Digital twinning of the human ventricular activation sequence to Clinical 12-lead ECGs and magnetic resonance imaging using realistic Purkinje networks for in silico clinical trials.

Cardiac in silico clinical trials can virtually assess the safety and efficacy of therapies using human-based modelling and simulation. These technologies can provide mechanistic explanations for clinically observed pathological behaviour. Designing virtual cohorts for in silico trials requires exploiting clinical data to capture the physiological variability in the human population. The clinical characterisation of ventricular activation and the Purkinje network is challenging, especially non-invasively. Our study aims to present a novel digital twinning pipeline that can efficiently generate and integrate Purkinje networks into human multiscale biventricular models based on subject-specific clinical 12-lead electrocardiogram and magnetic resonance recordings. Essential novel features of the pipeline are the human-based Purkinje network generation method, personalisation considering ECG R wave progression as well as QRS morphology, and translation from reduced-order Eikonal models to equivalent biophysically-detailed monodomain ones. We demonstrate ECG simulations in line with clinical data with clinical image-based multiscale models with Purkinje in four control subjects and two hypertrophic cardiomyopathy patients (simulated and clinical QRS complexes with Pearson's correlation coefficients > 0.7). Our methods also considered possible differences in the density of Purkinje myocardial junctions in the Eikonal-based inference as regional conduction velocities. These differences translated into regional coupling effects between Purkinje and myocardial models in the monodomain formulation. In summary, we demonstrate a digital twin pipeline enabling simulations yielding clinically consistent ECGs with clinical CMR image-based biventricular multiscale models, including personalised Purkinje in healthy and cardiac disease conditions.

Intramural Purkinje fibers facilitate rapid ventricular activation in the equine heart.

BACKGROUND: The Purkinje fibers convey the electrical impulses at much higher speed than the working myocardial cells. Thus, the distribution of the Purkinje network is of paramount importance for the timing and coordination of ventricular activation. The Purkinje fibers are found in the subendocardium of all species of mammals, but some mammals also possess an intramural Purkinje fiber network that provides for relatively instantaneous, burst-like activation of the entire ventricular wall, and gives rise to an rS configuration in lead II of the ECG. AIM: To relate the topography of the horse heart and the distribution and histology of the conduction system to the pattern of ventricular activation as a mechanism for the unique electrical axis of the equine heart. METHODS: The morphology and distribution of the cardiac conduction system was determined by histochemistry. The electrical activity was measured using ECG in the Einthoven and orthogonal configuration. RESULTS: The long axis of the equine heart is close to vertical. Outside the nodal regions the conduction system consisted of Purkinje fibers connected by connexin 43 and long, slender parallel running transitional cells. The Purkinje fiber network extended deep into the ventricular walls. ECGs recorded in an orthogonal configuration revealed a mean electrical axis pointing in a cranial-to-left direction indicating ventricular activation in an apex-to-base direction. CONCLUSION: The direction of the mean electrical axis in the equine heart is determined by the architecture of the intramural Purkinje network, rather than being a reflection of ventricular mass.

Accurate in silico simulation of the rabbit Purkinje fiber electrophysiological assay to facilitate early pharmaceutical cardiosafety assessment: Dream or reality?

As a branch of quantitative systems toxicology, in silico simulations are of growing attractiveness to guide preclinical cardiosafety risk assessments. Traditionally, a cascade of in vitro/in vivo assays has been applied in pharmaceutical research to screen out molecules at risk for cardiac side effects and prevent subsequent risk for patients. Drug cardiosafety assessments typically employ early mechanistic, hazard-oriented in silico/in vitro assays for compound inhibition of cardiac ion channels, followed by induced pluripotent stem cells (iPSCs) or tissue-based models such as the rabbit Purkinje fiber assay, which includes the major mechanisms contributing to action potential (AP) genesis. Additionally, multiscale simulation techniques based on mathematical models have become available, which are performed in silico 'at the heart' of compound triage to substitute Purkinje tests and increase translatability through mechanistic interpretability. To adhere to the 3R principle and reduce animal experiments, we performed a comparative benchmark and investigated a variety of mathematical cardiac AP models, including a newly developed minimalistic model specifically tailored to the AP of rabbit Purkinje cells, for their ability to substitute experiments. The simulated changes in AP duration (dAPD90) at increasing drug concentrations were compared to experimental results from 588 internal Purkinje fiber studies covering 555 different drugs with diverse modes of action. Using our minimalistic model, 80% of the Purkinje experiments could be quantitatively reproduced. This result allows for significant saving of experimental effort in early research and justifies the embedding of electrophysiological simulations into the DMTA (Design, Make, Test, Analyze) cycle in pharmaceutical compound optimization.

