Blocking Nuclear Receptor Nr4a3 Unlocks the Senescence Barrier to Promote Direct Cardiac Reprogramming [neonatal scRNA-seq]

Direct cardiac reprogramming of fibroblast to cardiomyocytes represents a potential means of restoring cardiac function following injury. However, aged and adult fibroblast are less efficient for reprogramming compared with neonatal fibroblast. In this study, through the joint analysis of RNAseq and ATACseq, we found that compared with Neo-iCM, cardiac related features were down-regulated with aging, while fibrosis and SASP related genes were significantly up-regulated with aging. Single-cell transcriptomics reveals a bifurcated trajectory of aged iCM reprogramming. Then, we screened 51 transcriptomic and epigenomic regulators of the barriers to aged-iCM reprogramming and found that Nuclear receptor subfamily 4 group A member 3 (Nr4a3), an pro-inflammatory transcription factor, induced cellular senescence to inhibit direct reprogramming during aging. Mechanistically, knockdown of Nr4a3 enhanced direct cardiac reprogramming by promoting cardiac gene programs and inhibiting fibrosis pathway and secretes SASP. In addition, knockdown of Nr4a3 promoted human reprogramming in primary ventricular human cardiac fibroblasts (HCFs) and improved heart function after MI. Overall design: Single cell RNA-seq profiles of MGT-transduced neonatal FBs cultured at reprogramming day 14 by 10x Genomics Chromium System.

成纤维细胞（fibroblast）向心肌细胞的直接心脏重编程，是损伤后恢复心脏功能的潜在治疗策略。然而，与新生成纤维细胞（neonatal fibroblast）相比，成年及衰老成纤维细胞的重编程效率显著更低。本研究通过RNA测序（RNAseq）与转座酶可及性测序（ATACseq）的联合分析发现：与新生诱导型心肌细胞（Neo-iCM）相比，心脏相关特征随衰老进程显著下调，而纤维化及衰老相关分泌表型（SASP）相关基因则随衰老显著上调。单细胞转录组学分析揭示了衰老诱导型心肌细胞重编程的分叉轨迹。随后，我们筛选得到51个调控衰老诱导型心肌细胞重编程障碍的转录组与表观组调控因子，并发现核受体亚家族4A组3成员（Nr4a3）作为一种促炎转录因子，可通过诱导细胞衰老抑制衰老过程中的直接心脏重编程。机制层面，敲低Nr4a3可通过激活心脏基因程序、抑制纤维化通路及衰老相关分泌表型（SASP）的分泌，增强直接心脏重编程效率。此外，在原代人心室成纤维细胞（HCFs）中敲低Nr4a3可促进人类细胞的重编程，并改善心肌梗死（MI）后的心脏功能。实验整体设计：借助10x Genomics Chromium系统，对重编程第14天的MGT转导新生成纤维细胞（neonatal FBs）开展单细胞RNA测序（RNAseq）分析。