| Objective: Exercise preconditioning(EP)is a phenomenon of exercise protection that causes intermittent relative or absolute myocardial ischemia/hypoxia through a repeated short period of high-intensity intermittent aerobic exercise,induces the body to produce endogenous protective effects,improves the ability of the myocardium to tolerate ischemia/hypoxia,reduces the subsequent long-term ischemia/hypoxia injury of the myocardium,and exerts myocardial protective effects.The myocardial protective of EP has been confirmed.In the past,the myocardial protective effect of EP was often comprehensively evaluated by blood myocardial injury markers and morphological indicators of myocardial injury.Electrocardiogram(ECG)has not been used to study the myocardial protective effect of EP from the perspective of myocardial electrophysiology.Autophagy is one of the mechanisms of EP myocardial protection,which acts by ensuring cellular homeostasis through digestion and recycling of misfolded proteins,damaged organelles,and invasive pathogens.When cells appear ischemia/hypoxia,oxidative stress,starvation will induce the up-regulation of autophagy,play a protective role in cells.In this study,ECG,myocardial injury markers and morphological indicators of myocardial injury were used to comprehensively evaluate the degree of myocardial injury and protection,in an attempt to confirm that EP is involved in the protection of exercise-induced myocardial ischemia and hypoxia injury by optimizing myocardial energy metabolism,and then inducing autophagy,enriching the mechanism of autophagy involved in the myocardial protective effect of EP.This study will further improve the research methods of EP myocardial protection effect evaluation,will further enrich the research theory of EP endogenous myocardial protection effect mechanism,and provide updated ideas and methods for exercise training monitoring,exercise to improve health and prevent and treat cardiovascular diseases.Methods: 120 male SD rats were randomly divided into 6 groups,20 rats in each group,including control group(group C),early exercise preconditioning group(group EEP),late exercise preconditioning group(group LEP),exhaustive exercise group(group EE),early exercise preconditioning + exhaustive exercise group(group EEP+EE),and late exercise preconditioning + exhaustive exercise group(group LEP+EE).(1)The model of exercise-induced myocardial ischemia/hypoxia injury was established by exhaustive exercise.The protective effect of EP on exercise-induced myocardial ischemia/hypoxia injury was comprehensively evaluated by ST segment and T wave of ECG,c Tn I,myocardial injury blood marker,combined with HBFP staining for myocardial ischemia and hypoxia;(2)ECG waveform,PR interval,QRS complex and QT interval were analyzed to investigate the incidence of arrhythmia induced by EP in reducing exhaustive exercise.(3)Correlative analysis was used to investigate the relationship between ST segment and T wave of ECG and HBFP staining in ischemia/hypoxia as well as HBFP staining in ischemia/hypoxia and the degree of co-localization of GLUT4 and cytomembrane.(4)Immunoblotting assay was used to detect the expression levels of AMPKα,p-AMPKα,total GLUT4 in myocardial tissue,cytoplasmic GLUT4,cytomembrane GLU4,and autophagy-related proteins LC3,Beclin1,and p62;(5)Immunofluorescence analysis was used to analyze the mean fluorescence intensity of GLUT4 and GLUT4 translocation from the cell to the cell membrane.Results:(1)The results of ECG showed that compared with group C,the ECG waveforms of rats in group EEP and group LEP were normal,and no ischemia/hypoxia changes were found;the ECG waveforms of rats in group EE had significant ischemia/hypoxia changes,ST segment elevation(P<0.05),T wave elevation(P < 0.05),and the ECG waveforms of rats in group EEP+EE and group LEP+EE were significantly improved,compared with group EE,ST segment elevation was significantly improved(P<0.05),and T wave elevation was significantly reduced(P< 0.05);the heart rate results showed that compared with group C,the heart rate of rats in group EE was significantly increased(P<0.05),and compared with group EE,the heart rate of rats in group EEP+EE and group LEP+EE was significantly decreased(P<0.05).