Research rationale
The heterogeneity of functional cardiomyocytes arises during heart development, which is essential to the complex and highly coordinated cardiac physiological function1. Yet the biological and physiological identities and the origin of the specialized cardiomyocyte populations have not been fully clarified.
Methodology
Single-cell RNA-seq and spatial transcriptomic technologies are applied to investigate cardiogenesis and the genetic profile of the cells of interest. The genetic mouse model DbhCre/Td-ChR2 was generated for lineage tracing and cell fate mapping. Transmission electron microscopy was applied for structural and functional study.
Results
Using single-cell RNA sequencing (scRNA-Seq), we mapped the transcriptional landscape of CCS (Cardiac Conduction System) formation and maturation in the murine heart. This led to the identification of an unreported cardiomyocyte population expressing Dbh gene encoding Dopamine-beta-hydroxylase which is key enzyme for biosynthesis of neurotransmitter Noradrenaline. We determined how these myocytes are distributed across the heart by utilising advanced single-cell and spatial transcriptomic analyses, genetic fate mapping and molecular imaging with computational reconstruction. From E14.5 to adulthood, Dbh+ CMs were abundant in the CCS regions where sympathetic innervation is enriched, as detected by immunostaining with anti-Th antibody, particularly in the adult heart, revealing their close relationship with sympathetic innervation. Immuno-Electron Microscopy further unveiled the presence of high electron-density vesicles in adult Dbh+ cardiomyocytes, which strongly indicates the secretory function of Dbh+ cardiomyocytes.
Conclusions
We discovered a uniquely distributed group of unreported catecholaminergic cardiomyocytes with key regulatory roles in cardiac excitation conduction, providing new insights into neuron-endocrine function of cardiomyocytes. The physiological and pathophysiological implications of such function need to be further explored in the future.
We discovered a uniquely distributed group of unreported catecholaminergic cardiomyocytes with key regulatory roles in cardiac excitation conduction. We also revealed their close relationship with sympathetic innervation during cardiac conduction system (CCS) formation.
Our study thus provides new insights into the development and heterogeneity of the mammalian cardiac conduction system by revealing a new cardiomyocyte population with potential catecholaminergic endocrine function.
Ethics declarations
Competing interests: The chip, procedure, and application of Stereo-seq are covered in pending patents. The remaining authors declare no other competing interests.
Animals and ethical approval
All animal experiments were performed on mice neonatal or adult mice (both genders) in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and were approved by the University of Oxford Pharmacology ethical committee (approval ref. PPL: PP8557407) or Animal Care and Use Committee of the Southwest Medical University, Sichuan (China) (No: 20160930) in conformity with the national guidelines under which the institution operates.
Statistics
For the spatial transcriptomics, we had at least n = 3 sections for hearts from DbhCre/Rosa26-tdTomato mice at E12.5, E14.5, P3, and P56, respectively.
Whole embryos (E8.5, E9.5, E10.5, E12.5, E14.5, n = 5 embryos per stage) or isolated hearts (E12.5, E13.5, E14.5, E16.5, P3, P56, n = 5 hearts per stage) were analyzed by using multiplex nucleic acid in situ hybridization (RNAscope), immunohistological staining, confocal microscopic imaging and EM.