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Research Article

2018; 11(3): 88-96

Published online June 1, 2018 https://doi.org/10.1016/j.jams.2018.03.002

Copyright © Medical Association of Pharmacopuncture Institute.

The Beneficial Effects of Electroacupuncture at PC6 Acupoints (Neiguan) on Myocardial Ischemia in ASIC3 −/− mice

Ying-Wang1*, Wan-shuang Zhao1, Di Li1, Ya-han Xu1, Meng-di Li1, Jin Chen1, Zhi-jun Kou2, Qi-ge Wang1, Yi-guo Chen1*, Nsoa dimitri Joseph1

1Dept. of Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China
2Dept. of Zhejiang University of Traditional Chinese Medicine, Hangzhou 310053, China

Correspondence to:Yi-guo Chen

Received: September 11, 2017; Revised: February 22, 2018; Accepted: March 19, 2018

http://creativecommons.org/licenses/by-nc-nd/4.0/

Abstract

This study aims to investigate the possible mechanisms of electroacupuncture (EA) at PC6 to improve myocardial ischemia (MI) by regulating the cardiac transient outward potassium current channel (Ito). According to the random number table, the mice were divided into six groups of six mice each: control group, MI group, PC6, LU7 (Lieque-point), ST36 (Zusanli-point), and nonacupoint group. Mice in the control group were injected with saline (20 mg/kg, 24 hours interval), and the other ASIC3 −/− mice were injected subcutaneously twice with isoproterenol (ISO) (20 mg/kg, 24 hours interval). In the preexperiment, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg of ISO were used, and the results showed that 5 mg/kg and 10 mg/kg of ISO both could induce acute MI, but shorter duration of sustained MI. On the other hand, an injection of 30 mg/kg can make the mice experience arrhythmia or die immediately, and EA was operated at PC6, LU7, ST36 acupoints, and nonacupoint in the mice of PC6, LU7, ST36, and nonacupoint groups, respectively, after injecting twice. Then Western blotting techniques (Western Blot) were used to analyze the protein expressions of Kv1.4, Kv4.2, Kv4.3, and KchIP2. The results of this experiment showed that the protein expressions of Kv1.4, Kv4.2, Kv4.3, and KChIP2 in MI group were significantly lower than those in the control group (p < 0.01). Compared with MI group, the results of PC6, LU7, and ST36 groups obviously increased (p < 0.05). Furthermore, the expressions of PC6 group were higher than LU7 group and ST36 group (p < 0.05). And electrocardiogram's T-waves showed obvious pathological changes in the MI group compared to the control group (p < 0.01). After EA, the abnormal T-waves voltage of ECG in PC6, LU7, and ST36 groups was improved (p < 0.05). In addition, the rate change of PC6 group was larger than that of both LU7 and ST36 groups (p < 0.05). But the T-waves voltage of the nonacupoint group was not significantly different than that of the MI group (p > 0.05).

Keywords: ASIC3 &minus,/&minus, mice, cardiac transient outward potassium current channel, electroacupuncture, myocardial ischemia, PC6 (Neiguan-point), protein

1. Introduction

The heart is a vital organ for the human body, which regulates blood circulation for all the other organs by causing blood to flow continuously in the blood vessels, thereby maintaining normal physiological functions of human body. At the same time, it is an aerobic organ, and its main source of energy is entirely derived from the oxidation (of free fatty acids and glucose) and the metabolism (of the myocardium). Normally, the heart takes a large amount of oxygen from coronary circulation to maintain its normal physiological functions. If the heart activity increases, the coronary arteries often dilate to increase blood supply to the body [1]. In recent years, many changes in peoplés lifestyle, such as increased intake of foods high in saturated fats, high-cholesterol foods, decreased physical activity, smoking, and other bad habits, further increased the incidence of cardiovascular diseases which have become a very serious threat to the global health. In China, about 290 million people are suffering from cardiovascular diseases, and there are still rising morbidity and mortality rate among people suffering from these diseases. It is accounts for 40% of all cases of mortality, even higher than cancers and other diseases death rate [2]. Myocardial ischemia (MI) is a common cardiovascular disease, mostly caused by coronary atherosclerosis, vascular spasm stenosis, or heart valves diseases and others causes of coronary blood flow reduction. And it also has typical clinical manifestations, such as angina pectoris, arrhythmia, myocardial infarction, cardiac dysfunction, and even sudden death. This is why it has a higher mortality rate. Patients often complain of chest tightness and chest pain which sometimes radiates to the left shoulder or left arm. These symptoms can be mitigated after taking medication or a period of rest. But in severe cases, they may be persistent and cause severe pain such that rest or oral medications cannot relieve them [3].

Voltage-gated potassium channels (Kvs) are potassium channels sensitive to voltage changes in cell membrane, and they are widely distributed in the cell membrane [4]. Kvs on myocardial cell membrane are involved in the recovery process of myocardium electrical excitability, which performs a decisive role in action potential duration. Studies have shown that the magnitude of the current determines the magnitude of the action potential in phase 1, and it can also affect the duration of action potential in phase 2. It means that it is possible to adjust the action potential duration and shape in order to maintain the normal electrical signal transduction of the heart or pathological conditions caused by heart diseases. The down-regulation of cardiac transient outward potassium current can lead to prolongation of action potential duration which is a common manifestation of serious heart diseases, such as myocardial infarction, atrial fibrillation, and heart failure [5-7].

