2.10. Neonatal rat cardiomyocyte isolation and treatment

Neonatal rat ventricular myocytes (NRVMs) were isolated from 1–2 days old neonatal rat pups as previously described by Kapoor et al. [21]. The isolated NRVMs were cultured in M199 medium supplemented with 10 mM HEPES, 0.1 mM non-essential amino acids, 3.5 mg/mL glucose, 2 mM L-glutamine, 4 μg/ml vitamin B12, 100 U/ml penicillin, and heat-inactivated FBS. The cells were treated with 1μM acetaldehyde (402788, Sigma-Aldrich) and 10μM BMS 493 (B6688, Sigma-Aldrich) for 48 hours using in 2% FBS supplemented medium. The cells treated with ddH₂O or DMSO were used as controls. Total RNA isolation for real-time PCR analysis was performed using RNeasy^® Mini Kit (74106, Qiagen, Venlo, Netherlands).