| Part one:Study on The Mechanism of Shear Stress on Mitral Valve Lesion Combined with Atrial Fibrillation in Pig Left Atrial Flow Field analysis was performed to compare the differences between the each group and the control group.Objective:The Models of mitral valve stenosis,mitral valve insufficiency,atrial fibrillation and valvular atrial fibrillation were established respectively to clarify the relationship between TurBulent Shear Stress(TSS)and flow field uniformity in left atrial flow field of each model and pathological changes of left atrial tissue.To lay a foundation for further study on the molecular biology of left atrial tissue injury induced by TSS.Methods:1.An animal model of simple mitral stenosis,mitral regurgitation,atrial fibrillation,mitral stenosis with atrial fibrillation,mitral regurgitation with atrial fibrillation was established using a small group of Yunnan small ear pigs.Model grouping:MS group(n=5),MR group(n=5),AF group(n=5),MS+AF group(n=5),MR+AF group(n=5)and normal control group(n=5),and carry out 1 year of dynamic observation and monitoring.Echocardiography was performed on each group before modeling,immediately after surgery,1 month,6 months,and 12 months after surgery;Detect conventional Doppler indicators(MVmax,MPGmean,RFMV,LAD,LAAD).,LAEF,LAAEF);flow field uniformity index(MVA,MVRV,PVVmean,LAAVmean,Vmax,?Vmax);application of echocardiography combined with computer-aided images,computatioinal fluid dynamics,and analysis of turbulent shear stress(TSS).Statistical2.After the modeling of 12 months and completing the echocardiographic examination,all animals were sacrificed after surgery and took the pulmonary vein opening,left atrial appendage,atrial septum,mitral valve,and papillary muscles.According to the TSS test results,the most stressed tissue was neutralized.After formalin fixation,paraffin-embedded sections were stained with HE and Masson staining.The pathological changes of the atrial tissue were observed under light microscope.Right atrial homogenate was cut and the mRNA expression of KCa2.3/3.1 in left atrial tissue was detected by Real-Time PCR.The expression of KCa2.3/3.1 in left atrial tissue was detected by Western Blot.Immunofluorescence was used.Techniques to determine the distribution characteristics of KCa2.3/3.1 in left atrial tissue.Statistical analysis was performed and compared with normal control group and AF alone animal model group.Results:1.The models were well-produced.Among them,1 case died in the MR group,MS+AF group and the MR+AF group.In the AF group,1 case died during the observation.2.There was no statistical difference in the indexes before and after surgery in the control group(P<0.05).3.Routine doppler indicators,flow field uniformity and TSS results:there were statistically significant differences in the changes of the indicators of LAD,LAAD,LAEF and LAAEF after the establishment of each model compared with the control group(P<0.05),and they were consistent with the echocardio graphic features of corresponding diseases.MVA,MR and MVRV of MS and MS+AF group showed immediate changes after surgery,and gradually increased with the extension of observation time(P<0.05).Other indicators of PVVmean LAAVmean,Vmax and endovascular Vmax were all significantly changed after modeling,and the flow field uniformity in the left atrium was significantly reduced.There was no difference in TSS between each model group and the control group in Lc core area(P>0.05),while both Lm and L1 sites were significantly increased compared with the control group(P<0.01).4.The LAD,LAAD,LAEF,and LAAEF of each model group were compared immediately after modeling and one month after modeling,and the difference was not statistically significant(P>0.05);After 6m and 12m after modeling,the above indicators were compared statistically(P<0.05).The PVVmean,LAAVmean,Vmax,and AVmax of each model group were compared immediately after modeling and one month after modeling,and the difference was not statistically significant(P>0.05);after 6m and 12m after modeling,the above indicators were compared statistically(P<0.05);among them,the MS+AF group was significantly different from the other groups at 6m and 12m after modeling(P<0.01).The TSS detection index Lm of each model group was compared with that before and after modeling,and each time point after modeling were not statistically significant(P>0.05).The TSS detection indexes Lc and L1 of each model group were compared immediately after modeling and one month after modeling,and the difference was not statistically significant(P>0.05).The differences between the two indicators were statistically significant(P<0.05)at 6m after modeling and 12m after modeling.Among them,the Lc and L1 indicators at the 12m time point after modeling in the MS+AF group were significantly different from the other groups(P<0.01).5.HE and Masson staining of left atrial myocardium showed that the left atrial myocardium in the normal group was structurally intact and neatly