84 consecutive patients with rheumatic heart disease (RHD) who were going to undertake cardiac surgery were enrolled in this study at First Affiliated Hospital of Xi’an Jiaotong University, Xijing Hospital and Shanxi Provincial People’s Hospital in 2012. All patients consecutively underwent simple mitral valve replacement surgery.

Baseline demographics, physical examination, routine laboratory testing, echocardiography, and additional clinical data were available for all patients before surgery.

84 selected patients were divided into two group, they were atrial fibrillation (AF) group (the sample size is thirty-nine) and sinus rhythm (SR) group (the sample size is forty-five). The subjects were considered eligible to be enrolled into the AF group if they had obvious AF history and had been documented by electrocardiogram with AF for more than 6 months. The SR group is composed of patients who is sinus rhythm and without history of atrial fibrillation.

Before surgery, no patients received any type of angiotensin receptor blockers (ARB) or angiotensin-converting enzyme inhibitors (ACEI) for at least six months, and none of the subjects had taken anti-inflammatory drugs previously more than two weeks before the study. In the present study, we excluded the patients as follows: 1) patients who exceeded 65 years old or had a history of cancer; 2) patients with complicated diabetes, renal or liver failure; 3) patients with hyperthyroidism, hypertension, autoimmune disease; 4) patients with heart failure over New York Heart Association (NYHA) III or left ventricular ejection fraction (LVEF) less than 40%, and other heart diseases; 5) patients who suffered rheumatic fever in active stage.

Before they take part in this investigation, every selected patient has signed informed consent forms that obtained from every selected patient or their family members. All informed consent forms and the procedure protocols were authorized by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University. The investigation tallies with the principles outlined in the Declaration of Helsinki.

All patients underwent cardiac surgical operation with moderate hypothermia (33˚C - 34˚C) and the right atrium (RAA) tissue (0.3 - 0.5 mm³) was disserted during the surgery. Each piece of tissue was cut immediately into three parts. One was dropped into liquid nitrogen and then stored in the –80˚C for RT-PCR and Western blot. Samples for immunofluorescence were promptly implanted into Tissue OCT-Freeze compound, flash frozen, and cut into 10-μm sections using Cryostat at optimal cutting temperature. All tissue sections were stored at –80˚C until they were used for immunostaining and analysis. The third was fixed in 4% paraformaldehyde solution for 12 - 24 hours, imbedded in paraffin with Masson’s trichrome staining.

The specimens fixed in 4% paraformaldehyde were subjected to alcoholic dehydration and embedded in paraffin. 4 μm serial sections were sliced and subjected to Masson’s trichrome staining to highlight collagen fibers. Collagen volume fraction (CVF) assay was performed in tissue sections of the right atrium. Masson’s trichrome a was obtained from Boster Biological Engineering Corporation (Wuhan, China). Two slides of each sample were randomly selected and observed under polarization microscope. Six different vessel-free fields (×200) of each slide were captured, and the images were analyzed using Image Pro Plus 6.0 (IPP 6.0) software. Collagen volume fraction was showed as the percentage of area of positive collagen staining in the total area of the image. The following formula was used to calculate the fibrosis score: collagen fiber area/total view area × 100%.

The right atrial tissue samples were fixed in 4 per centum paraformaldehyde for 24 hour, afterwards in 30 per centum sucrose at 4˚C, until these tissues sank. After embedded in embedding reagent for frozen sections, 6-μm sections were obtained onto polylysine-coated slides. These slides were treated with acetone at 4˚C for 15 min and then with PBS (phosphate buffered solution). After treatment with 0.5% Triton X-100 at 37˚C for 30 minute, sections were hatched with 10% normal goat serum for 45minute at 37˚C. Subsequently, these sections were treated with foremost antibody (PDGF-A: 1:200; vimentin: 1:2500) at 4˚C overnight and secondary antibody (FITC conjugated goat anti-mouse antibody: 1:200; rhodamine red conjugated goat anti-rabbit antibody: 1:200; DAPI: 1:2000) at 37˚C for 30 min. After washing in PBS 5 times (5 min for each), mounting was done with anti-quencher, and sections were observed under a fluorescence microscope and photographed (×200). ImageJ image analysis software was employed to analyze and merge these photographs. Negative controls were obtained by omitting the incubation with primary antibodies.

In brief, frozen human right atrial tissue samples were thawed and homogenized on ice, then overall RNA was isolated using RNA-simple Total RNA Kit (Tiangen Biotechnology, China) according to the corporation’s directions and quantified. Then, using ReverTra Ace qPCR RT Kit (Tiangen Biotechnology) and according to the manufacturer’s instructions, RNA was reversely transcribed into cDNA successfully. Amplification of cDNA was done on thermal cycler (Applied Biosystems Step One Plus System). The volume of the reaction mixture was 20 μl, according to merchant description, amplification was executed successfully (SYBR^® Premix Ex Taq^TM II PCR kit and Applied Biosystems Step One Plus System). The reaction conditions are as follows: pre-denaturation for 30 second at 95˚C and 40 cycles of 95˚Cfor 5 second and 60˚C for 30 second. The melt curve was applied to define the specificity of products. The supporting software was applied to analysis the Ct value of products. According to the following formula: ΔCt = Cttarget gene − Ctinternal reference, the ΔCt was Table 1 Oligonucleotide probes calculated in two groups, respectively. The relationship between the CT value of the target gene and the copies of this gene is negative, consequently, with the increase of the ΔCt, the gene expression decreased. Then, 2^−ΔΔCt method was applied to calculate the relative mRNA expression of target genes. Oligonucleotide probes were in Table 1.

