Various models of atrial fibrillation induced by acetylcholine and its application in the field of traditional Chinese medicine

Qin Zhang, Ping Wang, Lin Wu

1The Key laboratory of Chinese internal medicine of MOE, BUCM, Beijing, China. 2Tianjin University of Traditional Chinese Medicine. Tianjin, China. 3The first Hospital of Peking University, Beijing, China.

Abstract Atrial fibrillation is of public health importance and profoundly increases morbidity, mortality and health-related expenditures. Morbidities include the increased risks of cardiovascular outcomes such as heart failure and stroke, and the deleterious effects on quality of life, functional status and cognition. Our purpose is to understand the molecular mechanism of atrial fibrillation, a model under pathological conditions should be established. Acetylcholine, a neurotransmitter in the vagus nerve, has been established with numerous atrial fibrillation models. We will discuss the established model of atrial fibrillation which induced by acetylcholine, aiming to seek a model which are more close to the clinical condition. The underlying mechanisms will be discussed in order to observe the mechanism of antiarrhythmic drugs.

Keywords: Acetylcholine, Atrial fibrillation, In vivo model, In vitro model

Background

Atrial fibrillation (AF) has profound clinical and public health burdens, which have grown over the last several decades [1]. The prevalence of AF is expected to increase markedly with the aging population. A study for dog’s heart showed that the vagus nerve was distributed in the epicardial fat pad which was in the junction of the right pulmonary vein and the left atrium. Evidences pointed that the vagus nerve played an important role in the occurrence and maintenance of AF. Effective refractory period (ERP) acts as a protective mechanism and keeps the heart rate in check and prevents arrhythmias. One of the mechanism may be an effective short period of refractory of the atrial and pulmonary veins. Another mechanism is the reduction of internal conduction of the atrium. In addition, vagal nerve stimulation can shorten AF cyclelength which is more obvious in the pulmonary vein. Acetylcholine (Ach) is a vagus neurotransmitter. AF model induced by Ach is consistent with the clinical vagus idiopathic atrial fibrillation. Therefore, establishing a stable atrial fibrillation model, understanding the mechanism of acetylcholine induced atrial fibrillation,and observing the effect of antiarrhythmic drugs on this model are beneficial to optimize the drugs and improve the therapeutic effect and reduce the side effects.

The relationship between acetylcholine and atrial fibrillation

In 1978, it had been shown that autonomic nerve contributed to the occurrence of AF in individual with normal cardiac function [2]. In 1960, Moeet aldeveloped a two-dimensional computer model of cholinergic AF using a model of canine atrial tissue (12.4 * 12.8cm). The theory of multiwave reentry was proposed, and AF was redefined from experimental and clinical perspectives.The pathological changes of AF in experiment are not coincident with those in clinic because of the model of canine atrial tissue. Subsequently, it has been well known that the vagus nerve, was postulated to play an important role in the initiation and maintenance of AF and used for the guidance of the treatment of AF. Wijffelset al[3]found that persistent AF can be produced by rapid atrial pacing, and then the episodes of AF can be produced by continuous infusion of acetylcholine and rapid pacing.

Ach is an organic chemical that functions in the brain and body in many types of animals, including humans, as a neurotransmitter—a chemical released by nerve cells to send signals to other cells. Acetylcholine is the neurotransmitter found at all nervemuscle junctions and many other sites in the nervous system. Ach is released by vagus nerve and has shown importance in occurrence and maintenance of AF [4]. Acetylcholinesterase can be effectively decomposed at the site where acetylcholine releases to prevent the increase of acetylcholine.Therefore, the effect of acetylcholine is limited. The spatial heterogeneity of the acetylcholine effect under the activation of the vagus nerve is very important in the occurrence of AF.

