Efeitos do estradiol mediados pelo GPER na hipertrofia de cardiomiócitos induzida por Endotelina-1

Abstract

Previous studies have shown that 17β-estradiol (E2) prevents cardiac hypertrophy in females. These cardioprotective actions exerted by E2 have been mainly related to activity of estrogen receptor alpha (ERα) and beta (ERβ). Since 2005, the G protein-coupled estrogen receptor 1 (GPER1), an E2 membrane receptor, has been studied and identified as a mediator of many estrogenic actions of E2 in vivo and in vitro models. However, the mechanisms by which the GPER1 exerts its estrogenic actions in cardiomyocytes are still poorly known. In this work, we investigated the participation of GPER1 on anti-hypertrophic actions performed by E2 in cardiomyocyte hypertrophy endothelin-1(ET-1)-induced. To perform this study, we used cardiomyocytes culture methodology of neonate female rats with 1-3 days life. Cardiomyocytes were isolated and treated with ET-1 (100 nmol/L) for 48 h in the presence or absence of E2 (10 nmol/L), GPER1 antagonists (G15 or G36) or agonist GPER1 (G1) (10 nmol/L). At the end of treatment protocol, the cells were used to analyze the frequency of contraction, mobility range of membrane, Western blot, RT-PCR and immunofluorescence. The treatment with ET-1 was able to produce a significant increase in surface area of cardiomyocytes and this response was associated with increased mRNA expression levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). Treatment with 10 nM E2 prevented the development of cardiomyocytes hypertrophy of cells treated with ET-1 and also a reduction in the expression of ANP and BNP. These effects mediated by E2 were blocked with pretreatment of the cells with the antagonist GPER1 (G15). A similar effect of E2 in the anti-hypertrophic activity was observed when the cardiomyocytes were pretreated with G1, the GPER1 agonist. Notably, the analyzes of the data pointed to the participation of GPER1 in anti-hypertrophic activity exerted by E2. This effect was proven by the silencing technique GPER1 (siRNA), which showed a partial blockage of the anti-hypertrophic activity of E2 when GPER1 was silenced by 65%. Treatment of cardiomyocytes with E2 was able to reverse the reduction in the contraction frequency induced by ET-1, an effect that was independent of GPER1. Nevertheless, GPER1 participated in reducing membrane amplitude of contractions E2-induced. ET-1 led to an increase in the nuclear translocation of G proteincoupled receptor kinase 5 (GRK5), a kinase related to activation of hypertrophic genes. The pre-treatment with E2 was able to prevent the increase in GRK5 translocation, an independent effect of GPER1. The ET-1 treatment resulted in increased phosphorylation of Extracellular signal-regulated protein kinases 1 and 2 (ERK 1/2), an effect reversed by treatment with E2 and G1 and abolished when cells were treated with G15. Likewise, pretreatment with E2 reversed the decreased levels of phosphorylated Akt and its activating protein Phosphoinositide-dependent kinase-1 (PDK1) induced by ET-1, and this effect was mediated by GPER1. In conclusion, the GPER1 plays an important role in the anti-hypertrophic activity of E2 in cardiomyocytes, preventing activation of hypertrophic pathways and stimulating cell survival pathways. The data suggest that GPER1 can be considered as a strategy for understanding non-genomic cardioprotective actions of E2 in a cardiomyocyte hypertrophy condition.