Myocyte growth media

Primary cardiomyocytes from 5 different donors (Mean age = 55, range 50–61, Caucasian) with cardiomyopathy were obtained from Promocell (Heidelberg, Germany) and maintained in myocyte growth media (Promocell, Heidelberg, Germany) at 37 °C. For perturbation experiments cells were plated overnight (24 h) then incubated in media with high glucose (20 mM) and free fatty acids [oleic acid 3.33 μM (Sigma), linoleic acid 3.33 μM (Sigma), and l-carnitine 1 mM (Sigma)] for 6 h prior to harvesting for proteomic analysis. Rat embryonic cardiomyocytes [H9C2(2–1)] were obtained from ATCC (ATCC^® CRL-1446™) and maintained in DMEM growth media (Lonza-12-604F) supplemented with 10% fetal bovine serum (Gibco) at 37 °C. H9C2 myocytes were seeded 24 h prior to differentiation into myotubes by reducing serum concentration to 1% serum within the culture media and incubated for 48 h.

Human Cardiac Fibroblasts (HCF) or Human Cardiomyocytes (HCM) were cultured at a cell density of 5 × 10⁶ cells in a specialized Fibroblast medium 3 (Promocell) and Myocyte growth media (Promocell) respectively at 37 °C in a 5% CO₂ incubator. The cells were subcultured every 6–7 days or until confluent.

Cells were maintained at 37 °C in 5% CO₂. Baby hamster kidney (BHK-21, ATCC CCL-10) were grown in Dulbecco’s modified Eagle’s medium (DMEM; Corning) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals) and 1% nonessential amino acids (NEAA, Gibco). Vero cells (ATCC CCL-81) were grown in DMEM with 10% newborn calf serum (NBCS; Gibco). Human primary cardiac cells were obtained from PromoCell, used up to passage 10, and grown following the company recommendations. Human Cardiac Fibroblast (C-12375), human Cardiac Microvascular Endothelial Cells (C-12285), and human Cardiac Myocytes (C-12810) were grown in Fibroblast Growth Medium 3 (PromoCell, C-23025), Endothelial Cell Growth Media MV (PromoCell, C-22020), and Myocyte Growth Media (PromoCell, C-22070), supplemented with 1% Penicillin-Streptomycin (Cellgro, 30-002-CI), respectively. All the cell types were confirmed free of mycoplasma using Lookout Mycoplasma PCR detection kit (Sigma-Aldrich).