Cell-Type-Resolved Transcriptomics Defines Stable and Accessible Markers of the Cardiac Purkinje Fiber in Sheep and Human Translation
Sabine CharroniD, Néstor Pallares‐LuponiD, Marion ConstantiniD, Jason D. BayeriD, Philippe PasdoisiD, Fanny VaillantiD, Richard D. WaltoniD
Abstract
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.
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In-depth summary
Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (25 Aug 2026) · CC BY 4.0
Laser-capture RNA-seq of sheep Purkinje fibers yields a 331-gene enriched panel; thirteen of sixteen ranked candidates confirmed by RT-qPCR, and CNTN5 is Purkinje-enriched in sheep and human.
The His-Purkinje network drives ventricular activation and is a major arrhythmia substrate, yet its markers come mostly from rodents, transfer poorly to large mammals and humans, and are often shared with other tissues or intracellular. The authors describe this as the first genome-wide, cell-type-resolved Purkinje transcriptome in a large-animal model, and they rank markers on cross-individual stability and predicted surface accessibility as well as enrichment.
Key results
ShownLaser-captured sheep Purkinje fibers have a transcriptome that separates from both left-ventricular cardiomyocytes and peri-Purkinje stroma. Genes enriched against both comparators form a 331-gene panel.
Principal component analysis split the three populations into discrete clusters (PC1 45.3%, PC2 12.0% of variance; n = 6 animals per population). DESeq2 (adjusted P < 0.01, |log2 fold-change| > 1) found 1,795 genes up versus cardiomyocytes and 2,276 versus stroma. Their expression-filtered intersection gave 331 genes, 286 of them with a mappable human ortholog. · PFs possess a transcriptome distinct from…; Definition of the Purkinje-enriched panel; Read processing, alignment, quality control and…; Figure legends
ShownA weighted score combining specificity, stability and predicted compartment ranked the 286 scored genes (CSRP1 first, then PTGR1, TAGLN, CNN1, MYL4). Most of the prioritized candidates that were tested proved Purkinje-enriched in sheep.
Thirteen of sixteen candidates were higher in Purkinje fibers than in cardiomyocytes, with complete group separation, roughly three-fold to over two hundred-fold, all P < 0.005 (Welch's t test, n = 4 per group). THSD4 was unchanged (P = 0.72); GYS1 and TUBA1A were higher in cardiomyocytes (P = 0.04, P = 0.005). Immunohistochemistry placed MYL4, CNN1, TAGLN and DKK3 in Purkinje fibers. · A composite framework prioritizes specific and…; Targeted RT-qPCR validates prioritized…; Protein-level validation of lead candidates
ShownCanonical conduction-system markers are enriched in sheep Purkinje fibers, but contactin-2 (CNTN2), described as the rodent gold-standard marker, is not.
RT-qPCR, n = 4 per group: GJA5, HCN4, NEFM and MYL4 were enriched with complete separation, from roughly ten-fold (connexin-40) to over two hundred-fold (MYL4), all P < 0.012. TBX5 was about two-fold higher (P = 0.02); CNTN2 did not differ (P = 0.25). Immunofluorescence with cTnT co-staining localized connexin-40 and MYL4 to Purkinje fibers, with negligible signal in working myocardium. · Ovine Purkinje fibers recapitulate canonical…; Figure legends
ShownContactin-5 (CNTN5) marks sheep Purkinje fibers at both transcript and protein level, whereas its relative CNTN4 does not.
RT-qPCR enrichment versus cardiomyocytes, P < 0.001. Cardiomyocyte expression was at or below the detection limit in two of four cardiomyocyte replicates (only two were quantifiable). CNTN4 was not enriched (P = 0.15). CNTN5 immunofluorescence was confined to Purkinje fibers and absent from surrounding cardiomyocytes. · Contactin-5 (CNTN5) is a transcript-and…; RT-qPCR validation; Figure legends
SuggestedIn human myocardium, a subset of the sheep markers (MYL4, connexin-40, CNTN5, TAGLN) is Purkinje-enriched at the transcript level. Several others are not enriched, and CAPN3 reverses direction.
