G protein-coupled bile acid receptor 1 activation attenuates endothelin-1-induced rat cardiomyocyte hypertrophy by inhibiting CaN/NAFT3 pathway

Abstract

Objective To investigate the inhibitory effect of G protein-coupled bile acid receptor 1 (TGR5) on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore the mechanism. Methods Primary cultured neonatal rat cardiomyocytes were exposed to ET-1 at 10-6, 10-7, or 10-8 mmol/L for 12, 24, 36, or 48 h, and the surface area and total protein concentration of the cells were measured to verify the establishment of cardiac mast cell models. In the subsequent experiment, rat cardiomyocytes were treated with ET-1, ET-1+INT-777 (a TGR5 receptor agonist), ET-1+INT-777+TGR5 siRNA, or ET-1+ INT-777+TGR5 siRNA-NC (an empty viral vector). The surface area of the cardiomyocytes was measured using an image analysis system, and the total protein content in the cells was determined with BCA method; The mRNA levels of TGR5 and calcineurin (CaN) were detected using RT-PCR, and the protein levels of atrial natriuretic factor (ANF), β-myosin heavy chain (β-MHC), TGR5, CaN and activated T cell nuclear factor 3 (NFAT3) were determined using Western blotting. Results Within 48 h, ET-1 concentration- and time-dependently induced cardiomyocyte hypertrophy in the concentration range of 1×10-8 to 1×10-6 mmol/L (P < 0.05). ET-1 treatment resulted in significantly increased surface area (3 624.7±71.60 μm2) and total protein content (51.810±1.47 μg) in the cardiomyocytes at 48 h as compared with the those in the control cells (1 560.8±3 188.94 μm2 and 37.827±0.47 μg, respectively; P < 0.05). ET-1 exposure of the cells caused also significantly increased expression of ANF, β-MHC, CaN and NFAT3 (P < 0.05). Compared with the cells treated with ET-1 alone, the cells with both ET-1 and INT-777 treatment showed significantly reduced total cell surface area, total protein content, and protein expressions of ANF, β-MHC, CaN and NFAT3 (P < 0.05). Transfection with siTGR5 partially eliminated the inhibitory effects of INT-777 on ET-1-induced cardiomyocyte hypertrophy (P < 0.05). Conclusion Activation of TGR5 may ameliorate ET-1-induced cardiomyocyte hypertrophy, the mechanism of which is related to the inhibition of CaN/NFAT3 signaling pathway.

Keywords

• g protein-coupled bile acid receptor 1
• endothelin-1; cardiomyocyte hypertrophy
• calcineurin
• activated t cell nuclear factor 3