Trichostatin A Promotes Cardiomyocyte Differentiation of Rat Mesenchymal Stem Cells After 5-Azacytidine Induction or During Coculture With Neonatal Cardiomyocytes Via a Mechanism Independent of Histone Deacetylase Inhibition

This study was to investigate the effect of trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, on cardiac differentiation of bone marrow mesenchymal stem cells (MSCs) in vitro. Rat MSCs were isolated and divided into six groups: 1) control; 2) 5-azacytidine treatment (5-aza, 10 mu M); 3) treatment with TSA (100, 3(10, and 500 nM); 4) treatment with 5-aza followed by incubation with TSA; 5) coculture with neonatal cardiomyocytes (CMs); and 6) treatment with TSA then coculture with CMs. HDAC activity was significantly inhibited in TSA-treated cells with the maximal inhibition after 24 h of exposure to TSA at 300 nM. No changes in HDAC activity were observed in control, 5-aza-treated, or coculture groups. Following 7 days of differentiation, the expression of early cardiac transcription factors GATA-4, NKx2.5, MEF2c, and cardiac troponin T (cTnT) was increased by 6-8 times in the cells in 5-aza-treated, coculture, or TSA-treated groups over control as determined using real-time PCR, immunofluorescence staining, and Western blotting. However, the percent cTnT-positive cells were dramatically different with 0.7% for control, 10% for 5-aza-treated, 25% for coculture, and 4% for TSA-treated group (500 nM). TSA treatment of the cells pretreated with 5-aza or cocultured with CMs dramatically increased the expression of GATA-4, NKx2.5, and MEF2c by 35-50 times over control. The cTnT protein expression was also significantly increased by over threefold by TSA treatment (500 nM) in both 5-aza-treated and coculture group over control. The percent cTnT-positive cells in both 5-aza-pre-treated and coculture groups were significantly increased by TSA treatment after 1 week of differentiation by up to 92.6% (from 10.3% to 19.8%) and 23.9% (from 24.5% to 30.2%), respectively. These data suggested that TSA enhanced the cardiac differentiation of MSCs after 5-aza induction or (luring coculture with CMs through a mechanism beyond the inhibition of HDAC activity.