A Study On Molecular Mechanism Of Atrial Structure Remodeling In Human Rheumatic Atrial Fibrillation

Part I The Atrial Structure Changes of Patients with Rheumatic Atrial FibrillationAim: The aim of this study is to observe the structural changes of left atrium in patients with rheumatic heart diseases(RHD),and to investigate the role of atrial remodeling of structure on the mechanism for the onset and maintenance of atrial fibrillation(AF).Methods: 43 patients with RHD who required valve replacement were included, who were consist of 15 patients with regular sinus rhythm(Group RSR), and 8 patients with paroxysmal AF(Group PAF) and 20 patients with chronic AF(Group CAF). 10 patients with congenital heart disease with regular sinus rhythm were selected as control group (Group CONT).The left and right atrial dimension, left ventricular end-diastolic dimension and ejection fraction of left ventricular were measured by UCG before operation. After the left atrial tissues obtained during cardiac surgical operation, the structure and ultrastructure changes were observed by light microscope through HE staining and by electron microscope.Results: The left and right atrial dimension of group CAF(Lad:61.1±10.1 mm, Rad:48.3±15.4mm) and PAF(Lad:55.0±7.8mm, Rad:41.6±6.3mm) were significantly higher than that of group RSR(Lad:45.4±6.3mm, Rad:35.9±4.6mm) and CONT (Lad:38.2±5.8mm, Rad:36.7±5.2mm) (all P value﹤0.05).Through HE staining, disorder myocytes and loose myocardium fibres can be shown in group PAF.The left atrial myocyte of group CAF disarranged, and myocardium fibres is loose and inconsecutive.The remarked changes can be seen by electrical microscope in group CAF, such as disorganized sarcomere, enlarged mitochondria, fragmentation of sarcoplasmic reticulum, tortuous intercalated disc.Conclusions: The obvious changes happened in the left atria at the general level, tissue and cell level, and ultrastructure level, respectively. The structural remodeling may play a key role on the mechanism for incidence and maintenance of AF.Part II The Expression and Significance of Caspase-3 and Bcl-2 in Human Rheumatic Fibrillating AtriaAims: The aim of this study is to detect the protein content of Bcl-2,caspase-3 and apoptosis at the left atria of patients with RHD, and to investigate the role of the two proteins on the atrial remodeling.Methods: 43 patients with RHD who required valve-replacement were included, who were consist of 15 patients of RHD with regular sinus rhythm(Group RSR), and 8 patients with paroxysmal AF(Group PAF) and 20 patients with chronic AF(Group CAF). Western blot was used to examine the protein content of caspase-3 and Bcl-2. The apoptosis index(AI) were measured by TUNEL.Results: (1)Compared to Group RSR ,the content of caspase-3 of group CAF were up to 394%±99.4%(P﹤0.001);But the protein content of Bcl-2 of group CAF reduced to 32.8%±15.9%(P<0.001). The expression of these proteins did not changed in group PAF. (2) AI of group CAF is 24.6%,which is positively higher than that of group RSR and PAF(P<0.01). (3) In group CAF, the protein content of caspase-3 and AI were positively relative to left atrial dimension and AF duration, respectively(all P value<0.005); The protein content of Bcl-2 were negatively correlated with left atrial dimension and AF duration(all P value<0.001).(4) The protein content of caspase-3 positively relate with AI (r=0.794,P <0.001), and the content of BCL-2 negatively relate with AI(r=-0.734,P <0.001). The protein content of caspase-3 negatively relate with that of Bcl-2(r=-0.848,P <0.001).Conclusions: Apoptosis increased in the left chronic fibrillating atrium. The key two proteinases of caspase-3 and Bcl-2 have interaction to regulate apoptosis in left atria. This course causes irreversibly deteriorated atrial structure and dysfunction to promote the onset and maintenance of AF. This may be important for the mechanism of AF.Part III The Expression and Significance of Calpain-I in Left Rheumatic Fibrillating Human AtriaAims: The aim of this study is to detect the expression of calpain-I at the left atria of patients with RHD, and to investigate the relationship between calpain-I,calpastatin, caspase-3 and apoptosis and the role of calpain-I on the mechanism of AF.Methods: 43 patients with RHD who required valve-replacement were included, of whom, 15 patients with regular sinus rhythm(Group RSR), 8 patients with