| BackgroundRheumatic aortic stenosis,a common disease which leads to left ventricular overload,is one of the main causes of heart failure.However,relieving ventricular overload by artificial valve replacement can not benefit all patients equally.Postoperative recovery of cardiac shape and function was positively correlated with preoperative cardiac function.In previous animal experiments,we also found that the left ventricular overload rats with 50% ascending aorta coarctation experienced a relatively normal or even slightly increased compensatory period and a progressively decreased decompensation period,and the left ventricular overload rats recovered well in the modern compensatory phase during unload surgery.In decompensated stage,cardiac function can not be effectively restored.Although our previous studies have proved the clinical phenomenon well,the specific mechanism of the transformation from cardiac function compensation to decompensation caused by left ventricular overload remains unclear and needs further study.The mechanical stress induced by overload leads to hypertrophy of myocardial cells,interstitial fibrosis,destruction of microvascular structure and deposition of interstitial collagen.These changes are collectively called myocardial remodeling,and are the main pathological characteristics of myocardial structural changes.In myocardial tissue,about 70% of the cells are non-cardiac cells,and fibroblasts and endothelial cells are the most abundant of them.We speculate that besides adaptive hypertrophy,cardiac myocytes may also play an important role in regulating the occurrence and development of interstitial remodeling,and the change of matrix structure caused by interstitial remodeling may be a sign of the transformation of cardiac function from compensatory to decompensated.Therefore,it is of great significance to study the characteristics and molecular mechanism of myocardial remodeling in different periods of overload.A large number of studies have shown that KLF15,a member of Kruppel-like factor family,has an important inhibitory effect on interstitial fibrosis,but the specific mechanism is unclear.Some studies have shown that the expression of TGF-? in myocardium of patients with chronic aortic stenosis is significantly increased.Meanwhile,the expression of CTGF regulated by TGF-? is also enhanced.It is confirmed that the phosphorylation of Smad3 is the key link in the regulation of CTGF expression by TGF-?.It is speculated that KLF15 may competitively inhibit the phosphorylation of Smad3 promoting the transcription promoter of CTGF.On the other hand,it has been found that chonic LV overload can inhibit the expression of KLF15 by activating p38-MAPK.It is speculated that the low expression of KLF15 induced by p38-MAPK activation may be a key factor in the regulation of interstitial fibrosis.Recent studies have shown that KLF15 plays an important role in myocardial interstitial angiogenesis,which may be another key way for KLF15 to inhibit cardiac decompensation.Its mechanism may be related to the positive regulation of the secretion of vascular endothelial growth factor by KLF15,which needs further study.Based on previous studies and progress,we speculate that the changes of myocardial remodeling in different stages in overload disease have different characteristics,and interstitial remodeling is an important link leading to heart failure.As a key negative regulator,KLF15 inhibits interstitial remodeling and heart failure.In this study,KLF15 overexpressed transgenic mice were introduced and the mechanism of KLF15 inhibiting left ventricular pressure overload-induced heart failure was studied by constructing a model of left ventricular pressure overload mice.It is helpful for us to understand the pathophysiological process of pressure overload diseases.Methods1.C57/BL6 J mice with healthy male SPF class 8-10 weeks old were chose to construct left ventricular pressure overload models induced by ascending aortic coarctation without artificial ventilation.Ejection fraction(EF)and short axis shortening rate(FS)were examined by echocardiography before operation and 1,2,3,4,5 and 6 weeks after operation,respectively,to determine the cardiac function of left ventricular overload mice at different time points.After determining the specific time points of the compensatory and decompensated period of cardiac function in mice,the left ventricular thickness,left ventricular internal diameter,cardiac weight and lung weight of mice with left ventricular overload at different stages were measured by M-mode echocardiography and electronic balance.The morphological changes of the heart in mice with left ventricular overload at different stages were evaluated in terms of general morphology.2.HE,Masson staining and CD31 immunohistochemical staining were performed on myocardial tissue of left ventricular overload wild-type mice at compensatory and decompensated stages respectively;collagen type ? and collagen type ? were detected by ELISA;expression of KLF15 and its related proteins CTGF and VEGF and phosphorylation levels of p38 and Smad3 in myocardial tissue was detected by WB.Sham operation group were set as control.The different characteristics of compensatory and decompensated cardiac function were further elucidated in terms of myocardial ultrastructure and molecular expression.The intrinsic relationship between KLF15 and its related proteins CTGF,VEGF and phosphorylation levels of p38 and Smad3 was further studied.3.Left ventricular pressure overload model was established by introducing