Modeling the His-Purkinje Effect in Non-invasive Estimation of Endocardial and Epicardial Ventricular Activation.

Inverse electrocardiography (iECG) estimates epi- and endocardial electrical activity from body surface potentials maps (BSPM). In individuals at risk for cardiomyopathy, non-invasive estimation of normal ventricular activation may provide valuable information to aid risk stratification to prevent sudden cardiac death. However, multiple simultaneous activation wavefronts initiated by the His-Purkinje system, severely complicate iECG. To improve the estimation of normal ventricular activation, the iECG method should accurately mimic the effect of the His-Purkinje system, which is not taken into account in the previously published multi-focal iECG. Therefore, we introduce the novel multi-wave iECG method and report on its performance. Multi-wave iECG and multi-focal iECG were tested in four patients undergoing invasive electro-anatomical mapping during normal ventricular activation. In each subject, 67-electrode BSPM were recorded and used as input for both iECG methods. The iECG and invasive local activation timing (LAT) maps were compared. Median epicardial inter-map correlation coefficient (CC) between invasive LAT maps and estimated multi-wave iECG versus multi-focal iECG was 0.61 versus 0.31. Endocardial inter-map CC was 0.54 respectively 0.22. Modeling the His-Purkinje system resulted in a physiologically realistic and robust non-invasive estimation of normal ventricular activation, which might enable the early detection of cardiac disease during normal sinus rhythm.

A novel transgenic Cre allele to label mouse cardiac conduction system.

The cardiac conduction system is a network of heterogeneous cell population that initiates and propagates electric excitations in the myocardium. Purkinje fibers, a network of specialized myocardial cells, comprise the distal end of the conduction system in the ventricles. The developmental origins of Purkinje fibers and their roles during cardiac physiology and arrhythmia have been reported. However, it is not clear if they play a role during ischemic injury and heart regeneration. Here we introduce a novel tamoxifen-inducible Cre allele that specifically labels a broad range of components in the cardiac conduction system while excludes other cardiac cell types and vital organs. Using this new allele, we investigated the cellular and molecular response of Purkinje fibers to myocardial injury. In a neonatal mouse myocardial infarction model, we observed significant increase in Purkinje cell number in regenerating myocardium. RNA-Seq analysis using laser-captured Purkinje fibers showed a unique transcriptomic response to myocardial infarction. Our finds suggest a novel role of cardiac Purkinje fibers in heart injury.

Gap Junctional Communication via Connexin43 between Purkinje Fibers and Working Myocytes Explains the Epicardial Activation Pattern in the Postnatal Mouse Left Ventricle.

The mammalian ventricular myocardium forms a functional syncytium due to flow of electrical current mediated in part by gap junctions localized within intercalated disks. The connexin (Cx) subunit of gap junctions have direct and indirect roles in conduction of electrical impulse from the cardiac pacemaker via the cardiac conduction system (CCS) to working myocytes. Cx43 is the dominant isoform in these channels. We have studied the distribution of Cx43 junctions between the CCS and working myocytes in a transgenic mouse model, which had the His-Purkinje portion of the CCS labeled with green fluorescence protein. The highest number of such connections was found in a region about one-third of ventricular length above the apex, and it correlated with the peak proportion of Purkinje fibers (PFs) to the ventricular myocardium. At this location, on the septal surface of the left ventricle, the insulated left bundle branch split into the uninsulated network of PFs that continued to the free wall anteriorly and posteriorly. The second peak of PF abundance was present in the ventricular apex. Epicardial activation maps correspondingly placed the site of the first activation in the apical region, while some hearts presented more highly located breakthrough sites. Taken together, these results increase our understanding of the physiological pattern of ventricular activation and its morphological underpinning through detailed CCS anatomy and distribution of its gap junctional coupling to the working myocardium.

His bundle pacing shows similar ventricular electrical activation as sinus: selective and nonselective His pacing indistinguishable.

