The cardiac Purkinje network plays a critical role in maintaining synchronized ventricular activation but remains difficult to image due to its fine and complex structure. Conventional MRI techniques lack sufficient contrast to distinguish the structural composition of Purkinje fibers (PFs). This study investigates the potential of inhomogeneous magnetization transfer (ihMT) as a novel contrast mechanism for visualizing and differentiating subregions of the Purkinje network. Five fixed ex vivo sheep hearts containing free-running PFs were scanned using a 9.4 T MRI system with a 2D ihMT RARE sequence. ihMTR maps were analyzed using manually defined regions of interest (ROIs) corresponding to free-running fibers, the Purkinje-myocardial junction (PMJ), and the surrounding myocardium. Histological analysis was performed on matched tissue sections to quantify collagen types I and III, adipocytes, Purkinje cells, and cardiomyocytes. Three ihMT protocols that produced high ihMTR values in free-running fibers (9.25-10.83%) and strong contrast relative to myocardium (2.00-2.17%) and the PMJ (2.99-3.40%) in 1 sample were selected and applied to all samples. Across all hearts, mean ihMTR values were consistently higher in free-running fibers compared to the PMJ (11.5 ± 1.5% vs 9.0 ± 2.9%). Histological analysis revealed significantly greater collagen content in free-running regions compared with the PMJ (72.4 ± 15.9% vs 31.1 ± 13.1%; p = 0.001), along with higher adipocyte content at the PMJ compared to free-running regions (12.3 ± 6.1% vs 3.8 ± 2.7%, not significant). Collagen type III was more prominent at the PMJ but remained a minor component overall. These findings demonstrate that ihMT imaging can distinguish PF subregions based on underlying microstructural differences, particularly collagen and adipocyte distribution. This study lays the groundwork for developing biophysical models to interpret ihMT signals in terms of tissue composition and microstructure, providing a foundation for future studies.

BACKGROUND:Early repolarization syndrome (ERS) exhibits diverse clinical and mechanistic origins within the J-wave spectrum. Conduction, repolarization, and structural factors may shape its electrocardiography phenotype. OBJECTIVES:This study sought to determine the contribution of endocardial electrical and structural remodeling to malignant ERS phenotypes and arrhythmogenesis in a familial case. METHODS:Two siblings with malignant and drug-refractory ERS underwent in vivo electrocardiographic imaging and ex vivo analyses of the right and/or left explanted ventricles. Optical mapping, microelectrode recordings, high-field cardiac magnetic resonance, histology, guide DNA/complementary DNA sequencing, reverse-transcription quantitative polymerase chain reaction, and Western blot analyses were compared with ERS-absent control hearts. Findings were integrated with multiscale simulations to test mechanistic hypotheses. RESULTS:Long pacing cycle lengths or pauses induced endocardial biphasic optical action potential upstrokes and amplification of the phase 1 notch in ERS. Their rate dependence, suppression by 4-aminopyridine, and insensitivity to flecainide implicated slow transient outward potassium current (Ito,slow)-mediated repolarization mechanism. Pause-dependent conduction slowing appeared in the second ERS sibling, pointing to an additional Ito,slow-related conduction mechanism in this case. Molecular profiling revealed endocardial KV1.4 overexpression, supporting Ito,slow involvement. Diffuse collagen deposition found in both right ventricles may provide a synergistic proarrhythmic substrate. In silico, Ito,slow enhancement alone reproduced the pause-dependent repolarization phenotype and associated conduction abnormalities, whereas its combination with diffuse structural alterations was required to generate premature beats. CONCLUSIONS:In these ERS cases, endocardial Ito,slow-driven abnormalities underlie pause-dependent J waves, while structural remodeling synergizes to promote arrhythmogenesis. Distinct mechanisms between siblings highlight the mechanistic heterogeneity of J-wave syndromes and refine rather than refute previous hypotheses.

