| Purpose:Mitochondrial dysfunction is an important cause of heart failure.Clearing dysfunction of mitochondria in time will contribute reduce oxidative stress,promote the recovery of mitochondrial membrane potential and function,improve energy supply,and protect myocardial function.Mitophagy,a process that selectively removes damaged mitochondria via a specialized form of autophagy,is essential for mitochondrial quality control and metabolic homeostasis.Studies have found that chronic intermittent hypobaric hypoxia(CIHH)pretreatment can reduce the area of myocardial ischemia and/ or reperfusion infarction,improve myocardial contractile function,and delay the development of heart failure.However,it is unclear whether CIHH exerts a protective effect through the regulation of mitochondrial autophagy.Because mitochondrial autophagy receptor FUN14 domain containing 1(FUNDC1)is a key protein in response to hypoxia stress.Therefore,this study explores the role and mechanism of FUNDC1 in CIHH preconditioning to protect heart function and delay heart failure after MI.Methods:Male C57BL/6 mice were randomly divided into control(CON),CIHH(treated with 4 weeks hypobaric hypoxia simulating an altitude of 5000 m,8h daily),MI(received ligation of left anterior descending coronary artery of the heart)and CIHH+MI group.The weight of mice was measured weekly,echocardiography was performed before and after MI to evaluate cardiac function.Measurement of serum myocardial injury markers and expression of BNP in left ventricular myocardial tissue to assess the extent of cardiac injury.HE and Masson staining were used to evaluate myocardial pathological changes and infarct fibrosis,respectively.Western blot and RT-PCR were used to detect the expression of mitochondrial autophagy receptor FUNDC1 in myocardial tissue after infarction.At the same time,cardiomyocytes(H9C2)transfected with adenovirus Fundc1-shRNA were used to inhibit the expression of FUNDC1 gene in cell experiments,and the effect of changes in FUNDC1 level on hypoxic preconditioning on myocardial ischemic injury was observed.Then Seahorse was used to measure mitochondrial oxygen consumption and flow cytometry to measuremitochondrial ROS to assess mitochondrial function.Finally,the mitochondrial structure was observed with an electron microscope and Western blot were used to observe the effects of FUNDC1 on autophagy-related proteins and mitochondrial dynamic proteins.Result:1.Changes of mice body weight,left ventricular function,and myocardial histopathology after CIHH treatment :After CIHH treatment for 4 weeks,compared with the CON group,there were no significant difference in body weight and cardiac function(EF,FS);Compared with the CON group,No pathological damage was observed in the CIHH group by H & E staining.2.Effects of CIHH on left ventricular cardiac function parameters,myocardial injury markers,and myocardial infarction area after myocardial infarction in mice:After4 weeks of MI in mice,compared with the CON group,the measured values of EF and FS in the MI group were significantly reduced(P<0.01);Compared with the CIHH group,the measured values of EF and FS in the CIHH+MI group were also significantly reduced(P<0.01);Compared with the MI group,the EF and FS measurements in the CIHH+MI group increased(P<0.05);The EF and FS measurements were not statistically significant in the CON and CIHH groups(P>0.05);Similarly,mice serum LDH and CK-MB were not statistically significant in the CON and CIHH groups(P>0.05).Compared with the MI and CIHH+MI groups,the levels of Compared with the MI group,the expression levels of LDH and CK-MB in the CIHH+MI group were reduced(P<0.05);Compared with the CON group,the BNP expression in the MI group was significantly increased(P<0.01);Compared with the MI group,the expression of BNP in CIHH+MI group decreased(P<0.05);Compared with the CON group,the myocardial infarct area increased in the MI group(P<0.01);Compared with the CIHH group,the area of myocardial infarction increased in the CIHH+MI group(P<0.01);However,compared with the MI group,the myocardial infarction area in the CIHH group was reduced(P<0.01).3.Expression of FUNDC1 protein and mRNA in cardiac tissue:Compared with the CON group,the expression of FUNDC1 protein and mRNA in the CIHH groupwere not statistically significant(P>0.05);Compared with the CON group,the expression of FUNDC1 protein and mRNA in the MI group were reduced(P<0.05);Compared with the MI group,the expression of FUNDC1 protein and mRNA in the CIHH+MI group were significantly increased(P <0.01 and P <0.05);4.Effect