Primary cell culture

Neonatal rat ventricular cardiomyocytes (NRVCs) were harvested from both ventricles of 1- to 2-day-old Sprague Dawley rat hearts. The ventricles were chopped into small pieces, and collagen digestion was performed in 0.1% type-II collagenase in ADS buffer (20 mmol/L HEPES, pH 7.4, 120 mmol/L NaCl, 5.5 mmol/L glucose, 11 mmol/L NaH₂PO₄, 5.4 mmol/L KCl, and 0.44 mmol/L MgSO₄ in distilled water) for 30 min at 37 °C with gentle agitation. By adding growth medium (DMEM high glucose supplemented with 10% fetal bovine serum and 1% antibiotics), enzyme digestion was completed. Cells were collected by a brief centrifugation at 1000 RCF for 10 min. Then, the supernatant was removed, and the pellet was resuspended in growth medium. The cells were pre-plated for 1 h to remove fibroblasts. After counting, the cells were plated in culture dishes coated with 0.2% gelatin.