Determinants of electrical propagation and propagation block in Arrhythmogenic Cardiomyopathy.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

Nano-scale solution of the Poisson-Nernst-Planck (PNP) equations in a fraction of two neighboring cells reveals the magnitude of intercellular electrochemical waves.

The basic building blocks of the electrophysiology of cardiomyocytes are ion channels integrated in the cell membranes. Close to the ion channels there are very strong electrical and chemical gradients. However, these gradients extend for only a few nano-meters and are therefore commonly ignored in mathematical models. The full complexity of the dynamics is modelled by the Poisson-Nernst-Planck (PNP) equations but these equations must be solved using temporal and spatial scales of nano-seconds and nano-meters. Here we report solutions of the PNP equations in a fraction of two abuttal cells separated by a tiny extracellular space. We show that when only the potassium channels of the two cells are open, a stationary solution is reached with the well-known Debye layer close to the membranes. When the sodium channels of one of the cells are opened, a very strong and brief electrochemical wave emanates from the channels. If the extracellular space is sufficiently small and the number of sodium channels is sufficiently high, the wave extends all the way over to the neighboring cell and may therefore explain cardiac conduction even at very low levels of gap junctional coupling.

A detailed analysis of single-channel Nav 1.5 recordings does not reveal any cooperative gating.

Cardiac voltage-gated sodium (Na+ ) channels (Nav 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 Nav 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 Nav 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: Nav 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 Nav 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 Nav 1.5 channels. Our unbiased analysis may be useful in further studies examining how Na+ channel interactions are affected by mutations and auxiliary proteins.

Active force generation contributes to the complexity of spontaneous activity and to the response to stretch of murine cardiomyocyte cultures.

Cardiomyocyte cultures exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. In such preparations, beat rate variability exhibits features similar to those of heart rate variability in vivo. Mechanical deformations and forces feed back on the electrical properties of cardiomyocytes, but it is not fully elucidated how this mechano-electrical interplay affects beating variability in such preparations. Using stretchable microelectrode arrays, we assessed the effects of the myosin inhibitor blebbistatin and the non-selective stretch-activated channel blocker streptomycin on beating variability and on the response of neonatal or fetal murine ventricular cell cultures against deformation. Spontaneous electrical activity was recorded without stretch and upon predefined deformation protocols (5% uniaxial and 2% equibiaxial strain, applied repeatedly for 1 min every 3 min). Without stretch, spontaneous activity originated from the edge of the preparations, and its site of origin switched frequently in a complex manner across the cultures. Blebbistatin did not change mean beat rate, but it decreased the spatial complexity of spontaneous activity. In contrast, streptomycin did not exert any manifest effects. During the deformation protocols, beat rate increased transiently upon stretch but, paradoxically, also upon release. Blebbistatin attenuated the response to stretch, whereas this response was not affected by streptomycin. Therefore, our data support the notion that in a spontaneously firing network of cardiomyocytes, active force generation, rather than stretch-activated channels, is involved mechanistically in the complexity of the spatiotemporal patterns of spontaneous activity and in the stretch-induced acceleration of beating. KEY POINTS: Monolayer cultures of cardiac cells exhibit spontaneous electrical and contractile activity, as in a natural cardiac pacemaker. Beating variability in these preparations recapitulates the power-law behaviour of heart rate variability in vivo. However, the effects of mechano-electrical feedback on beating variability are not yet fully understood. Using stretchable microelectrode arrays, we examined the effects of the contraction uncoupler blebbistatin and the non-specific stretch-activated channel blocker streptomycin on beating variability and on stretch-induced changes of beat rate. Without stretch, blebbistatin decreased the spatial complexity of beating variability, whereas streptomycin had no effects. Both stretch and release increased beat rate transiently; blebbistatin attenuated the increase of beat rate upon stretch, whereas streptomycin had no effects. Active force generation contributes to the complexity of spatiotemporal patterns of beating variability and to the increase of beat rate upon mechanical deformation. Our study contributes to the understanding of how mechano-electrical feedback influences heart rate variability.

