Cell culture

Neonatal rat ventricular myocytes (NRVM) were isolated from 1- to 2-day-old Sprague-Dawley rats as previously described¹⁵. Briefly, ventricles were harvested and digested. Fibroblasts were cleared by pre-plating the cell suspension for 2 hours. Cardiomyocytes were then plated at a density of 1,250 cells per 1 mm² in plating medium containing 10% fetal bovine serum with 100 μM bromodeoxyuridine. The culture contained ≈95% cardiomyocytes (data not shown). Twenty-four hours after plating, NRVM were maintained in NRVM culture medium (DMEM high glucose and Medium 199 in 3:1 ratio) containing 3% fetal bovine serum. On the fourth day after plating, experiments were initiated.

Adult rat ventricular myocytes (ARVM) were isolated and cultured as previously described¹⁶. Four hours after plating, ARVM were maintained in culture medium (DMEM supplemented with 1x ITS, 10 mM 2,3-butanedione monoxime and 100 U/mL penicillin-streptomycin). On the second day after plating, experiments were initiated.

Cardiomyocyte differentiation from H9 human embryonic stem cells was performed as previously reported^{17, 18}. Briefly, embryonic stem cells (H9 cell line) were maintained on matrigel in mTeSR culture medium. When H9 cells reached 85–90% confluency, the medium was changed to RPMI 1640 with B27 supplement, minus insulin (Life Technologies) (day 0 to 8), supplemented with CHIR99021 (6 μM, Selleckchem) for the first 48 hours, and IWR-1 (5 μM, Sigma) on day 4 for 48 hours. On day 8, the medium was switched to RPMI 1640 with B27 supplement, and the cells were metabolically selected for 10 days. Then, cells were replated and used for experiments after day 30. H9c2 and C2C12 cells were cultured in Hyclone high glucose DMEM supplemented with 10% fetal bovine serum.