Effects of Ang Ⅱ perfusion on transmural heterogeneous of Cx43 in acute myocardial ischemia reperfusion

Xiao-Pan Chen, Shi-Juan Lu, Kang Huang, Wei Zhang, Zheng-Wang Liu, Jiang-Hua Zhong*Hainan Medical University Affiliated Hospital, Haikou 5700, ChinaCentral South University Xiangya School of Medicine Affiliated Haikou Hospital, Haikou People’s Hospital, Haikou 57008, China



Effects of Ang Ⅱ perfusion on transmural heterogeneous of Cx43 in acute myocardial ischemia reperfusion

Xiao-Pan Chen1, Shi-Juan Lu2, Kang Huang2, Wei Zhang2, Zheng-Wang Liu1, Jiang-Hua Zhong2*
1Hainan Medical University Affiliated Hospital, Haikou 570102, China
2Central South University Xiangya School of Medicine Affiliated Haikou Hospital, Haikou People’s Hospital, Haikou 570208, China

ABSTRACT

Objective: To observe the effects of angiotensin Ⅱ (Ang Ⅱ) pefusion on transmural heterogeneity of Cx43 expression in the rabbit model with acute myocardial ischemia reperfusion (MIR), and investigate the role of rennin-angiotensin system in malignant ventricular arrhythmia induced by MIR. Methods: Twenty rabbits were randomly divided into MIR group (n=10) and Ang Ⅱ group (n=10). MIR model was produced with traditional ligation and opening of the anterior descending coronary artery in all animal. The hearts in vitro in the MIR group and the Ang Ⅱ group were perfused with simply improved Tyrode’s solution and containing Ang Ⅱ Tyrode’s solution respectively. 90% monophasic action potential repolarization duration, transmural dispersion of repolarization, Cx43 protein (Cx43-pro) and mRNA (Cx43-Cq) expression in subepicardial, midmyocardial and subendocardial myocardium were measured in both groups. The greatest differences of Cx43-pro and Cx43-Cq among three myocardial layers were calculated and shown with △Cx43-pro and △Cx43-Cq respectively. Results: After Ang Ⅱ perfusion, 90% monophasic action potential repolarization duration among three myocardial layer were significantly prolonged (P ＜ 0.05 and P ＜ 0.01), and transmural dispersion of repolarization also significantly increased compared with the MIR group (P ＜ 0.05). Compare with the MIR group, three myocardial Cx43-pro and Cx43-Cq expression in the Ang Ⅱ group were significantly decreased (P ＜ 0.05 and P ＜ 0.01), but △Cx43-pro and △Cx43-Cq were significant increased. Conclusions: Renin-angiotensin system increases transmural heterogeneity of Cx43 expression in the rabbit model with MIR by Ang Ⅱ, and enlarge transmural dispersion of repolarization among three myocardial layers of left ventricular which induces malignant ventricular arrhythmia.

ARTICLE INFO

Article history:

Received 15 October 2015

Received in revised form 20 November 2015

Accepted 15 December 2015

Available online 20 January 2016

Keywords:

Acute myocardial ischemia reperfusion

Dispersion of repolarization

Cx43

Angiotensin Ⅱ

E-mail: zhong3882@163.com

Foundation project: It is supported by National Natural Science Foundation of China (NO. 81160024) and Natural Science Foundation of Hainan Province (NO. 814371).

1. Introduction

Acute myocardial ischemia reperfusion easily causes malignant ventricular arrhythmia (MVA) and sudden cardiac death, whose mechanism is in need of further investigation. Besides, angiotensin Ⅱ (Ang Ⅱ), the main element of rennin-angiotensin systems (RAS), is actively involved in the cause and development of cardiovascular diseases, such as hypertension, heart failure and myocardial infarction. According to recent researches, Ang Ⅱ can influence Cx43 remodeling and change myocardial electrophysiology parameter and it is closely related to MVA and sudden death[1,2], which indicates that Ang Ⅱ-mediated gap reconstruction caused abnormal electrophysiology.

We observe effects of Ang Ⅱ perfusion on myocardial ischemiareperfusion (MIR) rabbit model transmural dispersion of repolarization and expression heterogeneity of Cx43, and discuss occurrence mechanism of MVA in MIR and the therapeutic target through making MIR rabbit model in our research.

