| Research background and purposeThe incidence rate and mortality rate of acute myocardial infarction are increasing in China and abroad year by year.With the continuous development of treatment methods,the life span of patients after myocardial infarction is prolonged.At present,acute myocardial infarction is one of the most important causes of heart failure in the world.Studies have shown that the incidence of heart failure after myocardial infarction in China is high,the prognosis is poor,and it is on the rising.Heart failure after myocardial infarction increases the risk of short-term and long-term adverse events.The pathophysiological basis of heart failure after myocardial infarction is cardiac structural remodeling and energy remodeling.In the process of energy metabolism,the heart can effectively ensure the stability of the inner environment of the heart,promote the renewal of tissue structure,in which adenosine triphosphate(ATP),the product of heart energy metabolism,plays a key role in this process,and ATP plays an important role in ensuring the heart function,and it is also its basic energy.With the progress of myocardial structural remodeling after myocardial infarction,the metabolic process of myocardial cells is constantly changing.Once metabolic disorder occurs in the process of myocardial energy metabolism,this situation will have a negative impact on myocardial remodeling.Mitochondria are also the key sites of energy formation in eukaryotic cells.Based on oxidative phosphorylation,providing ATP in the process of body action is one of the research directions of preventing and treating heart failure after myocardial infarction.Mitophagy is closely related to myocardial energy metabolism.Mitophagy can directly remove the structural and functional abnormalities of mitochondria,regulate the quantity and quality of mitochondria,and ensure the effectiveness of mitochondrial function,which is one of the important factors to maintain the normal and balanced energy metabolism of cells.Studies have shown that Mitophagy plays an important role in ventricular remodeling and heart failure after myocardial infarction.Studies have shown that the moderate increase of Mitophagy in myocardial cells after myocardial infarction can directly eliminate the aging or abnormal mitochondria in the body,reduce the level of endogenous reactive oxygen species(ROS),provide materials for the normal function of mitochondria to synthesize energy,ensure the normal ATP level of myocardium and meet the needs of myocardium.After myocardial infarction,the autophagy level of myocardial cells increased significantly in the early stage(within 1～3 days to 1 week)to support the survival of myocardial cells.With the gradual prolongation of ischemia time,mitochondria were damaged,oxidative stress response was enhanced,autophagy level was unbalanced,and myocardial cell necrosis increased,which aggravated the progress of heart failure after myocardial infarction.Autophagy worsens ventricular remodeling by mediating cardiomyocyte apoptosis,at the same time,it also affects the function of normal mitochondria,which leads to abnormal metabolism of mitochondrial capacity and further aggravates heart damage.Mitophagy is involved in the occurrence of heart failure after myocardial infarction,ventricular remodeling,energy metabolism and so on.It is a potential target for exploring the treatment of heart failure at present.Recent studies have shown that ATP-sensitive potassium channel(KATP)plays an important role in Mitophagy.The KATP channel in mitochondria is opened,which can regulate the membrane potential in mitochondria,reduce Ca2+overload in mitochondria,directly affect ROS production,improve the respiratory chain of mitochondria,inhibit apoptosis and promote the occurrence of Mitophagy.Studies have found that KATP channel in ischemic myocardial cells of rats with heart failure after myocardial infarction is positively correlated with angiotensin Ⅱ in tissues.The expression of KATP was increased by the action of AngⅡ on transcription factor FOXO3 through TNF-α.Angiotensin Ⅱ receptor antagonist can improve heart failure and inhibit ventricular remodeling after myocardial infarction by blocking the combination of Ang Ⅱ and AT1.Therefore,we hypothesized that losartan,an ARB drug,might inhibit Mitophagy and improve cardiac function and myocardial energy metabolism through KATP channel.In this study,the effects of losartan on heart function,mitochondrial function and Mitophagy level in rats with heart failure