| BackgroundAtrial fibrillation (AF) is one of the most common arrhythmias in clinical practice nowadays, which is associated with significant morbidity in the general population. AF can induce many complications, such as cardiac dysfunction, cerebral embolism and peripheral vascular embolism, which could cause the mortality of patients with AF is more than twice higher than normal subjects. In China, with the aging of the population, the prevalence of AF will be greatly increased in the future, which must be a heavy burden on the patient’s family and society. Although multiple factors for the etiology of AF, including coronary heart disease, hyperthyroidism and hypertension, have been recognized, the precise mechanisms that cause AF are not completely understood and the treatment of AF in clinical practice is unsatisfactory.It is recently reported that atrial remodeling, including atrial electrical remodeling (AER) and atrial structural remodeling (ASR), is tightly associated with the initiation, maintenance, and perpetuation of AF. As one of the most important manifestations in ASR, atrial interstitial fibrosis could increase space between cardiomyocytes, which is likely due to the loss of cells and fibrotic replacement and expansion of the extracellular matrix (ECM) may also cause conduction delays between cardiomyocytes and allow for alternate pathways of conduction. These changes are electrically apparent as ectopic foci and anisotropic conduction (directional variations), which create non-uniform wave fronts that allow for abnormal re-entrant arrhythmias. For this reason, atrial interstitial fibrosis is thought to playing a significant role in AF, and it is meaningful to investigate the mechanism of atrial interstitial fibrosis for clarifying the mechanism of AF and its effective treatment.Caveolae is50-to100-nm omega-shaped invagination of the plasma membrane which was first reported as early as the middle of the last century. Caveolae have been identified as an important member participating in the transcytosis of macromolecules, cholesterol transport and signal transduction in the various cells. Recent studies have indicated that, caveolin-1, the principal structural component and marker protein of caveolae, can attenuate many signal pathways in the process of fibrosis. However, these researches mainly focus on the diseases such as pulmonary fibrosis, connective tissue disease and systemic sclerosis. Utill now, there is no study on the relationship between caveolin-1and atrial interstitial fibrosis in patients with AF in the domestic and overseas. To investigate the mechanism of caveolin-1might be a key to understand the occurrence of atrial interstitial fibrosis in AF, which will probably provide a new target for treatment of AF.Objectives(1) To observe the expressions of caveolin-1and TGF-β1in atrial tissue samples from patients with AF;(2) To investigate the effect of TGF-β1on caveolin-1in cardiac fibroblasts.MethodsThe object was comprised of patients with rheumatic heart disease undergoing mitral/aortic valve replacement in department of cardiac surgery, including sinus rhythm group (SR) and chronic or persistent AF (>6months). After written informed consents were given, atrial tissue samples from right atrial appendage were obtained from the all subjects. Part of the samples were then fixed in4%formaldehyde solution for24hours and processed for paraffin embedding. Paraffin blocks were cut into5-mm sections and mounted to slides. Sections of atrial tissue were stained with Masson-trichrome to assess the degree of fibrosis in AF and SR group. Atrial tissue samples were investigated by Western-blot for the protein expressions of caveolin-1, TGF-β1, Collagen I and III, and qRT-PCR for the mRNA expressions of caveolin-1and TGF-β1.Cardiac fibroblasts were obtained by enzymatic digestion and differential attachment from the hearts of15SpragueDawley rats. After seeded in six-well tissue culture plates and reaching about90%confluence, cells were treated with recombinant human TGF-β1according to the experimental design. Fibroblasts were incubated for different lengths of time (0,6,12,24and48h) with100ng/ml recombinant human TGF-β1, and with different concentrations of TGF-β1(0,0.1,1,10,100and1000ng/ml) for48hours. Cells were then harvested from each experimental group and processed to obtain cell lysates for Western blot to measure caveolin-1protein level. Each experimental condition was tested in nine replicate wells and the mean was taken to represent an individual experiment.Results(1) There were23patients recruited in this study, including10with no history of AF (assigned into SR group) and13with chronic or persistent AF (assigned into AF group). Left atrial diameter (LAD) in AF group is larger than in SR group (59.41±10.54vs.47.83±4.92, P<0.01).(2) To evaluate the fibrosis in AF and SR group, western blot for collagen I and collagen III and Masson staining were performed. The results showed that there was higher degree of fibrosis in AF patients (P<0.05).(3) Compared with SR group, the protein expression of caveolin-1in AF group was decreased significantly (0.65±0.17vs.1.00±0.14, P=0.01), meanwhile TGF-β1elevated (2.31±0.65vs.1.00±1.78, P=0.01), consisting with the result of qRT-PCR (0.28±0.13vs.1.02±0.20,3.29±1.64vs.1.03±0.27, P=0.00). The expression of caveolin-1was significantly correlated with the expression of TGF-β1(r=-0.34P=0.00).(4) Rat cardiac fibroblasts were successfully cultured, and the purity was95%.1) After incubated with100ng/ml TGF-β1for the time dependency (0,6,12,24, or48h), the caveolin-1expression showed as follows:in the first12h, TGF-β1elevated caveolin-1level to1.4±0.1fold than in control cells (P=0.00), however, after 48h incubation, the caveolin-1decreased to0.58±0.02fold than in control cells (P=0.00).2) After incubated for48h for the concentration dependency (0,0.1,1,10,100, or1000ng/ml of TGF-β1), the caveolin-1expression showed as follows:10,100and1000ng/ml of TGF-β1group was, respectively,0.73±0.23,0.65±0.27and0.58±0.32fold lower than in control cells (P=0.08,0.03and0.01).Conclusions(1) The expression of caveolin-1decreased in atrial tissue of patients with AF, which could promote the development of atrial interstitial fibrosis.(2) TGF-β1markedly decreased caveolin-1expression in a dose-and time-dependent manner in cardiac fibroblasts. |
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