Acupuncture has been used clinically for more than 2000 years in China. As time goes by, the main scope of indications have been spread [8]. Acupuncture has on the body a variety of regulatory mechanisms. As a physical therapy for the treatment of diseases with minimal damage and no side effects, it has been widely used as a kind of complementary or alternative medicine to treat cardiovascular diseases all over the world [9-10]. In this experiment, we injected ASIC3 −/− mice with isoproterenol (ISO) to obtain models with myocardial ischemia. Using electroacupuncture (EA) on mice from PC6, LU7, ST36, and nonacupoints groups, we observed the expression of related proteins in Ito, and we explore the possible mechanisms of EA at PC6 against myocardial ischemia.

2. Materials and methods

2.1. Animals

Eight-week-old male ASIC3 −/− mice (C57BL/6), SPF grade, and with body weight of 22 ± 5.5 g were used. They were all provided by the Liaoning Biotechnology Co., Ltd. [License No. SCXE (Liao) 2010-0001]. The animal experiments and care were designed and conducted according to the National Research Council Guide for the Care and Use of Laboratory Animals.

2.2. Main reagents

Isoproterenol hydrochloride (ISO, batch number: PTAPE-LJ); Kir1.4 antibody (Item: ab-16718); Kir4.2 antibody (Item: ab99040); Kir4.3 antibody (Item: ab99045); KChIP2 antibody (Item: ab99041); β-actin; protein extraction kit; protein marker; polyvinylidene fluoride membrane; electro-chemi-luminescence (ECL) kit; sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel kit.

2.3. Main instrument

Hua Tuo brand acupuncture needles (φ0.19 × 13 mm); electric needle instrument (G6805-A type); electronic balance scales (BS223S type); Powerlab Eight Channel Physiological Recorder (PL3508 type); high—speed desktop refrigerated centrifuge (TGL-20M type); constant temperature water bath oscillator (SHA-B type); horizontal decolorization shaker (TS-1000 type); mini VE protein electrophoresis and transfer system; microplate reader; and gel imaging analysis system.

2.4. Experimental models and groups

Before the experiment, the electrocardiograms (ECGs) of all the mice were recorded, and all the abnormalities observed on the mice ECGs have been removed. According to the random number table, 36 mice were divided into six groups: control group, MI group, PC6 group, LU7 group, ST36 group, and nonacupoint group. Mice in the control group were injected with saline (20 mg/kg, 24 hours interval), and the other ASIC3 −/− mice were injected subcutaneously twice with ISO (20 mg/kg, 24 hours interval) [11]. After the second injection, the ECGs of each group were recorded during 20 minutes. We screened out the mice which met the standards, and they were randomly divided into three groups, with six mice each: MI group, PC6 group, LU7 group, ST36 group, and nonacupoint group (Fig. 1).

Figure 1. Diagrams of experimental steps. ECG = electrocardiogram; ISO = isoproterenol.

2.5. Acupuncture treatment

Some researches show that using acupuncture on SD mice for 20 minutes during 7 days has a therapeutic effect on myocardial ischemia [12]. Therefore, we followed this procedure to complete our experiment. Because of the small size of the mice, we chose acupuncture needles of 13 mm long and 0.19 mm in diameter (φ0.19 × 13 mm) and a needle insertion depth of about 3 mm, thereby avoiding piercing through to the opposite side which might also be another acupoint.

The positioning method of PC6, LU7, and ST36 reference the description of the corresponding points of “experimental acupuncture” and compare to the length of rat and mice, according to the proportion of acupoints finally [13].

Mice were treated every day at 8:30 am. All mice were immobilized on the experimental table while they are awake. The mice in the control group and the MI group were not treated, and those from the other four groups were given EA treatment. Nonacupoints were located in between and along the line connecting CV8 (Shenque-point) and ST25 (Tianshu-point), on the abdomen of the mice. We inserted acupuncture needles 2 mm into acupuncture points of mice from each groups, the electrical current with a frequency of 4/20 Hz and an intensity level of 2 mA was applied to the needles for 20 minutes daily for a total of 7 days (Fig. 2) (See Fig. 3).

Figure 2. Electroacupunture. (A) EA at PC6. (B) EA at LU7. (C) EA at nonacupoint. (D) EA at ST36. EA = electroacupunture.

Figure 3. The locations of acupoints on human and mice. (A) The location of PC6 and LU7. In human, PC6: 2 cun above the transverse crease of the wrist, between the tendons of m. palmaris and m. flexor radialis; LU7: Superior to the styloid process of the radius, 1.5 cun above the transverse crease of the wrist between the brachioradial muscle and the tendon of the long abductor muscle of the thumb. In mice, PC6: 1 mm above the transverse crease of the wrist, between the ulna and the radial bones. LU7: on the radial side of the forelimb, 1 mm above the transverse crease of the wrist. (B) The location of ST25, RN8, and nonacupoint. In human, RN8: in the center of the umbilicus. ST25: 2 cun lateral to the center of the umbilicus. Nonacupoint: in the middle, between ST25 and RN8. In mice, RN8: On the chest sword joint. ST25: when locating the point, put the rat in the supine position, it is on the middle of the abdomen, 2 cun laterally at the same level of RN8. Nonacupoint: In the middle, between ST25 and RN8. (C) The location of ST36. In human, ST36: on the front of the leg, one hand width (four fingers) below the kneecap, on the outside, in the depression between the shinbone and the leg muscle. In mice, ST36: 3 mm below the fibular head, in the posterolateral part of the knee.