paroxysmal AF(Group PAF) and 20 patients with chronic AF(Group CAF). Western blotting was used to examine the protein amount of calpain-I, calpastatin, caspase-3 and Troponin-I.RT-PCR was used to detect mRNA expression of calpain-I. The relationship between calpain-I,calpastatin, caspase-3 and apoptosis were analyzed.Results:1.Compared with that of Group RSR by being measured through western blotting, the protein content of calpain-I of group CAF increased to 344%±101.9%(P<0.001).The contents of Troponin-I of group CAF decreased to 45.0%±13.4% (P<0.001), and that of caspase-3 of group CAF were up to 394%±99.4%(P﹤0.001);But the content of calpastatin of group CAF reduced to 27%±12.8%(P<0.001).The expression of these proteins had not changed in group PAF.2. The expression of calpain-I in mRNA level reach at 2.49±0.86,which was significantly higher than that of Group RSR(0.89±0.23)and PAF(1.23±0.31)(P<0.001)3. In group CAF, the protein content and the amount in mRNA level of calpain-I were positively relative to left atrial dimension and AF duration, respectively(all P value<0.05). The protein content of calpastatin were negatively relative to left atrial dimension and AF duration, respectively(all P value<0.05).4. There was a negative relationship between the protein content of calpain-I and Troponin-I.5.The protein content of calpain-I positively related with that of caspase-3 and AI (all P<0.001). and the content of calpastatin negatively relate with that of calpain-I and caspase-3(all P <0.001).Conclusions: In left atria with chronic fibrillation the protein content of calpain-I,caspase-3 and calpastatin had changed. There is close relationship between these factors, and they influence each other and may compose a system to cause the structure remodeling and dysfunction of atria. This course may promote the onset and maintenance of AF, and plays a key role on the mechanism of AF.Part IV The Role of Matrix Metalloproteinases-1,-9 and Tissue Inhibitor of Metalloproteinase-1 on the Atrial Structural RemodelingAims: The aim of this study is to detect the expression of matrix metalloproteinases -1,-9(MMP-1,-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) at the left atria of patients with RHD,and to investigate the role of MMP-1,-9 and TIMP-1 on the mechanism of AF.Methods: 43 patients with RHD who required valve-replacement were included, of whom, 15 patients with regular sinus rhythm(Group RSR), 8 patients with paroxysmal AF(Group PAF) and 20 patients with chronic AF(Group CAF). Western blotting was used to examine the protein content of MMP-1,-9 and TIMP-1.RT-PCR was used to detect mRNA level expression of MMP-1,-9 and TIMP-1.Results:1. The protein amout of proteins tested by western blotting.(1) Compared with Group RSR, the contents of MMP-9 of group CAF increased to 183%±629% (P<0.001), but the contents of TIMP-1 of group CAF decreased to 68%±28% (P<0.001). There were no changes in the expression of the above two proteins in Group PAF. It also was not diffrernce in the expression of the protein MMP-1 in every group.(2) In the group of CAF, the protein contents of MMP-9 were positively relative to left atria dimension and AF duration respectively(all P value<0.05), and that of TIMP-1 were negatively relative to left atrial dimension and AF duration respectively (all P value<0.05). The protein contents of MMP-9 was negatively relative to that of TIMP-1.2. The mRNA level expression of proteins tested by RT-PCR.(1) Compared with that of Group RSR, the mRNA level expression of MMP-9 of group CAF statistically increased (P<0.001), but that of TIMP-1 of group CAF statistically decreased (P<0.001). There were no changes in the expression of the above two proteins in Group PAF. It also was not diffrernce in the mRNA level expression of MMP-1 in every group.(2) In the group of CAF, the mRNA level expression of MMP-9 were positively relative to left atrial dimension and AF duration respectively(all P value<0.05), and that of TIMP-1 were negatively relative to left atrial dimension and AF duration respectively (all P value<0.05). The mRNA level expression of MMP-9 was negatively relative to that of TIMP-1. Conclusions: The expression of MMP-1 upregulated and that of TIMP downregulated in left fibrillating atria. Losing the balance of MMP-1 and TIMP-1 promotes the structural changes and dysfunction of the atria, and involve with the mechanism of AF.