Abstract
4-phenylbutyrate (4-PBA), a terminal aromatic substituted fatty acid, is used widely to specifically attenuate endoplasmic reticulum (ER) stress and inhibit histone deacetylases (HDACs). In this study, we investigated the effect of 4-PBA on cardiac differentiation of mouse embryonic stem (ES) cells. Herein, we found that 4-PBA regulated cardiac differentiation in a stage-specific manner just like trichostatin A (TSA), a well-known HDAC inhibitor. 4-PBA and TSA favored the early-stage differentiation, but inhibited the late-stage cardiac differentiation via acetylation. Mechanistic studies suggested that HDACs exhibited a temporal expression profiling during cardiomyogenesis. Hdac1 expression underwent a decrease at the early stage, while was upregulated at the late stage of cardiac induction. During the early stage of cardiac differentiation, acetylation favored the induction of Isl1 and Nkx2.5, two transcription factors of cardiac progenitors. During the late stage, histone acetylation induced by 4-PBA or TSA interrupted the gene silence of Oct4, a key determinant of self-renewal and pluripotency. Thereby, 4-PBA and TSA at the late stage hindered the exit from pluripotency, and attenuated the expression of cardiac-specific contractile proteins. Overexpression of HDAC1 and p300 exerted different effects at the distinct stages of cardiac induction. Collectively, our study shows that timely manipulation of HDACs exhibits distinct effects on cardiac differentiation. And the context-dependent effects of HDAC inhibitors depend on cell differentiation states marked by the temporal expression of pluripotency-associated genes.
Highlights
Cardiac differentiation of pluripotent stem cells in vitro mimics cardiomyogenesis in vivo, exhibiting several developmental stages
Our study shows that inhibition of histone deacetylases (HDACs) via 4-PBA or trichostatin A (TSA) can modify epigenetic program to affect cardiac differentiation in a stage-dependent manner
Our results strongly indicated that the conversion of Oct4 gene from an accessible demethylated state to a condensed methylation one happened during the late-stage cardiac differentiation
Summary
Cardiac differentiation of pluripotent stem cells in vitro mimics cardiomyogenesis in vivo, exhibiting several developmental stages. Pharmacologically blocking or activating specific signaling pathways, are an alternative approach to achieve a satisfactory quantity and purity of pluripotent stem cells-derived cardiomyocytes. KY02111 and XAV939, small molecules inhibiting Wnt signaling, respectively promoted differentiation of induced pluripotent stem (iPS) cells and ES cells to cardiomyocytes [13,14,15]. Further investigation revealed that 4-PBA displayed similar stage-specific effects on cardiac differentiation as trichostatin A (TSA), a histone deacetylase inhibitor. At the early stage of cardiac differentiation, 4-PBA and TSA promoted histone acetylation, thereby enhanced the expression of Isl and Nkx2.5, two of the early markers for cardiac progenitors. The treatment with 4-PBA and TSA at the late stage attenuated the expression of cardiac-specific contractile proteins, decreasing the generation of spontaneously beating cardiomyocytes. Our study shows that inhibition of HDACs via 4-PBA or TSA can modify epigenetic program to affect cardiac differentiation in a stage-dependent manner
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