Introduction Idiopathic ventricular fibrillation (IVF) is a major cause of sudden cardiac death in young adults. A distinct subtype of IVF, triggered by short-coupled premature ventricular contractions (coupling interval<350ms) arising from Purkinje fibers, has recently been described and frequently presents as sudden death, which is the initial manifestation in more than 60% of reported cases. Approximately 15% of patients with Purkinje-related IVF have a family history, supporting a potential genetic contribution. However, the pathophysiological mechanisms underlying this arrhythmia remain poorly understood, in part due to the limited availability of experimental models of human cardiac Purkinje cells. Objective To elucidate the mechanisms underlying short-coupled Purkinje-related IVF and to establish a human cellular model using patient-specific induced pluripotent stem cell-derived cardiomyocytes, with a particular focus on familial forms of the disease. Method Genomic DNA and peripheral blood samples were obtained from a patient with Purkinje-related IVF and a strong family history of sudden cardiac death, including three affected siblings. Whole exome sequencing was performed and analysed according to current guidelines. Peripheral blood mononuclear cells (PBMCs) were reprogrammed into induced Pluripotent Stem Cells (iPSCs) and subsequently differentiated into cardiomyocytes (iPSC-CMs) and Purkinje-like cells (iPSC-PKs). Results Whole-exome sequencing identified a splice variant in a gene encoding a potassium channel anchoring protein. RNA sequencing of iPSC-CMs confirmed that this variant leads to exon skipping without shifting the reading frame. PBMCs from the patient were successfully reprogrammed into iPSCs and differentiated into Purkinje-like cells. These cells exhibited increased expression of Purkinje cell markers, including IRX3, SCN5A and ETV1, compared with control iPSC-CMs, supporting successful Purkinje-like differentiation. Electrophysiological properties of patient-derived Purkinje-like cells are currently being assessed using optical mapping. Conclusion This study identified a pathogenic splice variant associated with familial Purkinje-related IVF and established a patient-specific human iPSC-derived Purkinje cell model that provides a novel platform to investigate disease mechanisms.

Introduction Purkinje Fibers (PF) play a critical role in cardiac function, but they have been identified to trigger lethal arrhythmias. Recently, PF ectopy have been inducible by sodium channel blockers (SCBs). Objective However, the mechanisms underlying drug-induced PF ectopies remain unknown. Method We investigated the effects of two SCBs, ajmaline (15μM) and flecainide (2μM) using optical mapping on ex-vivo coronary-perfused ovine left ventricles (n=8). We measured ventricular activation and repolarization properties at the Purkinje-Muscle Junctions (PMJ) vs ventricular myocardium under increased pacing frequencies (1 to 5Hz) and various pacing sites (His, PF and myocardium) before and after introduction of SCBs. We also quantified and characterized arrhythmias for these conditions. Finally, myocardial and PF samples were collected to investigate voltage-gated channels and regulatory subunits expression by RTqPCR. Results Ajmaline significantly increased activation times (AT, 57.1±18.2 vs 220.2±62.1ms, P<0.01) for all pacing sites. Furthermore, a significant increase in action potential duration (APD80, 254.7±11.2 vs 380.5±82.9ms, P<0.05) was observed specifically at the PMJs, which led to an increased heterogeneity of repolarization time (RT, 0.045±0.022ms/mm2 vs 0.157±0.069ms/mm2, P<0.01). Flecainide increased AT (64.5±10.6ms vs 148.9±32.4ms, P<0.05) but had no significant impact on APD or RT dispersion. An increase in spontaneous arrhythmias was observed at low pacing frequencies (≤2Hz) for ajmaline (0.13±0.35 episodes vs 2.25±2.12 episodes, P<0.05) with a lower dominant frequency (4.98±1.0 vs 1.71±0.57, P<0.0001) whereas for flecainide, arrhythmias only occurred at high pacing frequencies (2-3.5Hz, 0.0±0.0 vs 1.11±1.67, P=0.625). RTqPCR revealed a higher expression of SCN5A, KCNA4 and KCND2 (×4, ×11.37, ×11.32 respectively) and a 10-fold lower expression of the regulatory subunit KCNIP2 in PF, compared to cardiomyocytes. Conclusion In conclusion, despite its expected effect on AT like flecainide, we demonstrate that ajmaline prolongs APD and increase RT dispersion of the PMJs leading to arrhythmic events even at low heart rates. Our molecular biology screening suggests that inhibition of the Ito current mediated by potassium channels may underly the effect of ajmaline on repolarization. These findings open new perspectives in terms of risk stratification and prevention.

