| Objective:Studies have shown that endoplasmic reticulum stress(ERS)and cardiomyocyte apoptosis caused by mitochondrial damage play an important role in the pathogenesis of Myocardial infarction(MI).Enalapril can significantly improve the prognosis of MI patients by improving myocardial cell remodeling and cardiac systolic function in clinical application.Further studies have also shown that enalapril can reduce cardiomyocyte apoptosis and ERS in rats with cardiac hypertrophy.Does enalapril play the same role in MI?No related studies have been reported in the current research.In addition,studies have found that transforming growth factor-activated kinase 1(TAK1)is significantly up-regulated in MI animal models,and TAK1 can play an anti-apoptotic effect in vivo,and activate T cell nuclear factor 3(Nuclear factor of activated T cells 3(NFAT3)is an important downstream molecule that TAK1 plays a role.What role does TAK1/NFAT3 pathway play in MI?Does enalapril have a regulatory effect on it?No related studies have been seen so far.The purpose of this investigation:1.To observe whether there is mitochondrial damage and ERS in heart failure after MI,and to study the expression of TAK1/NFAT3 pathway.2.To study whether enalapril can inhibit or reduce mitochondrial damage and endoplasmic reticulum stress in heart failure after MI,while regulating the TAK1/NFAT3 signaling pathway.3.To study the effect of TAK1/NFAT3 pathway on endoplasmic reticulum stress,mitochondrial damage and apoptosis.Methods:1.Clinical research:Compare the left ventricular ejection fraction(LVEF)and serum NT-proBNP content of 32 patients with heart failure after myocardial infarction and 30 age-matched healthy personb and detecting the levels of mitochondrial damage marker protein cytochrome C(Cytochrome C,Cyt C)and ERS damage marker protein PERK by ELISA method.2.Animal experiment:The mouse heart failure model after MI was replicated by ligating the mouse left anterior descending(LAD)coronary artery.Shamoperated mice were subjected to the same procedure,but the suture around the LAD was not tied.After the model was stable for one week,Mice that survived surgery were randomly assigned to different treatment groups(n=10 per group).MI mice were treated with a dose of 20 mg/kg enalapril(Sigma-Aldrich;Merck KGaA)or an equal amount of drinking water daily through gastric gavage for 3 weeks.These groups were termed MI+Ena and MI groups,respectively.The small animal echocardiography was used to measure the LV parameters,including the internal diastolic diameter(LVIDd)and internal systolic diameter(LVIDs),were obtained from M-mode interrogation in the long-axis view.Then the mice were sacrificed and heart specimens were collected.TUNEL method was used to detect the degree of cardiomyocyte apoptosis;Masson’s trichrome staining was used to observe the degree of myocardial cell fibrosis;The immunohistochemical method was used to detect the expression of cleaved caspase 3 and NFAT3 protein;The Western blot method was used to detect the protein expression levels of GRP78,ATF6,TAK1,NFAT3,Bcl-2,Bax,PARP,cleaved-PARP,caspase-9,cleaved caspase-9,caspase-3 and cleaved caspase-3.3.Cell experiment:Based on the glucose and oxygen deprivation culture system,the OGD model,the MI model was replicated using primary rat neonatal rat cardiomyocytes.Western blot was used to detect changes in the expression levels of fibrosis-related proteins Collagen I,Collagen Ⅲ and α-SMA.After intervention with enalapril,the changes in the levels of apoptosis-related proteins Cleaved-PARP-1,Cleaved-casppase 9,Cleaved-casppase 3,Bax and Bcl-2 were observed.In addition,the endoplasmic reticulum stress-related proteins GRP78 and ATF6 and the protein content of signaling pathway TAK1/NFAT3 were detected.After designing the siRNA of NFAT3 to inhibit its expression,observe the changes of the above-mentioned fibrosis-related proteins,apoptosis-related proteins and endoplasmic reticulum stress-related proteins.Result:1.Clinical results:The LVEF(33.1±3.38%)of HF patients was significantly lower than healthy control group(63.9±3.35%,n=30),P<0.01;while the serum NT-proBNP concentration(7008.5±3303.8 pg/ml)was significantly higher than control group(111.1±51.22 pg/ml),P<0.01.ELISA results:Compared with the healthy control group,the content of Cyt C in the serum of patients with heart failure after MI(1.37±0.65 ng/ml)was significantly higher than that of the control group(0.34±0.33 ng/ml),P<0.01;in addition,the serum PERK The content of(599.4±101.1pg/ml)was also significantly higher than that of the control group(403.3±79.81 ng/ml),P<0.01.ROC curve analysis shows that when the concentration of Cyt C is 0.72ng/ml and the concentration of PERK is 535.5pg/ml,it has higher sensitivity and specificity.2.The effect of enalapril on the systolic function of mice in each group:Cardiac systolic function was normal in the control group(EF,83.1 