Whole-heart computational modelling provides further mechanistic insights into ST-elevation in Brugada syndrome.

Background: Brugada syndrome (BrS) is characterized by dynamic ST-elevations in right precordial leads and increased risk of ventricular fibrillation and sudden cardiac death. As the mechanism underlying ST-elevation and malignant arrhythmias is controversial computational modeling can aid in exploring the disease mechanism. Thus we aim to test the main competing hypotheses ('delayed depolarization' vs. 'early repolarization') of BrS in a whole-heart computational model. Methods: In a 3D whole-heart computational model, delayed epicardial RVOT activation with local conduction delay was simulated by reducing conductivity in the epicardial RVOT. Early repolarization was simulated by instead increasing the transient outward potassium current (Ito) in the same region. Additionally, a reduction in the fast sodium current (INa) was incorporated in both models. Results: Delayed depolarization with local conduction delay in the computational model resulted in coved-type ST-elevation with negative T-waves in the precordial surface ECG leads. 'Saddleback'-shaped ST-elevation was obtained with reduced substrate extent or thickness. Increased Ito simulations showed early repolarization in the RVOT with a descending but not coved-type ST-elevation. Reduced INa did not show a significant effect on ECG morphology. Conclusions: In this whole-heart BrS computational model of both major hypotheses, realistic coved-type ECG resulted only from delayed epicardial RVOT depolarization with local conduction delay but not early repolarizing ion channel modifications. These simulations provide further support for the depolarization hypothesis as electrophysiological mechanism underlying BrS.

Directed Graph Mapping exceeds Phase Mapping for the detection of simulated 2D meandering rotors in fibrotic tissue with added noise.

Cardiac arrhythmias such as atrial fibrillation (AF) are recognised to be associated with re-entry or rotors. A rotor is a wave of excitation in the cardiac tissue that wraps around its refractory tail, causing faster-than-normal periodic excitation. The detection of rotor centres is of crucial importance in guiding ablation strategies for the treatment of arrhythmia. The most popular technique for detecting rotor centres is Phase Mapping (PM), which detects phase singularities derived from the phase of a signal. This method has been proven to be prone to errors, especially in regimes of fibrotic tissue and temporal noise. Recently, a novel technique called Directed Graph Mapping (DGM) was developed to detect rotational activity such as rotors by creating a network of excitation. This research aims to compare the performance of advanced PM techniques versus DGM for the detection of rotors using 64 simulated 2D meandering rotors in the presence of various levels of fibrotic tissue and temporal noise. Four strategies were employed to compare the performances of PM and DGM. These included a visual analysis, a comparison of F2-scores and distance distributions, and calculating p-values using the mid-p McNemar test. Results indicate that in the case of low meandering, fibrosis and noise, PM and DGM yield excellent results and are comparable. However, in the case of high meandering, fibrosis and noise, PM is undeniably prone to errors, mainly in the form of an excess of false positives, resulting in low precision. In contrast, DGM is more robust against these factors as F2-scores remain high, yielding F2≥0.931 as opposed to the best PM F2≥0.635 across all 64 simulations.

Disease severity, arrhythmogenesis, and fibrosis are related to longer action potentials in tetralogy of Fallot.

BACKGROUND: Arrhythmias may originate from surgically unaffected right ventricular (RV) regions in patients with tetralogy of Fallot (TOF). We aimed to investigate action potential (AP) remodelling and arrhythmia susceptibility in RV myocardium of patients with repaired and with unrepaired TOF, identify possible correlations with clinical phenotype and myocardial fibrosis, and compare findings with data from patients with atrial septal defect (ASD), a less severe congenital heart disease. METHODS: Intracellular AP were recorded ex vivo in RV outflow tract samples from 22 TOF and three ASD patients. Arrhythmias were provoked by superfusion with solutions containing reduced potassium and barium chloride, or isoprenaline. Myocardial fibrosis was quantified histologically and associations between clinical phenotype, AP shape, tissue arrhythmia propensity, and fibrosis were examined. RESULTS: Electrophysiological abnormalities (arrhythmias, AP duration [APD] alternans, impaired APD shortening at increased stimulation frequencies) were generally present in TOF tissue, even from infants, but rare or absent in ASD samples. More severely diseased and acyanotic patients, pronounced tissue susceptibility to arrhythmogenesis, and greater fibrosis extent were associated with longer APD. In contrast, APD was shorter in tissue from patients with pre-operative cyanosis. Increased fibrosis and repaired-TOF status were linked to tissue arrhythmia inducibility. CONCLUSIONS: Functional and structural tissue remodelling may explain arrhythmic activity in TOF patients, even at a very young age. Surprisingly, clinical acyanosis appears to be associated with more severe arrhythmogenic remodelling. Further research into the clinical drivers of structural and electrical myocardial alterations, and the relation between them, is needed to identify predictive factors for patients at risk.

