Compound c

Mice were given 100 units of heparin i.p. (Sagent Pharmaceuticals, Schaumburg, IL) for anticoagulation before anesthetized with 100 mg/kg sodium pentobarbital i.p. (Sigma, St. Louis, MO). The heart was excised and fastened onto the cardiomyocyte perfusion apparatus (Radnoti, Monrovia, CA) and perfusion was initiated in the Langendorff mode. Hearts were perfused at 37 °C with a Ca^{2 +}-free Krebs–Henseleit based buffer (pH 7.3) containing: 0.6 mM KH₂PO4, 0.6 mM Na₂HPO₄, 10 mM HEPES, 14.7 mM KCl, 1.7 mM MgSO₄, 120.3 mM NaCl, 4.6 mM NaHCO₃, 30 mM taurine, 10 mM glucose, and 10 mM 2,3-butanedione monoxime that was bubbled with 95% O₂/5% CO₂. After a few minutes of stabilization, the heart was then digested with the same perfusion buffer containing 0.067 mg/mL Liberase Blendzyme 4 (Roche, Indianapolis, IN). After digestion, the heart was removed and minced. Extracellular Ca²⁺ was added back to the cells to reach a final concentration of 1 mM. Cardiomyocytes were then subjected to pharmacological drug treatment with either vehicle (DMSO), 10 mM Compound C (Enzo Life Sciences, Farmingdale, NY) or A-769662 (SelleckBio) for 20 min at room temperature (20–25 °C).