Chuan-wu was obtained from the Anguo medicine market (Shijiazhuang, China). AC was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Ber was obtained from Acros Organics (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Pentobarbital was obtained from Sigma-Aldrich (Merck KGaA). Creatine kinase (CK) and CK-MB isoenzyme diagnostic kits were all from Sysmex Corporation (Kobe, Japan). Lactate dehydrogenase (LDH) assay kit was obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Sprague Dawley (SD) rats and guinea pigs were obtained from the Experimental Animal Center of Hebei Medical University [license no. SCXK (Hebei) 2013-003]. All experiments were approved by the Ethics Committee for the Use of Experimental Animals at Hebei Medical University (Shijiazhuang, Hebei, China).

Male SD rats (8-week-old; certificate no. 1109105), weighing 250–300 g, were housed under controlled atmosphere (12-h light/dark cycle, relative humidity 50–60%, and 24±3°C) and food and water were provided ad libitum. Rats (n=40) were randomly divided into Control group (A), AC group (B), AC + Ber (8 mg/kg/d) group (C), and AC + Ber (16 mg/kg/d) group (D). Ber was administered intragastrically once a day for 7 consecutive days in groups C and D. Group A and B rats were administrated with an equal volume of distilled water. Chuan-wu (0.08 mg/kg) was administrated from day 4 during the Ber administration period by dissolving the Chuan-wu powder into distilled water; this was performed once a day for 3 days, while an equal volume of distilled water was given to the control group.

On days 1 and 3 following Chuan-wu administration, 30 min following Ber administration, the rats were anesthetized with 45 mg/kg pentobarbital sodium via an intraperitoneal injection (i.p.). The electrodes were inserted into the subcutaneous tissue of the rat limb, and the ECG parameters (day 1) and the different types of arrhythmia (day 3) were recorded in each group using an RM 6240CD multi-channel physiological signal acquisition and processing system (Chengdu Instrument Factory, Chengdu, China) via standard limb lead II.

On day 3 following Chuan-wu administration, blood samples were collected from the abdominal aorta and stored on ice prior to centrifugation at 2,100 × g for 10 min within 1 h of collection. Subsequently, the supernatant was used for the determination of biochemical indicators. The levels of LDH, CK and CK-MB in the serum were assayed as sensitive indicators of myocardial damage, according to the manufacturer's protocol of the diagnostic kits (cat. nos., LDH, R1 1000331, R2 1000312; CK, R1 1000531, R2 1000512; CK-MB R1 1001611 and R2 1001612; Sysmex Corporation) using a CHEMIX-180 automatic biochemistry analyzer (Sysmex Corporation).

At day 3 after Chuan-wu administration, the heart was rapidly removed and rinsed with ice-cold saline following the collection of blood samples. Heart samples were fixed in 4% formaldehyde immediately following excision for 7 days at room temperature, then dehydrated in an ascending series of ethanol (70, 80, 96 and 100%). Following paraffin embedding, transverse slides (5 µm thickness) were stained with hematoxylin and eosin at room temperature for 2 min respectively, and histological examination was conducted using a light microscope (Olympus BX-50; Olympus Corporation, Tokyo, Japan).

Male SD rats (n=40, certificate no.1107071), weighing 250–300 g were randomly divided into 4 groups: Control group (A), AC group (B), AC + Ber (8 mg/kg/d) group (C), and AC + Ber (16 mg/kg/d) group (D). Ber was administered intragastrically once a day for 7 consecutive days in groups C and D. Rats in groups A and B received an equal volume of distilled water. On day 7, 30 min following Ber administration, the rats were anesthetized with 45 mg/kg pentobarbital sodium (i.p.). The surface lead II ECG was recorded continuously with three subcutaneous limb electrodes using the RM 6240CD multi-channel physiological signal acquisition and processing system. The femoral vein was cannulated for administration of AC (10 µg/ml) at a rate of 0.08 ml/min in groups B, C and D, while an equal volume of normal saline was given to group A. The time onset of the first ventricular premature contraction discrete (VPCD), ventricular premature contraction bigeminy (VPB), successive ventricular tachycardia (VT), ventricular fibrillation (VF) and mortality (DD) was recorded, and the amount of AC required to induce arrhythmia was calculated.

Male guinea pigs (4–5 weeks of age, n=6), weighing 200–250 g, were housed under controlled atmosphere (12-h light/dark cycle, relative humidity 50~60% and 22±2°C), and food and water were provided ad libitum. The animals were anesthetized with 40 mg/kg pentobarbital sodium (i.p), then the heart was rapidly removed and put into a preparation dish containing ice-cold K-H solution (140 mmol/l NaCl, 5.4 mmol/l KCl, 1 mmol/l MgCl₂, 2 mmol/l CaCl₂, 10 mmol/l HEPES and 10 mmol/l glucose). The right ventricle was opened and columnar papillary muscles were removed. The papillary muscles were then transferred into the tissue chamber and perfused with K-H solution, which was continually gassed with 100% O₂. The temperature was maintained at 36°C. A conventional microelectrode recording technique was used to record the action potential of the papillary muscle of guinea pigs under the stimulation frequency of 1 Hz. The following action potential parameters were calculated: Action potential amplitude (APA), duration of 30% repolarization (APD₃₀) and duration of 90% repolarization (APD₉₀). The hearts were divided into two groups (n=3 in each group): Control and Ber treatment groups. Once a normal action potential was continuously recorded in the control group, Tyrode's solution containing AC (3×10⁻⁷ mol/l) was perfused, and DAD and TA by AC were observed. While in the Ber treatment group, once a normal action potential was continuously recorded, solution containing Ber (1×10⁻⁵ mol/l) was perfused. Following 5 min, the action potential was recorded. Then, AC (3×10⁻⁷ mol/l) was perfused, and the changes in action potential following AC perfusion were recorded. If no change appeared, 15 min later, 1×10⁻⁶ mol/l AC was added to observe the change in the action potential.

