Effect of G Protein Coupled Estrogen Receptor 1 on Angiotensin Ⅱ-induced Hypertrophy of Cultured Neonatal Rat Cardiomyocytes / 中国循环杂志

ABSTRACT

Objectives: To observe the effect of activated G-protein coupled estrogen receptor 1 (GPER1) on Angiotensin II (AngII)-induced hypertrophy of cultured neonatal rat cardiomyocytes and explore related mechanisms. Methods: Primary cardiomyocytes derived from 2-to 3-day-old neonatal rats were cultured in vitro. Tandem mass tags (TMT) protein mass spectrometry was used to examine protein expressions; relevant bioinformatics analysis was performed to screen the possible regulatory mechanisms. Cardiomyocytes were divided into 6 groups (1)Blank control group, (2) AngII group, (3)AngII+G1 (GPER1 activator) group, (4)AngII+G1+G15 (GPER1 inhibitor) group, (5)AngII+G1+U0126 (extracellular ERK inhibitor) group and (6)AngII+G1+MK2206 (AKT inhibitor) group (n=3 for each group). Cardiomyocytes GPER1 expressions, mRNA levels of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), protein levels of ERK, AKT with their interactions, autophagy-related proteins LC3II and LC3I were compared among different groups;impact of GPER1 on cardiomyocytes apoptosis was detected by flowcytometry. Results: AngII induced cardiomyocytes hypertrophy and upregulation of ANP and BNP mRNA levels in a dose-dependent manner (P<0.05). GPER1 expression could be detected on cardiomyocytes by Immunofluorescence technique. qRT-PCR results showed that GPER1 was activated by the specific activator G1 and mRNA expressions of ANP and BNP were inhibited in a dose-dependent manner by the specific activator G1 (P<0.05); mRNA levels of ANP and BNP were re-elevated in AngII+G1+G15 group (P<0.05). Western blotting results showed that protein expression of p-ERK and p-AKT was significantly higher in AngII group and AngII+G1 group than in blank control group (P<0.05), significantly reduced in AngII+G1+G15 group compared with AngII+G1 group (P<0.05); decreased expressions of p-ERK, p-AKT and mRNA levels of ANP,BNP were also detected in AngII+G1+MK2206 group (P<0.05). G1 induced protein expression was similar between AngII+G1 group and AngII+G1+U0126 group. Flowcytometry results indicated that cardiomyocytes apoptosis was similar between AngII+G1 group and AngII group (P>0.05). Ratio of LC3II/LC3I was significantly higher and autophagy levels were significantly enhanced in AngII group than in blank control group (P<0.01), these changes could be significantly reversed in AngII +G1 group (P<0.01 vs. AngII). Conclusions: Activation of GPER1 could inhibit neonatal cardiomyocytes hypertrophy, this effect might be related to AKT and ERK signaling pathway and cell autophagy.