Experiments were approved by the Institutional Animal Care and Use Committee of Wannan Medical College (Wuhu, China). A total of 50 Sprague-Dawley rats (250 to 300 g) were randomly assigned to five groups: Control group, IR group and three taxol pretreatment groups. In the control and pretreatment groups, normal saline and taxol (Sigma-Aldrich, St. Louis, MO, USA; 0.1, 0.3 and 0.9 μM·kg⁻¹ in 0.5 ml saline) were injected intraperitoneally (IP) 30 min prior to ischemia induction.

Each rat was anesthetized (pentobarbital sodium, 50 mg/kg IP; Sigma-Aldrich) and artificially ventilated. Once the pericardium was opened, a 6.0 silk atraumatic suture (Jinghua Co., Shanghai, China) was passed around the left anterior descending coronary artery at the level of the left atrial appendage. Ischemia was induced for 20 min by ligating the suture, following which it was confirmed by an elevated ST segment in the electrocardiogram (ECG) and changes in myocardial color from red to pale. Reperfusion was initiated by reopening the ligation for 20 min. The control group was established by following the same procedure, but without closing the suture.

ECG traces were recorded continuously, and signals <10 Hz and >100 Hz were filtered out. Epicardial MAPs were recorded at specific time points intermittently in normal sinus rhythm (10). MAP signals were amplified and recorded on a computer for subsequent analysis. A commercially available physiological signals recording and analysis system (RM6240; Chengdu Instrument Company, Chengdu, China) was used to digitalize, store and analyze MAP signals. The software was used to analyze action potential durations (APD₉₀) at 90% of repolarization. Dispersion of action potential duration (APDd) represented the absolute value of the time difference between the maximum and minimum APD.

VAs were analyzed according to Lambeth conventions (11). The score system (9) used to quantify arrhythmias was as follows: 0, <10 ventricular premature contractions (VPCs); 1, ≥10 VPCs; 2, 1–5 episodes of ventricular tachycardia (VT); 3, >5 episodes of VT or 1 episode of ventricular fibrillation (VF); 4, 2–5 episodes of VF; 5, >5 episodes of VF; 6, VT or VF, or the two together with total combined duration ≤300 sec; 7, VT or VF, or the two together with total combined duration >300 sec.

A tubulin polymerization assay was performed using a previously described method (6). Myocardial tissue from the ischemic area (1 g) was minced and homogenized in different buffers. For the total tubulin fraction, the myocardium was homogenized in 1% SDS buffer containing 10 mM Tris·HCl (pH 7.4), 0.5 mM dithiothreitol and 1 mM Na₃VO₄. The mixture was boiled for 5 min and centrifuged at 16,000 × g at 4°C for 10 min and then the supernatant was saved as the total protein fraction. For the free tubulin heterodimer and polymerized tubulin (microtubule) fractions, the myocardium was homogenized in a microtubule stabilization buffer containing 50% glycerol, 5% dimethyl sulfoxide, 10 mM Na₂HPO₄, 0.5 mM ethylene glycol tetraacetic acid and 0.5 mM MgSO₄ and then centrifuged at 100,000 × g at 25°C for 20 min. The supernatant was saved as the tubulin heterodimer fraction, whereas the pellet was saved as the tubulin microtubule fraction. For immunoblotting, rabbit polyconal α-tubulin (1:2,000; ab125267; Abcam, Cambridge, MA, USA) antibody was used to detect the free tubulin heterodimer and polymerized tubulin.

Protein (100 μg) was denatured by heating at 95°C for 5 min prior to resolution by SDS-PAGE and transferring to a polyvinylidene difluoride membrane (Shanghai Threebio Technology Co., Ltd, Shanghai, China). The membrane was blocked in phosphate-buffered saline (Shanghai Threebio Technology Co., Ltd) containing 0.2% Tween-20 (Shanghai Threebio Technology Co., Ltd) and 5% skimmed milk for 2 h at 37°C and incubated overnight at 4°C with primary polyclonal antibodies (rabbit anti-Cx43; 1:2,000; ab11370; Abcam). The housekeeping protein, glyceraldehyde-3-phosphate dehydrogenase (rabbit anti-GAPDH polyclonal antibody; ab9485; Abcam) was used as a loading control. Antibody binding was detected using horseradish peroxidase-conjugated secondary antibody (1:2,000; Sigma-Aldrich) and visualized using an enhanced chemiluminescence kit (Chemicon, Temecula, CA, USA).

Frozen tissue sections fixed in cold acetone (−20°C for 5 min) were used for immunofluorescence staining. Nonspecific antibody binding sites were blocked with 5% fetal bovine serum for 60 min. Tissue sections were labeled for 16 h at 4°C with rabbit polyclonal anti-Cx43 (Cx43; 1:200, Abcam). Anti-rabbit immunoglobulin G conjugated with fluorescein isothiocyanate was used for detection and images were observed using a Zeiss confocal microscope (LSM-510; Carl Zeiss, Gottingen, Germany).

Statistical analysis was performed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard error of the mean and were analyzed using analysis of variance followed by Newman-Keul’s t-test. P<0.05 was considered to indicate a statistically significant difference.

In a preliminary experiment, it was observed that >2 μM taxol may lead to lethal ventricular bradycardias. Thus, the doses of taxol were reduced to 0.1, 0.3 and 0.9 μM. In the control group, 6/10 rats had ≥10 VPCs and only 1 had an episode of VT. In the IR group, all animals had >5 episodes of VT or several episodes of VF. The spontaneous VTs/VFs appeared most frequently (5 to 10 min) after ligation and then recovered gradually. Within 2 min after reperfusion, VPCs/VTs/VFs occurred transiently and automatically recovered rapidly. In three taxol treatment groups, fewer animals had VTs and VFs than in the IR group. However, no difference in VAs during reperfusion periods between the IR group and taxol-treated groups was observed (Fig. 1; Table I). The severity of VAs was significantly decreased in taxol-treated groups in a dose-dependent manner compared with the IR group based on the VA score system (P<0.01; Fig. 1).

The MAP duration at APD₉₀ of the ischemic epicardium in the IR group was significantly shorter than that of the control group (Fig. 2). When perfusion was restored, the APD₉₀ rapidly recovered within 5 min and returned to baseline levels. The APDd was significantly increased in the IR group compared with that in the control group. Taxol treatment partially but significantly restored APD₉₀ and reduced APDd during ischemia in the pretreatment groups. However, no significant difference in APD₉₀ was observed during the reperfusion periods between the IR group and taxol-treated groups.

The relative levels of free tubulin in the IR group increased significantly after the heart was exposed to IR compared with the control group (Fig. 3). In addition, polymerized tubulin in the IR group significantly decreased. Taxol treatment maintained tubulin polymerization significantly and reduced free tubulin in a dose-dependent manner (P<0.01).

The Cx43 staining signal in the non-ischemic heart was concentrated at the IDs (red arrows, Fig. 4A), whereas little Cx43 was located in the lateral cardiomyocyte surfaces (yellow arrows, Fig. 4A). In the hearts of the IR group, the Cx43 signal was markedly reduced in ID and redistributed to the lateral cardiomyocyte surfaces (Fig. 4B). The hearts subjected to taxol pretreatment exhibited significantly improved Cx43 distribution compared with the IR groups (Fig. 4C, D and E).

The relative levels of Cx43 protein expression in myocardial tissue were reduced significantly in the IR group compared with the control group (Fig. 5). Taxol treatment significantly improved Cx43 expression in a dose-dependent manner (P<0.01).