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A Study on Endocardial Monophasic Action Potentials and Triggered Arrhythmias in Rabbit Heart in vivo

Gengsheng Yu, Li Zou, Wanzhen Li *, Xiaomei Li*, Jiarong Zhong

Children’s Hospital, Chong Qing University of Medical Sciences
Chong Qing, P.R China, 400014
* Department of Pediatrics, The First Hospital, Beijing Medicine University
Beijing 100034

Abstract
Introduction
Material and Methods
Results
Discussion
References

Abstract
Introduction: Monophasic action potential (MAP) recording by using a contact electrode tecnique allows us to provides an important bridge between basic and clinical electrophysiologicaly.
Objectives: This study was to investigate the mechanism, diagnosis and treatment of triggered arrhythmias.
Material and Methods: Twenty-one Rabbits entered the study and they were divided into three groups: Group A (control): received CsCl. alone, group B: received verapamil or motoprolol before CsCl-induced triggered activities, group C: received verapamil or ATP after CsCl-induced triggered activities.
Results: Group A : Early Afterdepolarizations (EADs) appeared in the middle-later period of phase 3 in MAP after CsCl; The amplitude of EAD was 6.3 ±0.67mV, The percentage of total MAP amplitude was 33.8±4.36%. A threshold cumulative dose of CsCl-induced sustained VT was 1.6±0+0.28 mmol/kg. Group B: CsCl injected first After administering metoprolol elicited EAD,too. The amplitude of the EAD or DAD gradually increased in size until ventricular arrhythmias happened (VPCs or VT). A threshold cumulative dose was 1.8±0.14 mmol/kg(P> O.05). CsCl injected first after administering verapamil elicited EADs, but ventricular arrhythmias didn’t happened. A threshold cumulative dose was 3.4+0.31mmol/kg(P<0.01). Group C:Verapamil inhihited the triggered activities slowly at beginning but it lasted longer . ATP inhibited the triggered activities fast at beginning, but it lasted shorter. And after effect of ATP was gone, EADs induced by CsCl appeared again. Then ATP terminated DAD induced by CsCl.
Discussion: The results showed that verapamil could not only prevent CsCl-induced triggered arrhythmias but also terminate it, which suggested that CsCl-induced triggered activity was related to Ca2++ closely and the abnormal calcium transport was probably involved in the mechanisms of induced early after depolarization. The fact that metoprolol could hardly terminate CsCl-induced triggered arrhythmias indicated the limited effect of sympathetic nervous tension on triggered activity. Fur the more, ATP could has biphasic effect on afterdepolarization by CsCl.
Conclusion: In conclusion, monophasic action potential is valuable in studying triggered activity in vivo.

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Introduction

Monophasic action potential (MAP) of the left ventricular endocardial in 21 rabbits was recorded by using a contact electrode. The present study had built the rabbit model of CsCl-induced triggered activities, for investigating the effects of verapamil, metoprolol and adenosine triphosphate (ATP) on the triggered arrhythmias. This study was designed to investigated the generation of the triggered arrhythmias, the diagnosis of the triggered arrhythmias and the evaluation of the drug effects on the triggered activities.

Materials and Methods

1.Animals and MAP Recording Methods

21 healthy rabbits are chosen, weighing from 2.8 to 3.8 kg, covering both male and female. 20% Urethane (1.0g/kg) was used for intravenous anesthesia. Contact electrode catheter was put in through the neck artery to record MAP of left ventricular endocardium. The signals were amplified through a Japanese Photoelectric AB-601G bioelectricity amplifier and then were sent to a physiologic recorder, recording simultaneously ECG, MAP and its zero-phase maximal upstroke velocity (Vmax). A nonpolarizable electrode was gently pressed against the endocardium of the rabbit. Drugs were applied after recording 5 minutes of a stable MAP recording.

2.Groups and Injection Methods

2.1.Control group (the model of CsCl-induced triggered activities): five rabbits were taken; one minute after injecting 5 ml of normal saline, CsCl (1.0mmol/kg, within 15 seconds) was put in through intravenous injection; Changes of all indicators were recorded 10,20,30 seconds, 1,3,5,10,15 minutes after; intravenous injection of CsCl 0.5 mmol/kg was repeated at 15 minutes intervals until sustained ventricular tachycardia was produced; CsCl threshold cumulative dose was recorded.

2.2.Drug-prevented Group referring to the influence of drugs on the formation of CsCl-induced triggered activities,divided further into two subgroups—one with Verapamil, the other with Metoprolol—each had four rabbits which had intravenous injection of Verapamil (0.5mg/kg) and Metoprolol (0.2mg/kg) within 2 minutes accordingly. One minute after that, CsCl was applied the same way as in the control group.

2.3.Drug-terminated Group referring to the influence of drugs on triggered activities already induced by CsCl, divided further into Verapamil and ATP subgroups. Each subgroup had four rabbits that had intravenous injection of Verapamil (0.25mg/kg, within 2 minutes) and ATP (1mg/kg, within 3 seconds) accordingly. Changes of all indicators were recorded before and after using the drugs.