Background Atrial fibrillation (AF) often progresses from paroxysmal to more stable forms. It is well-recognized that patients vary in their AF progression, but underlying mechanisms remain unclear. This work, performed in a sheep AF-model, aimed to identify atrial redox and energetic status differences between animals developing stable AF (AF-S) versus those resistant to AF-stabilization (AF-R). Methods AF was monitored with telemetry and maintained with bursts of atrial tachystimulation whenever sinus rhythm resumed. Electrophysiological remodeling was assessed via contact mapping. Structural remodeling was described by histology. Proteomic, metabolomic, enzymatic and bioenergetic remodeling were evaluated using frozen left atrial appendage (LAA) tissues and isolated LAA mitochondria. Healthy young rats were used to investigate if an induced metabolic challenge could stabilize AF episodes upon transesophageal atrial tachypacing challenge. Results AF-S sheep developed stable AF (>24-hours self-sustained) after 13 days on average, whereas AF-R sheep failed to develop self-sustained AF despite 120 days of electrically-maintained AF. Contact mapping and histological analysis revealed similar electro-structural remodeling in both groups. Metabolic analysis showed significant differences in tricarboxylic acid (TCA) cycle enzymes activities and a 45% increase in AF-S LAA succinate content versus AF-R. AF-S mitochondria showed abnormal mitochondrial succinate oxidation, associated with a significant 20% decrease in ATP synthesis rate, 22% increase in ROS emission and mitochondrial inner membrane hyperpolarization. The ratios of ATP to ADP, NAD+ to NADH, and Complex I/II were disturbed in AF-S compared to AF-R. Calculated mitochondrial NAD+ to NADH ratios suggest a reduced state of in-vivo AF-R mitochondria compared to the oxidized state of AF-S. Exogenous succinate was metabolized when incubated with rat atrial cardiomyocytes and altered redox balance, while intravenous succinate stabilized atrial arrhythmias induced by tachypacing in vivo . Conclusions Sheep resistant to AF-progression showed specific TCA cycle, energetic and redox adaptations compared to animals that developed self-sustained AF. In this animal model, mitochondrial TCA cycle remodeling and associated redox and energetic responses determined the resistance to AF domestication, with potential relevance to identify new mechanistic determinants of AF progression in humans. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, ANR-17-CE14-0029-01, ANR-10-IAHU-04

The oblique vein of the left atrium (OVLA), also known as the vein of Marshall, is an embryonic remnant of interest to cardiac electrophysiologists. The aim of this cadaveric study was to describe the anatomy of the OVLA and its relationship with the left atrial wall using micro-CT. The OVLA was selectively injected with a baryum-gelatin mixture, then the specimen was bathed in a Lugol's solution and imaged with micro-CT at an isotropic resolution of 42 µm. In addition to qualitative descriptions of the relationship between the vein and the surrounding musculature, quantitative measures of the veins’ dimensions were obtained. Eighteen hearts were analyzed. The mean length of the OVLA main axis was 36.5 ± 19.4 mm. Analysis of OVLA segmentations showed heterogeneous arborization. The main axis, oblique towards the ridge, was variable in length. It was short in 3 specimens and reached the roof of the atrium in 4 cases. The main branch received short collateral branches localized near the pulmonary veins (N = 5), or long collaterals from the posterior wall (N = 3). Myocardial tissue was consistently found at different level surrounding the vein, separated from the compact endocardium by fatty tissue. This is the first anatomical study of the oblique vein of the left atrium using micro-CT after selective injection. It enabled very high-resolution analysis of the OVLA and showed heterogeneous lengths and arborizations, up to the roof or posterior wall of the left atrium. The veins colocalizes with non-compact muscular tissue relevant to cardiac electrophysiology.

A gradient in the density of SR-Ca 2+ pumps appears from the center to the periphery of Purkinje cells (Pcells) after MI. We found that this post-MI rearrangement could result from the peripheral expression of SERCA2b pump, which is absent in healthy hearts. The additional expression of SERCA2b to the existing cardiac pump SERCA2a, and possibly more efficient Ca 2+ -transport properties of SERCA2b, are consistent with the proarrhythmic elevation of SR-Ca 2+ uptake previously proposed in Pcells after MI.

