Single-cell RNA-Sequencing Of The Murine Cardiac Conduction System Reveals Transcriptional Regulators Associated With Conduction Development And Disease

Rhythmic beating of the heart is driven by a small subset of heart cells, altogether known as the cardiac conduction system (CCS). The CCS consists of unique components including the sinoatrial node (SAN), the atrioventricular node (AVN), the bundle of His, bundle branches and Purkinje fiber (PF) system. Due to obstacles inherent in studying the CCS, including small cell numbers, large cell-type heterogeneity and complex anatomy, our understanding of the transcriptional networks driving CCS development remains limited. Here, we harnessed single-cell RNA-sequencing (scRNAseq) and bioinformatic analyses to uncover putative DNA-binding transcription factors (TFs) as well as transcriptional co-regulators enriched within the entire developing murine CCS. Comparison of multiple independent prior datasets, spanning all major components of the CCS, revealed previously known TFs necessary for conduction development including, Islet 1 ( Isl1 ; avg log fold change 0.34, p= 1E-192) in the SAN; T-box transcription factor 5 (Tbx5 ; avg log fold change 0.54, p= 2.1E-110) in the AVN; and ETS variant transcription factor 1 ( Etv1; avg log fold change 0.89, p= 6.7E-105) in the PFs, among others. Notably, an additional 16 novel DNA-binding transcription factors and 40 transcription co-regulators were enriched within specific CCS components at statistical significance. To further evaluate the transcriptional landscape of individual subcomponents within the SAN (compact versus transitional cells), we also performed scRNAseq on FACS-isolated SAN cells from embryonic day 16.5 Hcn4 -GFP transgenic mice revealing differential expression of a host of TFs, both established and novel. Finally, our murine findings were then cross-referenced to over 15 human GWAS studies related to cardiac rhythm abnormalities, revealing an association of a subset of these murine CCS-enriched transcription factors and cardiac arrhythmias in humans. In conclusion, this study provides a comprehensive gene expression atlas of transcription factors and transcriptional co-regulators within individual CCS components at single-cell resolution, laying the groundwork for future studies into the transcriptional regulation of cardiac conduction development and disease.