TGF-?1 And TIMP-4 Regulate Atrial Fibrosis In AF Secondary To RHD

Background Atrial fibrillation(AF) is a common clinical manifestation in rheumatic heart diseases(RHD) caused by valvular damage due to streptococcal infections. Atrial fibrosis occurs due to the proliferation of fibroblasts in the cardiac muscle and is a prominent manifestation of atrial structural remodeling in AF patients, which is an irreversible process.TGF-?1 is present at high levels in fibrotic cardiac tissue and apart from its role in collagen deposition, TGF-?1 is also involved in fibroblast differentiation to myofibroblasts.TIMP-4 is involved in the control of ECM degradation under normal conditions and its overexpression results in activation of signaling pathways associated with fibrosis and inflammation. Theregulationmechanismof two factors involvedin atrial fibrosisstillunclear,especiallynorelateddatawith TIMP-4.Objective To investigatetherelationshipwith tissue inhibitor of metalloproteinase 4(TIMP-4) and atrialfirbrosis, andtheregulation function andinteractionofthe transforming growth factor-?1(TGF-?1) and TIMP-4 in influencing the severity of atrial fibrosis in rheumatic heart disease(RHD) patients withatrial fibrillation(AF).Methods 1. Adopting systematic random sampling, a total of 53 patients(aged between 30-70 years old) with rheumatic heart disease who underwent heart valve replacement surgery orcardiac catheterization were selected. Based on routine preoperative electrocardiogram and 24 h Holter monitoring, all recruited patients were divided into sinus rhythm group group and atrial fibrillation group.2. The degree of myocardial fibrosis was evaluated using Masson staining. The collagen area was calculated using image analysis software, IPP 7.0. Ten fields were randomly selected from each section at high magnification and the total collagen area(green-stained areas) in each field was measured. The collagen volume fraction(CVF) = total collagen area/total field area and the mean values were calculated and recorded. 3. The expression levels of TGF-?1,TIMP-4, matrix metalloproteinase-2(MMP-2),type?collagen and type ? collagen were estimated by western blot analysis. Total cellular protein was extracted from each TGF-?1 treatment and the effect of TGF-?1 stimulation on TIMP-4 expression in atrial fibroblasts was estimated by western blot analysis. The results presented are a representative of three independent experiments. 4. Additionally, TGF-?1 and TIMP-4 m RNA levels were quantified by q RT-PCR. The effect of TGF-?1 stimulation on TIMP-4 expression was assessed by in vitro stimulation of freshly isolated human atrial fibroblasts with recombinant human TGF-?1, followed by western blot analysis to detect changes in TIMP-4 levels. 5. All statistical analyses were conducted applying SPSS19.0 software(SPSS Inc., Chicago, IL, USA), and the data were represented as means ± standard deviation(SD, ±s). The statistical comparison between two groups was performed using the two independent samples t-test and correlation analysis was conducted using the Pearson correlation analysis.P < 0.05 was considered as statistically significant.Results 1. Masson stain revealed that the left atrial diameter(LAD) and collagen volume fraction(CVF) were obviouslyincreased in AF patients, compared to sinus rhythm(SR) controls(both P< 0.05). 2. Western blot analysis showed significantly elevated levels of the AF markers MMP-2, type?collagen and type ?collagen in the AF group, in comparison to the SR controls(all P < 0.05). 3. In the AF group, TGF-?1 expression was relatively higher, while TIMP-4 expression was apparently lower than the SR group(all P< 0.05).4. TIMP-4 expression level showed a negative association with TGF-?1 expression level(r =-0.98, P< 0.01) and TGF-?1 stimulation of atrial fibroblasts led to a sharp decrease in TIMP-4 protein level.Conclusions 1. Increased TGF-?1 expression and decreased TIMP-4 expression correlated with atrial fibrosis and ECM changes in the atria of RHD patients with AF. 2. Notably, TGF-?1 suppressed TIMP-4 expression, suggesting that selective TGF-?1 inhibitors may be useful therapeutic agents. 3. Decreased TIMP-4 expression and increased TGF-?1 expression in the atrium correlates with atrial fibrosis and ECM changes in the atria of RHD patients with AF. 4. Our findings support the hypothesis that TIMP-4 and TGF-?1 are involved in the pathogenesis of AF in RHD patients, implicating that TGF-?1 and TIMP-4 and ECM molecules could be used as biomarkers of AF in RHD patients.