Acetylcholine receptors on the heart is a subtype of M2 muscari, acetylcholine acts on acetylcholine sensitive potassium current (IKach) through M2 receptor. IKach activates the outward potassium current and flows through the depolarizing stage of cardiac action potential(AP), resulting in a significant decrease in Action potential duration (APD), which is easy to induce AF.

Several AF models induced by acetylcholine

AF model induced by acetylcholine is divided intoin vivomodel andin vitromodel. In the two models, the mechanism, duration and stability of AF vary according to the experimental animals, the way of administration,the location of administration and the way of stimulation.

In vivo AF model

In order to study the mechanism and treatment of AF, we need to establish a stable and reproducible animal model of AF which can simulate the clinical situation based on different causes.

Anson M.Lee [5] used domesticated pig as the experimental animal. A median sternotomy and pericardial suspension were performed to expose the heart. Two bipolar electrodes were performed with suture fixation in the right atrium, a bipolar electrode for pacing.Another electrode was used to record the atrial electrogram, baseline pacing threshold each time at the beginning of the experiment. Then pacing at 2 times diastolic threshold with S1S2 stimulation was used to measure ERP. Catheter was inserted into the left atrium through a purse string suture. Then continuous infusion of acetylcholine (0.005 mg/kg/min) was begun, and ERP was measured per 5min. The concentration of Ach was gradually increased, respectively 0.005, 0.01, 0.015, 0.02 mg/kg/min. The relationship between ERP and Ach dose-responses curves were performed by venous infusion, and the mechanism may be the increase of atrial area and the decrease of ERP.

Zhou Zhiwenet al[6] put intravenous IV catheter system into rabbit’s ear for inputting of anesthesia and drugs, and subcutaneous implantating with ECG measuring. Under the fluoroscopic guidance, multipolar right ventricular electrode was put into the right atrium through the left external jugular vein to connect with the precordial V1 and cardiac electrophysiological stimulation system. And Ach (10 ml/h) of 10 mM were performed by venous infusion with rapid atrial pacing,and AF was successfully induced. AF was defined as completely irregular room rate and average perimeter of ventricular rate that is less than 150 ms. The mechanism of this model is that Ach induced AF by activating potassium currents through activating atrial muscle and shortening atrial repolarization and refractory period.

Yang Zhaoxuet al[7] performed continuous intravenous injection of Ach with rapid atrial pacing.Before giving Ach, the rapid atrial pacing failed to induce persistent AF. With the increasing of Ach, the duration of AF prolonged, when Ach up to a certain amount (18±8μg/ kg·min), persistent AF of 10 dogs were induced by rapid atrial pacing. The mechanism was shortening atrial ERP and a formation of reentrant ring. The self-discipline of the sinoatrial node was reduced, and the self-discipline of the ectopic pacing point was increased relatively, and the inhibitory effect of the sinoatrial node on ectopic excitability was weakened. These are many the factors that AF can be induced, and it is easy to maintain AF after the onset of AF. The mechanism is consistent with the reentry mechanism of the current clinical AF.

Geng Ning [8] put 0.5 ml acetylcholine (10 mmol/L)injection into fat pad between right pulmonary vein and right atrial of the rabbit. Gave Pre programmed stimulation at the right atrium. Before the injection of acetylcholine, only paroxysmal AF could be induced.After Ach injection, fast frequency AF could be induced and continued for a long time. The mechanism of shortening atrial effective refractory period may be related to the releasing of more neurotransmitters in the right atrial autonomic nerve fibers with the increasing of the intensity of the right atrium. In this model, Ach is injected into the right atrial fat pad to establish persistent focal atrial fibrillation without rapid atrial stimulation. It takes shorter time and is much easier.

