Mzf1 binding on Nkx2.5 cardiac enhancer – potential modulatory role of Mzf1 in heart development

Objectives: Pluripotent stem cells (PSC) are the most promising cell source for cardiac regenerative therapy. The understanding of the underlying mechanism for cardiac differentiation of PSC is crucial for efficient cardiac regeneration. Therefore, we investigated a panel of transcription factors (TF) for their potential role during differentiation of PSC into cardiac progenitor cells.

Methods: The activity of nine different TFs on an Nkx2.5 cardiac enhancer (CE) was analyzed by luciferase assays. For protein-DNA interaction EMSA and ChIP-assays were performed. The influence of overexpression of candidate TFs was investigated using an ES cell line carrying the Nkx 2.5 CE-GFP transgene and the Dox-inducible TF-cDNA. Differentiation assays were performed by standard “hanging drop” protocols with and without Dox. The amount of GFP-positive cardiac progenitor cells was analyzed by FACS and gene expression was confirmed by qRT-PCR. Furthermore, gene expression of selected TFs was compared in GFP-positive and negative in vitro differentiated Nkx2.5 CE GFP ES cells and the analogous populations in embryos of the corresponding transgenic mouse line.

Results: Luciferase assays identified Mesp1, Pax3 and Mzf1 as potential activators of the Nkx2.5 CE in HEK 293 and H9c2 cells. Binding of Mzf1 protein to the Nkx2.5 CE was confirmed in vitro by an EMSA and in vivo by a ChIP assay in murine ES cells differentiated for 5 – 6 days.

FACS sorting of GFP-positive cardiac progenitor cells after differentiation of Nkx2.5 CE-GFP ES cells on day 5, 7, and 9 revealed an up to 20-fold enhancement of Mzf1 expression in the GFP positive cardiac progenitor cell population. Similarly, FACS sorting of GFP-positive cells of mouse E9/10 embryos of an Nkx2.5 CE-GFP transgenic mouse line also yielded a more than 20-fold overexpression of Mzf1 in this population.

Mzf1 overexpression until day 5 of in vitro differentiation revealed a clear inhibitory effect on differentiation into cardiac progenitor cells. This was substantiated by the decrease of GFP-positive cells after FACS sorting and a modulation of gene expression of cardiac and hematopoietic markers. Overexpression of Mzf1 starting at day 5 of differentiation leads to an increase in GFP-positive cardiac progenitor cells.

Conclusions: The understanding of cardiogenesis is crucial for the development of cardiac regenerative strategies. Here, we identified Mzf1 as a new modulatory TF with potential influence on mesodermal development.