2.6. Sample extracting

We recorded the ECGs of all the mice after treatment. Part of the heart tissue of all the mice was separated quickly from their chests and was immediately placed on ice and cleaned with ice-cold saline. We cut off the atria and the great vessels and separated the left and right ventricles along the interventricular septum. The left ventricle tissue was cut into pieces and stored at −80°C until it was tested.

2.7. Western blot

The heart tissue was homogenized with a pellet pestle mixer in pyrolysis liquid based on the ratio of 1:10 between pyrolysis liquid and the weight of the mice myocardial tissue and fully grinded until there was no obvious solid visible particle on the ice. Samples were centrifuged at the rate of 12,000r/min at 4°C for 10 minutes, and the supernatant was collected. Then a portion of the supernatant was used to analyze the protein concentration, the rest was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After separation, proteins were transferred to polyvinylidene fluoride membranes and blocked in tris-buffered saline and Tween-20 (TBST) for at least 1h at 37°C in a water bath. When the TBST was removed and primary antibodies were added, membranes were rocked gently for 1h at 37°C in a water bath and then incubated overnight at 4°C. The experimental antibodies were used: Rabbit Anti-Kv 1.4 antibody (ab16718, Abcam, USA), Mouse Anti-Kv4.2 antibody (ab99040, Abcam, USA), Mouse Anti-Kv4.3 antibody (ab99045, Abcam, USA) and Mouse Anti-KChIP2 antibody (ab99041, Abcam, USA). The next day, membranes were rinsed three times in TBST for 5 minutes each and incubated with secondary antibodies [a mouse IgG or a rabbit IgG (V1008, Beijing dingguo changsheng biotech CO.LTD)] for 1h at 37°C in the water bath. The intensity of protein signals were quantified by densitometry. Values are expressed as the ratio of the objective blots to the internal reference blots (Fig. 4).

Figure 4. Western blot analysis.

2.8. Statistical analysis

Statistical calculations were performed with SPSS 17.0 (Institute Inc, Chicago, IL, USA). The results were presented as the means ± standard deviation (x_±s). Comparisons of the differences among the groups were performed by one-way analysis of variance, the sample means in each group are estimated by multiple comparison via least—significant difference (LSD) test, and a p < 0.05 or p < 0.01 were adopted as the statistical significance levels. (See Fig. 5)

Figure 5. The diagram of the meridians.

The pericardium meridian of Hand-Jueyin runs from the chest to the hand, and the triple energizer meridian of Hand-Shaoyang runs from the hand to the chest.

3. Results

3.1. ECG variations of T-wave

Table 1 shows that there were no T-wave changes in the control group during the whole experiment. After MI was induced, the T-waves of MI group, PC6 group, LU7 group, ST36 group, and nonacupoint group decreased of 75.6%, 76.0%, 75.1%, and 76.5%, respectively (p < 0.01). After EA at PC6, LU7, and ST36 acupoints and nonacupoint for 7 days, the T-waves increased of 181.4%, 95.2%, and 76.7%, respectively compared to the T-waves before EA treatment (p < 0.05, p < 0.01, and p > 0.05, respectively). But there were no significant changes in the nonacupoint group.

EA = electroacupunture; ECG = electrocardiogram; MI = myocardial ischemia..

*p < 0.05, **p < 0.01, comparison before modeling; p < 0.05, △△p < 0.01, comparison after modeling; #p < 0.05, ##p < 0.01, compared with control group; p < 0.05, ▼▼p < 0.01, compared with MI group. After injection with ISO, the T-waves of the mice were flatter than those before MI was induced. After EA, the T-waves of PC6 group, LU7 group, and ST36 group were taller than those of MI mice..

&md=tbl&idx=1' data-target="#file-modal"">Table 1

ECG variations of T-wave (x_±s)..

GroupnBefore MI induced (mV)After MI induced (mV)After EA (mV)
Control60.170 ± 0.0570.165 ± 0.0530.169 ± 0.055
MI60.180 ± 0.0250.042 ± 0.030**0.043 ± 0.036**##
PC660.176 ± 0.0480.043 ± 0.028**0.121 ± 0.025*△△#▾▾
LU760.175 ± 0.0590.042 ± 0.019**0.082 ± 0.027*##
ST3660.173 ± 0.0650.043 ± 0.031**0.076 ± 0.030**##
Nonacupoint60.179 ± 0.0220.042 ± 0.015**0.041 ± 0.018**##

EA = electroacupunture; ECG = electrocardiogram; MI = myocardial ischemia..

*p < 0.05, **p < 0.01, comparison before modeling; p < 0.05, △△p < 0.01, comparison after modeling; #p < 0.05, ##p < 0.01, compared with control group; p < 0.05, ▼▼p < 0.01, compared with MI group. After injection with ISO, the T-waves of the mice were flatter than those before MI was induced. After EA, the T-waves of PC6 group, LU7 group, and ST36 group were taller than those of MI mice..



Fig. 6 shows the changes of ECGs' T-waves in control group, MI group, PC6 group, LU7 group, ST36 group, and nonacupoint group. These changes are consistent with Table 1.

