31284824

PMC6675655

Rationale

The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node (SAN), atrioventricular node (AVN), His bundle, bundle branches (BB) and Purkinje fibers (PF). Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate due to inherent obstacles including small cell numbers, large cell type heterogeneity, complex anatomy and difficulty in isolation. Single-cell RNA-sequencing (scRNA-seq) allows for genome-wide analysis of gene expression at single-cell resolution.

Objective

Assess the transcriptional landscape of the entire CCS at single-cell resolution by scRNA-seq within the developing mouse heart.

Methods and Results

Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and three zones of microdissection were isolated, including: Zone I – SAN region; Zone II – AVN/His region; and Zone III – BB/PF region. Tissue was digested into single cell suspensions, isolated, reverse transcribed and barcoded prior to high-throughput sequencing and bioinformatics analyses. scRNA-seq was performed on over 22,000 cells and all major cell types of the murine heart were successfully captured including bona fide clusters of cells consistent with each major component of the CCS. Unsupervised weighted gene co-expression network analysis led to the discovery of a host of novel CCS genes, a subset of which were validated using fluorescent in situ hybridization as well as whole mount immunolabelling with volume imaging (iDISCO+) in three-dimensions on intact mouse hearts. Further, subcluster analysis unveiled isolation of distinct CCS cell subtypes, including the clinically-relevant but poorly characterized “transitional cells” that bridge the CCS and surrounding myocardium.

Conclusions

Our study represents the first comprehensive assessment of the transcriptional profiles from the entire CCS at single-cell resolution and provides a gene atlas for facilitating future efforts in conduction cell identification, isolation and characterization in the context of development and disease.

CDC Public Access

T32 EB009035/NIBIB NIH HHS/National Institute of Biomedical Imaging and Bioengineering/United States
P30 NS069375/NINDS NIH HHS/National Institute of Neurological Disorders and Stroke/United States
DP1 LM012179/NLM NIH HHS/National Library of Medicine/United States
R01 HL145676/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United States
T32 HL094274/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United States
R01 HL141371/NHLBI NIH HHS/National Heart, Lung, and Blood Institute/United States
S10 OD025091/ODCDC CDC HHS/Office of the Director/United States
DP1 LM012179/NLM NIH HHS/National Library of Medicine/United States