Inhibitory Effect Of Zacopride On Ouabain-induced Arrhythmias In Adult Rats And Investigation On Its Mechanism

Objective:1.To investigate the effects of the inward rectifier potassium channel agonist zacopride on ouabain-induced in vitro and in vivo arrhythmias in adult rats.2.To explore the underlying electrophysiological mechanism,whole-cell patch clamp technique was used to observe effects of zacopride on inward rectifier potassium current(IK1),resting membrane potential(RMP)and delayed afterdepolarization(DADs)in single rat ventricular myocyte.Methods:1.Establishment of ouabain-induced rat arrhythmic models in vitro and in vivo(1)Ouabain-induced in vitro arrhythmic rat modelHealthy SD rats were anesthetized with pentobarbital sodium(50 mg/kg,i.p.).The heart was rapidly excised and mounted on the Langendorff apparatus.Aorta retrograde perfusion was performed at a flow rate of 8 mL/min.Isolated hearts were perfused for at least 30 min for stblization before drugs were administered via micro-pump.ECG was recorded and number of premature ventricular beats,the duration and incidence of ventricular tachycardia and ventricular fibrillation were recorded and analyzed for 30 min during the continuous drug intervention.(2)Ouabain-induced in vivo rat arrhythmic model.Healthy SD rats were anesthetized with chloral hydrate(30 mg/kg,i.p.)and were randomly divided into 5 groups.Rats were placed on the mouse plate and limb leads were connected.BL-420F biological function experiment system was used to record the electrocardiogram.After 30 min continuous perfusion for stablization,drugs were applied via tail vein.The number of premature ventricular beats,the duration and incidence of ventricular tachycardia and ventricular fibrillation were recorded for 1 hour during the continuous drug intervention.2.Preparation of rat single ventricular myocyte(method of enzymatic dissociation).Healthy SD rats were anesthetized with pentobarbital sodium(50 mg/kg,i.p.).Rat was bleeded through the carotid artery.After opening the chest,heart was rapidly excised and put into 40C Tyrode's solution perfused with 100%O2.Heart was mounted via the aorta on Langendorff perfusion apparatus.Firstly,the heart was perfused with Ca2+-free Tyrode's solution for approximately 8-10 min to wash out the blood,then the perfusate was switched to Tyrode's solution with Collagenase P for 15-20 min until the heart was well digested.During the process of perfusion,constant temperature(37?),pressure(80 cmH2O)and 100%O2 ventilation were sustained.The left ventricle was separated and minced in the KB liquid when the ventricular myocardium became soft,then blowed gently for 3-5 min.The single ventricular myocyte was filtered through a 150 ?m pore size and stored in KB solution.Cells were stored at room temperature(25?)at least 3 hours before used.3.Whole-cell patch clamp recording.1-2 drops of the cell suspensed in KB liquid were added into the cell compartment of the inverted microscope(about 1 mL)for 8-10 min.After the cells were solidly adhered,the cells were perfused with calcium-containing Tyrode's liquid with flow rate of 2 mL/min.Glass electrode was filled with electrode solution,and its resistance is about 2-5 MQ.Ventricular myocytes with rod-shape,clear texture and no contraction were selected in high microscope.Glass electrodes were slowly moved to the cell surface,and negative pressure was applied to form a high-resistance seal between the cell membrane and the electrode tip(the resistance reached G?).After membrane rupture,membrane capacitance compensation and series resistance compensation were used before recording.The ion currents,resting membrane potential and induced DADs were recorded and analyzed by Axopatch 200B patch-clamp amplifiers,Digidate 1440A digital-to-analog converters and Pclamp 8.0.Results:1.Zacopride inhibited ouabain-induced arrhythmia significantly in rat in vitro1 ?mol/L zacopride significantly inhibited ouabain-induced