p63 silencing induces epigenetic modulation to enhance human cardiac fibroblast to cardiomyocyte-like differentiation

Abstract

Direct cell reprogramming represents a promising new myocardial regeneration strategy involving in situ transdifferentiation of cardiac fibroblasts into induced cardiomyocytes. Adult human cells are relatively resistant to reprogramming, however, likely because of epigenetic restraints on reprogramming gene activation. We hypothesized that modulation of the epigenetic regulator gene p63 could improve the efficiency of human cell cardio-differentiation. qRT-PCR analysis demonstrated significantly increased expression of a panel of cardiomyocyte marker genes in neonatal rat and adult rat and human cardiac fibroblasts treated with p63 shRNA (shp63) and the cardio-differentiation factors Hand2/Myocardin (H/M) versus treatment with Gata4, Mef2c and Tbx5 (GMT) with or without shp63 (p < 0.001). FACS analysis demonstrated that shp63+ H/M treatment of human cardiac fibroblasts significantly increased the percentage of cells expressing the cardiomyocyte marker cTnT compared to GMT treatment with or without shp63 (14.8% ± 1.4% versus 4.3% ± 1.1% and 3.1% ± 0.98%, respectively; p < 0.001). We further demonstrated that overexpression of the p63—transactivation inhibitory domain (TID) interferes with the physical interaction of p63 with the epigenetic regulator HDAC1 and that human cardiac fibroblasts treated with p63-TID+ H/M demonstrate increased cardiomyocyte marker gene expression compared to cells treated with shp63+ H/M (p < 0.05). Whereas human cardiac fibroblasts treated with GMT alone failed to contract in co-culture experiments, human cardiac fibroblasts treated with shp63+ HM or p63-TID+ H/M demonstrated calcium transients upon electrical stimulation and contractility synchronous with surrounding neonatal cardiomyocytes. These findings demonstrate that p63 silencing provides enhanced rat and human cardiac fibroblast transdifferentiation into induced cardiomyocytes compared to a standard reprogramming strategy. p63-TID overexpression may be a useful reprogramming strategy for overcoming epigenetic barriers to human fibroblast cardio-differentiation.

Two decades of heart regeneration research: Cardiomyocyte proliferation and beyond

“…Tang et al, 2022). In addition to the use of lenti-viral transduction to overexpress specific transcription factors to aid in the transdifferentiation of cardiac non-myocytes into iCMs, some have investigated the potential of utilizing short hairpin RNA to silence specific factors to enhance the generation of iCMs (Patel et al, 2018;Pinnamaneni et al, 2022). Downregulation of p63 further enhances the generation efficiency and functionality of iCMs and epigenetically regulates induced pluripotent stem cell (iPSC) reprogramming (Patel et al, 2018;Pinnamaneni et al, 2022).…”

“…In addition to the use of lenti‐viral transduction to overexpress specific transcription factors to aid in the transdifferentiation of cardiac non‐myocytes into iCMs, some have investigated the potential of utilizing short hairpin RNA to silence specific factors to enhance the generation of iCMs (Patel et al, 2018; Pinnamaneni et al, 2022). Downregulation of p63 further enhances the generation efficiency and functionality of iCMs and epigenetically regulates induced pluripotent stem cell (iPSC) reprogramming (Patel et al, 2018; Pinnamaneni et al, 2022). Others have reportedly enhanced the generation of iCMs with the addition of small‐molecule cocktails with the GMT cocktail supplemented with other transcription factors and entirely with small‐molecule cocktails alone (N. Cao, Huang, et al, 2016; Singh et al, 2020).…”

Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration

“…Further investigation revealed that epigenetic modulations played a key role as overexpression of the p63 motif transactivation inhibitory domain (TID) inhibited p63 binding with histone deacetylase 1 (HDAC1), and p63-TID+ Hand2/Myocardin resulted in better cardiac reprogramming of CFs into iCM compared to the shp63+Hand2/Myocardin combination. It was concluded that the p63-TID overexpression reprogramming strategy could have the potential to circumvent epigenetic hurdles to CF cardio-differentiation [ 65 ].…”

Nkx2.5: a crucial regulator of cardiac development, regeneration and diseases

“…During this process, genetic epigenetic factors, transcription factors, signaling pathways, and small RNAs are involved in regulating the developmental process of the heart ( 7 ). Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), myosin heavy chain (MHC), myosin light chain (MLC)-2a, MLC-2v, Nkx2.5, GATA-4, TEF-1, MEF2-A, MEF2-C, and MEF2-D are some of cardiomyocyte-selective genes that significantly aided myocardial regeneration ( 8 , 9 ). Among them, Nkx2.5 is a key transcription factor associated with human congenital heart disease and one of the earliest markers of cardiac progenitor cells, so it is a promising candidate factor for the regeneration of cardiomyocytes ( 7 ).…”