3.Measurement

3. 1 .MAP parameters:MAP amplitude (MAPA, mV); (2) MAP duration(MAPD, ms): MAPD 50(MAP duration of a repolarization level of 50%) and MAPD 90 (MAP duration of a repolarization level of 90%); (3) The maximal upstroke velocity (Vmax, V/s) of zero-phase MAP.

3.2.After depolarization parameters: after depolarization amplitude (EADA or DADA, mV); (2) DADA's percentage in MAPA (EADA% or DADA%).

3.3. ECG parameters: (1)Heart rate (HR, times/min); (2) Q-T interval (ms).

4. Statistic Analysis

Experimental data was represented as at means + standard errors (X+SE). Significance test adopted variance analysis and then had q test. Before and after using drugs pairing t test was applied and statistic analysis was realized with a Primer statistic software.A p<0.05 was considered indicative of statistically significantly difference.

Results:

1.CsC´s influence on the MAP of endocardium
For the control group, after the CsCl injection, which reached maximal effect at 20-30 seconds, heart rate became significantly slower with MAPA decreased gradually. There was a significant prolonging of the 3-phase (MAPD 90) and a gradual decreasing of zero-phase Vmax. Both were significant compared with the situations before using the drugs (P<0.05). Afterpolarization, mainly EADs, occurred at the 3-phase. Their EAD shape ranged from " tail", "plateau", to "hump" shape. The amplitude reached as high as 6.3+0.7mV. Its EADA% is 33.8%±0.4% and triggered arrhythmias (see appendix). A threshold accumulative dose of CsCl-induced sustained VT is 1.60+0.28mmol/kg.

2.Drug-prevented group’s effect on CsCl-induced triggered activities
The results showed that injecting verapamil and Metoprolol alone would have no significant influence on MAPA, MAPD90, Vmax, HR, and Q-T interval (P>0.05). After injecting CsCl, HR and MAPD90 of the verapamil subgroup decreased or prolonged(P<0.05).There were no significant difference between other parameters with those of the control group.

For the drug-prevented group, afterdepolarization also was induced. Their EADA% is: 29.7%+3.6% for the Verapamil subgroup, 26.2%+1.8%foe the Metoprolol subgroup, and 33.8%+4.4%for the control group. Compared with the control group, prevented group’s EADA% decreased a certain amount but not significantly. All the EADs induced by the first dose of Verapamil subgroup had "tail" shape, however, it could not trigger arrhythiams. This caused a sustainable VT’s CsCl threshold accumulative dose to reach as high as 3.4+0.3mmol/kg, which had significant difference with that of the control group (P<0.01). For the Metoprolol subgroup, the first-dose CsCl-induced EAD was somewhat restrained. It had "plateau" shape and high probability of secondary DAD, both of which can trigger arrhythiams, and polymorphic VT. Repeated injection of CsCl would more easily induce sustained VT, which made CsCl threshold accumulative dose of sustained VT have no significant difference from that of the control group.

3. Drug-terminated group’s effect on CsCl-induced triggered activities
Injecting CsCl alone produced no significant changes in parameters like MAPD50, MAPA, and Vmax, while MAPD90 and Q-T intervals were clearly prolonged. After injecting Verapamil or ATP, MAPD90 and Q-T intervals were shortened, reaching the level before injection of CsCl. What is worth noticing is ATP’s effect lasted only a short time. In several seconds MAPD90 was prolonged again, with accompanying EAD. For Vmax, MAPA’s influence was first restraining and then resuming gradually in several seconds.

After 20-30 seconds of the influence peak of CsCl, injecting both Verapamil and ATP could suppress EAD and ventricular arrhythiamia it induced. Verapamil’s restraining of EADs started slowly but reached ultimate control in the end. ATP’s restraining started faster but lasted also for shorter period of time, and EAD could reappear after its effect disappeared. When applying ATP to stop the Secondary DAD after CsCl-induced EAD, an instant accentuated DAD would occur, and arrhythiams and even ventricular fibrillation would be induced. Then DAD would be restrained and triggered activities would be eliminated.

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Discussion:

1.Characteristics of CsCl-induced triggered activities of hearts in vivo

Triggered activities include afterdepolarization and triggered arrhythiams. Afterdepolarization is a transient membrane oscillation potential, including EAD and DAD. When this occurs early ,before repolarization is complete ,its called EAD;when is comes after the completion of repolarization ,it is called delayed afterdepolarization (DAD)(1). According to its shapes, it is divided into "tail", "plateau", and "hump". We observed during the experiments that: (1) All CsCl-induced EAD occurred at the end of MAP’s three phases, featuring an obvious prolongion of MAPD90. (2) The process of EAD inducing goes from "tail" to "Plateau" or "Hump" shape. Arrhythiams is triggered at "plateau" or "hump". Neither is triggered at "tail" shape. (3) The Characteristics of CsCl-induced triggered arrhythmias are: Ventricular arrhythmia triggered at EAD’s peak , ectopic rhythm of heart corresponded with dominant EAD at matching intervals . Triggered during the decreasing of EAD, identical with DAD during matching interval which caused the inference of its being triggered by DAD ,ectopic rhythm of heart triggered at secondary DAD peaks. (4). The frequency of triggered arrythmias parallels with the accumulative dosage of CsCl, so a big dose can directly induce ventricular fibrillation.

