Hypoxic incubation chamber

HL-1 murine cardiomyocyte cells (Sigma-Aldrich, SCC065) were cultured in Claycomb medium (Sigma-Aldrich, 51800C). 4-OI or itaconate (Sigma-Aldrich, I29204) was added to the Claycomb medium for 2 hours. The medium was then changed to cold HTK solution and incubated in a hypoxic incubation chamber (STEMCELL Technologies). The chamber was filled with premixed gas containing 1% oxygen and 5% carbon dioxide gas (Cryogenic Gases, Detroit, MI) and incubated at 4°C for 2 hours. Subsequently, the medium was changed to 37°C Dulbecco’s modified Eagle’s medium at atmospheric conditions, and cells were incubated for an additional 2 hours. Cell lysates were then collected for Western blot analysis.

RL14 cells are a commercially available cell line of human fetal ventricular cardiomyocytes (CMs) derived from non-proliferative primary cultures of human fetal heart tissue (ATCC, Manassas, VA).^{17 (link)–19 (link)} RL14 cells were grown in DMEM/F-12 nutrient mixture (GE Healthcare Life Sciences, Logan, UT) supplemented with 12.5% (v/v) fetal bovine serum (Gibco, Grand Island, NY) and penicillin-streptomycin (10,000 U/mL; Gibco). Because RL14 cells do not have spontaneous electrical activity or inducible action potentials, human induced pluripotent stem cells (hiPSC)-derived CMs (iCell^® CMs) were obtained from Cellular Dynamics International (Madison, WI). iCell^® CMs were seeded and maintained using iCell^® Cardiomyocyte Plating Medium and Maintenance Medium (Cellular Dynamics International). For hypoxic conditions, the cells were cultured in an hypoxic incubation chamber (STEMCELL Technologies, Vancouver, BC) using pre-incubated culture media or were treated with hypoxic-mimetic chemicals, desferrioxamine (DFX) and cobalt chloride (CoCl₂) (Sigma, St. Louis, MO).