Biology subjects

Walton, R. D.

Publications and source records attributed to Walton, R. D..

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↗

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↗

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↗