Neonatal rat ventricular myocytes (NRVM) isolation and culture

NRVM were detached from ventricles of 1day old neonatal Sprague-Dawley rats. Ventricles were cut into pieces and digested with collagenase and trypsin. The cell suspension was collected and centrifuged to collect the cell pellets. To remove fibroblasts, cells were seeded and maintained routine incubation condition for 2 hr, and then the non-adherent CMs were collected, re-suspended and seeded for culture while the adherent fibroblasts were disposed.

The cells undergone starvation under serum-free medium condition for 12 h to synchronize before treatment with Ang II (1 µM) or PBS or ASK1 inhibitor GS-4997(iASK1 80 µM, 1148428-04-3, Selleck) or DMSO for another 48 h.