Transcriptional Heterogeneity Of The Postnatal Cardiac Conduction System

The cardiac conduction system (CCS) is a network of specialized cardiomyocytes responsible for the coordinated spreading of electrical impulses throughout the heart for synchronized contractions. The CCS is largely made up of the sinoatrial node (SAN), atrioventricular node (AVN) and the fast-conducting ventricular conduction system (VCS), which is further divided into the His bundle, Bundle branches and Purkinje fibers. Although these CCS structures were anatomically discovered >100 years ago, their molecular constituents are not fully known. In this study, CCS cells, making up ~0.05% of myocytes (~500 cells/heart), were isolated from early postnatal hearts (postnatal day 1 to 4) of fluorescent reporter mice (Cntn2-Cre;Rosa26TdTomato), purified using fluorescence-activated cell sorting, and subjected to single-cell RNA-sequencing. Unbiased cluster analysis on ~7000 CCS cells demonstrated distinct transcriptomic profiles in the SAN, AVN, proximal VCS ( His bundle, bundle branches) and distal VCS ( Purkinje fibers). Conforming to previous studies, global conduction markers, such as Cntn2 and Id2, were present throughout the entire CCS, while a VCS-specific transcription factor, Irx3 , was expressed only in the VCS. A comparison between proximal and distal VCS components revealed enrichments of Igfbp7 , Lyz2 and Sfrp5 in the proximal VCS, and Scn10a , Psd3 and Wisp1 in the distal VCS. Sub-clustering analysis followed by validation with immunostaining further identified unique molecular profiles of the SAN ( Shox2 , Vsnl1 ), AVN ( Rspo3 ), His bundle ( S100a6 , Pcp4 ) and bundle branches ( Nppa , Mest ). Data integrating publicly available ATAC-seq in SAN, AV junction, and atrial and ventricular cardiomyocytes as well as Tbx3-ChIP-seq, suggested that these regional markers are possibly regulated by two CCS-specific transcription factors, Irx3 and Tbx3 . More importantly, overexpressing Irx3 and/or Tbx3 in cultured neonatal mouse atrial and ventricular myocytes recapitulated these region-specific expression patterns in a dish. Together, our study provides a comprehensive view of the molecular profiles within the postnatal CCS at a high-resolution and show Irx3 and Tbx3 as genetic regulators establishing heterogeneities of the CCS.