For Western blot analysis, frozen human right atrial tissue samples were used for protein isolation. Protein extraction was followed by the instruction of the total protein extraction kit (Apply gen Technologies Inc, China). Proteins (10 µg) was isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (Stratagene, USA) followed by staining with Ponceau S solution (Sigma, USA). The membranes were blocked with 5% non-fat dry milk and then explored rabbit polyclonal anti-human PDEGF-A (Abcam, USA) antibodies. Horse radish peroxidase-conjugated rabbit anti-mouse or anti-rabbit IgG (1:5000, Santa Cruz Biotechnology) was used as secondary anti-body followed by incubation with ECL Western Blot Detection Kit (Amersham, The Netherlands). The amount of protein chosen was in the linear immunoreactive signal range. The immunoreactive signals were exposed to Kodak film for 5 min and analyzed with gelpro analyzer after analysis software (Bio-Rad, USA) normalized by the corresponding value of β-actin. Experiments were repeated three times and the mean was scored.

All statistical analysis was made by the SPSS version 15.0. If they were normally distributed, continuous variables were indicated as mean ± standard deviation (x ± s). Comparisons of means were applied to independent t test between AF group and SR group. Groups for categorical variables were analyzed by chi-square. Correlation coefficients were assessed between PDGF-A and LAD, including the protein and mRNA expression of PDGF-A. A value of P < 0.05 was considered statistically significant.

The reporting of this study conforms to STROBE guidelines.

Patients’ characteristics are shown in Table 2. As a whole, most characteristics were similar between two groups. Left and right atrial diameters, measured by echocardiography, were significantly larger in the AF group than the SR group. All drugs were stopped at least 12 h before surgery.

Representative examples of right atrial tissue stained with Masson’s trichrome from each group are shown in Figure 1. Although the marked interstitial alterations were present because of all patients with RMVD, an apparent difference was observed between the two groups.

Comparison with the SR (Figure 1(a)) patients, there were abundant collagen fibers in the AF (Figure 1(b)) group. On the contrary, only a small amount of collagen fibers was observed in the SR group. The CVF in the AF patients (45.4% ± 2.33%) increased more drastically than that in the SR patients (12.9% ± 1.02%) (P < 0.001; Figure 1(c)), indicating some relationships between AF and fibrosis.

SBP, systolic blood pressure; DBP, diastolic blood pressure; WBC, white blood cell; RBC, red blood cell; HB, hemoglobin; CR, serum creatinine; LVEF, left ventricular ejection fraction; LAD, left atrium diameter; RAD, right atrium diameter.

We performed qRT-PCR to test whether the type I collagen or type III collagen level increases in the AF patient’s right atrial tissues. After RT-PCR, the Ct value and number of cycles were applied to depiction, and finally, the mRNA’s amplification curve was acquired. The results demonstrated that the way has good repeatability and consistent amplification efficiency. 2^−ΔΔCt method was applied to represent the relative expressions of target genes. The results displayed that the expression of mRNA of type I collagen was 2.042 ± 0.177 in the AF group, which was markedly higher than those in the SR group (0.988 ± 0.099), P < 0.05 (Figure 2(a)).

Compared with the SR group, the expression of type III collagen mRNA in the AF group was greater, but there was no marked difference between the AF group and SR group (1.228 ± 0.151 VS 1.067 ± 0.068, P > 0.05), (Figure 2(b)).

Representative sections of the immunofluorescent stained right atrial tissue from each group are shown in Figure 3. Although PDGF-A was expressed in both groups, compared with the SR group, the level of the PDGF-A’s protein was apparently higher in the right atrial myocardium of the AF group (Figure 4).

To test whether the PDGF-A increases in the right atrial tissues of the AF patients, we performed qRT-PCR and Western blotting. After real time PCR, the Ct value and number of cycles were applied to depiction, finally, the amplification curve of mRNA was acquired. The results demonstrated that the way has good repeatability and consistent amplification efficiency. 2^−ΔΔCt method was applied to indicates the relative expressions of target genes. The results displayed that the PDGF-A’s mRNA expressions was 2.062 ± 0.184, the PDGF-A’s protein expressions was 1.282 ± 0.193 in AF group, which were markedly higher than those in SR group (0.991 ± 0.062 and 0.517 ± 0.067, respectively; P < 0.01).

A strong positive correlation existed between mRNA of PDGF-A and Type I collagen; western blotting revealed a strong positive correlation between protein of PDGF-A and mRNA of Type I collagen (Figure 5(a), r = 0.75; Figure 5(b), r = 0.72, respectively).