arranged,surrounded by a small amount of interstitial tissue;the nucleus was large and clear,and the regular fiber network was filled in the whole cardiomyocytes.In the MS,MR,AF,MS+AF and MR+AF groups,the myocardial cells in the left atrial tissue were disordered,elongated and corrugated,and the connective tissue between the myocardial fibers accumulated,which widened the interval between the cardiomyocytes.Compared with the control group,the myocardial cells in MS and MR groups were larger,loose and edematous,with enlarged nuclei,vacuoles and increased intercellular connective tissue.The above pathological manifestations of MS+AF and MR+AF groups were more prominent,especially in the MS+AF group.The vacuoles were more and larger,the nucleus was concentrated on the edge,and large number of fibrosis accumulated around the cardiomyocytes,which was rich and dense.6.Real-time PCR and Western Blot results showed that compared with the normal control group,the mRNA and protein expression levels of KCa2.3,KCa3.1,AKT1 and P300 in different parts of the model group increased to different degrees(P<0.05);among them,the MS+AF group had the largest change(P<0.05);followed by MR+AF and AF group(P<0.05).The mRNA and protein expression levels of KCa2.3,AKT1,KCa/3.1,and P300 in MS and MR groups were different but there was no statistical difference(P>0.05).The expression levels of KCa2.3,AKT1,KCa3.1,P300 mRNA and protein in pulmonary vein orifice,left atrial appendage,atrial septum,mitral valve and papillary muscle were compared in each group.Compared with the normal group,the expression levels of mRNA and protein in each site were increased;among them,the indexes of pulmonary vein opening and left atrial appendage showed the most significant change(P<0.01);compared with the left atrial appendage,there was a statistically significant difference in the opening of pulmonary veins(P<0.05);the expressions of KCa2.3,AKT1,KCa/3.1,P 3 0 0 mRNA and protein in the atrial septum,mitral valve and papillary muscle were also different from those in the control group,but the changes were significantly smaller than those in the pulmonary vein opening and left atrial appendage(P<0.05),there was no significant difference in left atrial appendage and atrial septal area(P>0.05).Conclusions:(1)The methods established by the five animal models are scientific,reliable,feasible,and reproducible,laying the foundation for further study of valvular disease and abnormal hemodynamics of valvular atrial fibrillation.(2)Increased turbulent shear stress in the left atrial flow field is one of the important factors leading to left atrial remodeling.(3)The expression distribution of KCa2.3/3.1 in different parts of myocardial tissue was different and the highest expression was found in the pulmonary vein opening,followed by left atrial appendage=interatrial septum>mitral valve>papillary muscle.(4)KCa2.3/3.1 and its related genes play a key role in the process of TSS leading to continuous damage of mitral valve disease and atrial fibrillation.(5)KCa2.3/3.1 is involved in the signal transduction of endothelial cells by TSS.Part two:Study on The Effect of Shear Stress in Left atrial Flow Field on Thrombosis in Valvular Atrial Fibrillation Model in Patients with Atrial FibrillationObjective:Simulate changes in flow fields and shear stress in the left atrium using computer techniques,and compare the pathological differences of myocardial tissue in patients with simple mitral valve disease,valvular atrial fibrillation,and control groups with KCa2.3/3.1 channel proteins and related genes differences in expression,explore the relationship between KCa2.3/3.1 channel expression and changes in shear stress.Methods:On the premise of ethical review and informed consent,75 patients undergoing surgical mitral valve replacement or mitral valve repair are randomly divided into 15patients with each mitral valve disease(MS,MR),and 15 patients with each valvular atrial fibrillation(MS+AF,MS+AF,MR+AF)and set up a normal control group with 15 patients.The changes of left atrial flow field and turbulent shear stress were simulated by computer.The left atrial flow velocity was detected by doppler ultrasound,and the atrial septum tissue near the open pulmonary vein was taken out during surgery.The pathological changes were detected by HE and Masson staining.The mRNA expression of KCa2.3/3.1 related gene CKNN3/4 in left atrial tissues was detected by real-time PCR.The expression of KCa2.3/3.1 in left atrial tissue protein was detected by Western Blot technique;The expression level of kca2.3/3.1 in left atrial tissue was detected by immunofluorescence technique.Statistical analysis was performed to compare the differences between the groups and the control group.Results:1.The thrombelastograph showed that the R and K values decreased,ard the increase of the Ma value and the CI value was the most significant in the MS+AF+thrombus group(p<0.01),followed by the MS+AF group and the MS group(p<0.05).There