Biology subjects

Pasdois, P.

Publications and source records attributed to Pasdois, P..

4 recordsLinked to original sources

bioRxiv2026

Cell-Type-Resolved Transcriptomics Defines Stable and Accessible Markers of the Cardiac Purkinje Fiber in Sheep and Human Translation

Background: The His-Purkinje network drives rapid ventricular activation and is a major substrate for ventricular arrhythmias, yet it is among the least molecularly characterized cardiac compartments. Markers validated in rodents transfer poorly across species, few are confirmed at the protein level in large mammals or humans, and most lack the stability and surface accessibility that demanding applications require. Methods: We combined histology-guided laser-capture microdissection with low-input, cell-type-resolved RNA-sequencing to profile Purkinje fibers, left-ventricular cardiomyocytes and peri-Purkinje stroma from adult sheep. Differentially expressed genes were ranked by a transparent composite framework weighting expression specificity, cross-individual stability and predicted subcellular accessibility; leading candidates were validated by RT-qPCR and immunolabelling in sheep and by RT-qPCR in human myocardium. Results: RNA-sequencing resolved a Purkinje transcriptome distinct from cardiomyocytes and stroma and defined 331 concordantly enriched genes, which the composite framework ranked into stable, specific candidates spanning intracellular and cell-surface compartments. By RT-qPCR, the canonical conduction markers connexin-40/GJA5, HCN4, NEFM and MYL4 were strongly enriched in Purkinje fibers, whereas the rodent gold-standard contactin-2 was not, underscoring species divergence. Thirteen of sixteen prioritized candidates were confirmed by RT-qPCR, and immunolabelling localized MYL4, CNN1, TAGLN and DKK3 to Purkinje fibers; contactin-5 emerged as a novel transcript- and protein-validated Purkinje marker. In human myocardium, a defined subset - MYL4, connexin-40/GJA5, contactin-5 and TAGLN - was conserved, while several markers proved species-restricted. Conclusions: We provide the first genome-wide, cell-type-resolved molecular portrait of the Purkinje fiber in a large-animal model and a generalizable strategy that selects markers for specificity, stability and accessibility. The resulting resource - including the cross-species marker contactin-5 and compartment-matched candidates - supplies validated tools to identify, isolate and target Purkinje cells and demonstrates the necessity of cross-species validation.

physiology↗

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↗

bioRxiv2025

Time-dependent Mitochondrial Remodeling in Experimental Atrial Fibrillation and Potential Therapeutic Relevance

BACKGROUNDChanges in mitochondria have been implicated in atrial fibrillation (AF), but their manifestations and significance are poorly understood. Here, we studied changes in mitochondrial morphology and function during AF and assessed the effect of a mitochondrial-targeted intervention in a large animal model. METHODS AND RESULTSAtrial cardiomyocytes (ACMs) were isolated from dogs in electrically-driven AF for periods of 24 hours to 3 weeks and from humans with/without longstanding persistent AF. Mitochondrial Ca2+-concentration ([Ca2+]Mito), reactive oxygen species (mtROS) production, membrane potential ({Delta}{Psi}m), permeability transition-pore (mPTP) opening and flavin adenine dinucleotide (FAD) were measured via confocal microscopy; nicotine adenine dinucleotide (NADH) under ultraviolet light. mtROS-production increased within 24 hours and superoxide-dismutase type-2 was significantly reduced from 3-day AF. [Ca2+]Mito and mPTP-opening frequency/duration increased progressively during AF. Mitochondrial depolarization was detectable 24 hours after AF-onset. NADH increased by 15% at 24-hour AF, concomitant with increased pyruvate-dehydrogenase expression, then gradually decreased. Mitochondria enlarged and elongated at 24-hour and 3-day AF, followed by progressive fragmentation, rupture and shrinkage. Mitochondrial fusion protein-1 (MFN1) was reduced from 3-day to 3-week AF and phosphorylated dynamin-related protein-1 (p-DRP1ser-616) increased after 1 week of canine AF and in human AF. Addition of the mitochondrial antioxidant MitoTempo attenuated action-potential shortening and L-type Ca2+-current (ICaL)-downregulation in canine and human AF ACMs in vitro. Administration of the orally-active mitochondrial-targeted ubiquinone mitoquinone to dogs during 3-week AF prevented mitochondrial Ca2+-overload, mtROS-overproduction, structural damage and abnormalities in {Delta}{Psi}m and respiration. Functionally, mitoquinone reduced AF-induced Ca2+-current downregulation, action-potential abbreviation, contractile dysfunction and fibrosis, preventing AF-substrate development and AF-sustainability. CONCLUSIONSMitochondria show a series of changes during AF, with early hyperfunction and enhanced ROS-generation, followed by progressive damage and dysfunction. Mitochondrial-targeted therapy prevents mitochondrial dysfunction and attenuates adverse AF-related remodeling, positioning mitochondrial protection as a potential novel therapeutic target in AF.

pathology↗