Neonatal rat ventricular myocytes primary culture

NRVMs were prepared and cultured using a Neonatal Rat/Mouse Cardiomyocyte Isolation Kit (nc-6031; Cellutron Life Technology, Baltimore, MD, USA) which was previously described [23]. Hearts were dissected from 1- to 3-day-old Sprague-Dawley rats and transferred into a sterile beaker. Each heart was digested and stirred in the beaker at 37?°C for 12?min. The supernatant was then transferred to a new sterile tube and spun at 1200?r.p.m. for 1?min. The cell pellets were then re-suspended in D3 buffer and pre-plated for 1?h by seeding on an uncoated plate at 37?°C in a CO₂ incubator to select the cardiac fibroblasts. The unattached cells were transferred onto plates that were precoated with NS medium (supplemented with 10% fetal bovine serum). After overnight culture, the NS medium was replaced with a serum-free NW (without serum) medium. The cardiomyocyte cultures were ready for experiments 48?h after the initial plating.