7±5.4%;FS,46.32±5.64%).Compared with the MI group(EF,53.76±4.07%;FS,23.48±2.25%),Heart function of MI+Ena group(EF,68.43±5.36%;FS,33.73±3.86%,all P<0.01)was improved significantly.3.The effect of enalapril on myocardial fibrosis:Masson’s staining of mouse animal models showed that compared with MI group,the proportion of myocardial fibrosis area in MI+Ena group was significantly reduced(P<0.01),while There was no obvious fibrosis in the myocardium in the sham group.The cell model showed that compared with the OGD non-intervention group,the fibrosis-related proteins Collagen I,Collagen III and α-SMA were significantly down-regulated in the Ena group(P<0.05).4.The effect of enalapril on cardiomyocyte apoptosis:Animal model:Myocardial apoptosis was detected via TUNEL staining in the infarct and border areas.The results showed that there were no apoptosis-positive cells in the sham group.The number of TUNEL-positive cells increased significantly at 4 weeks after MI and a protective effect was observed in the enalapril-treatment group(P<0.01).Immunohistochemical staining showed that cleaved-caspase-3 staining was significantly enhanced in the MI group,while the staining in the MI+Ena group was reduced,and no staining was found in the Sham group.Western blot results showed that compared with the Sham group,the contents of apoptotic proteins of cleaved-PARP,cleaved-caspase-9 and cleaved-caspase-3 increased significantly in the MI group(all P<0.01),but decreased significantly after treatment in the MI+Ena group(all P<0.01).Cell model:Western blot results showed that compared with the OGD non-intervention group,the expression levels of Cleaved-PARP-1,Cleaved-casppase 9,Cleaved-casppase 3,and Bax protein in the Ena group decreased significantly,while the expression of Bcl-2 increased significantly(P<0.05).TUNEL staining also showed that Ena can significantly reduce cardiomyocyte apoptosis.5.The effect of Enalapril on cardiomyocyte ERS:Animal model:Western blot results showed that compared with the sham group,ERS-related proteins GRP78 and ATF6 were significantly up-regulated in the MI group(P<0.05),while in the MI+Ena group was significantly down-regulated compared with MI group(P<0.01).Cell model:Western blot results showed that compared with the OGD non-intervention group,Ena treatment could significantly reduce the expression levels of ERS-related proteins GRP78 and ATF6(P<0.05).6.The effect of enalapril on the TAK1/NFAT3 signal pathway of cardiomyocytes:Animal model:Western blot results showed that compared with the sham group,phosphorylated TAK1 was significantly higher in the MI group(P<0.05),and increased more significantly in the MI+Ena group(P<0.05).The protein content of NFAT3 is similar to that of TAK1,that is,it is higher in MI group than sham group(P<0.05),and the content of MI+Ena group is more higher(P<0.05);immunohistochemistry also shows that NFAT3 protein is in The MI group had stronger staining than the sham group,and MI+Ena histone staining was the strongest,while the sham group had no obvious staining.In addition,Western blot results showed that the nuclear NFAT3 content in MI+Ena group was significantly higher than that in MI group(P<0.05),indicating that nuclear translocation was enhanced.Cell model:Western blot results showed that compared with the control group,phosphorylated TAK1 was significantly increased in the OGD group,but increased more significantly in the OGD+Ena group.The protein content of NFAT3 was similar to that of TAK1,that is,it was higher in the OGD group than the control group,and the OGD group treated with Ena had the highest content.In addition,the nuclear translocation of NFAT3 in the OGD+Ena group was significantly higher than that in the OGD group.P<0.05.7.The effect of intervention on TAK1/NFAT3 signaling pathway on cardiomyocyte function:After using siRNA to inhibit the expression of NFAT3,Western blot results show that Ena inhibits the expression of fibrosis-related proteins(Collagen Ⅰ,Collagen Ⅲ and α-SMA)and the effects of inhibiting the expression of proapoptotic factors Cleaved-PARP-1,Cleaved-casppase 9,Cleaved-casppase 3 and Bax protein,and inhibiting the expression of ERS proteins GRP78 and ATF6 were all reversed.TUNEL staining also showed that cardiomyocyte apoptosis increased significantly after inhibiting the expression of NFAT3.Conclusions:1.Patients with heart failure after myocardial infarction have ERS and mitochondrial damage.2.Enalapril can play a therapeutic role by improving myocardial fibrosis and improving cardiac contractility.3.The therapeutic effect of enalapril in heart failure after MI may be achieved by reducing mitochondrial damage and apoptosis caused by ERS.4.Enalapril can reduce cardiomyocyte fibrosis,mitochondrial damage and ERS by up-regulating the TAK1/NFAT3 signaling pathway and promoting nuclear translocation of NFAT3. |
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