ABC | Volume 112, Nº1, January 2019

Original Article Jevjdovic et al Prenatal stress affects rat heart ADRB1 Arq Bras Cardiol. 2019; 112(1):67-75 Figure 1 – Maternal plasma ACTH concentrations before (GD13) and following (GD21) exposure to CUMS during pregnancy. Data are expressed as mean ± SD, control group (open bars, n =5), stressed group (black bars, n = 6). In the stressed group on GD21 two samples were excluded due to hemolysis. ***p < 0.001, 2-way ANOVA and Bonferroni’s multiple comparison test 400 300 200 100 0 *** *** Control Stressed GD13 GD21 Maternal plasma ACTH concentrations (pg/ml) Table 2 – TaqMan expression assays Gene TaqMan assay ID Beta 1 adrenergic receptor ( Adrb1 ) Rn00824536_s1 Beta 2 adrenergic receptor ( Adrb2 ) Rn00560650_s1 Beta 3 adrenergic receptor ( Adrb3 ) Rn01478698_g1 Monoamine oxidase A ( Maoa ) Rn01430955_A1 Beta-actin ( Actb ) Rn01412977_g1 duplicate, using 1x TaqMan Master Mix (Applied Biosystems) and 1x TaqMan expression assays for each gene (Table 2: Adrb1, Adrb2, Adrb3, MaoA, ActB), with 2 µg of cDNA template in a total volume of 20 µl. Real-time PCR reactions were performed on an Applied Biosystem 7900 Real-Time PCR System with standard PCR conditions (50°C for 2 min; 95°C for 10 min; 95°C for 15 s, and 60°C for 1 min for 40 cycles). The relative gene expression levels were determined by comparative 2ˆ(- ∆∆ C T ) quantification method 22 using beta-actin as the reference gene. Statistical analysis Statistical analysis was performed using GraphPad Prism Software-version 6.01 (San Diego, USA). Parameters measured in mothers and offspring were expressed as means ± standard deviation (SD). Data were analyzed by unpaired Student’s t-test, unless otherwise indicated. Offspring data obtained by real-time PCR analysis were expressed as median with interquartile range. Two-way ANOVA analysis with Bonferroni’s multiple comparison test was used to examine the effect of prenatal stress and pregnancy on maternal serum ACTH levels as well as the effects of prenatal stress on ADRB genes expression patterns in examined regions of offspring’s left ventricle. The statistical significance of differences among the real-time PCR data obtained from experimental groups was evaluated by nonparametric Mann-Whitney U-test. The level of significance was set to p < 0.05. Results Effects of CUMS on maternal and offspring parameters In order to determine whether the stress protocol applied activated HPA axis in pregnant females, maternal plasma ACTH levels were evaluated. Prior to the start of the stress protocol (GD13), maternal plasma ACTH levels were not significantly different between experimental groups (Figure 1). Following random and intermittent exposure of the pregnant female rats to a variety of stressors during the third week of gestation (GD13-21), maternal plasma ACTH levels increased compared to control pregnant females (Figure 1, p < 0.001, 2-way ANOVA with Bonferroni’s multiple comparison test). Additionally, in the group of stressed mothers, following exposure to diverse stressors, plasma ACTH levels increased compared to GD13 suggesting that HPA axis was activated in this experimental group (Figure 1, p < 0.001, 2-way ANOVA with Bonferroni’s multiple comparison test). CUMS did not affect maternal weight gain during the last week of pregnancy (Table 3) or water and food intake throughout pregnancy (data not shown). Maternal blood glucose levels were similar in both experimental groups before and after application of the CUMS protocol (Table 3). There was no effect of prenatal stress on litter size or offspring sex ratio (Table 3). Maternal stress during the last week of pregnancy did not affect offspring birth weight or weight gain during either pre- or post-weaning periods (Table 4). Effects of prenatal stress on regional ADRB subtype gene expression in left ventricle of female and male Using quantitative PCR analysis, relative mRNA levels of ADRB1, ADRB2, and ADRB3 were examined at the apical and the basal region of left ventricle harvested from control (C) and prenatally stressed (PS) adult female and male offspring. ADRB3 mRNA was undetectable at the examined regions of the left ventricle in male and female offspring. We detected higher ADRB1 mRNA expression at the apex and the base of left ventricle from control female offspring, in comparison to ADRB2 mRNA levels (Figure 2A 69