Directed graph mapping shows rotors maintain non-terminating and focal sources maintain self-terminating Torsade de Pointes in canine model.

Torsade de Pointes is a polymorphic ventricular tachycardia which is as yet incompletely understood. While the onset of a TdP episode is generally accepted to be caused by triggered activity, the mechanisms for the perpetuation is still under debate. In this study, we analysed data from 54 TdP episodes divided over 5 dogs (4 female, 1 male) with chronic atrioventricular block. Previous research on this dataset showed both reentry and triggered activity to perpetuate the arrhythmia. 13 of those TdP episodes showed reentry as part of the driving mechanism of perpetuating the episode. The remaining 41 episodes were purely ectopic. Reentry was the main mechanism in long-lasting episodes (>14 beats), while focal sources were responsible for maintaining shorter episodes. Building on these results, we re-analysed the data using directed graph mapping This program uses principles from network theory and a combination of positional data and local activation times to identify reentry loops and focal sources within the data. The results of this study are twofold. First, concerning reentry loops, we found that on average non-terminating (NT) episodes (≥10 s) show significantly more simultaneous reentry loops than self-terminating (ST) TdP (<10 s). Non-terminating episodes have on average 2.72 ± 1.48 simultaneous loops, compared to an average of 1.33 ± 0.66 for self-terminating episodes. In addition, each NT episode showed a presence of (bi-)ventricular loops between 10.10% and 69.62% of their total reentry duration. Compared to the ST episodes, only 1 in 4 episodes (25%) showed (bi-)ventricular reentry, lasting only 7.12% of its total reentry duration. This suggests that while focal beats trigger TdP, macro-reentry and multiple simultaneous localized reentries are the major drivers of long-lasting episodes. Second, using heatmaps, we found focal sources to occur in preferred locations, instead of being distributed randomly. This may have implications on treatment if such focal origins can be disabled reliably.

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 computational model of rabbit geometry and ECG: Optimizing ventricular activation sequence and APD distribution.

Computational modeling of electrophysiological properties of the rabbit heart is a commonly used way to enhance and/or complement findings from classic lab work on single cell or tissue levels. Yet, thus far, there was no possibility to extend the scope to include the resulting body surface potentials as a way of validation or to investigate the effect of certain pathologies. Based on CT imaging, we developed the first openly available computational geometrical model not only of the whole heart but also the complete torso of the rabbit. Additionally, we fabricated a 32-lead ECG-vest to record body surface potential signals of the aforementioned rabbit. Based on the developed geometrical model and the measured signals, we then optimized the activation sequence of the ventricles, recreating the functionality of the Purkinje network, and we investigated different apico-basal and transmural gradients in action potential duration. Optimization of the activation sequence resulted in an average root mean square error between measured and simulated signal of 0.074 mV/ms for all leads. The best-fit T-Wave, compared to measured data (0.038 mV/ms), resulted from incorporating an action potential duration gradient from base to apex with a respective shortening of 20 ms and a transmural gradient with a shortening of 15 ms from endocardium to epicardium. By making our model and measured data openly available, we hope to give other researchers the opportunity to verify their research, as well as to create the possibility to investigate the impact of electrophysiological alterations on body surface signals for translational research.

Docosahexaenoic acid normalizes QT interval in long QT type 2 transgenic rabbit models in a genotype-specific fashion.

AIM: Long QT syndrome (LQTS) is a cardiac channelopathy predisposing to ventricular arrhythmias and sudden cardiac death. Since current therapies often fail to prevent arrhythmic events in certain LQTS subtypes, new therapeutic strategies are needed. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, which enhances the repolarizing IKs current. METHODS AND RESULTS: We investigated the effects of DHA in wild type (WT) and transgenic long QT Type 1 (LQT1; loss of IKs), LQT2 (loss of IKr), LQT5 (reduction of IKs), and LQT2-5 (loss of IKr and reduction of IKs) rabbits. In vivo ECGs were recorded at baseline and after 10 µM/kg DHA to assess changes in heart-rate corrected QT (QTc) and short-term variability of QT (STVQT). Ex vivo monophasic action potentials were recorded in Langendorff-perfused rabbit hearts, and action potential duration (APD75) and triangulation were assessed. Docosahexaenoic acid significantly shortened QTc in vivo only in WT and LQT2 rabbits, in which both α- and ß-subunits of IKs-conducting channels are functionally intact. In LQT2, this led to a normalization of QTc and of its short-term variability. Docosahexaenoic acid had no effect on QTc in LQT1, LQT5, and LQT2-5. Similarly, ex vivo, DHA shortened APD75 in WT and normalized it in LQT2, and additionally decreased AP triangulation in LQT2. CONCLUSIONS: Docosahexaenoic acid exerts a genotype-specific beneficial shortening/normalizing effect on QTc and APD75 and reduces pro-arrhythmia markers STVQT and AP triangulation through activation of IKs in LQT2 rabbits but has no effects if either α- or ß-subunits to IKs are functionally impaired. Docosahexaenoic acid could represent a new genotype-specific therapy in LQT2.

