(VP119) TRANSCRIPTIONAL REGULATION OF THE POSTNATAL CARDIAC CONDUCTION SYSTEM HETEROGENEITY

Background: The cardiac conduction system (CCS) is a network of specialized cardiomyocytes coordinating electrical impulse propagation for synchronized heart contractions. The CCS is primarily 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 CCS components were anatomically discovered more than 100 years ago, their molecular constituents remain not fully understood.

METHODS AND RESULTS: Here, we demonstrated the transcriptomic landscape of the postnatal mouse CCS at a single-cell resolution. CCS cells, making up ~0.1% 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). Notably, CCS region-specific gene expression patterns were recapitulated in vitro using neonatal mouse atrial and ventricular myocytes overexpressing two CCS-specific transcription factors, Irx3 and/or Tbx3, which was supported by integrated data from ATAC-seq in different CCS regions, Tbx3-ChIP-seq, and Irx motifs.

Conclusion: In conclusion, our study offers comprehensive molecular profiles within the postnatal CCS and provides evidence of a regulatory mechanism involving Irx3 and Tbx3 that contributes to the molecular heterogeneities across the CCS.