Cardiomyocyte isolation and stimulation with isoprenaline

All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the University Medical Center Göttingen in compliance with the humane care and use of laboratory animals. Hearts were perfused by Langendorff solution [120.4 mM NaCl, 17.7 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 2,3-butanedione-monoxime, and 5.5 mM glucose (pH 7.4)] with a flow of 4 ml/min for a period of 4 min at 37°C. To digest the tissue, the heart was perfused with perfusion buffer supplemented with collagenase type II (2 mg/ml) and CaCl₂ (40 μM) for 8 min. The heart was removed from the Langendorff perfusion apparatus and placed in collagenase solution, and the tissue was mechanically disrupted with a scissor and by resuspension with a pipette. The collagenase was stopped with bovine calf serum (10%) and CaCl₂ (12.5 μM). Isolated cardiomyocytes were collected by filtration with a 100 μM cell sieve and centrifugation at 22g for 1.5 min. Isolated cardiomyocytes from one animal were split into two Eppendorf tubes in experimental buffer [10 mM Hepes, 136 mM NaCl, 40 mM KCl, 1 mM MgCl₂, 10 mM glucose, and 1 mM CaCl₂ (pH 7.35)], and one tube was treated with 100 nM isoprenaline for 30 s, whereas the other was not treated. After isoprenaline stimulation, the cells were collected by centrifugation and resuspend in sucrose homogenization buffer [50 mM NaCl, 320 mM sucrose, 2 mM EDTA, and 20 mM Hepes (pH 7.4)] supplied with protease and phosphatase inhibitors (Roche) and stored at −80°C.