Isolation and Culture of Cardiac Cells

Mice were sacrificed under deep isoflurane anesthesia and the hearts were rapidly cannulated. Cardiomyocytes and fibroblasts were isolated by liberase/trypsin digestion (protocol PP00000125 from The Alliance for Cellular Signaling) (17 (link)). Myocytes were plated for 4 hours on laminin-coated dishes. Fibroblasts were cultured in fibroblast growth medium (Promocell) during 5 days and then passaged once. For cGMP determinations the cells were incubated at 37°C with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM; MilliporeSigma) for 15 minutes and thereafter with synthetic CNP, ANP, or vehicle (saline) during an additional 10 minutes (17 (link), 26 (link)). For studies of phospholamban and titin phosphorylation, myocytes were incubated with CNP for 10 minutes (17 (link)).