Figure 6. ECG variations of T-wave. ECG = electrocardiogram; MI = myocardial ischemia; EA = electroacupunture.

3.2. The expression of Kv1.4, Kv4.2, Kv4.3, and KCHIP2 proteins of ASIC3 −/− mice in each group

The expression of Kv1.4, Kv4.2, Kv4.3, and KChIP2 proteins in myocardial tissues showed a similar trend in Western blot; the expression of four proteins in the MI group was significantly lower than that of the control group (p < 0.01). Compared to the MI group, the protein expression in the PC6, LU7, and ST36 groups increased 7 days after EA (p < 0.05), and the difference was significant (p < 0.05). The expression of the proteins in the PC6 group was higher than that of LU7 and ST36 groups (p < 0.05). The protein expression of nonacupoint group was not significantly different (p > 0.05) (Table 2, Table 3, Figs. 7 and 8).

MI = myocardial ischemia..

Compared with the control group:*p < 0.05, **p < 0.01; compared with the MI group: p < 0.05, △△p < 0.01..

&md=tbl&idx=2' data-target="#file-modal"">Table 2

Comparison of the changes of Kv1.4, Kv4.2, and Kv4.3 protein expressions of ASIC3 −/− mice in each group (x_±s)..

GroupnKv1.4Kv4.2Kv4.3
Control group60.692 ± 0.1010.683 ± 0.0650.741 ± 0.099
MI group60.245 ± 0.019**0.209 ± 0.037**0.226 ± 0.039**
PC6 group60.515 ± 0.045*△△0.578 ± 0.054*△△0.575 ± 0.077*△△
LU7 group60.381 ± 0.039**0.367 ± 0.032**0.370 ± 0.044**
ST36 group60.329 ± 0.014**0.291 ± 0.036**0.311 ± 0.020**△△
Nonacupoint group60.271 ± 0.045**0.232 ± 0.025**0.277 ± 0.055**

MI = myocardial ischemia..

Compared with the control group:*p < 0.05, **p < 0.01; compared with the MI group: p < 0.05, △△p < 0.01..



MI = myocardial ischemia..

Compared with the control group: *p < 0.05, **p < 0.01; compared with MI group: p < 0.05, △△p < 0.01..

&md=tbl&idx=3' data-target="#file-modal"">Table 3

Comparison of changes in KChIP2 expression of ASIC3 −/− mice accessory proteins in each group (x_±s)..

GroupnKChIP2
Control group60.693 ± 0.037
MI group60.224 ± 0.038**
PC6 group60.578 ± 0.062*△△
LU7 group60.383 ± 0.049**
ST36 group60.285 ± 0.028**△△
Non-acupoint group60.246 ± 0.014**

MI = myocardial ischemia..

Compared with the control group: *p < 0.05, **p < 0.01; compared with MI group: p < 0.05, △△p < 0.01..



Figure 7. The changes of Kv1.4, Kv4.2, Kv4.3, and KCHIP2 protein expressions in each group. MI = myocardial ischemia.

Figure 8. The bands of Kv1.4, Kv4.2, Kv4.3, and KChIP2 of ASIC3 −/− mice in each group. MI = myocardial ischemia.

4. Discussion

Potassium channels are one of the ion channels very important for the heart and maintain the normal physiological function of the heart together with sodium ion channels and calcium ion channels. When the function or expression of potassium channel is abnormal or its balance with other channel is destroyed, the heart will be in a pathological state. Potassium channels can control and limit myocardial excitability and therefore play a role in myocardial ischemia, arrhythmia, heart failure, myocardial infarction, or other lesions [14]. This is why the study of potassium channels has been a hot spot in the field of cardiac diseases in recent years [15-16].

Human Kv is a kind of transmembrane glycoprotein, a complex which is composed of an α superfamily subunit and β auxiliary subunit [17]. The transient outward potassium currents (Itos) of Kv play an important role in the repolarization of the cardiac action potential, and it appears in repolarization 1 which is the potassium ion current of quick activation and deactivation. Studies have shown that in cardiac tissue of some animals and humans, Ito is mainly formed of Kv4 subunit [18]. And the Kv4 family mainly includes Kv4.2, Kv1.4 and Kv4.3, which are expressed in high levels of neurons cells, cardiac muscle cells, and smooth muscle cells, and they even play an important role in modulating neuronal discharge frequency and cardiac excitation–contraction coupling [19]. Itos have two main components, fast transient outward potassium current (Ito.f) and slow transient outward potassium current (Ito.s) [20]. Kv4.2 and Kv4.3 are the main components of Ito.f channel [21], Kv1.4 is the major component of Ito.s channel [22]. In the myocardial tissue, Kv4.2 is mainly distributed in ventricular myocardium outer membrane, and Kv1.4 is mainly distributed in the lining of the ventricular myocardial cell membranes [23]. KChIP1 ∼ KChIP4 are included in KChIPs genes and are abundantly expressed in the brain. But KchIP2 is the only rich expression in the heart [24]. Studies have found that the ventricular Ito of the mice completely disappeared when there is a lack of the KChIP2 genes. Therefore, all these indicate that Kv4 constitute Ito with at least one auxiliary subunit KchIP2 and the mutual protein KchIP2 assists Kv4.2 or Kv4.3 inside to the cell membrane [25, 26]. Researches show that when the mice are suffering from ventricular ischemia and heart failure, Ito and the corresponding mRNA and protein channel (Kv4.2, Kv4.3 and Kv1.4) reduce their expressions significantly [27]. Therefore, we can predicate whether acupuncture has therapeutic effects on myocardial ischemia or not and predicate the therapeutic effects of acupuncture at different acupoints by observing the expression of Kv4.2, Kv4.3, Kv1.4, and KChIP2.

