The Antiarrhythmic Effect And Electrophysiological Mechanism Of Cyclovithoxin D

Arrhythmia refers to any abnormality in the frequency,rhythm and impulse conduction of the heart,which can be divided into rapidity(such as various types of premature beats and tachycardia)and slowness(such as various conduction blocks,etc).Arrhythmia,in addition to organic heart disease can cause a variety of arrhythmias,other system diseases,such as hyperthyroidism,chronic obstructive pulmonary disease,blood diseases,neurosis,etc.will also appear arrhythmia.According to the 2018 Chinese Health Big Data Statistics,22% of young people died of cardio-cerebrovascular disease,one of the four major chronic diseases,and80 % of them had various degrees of arrhythmia.There are about 20 million patients with arrhythmia in my country,and about 50 % of the patients die of sudden arrhythmia.The morbidity and mortality rate are increasing year by year,which seriously affects the lives and health of patients.The four types of classic antiarrhythmic drugs generally have potential risks of causing arrhythmia,have large side effects,and are not particularly ideal for improving patient survival and prognosis.Looking for new anti-arrhythmic drugs,especially from natural plants or Chinese herbal medicines,to screen safe,effective,mild-acting,and no or low-toxic active ingredients;and to explore its mechanism of action has become a hot spot in new drug research.Cyclovirobuxine D(CVB-D)is an alkaloid monomer component extracted from the stems and leaves of Buxus chinensis and its genus plants of the Buxaceae family.It is the main effective component of the preparation of Huangyangning tablets.Huangyangning tablets have been included in the Pharmacopoeia of the People’s Republic of China(2020 Edition).More and more clinical and laboratory evidences show that the drug has a wide range of therapeutic effects on cardiovascular diseases,and it can prevent arrhythmia,myocardial ischemia,and cardiac arrhythmia.It has good curative effect on decay and so on.As an active compound,CVB-D represents the antiarrhythmic activity of Buxus sinensis to a certain extent.Due to the slow development of anti-arrhythmic drugs,CVB-D is expected to become a candidate drug for anti-arrhythmics.Studying the electrophysiological characteristics of CVB-D and its anti-arrhythmic effect site is a necessary condition for elucidating its anti-arrhythmic mechanism.The ion channel on the myocardial cell membrane is the target of anti-arrhythmic drugs.Studies have found that CVB-D mainly inhibits potassium current and prolongs the action potential of the ventricular muscle,thereby achieving the purpose of anti-arrhythmic.Single-cell patch clamp is insufficient in studying the comprehensive effects of CVB-D,especially in cardiac electrical conduction.In order to develop anti-arrhythmic drug candidates,the detailed electrophysiological properties of CVB-D need to be further studied.Therefore,this article intends to study from the three levels of animal-organ-cell to clarify the anti-arrhythmic effect and electrophysiological mechanism of CVB-D,in order to provide experimental basis for drug development and clinical medication.Part 1 Verification of the anti-arrhythmic effect of Cyclovirobuxine DObjective:Using the rat model of arrhythmia induced by surgery and drugs,the effect of CVB-D on arrhythmia was clarified.Methods:According to the conversion coefficient of human and animal body surface area,the clinical equivalent dose is obtained.Using the rat coronary artery ligation and rat aconitine-induced classic arrhythmia model,the electrophysiological recorder was used to evaluate the effect of the drug on the appearance and duration of ventricular tachycardia,and the CVB-D anti-arrhythmia score was confirmed by the arrhythmia score role of aberrations.Results:1.Effect of CVB-D on rat model of arrhythmia induced by coronary artery ligationCompared with the model group,the CVB-D group(0.6 mg/kg)can reduce the arrhythmia score from 4 ± 1 in the model group to 1 ± 0(P < 0.05);the duration of ventricular tachycardia is reduced by 258.2 ± 117.7 s decreased to 0.6 ± 0.1 s(P < 0.05);the appearance time of ventricular tachycardia was extended from 644.6 ± 167.8 to 1515.0 ± 79.0 s(P < 0.001).2.The effect of CVB-D on aconitine-induced arrhythmia model in ratsCompared with the model group,the CVB-D group(0.6 mg/kg)can significantly reduce the duration of ventricular tachycardia(P < 0.001).Conclusions:CVB-D can significantly prolong the appearance time of ventricular tachycardia,reduce the duration of ventricular tachycardia and the arrhythmia score,and it is clear that CVB-D has a therapeutic effect on ventricular arrhythmia.Part 2 To study the electrophysiological mechanism of Cyclovirobuxine D against arrhythmia at the level of isolated organs and cellsObjective:Use isolated heart and ventricular myocytes to study the electrophysiological mechanism of CVB-D anti-arrhythmic effects,and explore its anti-arrhythmic targets,with a view to providing experimental evidence for new drug development and guiding clinical rational use of drugs.Methods:First,it is planned to use the Mapping Lab multi-channel matrix cardiac electrophysiological mapping system combined with the Langendorff isolated heart perfusion system to record electrical conduction signals at the level of the isolated heart,and then use the 700 B single-cell patch clamp system to record the guinea pig ventricular muscle in the current clamp mode.The action potential of the cell was recorded in the voltage clamp mode and the potassium current and calcium current of rat ventricular myocytes.Results:1.The effect of CVB-D on the electrical conduction of the heart CVB-D(10 μM,20 μM,and 30 μM)can reduce the heart rate of rats from 305± 27 bpm before administration to 223 ± 12 bpm(P < 0.05),197 ± 9 bpm(P <0.05)And 143 ± 42 bpm(P < 0.01).CVB-D has no effect on atrioventricular node conduction,but it can significantly prolong the atrial conduction velocity at(20μM,30 μM)and the ventricular conduction velocity at(30 μM)(P <0.05).2.The effect of CVB-D on Action Potential Time Course of Guinea Pig Ventricular MyocytesCVB-D(1μM)can extend 90%(APD 90)of the action potential duration of guinea pig ventricular myocytes from 647.5 ± 58.5 ms to 1062.4 ± 112.4 ms(P < 0.001).3.The effect of CVB-D on potassium current and current-voltage(I-V)curve of rat cardiomyocytes3.1 CVB-D(10 μM,20 μM,and 30 μM)inhibited the potassium current of rat cardiomyocytes in a concentration-dependent manner(P< 0.001).3.2 CVB-D(10 μM,20 μM,and 30 μM)can increase with the concentration to move the current-voltage(I-V)relationship curve upward without affecting the current-voltage relationship.4.The effect of CVB-D on the calcium current and current-voltage(I-V)curve of rat cardiomyocytes4.1 CVB-D(1 μM,3 μM,and 10 μM)inhibited the calcium current of rat cardiomyocytes in a concentration-dependent manner(P < 0.001).At a voltage of 5 m V,the normalized calcium currents of CVB-D(1 μM,3 μM,and 10 μM)are-0.85 ± 0.06,-0.70 ± 0.07 and-0.42 ± 0.06,respectively.4.2 CVB-D(1 μM,3 μM,and 10 μM)can increase with the concentration to move the current-voltage(I-V)relationship curve upward without affecting the current-voltage relationship.Conclusions:The results of isolated heart Mapping and patch clamp showed that CVB-D prolonged the action potential time course of cardiomyocytes by inhibiting the calcium current and potassium current of cardiomyocytes in a concentration-dependent manner;inhibiting the autonomy and conductivity of the heart,and slowing down the heart rate.Slow down the atrium and ventricular conduction velocity,and inhibit the negative conduction of the heart;play an anti-arrhythmic effect.