Angiotension 2 ang 2

Neonatal rat ventricular myocytes (NRCMs) were isolated from 1 to 3-day-old neonatal Sprague–Dawley rats as previously described [22 ], NRCMs were plated at a density of 1 × 10⁶ cells/mL and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with penicillin (Beyotime Biotechnology, Shanghai, China), streptomycin (Beyotime Biotechnology, Shanghai, China), 10% fetal bovine serum (FBS, Biological Industries, Kibbutz Beit-Haemek, Israel), 10 mM HEPES, and 0.1 mM 5-Bromo-2′-deoxyuridine (BrdU, Sigma-Aldrich, St. Louis, MO, USA). 36 h after the NRCMs were seeded, the adherent cells were infected with adv-Peli1 (MOI = 50) and adv-HNF4α (MOI = 50). Four hours later, the culture medium containing the virus was replaced with fresh medium. After 24 h, NRCMs were treated with angiotension II (Ang II, Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 1 μM for 24 h. In addition, NRCMs transduced with adv-HNF4α (MOI = 50), adv-Peli1 (MOI = 50) or adv-GFP (MOI = 50) for 48 h, followed by treatment with 100 μM cycloheximide (HY-12320, MedChem Express, Monmouth Junction, NJ, USA) for 0.5, 1, 2 and 4 h to detect the degradation levels of HNF4α.