In this single-center, prospective case-control study, 30 patients who underwent valve replacement for the treatment of RVHD in the Cardiac and Major Vascular Department, Yue Bei People’s Hospital between August 2012 and January 2015, were included. The patients were categorized equally into two groups according to the electrocardiogram (ECG) characteristics as follows: sinus rhythm (SR) group and chronic AF group.
Inclusion and exclusion criteria
The inclusion criteria of the patients were as follows: 1) RVHD patients with indications for cardiac valve replacement; AF patients also met the ECG diagnostic criteria; 2) NYHA grade of heart was grade II-III; 3) Vital signs and the disease condition was stable; 4) Clear consciousness, volunteered to participate in this study, and cooperated with the treatments. The exclusion criteria were as follows: 1) Other types of heart diseases, such as myocardial infarction and acute myocarditis, within the last month; 2) Complications such as a malignant tumor, chronic obstructive pulmonary disease (COPD), and uremia; 3) Cardiac function of grade IV.
This study was approved by the Ethics Committee of the Yue Bei People’s Hospital. The serum and tissues of the right auricle were collected during hospitalization. The collection of serum and tissues were performed after consent from the families. Written informed consent was also obtained.
Diagnosis of AF
For the diagnosis of AF, patients must meet at least one of the following criteria: 1) current ECG showed AF and 2) patients had been diagnosed with AF before.
According to the Guidelines for the Management of Atrial Fibrillation issued by the European Society of Cardiology (ESC) in 2010, AF was classified into the following five types : 1) first diagnosed AF: The patients are diagnosed with AF for the first time; 2) paroxysmal AF: the AF is self-terminating, usually within 48 h and does not persist for more than 7 days. If the AF persisted for > 48 h, the likelihood of spontaneous conversion is low, and anticoagulation must be considered; 3) persistent AF: the AF episode either lasts for > 7 days or requires termination by drug therapy or direct current cardioversion; 4) long-standing persistent AF: the AF lasts for > 1 year and requires rhythm control strategy; 5) permanent AF: both drug therapy and direct current cardioversion failed, and the presence of AF is accepted by patients and physicians.
According to the New York Heart Disease Association (NYHA), the cardiac functions were graded as follows : 1) grade I, physical activities are not limited, and general physical activities do not induce excessive weakness, palpitation, or shortness of breath; 2) grade II, physical activities are slightly limited, and no discomfort is reported during rest, while several daily activities induces weakness, palpitation, or shortness of breath; 3) grade III, physical activities are evidently limited, no discomfort is reported during rest, while the load less than daily activities induce weakness, palpitation, or shortness of breath; 4) could not perform any physical activities without symptoms; symptoms could appear during rest, and any physical activities could increase the discomfort.
After hospitalization, patients were also excluded if the medical history, physical examination, and laboratory examination results ruled out malignant tumors. Also, patients with the cardiac function of grade IV were excluded.
Equipment and materials
Modified Masson tri-staining dye was from the Maiwei Biotechnology Co. Ltd (Xiamen, China). Rabbit-anti-human anti-MMP-9 antibody, working solution of horseradish peroxidase (HRP)-labeled streptavidin, and enzyme-linked immunosorbent assay (ELISA) kits for human MMP-9/gelatinase-B were purchased from the Zhongshanjinqiao Biotechnology Co. Ltd (Beijing, China). Goat-anti-rabbit IgG-HRP antibody was purchased from Bioworld Technology (USA). Other reagents were obtained from the Department of Laboratory Medicine, Yue Bei People’s Hospital.
A volume of 5 mL venous whole blood was collected in an RNA-free dry vacuum tube by centrifugation at 3000 rpm for 20 min at room temperature. The serum was transferred into a nuclease-free Eppendorf tube and stored at -80 °C until further use.
The cardiac tissues of all patients were harvested by a chief surgeon who had worked in the Cardiothoracic Surgery Department for over 20 years. All the pathological examinations were performed by an attending pathologist with 10 years of experience. During the operation, an equivalent of 200 mg myocardial tissues of the right auricle was harvested, rinsed by normal saline to remove blood, and the adipose tissues removed. Then, the myocardial tissues were placed in liquid nitrogen until use. In addition, a part of myocardial tissues was fixed with 10% neutral formalin liquid, and embedded in paraffin.
Hematoxylin-eosin (H&E) staining of myocardial tissues
The myocardial tissues were routinely embedded, sliced, dewaxed by xylene, dehydrated by gradient ethanol, washed, and stained with hematoxylin. Then, the tissues were washed again, developed in a hydrochloric acid ethanol solution, counterstained by eosin, washed, dehydrated, cleared, and mounted by neutral balsam.
Masson’s staining of myocardial tissues
After Masson staining, the myocardial tissues were examined by light microscopy (× 400) to assess the contents and distribution of collagen fibers. For each sample, images of five random visual fields were captured. The Image Pro Plus, 6.0 image analysis system, was used for the analysis of the images and assess the percentage of collagen fibers in the overall area of myocardial tissues.
Immunohistochemistry of myocardial tissues
The myocardial tissues were embedded in paraffin and sliced, followed by dewaxing and rehydration. Then, the slices were incubated with 3% H2O2 for 5–10 min at room temperature, washed with distilled water, and immersed in phosphate-buffered saline (PBS) for 5 min. Subsequently, 5–10% normal goat serum (diluted in PBS) was used to block the sections for 10 min at room temperature and incubated with rabbit-anti-human MMP-9 antibody at 37 °C for 2 h and then with HRP-labeled streptavidin at room temperature for 30 min. Finally, the slices were washed with PBS and developed using Diaminobenzidine (DAB) for 3–15 min, rinsed under running water, counterstained, dehydrated, cleared, and mounted.
Measuring serum MMP-9 level with ELISA
Fasting peripheral venous blood was withdrawn in the morning when the patients were conscious. ELISA kits (Boster, Wuhan, China) were used to measure the MMP-9 level by the same investigator from the Department of Laboratory Medicine, according to the manufacturer’s instructions.
Measurement of LAD and ejection fraction (EF)
Echocardiography was performed by an attending physician at the Department of Sonography before the operation, and the LAD and EF were measured.
SPSS19.0 software was used for the statistical analysis. Quantitative data were described using means and standard deviations. Unpaired Student’s t-test was conducted for the comparisons between two groups of continuous data. Qualitative data were compared using χ2 test. Pearson’s linear correlation analysis was performed for the correlation test. Multivariate logistic regression analysis was performed to investigate the association between general characteristics, including age, sex, smoking history, LAD, systolic blood pressure (SBP), diastolic blood pressure (DBP), blood lipid, creatinine (Cr), uric acid (UA), and blood glucose with MMP-9 levels, and further assess the effect sizes. p < 0.05 indicated statistical significance.