Abstract Body: The development and function of cardiac conduction system (CCS) components are governed by specialized and unique gene expression programs. Although this transcriptional heterogeneity has been studied in mice, how these molecular profiles compare to those in humans remains unclear. Additionally, transcriptional changes during CCS maturation are not well understood. Here, we examine the conservation and divergence of CCS gene programs across species and development.

To achieve this, CCS cells were isolated and analyzed using published single-cell/nucleus RNA sequencing and spatial transcriptomic data from mouse (embryonic/postnatal) and human (fetal/adult) hearts. As expected, species-conserved markers of broad CCS zones included pacemaker ion channels in the nodal regions and fast-conducting ion channels in the ventricular conduction system, as well as many genes newly implicated in the CCS. Additionally, we found species-conserved markers specific to individual CCS components, such as GNAO1 and CDH11 in the sinoatrial node; RSPO3 and BMP2 in the atrioventricular node; FBN2 in the His bundle; and IRX1 and IRX2 in Purkinje fibers. Our analyses also identified species- and stage-specific markers, reflecting evolutionary divergence and developmental changes in the CCS. These included functional genes such as ion channels, as well as signaling molecules and transcription factors that may regulate CCS development and maturation. Further examination of rat and zebrafish sinoatrial and atrioventricular nodes revealed a core set of vertebrate-conserved markers, as well as those conserved within mammals or rodents only. We then reconstructed gene regulatory networks and identified conserved transcriptional mechanisms that establish CCS heterogeneity. Moreover, we examined associations of common and rare genetic variants of conserved CCS genes with conduction-related traits. This analysis identified significant associations of 55 conserved CCS genes with arrhythmias and electrophysiological measures, suggesting functional and translational relevance. To enhance data accessibility, we launched an interactive web application (ccsatlas.com) that visualizes CCS gene expression across species and development.

Our study provides a systematic comparative analysis of CCS transcriptional heterogeneity, identifying conserved gene programs relevant to human physiology and elucidating the molecular basis for species- and stage-dependent differences in CCS function.