Stable molecular signatures and age-related microstructural remodeling of the Purkinje network in sheep and human hearts

Background The ventricular Purkinje network is a recognized trigger and substrate of malignant ventricular arrhythmias, particularly with ageing, yet remains poorly characterized in large mammals due to limited molecular specificity and difficulty resolving its microstructure. Rodent-derived markers lack reproducibility in translational models and humans. We aimed to define stable Purkinje-specific molecular signatures and characterize age-associated remodeling using a quantitative multiscale prioritization framework relevant to arrhythmogenic substrate definition. Methods Purkinje fibre (PF)-enriched regions were isolated from ovine left ventricular endocardium by laser microdissection and compared with adjacent working myocardium and stromal sheath tissue using bulk RNA sequencing (DESeq2 normalization). Differential expression was assessed with FDR correction. A stability framework integrated: (1) transcript stability (expression rank, coefficient of variance, age persistence), (2) structural annotation enrichment, and (3) disease stability based on Human Phenotype Ontology gene–disease associations. Age comparisons were performed between young (1 year) and mature (>6 years) sheep. Genes with <10 reads per group were excluded. Validation included RT-qPCR in isolated free-running PF strands and immunofluorescence in ovine and human ventricular tissue. Results Comparisons identified 1,795 genes upregulated in PF vs cardiomyocytes (young), 2,276 in PF vs stroma (young), and 1,211 in PF vs cardiomyocytes (old). Intersection across all comparisons yielded 407 genes consistently enriched in PF relative to myocardium and sheath with persistence into adulthood. Transcript stability scoring prioritized low-variance, age-consistent genes, predominantly associated with cytostructural functions. Integrated stability scoring identified 10 high-priority PF-enriched candidates demonstrating strong differential expression across all comparisons (log₂FC 2.0–9.9; adjusted p<10-6). RT-qPCR confirmed 6–900-fold enrichment in isolated PF strands versus myocardium for 9/10 candidates (all p<0.01). Immunofluorescence demonstrated selective localization along PF bundles with minimal myocardial cross-reactivity in sheep and conserved enrichment patterns in human ventricular samples. Age-associated analysis revealed coordinated downregulation of adhesion and mitochondrial transcripts in mature PF tissue. Quantitative histology demonstrated increased collagen partitioning and fragmentation of PF bundles in older hearts, consistent with progressive microstructural remodeling of the conduction substrate. Conclusions We define a reproducible molecular signature of the ovine Purkinje network and demonstrate age-associated transcriptional and architectural remodeling. This multiscale framework establishes a biological foundation for phenotypic investigation of Purkinje substrate relevant to ventricular arrhythmogenesis in ageing hearts.