Cardiomyocyte-Endothelial Co-culture Assay

Adult primary cultured cardiomyocytes were isolated from mouse hearts following the protocol described by Ackers-Johnson et al [40 (link)] and C166 mouse yolk sac endothelial cells were from American Type Culture Collection (ATCC) (Manassas, VA, USA). Cells were incubated in the presence or absence of linagliptin (9 nmol/L) for 5 min for immunoblotting experiments and for 24 h for RNA sequencing. In addition, cardiomyocytes were incubated in the presence or absence of 100 µmol/L S-nitroso-N-acetylpenicillamine (SNAP) (Sigma-Aldrich, Oakville, Ontario, Canada) [41 (link)] for 5 min prior to immunoblotting. RNA isolation from cardiomyocytes was performed using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). For co-culture experiments, adult primary cultured cardiomyocytes and C166 cells were cultured separately on opposite sides of Millicell 6-well plate inserts with a 10 µm pore size PET membrane (MCRP06H48, EMD Millipore, Billerica, MA, USA) at a 1:2 density under control conditions or with culture media supplemented with 9 nmol/L linagliptin for 5 min. Cardiomyocyte cGMP concentrations were determined by direct immunoassay (ab65356, Abcam, Cambridge, MA, USA) according to the manufacturer’s instructions.