arrhythmias in the in vitro rat hearts.In ouabain group,100%rats showed ventricular tachycardia within 30 min after intervention,the duration of VT was(218.40±29.61)s.Moreover,87.5%of the rats developed ventricular fibrillation with a duration of(10.71±2.90)s.Counts of premature ventricular beats was 751±24(n=8,P<0.01).In zacopride-intervened group,ventricular fibrillation was completely eliminated within 30 min after intervention.Only 25%rats had ventricular tachycardia and its duration decreased to(18.85±12.40)s.Premature ventricular beats decreased to 125±14(n=8,P<0.01).When 1 ?mol/L BaCl2 was apllied,the number of premature ventricular beats,the duration and incidence of ventricular tachycardia and ventricular fibrillation all had no significant difference compared with ouabain group.2.Zacopride significantly inhibited ouabain-induced arrhythmias in the anesthesia rats.15 ?g/kg zacopride significantly inhibited ouabain-induced arrhythmias in anesthesia rats.In ouabain group,ventricular tachycardia occurred in 100%rats within 1 hour,with duration of(1250±59)s.71.4%of the rats developed ventricular fibrillation with duration of(19.14±6.49)s,and the count of premature ventricular beats was 2281±115(n=7,P<0.01).In zacopride+ouabain group,the ventricular fibrillation was completely eliminated within 1 hour after administration.Ventricular tachycardia occurred in 28.6%rats,and the duration decreased to(15.57±10.10)s.Premature ventricular beats decreased to 171±25(n=7,P<0.01).The antiarrhythmic effect was similar to that of the positive control lidocaine(7.5 mg/kg),and even better than lidocaine when compared to the premature ventricular beats(n=7,P<0.01).3.Zacopride reversed the inhibitory effect of ouabain on IK1 in rat ventricular myocytes.10 ?mol/L ouabain significantly decrease IK1 in rat ventricular myocytes,including inward and outward component of IK1.Inward current(at-100 mV)decreased from(-10.69±0.21)pA/pF to(-6.61±0.26)pA/pF,and the outward current(at-60 mV)from(3.11±0.20)pA/pF to(1.97±0.17)pA/pF(n=30,P<0.01).After addition of 1-10 ?mol/L zacopride in the perfusate,both inward and outward component of IK1 were restored to different levels by zacopride.Among them,1 ?mol/L zacopride showed the maximal effect,which restored inward and outward current of IK1 to(-11.09±0.37)and(3.29±0.31)pA/pF,respectively(n=12,P<0.01).This effect was blocked by 1 ?mol/L of BaCl2.After giving 1 ?mol/L of BaCl2,the inward and outward component of IK1 both decreased(n=12,P<0.01 and P<0.05 respectively).4.Zacopride reversed decrease effect of ouabain on RMP in rat ventricular myocytes10 ?mol/L ouabain significantly decrease the RMP of normal rat ventricular myocytes from(-74.79±0.19)to(-70.27±0.21)mV(n=30,P<0.01).After applying 0.1-10 ?mol/L zacopride,RMP decrease by ouabain was restored to different levels.1?mol/L showed the maximal effect,which increased RMP to(-74.98±0.44)mV(n=10,P<0.01).This effect was blocked by 1 ?mol/L of BaCl2.After giving BaCl2,RMP was reduced to(-70.67±0.37)mV(n=10,P<0.01),which was similar to value in ouabain group.5.Zacopride inhibited ouabain-induced DADs in rat ventricular myocytes.1 ?mol/L zacopride significantly inhibited occurrence of DADs in rat ventricular myocytes,(n=24,P<0.05).When 1 ?mol/L BaCl2 was added to perfusate,DADs reappeared.Conclusions:1.As the inward rectifier potassium channel agonist,zacopride significantly inhibits ouabain-induced in vitro and in vivo ventricular arrhythmia in adult rats.2.In the range of 0.1-10 ?mol/L,zacopride restors IK1 and RMP of rat ventricular myocytes decreased by ouabain,with 1 ?mol/L as its maximal effect concentration.In addition,1 ?mol/L zacopride significantly inhibits DADs in rat ventricular myocytes.In summary,this study confirms that Zacopride significantly inhibits ouabain-induced ventricular arrhythmias in adult rats,which is related to increase RMP level and inhibition of DADs by activation of IK1 channel by zacopride.