Oleg F [9] ligated the vagus nerve and sutured the bipolar electrode and the multi electrode epicardial template to the right atrium (RA). The sinus node was perfused with Ach (9 ml/min) to mimic the rapid and stable effect of neurotransmitters on the sinoatrial node region. All animals could induce AF when Ach was (2.8 +0.3 mol/L). There are two possible mechanisms, one is delayed after depolarization, the other is a transient perfusion of Ach in sinoatrial node after the advantages of non reactive pacemake (transiently unexcitable). And the transient reactive cells in the sinoatrial node area and pacemaker potential performance of the regional function disorder, and diffusion wave collision encounter the disorder may be broken, resulting in arrhythmias. This model is the first to demonstrate that the same effect can be produced with no electrical stimulation of the beta adrenaline analogue catecholamine, making spontaneous AF. This model cut the vagus nerve in the neck to prevent the vagus reflexing from reacting to the injection of neurotransmitters.

An minshenget alused adult mongrel dogs and opened the chest and pericardium suspended in the chest wall.Special needle electrodes were implanted into the limbs to record ECG. Multipolar electrode was stitched respectively to the left and right atria connecting polygraph. A group was deal with pulmonary vein ablation and fat pad injection Ach, B group was deal with the injection of Ach at superior vena cava, while giving BURST stimulation. The two groups were able to induce sustained and stable AF. This model has proved that the local concentration of ACH is associated with the complex fractionated atrial electrograms (CFAE), and CFAE has been shown to have a close relationship with the production of atrial fibrillation [10].

Isolated atrial fibrillation model

AF induced by acetylcholine Langendorff perfusion in vitro.Pan Lei [11] used Langendorff aortic perfusion,activating of cholinergic acetylcholine perfusion with burst stimulation, in order to establish a simple and feasible rabbit heart model of atrial fibrillation which is closed to clinic. Ach (8 μmmol) was perfused and burst stimulation can successfully induce stable AF. The underlying mechanism of AF is Ach, which can reduce the autonomic function of sinoatrial node, shorten the effective refractory period of atrium, and induce the electrical remodeling of atrial muscle. In addition,acetylcholine directly inhibited the calcium channel and reduced myocardial contractility by changing the homeostasis of intracellular calcium and triggering the remodeling of contractile function of the atrial muscle.The preparation of Langendorffin vitroperfusion device is relatively simple, repeatable, and has high induced rate of AF, relatively stable experimental conditions and less interference factors. It could be easily applied to the research and development of new drugs.

Four cavity perfusion model of isolated rabbit heart.After the heart was removed, the inferior vena cava and the left and right inferior pulmonary veins and the right superior pulmonary vein were ligated respectively [12].They were connected to the four chamber perfusion system respectively. The homemade bipolar ECG recording electrodes were fixed to the left atrial side wall,right atrium and pulmonary vein area. A large number of AF occurred in the pulmonary vein through Continuous infusion of Ach with rapid atrial pacing. The susceptibility to AF at all parts of the atrium is different.The pulmonary venous area was most likely to induce local tremor which followed by the left atrium. The heterogeneity of atrial electrophysiological characteristics and the dispersion of spatial distribution were the conditions of AF. Vagus nerve may induce AF through this mechanism, but it is more likely to play a role in enhancing the incidence of AF. This model is to understand the effect of ACh on AF by simulating coronary perfusion in a simulated physiological state. The perfusion mode is pumped by the heart itself into the coronary artery, rather than the Langendorff mode, which is injected by the external pump. And it ensures the uniform perfusion of the muscle cells in the whole atrium.The spatial dispersion caused by vagus nerve distribution is increased. Meanwhile, during the perfusion process,atrial periodic physiological tension changes, and the effect of tension change on atrial fibrillation is eliminated.

Frequency sampling technique to describe the spatial distribution of excitation frequency of AF.

Ravi Mandapati [13]et altook lamb as an experimental model. After anesthesia, the heart was perfused by Langendorff Ach (0.1-0.5 μmol/L) perfusion with rapid cardiac pacing, which successfully induced AF. There are two mechanisms for this model. The first one is the formation of reentrant activities. The second is the functional return wave around a fibrous circuit, which appears in the form of a spiral wave. This first one is the most likely mechanism. This model used a new frequency sampling technique to describe the spatial distribution of AF excitation frequency, which can accurately identify the loci of periodic activity during arrhythmia. This study confirms that these local points are associated with the maintenance of AF.

