Hydrochloride Guanfu Go In The Dog Fans Nervous Atrial Fibrillation Model The Role Of The Isolated Guinea Pig Atrial Muscle Action Potential And Effective Refractory Period

Acehytisine hydrochloride is a new anti-arrhythmic alkaloid with a structure of C20-diterpenoid isolated from the tuber of Aconitum coreanum(Levl) Raipaics. Acehytisine hydrochloride has been approved by SFDA to treat superventriular tachycardia in 2005.In pre-clinical study Acehytisine hydrochloride was effective in treating various arrhythmias in animal models.Previous electrophysiological study showed that Acehytisine hydrochloride may be a new agent to treat atrial tachycardias(including atrial fibrillation and atrial flutter),but more work should be done to investigate the mechanism and effect of Acehytisine hydrochloride on atrial fibrillation.Purpose of this study is to investigate the effect of Acehytisine hydrochloride on vagotonic atrial fibrillation in mongrel dogs and electrophysiological action on isolated guinea pig atrium,further to explore the optimal concentration of Acehytisine hydrochloride in treating atrial fibrillation. For this purpose following experiments were designed:Partâ… :Establishment of vagotonic atrial fibrillation model in mongrel dogs;Partâ…¡:Effect of acehytisine hydrochloride on vagotonic atrial fibrillation in mongrel dogs;Partâ…¢:Effect of acehytisine hydrochloride on action potential and effective refractory period in isolated guinea pig atrium.Partâ… :Establishment of Vagotonic Atrial Fibrillation Model in Mongrel DogsObjective:To induce vagotonic atrial fibrillation in mongrel dogs,and investigate the stability and lasting property of this animal model for evaluating the effect of antiarrhythmic agents on atrial fibrillation.Methods:Adult mongrel dogs of either sex were used for the study.Animals,fasted overnight,were anesthetized with a mixture of ketamine hydrochloride and diazepam (mixed at the ratio of 7:3) 1ml/kg and artificially ventilated with room air supplemented with oxygen using a veterinary respirator.During the experiment additional ketamine hydrochloride and diazepam mixture was administered to keep the animal deeply anesthetized.Four limb electrodes were positioned to obtain body electrocardiogram and connected to a physiograph recorder.Bilateral femoral veins and unilateral femoral artery(for monitoring arterial blood pressure) were cannulated and two quadrupolar electrodes(6F) were positioned in the right atrium appendage (RAA) and the tricuspid orifice(AVJ) via femoral vein at the guide of X ray for atrial pacing and recording the intra-cardiac electrocardiogram.These electrodes were connected to a programmed stimulator and a physiograph recorder.Baseline characteristics including intra-arterial blood pressure,body electrocardiogram,atrial electrocardiogram,and atrial-ventricular junction electrocardiogram were simultaneously recorded.The bilateral cervical vagal nerves were isolated and decentralized,prepared for electrical stimulation with a home-made pin-like electrodes.Threshold of atrium and atrial refractory period were measured.Atrial fibrillation was produced by a brief burst of atrial pacing in the presence of bilateral vagal stimulation delivered by a programmed stimulator with a pulse of 0.1 millisecond and an applied voltage.The vagal stimulation frequency was adjusted in each dog to the threshold for an asystole under control conditions.Bilateral vagal stimulation should not be terminated during atrial fibrillation persistence.Successful atrial fibrillation model was defined as a rapid and irregular atrial rhythm(400-600 bpm) lasting more than 30 minutes.Results:1.Total 38 mongrel dogs,21(55.3%) male,weighing 15～27kg,were used in the study,atrial fibrillation was successfully induced in 30(78.9%)dogs;2.In 11/12 (91.7%) dogs atrial fibrillation terminated within seconds after stopping vagal stimulation;3.Atrial fibrillation persisted in the presence of vagal stimulation for more than one and half an hour without any interruption;4.Median of vagal stimulation voltage and frequency were 4 V,13.3 Hz;5.Median of atrial pacing voltage and frequency were 3V,10Hz,mean of atrial pacing duration was 1.5s; median of atrial pacing times was 4. Conclusions:It was easy to induce vagotonic atrial fibrillation in mongrel dogs,atrial fibrillation could persist for one and half an hour at least and terminate itself in most of animals.Vagal atrial fibrillation in mongrel dogs could be a stable animal model mimicking paroxysmal atrial fibrillation,may be used to evaluate the effect and mechanism of antiarrhythmic agents on atrial fibrillation.Partâ…¡:Effect of Acehytisine Hydrochloride on Vagotonic Atrial Fibrillation in Mongrel DogsObjective:To investigate the effect of acehytisine