Localization of Na+ channel clusters in narrowed perinexi of gap junctions enhances cardiac impulse transmission via ephaptic coupling: a model study.

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.

Distribution of cardiac sodium channels in clusters potentiates ephaptic interactions in the intercalated disc.

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 Nav 1.5 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.

Stochastic pacing reveals the propensity to cardiac action potential alternans and uncovers its underlying dynamics.

KEY POINTS: Beat-to-beat alternation (alternans) of the cardiac action potential duration is known to precipitate life-threatening arrhythmias and can be driven by the kinetics of voltage-gated membrane currents or by instabilities in intracellular calcium fluxes. To prevent alternans and associated arrhythmias, suitable markers must be developed to quantify the susceptibility to alternans; previous theoretical studies showed that the eigenvalue of the alternating eigenmode represents an ideal marker of alternans. Using rabbit ventricular myocytes, we show that this eigenvalue can be estimated in practice by pacing these cells at intervals varying stochastically. We also show that stochastic pacing permits the estimation of further markers distinguishing between voltage-driven and calcium-driven alternans. Our study opens the perspective to use stochastic pacing during clinical investigations and in patients with implanted pacing devices to determine the susceptibility to, and the type of alternans, which are both important to guide preventive or therapeutic measures. ABSTRACT: Alternans of the cardiac action potential (AP) duration (APD) is a well-known arrhythmogenic mechanism. APD depends on several preceding diastolic intervals (DIs) and APDs, which complicates the prediction of alternans. Previous theoretical studies pinpointed a marker called λalt that directly quantifies how an alternating perturbation persists over successive APs. When the propensity to alternans increases, λalt decreases from 0 to -1. Our aim was to quantify λalt experimentally using stochastic pacing and to examine whether stochastic pacing allows discriminating between voltage-driven and Ca(2+) -driven alternans. APs were recorded in rabbit ventricular myocytes paced at cycle lengths (CLs) decreasing progressively and incorporating stochastic variations. Fitting APD with a function of two previous APDs and CLs permitted us to estimate λalt along with additional markers characterizing whether the dependence of APD on previous DIs or CLs is strong (typical for voltage-driven alternans) or weak (Ca(2+) -driven alternans). During the recordings, λalt gradually decreased from around 0 towards -1. Intermittent alternans appeared when λalt reached -0.8 and was followed by sustained alternans. The additional markers detected that alternans was Ca(2+) driven in control experiments and voltage driven in the presence of ryanodine. This distinction could be made even before alternans was manifest (specificity/sensitivity >80% for -0.4 > λalt  > -0.5). These observations were confirmed in a mathematical model of a rabbit ventricular myocyte. In conclusion, stochastic pacing allows the practical estimation of λalt to reveal the onset of alternans and distinguishes between voltage-driven and Ca(2+) -driven mechanisms, which is important since these two mechanisms may precipitate arrhythmias in different manners.

Nonlinear behaviour of conduction and block in cardiac tissue with heterogeneous expression of connexin 43.

Altered gap junctional coupling potentiates slow conduction and arrhythmias. To better understand how heterogeneous connexin expression affects conduction at the cellular scale, we investigated conduction in tissue consisting of two cardiomyocyte populations expressing different connexin levels. Conduction was mapped using microelectrode arrays in cultured strands of foetal murine ventricular myocytes with predefined contents of connexin 43 knockout (Cx43KO) cells. Corresponding computer simulations were run in randomly generated two-dimensional tissues mimicking the cellular architecture of the strands. In the cultures, the relationship between conduction velocity (CV) and Cx43KO cell content was nonlinear. CV first decreased significantly when Cx43KO content was increased from 0 to 50%. When the Cx43KO content was ≥60%, CV became comparable to that in 100% Cx43KO strands. Co-culturing Cx43KO and wild-type cells also resulted in significantly more heterogeneous conduction patterns and in frequent conduction blocks. The simulations replicated this behaviour of conduction. For Cx43KO contents of 10-50%, conduction was slowed due to wavefront meandering between Cx43KO cells. For Cx43KO contents ≥60%, clusters of remaining wild-type cells acted as electrical loads that impaired conduction. For Cx43KO contents of 40-60%, conduction exhibited fractal characteristics, was prone to block, and was more sensitive to changes in ion currents compared to homogeneous tissue. In conclusion, conduction velocity and stability behave in a nonlinear manner when cardiomyocytes expressing different connexin amounts are combined. This behaviour results from heterogeneous current-to-load relationships at the cellular level. Such behaviour is likely to be arrhythmogenic in various clinical contexts in which gap junctional coupling is heterogeneous.

