392 EFFECTS OF TRICHOSTATIN A, AN EPIGENETIC MODIFIER AGENT, ON DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO STRIATED MUSCLE CELLS

Abstract

In vitro generation of cardiomyocytes from embryonic stem cells (ES cells) is a promising approach to develop strategies for treatment of cardiac diseases. Epigenetic changes occur during ES cells differentiation, and by the first 5 days, the histone acetylation levels increase, promoting an improvement in gene expression. Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor and promotes histone hyperacetylation. In this study, we analyzed the effects of TSA treatment in ES cells differentiation into striated muscle cells. For that, murine ES cell line H106 was grown in hanging drops of 20 μL containing 2000 cells in DMEM medium supplemented with 15% FCS, 10 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 m L-glutamine, 10 mM nonessential amino acids, and 83.4 μg mL^-1 amikacin. After 5 days, embryoid bodies were transferred individually to a 96-well plate treated with 0.1% swine gelatin. Trichostatin A treatment was performed during hanging drop culture (group 15 nM d0-5), at Day 5 for 24 h after transfer to adherent culture (groups 50 nM d5 and 100 nM d5), and at Day 13 for 24 h (groups 50 nM d13 and 100 nM d13). Area of embryoid bodies and apoptosis rate from control and 15 nM d0-5 groups were analyzed at Day 5. Analysis of contractile structures was carried out at Day 14. Imunnocitochemistry reactions for desmin and troponin I were performed at Day 7 and 17, respectively. Results of apoptosis, desmin, and troponin I cell rates (positive cells/total cells) were analyzed by chi-square test, with a significance level of 5%, on MINITAB Release 14.1. Areas of embryoid bodies were submitted to one-way ANOVA and Tukey’s post-test, with a significance level of 5%, using GraphPad software. Embryoid bodies developed on TSA supplemented medium presented smaller areas (15 nM d0-5: 6.75 ± 0.93 mm²; control: 15.84 ± 1.64 mm²) and greater apoptosis rates (15 nM d0-5: 29.53%; control: 20.18%). Contractile structures were greater on 50 nM d5 (90%^c) and extremely less on the 15 nM d0-5 group (3.12%^b). Groups 100 nM d5 (66.6%), 50 nM d13 (70.93%), and 100 nM d13 (80.7%^a,c) were similar to the control group (68.25%^a). Rate of desmin positive cells was greater on the 50 nM d5 group (31.53^b) and less on the 100 nM d5 group (22.9^c). The 15 nM d0-5 group (26.03^a) was similar to control (25.25^a). Rate of troponin I positive cells was greater on 50 nM d5 (8.65^b) and 100 nM d13 (9.69^b) and less on the 100 nM d5 group (2.63^c). On the 15 nM d0-5 group, no positive cells were observed, and the 50 nM d13 group (6.67^a) was similar to control (6.44^a). In conclusion, the current study demonstrated that TSA improves striated muscle differentiation when supplemented at lesser concentrations at Day 5 (50 nM) and greater concentrations at Day 13 (100 nM) and promotes detrimental effects when used during embryoid body development, decreasing the area of structures and increasing apoptosis rate.

Acknowledgments are given to FAPESP 2007/55968-9 and 2008/58370-0.