Valproic acid (VPA) exposure during pregnancy has been proven to contribute to congenital heart disease (CHD). Our previous findings implied that disruption of planar cell polarity (PCP) signaling pathway in cardiomyocytes might be a factor for the cardiac teratogenesis of VPA. In addition, the teratogenic ability of VPA is positively correlated to its histone deacetylase (HDAC) inhibition activity. This study aimed to investigate the effect of the VPA on cardiac morphogenesis, HDAC1/2/3, and PCP key genes (Vangl2/Scrib/Rac1), subsequently screening out the specific HDACs regulating PCP pathway.
VPA was administered to pregnant C57BL mice at 700 mg/kg intraperitoneally on embryonic day 10.5. Dams were sacrificed on E15.5, and death/absorption rates of embryos were evaluated. Embryonic hearts were observed by hematoxylin-eosin staining to identify cardiac abnormalities. H9C2 cells (undifferentiated rat cardiomyoblasts) were transfected with Hdac1/2/3 specific small interfering RNA (siRNA). Based on the results of siRNA transfection, cells were transfected with Hdac3 expression plasmid and subsequently mock-treated or treated with 8.0 mmol/L VPA. Hdac1/2/3 as well as Vangl2/Scrib/Rac1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Total HDAC activity was detected by colorimetric assay.
VPA could induce CHD ( P < 0.001) and inhibit mRNA or protein expression of Hdac1/2/3 as well as Vangl2/Scrib in fetal hearts, in association with total Hdac activity repression (all P < 0.05). In vitro, Hdac3 inhibition could significantly decrease Vangl2/Scrib expression ( P < 0.01), while knockdown of Hdac1/2 had no influence ( P > 0.05); VPA exposure dramatically decreased the expression of Vanlg2/Scrib together with Hdac activity ( P < 0.01), while overexpression of Hdac3 could rescue the VPA-induced inhibition ( P > 0.05).
妊娠期丙戊酸(VPA)暴露被证实与先天性心脏病(CHD)发生有关。本团队前期实验研究发现,干扰心肌细胞平 面细胞极性(PCP)信号通路是VPA心脏致畸性的可能因素。此外,相关研究证实VPA的致畸作用与其对组蛋白去乙酰化酶 (HDAC)抑制作用成正相关。本研究旨在观察VPA对心脏形态发生、HDAC1/2/3及PCP通路关键基因(Vangl2/Scrib/Rac1) 的影响,进而筛选出参与PCP通路调控的特异性HDAC类型。
以VPA 700mg/kg于妊娠第10.5天(E10.5)腹腔注射C57BL孕鼠。于E15.5处死孕鼠,统计胚胎死亡/流产率。采 集心脏标本以苏木精-伊红染色观察心脏畸形情况。以Hdac1/2/3特异性siRNA转染H9C2细胞(大鼠未分化心肌细 胞)。根据转染siRNA相关结果,以Hdac3表达质粒转染细胞,随后以假处理或VPA 8.0mmol/L干预细胞。分别采 用实时定量PCR及Western-blot检测Hdac1/2/3及Vangl2/Scrib/Rac1 mRNA及蛋白表达水平。利用比色法测定总 HDAC 活性。