2. The mechanism of triggered activities

The mechanism of CsCl-induced afterdepolarization depends on that suppression of extroverted potassium current, prolongion of action potentials, and relative increase of introverted current resulted in EAD(2). This introverted current’s characteristics are considered closely correlated with Ca++(3). As the secondary DAD’s mechanism, it is considered to be correlated with prolongion of action potentials, extending of Ca++inversion time, and intracellular Ca++ overloading because of delayed discharge(4).

Our experiment also shows: CsCl-induced EAD, DAD, and triggered arrhythmia can all be terminated by Verapamil the Calcium current blocker; ATP also works well to terminate them. ATP can restrain L-type Calcium current, and reduce Calcium introversion, both suggesting that CsCl-induced triggered activities be closely connected with reduction of Ca++ introversion. The fact ß receptor blocker Metoprolol cannot effectively prevent CsCl from triggering activities indicates that a sympathetic nerve has only a limited influence.

3.Adenosine’s influence on triggered activities

ATP dissolves quickly into adenosine upon being injected into human blood. Many researchers have studied adenosine's action on triggered activities. It has already been proved that adenosine can terminate the triggered activities mediated by cyclic adenosine monophosphate (cAMP)(5). Investigation on isolated myocardial cells showed that ATP can promote the formation of afterdepolarization and triggered activities when the input of Ca++ is increased(6), but no report had been seen about the influence of adenosine over hearts in vivo with directly recorded MAP triggered activities. This experiment demonstrates: ATP is effective in preventing CsCl-induced EAD, while has a biphasic effects on CsCl-induced DAD (aggravating DAD at the beginning briefly, inducing arrhythmia even ventricular fibrillation); then it quickly turns to suppress and terminate the triggered activities.

ATP’s biphasic effects on CsCl-induced DAD are probably because that the occurrence of DAD is related to the overloading of Calcium. As the introverted current of Calcium increases(7), ATP will activate the adenosine-sensitive calcium channel in sarcoplasm reticulum(8), which will result in a short period of Ca++ release improvement. This would aggravate the calcium overloading and increase DAD and trigger ventricular rhythm of heart, even ventricular fibrillation. Then ATP quickly dissolve into adenosine, depress L-type calcium channels and reduces their number. Then the permeability of cell membrane for Ca++ will decrease(9,10), and calcium inward current will be reduced, and terminate triggered activities.

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References

1. Yuan S, Blomstrom Lundqvist C. Olsson SB. Monophasic action potentials, concepts to Practical applications. J Cardiovasc Electrophysiol, 1994 . 5: 287
2. Patterson E, Scabo B. Scherlag BJ, et al. Early and delayedafterdepoterizations associated with cesium chloride-induced arrhymias in the dog. J Cardiovasc Pharmacol, 1990, 15 :323
3. January CT, Riddle JM. Early afterdepolarizations mechanism of induction and block, A role for L-type Ca++ cur-rent. Circ Res, 1989, 64 : 977
4.Wang Zhengxing, Delayed Afterdepolarization and Triggered Activities of Myocardium and the Medicine Basis for its Treatment. Chinese Journal of Pharmacology , 1992, 8:178
5.Song Y, Thedford S, Lerman BB, et al. Adenosine-sensitive afterdepolarizations and triggered activity in guinea pig ventricular myocytes. Circ Res. 1992, 70:743
6. Song Y, I3elardinelli L. ATP promotes development of afterdepolatications and triggered activity in cardiac myocytes. Am J Physical, 1994, 267: 2005
7. Smith JS, Coronado R, Messier G. Sarcoplasmic reticulum contains adenine nucleotied-ativated calcium channels. Nature, 1985, 316: 446
8. Belardinelli L, Wu AN, Visenitin S. Adenosine regulation of cardiac electrode activity. In: Zipe DP, Jalife, eds. Cardiac electrophsiology from cell to bedside. Phdsdephiaz Saunders WB, 1990:284.
9. Lerman BB, Belardinelli L. Cardiac electrophysiology of adenosine. Basic and clinical concepts. Circulation, 1991, 83 : 1499
10. Wesly RC Jr, Turnquesi P. Torsades de pointe after intravenous adenosine in the presence of prolonged QT syndrome. Am Heart J, 1992, 123:794

 

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Update
Oct/30/1999