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Circ Res. 2019 Aug 2;125(4):379-397. doi: 10.1161/CIRCRESAHA.118.314578. Epub 2019 Jul 9.

Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution.

Author information

1
From the Cardiovascular Institute (W.R.G., B.M.B., D.T.P., L.T., G.L., J.W.B., O.C., S.C., S.V., J.C.W., S.M.W.), Stanford University School of Medicine, CA.
2
Department of Pediatrics (W.R.G., S.M.W.), Stanford University, CA.
3
Department of Cardiovascular Surgery, Institute Insure (Institute for Translational Cardiac Surgery), German Heart Center Munich at the Technical University of Munich, Germany (B.M.B.).
4
Department of Developmental Biology, University of Pittsburgh School of Medicine, PA (G.L.).
5
Department of Cardiology, Utrecht Regenerative Medicine Center, University Medical Center Utrecht, Utrecht University, The Netherlands (J.W.B.).
6
Department of Molecular, Cellular, and Developmental Biology, UC Santa Barbara, CA (O.C.).
7
Department of Biology (E.L.A., M.T.-L.), Stanford University, CA.
8
Department of Medicine, Cardiovascular Medicine (J.C.W., S.M.W.), Stanford University School of Medicine, CA.

Abstract

RATIONALE:

The cardiac conduction system (CCS) consists of distinct components including the sinoatrial node, atrioventricular node, His bundle, bundle branches, and Purkinje fibers. Despite an essential role for the CCS in heart development and function, the CCS has remained challenging to interrogate because of inherent obstacles including small cell numbers, large cell-type heterogeneity, complex anatomy, and difficulty in isolation. Single-cell RNA-sequencing 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 single-cell RNA-sequencing within the developing mouse heart.

METHODS AND RESULTS:

Wild-type, embryonic day 16.5 mouse hearts (n=6 per zone) were harvested and 3 zones of microdissection were isolated, including: Zone I-sinoatrial node region; Zone II-atrioventricular node/His region; and Zone III-bundle branch/Purkinje fiber region. Tissue was digested into single-cell suspensions, cells isolated, mRNA reverse transcribed, and barcoded before high-throughput sequencing and bioinformatics analyses. Single-cell RNA-sequencing was performed on over 22‚ÄČ000 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 coexpression 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 immunolabeling with volume imaging (iDISCO+) in 3 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 characterization in the context of development and disease.

KEYWORDS:

bioinformatics; cardiac conduction system; single-cell RNA-sequencing

PMID:
31284824
PMCID:
PMC6675655
[Available on 2020-08-02]
DOI:
10.1161/CIRCRESAHA.118.314578

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