His bundle pacing utilizes the His-Purkinje system to produce more physiological activation compared with traditional pacing therapies, but differences in electrical activation between pacing techniques are not yet quantified in terms of activation pattern. Furthermore, clinicians distinguish between selective and nonselective His pacing, but measurable differences in electrical activation remain to be seen. Hearts isolated from seven dogs were perfused using the Langendorff method. Electrograms were recorded using two 64-electrode basket catheters in the ventricles and a 128-electrode sock situated around the ventricles during sinus rhythm (right atrial pacing), right ventricular (RV) pacing, biventricular cardiac resynchronization therapy (biV-CRT), selective His pacing (selective capture of the His bundle), and nonselective His pacing (capture of nearby myocardium and His bundle). Activation maps were generated from these electrograms. Total activation time (TAT) was measured from the activation maps, and QRS duration was measured from a one-lead pseudo-ECG. Results showed that TAT, QRS duration, and activation sequence were most similar between sinus, selective, and nonselective His pacing. Bland-Altman analyses showed highest levels of similarity between all combinations of sinus, selective, and nonselective His pacing. RV and biV-CRT activation patterns were distinct from sinus and had significantly longer TAT and QRS duration. Cumulative activation graphs were most similar between sinus, selective, and nonselective His pacing. In conclusion, selective pacing and nonselective His bundle pacing are more similar to sinus compared with RV and biV-CRT pacing. Furthermore, selective pacing and nonselective His bundle pacing are not significantly different electrically.NEW & NOTEWORTHY Our high-density epicardial and endocardial electrical mapping study demonstrated that selective pacing and nonselective His bundle pacing are more electrically similar to sinus rhythm compared with right ventricular and biventricular cardiac resynchronization therapy pacing. Furthermore, small differences between selective and nonselective His bundle pacing, specifically a wider QRS in nonselective His pacing, do not translate into significant differences in the global activation pattern.

Different effects of amiodarone and dofetilide on the dispersion of repolarization between well-coupled ventricular and Purkinje fibers1.

Increased transmural dispersion of repolarization is an established contributing factor to ventricular tachyarrhythmias. In this study, we evaluated the effect of chronic amiodarone treatment and acute administration of dofetilide in canine cardiac preparations containing electrotonically coupled Purkinje fibers (PFs) and ventricular muscle (VM) and compared the effects to those in uncoupled PF and VM preparations using the conventional microelectrode technique. Dispersion between PFs and VM was inferred from the difference in the respective action potential durations (APDs). In coupled preparations, amiodarone decreased the difference in APDs between PFs and VM, thus decreasing dispersion. In the same preparations, dofetilide increased the dispersion by causing a more pronounced prolongation in PFs. This prolongation was even more emphasized in uncoupled PF preparations, while the effect in VM was the same. In uncoupled preparations, amiodarone elicited no change on the difference in APDs. In conclusion, amiodarone decreased the dispersion between PFs and VM, while dofetilide increased it. The measured difference in APD between cardiac regions may be the affected by electrotonic coupling; thus, studying PFs and VM separately may lead to an over- or underestimation of dispersion.

3D Bioprinting the Cardiac Purkinje System Using Human Adipogenic Mesenchymal Stem Cell Derived Purkinje Cells.

PURPOSE: The objective of this study was to reprogram human adipogenic mesenchymal stem cells (hADMSCs) to form Purkinje cells and to use the reprogrammed Purkinje cells to bioprint Purkinje networks. METHODS: hADMSCs were reprogrammed to form Purkinje cells using a multi-step process using transcription factors ETS2 and MESP1 to first form cardiac progenitor stem cells followed by SHOX2 and TBX3 to form Purkinje cells. A novel bioprinting method was developed based on Pluronic acid as the sacrificial material and type I collagen as the structural material. The reprogrammed Purkinje cells were used in conjunction with the novel bioprinting method to bioprint Purkinje networks. Printed constructs were evaluated for retention of functional protein connexin 40 (Cx40) and ability to undergo membrane potential changes in response to physiologic stimulus. RESULTS: hADMSCs were successfully reprogrammed to form Purkinje cells based on the expression pattern of IRX3, IRX5, SEMA and SCN10. Reprogrammed purkinje cells were incorporated into a collagen type-1 bioink and the left ventricular Purkinje network was printed using anatomical images of the bovine Purkinje system as reference. Optimization studies demonstrated that 1.8 mg/mL type-I collagen at a seeding density of 300,000 cells per 200 µL resulted in the most functional bioprinted Purkinje networks. Furthermore, bioprinted Purkinje networks formed continuous syncytium, retained expression of vital functional gap junction protein Cx40 post-print, and exhibited membrane potential changes in response to electric stimulation and acetylcholine evaluated by DiBAC4(5), an electrically responsive dye. CONCLUSION: Based on the results of this study, hADMSCs were successfully reprogrammed to form Purkinje cells and bioprinted to form Purkinje networks.