The heart pumps out blood to maintain the normal functions of the human body, each pulse of blood injection into the coronary artery also maintains the heart's own energy supply, and this cardiac output accounts for about 4%–5% [28]. If there is insufficiency of myocardial blood supply, myocardial cells will lack oxygen and nutrients, thereby causing serious myocardial dysfunctions or even necrosis. Patients may experience chest pain, shortness of breath, palpitations, fatigue, sudden death may happen or even other type of discomfort. Moreover it is consistent with the description of traditional Chinese medicine on angor pectoris and thoracic obstruction, etc. The theory of visceral outward manifestation holds the pericardium which is around the heart responsible for protecting the heart. Therefore, modern medicine believes that myocardial ischemia should be one of the pericardial diseases.

Ancient traditional Chinese medicine physicians thought that when evil invades the heart, the pericardium was the first to be attacked, so there was a “pericardial replacement of the heart by an evil” effect. It is well-known that the pericardium plays the role of protecting the heart, and the PC6 is an acupuncture point on the pericardium meridian; therefore, we chose PC6 to treat myocardial ischemia. Modern research shows PC6 has multichannel features in the treatment of myocardial ischemia, such as regulation of the central nervous system, cardiovascular effects of substances, improvement myocardial metabolism and antioxidant free radicals, etc [29]. The literature reported that sudden stimulation of sensory nerve cells can cause a terminal release reaction, just as pain occurs in the medial part of the forearm when there is a myocardial ischemic attack [30]. Liu Qiang studies show that parts of the nerve fibers from the heart and Neiguan area come from the same neuron in the spinal ganglia and cervical vagus nerve, which provide a reliable morphologicial basis for explaining the “pain” of the left arm and acupuncture at Neiguan point to improve cardiac functions during myocardial ischemia [31]. LU7 can improve myocardial ischemia by modulating lung functions as the hand lunar meridian points it out. Modern research found that PC6 and LU7 in the spinal cord ganglion innervated and superimposed on C8-T1 which dominates the heart [32]. ST36 belongs to the point of the stomach meridian with extensive role to reconcile the functions of the spleen and the stomach, of the blood, and other effects. Studies have shown that EA at ST36 can inhibit cardiomyocytes apoptosis and also can produce protective effect on myocardial ischemia–reperfusion injury of myocardial cells [33].

ASIC3 is an acid-sensitive cation channel that exists extensively in pain-mediated pathways. ASIC3 plays a significant role during the formation of hyperalgesia in the inflammatory environment. Therefore knocking out ASIC3 genes can reduce pain in the mice. Experimental ASIC3 −/− mice comes from the removal of relevant gene fragments in the mice embryonic development stage and followed by several rounds of hybridization to obtain homozygous ASIC3 knockout mice. Studies found that comparing ASIC3 −/− mice with normal strains of the same strain, ASIC3 −/− mice had a decreased mechanical susceptibility and a significant increase of the baseline of pain threshold [34]. During the experiment, we also found that ASIC3 −/− mice were less resistant to the experimental procedure and had a significantly lower rate of death compared to pretested mice. Therefore, it can reduce the stress response of myocardial ischemia mice to external injury, reduce the death rate of mice during the experiment, and eliminate the external factors that may interfere with the effect of acupuncture treatment.

In this research, it is one of the main techniques used to replicate mice model of myocardial ischemia. Nowadays, the main myocardial ischemia models are the coronary artery left anterior descending (LAD) ligation myocardial ischemia model, Langendorff in vitro myocardial ischemia model, and ISO-induced myocardial ischemia model [35]. ISO-induced mice acute myocardial ischemia model is commonly used in classical models, which were made by subcutaneous injections of ISO and increased myocardial oxygen consumption, leading to cardiac microcirculation overload and myocardial ischemia. Some studies systematically show that mice cardiomyocytes have different degrees of damages in the range of 680–0.02 mg/kg if they are injected with different doses of ISO on the cardiomyocytes [36]. By using this method, MI was induced by ISO through shrinking coronary artery, increasing the force of myocardial contraction and of myocardial oxygen consumption, etc. In the preexperiment, 5 mg/kg, 10 mg/kg, 20 mg/kg, and 30 mg/kg of ISO were used, respectively, and the results showed that 5 mg/kg and 10 mg/kg of ISO both could induce acute myocardial ischemia, which will be reversed after a short period of time. On the other hand, an injection of 30 mg/kg of ISO can make the mice experience arrhythmia or die immediately. Finally, 20 mg/kg of ISO was used to make MI model with subcutaneous multipoints injection. The method is simple and effective and can be used as a simple model of experimental myocardial ischemia, and it is closer to the pathological state of human myocardial ischemia [37]. In this study, a dose of 20 mg/kg of ISO was used and injected subcutaneously two times in an interval of 24 hours. This is not only led to a high success rate, but it also reduced animal mortality and saved experimental funds.