was no significant change between the MR+AF group and the MR group(p>0.05),but the difference was statistically significant(p<0.05).2.The HE staining sections of the left atrial tissue of the control group were observed,the nucleus was blue,and the cytoplasm,muscle fibers,collagen fibers and red blood cells were different in shades of red.In the control group,the left atrial myocardium was structurally intact,neatly arranged,surrounded by a small amount of interstitial tissue;the nucleus was large and clear,and the regular fiber network was filled in the whole cardiomyocyte.The myocardial cells in the left atrial atrial myocardium of each disease group were disordered,elongated and corrugated,and the connective tissue between the myocardial fibers accumulated,which widened the interval between the cardiomyocytes.The order from severe to slight was MS+AF+thrombus group,MS+AF group and MS group,while the MR+AF group and MR group did not change significantly.Masson staining showed that in MS+AF+thrombus group,MR+AF group and MS+AF group collagen fibers were blue,myocardial fibers were red,and collagen fibers were interconnected into a network.Compared with the control group,the blue collagen fibers in the valvular AF group were significantly increased,and the distribution of collagen fibers was disordered.The myocardial bundles were separated by collagen fibers.The blue collagen fibers were the most common in the valvular lesion group.The sequence from severe to slight was MS+AF+thrombus group,MS+AF group,MS group,and there is less blue color in the MR+AF group and MR group.3.Real-Time PCR results showed that compared with the control group,the expression levels of KCCN3,KCNN4,AKT1 and P300 mRNA in each group increased,the difference was statistically significant(P<0.05);Among them,MS+AF+thrombus group and MS+AF group had significant difference compared with the control group(P<0.01).The mRN A expressions of KCNN4 and KCNN4 were compared between the two groups.Except for MS group and MR group,MS+AF group and MR+AF group,the difference was not statistically significant(P>0.05);the differences between the other groups were statistically significant(P<0.05).The mRNA expression of AKT1 was compared between the two groups.Except for MS group and MS+AF group,MR group and MR+AF group,the difference was not statistically significant(P>0.05).The differences between the other groups were statistically significant(P<0.05).The mRNA expression of P300 was compared between the two groups.Except the difference between MS+AF+thrombotic group and each group was statistically significant(P<0.05),the other groups were compared between the two groups.The difference was not statistically significant(P>0.05).4.KCa2.3/3.1 channel protein was compared between the groups:compared with the control group,the protein expression levels of KCa2.3/3.1,AKT1 and P300 in each group were increased,the difference was statistically significant(P<0.05).Among them,MS+AF+thrombus group and MS+AF group had significant difference compared with the control group(P<0.01).There was no significant difference in KCa2.3/3.1 between MS group and MR group,MS+AF group and MR+AF group(P>0.05).MS+AF and MR+AF were significantly higher than MS and MR.That is,patients with VAF have higher expression of KCa2.3/3.1 channel protein in the myocardium than patients with simple mitral valve disease.Compared with MS+AF and MR+AF groups,the expression of KGa2.3/3.1 proteins in myocardium were statistically significant(P<0.05),and the expression of MS+AF+thrombus group was increased.The expression level of AKT1 protein was significantly different between the MS+AF+thrombotic group and the other groups(P<0.05).There was no significant difference between the other groups(P>0.05).The expression level of P300 protein was compared between the two groups.There was no significant difference between MS and MR group(P>0.05).The other groups were compared with each other,the difference was statistically significant(P<0.05).5.Immunohistochemistry showed that KCa2.3 expression was higher than KCa3.1 expression in myocardial tissue.Compared with the control group,kCa2.3/3.1 protein expression in the myocardium of each disease group was higher than that of the control group,and the difference was statistically significant(P<0.05).Among them,the expression of kca2.3/3.1 channel protein in VAF patients(MS+AF,MR+AF group),especially in VAF patients with thrombosis(MS+AF+thrombosis),was the most obvious,showing a significant difference(P<0.01)).And between MS and MR group,MS+AF group and comparison between MR+AF group,there was no statistically significant difference(P>0.05).6.In the MS+AF+thrombus group,MS+AF group and MS group,the systolic shear stress increased most significantly in the left atrium region at the left and right pulmonary vein entrances,followed by the MR+AF group.Comparing the two groups in each group,the area with the most obvious turbulent shear stress change in the left atrium,namely:the