A total of 80 healthy adult male and female Wistar rats (clean grade), aged 8–10 week and weighing 240–260 g, were purchased from the Experimental Animal Center of Jilin University (Changchun, China). Rats were maintained in an animal care facility at 21–23°C, relative humidity (55±5%) with a 12-h light/dark cycle and ad libitum access to food and chow for at least three days prior to the initiation of the experiment. The procedures used in the present study were approved by the Animal Ethics Committee of The First Hospital of Jilin University, in accordance with the Guide for the Care and Use of Laboratory Animals issued by the US National Institutes of Health (13). All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.

A total of 40 rats were randomly and equally divided into 4 groups: A sham-operated group, an AMI model group, an immune-enhanced group (treated with 1.25 mg/kg thymopeptide (H20003884; Livzon Pharmaceutical Group, Inc., Shanxi, China) by intraperitoneal injection) and an immune-suppressed group (treated with 15 mg/kg cyclophosphamide (H32020857; Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China) by intraperitoneal injection). For the 8 days leading up to the experimental procedures, the rats were maintained and fed, as outlined. The number of leukocytes was detected in the peripheral blood. Briefly, the rat tail was soaked at 45–50°C for ~3 min to dilate the blood vessels and a 5 mm incision was made to harvest 1 ml blood. Full blood count analysis was performed within 2 h of collection using a Sysmex XE 2100 hematology analyzer (Sysmex UK, Milton Keynes, UK). If the number of leukocytes in the immune-enhanced group was ≥1.2×10⁹/l and the number of leukocytes in the immune-suppressed group was ≤0.6×10⁹/l, then immune intervention was considered successful.

Rats were anesthetized with 20 g urethane dissolved in 100 ml saline solution (0.9%) (1 g/kg; 5 µl/g body weight; U2500) by intraperitoneal injection, and a thoracotomy was performed in the left 4–5 intercostal space. A total of 10 µl LPA (0.5 g/l dissolved in 0.9% NaCl saline; L7260) was intravenously injected into the hearts of the AMI group, the immune-enhanced group and the immune-suppressed group for <5 sec. The thoracotomy was completed in 30 sec. The left anterior descending artery of coronar was ligated. The heart was placed back in the chest prior to being closed with sutures. Rats in the 4 groups were analyzed by surface lead II ECG (DECG-03A; Mindray Medical International, Ltd., Nanshan, China). Successful construction of the acute myocardial infarction model was observed as ST-segment elevation and the formation of a flag-shaped waveform of the T wave. ECG recordings were obtained and analyzed using a BL-420S Biological Function Recording system (Taimeng Technology Co., Ltd., Chengdu, China).

A total of 40 rats were randomly and equally divided into 4 groups: A sham-operated group, an LPA group, an immune-enhanced + LPA group and an immune-suppressed + LPA group. Hearts were isolated and rapidly perfused with improved Krebs-Henseleit (K-H) solution in a Langendorff Perfusion system (Radnoti LLC, Monrovia, CA, USA). The perfusion pressure was 10.1 kPa, and the perfusion temperature was 37°C. The improved K-H solution was saturated with 95% O₂ and 5% CO₂, and the composition of the solution was as follows: 118.0 mmol/l NaCl, 4.7 mmol/l KC1, 1.2 mmol/l KH₂PO₄, 1.2 mmol/l MgSO₄, 25.0 mmol/l NaHCO₃, 1.3 mmol/l CaCl₂, and 1.1 mmol/l glucose, with pH titrated to 7.2–7.4. After 10 min the isolated heart was functioning in a stable manner. LPA (5 µmol/l) was added to the K-H solution in the hearts of the LPA model group, the immune-enhanced + LPA group and the immune-suppressed + LPA group. Surface electrocardiograms were analyzed by surface lead II ECG (the needle electrode was inserted subcutaneously into the pulmonary cone and the cardiac apex). ECG recordings were obtained and analyzed using the BL-420S Biological Function Recording system. Subsequently, the number of premature ventricular contractions were observed.