Intracardiac Delineation of Septal Conduction in Left Bundle-Branch Block Patterns.

BACKGROUND: Septal activation in patients with left bundle-branch block (LBBB) patterns has not been described previously. We performed detailed intracardiac mapping of left septal conduction to assess for the presence and level of complete conduction block (CCB) in the His-Purkinje system. Response to His bundle pacing was assessed in patients with and without CCB in the left bundle. METHODS: Left septal mapping was performed with a linear multielectrode catheter in consecutive patients with LBBB pattern referred for device implantation (n=38) or substrate mapping (n=47). QRS width, His duration, His-ventricular (HV) intervals, and septal conduction patterns were analyzed. The site of CCB was localized to the level of the left-sided His fibers (left intrahisian) or left bundle branch. Patients with ventricular activation preceded by Purkinje potentials were categorized as having intact Purkinje activation. RESULTS: A total of 88 left septal conduction recordings were analyzed in 85 patients: 72 LBBB block pattern and 16 controls (narrow QRS, n=11; right bundle-branch block, n=5). Among patients with LBB block pattern, CCB within the proximal left conduction system was observed in 64% (n=46) and intact Purkinje activation in the remaining 36% (n=26). Intact Purkinje activation was observed in all controls. The site of block in patients with CCB was at the level of the left His bundle in 72% and in the proximal left bundle branch in 28%. His bundle pacing corrected wide QRS in 54% of all patients with LBBB pattern and 85% of those with CCB (94% left intrahisian, 62% proximal left bundle-branch). No patients with intact Purkinje activation demonstrated correction of QRS with His bundle pacing. CCB showed better predictive value (positive predictive value 85%, negative predictive value 100%, sensitivity 100%) than surface ECG criteria for correction with His bundle pacing. CONCLUSIONS: Heterogeneous septal conduction was observed in patients with surface LBBB pattern, ranging from no discrete block to CCB. When block was present, we observed pathology localized within the left-sided His fibers (left intrahisian block), which was most amenable to corrective His bundle pacing by recruitment of latent Purkinje fibers. ECG criteria for LBBB incompletely predicted CCB, and intracardiac data might be useful in refining patient selection for resynchronization therapy.

Mechanisms of in utero cortisol effects on the newborn heart revealed by transcriptomic modeling.

We have identified effects of elevated maternal cortisol (induced by maternal infusion 1 mg·kg-1·day-1) on fetal cardiac maturation and function using an ovine model. Whereas short-term exposure (115-130-day gestation) increased myocyte proliferation and Purkinje fiber apoptosis, infusions until birth caused bradycardia with increased incidence of arrhythmias at birth and increased perinatal death, despite normal fetal cortisol concentrations from 130 days to birth. Statistical modeling of the transcriptomic changes in hearts at 130 and 140 days suggested that maternal cortisol excess disrupts cardiac metabolism. In the current study, we modeled pathways in the left ventricle (LV) and interventricular septum (IVS) of newborn lambs after maternal cortisol infusion from 115 days to birth. In both LV and IVS the transcriptomic model indicated over-representation of cell cycle genes and suggested disruption of cell cycle progression. Pathways in the LV involved in cardiac architecture, including SMAD and bone morphogenetic protein ( BMP) were altered, and collagen deposition was increased. Pathways in IVS related to metabolism, calcium signaling, and the actin cytoskeleton were altered. Comparison of the effects of maternal cortisol excess to the effects of normal maturation from day 140 to birth revealed that only 20% of the genes changed in the LV were consistent with normal maturation, indicating that chronic elevation of maternal cortisol alters normal maturation of the fetal myocardium. These effects of maternal cortisol on the cardiac transcriptome, which may be secondary to metabolic effects, are consistent with cardiac remodeling and likely contribute to the adverse impact of maternal stress on perinatal cardiac function.

