Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution
Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution
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DOI：
10.1161/circresaha.118.314578
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发表时间：
2019-08-02
影响因子：
20.1
通讯作者：
Wu, Sean M.
中科院分区：
文献类型：
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作者：
Goodyer, William R.;Beyersdorf, Benjamin M.;Wu, Sean M.
理由：心脏传导系统（CCS）由不同的组成部分组成，包括窦房结、房室结、希氏束、束支和浦肯野纤维。尽管CCS在心脏发育和功能中发挥着重要作用，但由于固有的障碍，包括细胞数量少、大细胞类型异质性、复杂的解剖结构和分离困难，CCS仍然具有挑战性。单细胞RNA测序允许以单细胞分辨率进行基因表达的全基因组分析。目的：通过在发育中的小鼠心脏内进行单细胞RNA测序，以单细胞分辨率评估整个CCS的转录景观。方法和结果：收获野生型胚胎16.5天小鼠心脏（n=6/区）并分离3个显微切割区，包括：I区-窦房结区; II区-房室结/His区;和III区-束分支/浦肯野纤维区。在高通量测序和生物信息学分析之前，将组织消化成单细胞悬浮液，分离细胞，mRNA逆转录并条形码化。对超过22000个细胞进行了单细胞RNA测序，成功捕获了鼠心脏的所有主要细胞类型，包括与CCS的每个主要组分一致的真实细胞簇。无监督的加权基因共表达网络分析导致发现了一系列新的CCS基因，其中一个子集使用荧光原位杂交以及整体安装免疫标记与体积成像（iDISCO+）在3维完整的小鼠心脏上进行了验证。此外，亚群分析揭示了不同CCS细胞亚型的分离，包括临床相关但表征不佳的桥接CCS和周围心肌的过渡细胞。结论：我们的研究代表了在单细胞分辨率下对整个CCS的转录谱进行的第一次全面评估，并提供了发育和疾病背景下的表征。
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.