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It has become evident that epitranscriptome events, mediated by specific enzymes, regulate gene expression and, subsequently, cell differentiation processes. We show that methyltransferase-like proteins METTL3/METTL14 and N6-adenosine methylation (m6A) in RNAs are homogeneously distributed in embryonic hearts, and histone deacetylase (HDAC) inhibitors valproic acid and Trichostatin A (TSA) up-regulate METTL3/METTL14 proteins. The levels of METTL3 in mouse adult hearts, isolated from male and female animals, were lower in the aorta and pulmonary trunks when compared with atria, but METT14 was up-regulated in the aorta and pulmonary trunk, in comparison with ventriculi. Aging caused METTL3 down-regulation in aorta and atria in male animals. Western blot analysis in differentiated mouse embryonic stem cells (mESCs), containing 10–30 percent of cardiomyocytes, showed METTL3/METTL14 down-regulation, while the differentiation-induced increased level of METTL16 was observed in both wild type (wt) and HDAC1 depleted (dn) cells. In parallel, experimental differentiation in especially HDAC1 wild type cells was accompanied by depletion of m6A in RNA. Immunofluorescence analysis of individual cells revealed the highest density of METTL3/METTL14 in α-actinin positive cardiomyocytes when compared with the other cells in the culture undergoing differentiation. In both wt and HDAC1 dn cells, the amount of METTL16 was also up-regulated in cardiomyocytes when compared to co-cultivated cells. Together, we showed that distinct anatomical regions of the mouse adult hearts are characterized by different levels of METTL3 and METTL14 proteins, which are changed during aging. Experimental cell differentiation was also accompanied by changes in METTL-like proteins and m6A in RNA; in particular, levels and distribution patterns of METTL3/METTL14 proteins were different from the same parameters studied in the case of the METTL16 protein. [ABSTRACT FROM AUTHOR]

Cell differentiation into cardiomyocytes requires activation of differentiation-specific genes and epigenetic factors that contribute to these physiological processes. This study is focused on the in vitro differentiation of mouse embryonic stem cells (mESCs) induced into cardiomyocytes. The effects of clinically promising inhibitors of histone deacetylases (HDACi) on mESC cardiomyogenesis and on explanted embryonic hearts were also analyzed. HDAC1 depletion caused early beating of cardiomyocytes compared with those of the wild-type (wt) counterpart. Moreover, the adherence of embryonic bodies (EBs) was reduced in HDAC1 double knockout (dn) mESCs. The most important finding was differentiation-specific H4 deacetylation observed during cardiomyocyte differentiation of wt mESCs, while H4 deacetylation was weakened in HDAC1-depleted cells induced to the cardiac pathway. Analysis of the effect of HDACi showed that Trichostatin A (TSA) is a strong hyperacetylating agent, especially in wt mESCs, but only SAHA reduced the size of the beating areas in EBs that originated from HDAC1 dn mESCs. Additionally, explanted embryonic hearts (e15) responded to treatment with HDACi: all of the tested HDACi (TSA, SAHA, VPA) increased the levels of H3K9ac, H4ac, H4K20ac, and pan-acetylated lysines in embryonic hearts. This observation shows that explanted tissue can be maintained in a hyperacetylation state several hours after excision, which appears to be useful information from the view of transplantation strategy and the maintenance of gene upregulation via acetylation in tissue intended for transplantation. [ABSTRACT FROM AUTHOR]