Prenatal di-(2-ethylhexyl) phthalate exposure induced myocardial cytotoxicity via the regulation of the NRG1-dependent ErbB2/ErbB4-PI3K/AKT signaling pathway in fetal mice

Abstract

Environmental sanitation of maternal contact during pregnancy is extremely important for the development of different fetal tissues and organs. In particular, during early pregnancy, any adverse exposure may cause abnormal fetal growth or inhibit the development of embryogenic organs. The potential risks of phthalate exposure, which affects the development of humans and animals, are becoming a serious concern worldwide. However, the specific molecular mechanism of di-(2-ethylhexyl) phthalate (DEHP)-induced cardiotoxicity in fetal mice remains unclear. In this study, animal models of DEHP gavage at concentrations of 250, 500, and 1000 mg/kg/day within 8.5–18.5 days of pregnancy were established. The cell proliferation, survival, and apoptosis rates were evaluated using CCK8, EdU, TUNEL and flow cytometry. The molecular mechanism was assessed via transcriptome sequencing, immunohistochemistry, immunofluorescence, reverse transcription-quantitative polymerase chain reaction, and Western blot analysis. In vivo, DEHP increased apoptosis, decreased Ki67 and CD31 expression, reduced heart weight and area, slowed down myocardial sarcomere development, and caused cardiac septal defect in fetal mice heart. Transcriptome sequencing showed that DEHP decreased NRG1 expression and downregulated the ErbB2/ErbB4-PI3K/AKT signaling pathway-related target genes. In vitro, primary cardiomyocytes were cultured with DEHP at a concentration of 150 μg/mL combined with ErbB inhibitor (AG1478, 10 μmol/L) and/or NRG1 protein (100 ng/mL) for 72 h. After DEHP intervention, the expression of NRG1 and the phosphorylation level of ErbB2, ErbB4, PI3K, and AKT decreased, and the apoptosis-related protein levels increased. Moreover, the apoptosis rate increased. After adding exogenous NRG1, the phosphorylation level of the NRG1/ERbB2/ERbB4-PI3K/AKT pathway increased, and the apoptosis-related protein levels decreased. Further, the apoptosis rate reduced. Interestingly, after exposure to DEHP and AG1478 + NRG1, the anti-apoptotic effect of NRG1 and cardiomyocyte proliferation decreased by inhibiting the NRG1/ERbB2/ERbB4-PI3K/AKT pathway. Hence, the NRG1-dependent regulation of the ERbB2/ERbB4-PI3K/AKT signaling pathway may be a key mechanism of DEHP-induced myocardial cytotoxicity.

Keywords

• DEHP
• Endocrine disruptors
• NRG1
• Myocardial cytotoxicity