of knocking down FUNDC1 on ischemic injury of cardiomyocytes:In cell experiments,using Fundc1-shRNA to knock down FUNDC1,compared with the Control-shRNA group,FUNDC1 gene expression in the Fundc1-shRNA group was significantly reduced(P<0.01);Chronic hypoxia glucose deprivation(OGD)after hypoxia preconditioning(HPC)was knocked down on FUNDC1 to simulate myocardial ischemic injury.Compared with the Control-shRNA+HPC+OGD group,the Fundc1-shRNA+HPC+OGD group had decreased FUNDC1 protein(P<0.05);Simultaneously,the Fundc1-shRNA+HPC+OGD group had increased LDH and cardiac myocyte apoptosis(P <0.01 and P <0.05).5.Effects of FUNDC1 on mitochondrial function:Compared with the CON group,there was no significant difference in the mitochondrial respiratory control rate induced by glutamate in the CIHH group.The mitochondrial respiration control rate in the MI group was decreased(P<0.01).In cellular experiments,after knocking down FUNDC1,compared with the Control-shRNA+HPC+OGD group,the maximum mitochondrial respiration in the Fundc1-shRNA+HPC+OGD group was significantly reduced(P <0.01),and the mitochondrial ROS production was increased(P <0.01).6.Changes of mitochondria under electron microscope: Compared with the CON group,the number of mitochondria in the heart tissue of mice in the MI group was reduced(P<0.05),and the mitochondria area was abnormally increased(P<0.01);compared with the CIHH group,the number of mitochondria in the CIHH+MI group did not decrease(P>0.05),and the mitochondrial area increased relatively(P<0.01);however,compared with the MI group,the number of mitochondria increased in the CIHH+MI group(P <0.05),and the mitochondrial area decreased(P <0.01).7.Changes in mitochondrial dynamic proteins and autophagy levels:Mitochondrial fusion protein 1(MFN1)and Mitochondrial fusion protein 2(MFN2)were not significantly different in the CON group,MI group,CIHH group,and CIHH+MI group(P>0.05);Compared with the MI group,the expression of Mitochondrialfission factor 1(FIS1)and Dynamin-related protein 1(DRP1)in the CIHH+MI group increased(P<0.05),and Mitochondrial fission factor(MFF)expression was not statistically significant(P>0.05);Compared with the CON group,the expression of LC3-? protein in the MI group was not statistically significant(P>0.05),and the expression of P62 protein was increased(P<0.05).Compared with the MI group,the expression of LC3-? protein was increased in the CIHH+MI group.(P<0.05),accompanied by a decrease in P62 protein(P <0.01).Conclusion:1.CIHH treatment had no effect on left heart function and myocardial histopathology in normal mice.2.CIHH treatment has a protective effect on the heart of mice with myocardial infarction,which can significantly improve left ventricular systolic and diastolic function,reduce serum LDH and CK-MB levels and BNP expression,decrease myocardial infarction area.3.The expression of FUNDC1 in myocardial infarction tissue decreased,and the expression of FUNDC1 in myocardial infarction myocardial tissue of CIHH treated mice increased;Knocking down FUNDC1 in cell experiments increased the apoptosis and LDH release of myocardial ischemic injury after hypoxia preconditioning,and reversed the protective effect of hypoxia preconditioning on ischemic cardiomyocytes..4.The mitochondrial respiration control rate of myocardial infarction tissue is reduced,but CIHH treatment increases the mitochondrial respiration control rate of myocardial infarction tissue;Knocking down FUNDC1 attenuates mitochondrial maximal respiration and increases mitochondrial ROS production,indicating that myocardial ischemic damage leads to impaired mitochondrial function.The protective effect of hypoxic preconditioning on ischemic myocardium may improve mitochondrial function by increasing FUNDC1.5.The number of mitochondria decreased in myocardial infarction tissue,the area of mitochondria increased abnormally,the expression of mitochondrial splitting proteins(DRP1 and FIS1)decreased,and the level of mitophagy decreased;the number of mitochondria in CIHH treated myocardial infarction tissue did not decrease,and the mitochondrial area increased,which increased DRP1 And FIS1 protein expression,mitophagy level increased.This study shows that during ischemic stress after myocardial infarction,CIHH treatment up-regulates mitophagy receptor FUNDC1 expression,promotes mitophagy,maintains mitochondrial mass homeostasis,improves mitochondrial function,reduces mitochondrial ROS production,and delays the deterioration of cardiac function after infarction. |
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