190 Effects of endocrine-disrupting chemical on calcium signalling in cardiomyocyte differentiation from mouse embryonic stem cells

Abstract

Endocrine-disrupting chemicals (EDC) are substances similar to steroid hormones that can disturb normal physiological functions of male and female reproductive organs. Endocrine-disrupting chemicals tend to bind to steroid hormone receptors. Sex steroid hormones modulate calcium signalling in the cardiac muscle in early embryo development. Among the steroid hormones, progesterone (P4) has been reported to affect blood pressure and other aspects of the cardiovascular system. The EDC that have similar structure to P4, such as octyl-phenol (OP) and bisphenol A (BPA), are potentially harmful to development of the heart. To confirm the effect of OP and BPA on early differentiation of mouse embryonic stem cells (mES) into cardiomyocytes, the hanging-drop method with mESC cell line (ES-E14TG2a) was used for forming embryoid bodies. Pluripotent mESC were cultured in basal medium with leukemia inhibitory factor and grown on mitomycin C-treated mouse embryonic fibroblasts in a 60-mm plate at 37°C in a 5% CO₂ humidified tissue culture incubator with basal medium consisting of DMEM/F-12 supplemented with nonessential amino acids, 10% heat-inactivated and certified fetal bovine serum (FBS), 2-mercaptoethanol, penicillin, and streptomycin. The mouse embryoid bodies (mEB) were suspended onto 6-well plates and cultured with differentiation medium containing steroid-free FBS without leukemia inhibitory factor. Progesterone, OP, and BPA were added on Day 2 from mEB attachment. In addition, mifepristone (RU486), an antagonist for progesterone receptor (PR), was used to confirm the effect of P4 through PR. To determine whether RU486 is capable of attenuating the inhibition effect of P4, RU486 was added for 1 day starting on Day 11. Assessment of cardiomyocyte differentiation was determined by checking the number of beating cell populations divided by the total number of attached mEB. Total RNA was extracted using Trizol reagent and synthesised to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase. Messenger RNA level was assessed using quantitative real-time PCR. To investigate calcium signalling, the mRNA levels of calcium channel gene Trpv2 and contraction-related genes Ryr2, Cam2, and Mylk3 were analysed. Beating ratio was decreased in P4, OP, and BPA treatments. Data were analysed by one-way ANOVA followed by Tukey’s multiple comparison tests, and P < 0.05 was considered statistically significant in least 3 different replicates. The mRNA level of Pgr was significantly increased in P4, OP, and BPA-treated group. However, the mRNA level of calcium channel gene Trpv2 was significantly decreased in the P4, OP, and BPA-treated group. Expression of contraction-related genes such as Ryr2, Cam2, and Mlck3 were significantly decreased in the P4, OP, and BPA-treated group. In addition, treatment with RU486 rescues altered calcium channel gene and contraction-related genes. Taken together, these results suggest that OP and BPA may affect the differentiation of mESC into cardiomyocyte and disrupts differentiation of cardiomyocytes.