Cardiomyocyte culture medium

Mouse cardiomyocytes isolated from Institute of Cancer Research mouse embryo hearts were purchased from COSMO Bio Co Ltd (Tokyo, Japan). The cells were seeded in a 24-well plate at a density of 100,000 cells per well, and cultured with cardiomyocyte culture medium (COSMO Bio) in fibronectincoated plates in an atmosphere of 95% air/5% CO2 at 37°C. Cells reaching 70-80% confluency were starved with serumfree M199 medium (Thermo Fisher Scientific, MA, USA) overnight, and incubated with the indicated concentrations of AngII (Sigma-Aldrich, MO, USA) and GIP (human GIP (1-42); AnaSpec, CA, USA) for 24 h for reverse transcriptionpolymerase chain reaction (RT-PCR) analysis.

All animal procedures were performed in accordance with the "Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions" stipulated by the Ministry of Education, Culture, Sports, Science and Technology and approved by the animal experiment committee of Cosmo Bio Co., Ltd. Primary rat cardiomyocytes were cultured as previously described. 15 Briefly, cardiomyocytes prepared from neonatal rat ventricles (Cosmo Bio, Japan) were seeded on 35-mm dishes that were coated with collagen at a concentration of 2×10 5 cells/dish. These cells were then incubated in cardiomyocyte culture medium (Cosmo Bio) at 37℃ in a humidified atmosphere containing 5% CO 2 . The medium was changed two days after seeding.
For SECM experiments, we used spontaneously beating cells within 25 days in a culture. Before making a beating measurement, the culture medium was replaced with 2 ml/dish of serum-free medium that contained 1 mM potassium ferrocyanide (K 4 [Fe(CN) 6 ]) that is an electron mediator of SECM measurement and 2% Knockout TM serum replacement (Thermo Fisher Scientific, USA) in DMEM/F-12 medium (Thermo Fisher Scientific, USA).