Small-conductance calcium-activated potassium current modulates the ventricular escape rhythm in normal rabbit hearts.

BACKGROUND: The apamin-sensitive small-conductance calcium-activated K (SK) current IKAS modulates automaticity of the sinus node. IKAS blockade by apamin causes sinus bradycardia. OBJECTIVE: The purpose of this study was to test the hypothesis that IKAS modulates ventricular automaticity. METHODS: We tested the effects of apamin (100 nM) on ventricular escape rhythms in Langendorff-perfused rabbit ventricles with atrioventricular block (protocol 1) and on recorded transmembrane action potential of pseudotendons of superfused right ventricular endocardial preparations (protocol 2). RESULTS: All preparations exhibited spontaneous ventricular escape rhythms. In protocol 1, apamin decreased the atrial rate from 186.2 ± 18.0 bpm to 163.8 ± 18.7 bpm (N = 6; P = .006) but accelerated the ventricular escape rate from 51.5 ± 10.7 bpm to 98.2 ± 25.4 bpm (P = .031). Three preparations exhibited bursts of nonsustained ventricular tachycardia and pauses, resulting in repeated burst termination pattern. In protocol 2, apamin increased the ventricular escape rate from 70.2 ± 13.1 bpm to 110.1 ± 2.2 bpm (P = .035). Spontaneous phase 4 depolarization was recorded from the pseudotendons in 6 of 10 preparations at baseline and in 3 in the presence of apamin. There were no changes of phase 4 slope (18.37 ± 3.55 mV/s vs 18.93 ± 3.26 mV/s, N = 3; P = .231, ), but the threshold of phase 0 activation (mV) reduced from -67.97 ± 1.53 to -75.26 ± 0.28 (P = .034). Addition of JTV-519, a ryanodine receptor 2 stabilizer, in 5 preparations reduced escape rate back to baseline. CONCLUSION: Contrary to its bradycardic effect in the sinus node, IKAS blockade by apamin accelerates ventricular automaticity and causes repeated nonsustained ventricular tachycardia in normal ventricles. ryanodine receptor 2 blockade reversed the apamin effects on ventricular automaticity.

Purkinje physiology and pathophysiology.

There has always been an appreciation of the role of Purkinje fibers in the fast conduction of the normal cardiac impulse. Here, we briefly update our knowledge of this important set of cardiac cells. We discuss the anatomy of a Purkinje fiber strand, the importance of longitudinal conduction within a strand, circus movement within a strand, conduction, and excitability properties of Purkinjes. At the cell level, we discuss the important components of the ion channel makeup in the nonremodeled Purkinjes of healthy hearts. Finally, we discuss the role of the Purkinjes in forming the heritable arrhythmogenic substrates such as long QT, heritable conduction slowing, CPVT, sQT, and Brugada syndromes.

Automatic estimation of Purkinje-Myocardial junction hot-spots from noisy endocardial samples: A simulation study.

The reconstruction of the ventricular cardiac conduction system (CCS) from patient-specific data is a challenging problem. High-resolution imaging techniques have allowed only the segmentation of proximal sections of the CCS from images acquired ex vivo. In this paper, we present an algorithm to estimate the location of a set of Purkinje-myocardial junctions (PMJs) from electro-anatomical maps, as those acquired during radio-frequency ablation procedures. The method requires a mesh representing the myocardium with local activation time measurements on a subset of nodes. We calculate the backwards propagation of the electrical signal from the measurement points to all the points in the mesh to define a set of candidate PMJs that is iteratively refined. The algorithm has been tested on several Purkinje network configurations, with simulated activation maps, subject to different error amplitudes. The results show that the method is able to build a set of PMJs that explain the observed activation map for different synthetic CCS configurations. In the tests, the average error in the predicted activation time is below the amplitude of the error applied to the data.

Numerical approximation of the electromechanical coupling in the left ventricle with inclusion of the Purkinje network.