Novel insights into the electrophysiology of murine cardiac macrophages: relevance of voltage-gated potassium channels.

AIMS: Macrophages (MΦ), known for immunological roles, such as phagocytosis and antigen presentation, have been found to electrotonically couple to cardiomyocytes (CM) of the atrioventricular node via Cx43, affecting cardiac conduction in isolated mouse hearts. Here, we characterize passive and active electrophysiological properties of murine cardiac resident MΦ, and model their potential electrophysiological relevance for CM. METHODS AND RESULTS: We combined classic electrophysiological approaches with 3D florescence imaging, RNA-sequencing, pharmacological interventions, and computer simulations. We used Cx3cr1eYFP/+ mice wherein cardiac MΦ are fluorescently labelled. FACS-purified fluorescent MΦ from mouse hearts were studied by whole-cell patch-clamp. MΦ electrophysiological properties include: membrane resistance 2.2±0.1 GΩ (all data mean±SEM), capacitance 18.3±0.1 pF, resting membrane potential -39.6±0.3 mV, and several voltage-activated, outward or inwardly rectifying potassium currents. Using ion channel blockers (barium, TEA, 4-AP, margatoxin, XEN-D0103, and DIDS), flow cytometry, immuno-staining, and RNA-sequencing, we identified Kv1.3, Kv1.5, and Kir2.1 as channels contributing to observed ion currents. MΦ displayed four patterns for outward and two for inward-rectifier potassium currents. Additionally, MΦ showed surface expression of Cx43, a prerequisite for homo- and/or heterotypic electrotonic coupling. Experimental results fed into development of an original computational model to describe cardiac MΦ electrophysiology. Computer simulations to quantitatively assess plausible effects of MΦ on electrotonically coupled CM showed that MΦ can depolarize resting CM, shorten early and prolong late action potential duration, with effects depending on coupling strength and individual MΦ electrophysiological properties, in particular resting membrane potential and presence/absence of Kir2.1. CONCLUSION: Our results provide a first electrophysiological characterization of cardiac resident MΦ, and a computational model to quantitatively explore their relevance in the heterocellular heart. Future work will be focussed at distinguishing electrophysiological effects of MΦ-CM coupling on both cell types during steady-state and in patho-physiological remodelling, when immune cells change their phenotype, proliferate, and/or invade from external sources.

CVAR-Seg: An Automated Signal Segmentation Pipeline for Conduction Velocity and Amplitude Restitution.

BACKGROUND: Rate-varying S1S2 stimulation protocols can be used for restitution studies to characterize atrial substrate, ionic remodeling, and atrial fibrillation risk. Clinical restitution studies with numerous patients create large amounts of these data. Thus, an automated pipeline to evaluate clinically acquired S1S2 stimulation protocol data necessitates consistent, robust, reproducible, and precise evaluation of local activation times, electrogram amplitude, and conduction velocity. Here, we present the CVAR-Seg pipeline, developed focusing on three challenges: (i) No previous knowledge of the stimulation parameters is available, thus, arbitrary protocols are supported. (ii) The pipeline remains robust under different noise conditions. (iii) The pipeline supports segmentation of atrial activities in close temporal proximity to the stimulation artifact, which is challenging due to larger amplitude and slope of the stimulus compared to the atrial activity. METHODS AND RESULTS: The S1 basic cycle length was estimated by time interval detection. Stimulation time windows were segmented by detecting synchronous peaks in different channels surpassing an amplitude threshold and identifying time intervals between detected stimuli. Elimination of the stimulation artifact by a matched filter allowed detection of local activation times in temporal proximity. A non-linear signal energy operator was used to segment periods of atrial activity. Geodesic and Euclidean inter electrode distances allowed approximation of conduction velocity. The automatic segmentation performance of the CVAR-Seg pipeline was evaluated on 37 synthetic datasets with decreasing signal-to-noise ratios. Noise was modeled by reconstructing the frequency spectrum of clinical noise. The pipeline retained a median local activation time error below a single sample (1 ms) for signal-to-noise ratios as low as 0 dB representing a high clinical noise level. As a proof of concept, the pipeline was tested on a CARTO case of a paroxysmal atrial fibrillation patient and yielded plausible restitution curves for conduction speed and amplitude. CONCLUSION: The proposed openly available CVAR-Seg pipeline promises fast, fully automated, robust, and accurate evaluations of atrial signals even with low signal-to-noise ratios. This is achieved by solving the proximity problem of stimulation and atrial activity to enable standardized evaluation without introducing human bias for large data sets.

