| Cardiac glycosides are widely used clinically to treat congestive heart failure. The basis of therapeutic and toxic effects of digitalis was thought to result from Na-K-ATPase inhibition. Digitalis-induced accumulation of intracellular calcium can cause myocardium transmembrane potential oscillatory and delayed afterdepolarization. These mechanisms were generally regarded as the basis of digitalis-induced ventricular tachycardia arrhythmia.However, this scheme is not sufficient to explain all effects of cardiac glycosides. Experiments evidence has suggested the possibility of additional actions by cardiac steroids. And not all arrhythmias caused by digitalis resulted from delayed afterdepolarization. Abnormal automaticity was thought to paly an important role in these arrhythmias. Zatebradine, a kind of pacemaker current inhibitor, could reduce the incidence of ventricular arrhythmias caused by digitalis intoxication. Therefore, pacemaker channel maybe another action site of digitalis, digitalis may enhance abnormal automaticity through pacemaker channel. This study is designed to confirmation this hypothesis.1. Character of pacemaker current from cultured newborn rat ventricular myocytes and the influence of culture time.Whole-cell pacemaker currents were recorded using patch-clamp techniques from singe ventricular myocytes acute-isolated from neonatal rats and matained in cell culture.Our results showed: membrane capacitances (Cm) were significantly decreased in myocytes even upon short term culture. The Cm of myocuytes cultured 1-3 day were 43±3 pF, decreased 23% compared with acute-isolated cells (56±7 pF) (p < 0.01). The Cm of cells from 4-6-day-cultured and 7-9-day-cultured myocytes were 44±3 pF and 45±4 pF, respectively. It seemed like the Cm increased slightly upon culture time, but no significant differences among various culture stages (p > 0.05). The reverse potential of pacemaker current was above -32mV upon different culture time and no significant difference with acute-isolated cells. Pacemaker currents density from acute-isolated, 1-3-day-cultured, 4-6-day-cultured and 7-9-day-cultured myocytes were 4.18±0.60, 3.85±0.42, 3.94±0.47 and 4.21±0.61, respectively, no significantly differences among cells from these groups (p> 0.05). Active threshold and V0.5 of pacemaker currents had no significant differences among cells from different group (p > 0.05). Therefore, the electrophysiological character of pacemaker currents did not change uponcell culture. Cultured myocytes can take place of acute-isolated cells used in pacemaker currents study.2. Action of ouabain on pacemaker current eletrophysiology.This experiment studied the influence of various dosage of ouabain on properties of pacemaker currents. The results showed: ouabain increased current density significantly even at concentration of 1 μM (control: 4.0 ± 0.41 pA/pF; 1 μM ouabain group: 4.76 ± 0.48 pA/pF. p < 0.01). The current density increased to 4.94 ±0.51 pA/pF when ouabain concentration was 20 μM (p < 0.01, compared with 1μM ouabain group) , to 4.96 ± 0.50 pA/pF with 40μM ouabain (p=0.053, compared with 20 /zM group) and to 4.97 ± 0.50 pA/pF with 80μM ouabain (p > 0.05, compared with 40 μM group). The V0.5 altered to depolarization direction, current conductance curves shifted to right and active velocity increased significantly with the action of ouabain. The pacemaker currents almost recovered to original level after the washout of ouabain. Ouabain increased amplitude of tail current, but had no influence on reversal potential.To investigate the relationship between intracellular calcium and activation of pacemaker current, the calcium chelator EGTA was excised from internal solution of patch pipette. This change resulted further increased current amplitude and channel active velocity. Current conductance curveshifted more to right. Thus, the modulation of ouabain on pacemaker current was enhanced when intracellular calcium was not chelated.3. Action of ouabain on pacemaker channel gene expression.We built successfully a kind of animal model for chronic ouabain intoxication with ouabain 0.045mg/Kg · d intraperitoneal injection lasted for 2 weeks. The ECG of rat showed elongation of PR interval, ventricular premature beats and even ventricular flutter. The blood pressure of rat did not increased upon two weeks ouabain action. There was no histological myocardium hypertrophy, too. HCN2 mRNA level of ouabain treatment group increased to almost 1.5 times of control group (p < 0.05). HCN4 mRNA levels had no significant change between ouabain group and control group (p > 0.05).It can be concluded from above study:1. Cultured myocardium could take place of acute-isolated cells in the electrophysiological study of pacemaker current. The cells cultured no more than one week were more suitable in this kind of electrophysiological experiment.2. Ouabain could stimulate pacemaker current from ventricular myocardium dose-dependly, shift the active threshold to more depolarization direction, increase current density and move theconductance curve to right. But the reversal potential did not change with ouabain action. These changes would become more significant when intracellular calcium chelated. Thus, ouabain can modulate pacemaker current directly and indirectly through cellular calcium overtake.3. Animal model of chronic ouabain intoxication could be built successfully with 2 weeks intraperitoneal injection of ouabian 0.045mg/Kg· d. HCN2 mRNA level were increased in ouabain-intoxicated myocardium, even more in left ventricle. HCN4 mRNA level did not change with ouabain action.
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