hydrochloride(AHH) on vagotonic atrial fibrillation in mongrel dog models,and its antiarrhythmic mechanism.Methods:30 adult mongrel dogs with either sex,weighing 15～27kg were used for the study.Animals were anesthetized and artificially ventilated using a veterinary respirator, and randomly assigned to normal saline group,AHH low dose group,and AHH high dose group.Four limb electrodes were positioned and connected to a physiograph recorder to obtain body electrocardiogram.Bilateral femoral veins and unilateral artery were cannulated(for monitoring arterial blood pressure) and two quadrupolar electrodes(6F) were positioned in the right atrium appendage(RAA) and the tricuspid orifice(AVJ) via the femoral veins at the guide of X ray for atrial pacing and recording the intracardiac electrocardiogram.These electrodes were connected to a programmed stimulator and a physiograph recorder.Baseline characteristics including intra-arterial blood pressure,body electrocardiogram,atrial electrocardiogram,and atrial-ventricular junction electrocardiogram were simultaneously recorded.The bilateral cervical vagal nerves were isolated and decentralized,prepared for electrical stimulation with a home-made pin-like electrodes.After measuring the threshold of atrium,the atrial refractory period was measured with a train of 8 basic(S₁) stimuli followed by a premature(S₂) stimulus at basic cycle lengths(BCL) of 200,250,300 milliseconds. AERP was defined as the longest S₁S₂ interval which could not pace the atrium.Finally, Atrial fibrillation was produced by a brief burst of atrial pacing in the presence of bilateral vagal stimulation.Bilateral vagal stimulation was delivered with a pulse of 0.1 millisecond and an applied voltage of 3V～5V.Frequency of vagal stimulation was adjusted in each dog to the threshold for an asystole under control conditions.After atrial fibrillation persisted for 30 minutes,normal saline or AHH was slowly administered in 5 minutes.If atrial fibrillation was converted to sinus rhythm within 30 minutes,recorded the time and collected blood samples,simultaneously recorded the post-drug body ECG,intra-cardiac ECG and arterial blood pressure.After measuring post-drug atrial refractory period,atrial fibrillation would be re-induced at the same condition,recorded the success rate of re-induction and duration of atrial fibrillation after drug administration.If atrial fibrillation did not terminate after first dose administration,second dose should be administrated as protocol allowed.Provided atrial fibrillation persisted even after second dose administration,bilateral vagal stimulation would be terminated and spontaneous conversion to sinus rhythm was expected(electrical conversion could be used if necessary).Repeat previous protocol and end the experiment.SPSS13.0 statistic software package was used for data analysis. Paired-sample t test was used for comparison before and after agent administration,and ANOVA was used for comparison between groups,a value of p＜0.05 was considered statistically significant.Results:1.AHH terminated atrial fibrillation in 9 dogs in low dose group after first drug administration,mean time was 3min32s±1min46s;7 after first dose,and 1 after second dose in high dose group,mean time was 8min44s±13min16s;only 1 atrial fibrillation was converted to sinus rhythm in control group;both conversion rate of low dose group and high dose group were higher compared with control group (p＜0.01),total conversion rate was higher too(17/20 vs 1/10,p＜0.01),there was no difference in conversion time between two AHH groups(p＞0.05);2.Persistent atrial fibrillation could be re-induced in all 10 dogs in control group;rate of re-induction was higher than that of 1/10 in both AHH groups(p＜0.01);atrial fibrillation could not be re-induced in 5 and 6 dogs of high dose group and low dose group,respectively (p＜0.05);3.Heart rate(HR) and blood pressure:Normal saline had no effect on HR (p＞0.05);AHH low dose group and high dose group decreased HR by 24.4,47.7 bpm, respectively(p＜0.05);systolic blood pressure(SBP) and diastolic blood pressure(DBP) were not changed in three groups;4.Body and intra-cardiac electrocardiogram:sinus cycle length(SCL),T wave duration and JT interval were increased in control group (p＜0.05);other parameters were significantly changed in AHH low dose group except for T wave and A wave duration;AHH high dose group significantly changed other parameters except for QRS wave and A wave duration;compared with control group, both AHH groups increased P wave duration,PR interval and PR segment by 9.6±1.5ms,9.1±1.3ms;15.2±2.8ms,25.5±7.5ms;8.7±2.9ms,16.2±4.6ms; respectively(p＜0.05),but there was no statistical difference between two groups; compared with control group,both AHH groups prolonged