2. Materials and methods

2.1. Animal grouping and model making

Twenty healthy rabbits, provided by Laboratory Animal Center of Hainan Medical University, weighting (2.0-3.0) kg, of either sex, were divided into MIR control group (n=10) and Ang Ⅱreperfusion group (n=10). Both groups of rabbits were weighted and anaesthetized with urethane, and their right common carotid arteries were separated. A plastic catheter was incubated through right common carotid arteries to measure mean arterial pressure. Their tracheas were incubated and connected to artificial respirators. The chests were open along the median line of breastbone. The pericardia were cut open to fully expose left anterior free walls of hearts and make pericardial cradles. Left ventricular branch of the coronary artery was separated and the anterior descending artery was threaded and ligated at 0.3 cm away from the lower margin of left atrial appendage. Observe the electrocardiogram and take ST-segment elevation＞0.5 mV in two adjacent leads as the indicator of myocardial ischemia. After 20 min observation, ligature was cut and myocardium was reperfused. The standard of reperfusion damage formation was that after reperfusion ST segment was decrease＞50%, and R wave amplitude was decreased with occurrence of arrhythmia and Q wave. After models were made, MIR control group was perfused with improved Tyrode’s solution, while Ang Ⅱperfusion group was perfused with Tyrode’s solution with 1 μmoL Ang Ⅱ.

2.2. Experiment solution preparation

Ingredients improved Tyrode’s solution for isolated heart perfusion (mmoL/L): NaCl 115, KCl 5.4, MgCl21, CaCl21.8, NaH2PO41, HEPES 5, glucose 10, adjusting pH value to 7.4[3]. Prepare eppendorf tubes containing buffer solution with pH value of 8, 1 mmoL/L NaHCO3, 5 mmoL/L EDTA, 1 mmoL/L EGTA, 1 μmoL/ L leupeptin, 1 μmoL/L pepstatin, 100 nmoL/L aprotinin, 1 mmoL/L benzamidine, 1 mmoL/L iodoacetamide and 1mmoL/L PMSF[4].

2.3. In vitro cardiac electrophysiology experiment

We referred to previous experiment method and made electrophysiological experiments as follow:

2.3.1. Pre-experiment preparation

Hearts were quickly cut down and put in 4 ℃ Tyrode’s solution to arrest. Washed by precool normal saline, the whole hearts were weighted. After aortic cannula, the hearts were perfused with Langendorff [pure oxygen saturation, constant temperature of (36.5-37.5) ℃, constant pressure of (8.52-8.78) kPa].

2.3.2. Electrophysiological experiment

Pacemaker electrode is consisted of two pin electrodes. The distance between the two electrodes is 0.5 cm, pricking into the position where the heartbeat was strongest. Reference electrodes were fixed on the root of aorta. Simplified electrodes of endocardium, epicardium and midmyocardium were placed in the needles of syringes respectively. The needles were fixed 10 mm away from apical and pricked in the position 5 mm away from interventricular septum. Endocardium electrodes were fixed in the subendocardial myocardium. Epicardium electrodes were fixed 3.0 mm away from lateral epicardium of left ventricular. Midmyocardium electrodes were fixed to make pinpoint of electrode 3.0 mm away from epicardial surface. Simplified electrodes of monophasic action potentials of three myocardial layers were fixed and connected to biological signal acquisition and processing system and the parameter was set as filter (500-1 000) Hz. Along the junction of free walls of right atrium and crista terminalis, sinoatrial node was cut by scissors, and slow nodal rhythm appeared. Cardiac electrophysiology stimulation was fixed at the wall of right ventricle and cardiac pacing was performed with pacing cycle length 1 000 ms. All rabbits were perfused by normal Tyrode’s solution for 20 min, and then steady monophasic action potentials of three myocardial layers were steadily recorded.

2.3.3. Post-experiment process

When experiments were finished, hearts were cut down and their wet weights were measured. Cardiac tissues of reperfused zone were obtained to produce pathological specimen.

