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

Abstract

Introduction: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Direct cardiac reprogramming; RB1; Senescence.

Fig. 1

Direct cardiac reprogramming accelerated cellular senescence and apoptosis. A Representative images of β-gal staining at different time points during cardiac reprogramming in hFCFs. Scale bar = 200 μm. B Relative mRNA expression of senescence markers P16 and GLB1 seven days after initiation of cardiac reprogramming in hFCFs. C Cellular apoptosis percentage was evaluated with Annexin V/PI Staining and analyzed by FACS at different time points upon initiation of cardiac reprogramming in hFCFs. D Representative images of β-gal staining three days after cardiac reprogramming in MEFs. Scale bar = 200 μm. E Relative mRNA expression of senescence marker P16 and GLB1 seven days after reprogramming in MEFs. Mean values + SEM of three independent experiments is shown (n = 3). Data were analyzed with two-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 and ****p ≤ 0.0001

Fig. 2

Analysis of differential expression on the RNA-Seq data of hFCFs. A PCA plot analysis was based on differentially expressed genes between GMT Dox (+) group and other control groups (FDR = 0.1). B Heatmap analyses demonstrated that genes in cellular senescence pathway are significantly upregulated in GMT Dox (+) group compared to control groups (FDR = 0.1). C GSEA was performed to elucidate whether the applied gene set is statistically enriched in certain pathways, such as cellular senescence pathway. Data were analyzed through Qlucore Omics Explorer

Fig. 3

shRNA screen identified RB1 as a critical modulator of cardiac reprogramming in hFCFs. A Schematic overview of the shRNA screen for identifying critical regulators of cardiac reprogramming in hFCFs. B Knockdown efficiency of indicated shRNAs measured by RT-PCR. Expression values for each gene were normalized to empty vector PLKO infected group. PLKO indicated as shRNA control. C Representative FACS image of α-actinin⁺ cells in hFCFs ten days after transduction of lentiviruses encoding shRNAs or PLKO (empty vector). D Quantification of α-actinin⁺ hFCFs upon Dox exposure. Mean values + SEM of three independent experiments is shown (n = 3). Data were analyzed with two-way ANOVA. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; ****P < 0.0001 vs. control

Fig. 4

shRNA screen identified RB1 as a critical modulator of cardiac reprogramming in MEFs. A Schematic overview of the shRNA screen for identifying critical regulators of cardiac reprogramming in MEFs. B Knockdown efficiency of indicated shRNAs measured by RT-PCR. Expression values for each gene were normalized to empty vector PLKO infected group. PLKO indicated as shRNA control. C Representative fluorescence images of MEFs with Dox exposure, scale bar 200 μm. D Representative FACS image of GFP⁺ cells in MEFs ten days after transduction of lentiviruses encoding shRNAs or PLKO. E Quantification of GFP⁺ MEFs upon Dox exposure. Mean values + SEM of three independent experiments is shown (n = 3). Data were analyzed with two-way ANOVA. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; ****P < 0.0001 vs. control

Fig. 5

Knock down of RB1 ameliorates cellular senescence. A Representative images of β-gal staining three days after cardiac reprogramming with RB1 inhibition in MEFs. B Relative mRNA expression of senescence marker P16 and GLB1 seven days after cardiac reprogramming with RB1 suppression in MEFs. C Representative images of β-gal staining three days after cardiac reprogramming with RB1 inhibition in hFCFs. D Relative mRNA expression of senescence marker P16 and GLB1 seven days after cardiac reprogramming with RB1 suppression in hFCFs. Mean values + SEM of three independent experiments is shown (n = 3). Data were analyzed with two-way ANOVA. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; ****P < 0.0001 vs. control

Fig. 6

RB1 represses the expression of GATA4 and MEF2C in hFCFs. A Schematic overview how RB1 potentially influences the expression of GATA4 and MEF2C. B Relative mRNA expression of RB1 in MEFs and hFCFs during cardiac reprogramming at different time point. C Relative mRNA expression of Gata4, Mef2c, and Tbx5 after RB1 suppression in hFCFs. Mean values + SEM of three independent experiments is shown (n = 3). Data were analyzed with two-way ANOVA. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; ****P < 0.0001 vs. control