Characterization of 2 genetic variants of Na(v) 1.5-arginine 689 found in patients with cardiac arrhythmias.

Hundreds of genetic variants in SCN5A, the gene coding for the pore-forming subunit of the cardiac sodium channel, Na(v) 1.5, have been described in patients with cardiac channelopathies as well as in individuals from control cohorts. The aim of this study was to characterize the biophysical properties of 2 naturally occurring Na(v) 1.5 variants, p.R689H and p.R689C, found in patients with cardiac arrhythmias and in control individuals. In addition, this study was motivated by the finding of the variant p.R689H in a family with sudden cardiac death (SCD) in children. When expressed in HEK293 cells, most of the sodium current (I(Na)) biophysical properties of both variants were indistinguishable from the wild-type (WT) channels. In both cases, however, an ∼2-fold increase of the tetrodotoxin-sensitive late I(Na) was observed. Action potential simulations and reconstruction of pseudo-ECGs demonstrated that such a subtle increase in the late I(Na) may prolong the QT interval in a nonlinear fashion. In conclusion, despite the fact that the causality link between p.R689H and the phenotype of the studied family cannot be demonstrated, this study supports the notion that subtle alterations of Na(v) 1.5 variants may increase the risk for cardiac arrhythmias.

Uncovering the dynamics of cardiac systems using stochastic pacing and frequency domain analyses.

Alternans of cardiac action potential duration (APD) is a well-known arrhythmogenic mechanism which results from dynamical instabilities. The propensity to alternans is classically investigated by examining APD restitution and by deriving APD restitution slopes as predictive markers. However, experiments have shown that such markers are not always accurate for the prediction of alternans. Using a mathematical ventricular cell model known to exhibit unstable dynamics of both membrane potential and Ca²âº cycling, we demonstrate that an accurate marker can be obtained by pacing at cycle lengths (CLs) varying randomly around a basic CL (BCL) and by evaluating the transfer function between the time series of CLs and APDs using an autoregressive-moving-average (ARMA) model. The first pole of this transfer function corresponds to the eigenvalue (λ(alt)) of the dominant eigenmode of the cardiac system, which predicts that alternans occurs when λ(alt) ≤ -1. For different BCLs, control values of λ(alt) were obtained using eigenmode analysis and compared to the first pole of the transfer function estimated using ARMA model fitting in simulations of random pacing protocols. In all versions of the cell model, this pole provided an accurate estimation of λ(alt). Furthermore, during slow ramp decreases of BCL or simulated drug application, this approach predicted the onset of alternans by extrapolating the time course of the estimated λ(alt). In conclusion, stochastic pacing and ARMA model identification represents a novel approach to predict alternans without making any assumptions about its ionic mechanisms. It should therefore be applicable experimentally for any type of myocardial cell.

Tortuous Cardiac Intercalated Discs Modulate Ephaptic Coupling.