2.4. Protein extracted from three myocardial layers of left ventricular

After electrophysiology experiment, free walls of left ventricular from rabbits were cut down and stored in liquid nitrogen. By the way of fast frozen section, from the surface of epicardium to endocadium, epicardial cardiomyocyte of left ventricular were obtained to make 10 serial section of 20 μm (altogether 200 μm). In the same way, from the surface of endocadium to epicardium, endocardial cardiomyocyte of left ventricular were obtained to make 10 serial section of 20 μm (altogether 200 μm). Mid myocardial cardiomyocyte, 3 mm away from the surface of epicardium, were obtained to make 10 serial section of 20 μm (altogether 200 μm)[3]. Cardiomyocyte of three layers were stored in prepared eppendorf tubes respectively.

2.5. CX43 expression detected by Western-blot

Samples in eppendorf tubes were homogenated, extractedprotein, loaded, and turned into PVDF membrane through SDS polyacrylamine gel electrophoresis. PVDF membrane were sealed in 5% TBST skim milk for 2 h in indoor temperature. Cx43 monoclonal antibody (Zymed 35-5000) was dropped in and kept over night at 4 ℃. Samples were added horseradish peroxidase labeled second antibody (Novogene company), and incubated for 2 h at indoor temperature. Colored by enhanced chemiluminescence, samples were developed on X-ray films. Protein signal strip image was obtained through gel image analysis system to calculate target Cx43 and optical density of internal reference protein GAPDH, taking their ratio as the expression level of Cx43.

2.6. Expression of CX43 mRNA detected by RT-qPCR

Frozen tissue samples of left ventricular of both groups in eppendorf tubes were ground into powder, from which total RNA was extracted. cDNA was compound by RevertAid First Strand reverse transcriptase kits, and amplified by qPCR, taking GAPDH as internal reference. Upstream primer: 5′-GAGGTGGCCTTCTTGCTGAT-3′, downstream primer: 5′-GTTTTCTCAGTGGGGCGAGA-3′,The length of estimated amplified fragment was 122 bp. GAPDH upstream primer: 5′-AGAGCACCAGAGGAGGACGA-3′, downstream primer: 5′-TGGGATGGAAACTGTGAAGAGG-3′, the length of estimated amplified fragment was 104 bp. Reverse transcription parameter: 42 ℃ 60 min, 70 ℃ 5 min. qPCR reaction parameter: 50 ℃ 3 min, 95 ℃ 15 min, 95 ℃ 10 s, 60 ℃ 20 s, 72 ℃30 s, repeated 40 cycles. During qPCR amplification, fluorescence was collected. When reaction was finished, cycle threshold detected by all fluorescence signals of samples during qPCR was analyzed and calculated. Cycle quantification of Cx43 mRNA of samples was calculated by 2-△△Ctmethod to present relative expression level.

2.7. Observed indicator

(1) APD parameter: ①90% repolarization of action potential duration (APD90): APD from phase 0, repolarization, to amplitude of 90%. ②Transmural dispersion of repolarization (TDR): The difference between the longest APD90and the shortest APD90among three myocardial layers. (2) Cx43 protein: Expression levels of Cx43 of every myocardial layer of left ventricular. (3) △Cx43-pro: The difference of Max Cx43-pro and Min Cx43-pro among three myocardial layers. (4) Cx43-Cq: Expression levels of Cx43 mRNA protein of every myocardial layer of left ventricular. (5) △Cx43-Cq: Greatest difference of Max and Min Cx43-Cq value among three myocardial layers.

2.8. Statistical method

SPSS 13.0 statistical software was used in statistic and detection. All experimental materials were randomly designed. One-way ANOVA was taken to performed significance analysis of difference among groups. SNK was used to test multiple comparison of variance homogeneity among groups. Variance heterogeneity was tested by Tamhane. If P ＜ 0.05, it has statistical meaning.

3. Results

3.1. Fundamental changes

Mean arterial pressure, heart/body weight, left ventricular weight of two groups did not differ significantly (P ＞ 0.05). However, in pathological section examination, disordered myocardial hypertrophy of left ventricular was observed, conforming to pathological manifestation of MIR.