A move in the light direction.

Computer simulations show how low-intensity illumination can be used to terminate cardiac arrhythmias.

Quantitative collagen assessment in right ventricular myectomies from patients with tetralogy of Fallot.

AIMS: Patients with tetralogy of Fallot (TOF) are often affected by right ventricular fibrosis, which has been associated with arrhythmias. This study aimed to assess fibrosis distribution in right ventricular outflow tract (RVOT) myocardium of TOF patients to evaluate the utility of single histology-section analyses, and to explore the possibility of fibrosis quantification in unlabelled tissue by second harmonic generation imaging (SHGI) as an alternative to conventional histology-based assays. METHODS AND RESULTS: We quantified fibrosis in 11 TOF RVOT samples, using a tailor-made automated image analysis method on Picrosirius red-stained sections. In a subset of samples, histology- and SHGI-based fibrosis quantification approaches were compared. Fibrosis distribution was highly heterogeneous, with significant and comparable variability between and within samples. We found that, on average, 67.8 mm2 of 10 µm thick, histologically processed tissue per patient had to be analysed for accurate fibrosis quantification. SHGI provided data faster and on live tissue, additionally enabling quantification of collagen anisotropy. CONCLUSION: Given the high intra-individual heterogeneity, fibrosis quantification should not be conducted on single sections of TOF RVOT myectomies. We provide an analysis algorithm for fibrosis quantification in histological images, which enables the required extended volume analyses in these patients.

Optogenetic targeting of cardiac myocytes and non-myocytes: Tools, challenges and utility.

In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.

Mini Electrodes on Ablation Catheters: Valuable Addition or Redundant Information?-Insights from a Computational Study.

Radiofrequency ablation has become a first-line approach for curative therapy of many cardiac arrhythmias. Various existing catheter designs provide high spatial resolution to identify the best spot for performing ablation and to assess lesion formation. However, creation of transmural and nonconducting ablation lesions requires usage of catheters with larger electrodes and improved thermal conductivity, leading to reduced spatial sensitivity. As trade-off, an ablation catheter with integrated mini electrodes was introduced. The additional diagnostic benefit of this catheter is still not clear. In order to solve this issue, we implemented a computational setup with different ablation scenarios. Our in silico results show that peak-to-peak amplitudes of unipolar electrograms from mini electrodes are more suitable to differentiate ablated and nonablated tissue compared to electrograms from the distal ablation electrode. However, in orthogonal mapping position, no significant difference was observed between distal electrode and mini electrodes electrograms in the ablation scenarios. In conclusion, catheters with mini electrodes bring about additional benefit to distinguish ablated tissue from nonablated tissue in parallel position with high spatial resolution. It is feasible to detect conduction gaps in linear lesions with this catheter by evaluating electrogram data from mini electrodes.

Macrophages Facilitate Electrical Conduction in the Heart.

Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.

Quantitative analysis of cardiac tissue including fibroblasts using three-dimensional confocal microscopy and image reconstruction: towards a basis for electrophysiological modeling.

Electrophysiological modeling of cardiac tissue is commonly based on functional and structural properties measured in experiments. Our knowledge of these properties is incomplete, in particular their remodeling in disease. Here, we introduce a methodology for quantitative tissue characterization based on fluorescent labeling, 3-D scanning confocal microscopy, image processing and reconstruction of tissue micro-structure at sub-micrometer resolution. We applied this methodology to normal rabbit ventricular tissue and tissue from hearts with myocardial infarction. Our analysis revealed that the volume fraction of fibroblasts increased from 4.83±0.42% (mean ± standard deviation) in normal tissue up to 6.51±0.38% in myocardium from infarcted hearts. The myocyte volume fraction decreased from 76.20±9.89% in normal to 73.48±8.02% adjacent to the infarct. Numerical field calculations on 3-D reconstructions of the extracellular space yielded an extracellular longitudinal conductivity of 0.264±0.082 S/m with an anisotropy ratio of 2.095±1.11 in normal tissue. Adjacent to the infarct, the longitudinal conductivity increased up to 0.400±0.051 S/m, but the anisotropy ratio decreased to 1.295±0.09. Our study indicates an increased density of gap junctions proximal to both fibroblasts and myocytes in infarcted versus normal tissue, supporting previous hypotheses of electrical coupling of fibroblasts and myocytes in infarcted hearts. We suggest that the presented methodology provides an important contribution to modeling normal and diseased tissue. Applications of the methodology include the clinical characterization of disease-associated remodeling.