(2)plasma c Tn I results showed that c Tn I levels did not change significantly in group EEP and group LEP compared with group C(P>0.05),and c Tn I levels increased significantly in the group EE,and c Tn I levels decreased significantly in the group EEP+EE and group LEP+EE compared with group EE(P<0.05)(3)HBFP staining results showed that individual cardiomyocytes in group EEP and group LEP showed ischemia/hypoxia changes compared with group C,a large number of cardiomyocytes in group EE showed ischemia/hypoxia changes,and the degree of ischemia/hypoxia was significantly aggravated(P<0.05);compared with group EE,the ischemia/hypoxia area decreased significantly in group EEP+EE and group LEP+EE(P<0.05),and the degree of ischemia/hypoxia was significantly alleviated(P<0.05).(4)Correlation analysis showed that ST segment and T wave of ECG were positively correlated with the degree of HBFP ischemia-hypoxia staining in myocardial tissue.With the increase of the degree of HBFP staining ischemia/hypoxia,ST segment of ECG was gradually increased,and T wave was gradually increased.The degree of HBFP ischemia/hypoxia staining was negatively correlated with the degree of GLUT4/cytomembrane co-localization.With the increase of GLUT4/cytomembrane co-localization,the degree of HBFP ischemia/hypoxia staining decreased.(5)Immunoblotting results showed that compared with group C,the proportion of AMPK phosphorylation was significantly increased in group EEP,the proportion of AMPK phosphorylation was significantly increased,cytoplasmic GLUT4 expression was significantly decreased in group LEP(P<0.05),cytomembrane GLUT4 expression was significantly increased in group EE(P<0.05),and cytomembrane GLUT4 was significantly increased(P<0.05).Compared with group EE,the proportion of AMPK phosphorylation was significantly increased in group LEP+EE,the total GLUT4 expression was significantly increased in group EEP+EE and group LEP+EE(P<0.05),cytoplasmic GLUT4 expression was significantly decreased(P<0.05),and cytomembrane GLUT4 was significantly increased(P<0.05).(6)Compared with group C,the expression levels of Beclin1 and p62 in the group EE,group EEP+EE,and group LEP+EE were significantly increased(P<0.05),while LC3-II/I in the group EEP+EE was significantly increased;Compared with the group EE,the LC3-II/I were significantly increased in the group EEP+EE,while the p62 level was significantly decreased in the group EEP+EE and group LEP+EE.(7)Compared with group C,the average fluorescence intensity of GLUT4 in the group EEP,group LEP,group EE,group EEP+EE,and group LEP+EE was significantly increased(P<0.05).Compared with the group EE,the average fluorescence intensity of GLUT4 in the group EEP+EE and group LEP+EE was significantly increased(P<0.05);Compared with group C,the degree of GLUT4 co localization with the cell membrane was significantly increased in the group EEP,group LEP,group EE,group EEP+EE,and group LEP+EE(P<0.05).Compared with the group EE,the degree of GLUT4 co localization with the membrane was significantly increased in group EEP+EE and group LEP+EE(P<0.05).Conclusions:(1)The electrocardiogram of myocardial ischemia/hypoxia injury caused by exhaustive exercise is characterized by ST segment elevation and T-wave elevation.EP can significantly improve the phenomenon of ST segment elevation,T-wave elevation,and high heart rate in the ECG caused by exhaustive exercise;Exhaustive exercise induced the occurrence of cardiac arrhythmias in rats,and EP effectively reduced the incidence of cardiac arrhythmias induced by exhaustive exercise and the risk of malignant arrhythmias in rats;The ST segment and T wave of ECG were positively correlated with the area and degree of HBFP ischemia/hypoxia staining,suggesting that there was a certain correlation between the changes of ECG ischemia/hypoxia and the ischemia and hypoxia of myocardial cells.(2)EP promotes the phosphorylation of AMPK,the expression of GLUT4,and the translocation of GLUT4 from cells to the cytomembrane.With the increase of GLUT4 translocation to the cytomembrane,the area and degree of myocardial ischemia/hypoxia gradually decrease;By promoting the phosphorylation of AMPK,EP promoted the increase of the expression level of autophagy related protein Beclin1,the increase of the LC3-Ⅱ/Ⅰ and the significant decrease of the expression level of p62,and induced autophagy and autophagy patency,suggesting that EP may participate in the protection of ischemic/hypoxic myocardium not only by promoting AMPK phosphorylation to induce autophagy,but also by promoting the translocation of myocardial cell GLUT4 to the cytomembrane,Furthermore,it may upregulate the proportion of glucose metabolism in ischemic and hypoxic myocardium,optimize myocardial energy metabolism,and exert myocardial protective effects. |
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