Roles And Molecular Mechanisms Of Oxidative Stress And Mitophagy In Cardioprotection Induced By Exercise Preconditioning

Objective: as a stimulative factor of intensity and work loading,exercise improves oxygen consumption to the heart extremely,leading to absolute or relative hypoxia.Intermittent high-intensity exercise results in repetitive and transient ischemia,similar to the process of ischemic preconditioning(IP),which allows the myocardium to be protected during subsequent myocardial injury induced by acute stress.The type of such exercise,is called exercise preconditioning(EP).Mitochondrial protection plays key role in maintaining stably physiological status to the heart,as such here has strong association with oxidative stress.It has been recently reported,macroautophagy and mitophagy to act on mitochondrial protection,also to have connection with oxidative stress.In the present study,oxidative stress,mitochondria,and autophagy are set as a researching line,combing with the principles of early and late protective phases of EP,animals being treated by additional exhaustive exercise creates damaging condition.Using autophagy inhibitor wortmannin,analyzing the translocational interaction between proteins by various experimental methods,the generational mechanisms of cardiac mitophagy is clarified.Further,the present study will provides freshly theoretical and experimental basis to the research of cardiacprotection and mechanisms by EP in future.Methods: 200 healthy male SD rats were randomly distributed in 10 groups including group control(C);group exhaustive exercise(EE),in which rats were subjected to one single bout exhaustive treadmill running;group EEP,in which rats underlying one single bout intermittent high-intensity running to establish EP protocol,killed at 0.5h after EP;group LEP,rats were killed at 24 h after EP;group EEP+EE,an exhaustive running was added at 0.5h after EP;group LEP+EE,the exhaustion was added at 24 h after EP;group W+EEP,rats received an intraperitoneal injection of wortmannin at 0.5h before EP,and killed at 0.5h after EP.group W+LEP,the injection is the same with W+EEP,killed at 24 h after EP.group W+EEP+EE,the injection is the same with the aforesaid,the exhaustion was added at 0.5h after EP;group W+LEP+EE,the injection is the same with the aforesaid,the exhaustion was added at 24 h after EP.The immunochemiluminometric assays were used to detect the levels of cardiac type troponin I(c Tn I),evaluating myocardial inury.Hypoxic-ischemic changes were assessed by using hematoxylin basic fuchsin picric acid(HBFP staining).Transmission electron microscopy was used to acquire the changes to myocardial ultrastructure,especially capturing mitochondria and autophagosomes.The concentrations of malondiadehyde(MDA),hydrogen peroxide(H2O2),and the activities of manganese superoxide dismutase(Mn SOD),total SOD,were acquired by using spectrophometric methods,combining with Mn SOD immune blotting analysis,these methods were together assessing extents of oxidative stress.The inhibition of mitochondrial permeability transition pore(m PTP)were assessed by detecting voltage-dependent anion-selective channel 1(VDAC1)phosphorylation trough Phos-tag methods.Using mitochondrial isolation and immune blotting to detect cytochrome c(Cyt-c),autophagic receptor p62,and mitophagic Parkin.Bnip3,assessing the changes of these proteins between cytosol and mitochondria.The crucial proteins of autophagy,Beclin1,Bcl-2,LC3 I,and LC3 II levels were detected by using immune blotting,combing with the calculation of ratios Beclin1/Bcl-2,and LC3II/LC3 I,the levels and characters of cellular autophagy were assessed.Using confocal laser scanning-based double-labeled fluorescence experiments to detect translocational levels between LC3 and inner mitochondrial membrane-located COX4/1,between Parkin and the outer mitochondrial membrane-located translocase(TOM)subunit TOM70,and between Bnip3 and another subunit TOM20,to also detect fluorescence intensity and distribution of these six proteins.Accordingly,through by these experiments,the effects and mechanisms of oxidative stress and mitophagy in EP-induced cardioprotection can be investigated deeply.Results:(1)compared with group C,group EE showed significant increases in the c Tn I levels and the extents of hupoxia-ischemia,showed obvious changes in damaged ultrastructures.Groups EEP and LEP are without changes to injury,Compared with group EE,groups EEP+EE and LEP+EE showed significant decreases in the c Tn I levels,and the damaged ultrastructural changes to be facilitated.Compared with group EEP+EE,group W+EEP+EE has slight decreases in the c Tn I levels,but has tendency in increased hypoxia-ischemia,in which mitochondria showed obvious hypertrophy.Compared with group LEP+EE,group W+LEP+EE showed increases in plasma c Tn I levels and hypoxia-ischemia,to be associated with significant damages to mitochondria.