Abstract Background Specialised cardiomyocytes in the cardiac conduction system (CCS) generate and spread the electrical impulse that initiates synchronised heartbeats. Disrupted signal transduction in the atrioventricular (AV) node, a key electrical connection between atria and ventricles, causes AV block. AV block potentially leads to electromechanical uncoupling and sudden cardiac death. Nkx2-5 encodes a cardiac transcription factor that is critical for embryonic CCS formation. Heterozygous loss-of-function mutation of Nkx2-5 (Nkx2-5+/–) results in a hypoplastic CCS in humans and mice and progressive AV block. Purpose We aimed to create a cell atlas of the adult mouse CCS and define the consequences of Nkx2-5 haploinsufficiency on the structural organisation and transcript expression of CCS cells. Methods We performed single-nucleus RNA sequencing on the sinoatrial nodes, AV nodes, His bundles, right bundle branches (RBB) and moderator bands of 17 wild type and 14 Nkx2-5+/– mice (aged 13 to 35 weeks, 5159 CCS cells). Employing single-molecule fluorescent RNA in situ hybridisation (smFISH), we validated selected transcripts and examined the spatial organisation of identified cell populations. Results In the AV node, we identified two subpopulations of pacemaker cells that were spatially separated in the proximal and distal pacemaker region (Fig1). Within these subpopulations, expressed transcripts indicated an atrial-to-ventricular gradient of electrical activity, corresponding to their respective topography. We determined two subpopulations in the His bundle and RBB that were enriched for either slow or fast conduction markers. Spatially, the slow-conducting populations seemed to surround the fast-conducting ones and might act as electrical isolation (Fig1). In Nkx2-5+/- cells of the pacemaker AV node, His bundle, RBB and Purkinje fibres, genes involved in Slit-Robo signalling were dysregulated. Slit-Robo signalling was previously reported in neuronal differentiation and embryonic heart lumen formation. Slit ligand 3 was downregulated in Nkx2-5+/- pacemaker AV node cells (Slit3, log2FC = -2.22, p = 7.0e-42), as was the roundabout guidance receptor 1 (Robo1, log2FC = -0.96, p = 1.6e-4). By contrast, there was upregulation of neural EGF-like 1 (Nell1, log2FC = 3.18, p = 5.3e-6), an alternative ligand of roundabout guidance receptors. Altered Nell1 and Slit3 expressions were confirmed by smFISH (Fig2). Conclusions Our cell atlas defines new CCS cell populations with informative transcriptomic profiles and spatial organisation, providing insights into subpopulations in the AV junction where electrical signals switch from slow to fast conduction. We identified disrupted Slit-Robo signalling in Nkx2-5+/-, possibly impairing differentiation and maturation of the CCS and altering electrophysiologic functions. Our normative anatomical and transcription data on the CCS can inform future studies of cardiac electrophysiology and arrhythmias.Fig1 - Subpopulations in AV junctionFig2 - Nell1 smFISH in pacemaker AV node