Abstract
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.
Highlights
Over the years, many techniques leading to cardiomyocyte differentiation and isolation have been established
HDAC1 dn mouse embryonic stem cells (mESCs) started to beat on the 10th day after differentiation, and when these cells began to beat in this early interval, the period of beating was 2.5 days (Figure 1C)
Epigenetically modified using epi-drugs, which could be an important observation from the viewpoint of transplantation strategies. It is well-known that the differentiation of ESCs into cardiomyocytes is regulated by the specific subset of genes that are regulated by epigenetic factors, including histone acetyltransferases (HATs) and histone deacetylases (HDACs) [30,31]
Summary
Many techniques leading to cardiomyocyte differentiation and isolation have been established. H9c2-derived cells from embryonic rat heart [1], embryonal avian heart [2], transgenic mice with myocardial tumors [3], or neonatal rat myocardial cells transfected with SV-40 large T antigen [4] have been tested In these cases, the established cell lines were not effective in studies of physiological processes leading to functional cardiomyocytes. The main reason for this result was that these cell lines were able to grow in culture for only a few passages because optimal cultivation conditions, including the composition of the cell culture media, for their growth in vitro had not been fully established [5] From this view, Wobus et al [6] studied the effect of retinoic acid on cardiomyocyte differentiation; these authors analyzed cell induction into ventricle-like cardiomyocytes. The authors induced cardiomyocytes with Purkinje- and ventricle-like markers while a reduced number of pacemaker- and atrium-like cells were observed
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