In the early stage of the experiment, we carried out preexperiment with the purpose of perfecting the experiment plan and personal acupuncture skills. Thus, no death occurred in the knockout mice. In the adaptive feeding stage, mice diet, toilet, and other physiological functions were in good condition. However, after the injection of ISO, we observed reduced diet, listlessness, and other performances. In the later stages of the treatment, the mice physiological functions were slightly different; the mice in PC6 group were more active and had an appetite which improved gradually during the course of treatment. The mice in ST36 and LU7 groups showed slight improvement, while no obvious improvement was found in mice from the nonacupoint group.

Western blot detection can be found: The expression of Ito-channel protein Kv1.4, Kv4.2, Kv4.3, and KChIP2 protein in mice myocytes decreased; the expression of the above proteins in the PC6 group was lower than that of the control group (p < 0.05) but higher than the protein expressions of the MI group (p < 0.01). The level of protein expressions in ST36 group and LU7 group increased (p < 0.05) but less than that of PC6 group; the expression of nonacupoint proteins was not significantly different compared to the MI group.

The experimental results show that LU7 has a certain regulatory role on Ito channel, but the adjustment range is less than PC6. Acupoints PC6 can make Ito channel–associated protein expression higher than other groups significantly. These findings suggest that acupuncture at PC6 can significantly improve the ISO-induced ischemic state in ASIC3 −/− mice by increasing the expression of Ito channel proteins.

5. Conclusion

This study indicates that EA at PC6 of the pericardium meridian of Hand-Jueyin can treat MI mice. Improvement in the amount of protein expressions of Kir4.2, Kir1.4, Kir4.3, and KChIP2 is the mechanism by which MI can be cured with EA from the level of potassium channels. But the expression of proteins is a reflection of channel structures that does not systematically correspond to channel functions. In the following researches, patch clamp technology will be a good way to test the variations of cells' electrophysiology and forward our research on potassium ion channel functions and their relationship with acupuncture treatment on MI [38].

Acknowledgments

This work is supported by Chinese National Essence Basic Research and Development 973 Program: The Effects of Meridian Specific target Organ Response on Biological Basic Research (No. 2012CB518503).

Conflict of interest


There is no conflict of interest.

Fig 1.

Figure 1.Diagrams of experimental steps. ECG = electrocardiogram; ISO = isoproterenol.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 2.

Figure 2.Electroacupunture. (A) EA at PC6. (B) EA at LU7. (C) EA at nonacupoint. (D) EA at ST36. EA = electroacupunture.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 3.

Figure 3.The locations of acupoints on human and mice. (A) The location of PC6 and LU7. In human, PC6: 2 cun above the transverse crease of the wrist, between the tendons of m. palmaris and m. flexor radialis; LU7: Superior to the styloid process of the radius, 1.5 cun above the transverse crease of the wrist between the brachioradial muscle and the tendon of the long abductor muscle of the thumb. In mice, PC6: 1 mm above the transverse crease of the wrist, between the ulna and the radial bones. LU7: on the radial side of the forelimb, 1 mm above the transverse crease of the wrist. (B) The location of ST25, RN8, and nonacupoint. In human, RN8: in the center of the umbilicus. ST25: 2 cun lateral to the center of the umbilicus. Nonacupoint: in the middle, between ST25 and RN8. In mice, RN8: On the chest sword joint. ST25: when locating the point, put the rat in the supine position, it is on the middle of the abdomen, 2 cun laterally at the same level of RN8. Nonacupoint: In the middle, between ST25 and RN8. (C) The location of ST36. In human, ST36: on the front of the leg, one hand width (four fingers) below the kneecap, on the outside, in the depression between the shinbone and the leg muscle. In mice, ST36: 3 mm below the fibular head, in the posterolateral part of the knee.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 4.

Figure 4.Western blot analysis.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 5.

Figure 5.The diagram of the meridians.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 6.

Figure 6.ECG variations of T-wave. ECG = electrocardiogram; MI = myocardial ischemia; EA = electroacupunture.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 7.

Figure 7.The changes of Kv1.4, Kv4.2, Kv4.3, and KCHIP2 protein expressions in each group. MI = myocardial ischemia.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Fig 8.

Figure 8.The bands of Kv1.4, Kv4.2, Kv4.3, and KChIP2 of ASIC3 −/− mice in each group. MI = myocardial ischemia.
Journal of Acupuncture and Meridian Studies 2018; 11: 88-96https://doi.org/10.1016/j.jams.2018.03.002

Table 1 . ECG variations of T-wave (x_±s)..

GroupnBefore MI induced (mV)After MI induced (mV)After EA (mV)
Control60.170 ± 0.0570.165 ± 0.0530.169 ± 0.055
MI60.180 ± 0.0250.042 ± 0.030**0.043 ± 0.036**##
PC660.176 ± 0.0480.043 ± 0.028**0.121 ± 0.025*△△#▾▾
LU760.175 ± 0.0590.042 ± 0.019**0.082 ± 0.027*##
ST3660.173 ± 0.0650.043 ± 0.031**0.076 ± 0.030**##
Nonacupoint60.179 ± 0.0220.042 ± 0.015**0.041 ± 0.018**##

EA = electroacupunture; ECG = electrocardiogram; MI = myocardial ischemia..

*p < 0.05, **p < 0.01, comparison before modeling; p < 0.05, △△p < 0.01, comparison after modeling; #p < 0.05, ##p < 0.01, compared with control group; p < 0.05, ▼▼p < 0.01, compared with MI group. After injection with ISO, the T-waves of the mice were flatter than those before MI was induced. After EA, the T-waves of PC6 group, LU7 group, and ST36 group were taller than those of MI mice..


