Isolation of cardiomyocytes

Single canine myocytes were obtained by enzymatic dispersion using the segment perfusion technique, as previously described²⁸. Briefly, a wedge-shaped section of the ventricular wall supplied by the LAD coronary artery was cannulated, dissected and perfused with a nominally Ca²⁺-free Joklik solution (Minimum Essential Medium Eagle, Joklik Modification, Sigma-Aldrich Co. St. Louis, MO, USA) for a period of 5 min. After this, the tissue was perfused with Joklik solution supplemented with 1 mg/ml collagenase (Type II, Worthington Biochemical Co., Lakewood, NJ, USA; representing final activity of 224 U/ml) and 0.2% bovine serum albumin (Fraction V., Sigma) containing 50 µM Ca²⁺ for 30 min. Finally, the normal external Ca²⁺ concentration was gradually restored and the cells were stored at 15 °C in Minimum Essential Medium Eagle until use. The chemicals used in the experiments were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA), except for GS967, which was purchased from MedKoo Biosciences, Inc. (Morrisville, NC, USA).