title: 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.
abstract: 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 μM); 3) treatment with TSA (100, 300, 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 during coculture with CMs through a mechanism beyond the inhibition of HDAC activity.
authors.affiliation: Department of Cell and Molecular Biology, Pediatric Institute of Chongqing Medical University, Chongqing, China.
authors.forename: Ge
authors.initials: G
authors.lastname: Yang
authors.forename: Jie
authors.initials: J
authors.lastname: Tian
authors.forename: Chuan
authors.initials: C
authors.lastname: Feng
authors.forename: Li-li
authors.initials: LL
authors.lastname: Zhao
authors.forename: Zhenguo
authors.initials: Z
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001374:Azacitidine / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D002478:Cells, Cultured; D018920:Coculture Techniques; D050991:GATA4 Transcription Factor / Q000378:metabolism; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006655:Histone Deacetylases / Q000737:chemistry / Q000378:metabolism*; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins / Q000378:metabolism; D006877:Hydroxamic Acids / Q000494:pharmacology*; D064326:MEF2 Transcription Factors; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018006:Myogenic Regulatory Factors / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014157:Transcription Factors / Q000378:metabolism; D020107:Troponin T / Q000378:metabolism
chemical_list: D050991:GATA4 Transcription Factor; D056572:Histone Deacetylase Inhibitors; D000072576:Homeobox Protein Nkx-2.5; D018398:Homeodomain Proteins; D006877:Hydroxamic Acids; D064326:MEF2 Transcription Factors; C578042:MEF2C protein, rat; D018006:Myogenic Regulatory Factors; C524161:Nkx2-5 protein, rat; D014157:Transcription Factors; D020107:Troponin T; C012589:trichostatin A; D006655:Histone Deacetylases; D001374:Azacitidine