Cells of the adult mouse cardiac conduction system in wild type and Nkx2-5 heterozygous loss-of-function mutation

Background:

Specialized cardiomyocytes in the cardiac conduction system (CCS) generate and spread the electrical impulse that initiates synchronized heart muscle contraction. A disruption of the signal transduction in the atrioventricular (AV) node, the only electrical connection between atria and ventricles, causes AV block. AV block potentially leads to electromechanical uncoupling and sudden cardiac death. Heterozygous loss-of-function mutation of Nkx2-5 (Nkx2-5^+/–), a core cardiac transcription factor, results in progressive AV blocks in humans and mice. 

Methods:

We used single nucleus RNA sequencing to study the transcriptional signature and cellular composition of the CCS in adult wild type (WT) and Nkx2-5^+/– mice. Employing single-molecule fluorescent RNA in situ hybridization (smFISH), we validated selected transcripts and examined the spatial organization of identified cell populations. 

Results:

We studied the sinoatrial (SA) node, AV node and His bundle from 31 mice at single cell resolution. We characterized the transcriptomes of pacemaker and transitional cells in the SA node and AV node, and re-identified five known subpopulations in the SA node.

Most interestingly, we identified two novel subpopulations in both the pacemaker AV node and the His bundle. In both cases, the two novel subpopulations are spatially separate. Their topography is suggestive of two gradients: an atrial-to-ventricular transcriptomic shift within the pacemaker AV node and a slow-to-fast conduction shift along the His bundle. 

We measured gene expression differences between WT and Nkx2-5^+/– in all cell populations. The AV node pacemaker of Nkx2-5^+/– mice showed downregulation of  slit ligand 3 (Slit3, log₂FC = -2.22, p = 7.0e^-42) and its roundabout guidance receptor 1 (Robo1, log₂FC = -0.96, p = 1.6e^-4), and upregulation of neural EGF like 1 (Nell1, log₂FC = 3.18, p = 5.3e^-6), an alternative ligand of roundabout guidance receptors. Slit-Robo signaling was previously reported in neuronal differentiation and axis formation in the developing heart. Altered Slit3 and Nell1 expressions were confirmed by smFISH.  

Conclusions:

Our findings provide insight into the transcriptomic profiles and spatial organization of the CCS cell populations that underlie electrical signal generation and transduction in the heart. We identified disturbed Slit-Robo signaling as a potential mechanism resulting in AV block. Future studies will focus on how Slit-Robo signaling affects the function of AV node pacemaker cells.