| Objective:To investigate the effect of NCX-2D2 antibody on ouabain-induced arrhythmias in normal and failing rat hearts,and to analyze the antiarrhythmia mechanism.Methods:1.Establishment of ouabain-induced rat arrhythmic models?1?Ouabain-induced in vitro arrhythmic rat modelHealthy SD rats were anesthetized with pentobarbital sodium(50 mg·kg-1,i.p.).Heart was rapidly excised and mounted on the Langendorff apparatus.Aorta retrograde perfusion was performed at a flow rate of 8 mL·min-1 using Tyrode's.Isolated hearts were perfused for at least 30 min for stblization before drugs were administered via micro-pump.The experimental groups were as follows:? Control group:administrate 2.5 mmol·L-1 CaCl2;? 2D2+CaCl2 group:administrate 10 ?g·ml-1 NCX-2D2 antibody and 2.5 mmol·L-1 CaCl2;? Oua+CaCl2 group:administrate 5 ?mol·L-1 Ouabain and 2.5 mmol·L-1 CaCl2;? 2D2+Oua+CaCl2 group:administrate 10 ?g·ml-1 NCX-2D2 antibody?5?mol·L-1 Ouabain and 2.5 mmol·L-1 CaCl2.ECGs were recorded and duration and incidence of ventricular tachycardia and ventricular fibrillation,and the total number of premature ventricular beats were recorded and analyzed during odservation for 30 min.?2?Ouabain-induced in vivo rat arrhythmic modelHealthy SD rats were anesthetized with chloral hydrate(30 mg·kg-1,i.p.).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.ECGs were recorded and duration and incidence of ventricular tachycardia and ventricular fibrillation,and the total number of premature ventricular beats were recorded and analyzed during odservation for 60 min.2.Establishment of rat heart failure model by abdominal aortic constrictionSD rats were randomly divided into sham group and HF group.Rats were anesthetized with chloral hydrate(30 mg·kg-1,i.p.)before surgery.After shaving,the abdominal cavity was opened,the abdominal aorta was constricted.Sham group was treated in the same way,but without constricting the abdominal aorta.Penicillin?200,000 units per day?was injected intraperitoneally for 3 days to prevent infection.3.Echocardiography and hematoxylin-eosin stainingEchocardiographic examination was performed 12 weeks after surgery.Detection indexs including left ventricular internal diameter at end-systole?LVIDs?,left ventricular internal diameter at end-diastole?LVIDd?,left ventricular ejection fraction?LVEF?,left ventricular fractional shortening?LVFS?.HE staining was used to observe the morphological characteristics of myocardial tissue in sham group and HF group.4.Establishment of ouabain-induced arrhythmia in heart failure model and heart function detectionSD rats were anesthetized with chloral hydrate(30 mg·kg-1,i.p.).Recorded the electrocardiogram using BL-420F biological function experiment system.Cardiac function was monitored through carotid artery cannulation into left ventricular cavity.Cardiac arrhythmia in heart failure rat model was established by administering ouabain(0.4 mg·kg-1)through carotid artery cannula.NCX-2D2 antibody(40,60 ?g·kg-1)or Amiodarone(3 mg·kg-1)were injected through carotid artery cannula 5 min before ouabain injection.The duration and incidence of ventricular tachycardia and ventricular fibrillation,the number of premature ventricular beats were recorded for 1 hour during the continuous drug intervention.Heart rate?HR?,left ventricular diastolic pressure?LVEDP?,maximum increase rate of left ventricular isovolumic systolic period?+dp/dt?and maximum decrease rate of left ventricular isovolumic diastolic period?-dp/dt?were also recorded.5.Preparation of rat single ventricular myocyte?method of enzymatic dissociation?.Healthy SD rats were anesthetized with pentobarbital sodium(50 mg·kg-1,i.p.).Rat was bleeded through the carotid artery.After opening the chest,heart was rapidly excised and put into 4? 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.6.Whole-cell patch clamp recording.Using whole-cell patch clamp technique to record L-type calcium current?ICa-L?,sodium-calcium exchange current?INa/Ca?at voltage-clamp configuration and delayed after-depolarizations?DADs?at current-clamp configuration.Results:1.Inhibitory effect of NCX-2D2 antibody on ouabain-induced arrhythmia in normal rats?1?10 ?g·ml-1 NCX-2D2 antibody significantly inhibited ouabain-induced arrhythmias in the in vitro rat hearts.In ouabain group,100%rats showed ventricular tachycardia within 30 min after intervention,and 71.43%rats had ventricular fibrillation.After administration of NCX-2D2 antibody at 10 ?g·ml-1,during observation for 30 min rats ventricular fibrillation was completely eliminated.Only 