3.2. Comparison of electrophysiological parameter of left ventricular

Compared with MIR control group, perfused by Ang Ⅱ, APD90of three myocardial layers were all further prolonged (P ＜ 0.05 and P ＜0.01), and TRD was increased (P ＜ 0.05), which indicated that Ang Ⅱ perfusion could enlarge transmural repolarization heterogeneity of MIR (Table 1).

Table 1 Electrophysiological parameter comparison between two groups of rabbits (mean±SD).

3.3. Comparison of left ventricular myocardial transmural Cx43 expression

Perfused by Ang Ⅱ, all Cx43 expression of three myocardial layers decreased than that of MIR control group (P ＜ 0.05 and P ＜ 0.01), and △Cx43-pro and △Cx43-Cq increased significantly (P ＜ 0.05), which indicated that Ang Ⅱ perfusion not only weakened MIR three myocardial layers Cx43 expression, but also increased heterogeneity of transmural Cx43 expression (Table 2).

Table 2 Comparison of left ventricular myocardial transmural Cx43 expression.

4. Discussion

Acute myocardial ischemia (AMI) is a clinical incident greatly threatening patients’ lives. The key to treatment of AMI is to open infarcted vessels as soon as possible. AMI reperfusion often accompanies with myocardial damage. Arrhythmia is the most common form of clinical reperfusion damage. Any kind of myocardial reperfusion will cause reperfusion arrhythmia with rate of 50%-80%. Arrhythmia, especially ventricular arrhythmia, is one of the main causes of exacerbation and sudden cardiac death by changing the hemodynamic of patients. However, at present, the physiological mechanism of easy-happening MVA is not totally clear.

Ang Ⅱ is the main ingredient of RAS, widely involved in the cause and development of cardiovascular diseases. According to recent researches, Ang Ⅱ also influence myocardial electrophysiology parameter and participate in the occurrence of MVA[4,5]. RAS antagonist, blocking Ang, can effectively reduce organic heart disease and tachyarrhythmia[6,7]. Further electrophysiological studies found that RAS antagonist can improve ventricular repolarization dispersion, inhibit the exchange of Na+/H+and Na+/Ca2+, and block L-type Ca2+channel[8]. However, specific mechanism of RAS’s role in malignant arrhythmia is not clear. Our results showed that, compared with MIR control group, APD90among three myocardial layers were significantly prolonged (P ＜ 0.05), and TDR also significantly increased compared with the MIR group (P ＜ 0.05). These indicated that, as Ang Ⅱ perfusion prolonged APD90among three myocardial layers, it further worsened transmural dispersion of ventricular repolarization. This may be the electrophysiologic mechanism of RAS’s participation in MIR and MVA.

Ang Ⅱ also got involved in the regulation of Cx by reducing Cx43 and slowing down conduction. Effect of Ang Ⅱ on expression of ventricular myocardium gap junction may be the important mechanism to cause ventricular arrhythmia[9]. According to recent studies[9], angiotensin converting enzyme 2 can turn Ang Ⅱ into Angl-7 through enzymatic hydrolysis, reduce Ang Ⅱ, and trigger reconstruction of ventricular protein GJ, especially Cx43, which is closely related to arrhythmia[10]. This also indicated that Ang Ⅱ-mediated gap reconstruction caused abnormal electrophysiology. Our results further showed that, compared with MIR control group, in Ang Ⅱ perfusion group expression of Cx43 and mRNA of endocardium, epicardium and midmyocardium decreased significantly (P ＜ 0.05), while △Cx43 further increased, which indicated that over activation of Ang Ⅱ increase the transmural expression heterogeneity of Cx43 in MIR. This may be the important molecule mechanism of RAS’s involvement in MIR MVA.

Conflict of interest statement

We declare that we have no conflict of interest.

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Contents lists available at ScienceDirect IF: 1.062
Asian Pacific Journal of Tropical Medicine
journal homepage:www.elsevier.com/locate/apjtm

doi:Document heading 10.1016/j.apjtm.2015.12.019

*Corresponding author:Jiang-Hua Zhong, Central South University Xiangya School of Medicine Affiliated Haikou Hospital, Haikou People’s Hospital, Haikou 570208 Hainan, China.