2.2 In vitro patch-clamp experiments
Cardiomyocytes were enzymatically isolated from the excised hearts of 26 rats after in vivo electrophysiological study. The isolated heart was retrogradely perfused for 10 minutes through the aorta in the Langendorff system with a modified Tyrode calcium free solution of the following composition (mM): 120 NaCl, 5.4 KCl, 5 MgSO4 · 7H2O, 5 sodium pyruvate, 20 glucose, 20 taurine, and 10 HEPES; pH 7.2, at 37˚C saturated with carbogen gas mixture (95% O2, 5% CO2). Then, perfusion was turned on the same solution supplemented with collagenase type II (0.5 mg/ml, Sigma Aldrich, USA), protease (0.01 mg/ml, Sigma Aldrich, USA) and 20 μM CaCl2 for 35 min. At the end of enzymatic perfusion, the ventricles were minced with fine scissors and triturated with the transfer pipettes. After that the cardiomyocytes were isolated from the chunks by filtering through a 200 μm mesh net in Kraft-Brühe solution containing (mM): 30 KCl, 50 glutamic acid, 30 K2HPO4 · 2H2O, 3 MgSO4 · 7H2O, 0.5 EGTA, 10 glucose, 20 taurine, and 10 HEPES; pH 7.2.
Whole-cell patch-clamp experiments were performed at room temperature (22-24˚ C) with the Axopatch 200B (Axon instrument, Burlingham, CA, USA). Ventricular cardiomyocytes were placed into an RC26 experimental chamber (Warner Instrument Corp, Brunswick, CT, United States). Patch pipettes were made of borosilicate glass (Sutter In-struments, United States) and pulled on a HEKA pipette puller type PIP 6 (HEKA Electronics, Germany).
To record the IK1 current (in a voltage-clamp mode) and action potentials (in a current-clamp mode), the pipettes were filled with a solution containing (mM) 140 KCl, 25 HEPES, 5 MgATP, 0.4 NaGTP, 0.5 EGTA; pH 7.2. To record IK(ATP), the pipette solution was modified so that MgATP and NaGTP were replaced by 5 MgCl2. A bath chamber was perfused with the solution containing (mM): 150 NaCl, 5.4 (or 1.3, to mimic hypokalemic conditions) KCl, 1.8 CaCl2, 1.2 MgCl2, 10 glucose, 10 HEPES, pH 7.4.
The IK1 current was evoked by a ramp voltage protocol (voltage range +60 to -120 mV) in the presence of 10 μM nifedipine and 2 mM 4-aminopyridine in solution. IK(ATP) was evoked by the same voltage ramp in the presence of 10 µM pinacidil. To determine the magnitude of the leakage current, 3 mM BaCl2 was added to the bath solution at the end of measurements.
Acute hypoxia was produced by bubbling the extracellular perfusate (normal extracellular solution for current recording, without glucose) with 100% N2 gas for 30 min with pH being continuously controlled. The bath solution PO2 was reduced thereby to 10 mm Hg and controlled by a multimeter Mettler Toledo Seven Duo Pro with the oxygen probe InLab 605-ISM-5m. The recorded traces were analyzed using Clampfit 9.2 (Molecular Devices, San Jose, CA, United States).
Statistical Analysis
Statistical analysis was performed with SPSS package (IBM SPSS Statistics 23, Armonk, North Castle, NY, USA). Parametric and nonparametric tests were used according to the Kolmogorov-Smirnov normality test. Parametric Student’s t-test and nonparametric Mann-Whitney test were used for comparison the groups of animals. The incidence of arrhythmia was compared by Chi-square test. Linear regression analysis was used to test associations between electrophysiological parameters and potassium concentration. All data are presented as mean ± SEM, the differences were considered significant at p < 0.05.