Enhancing cardiomyocyte reprogramming efficiency by targeting cellular senescence is mediated via Rb1 gene

Direct reprogramming of fibroblasts into cardiomyocytes by overexpressing cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) is a promising way for cardiac repair, however, the low reprogramming efficiency remains a significant challenge. Cellular senescence, an irreversible cell-cycle arrest occurring in mitotic cells, has been reported to influence the efficiency of induced pluripotent stem cell (iPSC) reprogramming.

We established an inducible GMT expression system in mouse embryonic fibroblasts (MEFs) and human fetal cardiac fibroblasts (hFCFs) using the PiggyBac transposon system. RNA sequencing was performed to identify genes associated with cellular senescence during reprogramming. Selected senescence-related genes were knocked down using shRNA, and their impact on reprogramming efficiency was assessed via flow cytometry, gene expression analysis, and staining for senescence and apoptosis markers.

Direct cardiac reprogramming induced cellular senescence and apoptosis, evidenced by enhanced β-Gal staining, elevated expression of senescence markers P16 and GLB1, and increased apoptosis rates. RNA sequencing and gene set enrichment analysis (GSEA) revealed significant upregulation of senescence-related genes (RB1, RBBP4, RBBP7, CBX8, and CDKN1B). Knockdown of these genes, particularly RB1, significantly enhanced reprogramming efficiency, increasing the proportion of GFP + cells in MEFs and α-actinin + cells in hFCFs. RB1 inhibition also reduced senescence marker levels and upregulated endogenous cardiac transcription factors GATA4 and MEF2C.

Our findings demonstrate that cellular senescence might serves as a barrier to direct cardiac reprogramming and offer novel insights into the regulatory mechanisms involved in this process.