Blocking Nuclear Receptor Nr4a3 Unlocks the Senescence Barrier to Promote Direct Cardiac Reprogramming

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: To determine the molecular basis by which knockdown Nr4a3 enhances iCM reprogramming, we conducted CUT&Tag profiling of two histone modifications including histone H3 acetylation on lysine 27 (H3K27ac, a mark for active enhancers and promoters) and histone H3 trimethylation on lysine 27 (H3K27me3, a repressive epigenetic mark) using shNr4a3-iCMs and shNT-iCMs collected at reprogramming day 7