Introduction Idiopathic ventricular fibrillation (IVF) is a significant cause of sudden cardiac death in young adults. A form of IVF triggered by short-coupled (coupling interval <350 ms) premature ventricular contractions originating in the Purkinje fibers has been recently identified, with death being the most frequent presentation (death is the first manifestation in >60% of cases in registries on this pathology). Approximately 15% of patients with Purkinje-related IVF have a family history, suggesting a potential genetic basis for the arrhythmia. However, the underlying mechanisms of IVF remain to be fully elucidated, partly due to the paucity of models currently available to study cardiac Purkinje cells. Objective The objective of this study is to establish a human cellular model of Purkinje-related IVF using iPS-derived cardiomyocytes from patients in order to characterize them in vitro and to identify the mechanisms underlying Purkinje arrhythmogenicity. The present project aims to focus on patients with a familial form of Purkinje-related IVF. Method A blood sample was obtained from a patient diagnosed with Purkinje-related IVF, who had three brothers who died prematurely from sudden cardiac death. Peripheral blood mononuclear cells (PBMCs) were isolated and reprogrammed into induced Pluripotent Stem Cells (iPSCs). These cells were then differentiated into Purkinje-like cardiac cells using a combination of specific drugs. Results PBMCs from the patient have been reprogrammed into iPSCs, and cardiomyocytes derived from the patient's iPSCs were differentiated into Purkinje-like cells. Preliminary results indicate an overexpression of Purkinje markers (IRX3, SCN5A and ETV1, for example) when compared to the vehicle condition, suggesting the success of our approach. Action potential from the patient's Purkinje-like cells will be characterized using optical mapping. Conclusion This project will provide novel insights into the key pathophysiological features of the hereditary form of a frequent cause of sudden cardiac death in young adults.

BACKGROUND Purkinje Fibers (PFs) are essential to the cardiac conduction system for synchronizing ventricular contractions. However, emerging evidence highlights their implication in the development of ventricular tachyarrhythmias. Nevertheless, isolating and studying the cellular mechanisms of PFs presents a significant challenge due to their intricate arborizing structure, heterogeneous cardiomyocytes (CMs) phenotype, and relatively small proportion within the ventricular mass, all of which hinder detailed functional investigations and comprehensive analysis of the conduction system network. OBJECTIVE To develop a new methodology for dissociation and classification of cell populations related to the ventricular conduction system from adult sheep. This workflow establishes, in part, a proof-of-concept deep learning-based classification strategy that leverages standard cellular imaging data. METHODS We developed a multi-tiered workflow to isolate and classify cardiac cell populations from adult sheep hearts. Coronary perfusion and enzymatic digestion were used to dissociate CMs from the left ventricular free wall (LVMs) and Purkinje-rich free-running false tendons (FTs). A three-pronged classification strategy was developed and implemented: (1) expert-guided visual phenotyping based on distinctive morphological traits; (2) rule-based morphometric quantification using automatic image analysis; and (3) deep learning-based classification with a retrained YOLOv9 model trained on augmented brightfield image datasets. This pipeline enabled accurate discrimination between LVM and FT-derived cells. Independent validation was performed using patch-clamp electrophysiology, T-tubule structure imaging with di-8-ANEPPS, and gene expression profiling (RT-qPCR) for Purkinje-specific biomarkers (Tbx5 and Cx40). RESULTS During the qualitative inspection, FT-dissociated cells had distinct morphological features, including an elongated or slender shape, finger-like projections, curves and tortuous shapes, and a new feature: the presence of spurs along the lateral membrane. Subsequently, a YOLOv9 model achieved an accuracy of 98% in distinguishing LVM and FT cells, based on the initial visual selection made by the operator. In addition, FT-cells exhibit a lower organization and density of T-Tubules compared to LVM. This classification was confirmed by the characterization of the typically longer action potential (AP) durations in FT cells. Finally, higher mRNA expression of the transcription factor Tbx5 and connexin40 (Cx40) was observed in FTs compared to left ventricular tissues. CONCLUSIONS We present a robust and scalable workflow for isolating and classifying cardiac Purkinje fiber cells from adult sheep, integrating manual phenotyping, rule-based morphometrics, and AI-driven deep learning. This multimodal approach enables high-accuracy identification of PF cells within heterogeneous tissue, confirmed through structural, molecular, and electrophysiological validation. Our findings overcome long-standing barriers in Purkinje fiber research and provide a powerful platform for advancing the study of ventricular conduction system biology and its role in arrhythmogenesis. ![Figure][1] WHAT IS KNOWN WHAT THE– STUDY ADDS ### Competing Interest Statement The authors have declared no competing interest. * AP : Action Potential CMS : Cardiomyocytes FTs : False tendons LV : Left ventricle LVM : Left Ventricular Myocyte PBS : Phosphate Buffer Saline PF : Purkinje Fiber RV : Right Ventricle SCD : Sudden Cardiac Death VF : Ventricular Fibrillation VT : Ventricular Tachycardia French National Research Agency (ANR) grants ANR-10-IAHU-04 ERC Advanced Grant n°322886 Fédération française de cardiologie, https://ror.org/039w1jh10 [1]: pending:yes

