Sodium valproate-induced congenital cardiac abnormalities in mice are associated with the inhibition of histone deacetylase

Abstract

Background: Valproic acid, a widely used anticonvulsant drug, is a potent teratogen resulting in various congenital abnormalities. However, the mechanisms underlying valproic acid induced teratogenesis are nor clear. Recent studies indicate that histone deacetylase is a direct target of valproic acid.

Methods: In the present study, we have used histological analysis and RT-PCR assays to examine the cardiac abnormalities in mice treated with sodium valproate (NaVP) and determined the effects of NaVP on histone deacetylase activity and the expression of heart development-related genes in mouse myocardial cells.

Results: The experimental data show that NaVP can induce cardiac abnormalities in fetal mice in a dose-dependent manner. NaVP causes a dose-dependent inhibition of hitone deacetylase (HDAC) activity in mouse myocardial cells. However, the expression levels of HDAC (both HDAC1 and HDAC2) are not significantly changed in fetal mouse hearts after administration of NaVP in pregnant mice. The transcriptional levels of other heart development-related genes, such as CHF1, Tbx5 and MEF2, are significantly increased in fetal mouse hearts treated with NaVP.

Conclusions: The study indicates that administration of NaVP in pregnant mice can result in various cardiac abnormalities in fetal hearts, which is associated with an inhibition of histone deacetylase without altering the transcription of this enzyme.

Figure 1

Dose-dependent cardiac abnormalities in fetal mice prenatally exposed to NaVP. Cardiac abnormality rate increased significantly in fetal mice (gestation day 19) after prenatal exposure to NaVP (pregnant female mice received a one-time injection of NaVP on gestation day 7 at various doses). The cardiac abnormality caused by NaVP shows a dose-dependent pattern with the highest rate in the group of a treatment dose at 700 mg/kg body weight. * P < 0.05 vs. control without sodium valproate treatment.

Figure 2

Histological examination of cardiac sections from fetal hearts with or without exposure to NaVP. Cardiac sections were examined from the fetal mice (gestation day 19) with or without prenatal exposure to NaVP (exposure group, the pregnant female mice received a one-time injection intraperitoneally of NaVP on gestation day 7 at a dose of 700 mg/kg body weight; control group, the pregnant female mice received a one-time injection of saline solution at the same time). (A) A representative cross section image of normal fetal hearts from the control groups. (B) A representative image of the fetal hearts from the NaVP treated group showing an interventricular septal interruption indicated by an arrow. RV, right ventricle; LV, left ventricle; IVS, interventricular septum.

Figure 3

Inhibition of NACD activities by NaVP in cardiac myocytes. HDAC activities were measured as described in Materials and Methods and HDAC activities were presented as absorbance fluorescent unit (AFU). Values are Mean ± SEM from 3 separated experiments. * P < 0.05 vs. control without sodium valproate treatment.

Figure 4

Effect of NaVP on transcriptional regulation of various cardiac development-related genes. Gene expression levels were analyzed using RT-PCR techniques as described in Materials and Methods. (A) A representative RT-PCR analysis of HDAC1, HDAC2, MEF2C, CHF1, GATA4, Nkx2.5 CHF1, Tbx5 and MEF2C mRNA expression in E16 fetal hearts with NaVP treatment on E7. Saline treated mice hearts served as controls. β-actin was used as an internal control. (B) Summary of real-time PCR data from 3 separate experiments. The values from each sample were normalized to that of β-actin mRNA level. Values are presented as means ± SEM of triplicate experiments. * P < 0.05 vs. controls.