| Rheumatic heart disease (RHD) is an autoimmune disease due to group A hemolytic streptococcus infection, which is the common cause of chornic heart failure (CHF). Ventricular pump dysfunction caused by abnormal hemodynamic disorders, decreased myocardial contractility, systemic and pulmonary circulation congestion, the blood output of heart is reduced and cannot pump out the venous retune and the various organs of the body tissue metabolism required to match the blood supply, which will generate a series of signs and symptoms. A variety of pathological changes such as hypertrophic changes of the myocardial cells, cardiac structural changes, cardiac interstitial fibrosis and inflammatory factors accumulation are associated with the development and progression of heart failure. The pathogenesis of heart failure caused by rheumatic heart disease is more complex and involving a wide range of molecular pathological changes. Recent studies have found that epigenetic mechanisms are involved in the onset and development of cardiovascular disease and are gradually being revealed. Epigenetic regulation include DNA methylation and histone modifications, in which DNA methylation is a hotspot of current research in the field of epigenetics. Studies show that gene expression was caused by the changes of cardiovascular disease-related gene promoter methylation status. In addition, the level of global genomic DNA methylation level and DNA methyltransferase is also involved in the process and outcome of disease. The three abnormal methylation status knows as "methylation imbalance". This kind of methylation imbalance is observed in coronary heart disease (CAD), atherosclerosis and congenital heart disease (CHD), however, the related epigenetic mechanism is still remains unknown in rheumatic heart disease. In this study, we firstly detected the mRNA expressions and the promoter methylation levels of three HF-related genes (BNP, NPRA and ICAM-1) in right atrial myocardial tissue of RHD patients and healthy controls by using real-time RT-PCR and the Bisulfite sequencing PCR. Then we measured the global DNA methylation status by ELISA and finally detected the expressions of DNMTs mRNA by real-time RT-PCR. We also explore the relationship between the clinical data and the above parameters meanwhile in order to discover the mechanisms and characteristics of epigenetics in rheumatic heart disease. Part I The inRNA expression levels and promoter methylation patterns of three HF-related genes in myocardium tissue of RHD-HF patientsObjective:To investigate the mRNA expression levels and the promoter methylation patterns of three HF-related genes and potential clinical significance in right atrial myocardium tissue of RHD-HF patients.Methods:Real-time RT-PCR and Bisulfite sequencing PCR (BSP+Sequencing) were performed for evaluated the mRNA expression levels and promoter region methylation patterns of three HF-related genes (BNP, NPRA and ICAM-1) in right atrial myocardial tissue of RHD patients and healthy controls. The correlation between the three HF-related genes mRNA, promoter methylation levels and clinical data were analyzed.Results:Compared with healthy controls, the mRNA expression levels of BNP and ICAM-1gene were significantly increased in right atrial myocardial tissue of RHD patients (BNP, p<0.001; ICAM-1, p <0.001); the mRNA expression level of NPRA gene was significantly reduced (p=0.044). The mRNA expressions of these genes were further changes with the progression of RHD disease. Compared with NYHA â…¡ group, the mRNA expression levels of BNP and ICAM-1gene were significantly increased in NYHA III group (BNP, p<0.001; ICAM-1, p=0.004); the mRNA expression level of NPRA gene was significantly reduced (p=0.001). For promoter methylation patterns, compared with healthy controls, the promoter methylation levels of BNP and NPRA genes were significantly increased (BNP, p<0.001; NPRA, p<0.001). The promoter methylation level of ICAM-1was significantly reduced (p=0.004). The promoter methylation levels of these genes were further changes with the progression of RHD disease. Compared with NYHA â…¡ group, the promoter methylation levels of BNP and NPRA gene were significantly increased in NYHA â…¢ group (BNP, p=0.005; NPRA, p <0.001); the promoter methylation level of NPRA gene was significantly reduced (p=0.004). The promoter