Deletion of activating transcription factor 3 (Atf3) promotes cardiomyocyte differentiation from embryonic stem cells.

Myocardial infarction is associated with massive cardiomyocyte loss, and embryonic stem cells (ESCs), owing to their cardiomyocyte differentiation ability, have emerged as a promising therapy. One potential factor involved in regulating ESC-to-cardiomyocyte differentiation is activating transcription factor 3 (Atf3), and this study clarifies its involvement. Atf3 was knocked out (ko) in mouse ESCs (mESCs) with CRISPR/Cas9. Both wild-type (WT) and Atf3-KO mESCs formed embryoid bodies (EBs) over 6 days, followed by adherent culture to induce cardiomyocyte differentiation from days 7-12; there, Atf3-KO had more beating EBs, at higher frequencies, from Day 8 of differentiation than WT. They also had increased expression of mesodermal markers on Days 3-6 (ex. T), cardiac progenitor markers on Days 6-9 (ex. Pdgfra), and cardiomyocyte differentiation marker on Days 9-12 (ex. cardiac troponin T), as measured by RT-qPCR and flow cytometry. Furthermore, cardiomyocyte differentiation-associated differentially-expressed genes were significantly upregulated in Atf3-KO EBs, compared to WT, under Gene Set Enrichment Analysis of RNA sequencing. All these effects in Atf3-KO mESCs were reversed upon "rescue", where Atf3 expression was restored in these cells. Therefore, Atf3-KO in mESCs promotes differentiation into mesodermal lineages, which further differentiate into cardiac progenitors, serving as a target for cell-based cardiac regeneration therapies.