Mechanisms Of Endogenous Myocardial Protection:Exercise Preconditioning Induces Cardiomyocyte Autophagy And Promotes Autophagosome Formation

Objective: Repeated short-term high-intensity,intermittent aerobic exercise can improve the ability of myocardial tolerance to ischemia-hypoxia and reduce the myocardial injury caused by subsequent long-term ischemia and hypoxia.This way of inducing endogenous myocardial protection by exercise is called exercise preconditioning(EP).Autophagy is a physiological process in which abnormal proteins and aging and damaged organelles in cells are degraded and reused.Autophagy is activated or enhanced at the stimulation by energy deficiency,ischemia,hypoxia,and oxidative stress.This is an endogenous protective mechanism to maintain cell homeostasis.At present,there are numerous research results on the myocardial protection and on the mechanism of EP,and there are also many reports on the mechanism of autophagy involved in the myocardial protection of EP.But many scientific problems are still to be discussed further.Taking autophagy as the starting point,this study further verified the protection of EP on exercise-induced myocardial ischemia-hypoxia injury and compared the application characteristics of HE,HBFP,and C-2R BG staining techniques.It explored the possible mechanism of EP-induced autophagy in promoting autophagosome formation,researching on the chan ges of autophagy inducment-related proteins Beclin1/Bcl-2 and Beclin1/LC3 and on the changes of Atg12-Atg5 and Atg16L1,in the hope of providing an updated theoretical and experimental basis for studies of the mechanism of EP’s myocardial protection.Methods: 120 healthy male SD rats were randomly divided into 6 groups with20 rats in each group,namely the control group(C group),early EP group(EEP group),late EP group(LEP group),exhaustive exercise group(EE group),early EP plus exhaustive exercise group(EEP+EE group),and late EP plus exhaustive exercise group(LEP+EE group).(1)Electrophysiology of heart activity of rats was recorded by ECG,and the myocardial ischemia and hypoxia situations of rats in each group were analyzed by the changes of ST segments and T waves.(2)Plasma markers of myocardial injury were detected,the content of plasma c Tn I was detected by chemiluminescence immunoassay,and the content of plasma H-FABP was detected by enzyme-linked immunosorbent assay.(3)The changes of myocardial ischemia and hypoxia were shown by HE,HBFP,and C-2R BG IV staining techniques,and the three staining characteristics were compared through adjacent sections.(4)The ultrastructure of cardiomyocytes and autophagy were observed by transmission electron microscope(TEM).ECG,plasma myocardial injury markers and myocardial histomorphological indexes were used to comprehensively evaluate the degree of myocardial injury and protection.(5)By using double-labeling immunofluorescence technique,the changes of Beclin1/Bcl-2,Beclin1/LC3 and Atg12-Atg5/Atg16L1 expression in myocardium were observed by laser confocal scanning microscope,and the degree of colocation was analyzed.(6)The changes of Atg12-Atg5 and Atg16L1 expression in myocardium were detected by Western blot.(7)The relationship between Atg12-Atg5 and Atg16L1 in myocardium was analyzed by immunoprecipitation assay.Results:(1)Compared with C group,ECG waveforms in EEP group and LEP group were normal,ST segments elevated and T waves in EE group were significantly higher(P<0.05);Compared with EE group,ST segments depressed and T waves lower in EEP+EE group and LEP+EE group decreased significantly(P<0.05).(2).Compared with C group,the detection of plasma myocardial injury markers showed that the levels of plasma c Tn I and H-FABP in EE group were significantly higher(P<0.05);compared with EE group,those levels in EEP+EE group and LEP+EE group were significantly lower(P<0.05).(3)The results of myocardial ischemia-hypoxia staining showed that the degree of myocardial ischemia-hypoxia in EE group was significantly worse than that in C group;compared with EE group,the degree of myocardial ischemia and hypoxia in EEP+EE group and LEP+EE group was significantly reduced.Compared with the three staining methods,the sites of ischemia and hypoxia were almost at the same locations.Compared with HE staining,HBFP and C-2R BG staining were specific for ischemia and hypoxia,and the contrast of staining results was obvious.(4)TEM showed that the myofibrils in cardiomyocytes in C group were arranged orderly;mitochondria are spherical or short-rod-shaped,with uniform electron density and evenly distributed among myofibrils;the nuclear membrane was complete and smooth,and the chromatin was evenly distributed.In EE group,myofibrils were disordered,and myofilaments were broken and dissolved;mitochondria edema,matrix thinning,electron density decreased significantly,and vacuolar changes were observed;nuclear membrane invagination can be seen in the nucleus,but chromatin was evenly distributed,and nuclear pyknosis and nuclear disintegration were not seen.Compared with EE group,m yocardial ultrastructure in EEP+EE group and LEP+EE group was significantly improved.Typical autophagosomes were observed in EEP group,LEP group and LEP+EE group,and a large number of substrates to be degraded were w rapped in autophagosomes in LEP+EE group.(5)Double-labeling immunofluorescence showed that Beclin1 and Atg12-Atg5 showed red fluorescence,Bcl-2,LC3 and Atg16L1 showed green fluorescence,and the co-localization of Beclin1/Bcl-2,Beclin1/LC3 and Atg12-Atg5/Atg16L1 showed yellow fluorescence.The co-localization analysis showed that compared with C group,the co-localization degree of Beclin1/Bcl-2 in myocardium of other groups decreased significantly(P<0.05),and the co-localization degree of Beclin1/LC3 and Atg12-Atg5/Atg16L1 in EEP group,EE group,EEP+EE group and LEP+EE group increased significantly(P<0.05).Compared with EE group,the co-localization of Beclin1/LC3 and Atg12-Atg5/Atg16L1 was significantly increased in EEP+EE group(P<0.05).(6)Western blotting results showed that the expression of Atg12-Atg5 in EEP group,LEP group and EEP+EE group was significantly higher than that in C group(P<0.05).The expression of Atg16L1 increased significantly in EEP group and in EEP+EE group(P<0.05).The expressions of Atg12-Atg5 and Atg16L1 in EEP+EE group were significantly higher than those in EE group(P<0.05).(7)Co-immunoprecipitation showed that there was a binding relationship between Atg12-Atg5 and Atg16L1 in myocardium of rats in each group.Conclusion: EP could significantly reduce the ST segment elevation and T wave towering of ECG in rats induced by exhaustive exercise,and decreas e the content of markers of plasma myocardial injury and the degree of myocardial ischemia-hypoxia.This further confirms that EP has protection on exercise-induced myocardial ischemia-hypoxia injury.HE,HBFP,and C-2R BG staining can all be used to show the changes in myocardial ischemia and hypoxia.HBFP and C-2R BG staining are specific for ischemia and hypoxia.The staining results are in obvious contrast and are easy for image processing.EP can significantly decrease the co-localization of Beclin1/Bcl-2 to reduce the binding of Beclin1/Bcl-2,suggesting that the level of free Beclin1 increases,which induces cardiomyocyte autophagy,promotes the degree of Beclin1/LC3co-localization and improves autophagy activity.EP can increase the expression of Atg12-Atg5 and Atg16L1,to enhance their interaction and increase the degree of co-localization.Typical autophagosomes in EEP group,LEP group,and LEP+EE group which suggest that EP can induce autophagy,further promotes autophagosome formation,and plays a role in endogenous myocardial protection.