Biology subjects

Bernus, O.

Publications and source records attributed to Bernus, O..

7 recordsLinked to original sources

bioRxiv2026

Structural Characterization of Cardiac Purkinje Fibers Using Inhomogeneous Magnetization Transfer (ihMT): A proof of Concept MRI-Histology Approach

BACKGROUNDThe cardiac Purkinje network plays a critical role in maintaining synchronized activation of ventricles but remains challenging to image due to its fine and unique structure. Conventional MRI techniques lack sufficient contrast to distinguish the underlying structural composition of Purkinje Fibers (PF). PURPOSEThis study investigates the potential of inhomogeneous Magnetization Transfer (ihMT) as a novel contrast mechanism for visualizing and differentiating subregions of the PF. METHODSFive fixed ex-vivo sheep hearts (n = 5) containing free running PF were scanned with a 9.4T MRI using a 2D ihMT RARE sequence. ASSESSMENTihMTR maps were analyzed using manually defined regions-of-interest (ROIs) corresponding to free-running PF, insertion points, and myocardium. Histological analysis (light and polarized microscopy) was performed on matched sections to quantify collagen types I and III, adipocytes, Purkinje cells, and cardiomyocytes. RESULTSThree ihMT protocols, which produced high ihMTR values in free-running PF (9.25-10.83%) and strong absolute contrast relative to the myocardium (2.00-2.17%) and insertion points (2.99-3.40%) in one sample were selected and applied to all samples. Across all samples, mean ihMTR in free-running was consistently higher than in insertion points (11.5 {+/-} 1.5% vs. 9.0 {+/-} 2.9%). Histological analysis revealed a significantly greater collagen content in free-running regions compared with insertion points (72.4 {+/-} 15.9% vs. 31.1 {+/-} 13.1%; p = 0.001), along with higher adipocyte content at insertion points vs. free-running regions (12.3 {+/-} 6.1% vs. 3.8 {+/-} 2.7%, non-significant). Collagen type III was more prominent at insertion points but remained a minor component overall. CONCLUSIONihMT imaging can distinguish PF subregions based on 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. SponsorThis study received financial support from the French Government by the National Research Agency (ANR; SYNATRA ANR-21-CE19-0014-01) and Region Nouvelle Aquitaine (convention N{degrees}AAPR2022-2021-16609210).

biophysics↗

bioRxiv2025

Resistance to Atrial Fibrillation Domestication and Mitochondrial Dysfunction in Sheep: a potential key role of the TCA Cycle and mitochondrial redox state

BackgroundAtrial 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). MethodsAF 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. ResultsAF-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. ConclusionsSheep 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.

pathology↗

bioRxiv2025

AI-based identification of cardiac Purkinje fiber cells isolated from whole adult sheep hearts

BACKGROUNDPurkinje 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. OBJECTIVETo 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. METHODSWe 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). RESULTSDuring 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. CONCLUSIONSWe 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. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=114 HEIGHT=200 SRC="FIGDIR/small/653917v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@18c0154org.highwire.dtl.DTLVardef@ab67eeorg.highwire.dtl.DTLVardef@1346eeaorg.highwire.dtl.DTLVardef@15e05e4_HPS_FORMAT_FIGEXP M_FIG C_FIG WHAT IS KNOWNO_LIThe PFs network constitutes a small part of the ventricular mass (<2%) but ensures spatio-temporal dynamic of ventricular activation. C_LIO_LIThe PFs are known to have distinct electrophysiological and Ca2+ dynamic compared to surrounding myocardial tissue. C_LIO_LIVentricular arrhythmias are the most common cause of sudden cardiac death (SCD), and recent evidence points to an essential contribution of PFs. C_LIO_LIHowever, little is known about the molecular mechanisms of PF-induced arrhythmias, partly because their isolation remains challenging. C_LI WHAT THE- STUDY ADDSO_LIA new dissociation technique combined with advanced AI methods to accurately dissociate and discriminate LVM and PF cells derived from free FT dissociation. C_LIO_LIThis improves the classification system for distinguishing morphological LVM cells from PF cells in sheep. C_LIO_LIOur model opens up new perspectives in the automatic analysis of various cell parameters. C_LI

physiology↗

bioRxiv2024

The Ithildin library for efficient numerical solution of anisotropic reaction-diffusion problems in excitable media

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 librarys 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. Author summaryWe present Ithildin, an open-source library for reaction-diffusion systems such as the electrical waves in cardiac tissue controlling the heart beat. We demonstrate the versatility of Ithildin by example simulations in various tissue models and geometries, from simple 2D simulations to detailed ones in ventricular geometry. Our simulations highlight key features of Ithildin, such as recording pseudo-electrograms or filament trajectories. We hope that our work will contribute to the growing understanding of cardiac electrophysiology and inform future experimental and theoretical studies.

systems biology↗

bioRxiv2023

Increased spontaneous Ca2+ activity in Cardiac Purkinje cells after myocardial infarction; A consequence of a dramatic shift of SERCA isoforms as potential adaptation to acute ischemia?

