Supplementary Material for: Decreased Amino Acid Concentrations are Involved in Congenital Heart Disease

Objective: Congenital heart disease (CHD) is the most common malformation in China. In this study, we determined whether amino acids (AAs) in the amniotic fluid (AF) of patients with CHD changed and clarified whether AAs would affect the insulin-like growth factor type 1 receptor (IGF1R). Method: Fifty-seven AF samples from pregnant women carrying CHD-affected (n = 17) or normal (n = 40) fetuses were collected. The AA concentrations were measured in AF by liquid chromatography-tandem mass spectrometry. The IGF axis-related and epigenetic marker proteins in AF after serial treatments were quantified using a multiple reaction monitoring approach. IGF1R and P300 were also confirmed by Western blot in AF without any treatment. Results: Most AAs decreased in the AF of patients with CHD. P300 and IGF1R decreased significantly in the CHD group. When H9C2 cells were cultured in one-half AA concentrations, the expression of P300 and IGF1R was reduced. Histone acetylation of the IGF1R promoter also decreased. Conclusion: Our data suggest that AAs decreased in the AF of patients with CHD. AAs may partly regulate the IGF1R through P300, which may be involved in heart development.研究目的：先天性心脏病（Congenital heart disease, CHD）是我国最常见的先天畸形。本研究旨在明确怀有先天性心脏病胎儿的孕妇的羊水（amniotic fluid, AF）中氨基酸（amino acids, AAs）是否发生变化，并阐明氨基酸是否会影响1型胰岛素样生长因子受体（insulin-like growth factor type 1 receptor, IGF1R）。 研究方法：本研究共收集57份羊水样本，其中17份来自怀有先天性心脏病胎儿的孕妇（n=17），40份来自怀有正常胎儿的孕妇（n=40）。采用液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry）检测羊水中的氨基酸浓度。通过多反应监测（multiple reaction monitoring）技术定量分析经系列处理后羊水中的胰岛素样生长因子轴相关蛋白及表观遗传标记物。同时，采用蛋白质印迹法（Western blot）对未经任何处理的羊水中的IGF1R与P300蛋白进行验证。 研究结果：先天性心脏病组孕妇的羊水中多数氨基酸水平呈下降趋势。该组羊水中的P300与IGF1R蛋白表达量显著降低。当用浓度为常规一半的氨基酸培养液培养H9C2细胞时，P300与IGF1R的表达水平均出现下调，IGF1R启动子的组蛋白乙酰化水平也随之降低。 研究结论：本研究数据表明，先天性心脏病胎儿孕妇的羊水氨基酸水平降低。氨基酸可能通过P300对IGF1R实现部分调控，这一过程可能参与心脏发育进程。