Transient Post-Natal Exposure to Xenoestrogens Induces Long-Term Alterations in Cardiac Calcium Signaling

Abstract

Today, non-communicable disorders are widespread worldwide. Among them, cardiovascular diseases represent the main cause of death. At the origin of these diseases, exposure to challenges during developmental windows of vulnerability (peri-conception, in utero, and early infancy periods) have been incriminated. Among the challenges that have been described, endocrine disruptors are of high concern because of their omnipresence in the environment. Worrisomely, since birth, children are exposed to a significant number of endocrine disruptors. However, the role of such early exposure on long-term cardiac health is poorly described. In this context, based on a model of rats exposed postnatally and transiently to an estrogenic compound prototype (estradiol benzoate, EB), we aimed to delineate the effects on the adult heart of such transient early exposure to endocrine disruptors and identify the underlying mechanisms involved in the potential pathogenesis. We found that this transient post-natal exposure to EB induced cardiac hypertrophy in adulthood, with increased cardiomyocyte size. The evaluation of cardiac calcium signaling, through immunoblot approaches, highlighted decreased expression of the sarcoplasmic reticulum calcium ATPase 2 (SERCA2) and decreased Nuclear Factor of Activated T Cells (NFAT3) phosphorylation as a potential underlying mechanism of cardiac hypertrophy. Furthermore, the treatment of cardiomyocytes with EB in vitro induced a decrease in SERCA2 protein levels. Overall, our study demonstrates that early transient exposure to EB induces permanent cardiac alterations. Together, our data highlight SERCA2 down-regulation as a potential mechanism involved in the cardiac pathogenesis induced by EB. These results suggest programming of adult heart dysfunctions such as arrhythmia and heart failures by early exposure to endocrine disruptors and could open new perspectives for treatment and prevention.

Keywords: calcium signaling; endocrine disruptor; heart; post-natal; programming.

Figure 1

Effects of transient post-natal exposure to estradiol benzoate on adult heart. (A) Longitudinal sections of whole heart stained with Masson’s trichrome, from adult rats exposed to corn oil (vehicle, CTRL) or estradiol benzoate (EB) (scale bar, 1 cm). (B) Heart versus body weight ratio of animals exposed to corn oil (vehicle, CTRL) or estradiol benzoate (EB). Data are expressed as mean ± SEM; n = 17–18 per group. Heart sections from 6 rats exposed to corn oil (CTRL) or estradiol benzoate (EB) were stained with wheat germ agglutinin (WGA) to evaluate cell surface (scale bar, 50 μm) (C) and Masson’s trichrome to evaluate extracellular matrix depot (scale bar, 100 μm) (D). Student’s t-test was performed to compare control and EB-treated animals. * indicates statistical significance (p < 0.05).

Figure 2

Post-natal exposure to estradiol benzoate alters calcium signaling in the adult heart. Protein levels of (A) phospho-PKA C-αPKA C-α, (B) phospho-PLN, PLN, and (C) SERCA2 protein levels were analyzed at post-natal day 77 in the heart of male rats exposed to corn oil (CTRL) or estradiol benzoate (EB) via Western blot. GAPDH protein levels were used as loading control. Data are expressed as mean ± SEM; n = 13 per group. (D) Serca2 mRNA levels were measured by RT-qPCR in the heart of CTRL and EB-treated rats at post-natal day 77. Data are expressed as mean ± SEM; n = 11 per group. (E) NFAT3 (phospho Ser289) and total NFAT3 protein levels were analyzed at post-natal day 77 in the heart of male rats exposed to corn oil (CTRL) or estradiol benzoate via Western blot. GAPDH protein levels were used as loading control. Data are expressed as mean ± SEM; n = 13 per group. Student’s t-test was performed to compare control and EB-treated animals. ns: Not statistically significant, ** p  <  0.01.

Figure 3

Estradiol benzoate influences SERCA2 expression in cardiomyocytes. H9C2 cells plated in 6-well plates were treated for 72 h with estradiol benzoate at 10 nM, 100 nM, and 1000 nM, or ethanol (EB0). In these cells, SERCA2 protein levels were measured via Western blot. β-ACTIN was used as loading control. Data are expressed as mean ± SEM; n = 3 per group. ANOVA with a Fisher’s LSD test was performed to compare control and EB-treated cells. * p  <  0.05. The results are representative of two independent experiments.

Figure 4

Effects of estradiol benzoate on oxidative stress in cardiomyocytes. H9C2 cells grown on coverslips were incubated with estradiol benzoate at 100 nM and 1000 nM or ethanol (EB0) for 72 h. (A) Cells grown on coverslips were incubated with an antibody targeting 8-OHdG (green) (Scale bar = 100 μm). (B) In these cells, antioxidant enzymes (Superoxide dismutase 1 and 2, SOD1, SOD2) and the regulator of mitochondrial oxidative phosphorylation COX2 were measured via Western blot. GAPDH protein levels were used as loading control. Data are expressed as mean ± SEM; n = 4 per group. ANOVA with a Fisher’s LSD test was performed to compare control and EB-treated cells. ns: not statistically significant.