Misoprostol

Isotretinoin, misoprostol, methotrexate, mifepristone and levonorgestrel were purchased from Sigma-Aldrich (St. Louis, MO, USA; Table I). The test drugs were dissolved/suspended in 0.75% Tween-80 solution (Sigma-Aldrich), and dosing solutions were prepared daily prior to treatment. The application volume of the drugs was 10 ml/kg body weight and was calculated based on the most recently recorded body weight of each individual animal. The test mixture was administered daily by gavage to female mice for 3 days. Control mice received an equivalent volume of 0.75% Tween-80 solution alone. The oral administration method was selected for this study since these drugs are administered orally in a clinical setting.

Rat primary ventricular neonatal cardiomyocytes (PVNC) were isolated from 1–2-day old pups using the Pierce Primary Cardiomyocyte Isolation Kit (#88281), which includes a cardiomyocyte growth supplement to reduce fibroblast contamination. H9c2 cells (ATCC CRL-1446) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone), containing penicillin, streptomycin, and 10% fetal bovine serum (Hyclone). Media was supplemented with MEM Non-Essential Amino Acids Solution (Gibco) for MEFs. Cells were incubated at 37 °C and 5% CO2. Human induced pluripotent stem cell-derived cardiomyocytes (H-iPSC-CMs) were obtained from Cellular Dynamics (iCell Cardiomyocytes #01434). iCell Cardiomyocytes were cultured in maintenance medium as per the manufacturer’s protocol and differentiated for 72 h. Cell lines were transfected using JetPrime Polyplus reagent, as per the manufacturer’s protocol. For misoprostol treatments, 10 mM misoprostol (Sigma) in phosphate buffered saline (PBS; Hyclone) was diluted to 10 μM directly in the media and applied to cells for 24 h. To achieve hypoxia, cells were held in a Biospherix incubator sub-chamber with 1% O₂ (±1%), 5% CO₂, balanced with pure N₂ (regulated by a Biospherix ProOx C21 sub-chamber controller) at 37 °C for 24 h. BvO2, L161-982, L798-106, and H89 dihydrochloride (H89) were purchased from Sigma.