cardiac overexpression KLF15 transgenic(TG)mice.Left ventricular pressure overload model of wild type mice was used as negative control,and sham operation group was used as blank control.By using echocardiography,cardiac weight,lung weight,pathological and immunohistochemical staining,ELISA and WB methods,the specific mechanism of how KLF15 regulates the expression of related factors and inhibits heart failure caused by left ventricular overload is further studied.Results1.Echocardiography showed that EF and FS in the overload group increased to a certain extent and reached the peak of compensation at 2 weeks after ascending aortic coarctation compared with the sham operation group,but there was no significant difference.After 2 weeks,the cardiac function of the overload group decreased with the extension of constriction time,and there were significant differences between the overload group and the sham-operated group at 5 and 6 weeks of constriction(P < 0.05).From the view of cardiac morphological changes,the heart of mice in the compensatory period of cardiac function mainly showed centripetal hypertrophy characterized by the increase of IVS and LVPW,while in the decompensated period,the heart of mice showed centrifugal hypertrophy characterized by the increase of LVID.The results of heart weight and lung weight showed that the increase of heart weight was the main manifestation in the compensatory period of cardiac function,while the increase of heart weight and lung weight was more significant in the decompensated period of cardiac function.2.HE staining showed that in the compensatory period of cardiac function,the myocardial cells showed hypertrophy,such as enlargement of cross section and spacing of myofibrils and deep dyeing of nuclei.In the decompensated period,the spacing of myofibrils increased further,and cytoplasmic breakage,fragmentation and irregular arrangement of myofibrils appeared.Masson staining showed that severe fibrosis occurred in myocardial tissue of mice during the decompensated period of cardiac function.Immunohistochemical staining of CD31 showed that the density of CD31 increased in myocardium of mice with cardiac decompensation,but decreased significantly in mice with cardiac decompensation.ELISA showed that there was no significant difference in myocardial collagen ? and ? between the compensatory period and the sham-operated group(P > 0.05).Myocardial collagen I and III in the decompensated period increased significantly compared with the sham-operated group(P < 0.01).WB showed that the myocardial phosphorylation of Smad3 increased in the compensated period of cardiac function(P < 0.01),while the expression of KLF15 and VEGF decreased in the decompensated period of cardiac function(P < 0.01),the expression of CTGF and the phosphorylation of p38 and Smad3 increased significantly(P < 0.01).3.Under the condition of left ventricular overload for 6 weeks,echocardiography showed that compared with WT mice,the increase of LVID and the decrease of EF and FS in TG mice were significantly reduced.Lung weight gain was relatively slight.Pathological staining showed that wild-type mice showed more severe interstitial fibrosis.CD31 immunohistochemical staining showed that the density of CD31 in myocardium of WT and TG mice decreased significantly,and that of CD31 in myocardium of TG mice decreased slightly.ELISA showed that myocardial collagen ? and ? levels in WT mice with left ventricular overload were significantly higher than those in TG mice with left ventricular overload(P < 0.01).WB showed that compared with WT mice with left ventricular overload,the expression level of CTGF in TG mice with left ventricular overload decreased significantly(P < 0.01),and the expression levels of VEGF and KLF15 increased significantly(P < 0.01).Conclusion1.The left ventricular overload animal model can be successfully constructed by the non-artificial ventilation ascending aortic coarctation.At two and six weeks after the application of this method,the compensatory model of cardiac function and the decompensated model of cardiac function can be successfully constructed respectively.2.In the compensatory period of cardiac function,cardiac remodeling is characterized by hypertrophy of cardiac myocytes and increased microvessel density in the interstitium without obvious interstitial fibrosis,and cardiac function can maintain normal or even increase compensatory.Its mechanism is related to the inhibition of KLF15 on p-Smad3 promoting CTGF transcription and the positive regulation of vascular endothelial growth factor.3.In the decompensated period of cardiac function,besides the hypertrophy of cardiomyocytes,a complex remodeling was displayed such as severe fibrosis of myocardial interstitium,collagen deposition and interstitial angiogenesis disorder,accompanied by the decrease of cardiac function.The mechanism is phosphorylation of P38 inhibits the expression of KLF15,and the low expression of KLF15 loses its inhibitory effect on the transcription of CTGF and motivating effect on the transcription of VEGF.4.Cardiac-specific KLF15 overexpression transgenic mice can significantly alleviate heart failure induced by left ventricular pressure overload for 6 weeks,which is related to the inhibition of interstitial fibrosis,collagen deposition and the promotion of angiogenesis.The mechanism is related to KLF15 overexpression compensated for the inhibition of upstream phosphorylated p38,and regulated the expression of VEGF positively and CTGF negatively. |
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