methylation level and the mRNA expression level of BNP gene had positive correlation (r=0.680, P <0.001). The promoter methylation level and the mRNA expression level of NPRA gene and ICAM-1gene had negative correlation (NPRA, r=-0.233, P=0.041ICAM-1, r=-0.459, P<0.001). There were no correlations between mRNA expression levels of the three HF-related genes with age, sex and the occurrence of atrial fibrillation (p>0.05).Conclusion:Our findings suggest that the mRNA expression levels of BNP, NPRA and ICAM-1gene were changed in RHD-HF patients and consistent with the severity of the disease and disease progression. The promoter methylation levels could play an important role in these expression level changes, DNA methylation is involved in the development of RHD-HF. Part II Global DNA methylation status and its clinic significance in myocardium tissue of RHD-HF patientsObjective:To evaluate the global DNA methylation status in right atrial myocardial tissue of RHD-HF patients.Methods:ELISA like method with an antibody against5-methylcytosi.ne (5-MC) were performed to observe the global DNA methylation status in right atrial myocardial tissue of RHD patients and healthy controls. The results were compared with the clinical data.Results:Compared with healthy controls, the global DNA methylation status were significantly higher in right atrial myocardial tissue of RHD patients (p=0.044), and the global DNA methylation status were further increased with the progression of RHD disease. Compared with NYHA II group, NYHA III group had higher global DNA methylation status (p=0.029). The global DNA methylation status had positive correlation with age, the global DNA methylation status gradually increased with age increased. Comparison of different ages (<40years,40-50years,50-60years and>60years), the global DNA methylation status of60-year-old age group was significantly higher than the other three age groups. There were no correlations between global DNA methylation status with sex and the occurrence of atrial fibrillation (p>0.05).Conclusion:Global DNA hypermethylation was observed in right atrial myocardial tissue of RHD patients compared with healthy controls. The global DNA methylation status was gradually increased with the progression of disease. The global DNA methylation status had positive correlation with age; the global DNA methylation status of60-year-old age group was significantly higher than the other age groups. Part III The mRNA expressions of DNMTs and clinic significance in myocardium tissue of RHD-HF patientsObjective:To investigate the mRNA expression of DNMTs gene and its potential clinical significance in right atrial myocardial tissue of RHD-HF patients.Methods:The real-time RT-PCR was performed to evaluate the expression of the DNMTs. The correlations between DNMTs mRNA expression with global DNA methylation status and clinical data were analyzed.Results:Compared with healthy controls, the mRNA expression levels of all three DNMTs were significantly higher in right atrial myocardial tissue of RHD patients.(DNMT1, p=0.02; DNMT3a, p=0.043; DNMT3b, p=0.035) and the mRNA expression levels of all three DNMTs were further increased with the progression of RHD disease, Compared with NYHA â…¡ group, NYHA â…¢ group had higher mRNA expression levels of the three DNMTs (DNMT1, p=0.019; DNMT3a, p=0.019; DNMT3b, p=0.002). These had positive correlations between mRNA expression levels of DNMT1and DNMT3a, DNMT3a and DNMT3b (DNMT1and DNMT3a, r=0.618, p<0.001; DNMT3a and DNMT3b, r=0.509, p<0.001). Moreover, a significant positive correlation was found between DNMT1mRNA levels and the OD value of global methylation levels (r=0.350, p=0.002). This correlation did not occur between OD values and DNMT3a or DNMT3b mRNA expression levels (r=0.221, p=0.053, for DNMT3a; r=0.181, p=0.115, for DNMT3b). There were no correlations between mRNA expression levels of DNMTs with age, sex and the occurrence of atrial fibrillation (p>0.05).Conclusion:DNMTs genes were over-expressed in right atrial myocardial tissue of RHD patients and the over-expressions of three DNMTs were further increased with the progression of RHD disease. These had correlations between the three DNMTs and DNMT1was closely related with the global DNA hypermethylation status in RHD-HF patients.
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