The Inhibitory Effect Of I_k1 Channel Agonist On Isoproterenol-induced Arrhythmia In Rat And Investigation On Its Mechanism

Objective:1.To observe the inhibitory effect of zacopride as IK1 agonist on isoproterenol(ISO)-induced arrhythmia in rat and the underlying electrophysiological mechanisms.2.To observe the inhibitory effect of zacopride on rat arrhythmia in ISO-induced cardiac hypertrophy and the underlying electrophysiological mechanisms.Methods:Acute animals experiments1.ECGs recording in rat ISO-induced arrhythmic model.The experimental groups were as follows:(1)Control group: intravenous injection 1ml normal saline;(2)Zac group: intravenous injection 15 μg/kg zacopride;(3)ISO group: intravenous injection 1280 μg/kg ISO;(4)ISO + Zac group: intravenous injection 1280 μg/kg ISO 3 min after administrating15 μg/kg zacopride.The ECGs were recorded and analyzed to determine the incidence of ventricular arrhythmia, the total of ventricular premature beats(VPB) and the amplitude of ST-segment depression.2.Intracellular membrane potential recording in rat right ventricular papillary muscle using intracellular microelectrode technique.SD rats were anesthetized with pentobarbital sodium(40 mg/kg, i.p.). The heart was rapidly excised, then the right ventricular papillary muscle was isolated. Intracellular membrane potential was recorded with a 3 mol/L KCl-filled micropipette(10-20 M?).DADs and TA were observed following conditioning train stimulation(5 Hz, 20 pulses).Experimental groups were as follows:(1)Control group: administrate Tyrode’s solution;(2)ISO + Ca Cl2 group: administrate 1 μmol/L ISO and 3.6 mmol/L Ca Cl2;(3)ISO + Ca Cl2 + Zac group: administrate 1 μmol/L ISO、3.6 mmol/L Ca Cl2 and 1μmol/L zacopride;(4)ISO + Ca Cl2 + Zac + Ba Cl2 group: administrate 1 μmol/L ISO、3.6 mmol/L Ca Cl2、1 μmol/L zacopride and 1 μmol/LBa Cl2.Chronic animals experiments3.Preparation of ISO-induced cardiac hypertrophy rat modelRats(220-260 g) were randomly divided into 3 groups:(1)control group: intraperitoneally administered 0.5 ml normal saline;(2)ISO group: intraperitoneally administered ISO 5 mg/kg/day;(3)ISO+Zac group: intraperitoneally administered ISO(5 mg/kg/day) and zacopride(15 μg/kg/day).All rats in three groups were continuously administered for 7 days with saline for control group and ISO/Zac in other two groups. Rats were used for experiments 24 h after the last injection. Cardiac structure and function were assessed by echocardiography analysis. ECGs were recorded and analyzed to determine the incidence of ventricular arrhythmia, the incidence of ST-segment elevation and the amplitude of ST-segment elevation.4.Isolation of rat single ventricular myocyte using Collagenase PRat single left ventricular myocyte was isolated from rats in different groups. SD rat was anesthetized with pentobarbital sodium(40 mg/kg, i.p.), the heart was then rapidly excised, and mounted on a Langendorff retrograde perfusion apparatus via the aorta. Firstly,the heart was perfused with 100% O2 saturated Ca2+-free Tyrode’s solution(at 37°C,75 cm H2O) for approximately 8 minutes to wash out the blood. Secondly, the perfusate was switched to enzyme solution(contained Collagenase P 0.07-0.1 g/L) for 15-20 minutes until the heart were well digested. The left ventricle wall was then separated and minced in the KB solution. The dispersed cells were filtrated in KB solution at room temperature(25°C) at least 3 hours before use.5.Whole-cell patch clamp recordingWhole-cell patch clamp technique was used to record action potential(AP), delayed afterdepolarizations(DADs), resting membrane potential(RMP) at current-clamp configuration, and L-type calcium current(ICa-L), inwardly rectifier potassium current(IK1)were recorded at voltage-clamp configuration.Results:Acute animals experiments1.In ISO group, ventricular premature beats(VPB) occurred frequently with ST-segment depression. Compared with ISO group, the incidence of ventricular arrhythmia in ISO+Zac group decreased from 100% to 50%(n = 6, P <0.05). Total number of VPB recorded in 1 hour significantly reduced from 1574 ± 521 to 33±40(n = 6, P<0.05), while there was no