Department of Pediatrics, West China Second Hospital of Sichuan University/Cardiac Development and Early Intervention Unit, West China Second Hospital of Sichuan University/Key Laboratory of Birth Defects and Related Diseases of Women and Children, Ministry of Education, Chengdu 610041, China
Abstract:Objective To investigate the effect of histone acetylation/deacetylation imbalances on embryonic hearts of mice and its effect on key genes of planar cell polarity (PCP) pathway-Vangl2, Scrib and Rac1 in H9C2 cells. Methods Forty pregnant C57/B6 mice were randomly assigned into three groups:blank group (n=10), vehicle group (n=10), and valproic acid (VPA)-treated group (n=20). In the VPA-treated group, VPA, a histone deacetylase (HDAC) inhibitor, was administered to each individual dam intraperitoneally at a single dose of 700 mg/kg on embryonic day 10.5 (E10.5). The vehicle and blank groups received equivalent saline or no interventions, respectively. Dams were sacrificed on E15.5, and death rates of embryos were evaluated. Subsequently, embryonic hearts of survival fetus were removed to observe cardiac abnormalities by hematoxylin-eosin (HE) staining. H9C2 cells were cultured and allotted to the blank, vehicle, and VPA-treated groups:the VPA treated group received VPA exposure at concentrations of 2.0, 4.0 and 8.0 mmol/L; the vehicle and blank groups received equivalent saline or no interventions, respectively. HDAC1-3 as well as Vangl2, Scrib and Rac1 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blot, respectively. The total HDAC activity was analyzed by colorimetric assay. Results The fetus mortality rate after VPA treatment was 31.7%, with a significantly higher rate of cardiac abnormalities in comparison with the controls (P < 0.05). In comparison with the blank and vehicle groups, HDAC1 mRNA was significantly increased at various concentrations of VPA treatment at all time points of exposure (P < 0.05), together with a reduction of protein level after 48 and 72 hours of exposure (P < 0.05). The inhibition of HDAC2 mRNA after various concentrations of VPA incubation was pronounced at 24 hours of exposure (P < 0.05), while the protein levels were reduced at all time points (P < 0.05). HDAC3 mRNA was prominently induced by VPA (4.0 and 8.0 mmol/L) at all time points of treatment (P < 0.05). In contrast, the protein level was inhibited after VPA treatment (P < 0.05). In comparison with the blank and vehicle groups, Vangl2 mRNA as well as Scrib mRNA/protein expression levels were markedly reduced after 48 and 72 hours of VPA treatment (P < 0.05), together with a reduction of protein level in Vangl2 at 72 hours (P < 0.05). Compared with the blank and vehicle groups, a significant repression in the total HDAC activity was observed in the VPA-treated group at concentrations of 4.0 and 8.0 mmol/L after 24 hours of treatment (P < 0.05), and the effect persisted up to 48 and 72 hours, exhibiting pronounced inhibition at all concentrations (P < 0.05). Conclusions VPA might result in acetylation/deacetylation imbalances by inhibiting HDAC1-3 protein expression and total HDAC activity, leading to the downregulation of mRNA and protein expression of Vangl2 and Scrib. This could be one of the mechanisms contributing to congenital heart disease.
Davis EE, Katsanis N. Cell polarization defects in early heart development[J]. Circ Res, 2007, 101(2):122-124.
[2]
Gallinari P, Di Marco S, Jones P, et al. HDACs, histone deacetylation and gene transcription:from molecular biology to cancer therapeutics[J]. Cell Res, 2007, 17(3):195-211.
Wu G, Nan C, Rollo JC, et al. Sodium valproate-induced congenital cardiac abnormalities in mice are associated with the inhibition of histone deacetylase[J]. J Biomed Sci, 2010, 17:16.
[5]
Kwiecińska P, Wróbel A, Taubøll E, et al. Valproic acid, but not levetiracetam, selectively decreases HDAC7 and HDAC2 expression in human ovarian cancer cells[J]. Toxicol Lett, 2014, 224(2):225-232.
[6]
Barbetti V, Gozzini A, Cheloni G, et al. Time-and residuespecific differences in histone acetylation induced by VPA and SAHA in AML1/ETO-positive leukemia cells[J]. Epigenetics, 2013, 8(2):210-219.
