Antenatal Hypoxia and Programming of Glucocorticoid Receptor Expression in the Adult Rat Heart

Abstract

Glucocorticoid receptor (GR) signaling is critical for development and function of the heart. Our previous study demonstrated that gestational hypoxia induced epigenetic repression of the GR gene in the developing heart. The present study aims to determine that the alterations of promoter methylation level and epigenetic repression of the GR gene in the developing heart in response to maternal hypoxia is sustained in adult offspring and potential gender differences in the programming of GR gene. Pregnant rats were treated with 10.5% O₂ from gestational day 15 (E15) to 21 (E21). Hearts were isolated from 5-month-old male and female offspring with the developing stage being equivalent to 18-year-old human. GR mRNA and protein abundance was determined with real time qRT-PCR and Western blot. GR gene promoter methylation and binding of transcription factors were measured with methylated DNA immunoprecipitation (MeDIP) and Chromatin immunoprecipitation (ChIP). The results showed that antenatal hypoxia significantly decreased the expression of GR mRNA and protein in the hearts of adult male offspring, but not in females, which is ascribed to the differential changes of alternative exon1 mRNA variants of GR gene in male and female hearts in response to prenatal hypoxia. In addition, the downregulation of GR expression in the male heart was correlated with increased methylation levels of CpG dinucleotides in promoters of exon 1₄, 1₅, 1₆, 1₇, and 1₁₀, which resulted in a decrease in the binding of their transcription factors. Thus, the study reveals that antenatal hypoxia results in a reprogramming and long-term change in GR gene expression in the heart by hypermethylation of GR promoter in a sex-differential pattern, which provides a novel mechanism regarding the increased vulnerability of heart later in life with exposure of prenatal hypoxia.

Keywords: DNA methylation; glucocorticoid receptor; heart; hypoxia; programming; sex.

FIGURE 1

Maternal hypoxia decreased the expression of GR protein and mRNA. Left ventricle of hearts was isolated from 5-month-old male and female offspring exposed to normoxia or hypoxia from E15 to E21. (A) GR mRNA abundance was determined by quantitative real-time RT-PCR. (B) GR protein abundance was determined using Western Blot analysis. Data are mean ± SEM, n = 4–5 (^∗p < 0.05, hypoxia vs. control).

FIGURE 2

Effects of prenatal hypoxia on the expression of GR exon 1 variants in the hearts of adult male and female offspring. Up panel, diagrammatic representation of GR gene structure. The mRNA abundance of GR exon 1 variants of the 5-month-old male was determined by quantitative real-time RT-PCR. Data are mean ± SEM, n = 5 (^∗p < 0.05, hypoxia vs. control).

FIGURE 3

DNA methylation levels of GR exon 1 promoters in the hearts of male adult offspring. Methylation of promoter region of GR exon 1₄, 1₅, 1₆, 1₇, and 1₁₀ in the heart was determined by MeDIP. F, forward primer, R, reverse primer. Data are mean ± SEM, n = 5 (^∗p < 0.05, hypoxia vs. control).

FIGURE 4

Effects of prenatal hypoxia on the binding of CREB and Sp1 to GR exon 1 promoters in the hearts of male adult offspring. Up panel, diagrammatic representation of the putative transcription factor binding sites at GR promoter and the relative location of primer set. Binding of CREB and Sp1 to GR promoter was determined by ChIP assay. Data are mean ± SEM, n = 5 (^∗p < 0.05, hypoxia vs. control).