Cardiac ephaptic coupling, a mechanism mediated by negative electric potentials occurring in the narrow intercellular clefts of intercalated discs, can influence action potential propagation by modulating the sodium current. Intercalated discs are highly tortuous due to the mingling of plicate and interplicate regions. To investigate the effect of their convoluted structure on ephaptic coupling, we refined our previous model of an intercalated disc and tested predefined folded geometries, which we parametrized by orientation, amplitude and number of folds. Ephaptic interactions (assessed by the minimal cleft potential and amplitude of the sodium currents) were reinforced by concentric folds. With increasing amplitude and number of concentric folds, the cleft potential became more negative during the sodium current transient. This is explained by the larger resistance between the cleft and the bulk extracellular space. In contrast, radial folds attenuated ephaptic interactions and led to a less negative cleft potential due to a decreased net cleft resistance. In conclusion, despite limitations inherent to the simplified geometries and sodium channel distributions investigated as well as simplifications regarding ion concentration changes, these results indicate that the folding pattern of intercalated discs modulates ephaptic coupling.

Effects of stochastic channel gating and distribution on the cardiac action potential.

Ion channels exhibit stochastic conformational changes determining their gating behavior. In addition, the process of protein turnover leads to a natural variability of the number of membrane and gap junctional channels. Nevertheless, in computational models, these two aspects are scarcely considered and their impacts are largely unknown. We investigated the effects of stochastic current fluctuations and channel distributions on action potential duration (APD), intercellular conduction delays (ICDs) and conduction blocks using a modified ventricular cell model (Rudy et al.) with Markovian formulations of the principal ion currents (to simulate their stochastic open-close gating behavior) and with channel counts drawn from Poisson distributions (to simulate their natural variability). In single cells, APD variability (coefficient of variation: 1.6% at BCL=1000ms) was essentially caused by stochastic channel gating of I(Ks), persistent I(Na) and I(Ca,L). In cell strands, ICD variability induced by stochastic channel gating and Poissonian channel distributions was low under normal conditions. Nonetheless, at low intercellular coupling levels, Poissonian gap junctional channel distribution resulted in a large ICD variability (coefficient of variation >20%), highly heterogeneous conduction patterns and conduction blocks. Therefore, the stochastic behavior of current fluctuations and channel distributions can contribute to the heterogeneity of conduction patterns and to conduction block, as observed previously in experiments in cardiac tissue with altered intercellular coupling.

Kinetic analysis of ASIC1a delineates conformational signaling from proton-sensing domains to the channel gate.

Acid-sensing ion channels (ASICs) are neuronal Na+ channels that are activated by a drop in pH. Their established physiological and pathological roles, involving fear behaviors, learning, pain sensation, and neurodegeneration after stroke, make them promising targets for future drugs. Currently, the ASIC activation mechanism is not understood. Here, we used voltage-clamp fluorometry (VCF) combined with fluorophore-quencher pairing to determine the kinetics and direction of movements. We show that conformational changes with the speed of channel activation occur close to the gate and in more distant extracellular sites, where they may be driven by local protonation events. Further, we provide evidence for fast conformational changes in a pathway linking protonation sites to the channel pore, in which an extracellular interdomain loop interacts via aromatic residue interactions with the upper end of a transmembrane helix and would thereby open the gate.

Alternans resonance and propagation block during supernormal conduction in cardiac tissue with decreased [K(+)](o).

Cardiac restitution is an important factor in arrhythmogenesis. Steep positive action potential duration and conduction velocity (CV) restitution slopes promote alternans and reentrant arrhythmias. We examined the consequences of supernormal conduction (characterized by a negative CV restitution slope) on patterns of conduction and alternans in strands of Luo-Rudy model cells and in cultured cardiac cell strands. Interbeat intervals (IBIs) were analyzed as a function of distance during S1S2 protocols and during pacing at alternating cycle lengths. Supernormal conduction was induced by decreasing [K(+)](o). In control [K(+)](o) simulations, S1S2 intervals converged toward basic cycle length with a length constant determined by both CV and the CV restitution slope. During alternant pacing, the amplitude of IBI alternans converged with a shorter length constant, determined also by the action potential duration restitution slope. In contrast, during supernormal conduction, S1S2 intervals and the amplitude of alternans diverged. This amplification (resonance) led to phase-locked or more complex alternans patterns, and then to distal conduction block. The convergence/divergence of IBIs was verified in the cultured strands, in which naturally occurring tissue heterogeneities resulted in prominent discontinuities of the spatial IBI profiles. We conclude that supernormal conduction potentiates alternans and spatial analysis of IBIs represents a powerful method to locate tissue heterogeneities.