BackgroundStudies of Purkinje cells (Pcells) from canine hearts have suggested an increase of Ca2+-release by the sarcoplasmic reticulum (SR) but also reported a potential augmentation of SR-Ca2+-uptake after MI. Abnormal increase of SR-Ca2+-uptake in heart cells is novel and contrasts with the reduction of this function in cells of failing heart. Our study examined the origin of this increased SR-Ca2+-uptake by considering a change in SR-Ca2+ pump (SERCA2) expression in Purkinje fibers (PFs) post MI. MethodsPcells were isolated from canine hearts 48Hrs post MI. Intracellular Ca2+-activity was captured by confocal microscopy. Purkinje-typical Ca2+ events were analyzed to probe the regional Ca2+-dynamics within Pcells. A Purkinje-specific numerical model assisted in the interpretation of Ca2+-anomalies detected in Pcells Ca2+-transients. SR-Ca2+-uptake system was studied by immunofluorescence in Pcells from canine, ovine and human hearts post MI. SERCA protein and gene expressions in PFs and myocardium were measured by Western Blots and RT-qPCR in a classical porcine model of MI. Results48Hrs after MI, Pcells showed 60% increase in spark-rate and 37% acceleration of Ca2+ wave decay. In the model of normal wave, 35% increase of Ca2+-uptake rate reproduced the actual post-MI wave alterations. In apparent contrast with increased Ca2+-uptake rate, SERCA2 protein expression was reduced in canine, sheep, and human Pcells after MI. In pig MI model, the protein level of cardiac-specific SERCA2-splicing variant SERCA2a was reduced by 52% in the whole infarcted ventricle whereas the "non-cardiac" SERCA2b level was increased by 120%. In the infarcted regions, PFs showed 30% downregulation of SERCA2a gene expression and 630% upregulation of SERCA2b. ConclusionOur results confirm that elevated spontaneous Ca2+-activity in post-MI PFs is due to increased SR-Ca2+-uptake within Pcells. Data suggest that a replacement of "cardiac" SERCA2a by the "non-cardiac" SERCA2b sub-isoform in cardiac cells in response to ischemia is implicated in this alteration.

cell biology↗

bioRxiv2021

Optimizing large organ scale micro computed tomography imaging in pig and human hearts using a novel air-drying technique

Underlying electrical propagation in the heart and potentially fatal arrhythmia is the cardiac microstructure. Despite the critical role of muscle architecture, a non-destructive approach to examine not only myocyte orientation, but cellular arrangement in to laminar organization is lacking in hearts from translational animal models and humans. X-ray micro computed tomography using contrast enhancing agents achieves three-dimensional images at near-histological resolutions. However, imaging large mammalian hearts presents challenges including X-ray over-attenuation and loss of image contrast. The goal of this study was to rethink tissue pre-treatment to optimize, and benefit from micro computed tomography imaging resolution in large tissues. Whole pig and human hearts were dehydrated and perfused with a tissue reinforcing agent, hexamethyldisilazane, and slowly air-dried. Heart morphology was conserved and temporally stable. This enabled direct air-mounting for micro computed tomography imaging. Moreover, the desiccated tissue density was significantly reduced compared to the initial hydrated state (P=0.04). Three-dimensional image reconstructions of air-dried hearts segmented using a single intensity threshold revealed detailed microstructural architecture of myolaminae. Conversely, one-step segmentation of hearts loaded with contrast agents poorly estimated the gross anatomical morphology of the heart and lacked identification of tissue microarchitecture. Air-drying large mammalian hearts optimizes X-ray imaging of cardiac microstructure.

physiology↗

bioRxiv2020

Mechanical stimulation of endocardial Purkinje fibres can trigger ventricular arrhythmias

Acute ventricular dilation can evoke mechanically-induced arrhythmias, our study investigated whether Purkinje fibres (PFs) may play a role. Changes in left ventricular (LV) pressure and pseudo-ECGs were measured in isolated, Langendorff-perfused, male Wistar rat hearts in sinus rhythm. The LV endocardial surface was irrigated with experimental agents, via an indwelling catheter. Mechanically-induced arrhythmias were triggered by LV lumen inflation (100l in 2s) via an indwelling balloon. Arrhythmias occurred as the LV volume was increased and spontaneously ceased within 20s of the onset of LV inflation. Arrhythmias were indexed as an increase in the standard deviation of all R-R intervals (SDRR), the number of ectopic activations and the period of these activations. Following 10s LV endocardial irrigation with Lugols solution (IK/I2) to chemically ablate surface PFs or with 0 Na+ Tyrode, there was a statistically significant attenuation of mechanically-induced arrhythmias. Lugols reduced the mechanically-induced increase in SDRR (Tyrode pre-stretch 3.5{+/-}1.7ms to 113.8{+/-}15.1ms during stretch vs Lugol pre-stretch 3.3{+/-}0.5ms to 39.9{+/-}14.5ms during stretch n=8, P < 0.05). There was also a reduction in the number (21.2{+/-}2.0 to 1.5{+/-}0.7, P<0.001) and period (5.9{+/-}0.71s to 1.7{+/-}0.85s, P< 0.01) of ectopic activations. The experiment was repeated using LV lumen irrigation with either 1{micro}M GsMTx4, a peptide that blocks stretch-activated channels or 50{micro}M 9-Phenanthrol (9-Phen), a blocker of TRPM4 channels. GsMTx4 did not attenuate mechanically-activated arrhythmias while 9-Phen had a partial effect. 9-Phen statistically reduced the number and period of ectopic activations but did not attenuate the mechanically-induced increase in SDRR (n=6-11 for each intervention). In further studies, in situ focal mechanical stimulation of individual PFs, caused ectopic activations, in each of 4 sheep LV preparations (0.2{+/-}0.1 ectopics in 10s pre-mechanical stimulation vs 2.1{+/-}0.2 ectopics in 10s during mechanical stimulation, P<0.001). We interpret our observations in rat and sheep hearts as evidence for a role of PFs in the generation of some mechanically-induced arrhythmia.

physiology↗