[7]
Fedier A, Dedes KJ, Imesch P, et al. The histone deacetylase inhibitors suberoylanilide hydroxamic (Vorinostat) and valproic acid induce irreversible and MDR1-independent resistance in human colon cancer cells[J]. Int J Oncol, 2007, 31(3):633-641.
[8]
Ramsbottom SA, Sharma V, Rhee HJ, et al. Vangl2-regulated polarisation of second heart field-derived cells is required for outflow tract lengthening during cardiac development[J]. PLoS Genet, 2014, 10(12):e1004871.
[9]
Phillips HM, Rhee HJ, Murdoch JN, et al. Disruption of planar cell polarity signaling results in congenital heart defects and cardiomyopathy attributable to early cardiomyocyte disorganization[J]. Circ Res, 2007, 101(2):137-145.
[10]
Leung C, Lu X, Liu M, et al. Rac1 signaling is critical to cardiomyocyte polarity and embryonic heart development[J]. J Am Heart Assoc, 2014, 3(5):e001271.
[11]
Sadowski SL. Congenital cardiac disease in the newborn infant:past, present, and future[J]. Crit Care Nurs Clin North Am, 2009, 21(1):37-48.
[12]
Huhta J, Linask KK. Environmental origins of congenital heart disease:The heart-placenta connection[J]. Semin Fetal Neonatal Med, 2013, 18(5):245-250.
[13]
Abend A, Kehat I. Histone deacetylases as therapeutic targets-from cancer to cardiac disease[J]. Pharmacol Ther, 2015, 147:55-62.
[14]
Eom GH, Kook H. Posttranslational modifications of histone deacetylases:implications for cardiovascular diseases[J]. Pharmacol Ther, 2014, 143(2):168-180.
[15]
Kim YS, Kim MJ, Koo TH, et al. Histone deacetylase is required for the activation of Wnt/β-catenin signaling crucial for heart valve formation in zebrafish embryos[J]. Biochem Biophys Res Commun, 2012, 423(1):140-146.
[16]
Reller MD, Strickland MJ, Riehle-Colarusso T, et al. Prevalence of congenital heart defects in metropolitan Atlanta, 1998-2005[J]. J Pediatr, 2008, 153(6):807-813.
[17]
Peterson GM, Naunton M. Valproate:a simple chemical with so much to offer[J]. J Clin Pharm Ther, 2005, 30(5):417-421.
[18]
Jentink J, Dolk H, Loane MA, et al. Intrauterine exposure to carbamazepine and specific congenital malformations:systematic review and case-control study[J]. BMJ, 2010, 341:c6581.
[19]
Bradbury CA, Khanim FL, Hayden R, et al. Histone deacetylases in acute myeloid leukaemia show a distinctive pattern of expression that changes selectively in response to deacetylase inhibitors[J]. Leukemia, 2005, 19(10):1751-1759.
[20]
Krämer OH, Zhu P, Ostendorff HP, et al. The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2[J]. EMBO J, 2003, 22(13):3411-3420.
[21]
Thapar R, Denmon AP. Signaling pathways that control mRNA turnover[J]. Cell Signal, 2013, 25(8):1699-1710.
[22]
Wakabayashi-Nakao K, Tamura A, Furukawa T, et al. Quality control of human ABCG2 protein in the endoplasmic reticulum:ubiquitination and proteasomal degradation[J]. Adv Drug Deliv Rev, 2009, 61(1):66-72.
[23]
Koraichi F, Videmann B, Mazallon M, et al. Zearalenone exposure modulates the expression of ABC transporters and nuclear receptors in pregnant rats and fetal liver[J]. Toxicol Lett, 2012, 211(3):246-256.
[24]
Chen G, Gharib TG, Huang CC, et al. Discordant protein and mRNA expression in lung adenocarcinomas[J]. Mol Cell Proteomics, 2002, 1(4):304-313.
[25]
Tian Q, Stepaniants SB, Mao M, et al. Integrated genomic and proteomic analyses of gene expression in Mammalian cells[J]. Mol Cell Proteomics, 2004, 3(10):960-969.
[26]
Zupkovitz G, Tischler J, Posch M, et al. Negative and positive regulation of gene expression by mouse histone deacetylase 1[J]. Mol Cell Biol, 2006, 26(21):7913-7928.
[27]
Cao Y, Lu L, Liu M, et al. Impact of epigenetics in the management of cardiovascular disease:a review[J]. Eur Rev Med Pharmacol Sci, 2014, 18(20):3097-3104.