Les fibrillations auriculaires ou atriales (FA) et les fibrillations ventriculaires (FV) constituent les arythmies cardiaques les plus graves rencontrées en clinique. Les FA affectent des millions d’individus, et augmentent les risques d’accident vasculaire cérébral embolique, d’insuffisance cardiaque, et de mortalité cardiaque. Les FV sont responsables de mort subite, soit la moitié de la mortalité cardiaque, principale cause mondiale de décès (OMS, 2020). Les fibrillations cardiaques ont en commun d’être liées à des mouvements électriques tourbillonnants, multiples et diffus dans l’organe affecté. Ces phénomènes électriques sont dynamiquement changeants et résistent aux agents pharmacologiques. En dépit de cette complexité, les fibrillations cardiaques prennent naissance dans des sources localisées : les veines pulmonaires pour les FA, et le tissu de Purkinje ou des zones altérées individuelles pour les FV. L’isolation des veines pulmonaires est devenue le traitement de référence international pour l’ablation des FA, et sa technique s’améliore par des innovations méthodologiques ou technologiques. Les essais thérapeutiques menés ont montré une réduction des AVC, des altérations cognitives, et de la mortalité cardiaque. Les sources générant les FV sont aussi des cibles d’ablation efficace, qui viennent en complément des traitements pharmacologiques et des défibrillateurs implantables. Cependant le problème fondamental des morts subites n’est pas thérapeutique, mais préventif. Il réside dans l’identification des sujets à risque et la prévention des cardiopathies. Le phénotypage (basal et dynamique) des sources de FV sera une clé essentielle pour la mise en œuvre d’une méthode efficace de détection.

The knowledge of the cardiac microstructure and the 3D myofiber architecture grow years after years with the multiplication and the upgrade of imaging technologies. However, the course of events of pathophysiological processes like cardiac remodeling, and the link with clinical phenotypes are not yet clearly understood. Some concerns have been raised regarding the interpretation of the late gadolinium enhancement (LGE) at the right ventricle attachment or insertion point (RVIP) however 3D microstructure organization of the RVIP has not been extensively described in the literature.

Ithildin is an open-source library and framework for efficient parallelized simulations of excitable media, written in the C++ programming language. It uses parallelization on multiple CPU processors via the message passing interface (MPI). We demonstrate the library's versatility through a series of simulations in the context of the mono-domain description of cardiac electrophysiology, including the S1S2 protocol, spiral break-up, and spiral waves in ventricular geometry. Our work demonstrates the power of Ithildin as a tool for studying complex wave patterns in cardiac tissue and its potential to inform future experimental and theoretical studies. We publish our full code with this paper in the name of open science.

The international Working Group of the Signal Summit is a consortium of experts in the field of cardiac electrophysiology dedicated to advancing knowledge on understanding and clinical application of signal recording and processing techniques. In 2023, the working group met in Reykjavik, Iceland, and laid the foundation for this manuscript. Atrial fibrillation (AF) is the most common arrhythmia in adults, with a rapidly increasing prevalence worldwide. Despite substantial research efforts, advancements in elucidating the underlying mechanisms of AF have been relatively modest. Since the discovery of pulmonary veins as a frequent trigger region for AF initiation more than 2½ decades ago, advancements in patient care have primarily focused on technologic innovations to improve the safety and efficacy of pulmonary vein isolation (PVI). Several factors may explain the limited scientific progress made. First, whereas AF initiation usually begins with an ectopic beat, the mechanisms of initiation, maintenance, and electrical propagation have not been fully elucidated in humans, largely owing to suboptimal spatiotemporal mapping. Second, underlying structural changes have not been clarified and may involve different types of reentry. Third, inconsistent definitions and terminology regarding fibrillatory characteristics contribute to the challenges of comparing results between studies. Fourth, a growing appreciation for phenotypical differences probably explains the wide range of clinical outcomes to catheter ablation in patients with seemingly similar AF types. Last, restoring sinus rhythm in advanced phenotypic forms of AF is often not feasible or may require extensive ablation with minimal or no positive impact on quality of life. The aims of this international position paper are to provide practical definitions as a foundation for discussing potential mechanisms and mapping results and to propose pathways toward meaningful advancements in AF research, ultimately leading to improved therapies for AF.