Patch clamp recording Kach current.

Kyoung-Im Cho [14] put mice heart into 37 degrees Tyrode’s solution which did not contain calcium(containing collagenase perfusion). Isolated atrial myocytes were given acetylcholine (100μmol, 2 min) to induce Kach current and whole cell recording Kach through patch clamp. Kach plays an important role in the occurrence of AF.

In these animal models, the dog’s heart is a good animal model, because the dog’s heart provides distribution of ion channel, and the activity of which is similar to humans. The electrocardiogram and conduction time are also similar to those of humans. So dogs are considered to be the best animal model for preclinical trials of human cardiac electrophysiological activity [15].The mechanism underlying atrial fibrillation that induced by Ach is important to the understanding of atrial fibrillation and the treatment of atrial fibrillation. The current model of AF induced by Ach has gradually matured. The effect of the drug can be observed by the establishment ofin vivomodel and anin vitromodel of different mechanisms.In vivomodel of AF which is regulated by neurohumoral factors is much closer to clinical status. The acute AF model induced by Ach atrial rapid pacingin vivohas the characteristics of high induced rate and long duration of AF and safety and simple fabrication. The mechanism of AF and the effect on cardiac function can partially simulate the pathophysiology process of AF in clinical practice. The experimental condition of the AFin vitrois easy to control, avoiding many internal influences including nerve and endocrine, eliminating the interference of other factors, controlling variables better under artificial control conditions, so as to observe the influence of exerting factors.

The application of traditional Chinese medicine in the acetylcholine atrial fibrillation model

At present, the treatment of AF in western medicine mainly includes: rate control and rhythm control. The effect of western medicine in the treatment of acute atrial fibrillation is obvious, but it is limited because of the recurrence of atrial fibrillation, visceral injury and side effects such as severe arrhythmia and so on [16],especially in the long-term application. Another effective treatment is catheter ablation, but it is not the best choice because AF is prone to recurrent attacks and the patient has to undergo multiple operations. In addition, there is a risk of antithrombotic therapy, such as bleeding,especially in the elderly. Chinese medicine which acts as a complementary and alternative treatment has made great progress in the treatment of AF. Some single Chinese patent medicines or traditional Chinese medicine active ingredients or non pharmacological treatment methods (acupuncture, Tai Chi, etc.) can improve symptoms such as palpitation and chest tightness in patients with AF, improving their tolerance to disease and quality of life. In addition, it is reported that many Chinese medicine components participate in the mechanisms of multiple ion channels, including inhibiting inflammatory factors, active antioxidants and anti platelet aggregation.

Extract of Chinese Medicine

Berberine is an important alkaloid that is extracted from the Rhizoma Coptidis. Modern pharmacological studies confirmed that berberine had significant anti heart failure,arrhythmia, lower cholesterol, anti-proliferation of vascular smooth muscle cells, improving insulin resistance, antiplatelet, anti-inflammatory and other effects. Disease of cardiovascular system and nervous system may have wide and important application prospects, and it gets more and more attention. A patch clamp technique was used to record the isolated cardiomyocytes which isolated from the heart of the rabbit. Acetylcholine perfusion significantly shortened the APD in isolated atrial myocytes (APD 50:152 + 13 / 81 +10ms; APD 90:256 + 19 /132 to 13ms,P＜ 0.01). The use of berberine (2 mg/kg) reverses the acetylcholine (APD 50:81 + 10 vs.134 + 15ms; APD 90:132 + 1vs:213 +17ms,P＜ 0.01) induced by APD shortening [17].Flavonoids from Saussurea involucrata and Acacia can inhibit the occurrence of AF of dog by prolonging APD and ERP. But they have no effect on the QT interval. The possible mechanism is to inhibit acetylcholine sensitive potassium current (IKach) [18].

