The main drugs and reagents used in the study included rosuvastatin (H20060406; AstraZeneca, London, UK), the hydroxyproline assay kit and Masson staining kit (Njjcbio, Nanjing, China), an anti-type I collagen antibody (Abcam, Cambridge, UK) and the Bradford Protein Assay kit (Blkwbio, Beijing, China).

Healthy New Zealand white rabbits of either gender, weighing 2.2±0.3 kg, were purchased from the Experimental Animal Center of Shandong University of Traditional Chinese Medicine (Shandong, China). The animal research protocol complied with ‘The Guide for the Care of Use of Laboratory Animals’ published by the National Institutes of Health (publication no. 85-23, revised 1996) and was approved by the Animal Care Committee of Medical College of Shandong University. The 30 surviving rabbits were randomly divided into the sham operation control group (S; n=8), the MI group (MI; n=7), the low-dose rosuvastatin group [R_s; 2.5 mg/(kg/d), n=7] and the high-dose rosuvastatin group [R_l; 5 mg/(kg/d), n=8]. The S group received thoracotomy only. Ligation of the left anterior descending coronary artery was performed to induce MI. The rabbits in the R_s and R_l groups received intragastric administration of rosuvastatin following MI for 8 weeks, while the rabbits in the S and MI groups did not receive any drug intervention.

The rabbits were anesthetized with 3% sodium pentobarbital (40 mg/kg) by intraperitoneal injection and were fixed on a small animal surgery table. Following a routine preoperative electrocardiogram (ECG), a longitudinal incision was made along the left sternal border, using the 3rd and 4th intercostal spaces as a midpoint, to fully expose the heart. Subsequent to opening the pericardium, the heart was lifted to expose the anterior descending left coronary artery. At the midpoint, ligation was conducted using a 6–0 suture. The operators directly observed that the anterior and apical left ventricular myocardium gradually turned to a dull and even pale color, which was accompanied with a weakened pulse. ECG showed a gradual ST segment elevation in I and aVL, indicating the formation of MI. Fully intraoperative hemostasis was achieved, followed by a layer-by-layer sternal closure subsequent to the ligation. For the S group, a suture was placed under the left anterior descending coronary artery without ligation. To prevent postoperative wound infection, all the rabbits were injected with penicillin at 100,000 U/d for 3 consecutive days.

Cardiac ultrasound was performed 8 weeks postoperatively using an ultrasound probe (5–12 MHz). The left ventricular end-systolic diameter, end-diastolic diameter, LVEF and LAD were measured. Real-time video recorded during the measuring was documented for future analysis. The final data represented the average of 3 parallel measurements.

Subsequent to measuring their weights 8 weeks postoperatively, the rabbits were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (40 mg/kg), followed by thoracotomy. The heart was quickly harvested and rinsed with saline. The large blood vessels, right atrium, left ventricle and right ventricle were removed. The left atrium was separated and divided into two portions; one portion was stored at −80°C for the measurement of collagen and type I collagen protein levels and another portion was fixed with 4% paraformaldehyde, embedded in paraffin and sectioned for hematoxylin and eosin (HE) and Masson staining.

The content of myocardial hydroxyproline was measured to derive the content of myocardial collagen. First, 30–50 mg of left atrial tissue was homogenized and processed according to the manufacturer's instructions provided with the hydroxyproline assay kit. Subsequently, the supernatant in each tube was collected and the optical density (OD) value at 550 nm was measured with a spectrophotometer. The content of collagen (mg/l) was calculated according to the following formula: Collagen content = [(OD value of the sample tube - OD value of the blank tube) / (OD value of the standard tube - OD value of the blank tube)] x 7.46 x dilution factor.

The tissue stored at −80°C, as mentioned in ‘Specimen collection,’ was weighed and dissolved in lysate buffer. The proteins were extracted, quantified, separated by SDS-PAGE electrophoresis and electrotransferred following denaturation. The nitrocellulose (NC) membrane bearing the transferred proteins was incubated with the blocking solution at room temperature for 1 h. Subsequently, the NC membrane was placed in a hybridization bag, agitated in the primary antibody solution at room temperature for 30 min and incubated in a 4°C refrigerator overnight. Subsequent to washing the polyvinylidene fluoride (PVDF) membrane with Tris-Buffered saline and Tween-20 (TBST) 3 times, with 10 min for each wash, the secondary antibody was added, followed by an incubation at room temperature for 60 min. Subsequent to washing with TBST 3 times for 10 min each, the PVDF membrane was developed using chromogenic substrate solution in a darkroom and the image was captured to measure the gray value for data analysis. The GAPDH level served as the internal control.

The SPSS version 17.0 (SPSS, Inc., Chicago, IL, USA) statistical package was applied for statistical analysis. All the data are expressed as mean ± standard deviation. Comparisons among multiple groups were performed using an analysis of variance and pairwise comparisons were carried out using the least significant difference method. P<0.05 was considered to indicate a statistically significant difference.

HE staining showed that the atrial myocytes in the S group were arranged regularly and that infiltration of a small amount of inflammatory cells had occurred, although neither myocardial necrosis and hypertrophy nor stromal hyperplasia was observed. In the MI group, the number of atrial myocytes was decreased, their arrangement was irregular, the nuclei were of different sizes, with a few dissolved or ruptured, and there was an accumulation of interstitial collagen. Compared to the MI group, the R_s and R_l groups exhibited reduced myocardial necrosis and reduced infiltration of inflammatory cells, with a significant improvement in compensatory hypertrophy, stromal hyperplasia and collagen accumulation in myocardial cells, as shown in Fig. 1. Masson staining showed normal cardiac tissue stained in red and collagen fiber stained in blue. The expression of collagen fiber in the left atrial tissue of MI rabbits significantly increased, with fibrosis and interstitial collagen accumulation in certain cardiomyocytes, as shown in Fig. 2.

The LAD and the left atrial collagen content in the MI group were significantly greater than those observed in the S group (P<0.01), while the LVEF value in the MI group was significantly lower than that in the S group (P<0.01). The LAD and the left atrial collagen content in the R_s and R_l groups were significantly lower than those in the MI group (P<0.01), while the LVEF values in these groups were significantly higher than that in the MI group (P<0.01). Compared to the R_s group, all the indicators in the R_l group were significantly lower, although the differences were not statistically significant, as shown in Table I.

The expression of left atrial type I collagen in the MI group was significantly higher than that observed in the S group (P<0.01) and the expression of left atrial type I collagen in the R_s and R_l groups was significantly lower compared to the MI group (P<0.05). However, the difference between the R_s and R_l groups was not statistically significant (P>0.05), as shown in Fig. 3.