QT interval by 27.8±10.8ms and 48.9±9.4ms,respectively(p＜0.01),this action was stronger in high dose group(p＜0.05);5.Atrial effective refractory period(AERP):AERP at the BCL of 200ms was prolonged by 10ms in control group(p＜0.05);AHH low dose group and high dose group significantly prolonged AERP by 44.0±11.4ms and 53.8±33.8ms, respectively(p＜0.01);compared with control group,AHH low dose group further ncreased AERP at BCL of 250ms,200ms by 28.0±6.6ms,29.9±5.0ms(p＜0.01);and AHH high dose group increased AERP by 33.8±10.6ms,33.3±9.6ms,27.7±6.2ms at BCL of 300ms,250ms,200ms(p＜0.01).Conclusions:1.AHH could terminate atrial fibrillation in 87.5%mongrel dogs, possibly due to its action of prolonging AERP;2.AHH could prevent atrial fibrillation re-induction in more than 50%mongrel dogs,showing no difference between two doses;3.AHH slowed intra-atrial and atria-ventricular conduction,and prolonged ventricular repolarization duration. Partâ…¢:Effect of Acehytisine Hydrochloride on Action Potential and Effective Refractory Period in Isolated Guinea Pig AtriumObjective:To investigate the effect of acehytisine hydrochloride(AHH) on action potential(AP) and effective refractory period(ERP) in atrium of guinea pig,and analyze the antiarrhythmic mechanism of AHH.Methods:Adult guinea pigs weighing 350g-500g were killed by cervical dislocation and the heart were immediately excised and bathed in Tyrode's solution with following composition(mmol/L):NaCl 150,KCl 5.4,glucose 10,Hepes 5,CaCl₂ 1.8,MgCl₂ 2,pH 7.35～7.40.The solution was continuously gassed with 95%O₂ and 5%CO₂ to give a pH of 7.4 and was maintained at 36.5℃±0.5℃.The obtained left atrial appendage preparations at size of 3mm×10mm were superfused with Tyrode's solution at a rate of 10ml/min,simultaneously driven by exterual electrical stimulation with rectangular current pulses(2ms duration,1.5×threshold strength) at a constant frequency(1Hz) generated by an electronic stimulator.Conventional microelectrode penetrations were made with glass microelectrodes filled with 3mol KCl for recording AP.Preparations were equilibrated in the bathing solution for at least 45 min,and measurements were performed.To determine the ERP,an extra stimulation S₂ of double the threshold voltage was inserted in the regular train of S₁ pulses with 5ms steps.The longest interval resulting no contractile response was defined as the ERP.After measuring baseline AP and ERP,atrial preparations were superfused with AHH(25μmol/L,75μmol/L,250μmol/L,500μmol/L) for 5 minutes,solved in normal saline or Tyrode's solution,respectively.Post-drug AP and ERP were measured.SPSS13.0 statistic software package was used for data analysis.Baseline values were compared with ANOVA,and paired t test was used for comparing the difference before and after drug administration.T test was used for comparing the difference at the same concentration between two groups.A value of p＜0.05 was considered statistically significant. Results:1.AHH in normal saline group:showed no effect on resting membrane potential(RMP);reduced maximum velocity of depolarization of phase 0(Vmax) by 28.3%,25.4%,17.1%,25.8%;reduced APA by 31.0%,23.5%,18.2%,23.1%; decreased overshoot(OS) by 80.2%,70.7%,86.5%,100%;significantly prolonged action potential duration(APD);decreased maximum velocity of repolarization (MAVRP) by 32.8%,18.4%,20.4%,31.5%;decreased average slope of repolarization(ASRP) by 50.2%,37.0%,38.1%,44.5%;all these changes reached statistical difference(p＜0.01);AHH in Tyrode's solution:decreased Vmax by 12.4% at 500μmol/L(p＜0.05);decreased ASRP by 19.2%(p＜0.01).Compared with Tyrode's group,25μmol/L AHH in normal saline group showed stronger effect on all parameters of APD;75μmol/L influenced all other parameters except for Vmax, APA,APD30 and MAVRP;250μmol/L for OS,Slope₂,Slope₃,APD,APD90 and ASRP;500μmol/L influenced all other parameters except for Vmax,APA and MAVRP.2.AHH in normal saline group prolonged atrial effective refractory period (AERP) at four different concentrations,all reached statistical difference;AHH in Tyrode's solution group also prolonged AERP at different concentration respectively, but only 250μmol/L,500μmol/L reached statistical significance,and this action was rate-dependent.Compared with Tyrode's solution group,AHH in normal saline group significantly prolonged AERP,except for 75μmol/L at 350ms(BCL).Conclusions:1.AHH at higher concentration reduced Vmax and tended to decrease APA,leading to the result of slowing intra-atrial conduction;2.AHH at higher concentration prolonged AERP in a rate-dependent manner,possibly being its main antiarrhythmic mechanism;3.AHH had no significant effect on atrial APD,but slightly reduced Slope of phase 2 repolarization so as to slow atrial repolarization relatively.