after myocardial infarction were observed by establishing myocardial infarction models.Meanwhile,Ang II-induced injury model of H9C2 cardiomyocytes was adopted,and opening agents and blocking agents were used to regulate mitochondrial KATP channel,so as to explore the molecular mechanism of losartan regulating Mitophagy.Method1.Construct the heart failure model after myocardial infarction:Ligate the coronary artery of rats and induce subsequent heart failure after myocardial infarction in this way.Echocardiography was performed the second day after operation.Left ventricular ejection fraction(LVEF)<50%was used as the standard for the success of the model.Three groups were randomly analyzed:sham group(sham group,n=15)9 heart failure group(HF group,n=15)and losartan treatment group(HF+ARB,n=15,l0mg/kg/d).After 4 weeks of intervention,hemodynamics,echocardiography and ECG,pathological staining and molecular biological indexes were used to detect the degree of mitochondrial autophagy and the expression level of KATP channel.2.Ang Ⅱ was used to induce H9C2 cardiomyocytes,and the injury model was constructed based on it.The cells were randomly grouped and analyzed,including Control group and model group(Ang Ⅱ),diazoxide group(Ang Ⅱ+Diazoxide,specific mitoKAPT channel opener),5-hydroxyquidone(Ang Ⅱ+5-HD,mitKATP specific blocker)and losartan group(Ang Ⅱ+ARB).Western blot was used to screen the concentration and action time of Ang Ⅱ which induced the highest autophagy level in H9C2 cardiomyocytes.The apoptosis rate of cells was measured by flow cytometry,and the activities of reactive oxygen species(ROS)and NADPH oxidase were measured to evaluate the level of.oxidative stress.Meanwhile,the expression levels of proteins corresponding to Atg5,Beclin-1 and LC3B Ⅱ/Ⅰ were measured in this way.Result1.Animal experiment(1)cardiac ultrasound:the results were analyzed and compared with Sham group.according to the situation of HF group rats,the left ventricular ejection coefficient left ventricular end diastolic inner diameter and left ventricular short axis shortening rate all showed obvious decrease(P<0.05).Compared with HF group,LVEF and LVFS in losartan intervention group(HF+ARB group)increased significantly,while LVIDd increased significantly(P<0.05).(2)Hemodynamics:Compared with Sham group,in HF group,the highest rate of increase of left ventricular pressure(+dp/dtmax),left ventricular end systolic pressure(LVEPs)and other parameters decreased significantly,and from the data of left ventricular end diastolic pressure(LVEPd),it increased significantly(P<0.05).Compared with-dp/dtmax and HF group,LVEPs and+dp/dtmax increased significantly after losartan treatment(all P<0.05).(3)Electrocardiogram:No arrhythmia occurred in Sham group rats during postoperative monitoring.The incidence of arrhythmia in HF group was 65.23%,and that in HF+ARB group was 33.86%.Compared with Sham group,the ventricular rate in HF group increased significantly(P<0.05),and losartan could significantly reduce the ventricular rate(P<0.05).In HF group,PR interval and QTc interval were significantly prolonged(P<0.05),while losartan shortened QTc prolongation in rats(P<0.05).(4)HE staining:The whole structure of myocardial tissue in Sham group was normal.Cardiomyocytes were distributed regularly,with clear transverse stripes and centered nuclei,and no inflammatory cell infiltration occurred.In HF group,left ventricular transmural necrosis of myocardium and fibrous tissue can be seen in myocardial tissue,and irregular arrangement of myocardial cells can be clearly seen in the ischemic area at the edge of myocardial scar tissue.At the same time,compensatory hypertrophy occurs in myocardial cells,and the overall arrangement of myocardial cells is irregular,with irregular stripes and obvious infiltration of inflammatory cells.In HF+ARB group,the infarct area of left ventricle was obviously reduced,the tissue structure of myocardium was basically intact,and the muscle fibers were arranged in order.(5)MASSON staining:Compared with Sham group,a large amount of scar collagen tissue can be seen in left ventricular myocardial infarction area of HF group rats,while the structure of myocardial cells in ischemic area is disordered,in which a large amount of collagen accumulates and collagen fibers in myocardium are distributed alternately.Compared with rats in heart failure group,the degree of fibrosis in ischemic myocardium of HF+ARB group was less,and most of them were encapsulated in myocardium.