William R Goodyer1,2, Benjamin M Beyersdorf1,3, David T Paik1, Lei Tian1, Guang Li1,4, Jan W Buikema1,5, Orlando Chirikian1,6, Shannon Choi1, Sneha Venkatraman1, Eliza L Adams7, Marc Tessier-Lavigne7, Joseph C Wu1,8, Sean M Wu1,8,2. 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 22 000 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 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.
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 22 000 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 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.
Authors: James O Tellez; Halina Dobrzynski; Ian D Greener; Gillian M Graham; Emma Laing; Haruo Honjo; Simon J Hubbard; Mark R Boyett; Rudi Billeter Journal: Circ Res Date: 2006-11-02 Impact factor: 17.367
Authors: Martijn L Bakker; Bastiaan J Boukens; Mathilda T M Mommersteeg; Janynke F Brons; Vincent Wakker; Antoon F M Moorman; Vincent M Christoffels Journal: Circ Res Date: 2008-05-08 Impact factor: 17.367
Authors: Jue Li; Ian D Greener; Shin Inada; Vladimir P Nikolski; Mitsuru Yamamoto; Jules C Hancox; Henggui Zhang; Rudi Billeter; Igor R Efimov; Halina Dobrzynski; Mark R Boyett Journal: Circ Res Date: 2008-02-28 Impact factor: 17.367
Authors: Ivan P G Moskowitz; Jae B Kim; Meredith L Moore; Cordula M Wolf; Michael A Peterson; Jay Shendure; Marcelo A Nobrega; Yoshifumi Yokota; Charles Berul; Seigo Izumo; J G Seidman; Christine E Seidman Journal: Cell Date: 2007-06-29 Impact factor: 41.582
Authors: Rajiv A Mohan; Fernanda M Bosada; Jan H van Weerd; Karel van Duijvenboden; Jianan Wang; Mathilda T M Mommersteeg; Ingeborg B Hooijkaas; Vincent Wakker; Corrie de Gier-de Vries; Ruben Coronel; Gerard J J Boink; Jeroen Bakkers; Phil Barnett; Bas J Boukens; Vincent M Christoffels Journal: Proc Natl Acad Sci U S A Date: 2020-07-16 Impact factor: 11.205
Authors: Peter Hanna; Michael J Dacey; Jaclyn Brennan; Alison Moss; Shaina Robbins; Sirisha Achanta; Natalia P Biscola; Mohammed A Swid; Pradeep S Rajendran; Shumpei Mori; Joseph E Hadaya; Elizabeth H Smith; Stanley G Peirce; Jin Chen; Leif A Havton; Zixi Jack Cheng; Rajanikanth Vadigepalli; James Schwaber; Robert L Lux; Igor Efimov; John D Tompkins; Donald B Hoover; Jeffrey L Ardell; Kalyanam Shivkumar Journal: Circ Res Date: 2021-02-25 Impact factor: 17.367
Authors: Ernesto Marín-Sedeño; Xabier Martínez de Morentin; Jose M Pérez-Pomares; David Gómez-Cabrero; Adrián Ruiz-Villalba Journal: Front Cell Dev Biol Date: 2021-05-12
Authors: Camila Delgado; Lei Bu; Jie Zhang; Fang-Yu Liu; Joseph Sall; Feng-Xia Liang; Andrew J Furley; Glenn I Fishman Journal: Development Date: 2021-06-07 Impact factor: 6.862