All values are presented as the mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance and Dunnett's test and SPSS v11.0 software (SPSS, Inc., Chicago, IL, USA). Comparisons of the same parameters prior to and following administration were performed using a paired t-test. P<0.05 was considered to indicate a statistically significant difference.

During the experiments, no animals succumbed in the control or Ber 16 mg/kg treatment groups; whereas, 3 animals in the Chuan-wu group and 1 animal in the Ber 8 mg/kg group perished.

On day 1 following Chuan-wu administration, the analysis of ECG parameters revealed that the heart rate was decreased (P<0.05) and the P-R, QRS and QT intervals were all prolonged in the Chuan-wu group (P<0.05 or P<0.01), compared with that observed in control group. The effects of Chuan-wu on heart rate, P-R, QRS and QT intervals in the 8 and 16 mg/kg Ber treatment groups were all reversed by different degrees when compared with the Chuan-wu group (P<0.05 or P<0.01; Fig. 1). On day 3 following Chuan-wu administration, there was no ECG abnormality in the control group. However, in the Chuan-wu group, ventricular premature beat, bigeminies or trigeminies were observed in 3 rats, typical ventricular tachycardia was observed in 4 rats, and 3 rats exhibited ventricular tachycardia and then succumbed following ventricular fibrillation. Additionally, in the Ber 8 mg/kg group, 4 rats exhibited ventricular premature beat-bigeminies, 3 rats presented typical ventricular tachycardia, and 1 rat perished from ventricular fibrillation. In the Ber 16 mg/kg group, 2 rats exhibited ventricular premature beat, bigeminies or trigeminies, and 1 rat presented with typical ventricular tachycardia, with no evident abnormalities. Typical arrhythmias are presented in Fig. 2. The summaries of the arrhythmias and mortalities in each group are presented in Table I.

Compared with the control group, the levels of serum CK, CK-MB and LDH were significantly increased in the Chuan-wu group (B; P<0.05 or P<0.01). Whereas, in the 8 and 16 mg/kg Ber treatment groups (C and D), the levels of serum CK, CK-MB and LDH were all markedly reduced when compared with the Chuan-wu group, particularly in the Ber 16 mg/kg group (P<0.05 or P<0.01; Fig. 3).

Histopathological examination of the heart tissue revealed that there were no evident abnormalities in the myocardium of the control group (Fig. 4A). Whereas, following Chuan-wu administration for 3 days, small vein dilation, congestion, ventricular dilatation and congestion were observed in myocardial tissue. Additionally, some muscle fibers exhibited mild atrophy and inflammatory cell infiltration was observed in the muscle. The representative images of congestion in veins and slight inflammatory cell infiltration are shown in Fig. 4B. Compared with the Chuan-wu group, the congestion was reduced in the Ber 8 and 16 mg/kg groups. Notably, in the Ber 16 mg/kg group, no marked congestion was observed and only minor infiltration of inflammatory cells in myocardial tissues was exhibited (Fig. 4C).

In the control group, an ECG was recorded continuously for 120 min with no evident abnormalities observed. In all of the other groups, no abnormal ECG features were observed prior to the administration of AC. However, in the AC group, VPCD, VPB, VT, VF and DD occurred successively following the administration of AC. Compared with the AC group, the occurrence time of VT, VF and DD was significantly prolonged in the Ber 8 mg/kg pretreatment group (P<0.05). Ber 16 mg/kg pretreatment also significantly prolonged the occurrence of all types of arrhythmias mentioned above (P<0.05 or P<0.01; Fig. 5A). Therefore, pretreatment with Ber 8 and 16 mg/kg increased the dosage of AC required to induce arrhythmias (P<0.05 or P<0.01; Fig. 5B).

In the ex vivo experiment, the APA of the papillary muscle action potential was decreased following the administration of 3×10⁻⁷ mol/l AC, and the APD₃₀ and APD₉₀ were also significantly shortened when compared with prior to AC treatment; DAD and TA was also subsequently observed (Fig. 6). However, when Ber 1×10⁻⁵ mol/l pre-administration was performed, there were no marked changes in APA, APD₃₀ and APD₉₀, and no DAD occurred following AC perfusion. Subsequently, when the concentration of AC was increased to 1×10⁻⁶ mol/l, a decrease in APA and a shortening in APD₉₀ and APD₃₀ were induced (P<0.01); and DAD and TA were also observed (Fig. 7).