28.5%rats had ventricular tachycardia and its duration reduced from?192.71±18.45?s to?11.85±20.27?s?P<0.05?.The total of premature ventricular beats was also reduced from 303±26 to 78±7 after NCX-2D2 antibody administration?P<0.05?.?2?NCX-2D2 antibody significantly inhibited ouabain-induced arrhythmia in anesthetized rat in vivo.Compared with ouabain group,after administration of NCX-2D2 antibody at 60?80 ?g kg-1,the rate of rats ventricular tachycardia were significantly dropped to 50%and 37.5%,respectively within the observation period?1h?,and rats ventricular fibrillation significantly dropped to 33.3%and 0,respectively.The duration of ventricular tachycardia reduced from?50.9±3.5?to?4.29±4.7?and?0.14±0.21?min,respectively?P<0.05?,and total number of premature ventricular beats and the duration of ventricular fibrillation also reduced?P<0.05?.The effect of NCX-2D2 antibody at 80?g·kg-1 on ouabain-induced arrhythmia was similar to that in positive control group.2.Rat heart failure model was successfully establishedCompared with sham group,left ventricular internal diameter at end-systole?LVIDs?,left ventricular internal diameter at end-diastole?LVIDd?were significantly increased from?2.05+0.08?,?5.25+0.19?mm to?5.42±0.29?,?7.59+0.36?mm,respectively after 12 weeks?P<0.05?.The LVEF,LVFS were significantly decreased from?92±1.4?,?58±2.5?%to?62±5.65?,?30±2.85?%,respectively?P<0.05?.The HF group rats myocardial cells arranged disorder,myocardial cells hypertrophy,and some of the myocardium appeared fibrosis.3.Inhibitory effect of NCX-2D2 antibody on ouabain-induced arrhythmia in heart failure rats in vivo0.4 mg-kg-1 ouabain enhanced cardiac function in heart failure rats,but also induced severe arrhythmia.40,60 ?g·kg-1 NCX-2D2 antibody inhibited arrhythmia in ouabain-induced heart failure rats and the rat heart function was significantly improved compared with rats in heart failure group.In ouabain group,ventricular tachycardia occurred in 100%rats within 1 hour,and 66.7%rats had ventricular fibrillation.After administration of NCX-2D2 antibody at 40 and 60 ?g·kg-1,ventricular tachycardia occurred in 66.7%and 33.3%rats,respectively within the observation period?1h?,and ventricular fibrillation occurred in 33.3%and 16.7%rats,respectively.The duration of ventricular tachycardia reduced from?1120.83±173.89?to?264.6±206.36?and?8.6±13.5?s,respectively?P<0.05?,and the duration of ventricular fibrillation reduced from?22.50±24.31?to?3.70±6.10?and?1.50±3.67?s,respectively?P<0.05?.The total of premature ventricular beats also decreased from 1640±157 to 488±19 and 138±3 after NCX-2D2 antibody administration,respectively?P<0.05?.Effect of NCX-2D2 antibody at 60 ?g·kg-1 on ouabain-induced arrhythmia was similar to that of positive control group.4.NCX-2D2 antibody partially reversed sodium-calcium exchange current?INa/Ca?increase by ouabain.INa/Ca in ouabain group was 9.20±0.09?pA/pF?at +50 mV and-11.42±0.27?pA/pF?at-100 mV,respectively,which was significantly higher than control group of 4.25±0.05 and-4.08±0.09?pA/pF?.After administration of 5 ?g·ml-1 NCX-2D2 antibody in the presence of 5 ?mol·L-1 ouabain,the outward and inward currents of INa/Ca both reduced to 6.67±0.27 and-7.42±0.26?pA/pF?,respectively,which were significantly lower than those in ouabain group?P<0.05?,although still higher than normal?P<0.05?.5.NCX-2D2 antibody attenuated increase of L-type calcium current?ICa-L?by ouabain in isolated rat ventricular myocyteICa-L?pA/pF?in ouabain group?-8.73±0.36?significantly increased than in control group?-6.30±0.53??P<0.05?.After administration of 5 ?g·ml-1 NCX-2D2 antibody,ICa-L decreased to-7.45± 0.29?pA/pF?,which was significantly lower than that in ouabain group?P<0.05?,but still higher normal?P<0.05?.6.Inhibitory effect of NCX-2D2 antibody on delayed after-depolarization induced by ouabain in isolated rat ventricular myocyteFollowing conditioned stimulis at 3 Hz,no delayed after-depolarization was observed in control and 2D2 groups.After NCX-2D2 antibody administration,the incidence of DADs significantly decreased from 83.33%in ouabain group to 8.3%?P<0.05?.Conclusion:NCX-2D2 antibody significantly inhibits ouabain-induced arrhythmias in normal and failing hearts.The underlying mechanism of its antiarrhythmic effects is mainly due to inhibition of sodium-calcium exchanger and L-type calcium channel related attenuation of cardiomyocyte calcium overload and markedly suppression of delayed after-depolarization in rat ventricular myocytes. |
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