Transcriptome profiling of neonatal heart regeneration. Transcriptome profiling of neonatal heart regeneration

Background: The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and non-regenerative mouse hearts over a 7-day time period following myocardial infarction. Methods: RNA-Seq, H3K27ac ChIP-Seq and H3K27me3 ChIP-Seq were performed on ventricular samples from regenerative P1 or non-regenerative P8 mouse hearts at +1.5d, +3d and +7d after MI or Sham surgery to assemble the transcriptome, active chromatin and repressed chromatin landscapes during neonatal heart regeneration. Dynamic enhancer landscapes from mouse hearts during cardiac development were analyzed using data from ENCODE. Effects on cardiomyocyte proliferation and cardiac function from selected factors identified in this study were tested using BrdU/EdU pulse-labeling or mouse models coupled with immunohistochemistry and echocardiography. Results: By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and an embryonic cardiogenic gene program that remains active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Conclusions: Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that might be modulated to promote heart regeneration. Overall design: We performed myocardial infarction (MI) or sham surgeries to mouse hearts at postnatal day 1 or day 8. At 1.5d, 3d or 7d post P1 and P8 surgery, ventricle samples below the surgery plane were collected in triplicate for mRNA-Seq.

背景：成年哺乳动物心脏损伤后再生能力极为有限，而新生小鼠心脏在出生后短期内即可高效完成再生。为揭示新生心脏再生的核心分子机制，我们对比了心肌梗死（myocardial infarction, MI）后7天周期内，具备再生能力与无再生能力的小鼠心脏的转录组与表观基因组特征。
方法：分别对出生后第1天（P1，具有再生能力）或第8天（P8，无再生能力）的小鼠心脏，在MI或假手术（Sham）后1.5天、3天、7天采集心室样本，开展RNA测序（RNA-Seq）、H3K27ac染色质免疫共沉淀测序（ChIP-Seq）及H3K27me3染色质免疫共沉淀测序，以构建新生心脏再生过程中的转录组、活性染色质与抑制性染色质景观。利用ENCODE数据库公开的小鼠心脏发育阶段动态增强子景观数据进行关联分析。通过BrdU/EdU脉冲标记法，或结合免疫组化与超声心动图检测的小鼠模型，对本研究筛选得到的候选调控因子在心肌细胞增殖及心脏功能中的作用进行验证。
结果：通过整合基因表达谱与活性/抑制性染色质相关的组蛋白修饰标记，我们鉴定出驱动新生心脏再生的转录程序，以及成年后心脏再生受阻的分子机制。研究结果揭示了再生心脏中独特的免疫应答模式，以及在新生心脏再生阶段持续激活的胚胎源性心脏发生基因程序。在与心脏再生相关的独特免疫因子及胚胎基因中，我们鉴定出两个此前未被报道的心肌细胞增殖调控因子：编码细胞因子的Ccl24，以及编码RNA结合蛋白的Igf2bp3。
结论：本研究数据为新生心脏再生的分子基础提供了全新视角，并鉴定出可通过靶向调控以促进心脏再生的潜在基因靶点。
整体实验设计：我们对出生后第1天或第8天的小鼠心脏实施MI或Sham手术。在P1和P8组小鼠术后1.5天、3天或7天，采集手术平面下方的心室样本，每组设置3次生物学重复，用于mRNA测序。