Chinese patent medicine

Wenxin Keli comprises with Dangshen (Salviae Miltiorrhizae Radix Et Rhizoma), Huangjing (Polygonati Rhizoma), Sanqi (Notoginseng Radix Et Rhizoma), Hupo(Succinum), Gansong (Nardostachyos Radix Et Rhizoma).Wenxin Keli has a clear theoretical basis for the treatment of AF. Modern clinical research of cardiac electrical physiological technology confirmed that the [19]mechanism of Wenxin Keli has a variety of anti-arrhythmia. The isolated heart of dog with Acetylcholine (0.5-1 μM) coronary perfusion can successfully induce AF, and then 5 g/L was added to the perfusion solution, which successfully terminated AF and could not be induced AF again. The ERP and APD90can be prolonged significantly.

Conclusion

AF is a complex and heterogeneous disease. The incidence rate increases with age. In 2012, epidemiology in Europe showed that the prevalence of AF in age group(50-59) was 0.5%, while almost 9% in group (80-89).Europe has more than 600 million of patients with AF,and USA has about 230 million of AF patients. It is estimated that patients with AF will increase exponentially in the next 50 years [20] with the increase of population. AF has a high incidence rate, and the harm is great. Stroke is the most common complication of AF,the probability of AF in patients with thromboembolic stroke is 5 times than normal. The probability of occurrence of congestive heart failure was 2 times higher than normal people. In addition, AF can induce some other neurodegenerative diseases, including cognitive impairment after adjustment, dementia, or ischemic stroke [21], which severely affect the life quality of patients. Current therapies are aiming at reducing heart rate, blocking reentry pathways and isolating ectopic lesions. In order to find better treatment, researchers are committed to finding new therapeutic targets and further understanding and classifying targets. Studies have shown that reducing autonomic innervation or outflow can reduce the incidence of spontaneous or induce atrial arrhythmia, suggesting that neural regulation may help in controlling AF [22-24]. We expect to find a mature acetylcholine model, which can better simulate the clinical state to observe the effect of drugs on this mechanism, so as to study the safety and efficacy of drugs.

AF is usually caused by rapid ectopic activity around the pulmonary veins, resulting in a stable reentry circuit,which is associated with short time wavelet propagation through the whole atrium [25]. There are a lot of vagus plexus in the fat pad of the pulmonary vein, so the activity of the vagus nerve may have a great relationship with the production of AF.

The cardiac autonomic nervous system is made up of endogenous and exogenous nervous system. These neurons stimulate the influence of plexus ablation on the pulmonary venous sleeves and myocardial cells, and provide important information for the occurrence and maintenance of AF [26]. The vagus nerve belongs to the endogenous autonomic nervous system and stimulates the vagus nerve system to deteriorate the electrical remodeling of the atrium. Ach is released from the postganglionic fibers of vagus nerve. When binding to the cholinergic receptor, acetylcholine produces negative time-varying, inotropic and variable conduction. It shortens the ERP of the atrium, making it easier to form exhumation. In addition, it can shorten the APD and improve the action potential dispersion, and make the atrial fibrillation more easily to be induced. In addition,acetylcholine can change the physiological characteristics of pulmonary vein It can significantly shorten the effective refractory period of various parts of pulmonary vein and reduce the threshold of AF [27].In clinic, more than 70% of AF is related to underlying diseases [28], including congestive heart failure,hypertension, valvular heart disease, and coronary artery disease [29]. These models are based on normal heart and still far away from the clinical state. A recent study found that vagus nerve underlying diseases of the human atrial muscle had no obvious effect on human atrium,suggesting that acetylcholine may have more influence on lone atrial fibrillation. Experiments have shown that AF induced by adrenaline and acetylcholine is more stable and longer duration. In the future, we can strengthen the research of these two aspects.