(6)Electron microscope:After analyzing the rats in Sham group,the structure of mitochondria in myocardial cells is clear,with regular distribution and dense number.The intercalated discs in mitochondria are clearly visible,and the boundary ridges are arranged regularly,clearly and densely.In HF group,mitochondria of myocardial cells in ischemic area were obviously swollen,intercalated disc was blurred,compensatory hyperplasia and autophagy increased.Compared with HF+ARB,the density of mitochondria in HF+ARB group increased,the swelling of mitochondria decreased and the intercalated disk structure was clear.(7)ATP concentration:Compared with Sham group,ATP content in HF group decreased significantly(P<0.05).Compared with HF group,ATP concentration of ischemic myocardial cells around infarction after ARB treatment increased significantly(P<0.05).(8)KATP channel proteins:Compared with Sham group,KATP channel proteins Kir6.2,SUR2A,MITOK and MITOSUR in HF group were all slightly increased compensatively(all P<0.05).Compared with HF,losartan decreased the expression of MITOK and MITOSUR(all P<0.05),but had no obvious effect on the expression of Kir6.2 and SUR2A.(9)Mitophagy protein:Compared with Sham group,the expression of P62 in cardiac myocytes in HF group decreased,while Beclin-1 and LC3B Ⅱ/LC3B Ⅰincreased(all P<0.05).Compared with HF group,the expression of P62 increased and the expression of Beclin-1 and LC3B Ⅱ/LC3B Ⅰ decreased in HF+ARB group.2.Cell experiment(1)Screening the drug concentration of losartan for intervention treatment:CCK8 was used to determine the effect of ARB on the activity of H9C2 cardiomyocytes at different concentrations and at different times.Results Losartan 10-4mol/L was selected as the intervention drug concentration.(2)Screening the concentration and time when Ang Ⅱ has the strongest mitophagy:Western blot was used to determine the protein expression levels of P62,Atg5,Beclin-1 and LC3B Ⅱ/Ⅰ in H9C2 cardiomyocytes at different concentrations,and the results showed that the highest level of Mitophagy occurred at 10-7mmol/L and 24h.(3)The myocardial apoptosis rate measured by flow cytometry:Compared with the Control group,the myocardial apoptosis rate in Ang Ⅱ group increased significantly(P<0.01).Compared with Control group,the apoptosis rate of myocardial cells in Ang II+Diazoxide group increased significantly,and 5-HD and losartan inhibited the apoptosis of myocardial cells induced by Ang Ⅱ(all P<0.01).(4)Oxidative stress response:Compared with Control group,ROS concentration and NADPHase activity in Ang Ⅱ group increased,while oxygen uptake decreased(all P<0.05).ROS concentration and NADPHase activity increased and oxygen uptake decreased in Ang II+Diazoxide group.Compared with Ang Ⅱ,the ROS concentration and NADPase activity of cardiac myocytes in Ang Ⅱ+5-HD and AngⅡ+ARB groups decreased,and the oxygen uptake capacity of cardiac myocytes increased(P<0.05).(5)ATP level:compared with Control group,ATP production of H9C2 cardiomyocytes in Ang Ⅱ group decreased(P<0.01).Intracellular ATP content of H9C2 myocardial cells in Ang II+Diazoxide group decreased significantly compared with Control group(P<0.01).ATP content increased after treatment with AngⅡ+5-HD and Ang Ⅱ+ARB,which were significantly different from those in Ang Ⅱgroup(all P<0.01).(6)Autophagy corpuscles:Compared with Control group,autophagy corpuscles in Ang Ⅱ group and Ang Ⅱ+Diazoxide group increased,while autophagy corpuscles in Ang Ⅱ+5-HD and Ang Ⅱ+LC3B group decreased significantly.(7)Mitophagy protein:Compared with the Control groups the expression of P62 protein decreased and the expression of Beclin-1 and LC3B Ⅱ/LC3B Ⅰ increased in Ang Ⅱ,Ang II+Diazoxide groups.In Ang Ⅱ+5-HD and Ang Ⅱ+ARB groups,P62 expression increased,while Beclin-1 and LC3B Ⅱ/LC3B Ⅰ expression decreased(all P<0.05).(8)KATP channel protein:Compared with the Control group,the expressions of Kir6.2,SUR2A,MITOK and MITOSUR in cardiac myocytes of Ang Ⅱ,AngⅡ+Diazoxide group increased significantly,while Kir6.2 and SUR2A decreased in Ang Ⅱ+5-HD group,and MITOK and MITOSUR decreased in Ang Ⅱ+ARB group,all with significant statistical differences compared with Ang Ⅱ group.Conclusion1.Losartan improved the cardiac function of heart failure after myocardial infarction,reduce the level of Mitophagy in ischemic cardiomyocytes;2.Losartan blocked mitoKATP opening and inhibited Mitophagy in cardiomyocytes;3.Losartan reduced the expression of mitoKATP channel in cardiomyocytes and inhibited Mitophagy. |
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