. In all cases, however, depolarizing current arises from the partial removal of sodium inactivation from at least part of the cell, because of either the natural process of repolarization or a localized acceleration of this process by the impressed field.

Wavelet formation in excitable cardiac tissue: the role of wavefront-obstacle interactions in initiating high-frequency fibrillatory-like arrhythmias

High-frequency arrhythmias leading to fibrillation are often associated with the presence of inhomogeneities (obstacles) in cardiac tissue and reduced excitability of cardiac cells. Studies of antiarrhythmic drugs in patients surviving myocardial infarction revealed an increased rate of sudden cardiac death compared with untreated patients. These drugs block the cardiac sodium channel, thereby reducing excitability, which may alter wavefront-obstacle interactions. In diseased atrial tissue, excitability is reduced by diminished sodium channel availability secondary to depolarized rest potentials and cellular decoupling secondary to intercellular fibrosis. Excitability can also be reduced by incomplete recovery between successive excitations. In all of these cases, wavefront-obstacle interactions in a poorly excitable medium may reflect an arrhythmogenic process that permits formation of reentrant wavelets leading to flutter, fibrillation, and sudden cardiac death. To probe the relationship between excitability and arrhythmogenesis, we explored conditions for new wavelet formation after collision of a plane wave with an obstacle in an otherwise homogeneous excitable medium. Formulating our approach in terms of the balance between charge available in the wavefront and the excitation charge requirements of adjacent medium, we found analytically the critical medium parameters that defined conditions for wavefront-obstacle separation. Under these conditions, when a parent wavefront collided with a primitive obstacle, the resultant fragments separated from the obstacle boundaries, subsequently curled, and spawned new "daughter" wavelets. We identified spatial arrangements of obstacles such that wavefront-obstacle collisions leading to spawning of new wavelets could produce high-frequency wavelet trains similar to fibrillation-like arrhythmias.

A model study of extracellular stimulation of cardiac cells.

Point source extracellular stimulation of a myocyte model was used to study the efficacy of excitation of cardiac cells, taking into account the shape of the pulse stimulus and its time of application in the cardiac cycle. The myocyte was modeled as a small cylinder of membrane (10 microns in diameter and 100 microns in length) capped at both ends and placed in an unbounded volume conductor. A Beeler-Reuter model modified for the Na+ dynamics served to simulate the membrane ionic current. The stimulus source was located on the cylinder axis, close to the myocyte (50 microns) in order to generate a nonlinear extracellular field (phi e). The low membrane impedance associated with the high frequency component of the make and break of the rectangular current pulse leads to a current flow across the membrane and an abrupt change in intracellular potential (phi i). Because the intracellular space is very small, phi i is nearly uniform over the length of the myocyte and the membrane potential (V = phi i-phi e) is governed by the applied field phi e. There is then a longitudinal gradient of membrane polarization which is the inverse of the gradient of extracellular potential. With an anodal (positive) pulse, for instance, the proximal portion of the myocyte is hyperpolarized and the distal portion is depolarized. Based on this principle and considering the voltage-dependent activation/inactivation dynamics of the membrane, it is shown that a cathodal (negative) pulse is the most efficacious stimulus at diastolic potentials, an anodal current is preferable during the plateau phase of the action potential, and a biphasic pulse is optimal during the relative refractory phase. Thus a biphasic pulse would constitute the best choice for maximum efficacy at all phases of the action potential.

Irregular Dynamics of Excitation in Biologic and Mathematical Models of Cardiac Cellsa

Excitation and impulse propagation in cardiac tissues are dependent on the heart rate and can occur in extremely complex patterns. In this chapter we present the results of Purkinje fiber experiments and of computer simulations using an ionic (Beeler & Reuter) model for the ventricular cell. We have studied the global rate-dependent behavior of cardiac cells through a systematic analysis of their response to single as well as repetitive depolarizing stimuli, and determined the role of nonlinearity in the mechanism(s) of their behaviors. To this end, we devised an analytical difference equation model of cardiac cell excitation which could be used to predict simple as well as chaotic behavior of both the Purkinje fiber and the Beeler & Reuter cell, depending on the stimulation rate. Both experimental and modeling results suggest that the presence of supernormal recovery in cell excitability establishes sufficient nonlinearity so that, during repetitive stimulation, the dynamics of cell response may be regular and predictable when the stimulus magnitude is either very small or very large, or they may be chaotic and very unpredictable when the stimulus magnitude is intermediate. The overall results suggest that the application of nonlinear systems theory to electrophysiology may have importance in the understanding of cardiac rhythm and conduction disturbances, and may have clinical implications as well.

Improved cardiac cell excitation with symmetrical biphasic defibrillator waveforms.

According to the most commonly accepted hypothesis, ventricular defibrillation is produced by exciting cells in a critical mass of the ventricle. For monophasic defibrillator waveforms, this hypothesis correctly predicts a direct correlation between defibrillation threshold in the transthoracic calf model and excitation threshold for extracellular field stimulation in the cultured cell model. To further test the hypothesis, we determined whether symmetrical biphasic waveforms, which reduce defibrillation threshold in the calf to approximately 65% of that of the corresponding monophasic waveform (14), decrease excitation threshold in the cultured cell model. Experiments were performed on 100- to 250-microns aggregates from 10- to 12-day-old chick embryos. Excitation threshold strength-duration curves obtained at extracellular potassium (Ko) = 6.5 mM and pacing interval of 1,000 ms showed a significant reduction for symmetrical biphasic rectangular waveforms, when compared with the corresponding monophasic waveforms for durations greater than 3 ms. At the rheobase, the threshold ratio between the biphasic and monophasic waveforms was 0.63 (SE = 0.02). Transmembrane potentials during stimulation showed that excitation takes place during the second portion of the biphasic waveform for intensities that are subthreshold for the monophasic waveform. The relative effectiveness of the biphasic waveform (5-ms duration) increases under "fibrillation conditions" of short pacing interval (300 ms) and high extracellular potassium (10.5 mM). These results show that symmetrical biphasic waveforms decrease excitation threshold in the cultured cell model and that the degree of threshold reduction is dependent on Ko and beat rate.