amelioration on amplitude of ST-segment depression induced by ISO(n = 6,P>0.05).2.Zacopride at 1 μmol/L hyperpolarized the resting membrane potential of right ventricular papillary muscle from(-74.42 ± 1.95) m V in normal rat to(-78.50 ± 2.07) m V(n = 6, P<0.05).3.Zacopride at 1 μmol/L significantly depressed the DADs and TA induced by ISO combining with 3.6 mmol/L Ca Cl2 in right ventricular papillary muscle. The incidence of DADs decreased from 93.75% in rats in ISO group to 25% in ISO+Zac group(n = 16, P<0.05), and this antiarrhythmic effect could be reversed by 1 μmol/L Ba Cl2.Chronic animals experiments4.Compared with control group, both the left ventricular end-systolic dimension(LVESD) and left ventricular end-diastolic dimension(LVEDD) significantly decreased in ISO group(n=6, P<0.05), whereas left ventricular posterior wall end-diastolic thickness(LVPWd), interventricular septum end-diastolic thickness(IVSd), left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS) all increased(n=6,P<0.05). These changes demonstrated that ventricular wall was thickened and ventricular cavity was narrowed in the rats of ISO group, suggesting formation of the concentric hypertrophy and enhanced systolic function. Compared with ISO group, all the indexes of cardiac morphology and function were at least partly reversed in ISO+Zac group, among which both LVESD and LVEDD increased, despite that there are still significant differences when compared with the related values in control group(n=6, P<0.05).Moreover, LVPWd, LVEF and LVFS did decrease to control level(n=6, P<0.05). These results suggested that zacopride could also ameliorate ventricular morphology remodeling in ISO-induced hypertrophy.5.Ventricular premature beats(VPB) occurred much more frequently with ST-segment elevation in rats from ISO group. Compared with ISO group, the incidence of ventriculararrhythmia decreased from 85.71% to 14.29% in ISO+Zac group(n=7, P<0.05), although there was no amelioration on amplitude of ST-segment elevation induced by ISO(n=7, P<0.05).6.Compared with control group, the resting membrane potential of rat single ventricular myocytes decreased from(-71.71±1.80) m V to(-69.83±1.76) m V in ISO group(n=10, P<0.05). After continuous administration of Zacopride, the resting membrane potential increased significantly to(-74.46±1.43) m V(n=10, P<0.05) compared with both control group and ISO group.7.Following conditioned stimulis at 3 Hz, the incidence of DADs in isolated rat ventricular myocytes increased from 2/14 in control group to 12/14 in ISO group, and then decreased to 1/14 in ISO+Zac group, which was very close to the level in control group(n=14, P>0.05).8.Zacopride inhibited the L-type calcium current(ICa-L) increase induced by ISO.Peak voltage value of ICa-L in each group was 0 m V. The current density(p A/p F) of ICa-L in isolated rat ventricular myocytes of ISO group was-16.6±1.4, which was much higher than the value in control group(-11.5±1.1)(n=6, P<0.05). ICa-L in ISO+Zac group was-11.3±0.5, with the value almost the same as that in control group(n=6, P>0.05)(Fig2-6,2-7). These results showed zacopride could completely prevent the enhancement of ICa-L by ISO.9.Zacopride increased the inward rectifier potassium current(IK1) in ISO group. IK1density(p A/p F) in ISO group was-24.4±3.0 at-100 m V and 2.4±0.4 at-60 m V,respectively, which showed no statistic significance when compared with control group(-22.6±3.0 and 2.3±0.8)(n=6, P>0.05). In ISO+Zac group, both inward and outward IK1 at-100 m V and-60 m V significantly increased to-33.1±5.5 and 3.6±1.2,which was significantly higher than control group(n=6, P<0.05)(Fig2-8, 2-9). These results suggested that ISO had no impact on IK1 enhancement by zacopride.Conclusions:Zacopride, a selective IK1 channel agonist, can significantly inhibit ISO-induced cardiac arrthymia in rat, the mechanism of which is mainly attributed to zacopride-induced resting membrane hyperpolarization and subsequent suppression of DADs and TA via enhancing IK1. These results provide further evidence that moderate enhancement of IK1 is afeasible pathway for antiarrthymic therapy.