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.