Cell culture and transfection for ion channel studies

For various aspects of cardiac ion channel study, human embryonic kidney (HEK293; ATCC, Manassas, VA, USA) cells were transiently transfected using lipofectAmin2000 (Gibco BRL, New York, NY, USA) according to the manufacturer’s instructions. The hERG (human ether-ago-go-related gene corresponding to I_Kr), KCNQ1/KCNE1 (the gene corresponding to I_Ks), KCNJ2 (the gene corresponding to I_K1) or SCN5A (the gene corresponding to I_Na) cDNA was co-transfected with green fluorescence protein, the surface marker protein, to allow assessment of the transfection efficiency. For the calcium current, enzymatically isolated single rat ventricular myocytes were used. Briefly, the hearts were rapidly excised from anaesthetized Sprague–Dawley rats (250–350 g) and perfused via the aorta on a Langendorff apparatus with an oxygenated normal Tyrode (NT) solution for 5 min. To clear the blood, then perfused with Ca²⁺-free normal Tyrode solution for 3 min. Next, the heart was perfused with enzyme solution containing 0.6 mg/mL collagenase (Worthington Biomedical Corp., Lakewood, NJ, USA) for 30–40 min. Finally, this enzyme-containing solution was washed out for 5 min. with a high-K⁺ and low-Cl^-) Kraft-Bruhe solution. Following the isolation procedure, the left ventricle was dissected out and agitated mechanically with a fire-polished Pasteur pipette in Kraft-Bruhe solution to obtain single myocytes. The isolated myocytes were stored at 4°C until use.