Isolation of adult rat ventricular myocytes

Adult rat ventricular myocytes were isolated from adult SD rats as described previously (19, 61). Briefly, after deep anesthesia with trichloroacetaldehyde monohydrate (0.5 g/kg, i.p.), the heart was quickly removed from the rat chest; cleaned and flushed with nominally Ca²⁺-free Tyrode solution consisting of 137 mm NaCl, 5.4 mm KCl, 1.2 mm MgCl₂, 1.2 mm NaH₂PO₄, 10 mm glucose, and 20 mm HEPES (pH 7.3, adjusted with NaOH); and perfused using a Langendorff apparatus at 37 °C. After 5 min, the solution was switched to the enzyme solution with 0.5 mg/ml collagenase (Worthington; Type II) and 0.06 mg/ml protease (Sigma; Type XIV) for 15 min. All solutions were equilibrated with 100% O₂. Then the heart was minced into small chunks, and single cells were shaken loose from the heart tissue and stored in HEPES-buffered external solution containing 137 mm NaCl, 5.4 mm KCl, 1 mm CaCl₂, 1.2 mm MgCl₂, 1.2 mm NaH₂PO₄, 20 mm glucose, and 20 mm HEPES (pH 7.4).