In this work, we consider the numerical approximation of the electromechanical coupling in the left ventricle with inclusion of the Purkinje network. The mathematical model couples the 3D elastodynamics and bidomain equations for the electrophysiology in the myocardium with the 1D monodomain equation in the Purkinje network. For the numerical solution of the coupled problem, we consider a fixed-point iterative algorithm that enables a partitioned solution of the myocardium and Purkinje network problems. Different levels of myocardium-Purkinje network splitting are considered and analyzed. The results are compared with those obtained using standard strategies proposed in the literature to trigger the electrical activation. Finally, we present a numerical study that, although performed in an idealized computational domain, features all the physiological issues that characterize a heartbeat simulation, including the initiation of the signal in the Purkinje network and the systolic and diastolic phases.

Specialized impulse conduction pathway in the alligator heart.

Mammals and birds have a specialized cardiac atrioventricular conduction system enabling rapid activation of both ventricles. This system may have evolved together with high heart rates to support their endothermic state (warm-bloodedness) and is seemingly lacking in ectothermic vertebrates from which first mammals then birds independently evolved. Here, we studied the conduction system in crocodiles (Alligator mississippiensis), the only ectothermic vertebrates with a full ventricular septum. We identified homologues of mammalian conduction system markers (Tbx3-Tbx5, Scn5a, Gja5, Nppa-Nppb) and show the presence of a functional atrioventricular bundle. The ventricular Purkinje network, however, was absent and slow ventricular conduction relied on trabecular myocardium, as it does in other ectothermic vertebrates. We propose the evolution of the atrioventricular bundle followed full ventricular septum formation prior to the development of high heart rates and endothermy. In contrast, the evolution of the ventricular Purkinje network is strongly associated with high heart rates and endothermy.

Genetic targeting of Purkinje fibres by Sema3a-CreERT2.

The maintenance of the heart rhythm and the conduction of excitatory signals require changing excitatory signals via electrical activity and coordination by communication between working and conductive cardiomyocytes. Understanding how the ventricular conduction system is established provides novel insights into the pathophysiological progress of cardiac arrhythmias. However, the major hurdle in this field is the lack of a specific genetic tool that targets the Purkinje fibres of the ventricular conduction system and no other types of cardiomyocytes or coronary vessels. Here, we generated a Sema3a-CreERT2 knock-in mouse line to test its specificity for genetically labelled Purkinje fibres. We found that Sema3a was expressed in the subendocardial layer of the trabecular myocardium in the embryonic heart and was restricted to the Purkinje fibres in the adult heart. A fate mapping study based on the Sema3a-CreERT2 line revealed that the Sema3a+ cardiomyocytes were restricted to the fate of Purkinje fibres in the perinatal but not the embryonic stage. Collectively, our study provides a new genetic tool, i.e., Sema3a-CreERT2, for studying the molecular mechanisms that regulate the function of Purkinje fibres.

Effects of new antiarrhythmic agent SS-68 on excitation conduction, electrical activity in Purkinje fibers and pulmonary veins: Assessment of safety and side effects risk.

The compound SS-68 has been selected among numerous new derivatives of indole and demonstrated antiarrhythmic effects in animal models. The present study concerns several aspects of SS-68 safety and efficacy as a potential antiarrhythmic drug. The first estimation of atrioventricular conduction in mammalian heart under SS-68 has been carried out; effects of SS-68 in Purkinje fibers and myocardium of pulmonary veins have been investigated. The drug weakly affects cardiac atrioventricular conduction: only high concentrations of SS-68 (≥10 µmol/L) significantly decrease this parameter. Also, the drug weakly affects Purkinje fibers automaticity, but effectively alters action potential waveform in Purkinje fibers in a concentration-dependent manner. SS-68 (0.1-100 µmol/L) failed to induce any early or delayed afterdepolarizations in Purkinje fibers both in basal conditions and under provocation of proarrhythmic activity by norepinephrine (NE). Moreover, 10 µmol/L SS-68 suppressed NE-induced extra-beats and rapid firing in Purkinje fibers. In pulmonary veins only high concentrations of SS-68 significantly increased action potential duration, while lower concentrations (0.1-1 µmol/L) were ineffective. Also, 0.1-100 µmol/L SS-68 was unable to elicit arrhythmogenic alternations of action potential waveform in pulmonary veins. In conclusion, SS-68 has no proarrhythmic effects, such as afterdepolarizations or abnormal automaticity in used experimental models.