Isolation of Mouse Ventricular Myocytes

Ventricular myocyte was isolated from the mouse hearts using a previously described method [43 (link)]. Mice were injected with heparin (50 units) and sacrificed by cervical dislocation. The heart was quickly removed from the chest, and the aorta was retrogradely perfused at 37 °C for 3 min with calcium-free Tyrode buffer (137 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, 10 mM glucose, 10 mM HEPES, 10 mM 2,3-butanedione monoxime (Sigma), and 5 mM taurine (Sigma) at pH 7.4) gassed with 95% O₂ and 5% CO₂. Enzymatic digestion was initiated by adding collagenase type B (0.35 U/ml; Roche, Basel, Switzerland) and hyaluronidase (0.1 mg/ml; Sigma) to the perfusion solution. When the heart became swollen after 10 min of digestion, the left ventricle was quickly removed, cut into several chunks, and further digested in the same enzyme solution on a shaker (60–70 rpm) for 10 min at 37 °C. The supernatant containing the dispersed myocyte was filtered through a cell strainer (100 µm diameter; BD Falcon, New York, USA) and gently centrifuged at 500 rpm for 1 min. Extracellular calcium was incrementally added back to a final concentration of 1.25 mM over 30 min to avoid the calcium paradox.