Table 2 . Comparison of the changes of Kv1.4, Kv4.2, and Kv4.3 protein expressions of ASIC3 −/− mice in each group (x_±s)..

GroupnKv1.4Kv4.2Kv4.3
Control group60.692 ± 0.1010.683 ± 0.0650.741 ± 0.099
MI group60.245 ± 0.019**0.209 ± 0.037**0.226 ± 0.039**
PC6 group60.515 ± 0.045*△△0.578 ± 0.054*△△0.575 ± 0.077*△△
LU7 group60.381 ± 0.039**0.367 ± 0.032**0.370 ± 0.044**
ST36 group60.329 ± 0.014**0.291 ± 0.036**0.311 ± 0.020**△△
Nonacupoint group60.271 ± 0.045**0.232 ± 0.025**0.277 ± 0.055**

MI = myocardial ischemia..

Compared with the control group:*p < 0.05, **p < 0.01; compared with the MI group: p < 0.05, △△p < 0.01..


Table 3 . Comparison of changes in KChIP2 expression of ASIC3 −/− mice accessory proteins in each group (x_±s)..

GroupnKChIP2
Control group60.693 ± 0.037
MI group60.224 ± 0.038**
PC6 group60.578 ± 0.062*△△
LU7 group60.383 ± 0.049**
ST36 group60.285 ± 0.028**△△
Non-acupoint group60.246 ± 0.014**

MI = myocardial ischemia..

Compared with the control group: *p < 0.05, **p < 0.01; compared with MI group: p < 0.05, △△p < 0.01..