Jurkat T cells were purchased from the National Platform of Experimental Cell Resources (Sci-Tech, Beijing, China) and plated in a 96-well plate (2×10⁶ cells/ml) in 200 µl RPMI-1640 medium (R8755) supplemented with 10% fetal bovine serum (F2442) and 100 mg/ml penicillin/streptomycin (V900929), and incubated at 37°C in atmosphere containing 5% CO₂ and 95% humidity. Jurkat T cells were divided into 3 groups: A control group, an LPA group (5 µmol/l), and a Ki16425 + LPA group (10 µmol/l and 5 µmol/l, respectively). Cell suspensions were obtained from each group following cell incubation for 1, 2, 4, 8, 12 and 24 h. The cell suspensions were centrifuged at 560 × g for 10 min at 22–23°C, and the supernatants were collected. The concentration of TNF-α was determined using an ELISA kit (ERT2010-1; Assaypro LLC, St. Charles, MO, USA).

Jurkat T cells (2×10⁴ cells/ml) were placed in a bath solution. Patch electrode pipettes were fabricated using a vertical pipette puller (Narishige PP-83; Narishige Scientific Instrument Lab., Tokyo, Japan). Pipettes with tip diameters of 1–2 µm had resistances of 3–6 MΩ when filled with the pipette solution. The pipette was placed on the cell surface using a microelectrode propeller (Narishige Scientific Instrument Lab.), and a high impedance seal (GΩ) was formed by vacuum suction. Subsequently, the negative pressure was increased in order to cause the clamp of the electrode tip to rupture, and the whole-cell patch clamp mode was formed. All experiments were performed at 20–22°C. The voltage-clamp circuit was provided by a patch/whole-cell clamp amplifier (Dagan Total Clamp 8800; Dagan Corporation, Minneapolis, MN, USA). Pulse protocols and data acquisition were performed using a digital interface (TL-1 DMA interface; Axon Instruments, Foster City, CA, USA) coupled to an IBM compatible computer with PCLAMP 6.0 software (Molecular Devices LLC).

Ionic currents of K_v were recorded under various voltage-clamp protocols of the step pulse: Holding potential −80 mV, test potentials −50 to +50 mV, step 10 mv, duration 300 ms, and frequency 1 Hz. The bath solution of K_v contained the following components: 140 mmol/l NaCl, 4 mmol/l KCl, 1 mmol/l MgC1₂, 1 mmol/l CaC1₂, 10 mmol/l glucose and 10 mmol/l HEPES, pH 7.2 (filtered). The pipette solution of K_v contained the following components: 90 mmol/l KCl, 50 mmol/l KF, 4 mmol/l NaCl, 4 MgCl₂, 0.5 mmol/l ethylene glycol tetraacetic acid (EGTA) and 10 mmol/l HEPES, pH 7.2 (filtered).

The bath solution of K_Ca contained the following components: 160 mmol/l NaCl, 4.5 mmol/l KC1, 2 mmol/l CaC1₂, 1 mmol/l MgC1₂, 5 mmol/l HEPES, pH 7.4 (filtered). The pipette solution of K_Ca contained the following components: 150 mmol/l K-asparatic acid, 2 mmol/l MgCl₂, 5 mmol/l HEPES, 10 mmol/l EGTA, 8.7 mmol/l CaCl₂, with a 1 µmol/l Ca²⁺ concentration and pH 7.2 (filtered).

Following the isolation of the hearts of the rats in the AMI, immune-enhanced and immune-suppressed groups were isolated (atria and blood vessels were discarded). The ventricles were fixed in 4% paraformaldehyde solution (252549) for 16–18 h. Fixed myocardial tissue samples were subsequently dehydrated in a graded ethanol series, cleared with xylene and embedded with paraffin (10152636; Guangjing Weiye Import & Export Co., Ltd., Tianjin, China). Tissue samples were cut into 3-µm sections using a sliding microtome (VT-1000S; Leica Microsystems GmbH, Wetzlar, Germany). One section was used for hematoxylin (H9627) and eosin (E4009) staining in order to observe the morphology of the myocardium, and the remaining two sections were used for immunohistochemical SP staining (SP-9000; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). Cx43 was observed in the tissue sections using an Olympus CKX41-A32PH inverted microscope (Olympus Corporation, Tokyo, Japan).

All data were analyzed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) and are presented as the means ± standard error of the mean (n=4). The results obtained were compared using a t-test. Plots were generated using GraphPad Prism 6.01 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of LPA on the incidence of arrhythmia in rats that had altered immune status, ECGs of myocardial infarction rats and isolated rat hearts were obtained; the results demonstrated the presence of arrhythmias and ventricular premature beats (VPBs). Endogenous LPA enhanced the incidence of VPBs in rats of the immune-enhanced group and reduced the incidence of VPBs in rats of the immune-suppressed group (Table I).