entrance to the pulmonary vein,in descending order:MS+AF+thrombus group,MS+AF group,MS group,MR+AF group,MR group,and control group.The order of left atrial wall pressure from large to small was MS+AF+thrombosis group,MS+AF group,MS group,MR+AF group,MR group and control group.The most significant increase of wall pressure was at the entrance of pulmonary vein and in the left atrial appendage,which coincided with the position of turbulent shear stress.The flow field uniformity in the left atrium was the worst in the MS+AF+thrombus group,followed by the MS+AF group,the MS group,the MR+AF group,and the MR group,which coincided with the turbulent shear stress and compressive stress.Among them,the velocity flow field in the opening of the left atrial appendage decreased most obviously,which was consistent with the predilection site of thrombosis in the left atrium.Conclusions:(1)The flow field simulation model of left atrium was successfully constructed,and the turbulence shear stress,wall pressure and velocity flow field in left atrium were directly analyzed.(2)Turbulent shear stress and turbulent flow field in the flow field of the left atrium of valvular AF are one of the important factors leading to progressive atrial injury and thrombosis.(3)Up-regulation of mRNA and protein expression of kca2.3/3.1,AKT1 and P300 is an important cause of valvular AF atrial tissue injury and thrombosis.Part three:To Investigate the Effects of Different Time and Shear Stress Changes on The Expression Changes of Cardiac Myocytes Kca2.3/3.1Objective:To investigate the effect of changes in shear stress at different times and different shear stress on the expression of KCa2.3/3.1 in cardiomyocytes.Methods:Cardiomyocytes.were placed in culture medium,and the shear stresses of different sizes(5dynes/cm2,15dynes/cm2,24dynes/cm2)were given at different time(0.5 h 1h 2 h)by fluid shear cell test system(TY-FSS110).Observe the morphological changes of cardiomyocytes under different in vitro shear stress at different time points,and use Real-Time PCR to detect the mRNA expression of KCa2.3/3.1 and AKT1 and P300 in cardiomyocytes;use Western Blot technique to detect expression of KCa2.3/3.1 and AKT1 and P300 in Cardiomyocytes.The concentration of TRAM-34 inhibitor(3?M)was added in 5dyne/cm2 group for 2h,to detect the mRNA expression of KCa2.3/3.1 and AKT1 and P300 in cardiomyocytes.After statistical analysis,the differences between each group and the control group were compared.Statistical analysis was performed to compare the difference between each group and the control group.Results:After the intervention of shear stress loading,the cells in the normal group proliferated with time,but no other obvious changes were observed.In 5dyne/cm2 and 15dyne/cm2 groups,the intercellular space was widened and the morphology became longer,and cells elongated along the long axis of the flow lumen.The arrangement of the cells is nearly parallel to the direction of the force.This change,along with the prolongation of the loading time,also changes gradually,showing a certain time dependence.There was no significant change in cell morphology in the 24dyne/cm2 group,but as the loading time increased,the proliferation of this group of cells became slower.Real-Time PCR and Western Blot results showed that compared with the normal group,the mRNA and protein expression levels of KCa2.3/3.1 were significantly up-regulated(P<0.01),and the mRNA and protein expression levels of AKT1 and P300 was up-regulated(P<0.05)in cardiomyocytes loaded with 5 dyne/cm2 and 15 dyne/cm2 for 1 h and 2 h,respectively.With the prolongation of loading time,the expression difference was significant and time-dependent.It is suggested that cardiomyocytes activate KCa2.3/3.1 ion channels when loaded with 5dyne/cm2,15dyne/cm2 shear stress.There was no significant difference in the mRNA and protein expression of KCa2.3/3.1,AKT1 and P300 between 24 dyne/cm2 group and normal group at 0.5,1 and 2 hours(P>0.05).It indicates that myocardial cell has no obvious effect on KCa2.3/3.1 ion channel when loading 24dyne/cm2 shear stress.After giving the inhibitor to the 5dyne/cm2/2h group,the results of Real-Time PCR and Western Blot showed that the mRNA and protein expression of KCa2.3/3.1 were significantly down-regulated(P<0.01),and the mRNA and protein expression of AKT1 and P 300 were down-regulated simultaneously(P<0.05).It indicates that AKT1 and P300 maybe downstream genes of KCa2.3/3.1 pathway.At the same time,the expression of KCa2.3 was also down-regulated after KCa3.1 was inhibited,indicating that there is an interaction between the two pathways KCa2.3 and KCa3.1.Conclusions:(1)KCa2.3/3.1 ion channel can respond to changes in mechanical signals of shear stress and is a bridge to convert hydrodynamic signals into biological behavior;(2)Low-layer flow shear stress can regulate cardiomyocyte injury caused by KCa2.3/3.1 ion channel in cardiomyocytes. |
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