Adult mice were anesthetized with 3% isoflurane and injected with 200 U of heparin 5 min before heart excision. The heart was quickly cannulated and retrogradely exposed to perfusion buffer (120.4 mM NaCl, 14.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Hepes, 4.6 mM NaHCO3, 30 mM taurine, 10 mM BDM (2,3-butanedione monoxime), and 5.5 mM glucose) at 37°C. Hearts were then perfused for 12 min with enzyme solution containing 50 mg of collagenase and 0.005% trypsin in perfusion buffer containing 12.5 μM CaCl2. Solution was then switched to perfusion buffer for another 3 min. Atria was removed, and ventricles were gently minced. Cardiomyocytes were dissociated by pipetting in stop solution (perfusion buffer containing 10% fetal bovine serum and 12.5 μM CaCl2). Cells were serially exposed to increasing amounts of calcium (62 μM to 1 mM).

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