Modeling the Interactions Between Sodium Channels Provides Insight Into the Negative Dominance of Certain Channel Mutations.

BACKGROUND: Nav1.5 cardiac Na+ channel mutations can cause arrhythmogenic syndromes. Some of these mutations exert a dominant negative effect on wild-type channels. Recent studies showed that Na+ channels can dimerize, allowing coupled gating. This leads to the hypothesis that allosteric interactions between Na+ channels modulate their function and that these interactions may contribute to the negative dominance of certain mutations. METHODS: To investigate how allosteric interactions affect microscopic and macroscopic channel function, we developed a modeling paradigm in which Markovian models of two channels are combined. Allosteric interactions are incorporated by modifying the free energies of the composite states and/or barriers between states. RESULTS: Simulations using two generic 2-state models (C-O, closed-open) revealed that increasing the free energy of the composite states CO/OC leads to coupled gating. Simulations using two 3-state models (closed-open-inactivated) revealed that coupled closings must also involve interactions between further composite states. Using two 6-state cardiac Na+ channel models, we replicated previous experimental results mainly by increasing the energies of the CO/OC states and lowering the energy barriers between the CO/OC and the CO/OO states. The channel model was then modified to simulate a negative dominant mutation (Nav1.5 p.L325R). Simulations of homodimers and heterodimers in the presence and absence of interactions showed that the interactions with the variant channel impair the opening of the wild-type channel and thus contribute to negative dominance. CONCLUSION: Our new modeling framework recapitulates qualitatively previous experimental observations and helps identifying possible interaction mechanisms between ion channels.

KCNQ1 Antibodies for Immunotherapy of Long QT Syndrome Type 2.

BACKGROUND: Patients with long QT syndrome (LQTS) are predisposed to life-threatening arrhythmias. A delay in cardiac repolarization is characteristic of the disease. Pharmacotherapy, implantable cardioverter-defibrillators, and left cardiac sympathetic denervation are part of the current treatment options, but no targeted therapy for LQTS exists to date. Previous studies indicate that induced autoimmunity against the voltage-gated KCNQ1 K+ channels accelerates cardiac repolarization. OBJECTIVES: However, a causative relationship between KCNQ1 antibodies and the observed electrophysiological effects has never been demonstrated, and thus presents the aim of this study. METHODS: The authors purified KCNQ1 antibodies and performed whole-cell patch clamp experiments as well as single-channel recordings on Chinese hamster ovary cells overexpressing IKs channels. The effect of purified KCNQ1 antibodies on human cardiomyocytes derived from induced pluripotent stem cells was then studied. RESULTS: The study demonstrated that KCNQ1 antibodies underlie the previously observed increase in repolarizing IKs current. The antibodies shift the voltage dependence of activation and slow the deactivation of IKs. At the single-channel level, KCNQ1 antibodies increase the open time and probability of the channel. In models of LQTS type 2 (LQTS2) using human induced pluripotent stem cell-derived cardiomyocytes, KCNQ1 antibodies reverse the prolonged cardiac repolarization and abolish arrhythmic activities. CONCLUSIONS: Here, the authors provide the first direct evidence that KCNQ1 antibodies act as agonists on IKs channels. Moreover, KCNQ1 antibodies were able to restore alterations in cardiac repolarization and most importantly to suppress arrhythmias in LQTS2. KCNQ1 antibody therapy may thus present a novel promising therapeutic approach for LQTS2.

Slowing of the Time Course of Acidification Decreases the Acid-Sensing Ion Channel 1a Current Amplitude and Modulates Action Potential Firing in Neurons.

