Integrated transcriptome and lineage analyses reveal novel catecholaminergic cardiomyocytes contributing to the cardiac conduction system in murine heart

Abstract

SummaryCardiac conduction system (CCS) morphogenesis is essential for correct heart function yet is incompletely understood. Here we established the transcriptional landscape of cell types populating the developing heart by integrating single-cell RNA sequencing and spatial enhanced resolution omics-sequencing (Stereo-seq). Stereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS. This led to the identification of a previously unrecognized cardiomyocyte population expressing dopamine beta-hydroxylase (Dbh+-CMs), which is closely associated with the CCS in transcriptomic analyses. To confirm this finding, genetic fate mapping by usingDbhCre/Rosa26-tdTomato reporter mouse line was performed with Stereo-seq, RNAscope, and immunohistology. We revealed thatDbh+-derived CMs first emerged in the sinus venosus at E12.5, then populated the atrial and ventricular CCS components at E14.5, with increasing abundance towards perinatal stages. Further tracing by usingDbhCFPreporter andDbhCreERT/Rosa26-tdTomato inducible reporter, we confirmed thatDbh+-CMs are mostly abundant in the AVN and ventricular CCS and this persists in the adult heart. By usingDbhCre/Rosa26-tdTomato/Cx40-eGFP compound reporter line, we validated a clear co-localization of tdTomato and eGFP signals in both left and right ventricular Purkinje fibre networks. Finally, electrophysiological optogenetic study using cell-type specific Channelrhodopsin2 (ChR2) expression further elucidated thatDbh+-derived CMs form a functional part of the ventricular CCS and display similar photostimulation-induced electrophysiological characteristics to Cx40CreERT/ChR2-tdTomato CCS components. Thus, by utilizing advanced transcriptomic, mouse genetic, and optogenetic functional analyses, our study provides new insights into mammalian CCS development and heterogeneity by revealing novelDbh+-CMs.HighlightsStereo-seq provided a spatiotemporal transcriptomic cell fate map of the murine heart with a panoramic field of view and in situ cellular resolution of the CCS.Established the transcriptional landscape of cell types populating the developing murine heart.Revealed previously unreported catecholaminergic cardiomyocyte populations contributing to the developing and mature murine cardiac conduction system.