ECGs of isolated rat hearts demonstrated that, following the addition of 5 µmol/l LPA to the K-H solution of the LPA model, immune-enhanced + LPA and immune-suppressed + LPA groups, the incidence of VPBs in the immune-enhanced + LPA group (90%) increased compared with that of the LPA model group (80%). The occurence of VPBs (7.4±3.7 times/5 min) also increased in the immune-enhanced + LPA group, as compared with the LPA model group (5.1±3.9 times/5 min). The incidence of VPBs in the immune-suppressed + LPA group (50%) significantly decreased compared with the LPA model group (80%; P<0.05), and occurrences (3.5±3.8 times/5 min) significantly increased compared with the LPA model group (5.2±3.9 times/5 min; P<0.05; Table II).

The levels of TNF-α secreted by T lymphocytes significantly increased in the LPA group compared with the control group (P<0.01). Ki16425, which is a specific inhibitor of LPA, was able suppress the secretion of TNF-α (Table III). The concentration of TNF-α increased markedly within a short period of time following the addition of LPA to the T lymphocytes. The TNF-α concentration reached its maximum at 4 h and stabilized within 24 h (Table III; Fig. 1).

A total of 28 voltage-dependent K⁺ currents of Jurkat T cells were obtained. To exclude the natural attenuation of K_v, K_v was continuously observed for 30 min, which determined that no other current was significantly attenuated. A total of 7 Jurkat T cells were observed in the control group (205.5±43.4 pA; Fig. 2A), 7 Jurkat T cells were observed in the 0.5 µmol/l LPA group (246.65±30.9 pA; Fig. 2B), 7 Jurkat T cells were observed in the 5 µmol/l LPA group (317.5±32.1 pA; Fig. 2C), and 7 Jurkat T cells were observed in the 50 µmol/l LPA group (361.8±46.7 pA; Fig. 2D).

The K_v current amplitude in the 5 µmol/l LPA group (317.5±32.1 pA) and the 50 µmol/l LPA group (361.8±46.7 pA) significantly increased compared with the control group (205.5±43.4 pA; P<0.01). The K_v current amplitude in the 0.5 µmol/l LPA group (246.65±30.9 pA) was significantly increased compared with the control group (P<0.05). The effect of LPA on K_v currents and the I–V curve of K_v currents are shown in Fig. 2E. It was deduced that LPA had a dose-dependent effect on the K_v current. The electrophysiological characteristics (current amplitude and activation, and inactivation voltage range) of K_v in the present study were concordant with those of previous reports (14,15).

The K_Ca of Jurkat T cells in the 5 µmol/l LPA group and the 10 µmol/l Ki16425 + 5 µmol/l LPA group were obtained to observe the effects of LPA on K_Ca and to examine the inhibitory effect of Ki16425. Ionic currents of K_Ca were recorded under the various voltage-clamp protocols of the step pulse: Holding potential −80 mV, test potentials −80 to +80 mV, step 10 mv, and duration 400 ms. Compared with the current amplitude prior to treatment with LPA (439.6±43.7 pA; Fig. 3A), the K_Ca current amplitude in the 5 µmol/l LPA group (628.5±46.1 pA; Fig. 3B) increased significantly (P<0.01). The K_Ca current amplitude in the 10 µmol/l Ki16425 + 5 µmol/l LPA group decreased to 507.5±71.4 pA (Fig. 3C), which suggests that Ki16425 was able to inhibit the effect of LPA (Fig. 3).

The expression of Cx43 was determined by immunohistochemical SP staining. Cx43 was abundantly expressed in the control group and displayed marked positive staining. Cx43 was uniformly distributed in the intercalated disk between adjacent cells and had a clustered distribution (Fig. 4). Cx43 in the LPA + AMI model group was clearly decreased and had a disordered, uneven and punctate distribution (Fig. 5). The expression of Cx43 in the Ki16425 + LPA group was very similar to the control group (Fig. 6). The LPA + immune-suppressed group expressed decreased Cx43 protein compared with the LPA + AMI model group. Cx43 was distributed relatively uniformly in the intercalated disk between adjacent cells and had a dotted distribution; however, both the density and coloring level of Cx43 were decreased as compared with the control group (Fig. 7). The expression of Cx43 in the LPA + immune-enhanced group was marginal (Fig. 8).