Acid-sensing ion channels (ASICs) are H+-activated neuronal Na+ channels. They are involved in fear behavior, learning, neurodegeneration after ischemic stroke and in pain sensation. ASIC activation has so far been studied only with fast pH changes, although the pH changes associated with many roles of ASICs are slow. It is currently not known whether slow pH changes can open ASICs at all. Here, we investigated to which extent slow pH changes can activate ASIC1a channels and induce action potential signaling. To this end, ASIC1a current amplitudes and charge transport in transfected Chinese hamster ovary cells, and ASIC-mediated action potential signaling in cultured cortical neurons were measured in response to defined pH ramps of 1-40 s duration from pH 7.4 to pH 6.6 or 6.0. A kinetic model of the ASIC1a current was developed and integrated into the Hodgkin-Huxley action potential model. Interestingly, whereas the ASIC1a current amplitude decreased with slower pH ramps, action potential firing was higher upon intermediate than fast acidification in cortical neurons. Indeed, fast pH changes (<4 s) induced short action potential bursts, while pH changes of intermediate speed (4-10 s) induced longer bursts. Slower pH changes (>10 s) did in many experiments not generate action potentials. Computer simulations corroborated these observations. We provide here the first description of ASIC function in response to defined slow pH changes. Our study shows that ASIC1a currents, and neuronal activity induced by ASIC1a currents, strongly depend on the speed of pH changes. Importantly, with pH changes that take >10 s to complete, ASIC1a activation is inefficient. Therefore, it is likely that currently unknown modulatory mechanisms allow ASIC activity in situations such as ischemia and inflammation.

The transfer functions of cardiac tissue during stochastic pacing.

The restitution properties of cardiac action potential duration (APD) and conduction velocity (CV) are important factors in arrhythmogenesis. They determine alternans, wavebreak, and the patterns of reentrant arrhythmias. We developed a novel approach to characterize restitution using transfer functions. Transfer functions relate an input and an output quantity in terms of gain and phase shift in the complex frequency domain. We derived an analytical expression for the transfer function of interbeat intervals (IBIs) during conduction from one site (input) to another site downstream (output). Transfer functions can be efficiently obtained using a stochastic pacing protocol. Using simulations of conduction and extracellular mapping of strands of neonatal rat ventricular myocytes, we show that transfer functions permit the quantification of APD and CV restitution slopes when it is difficult to measure APD directly. We find that the normally positive CV restitution slope attenuates IBI variations. In contrast, a negative CV restitution slope (induced by decreasing extracellular [K(+)]) amplifies IBI variations with a maximum at the frequency of alternans. Hence, it potentiates alternans and renders conduction unstable, even in the absence of APD restitution. Thus, stochastic pacing and transfer function analysis represent a powerful strategy to evaluate restitution and the stability of conduction.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome.

AIMS: Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation. METHODS AND RESULTS: SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block. CONCLUSION: Our data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.

Analysis of damped oscillations during reentry: a new approach to evaluate cardiac restitution.

Reentry is a mechanism underlying numerous cardiac arrhythmias. During reentry, head-tail interactions of the action potential can cause cycle length (CL) oscillations and affect the stability of reentry. We developed a method based on a difference-delay equation to determine the slopes of the action potential duration and conduction velocity restitution functions, known to be major determinants of reentrant arrhythmogenesis, from the spatial period P and the decay length D of damped CL oscillations. Using this approach, we analyzed CL oscillations after the induction of reentry and the resetting of reentry with electrical stimuli in rings of cultured neonatal rat ventricular myocytes grown on microelectrode arrays and in corresponding simulations with the Luo-Rudy model. In the experiments, P was larger and D was smaller after resetting impulses compared to the induction of reentry, indicating that reentry became more stable. Both restitution slopes were smaller. Consistent with the experimental findings, resetting of simulated reentry caused oscillations with gradually increasing P, decreasing D, and decreasing restitution slopes. However, these parameters remained constant when ion concentrations were clamped, revealing that intracellular ion accumulation stabilizes reentry. Thus, the analysis of CL oscillations during reentry opens new perspectives to gain quantitative insight into action potential restitution.