Study On The Relationship Of Endoplasmic Reticulum Stress And Autophagy With Exercise Preconditioning Induced Endogenous Cardioprotective Effects

Objective: Exercise preconditioning(EP)established by repetitive and brief bouts of high-intensity intermittent exercise before a severe,sustained myocardial ischemiahypoxia episode could initiate remarkable intrinsic cardioprotective effects for the preinduced intermittent ischemia-hypoxia changes in myocardium.EP is developed from the research background of the myocardial ischemic preconditioning(IP).Both the activated endoplasmic reticulum stress response and autophagy levels are cellular adaptive processes initiated under the stressing status.The activated endoplasmic reticulum stress response and autophagy levels are involved in the pro-survival cellular process during the cardioprotection of IP against ischemia/reperfusion(I/R)induced myocardial injury.However,whether the activating status of endoplasmic reticulum stress response and autophagy levels in myocardium is involved in the endogenous cardioprotective effects induced by EP remains unclear.The purpose of this study is to explore and discuss the relationship of endoplasmic reticulum stress response and autophagy alterative levels in myocardium with the EP-induced endogenous cardioprotective effects.Methods: Firstly,220 health male Sprague-Dawley(SD)rats were randomly divided into two parts to establish the animal research models of EP-induced cardioprotective effects and EP-induced cardioprotective phase.The animal research model of EPinduced cardioprotective effects including group C(control),group EEP(early EP),group LEP(late EP),group EE(exhaustive exercise),group EEP+EE(EP pretreatment at 0.5 hours before EE),group LEP+EE(EP pretreatment at 24 hours before EE),group W+EEP+EE and group W+LEP+EE(Wortmannin injection by intraperitoneal pretreatment at 0.5 hours before EP,Wortmannin is a PI3KC3 inhibitor which could inhibit cellular autophagy level).The EP protocol included 4 periods of 10 minutes of treadmill running each at 30 m/minute with intervening 10 minutes periods of rest,the hearts of rats in group EEP and group LEP were extracted at 0.5 hours and 24 hours after EP respectively.Plasma levels of cardiac troponin I(cTnI)and hematoxylin-basic fuchsin-picric acid(HBFP)staining were used to evaluate the ischemia-hypoxia injury and cardioprotective effects in rat myocardium.The rats were anesthetized and sacrificed at 0.5 hours,1 hour,2 hours,3 hours,5 hours,and 24 hours after EP to establish the animal research model of EP-induced cardioprotective phase.This research is based on the animal research models of EP-induced cardioprotective effects and EP-induced cardioprotective phase,alterative levels in endoplasmic reticulum stress response biomarker proteins(GRP78 and GRP94)in the left ventricular myocardium were analyzed by immuno-histochemical staining and Western blot.Alteration levels in several autophagy proteins(Beclin1?Atg7?Atg5?LC3 and p62)in the left ventricular myocardium were analyzed by Western blot.The relationship of endoplasmic reticulum stress response and autophagy levels in myocardium with the EP-induced endogenous cardioprotective effects was discussed by incorporating the protein level changes during EP-induced cardioprotective effects with the cardioprotective phase.Results:(1)Compared with group C,the cTnI levels in plasma,the ischemiahypoxia positive areas,integral optical density(IOD)value and mean optical density(MOD)value in HBFP-staining increased significantly in group EE,but no significant changes of plasma and HBFP-staining were found in group EEP and group LEP.Compared with group EE,the cTnI levels in plasma,the ischemia-hypoxia positive areas,IOD value and MOD value in HBFP-staining decreased significantly in group EEP+EE and group LEP+EE.Compared with group LEP+EE,the cTnI levels in plasma,the ischemia-hypoxia positive areas,IOD value and MOD value in HBFP-staining increased significantly in group W+LEP+EE.(2)Compared with group C,group EE showed no significant changes in the expression levels of GRP78 and GRP94 by Western blot and IOD value by immuno-histochemical staining.Compared with group EE,group EEP+EE and group LEP+EE showed no significant changes in the expression levels of GRP78 and GRP94 by Western blot and IOD value by immunohistochemical staining.(3)Compared with group C,the expression levels of GRP78 by Western blot decreased significantly at 5 hours and 24 hours after EP.In the results of immuno-histochemical staining,the IOD value of GRP78 decreased significantly at 0.5 hours,1 hour,2 hours,3 hours,5 hours and 24 hours after EP,positive areas of GRP78 decreased significantly at 0.5 hours,1 hour,2 hours,3 hours and 24 hours after EP,positive areas,IOD value and MOD value of GRP94 decreased significantly at 5 hours and 24 hours after EP.(4)Compared with group C,the LC3-II/LC3-I ratio,a marker of autophagosome formation,was reduced in group EE and group LEP+EE,but the LC3-II/LC3-I ratio remained unaltered in group EEP+EE.(5)Compared with group C,the LC3-II/LC3-I ratio,a marker of autophagosome formation,increased significantly at 2 hours during the cardioprotective phase after EP,Atg7 increased significantly at 1 hour during the cardioprotective phase after EP,Atg5 increased significantly at 3 hours and 5 hours during the cardioprotective phase after EP.Conclusions:(1)Exhaustive exercise could induce ischemia-hypoxia injury in rat myocardium.The ischemia-hypoxia injury in rat myocardium could not be induced at 0.5 hours and 24 hours after EP,EP is a relative safe exercise protocol.Pretreatment with EP at 0.5 hours and 24 hours prior to the exhaustive exercise initiate obvious cardioprotective effects,attenuate the ischemia-hypoxia injury.(2)Exhaustive exercise may not induce the accumulation of unfolded/misfolded proteins within endoplasmic reticulum lumen in cardiomyocytes of rat.EP-induced cardioprotection against ischemia-hypoxia injury may not involved the elevation of ER stress proteins(GRP78 and GRP94),and more studies need to be applied to verify this point of view.Decreased ER stress proteins(GRP78 and GRP94)were found during the cardioprotective phase after EP,more detailed mechanisms would be investigated in further research.(3)Exhaustive exercise could inhibit autophagy levels in rat myocardium,the activated autophagy level during the early cardioprotective phase after EP may be partly involved in the early cardioprotective effects by maintaining a normal autophagy basal level during subsequent exhaustive exercise in rat myocardium.