RT-qPCR of twelve markers in four donors (Purkinje fibers vs left-ventricular cardiomyocytes): MYL4 approximately eighteen-fold (P < 0.001), GJA5 approximately twenty-five-fold (P = 0.04), CNTN5 approximately fifteen-fold (P = 0.02), TAGLN approximately four-fold (P = 0.04). CNTN2 showed a trend (P = 0.07). CNN1, PTGR1, SCN4B, CNTN4, RIMS1 and TES were not enriched; CAPN3 was higher in cardiomyocytes (P = 0.01). · Conservation of candidate markers in the human…; Figure legends
Methods, data and reproducibility
Laser-capture microdissection isolated sheep Purkinje fibers, left-ventricular cardiomyocytes and peri-Purkinje stroma for low-input RNA-seq with unique molecular identifiers. Genes enriched against both comparators were ranked on specificity, stability and predicted compartment. Top candidates were then checked in sheep with RT-qPCR plus immunolabelling, and in human myocardium with RT-qPCR alone.
- Laser-captured Purkinje fibers, left-ventricular cardiomyocytes and peri-Purkinje collagen stroma from septobasal ventricle, low-input RNA-seq · sheep (Ovis aries, Charmoise breed) · n = 6 hearts; n = 6 animals per population
- False-tendon Purkinje fibers and sub-epicardial left-ventricular free wall, RT-qPCR · sheep (Ovis aries) · n = 7 hearts dissected; n = 4 per group in RT-qPCR (limitations state n = 4 to 5)
- Septobasal tissue blocks for immunofluorescence and immunohistochemistry · sheep (Ovis aries) · n = 2 additional hearts
- Free-running Purkinje fibers and left-ventricular myocardium from brain-dead donor hearts unsuitable for transplantation, RT-qPCR · human · n = 4 hearts (n = 4 donors per group)
- Methods & tools
- Zeiss P.A.L.M. MicroBeam (laser-capture microdissection), Takara SMART-Seq mRNA LP with UMIs, Illumina NextSeq 2000, Cogent NGS Analysis Pipeline v2.0.1, STAR, DESeq2, RNAseqQC, gage, Ensembl BioMart, KEGG, Reactome, WikiPathways, Gene Ontology, UniProt, matrisome, Bio-Rad CFX96 (SYBR RT-qPCR), GraphPad Prism 9.5
- Reagents & environment
- Cogent NGS Analysis Pipeline v2.0.1; STAR alignment to Ovis aries ARS-UI_Ramb_v2.0 (Ensembl release 113); ortholog mapping with Ensembl BioMart releases 114 and 115; GraphPad Prism 9.5. Other tool versions and QC metrics are in Table S5 (Supplemental Methods).
- Reproducibility gaps
- RNA-seq data not deposited (offered for GEO submission where necessary); other data on reasonable request. The in-house Python scoring suite has no public link; weights and formulae are in the Supplemental Methods.
Limitations (6)
- The stromal comparator was the peri-Purkinje collagen sheath, not purified vascular smooth muscle. Contractile candidates that smooth muscle also expresses should therefore be used in combination and with spatial confirmation. · stated by the authors
- Purkinje and stromal RNA came from low-input, cell-sparse material (Purkinje RIN 5.3-7.0; stromal RNA at the detection limit). · stated by the authors
- RT-qPCR cohorts were small (sheep n = 4 to 5, human n = 4), and P values for the pre-specified candidates were not corrected for multiple testing. · stated by the authors
- Scoring weights are hypothesis-driven, not trained, and subcellular compartment calls are computational, so surface and secreted candidates still need subcellular confirmation. Functional, electrophysiological and disease-state testing was not done. · stated by the authors
- The composite ranking is never compared with a fold-change-only ranking, so its benefit over enrichment alone is untested. Only sixteen of the 286 ranked genes were checked by RT-qPCR. · noted in this summary
- Human evidence is transcript-level only (RT-qPCR in four donors, no human immunolabelling), so CNTN5 protein is validated in sheep but not in human tissue. · noted in this summary
Who should read:Read this if you need validated markers to identify, isolate or target cardiac Purkinje fibers in sheep or human tissue, or want a template for ranking cell-type markers on stability and accessibility as well as enrichment.
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