References

  1. Zang YM, Zang WJ, Zhu MZ. Coronary circulation physiology and clinic. Chin J Cardiac Arrhythm (心功能杂志). 1999;11:260-262+275.
  2. Chen WW, Gao RL, Liu LS, Zhu ML, Wen W, Wang YJ, et al. Outline of china cardiovascular disease report 2016. Chin Circ J(中国循环杂志). 2017;32:521-30.
  3. Ge YB, Xu YJ. Internal Medicine [内科学]. Beijing: Peoplés Medical Publishing House, 2013:228-55.
  4. Tikhonov DB, Zhorov BS. Architecture and pore block of eukaryotic voltage-gated sodium channels in view of NavAb bacterial sodium channel structure. Mol Pharmacol. 2012;82:97-104.
    Pubmed CrossRef
  5. Cai Ben-zhi, Pan Zhen-wei, Yang Bao-feng. Transient outward potassium current and heart disease. Adv Cardiovasc Dis (心血 管病学进展). 2009;30:50-2.
  6. Yue Lixia, Melnyk Peter, Gaspo Rania, et al. Molecular mechanisms underlying ionic remodeling in a dog model of atrial fibrillation. Circ Res. 1999;84:776-84.
    Pubmed CrossRef
  7. Aggarwal N, Shi NQ, Makielski JC. ATP-sensitive potassium currents from channels formed by Kir6 and a modified cardiac mitochondrial SUR2 variant. Channels (Austin). 2013;7:493-502.
    Pubmed CrossRef
  8. Liu WH, Hao Y. Origin, development and prospect of treatment technique of acupuncture and moxibustion. J Tradit Chin Med (中医杂志). 2014;55:91-4.
  9. Wang Y, Wei W, Li D, Dai JY, Chun CR, Li JQ, et al. The beneficial effect of electro-acupuncture given at PC6 (PC6-point) by the increase in cardiac transient outward K+ current channel which depends on the gene and protein expressions in artificially induced myocardial ischemia rats. Acupunct Electro Therapeut Res. 2014;39:259-73.
    Pubmed CrossRef
  10. Bao L, Kefaloyianni E, Lader J, Hong M, Moreley G, Fishman G, et al. Unique properties of the ATP-sensitive K Channel in the mouse ventricular cardiac conduction system. Circ Arrhythm Electrophysiol. 2011;4:926-35.
    Pubmed CrossRef
  11. Faulx MD, Ernsberger P, Vatner D, Hoffman RD, Lewis W, Strachan R, et al. Strain-dependent ?-Adrenergic receptor function influences myocardial responses to isoproterenol stimulation in mice. Am J Physiol Heart Circ Physiol. 2005;289:H30-6.
    Pubmed CrossRef
  12. Wang Y, Zhang XL, Wang W, Li D, Dai JY, Li CR, et al. The beneficial effect of electro-acupuncture at PC6 (Neiguanpoint) of gene and protein expressions of classical inwardrectifier potassium channel in myocardial ischemia rats. Acupunct Electro Therapeut Res. 2015;40:335-53.
    Pubmed CrossRef
  13. Li Zhong-ren. Experimental Acupuncture and Moxibustion [实 验针灸]. 2nd ed. Beijing: China Traditional Chinese Medicine Publishing House, 2007:242-3.
  14. Nattel S, Maguy A, Bouter SL, Yeh YH. Arrhythmogenic ionchannel remodeling in the heart: heart failure, myocardial infarction, and atrial fibrillation. Physiol Rev. 2007;87:425-56.
    Pubmed CrossRef
  15. Li JQ, Wang W, Meng XY, Chen YG, Han ZY. Influence of needing acupoints along meridians on myocardialtransient outward potassium channel gene expression in rats of myocardial ischemia. Chin Arch Tradit Chin Med (中华中医药 学刊). 2015;30:78-81.
  16. Lampert Angelika, Korngreen Alon. Markov modeling of ion channels: implications for understanding disease. Progr Mol Biol Trans Sci. 2014;123:1-21.
    Pubmed CrossRef
  17. Decher N, Uyguner O, Scherer CR, Karaman B, Apak MY, Busch AE, et al. hKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant. Cardiovasc Res. 2001;52:255-64.
    Pubmed CrossRef
  18. Tian LL, Strauss HC, Wang SM, Huang CX, Jiang XJ. Potassium ion channel interacting protein with Kv4. Adv Cardiovasc Dis (心血管病学进展). 2006;27:109-11.
  19. Lin Lin. Kv potassium channel function of the regulatory mechanism. Chin J Microcirc (中国微循环杂志). 2007;11:404-7.
  20. Wettwer E, Amos GJ, Posival H, Ravens U. Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin. Circ Res. 1994;75:473-82.
    Pubmed CrossRef
  21. Nerbonne JM. Molecular basis of functional voltage? gated K+channel diversity in the mammalian myocardium. J Physiol. 2000;525:285-98.
    Pubmed KoreaMed CrossRef
  22. Kassiri Z, Haijar R, Backx PH. Molecular components of transient outward potassium current in cultured neonatal rat ventricular myocytes. J Mol Med. 2002;80:351-8.
    Pubmed CrossRef
  23. Brahmajothi MV, Campbell DL, Rasmusson RL, Morales MJ, Trimmer JS, Nerbonne JM, et al. Distinct transient outward potassium current (Ito) phenotypes and distribution of fastinactivating potassium channel alpha subunits in ferret left ventricular myocytes. J Gen Physiol. 1999;113:581-600.
    Pubmed KoreaMed CrossRef
  24. Boland LM, Jiang M, Lee SY, Fahrenkrup SC, Harnett MT, O'Grady SM. Functional properties of a brain-specific NH 2-terminally spliced modulator of Kv4 channels. Am J physiol Cell Physiol. 2003;285:C161-70.
    Pubmed CrossRef
  25. Biggin PC, Roosild T, Choe S. Potassium channel structure: domain by domain. Curr Opin Struct Biol. 2000;10:456-61.
    Pubmed CrossRef
  26. Ren Chong-yu, Cao Ji-min. Kv4 potassium channel and its interacting protein KChIP2 and arrhythmia. J Int Pathol Clin Med (国际病理科学和临床医学杂志). 2006;26:126-9.
  27. Rozanski GJ. Physiological remodeling of potassium channels in the heart. Cardiovasc Res. 2012;93:218-9.
    Pubmed CrossRef
  28. Peng Yi, Yang Zi-bin. Hemodynamic analysis of coronary circulation. Foreign Med Sci (国外医学). 2003;26:1-5.
    Pubmed CrossRef
  29. Liu JL, Cao QS, Luo MF, Wen C, Liu JL, Cui RL. Study on the mechanism of electroacupuncture at improving the acute myocardial ischemia by pericardium. Acupunct Study (针刺研 究). 1999;24:282-7.
  30. Holzer P. Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcItonin gene-related peptide and other neuropeptides. Neuroscience. 1988;24:739-68.
    Pubmed CrossRef
  31. Liu Q, Yang L, Li ZH, Lu G, Sun JH. Non - fluorescent double -labeling tracing of the Neural projection homology relation between the neural projection in rat heart and Neiguan area. Chin J Anat (解剖学杂志). 2002;25:39-42.
  32. Wang JJ, Jiang SH, Li SS, Jiang PW, Lou XF. The relationship between the distal acupoints of the twelve meridians and the spinal cord innervation. Chin Arch Tradit Chin Med (中华中医 药学刊). 2008;26:1272-3.
  33. Zhang Fan. Protective Effect of Electroacupuncture of Zusanli on Myocardial Ischemia Reperfusion Injury in Rats. Central South University (中南大学), 2009.
  34. Shi H, Ji CF, Shang HY, Xin JJ, Yang ZK, Su YS, et al. Comparison of Reproductive Performance and Pain Threshold of ASIC3, TRPV1 Knockout Mice Associated with Sensation. Chin J Comp Med (中国比较医学杂志). 2013;23:1-4.
  35. Tao Wu, Li Jian-an, Lu Xiao, et al. Establishment of animal model of controllable myocardial ischemia. Chin J Rehabilitat Med (中国康复医学杂志). 2006;21:1068-1071+1075.
  36. Rona G, Chappel CI, Balazs T, Gaudry R. An infract-like myocardial lesion and other toxic manifestations produced by isoproterenol in the rat. AMA Arch Pathol. 1959;67:443-55.
  37. Yan B, Wang GJ, Aa JY, Hao HP, Zheng YT, Liu LS, et al. Metabonomics of myocardial ischemic injury induced by isoproterenol in rats. Herald of Med (中国药理学会). 2007;26:66-7.
  38. Luo HY, Liang HM, Hu XW, et al. Expression of kir2.1, SCN5a and SCN1b channel genes in mouse cardiomyocytes with different electrical properties: patch clamp combined with single cell RT-PCR. Acta Physiol Sin (生理学报). 2012;64:82-6.