A subpopulation of Periostin-expressing fibroblasts is required for cardiac muscle and neuronal maturation after birth

During the postnatal period in mammals, the cardiac muscle transitions from hyperplasic to hypertrophic growth, the extracellular matrix (ECM) undergoes remodeling, and the heart loses regenerative capacity. While ECM maturation and crosstalk between cardiac fibroblasts (CFs) and cardiomyocytes (CM) have been implicated in neonatal heart development, not much is known about specialized fibroblast heterogeneity and functions in the early postnatal period. In order to better understand CF functions in heart maturation and postnatal cardiomyocyte cell cycle arrest, we have performed gene expression profiling and ablation of postnatal CF subpopulations. Fibroblast lineages expressing Tcf21 or Periostin were traced in transgenic GFP reporter mice and their biological functions and transitions during the postnatal period were examined in sorted cells using RNAseq. A subpopulation of highly proliferative Periostin (Postn)+ CFs was found from postnatal day (P)1 to P11 but was not detected at P30. This population was less abundant and transcriptionally different from Tcf21+ resident CFs, which persist in the mature heart. The Postn+ subpopulation preferentially expresses genes related to cell proliferation and neuronal development, while Tcf21+ CFs differentially express genes related to ECM maturation at P7 and immune crosstalk at P30. Ablation of the Postn+ CFs from P0 to P6 led to altered cardiac sympathetic nerve patterning and a reduction in CM binucleation, maturation, and hypertrophic growth. Thus, postnatal CFs are heterogeneous and include a transient proliferative Postn+ subpopulation required for cardiac nerve development and cardiomyocyte maturation soon after birth. Overall design: We have characterized expression and function of different subpopulations of cardiac fibroblasts during postnatal cardiac development. Use Tcf21MCM and PostnMCM with a tamoxifen-inducible R26eGFP reporter to trace different populations of fibroblast. These populations were GFP FACS sorted for RNA isolation and posterior RNAseq.

在哺乳动物出生后阶段，心肌会从增生性生长转向肥厚性生长，细胞外基质（extracellular matrix, ECM）发生重塑，同时心脏丧失再生能力。尽管细胞外基质成熟以及心脏成纤维细胞（cardiac fibroblasts, CFs）与心肌细胞（cardiomyocytes, CM）间的串扰已被证实与新生心脏发育相关，但人们对出生后早期阶段特化成纤维细胞的异质性及其功能仍知之甚少。
为深入阐明心脏成纤维细胞在心脏成熟以及出生后心肌细胞周期阻滞中的功能，本研究对出生后心脏成纤维细胞亚群开展了基因表达谱分析与消融实验。本研究在转染绿色荧光蛋白（green fluorescent protein, GFP）报告基因的小鼠中，示踪表达Tcf21或Periostin的成纤维细胞谱系，并通过RNA测序（RNA sequencing, RNAseq）对分选后的细胞进行出生后阶段的生物学功能及动态变化分析。研究发现，在出生后第1天（postnatal day, P）至第11天（P11）存在一群高增殖活性的Periostin阳性（Postn+）心脏成纤维细胞，但在出生后第30天（P30）未检测到该亚群。该亚群的丰度低于Tcf21阳性常驻心脏成纤维细胞，且转录组特征存在显著差异，后者可在成熟心脏中持续存在。Postn+亚群优先表达与细胞增殖及神经元发育相关的基因；而Tcf21+心脏成纤维细胞则在出生后第7天（P7）优先表达与细胞外基质成熟相关的基因，在出生后第30天（P30）则优先表达与免疫串扰相关的基因。在出生后第0天至第6天（P0至P6）消融Postn+心脏成纤维细胞，会导致心脏交感神经分布模式改变，并使心肌细胞双核化、成熟及肥厚性生长受到抑制。综上，出生后心脏成纤维细胞具有异质性，其中包含一群短暂存在的高增殖活性Postn+亚群，该亚群对出生后早期心脏神经发育及心肌细胞成熟至关重要。
实验整体设计：本研究对出生后心脏发育过程中不同心脏成纤维细胞亚群的表达特征与功能进行了系统解析。通过将Tcf21MCM、PostnMCM与他莫昔芬（tamoxifen）诱导型R26eGFP报告基因相结合，实现对不同成纤维细胞群体的示踪。随后通过绿色荧光蛋白荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分选目标群体以提取RNA，进而开展RNA测序（RNAseq）分析。