(2)Compared with group C,group EE showed significant increases in MDA,that is production of oxidative stress-injury,and showed decreases in total-SOD activity,taken together,indicating group EE to be associated with oxidative stress-injury,but without concentration or activity changes in H2O2,Mn SOD.Groups EEP and LEP showed no changes in activities of Mn SOD and total SOD,and in concentrations of MDA and Mn SOD,but group EEP is associated with decreases in H2O2.Compared with group EE,group EEP+EE showed significant decreases in MDA,and significant increases in the H2O2 and in the total SOD activity,group LEP+EE showed significant increases in the total SOD activity.Separately compared with groups EEP and LEP,groups EEP+EE and LEP+EE showed significant decreases in the activities of Mn SOD and total SOD.Compared with group EEP+EE,group W+EEP+EE showed significant increases in the MDA and in the Mn SOD activity.Compared with group LEP+EE,group W+LEP+EE showed significant increases in H2O2.(3)Compared with group C,groups EE and EEP+EE showed increases in the levels of VDAC1 phosphorylation to suppress m PTP opening.Compared with group EE,groups LEP+EE,W+EEP+EE,and W+LEP+EE showed significant decreases in the VDAC1 phosphorylation.Compared with group EEP+EE,group W+EEP+EE showed significant decreases in the VDAC1 phosphorylation,and showed significant increases in Cyt-c leakage from the mitochondria to the cytosol.(4)Compared with group C,group EE showed significant increases in the LC3 II and the ratio of LC3II/LC3 I,group EEP showed significant increases in the LC3II/LC3 I ratio,as such,group EEP+EE showed elevation in concentrations of LC3 II and Beclin1 to indicate the improvement to the cellular autophagy.An apoptotic autophagy can be identified in group W+LEP by the increased LC3 II,LC3II/LC3 I ratio,and the Beclin1/Bcl-2 ratio.Such two ratios were elevated in groups W+EEP+EE and W+LEP+EE also indicating increased apoptotic autophagy.Compared with group EE,group W+EEP+EE showed decreases in the LC3 II and in the LC3II/LC3 I ratio,indicating a lower levels in cellular autophagy,group W+EEP+EE showed increases in the Beclin1 levels.(5)Compared with group C,group EE showed significant decreases in the translocational percentage of LC3 to COX4/1,and in mitochondrial(mito)p62,group EEP showed a low level of LC3 translocation,and group W+LEP showed decreases in the fluorescence(fluor.)intensity of COX4/1.Compared with group EE,group EEP+EE showed increases in the LC3 translocation,in the fluor.intensity of LC3,and in the cytosolic(cyto)p62,group LEP+EE showed increases in the LC3 translocation and decreases in the fluor.intensities of LC3 and COX4/1,group W+EEP+EE showed decreases in the LC3 translocation and increases in the cyto,mito p62,group W+LEP+EE showed increases in the LC3 translocation and in the mito p62,but it showed significant decreases in the fluor.intensities of LC3 and COX4/1.Compared with EEP+EE,group W+EEP+EE showed significant decreases in the fluor.intensity of LC3.(6)Compared with group C,group EE showed significant decreases in the mito Parkin,in the translocational extents of Parkin to TOM70,and in the fluor.intensities of Parkin and TOM70,but it showed significant increases in the cyto,mito Bnip3,in the translocational extents of Bnip3 to TOM20,and in the fluor.intensity of Bnip3,without changes in the fluor.intensity of TOM20.Group EEP showed significant decreases in the Parkin translocation,and in the fluor.intensities of TOM70 and TOM20,but it showed increases in the cyto and mito Bnip3 levels.Group LEP showed significant increases in the Bnip3 translocation,and in the cyto,mito Bnip3 levels.Separately compared with groups EEP and LEP,groups W+EEP and W+LEP showed approach in the levels of mitophagic targets.Compared with group EE,group EEP+EE showed significant increases in the mito Parkin,in the Parkin translocation,and in the fluor.intensities of Parkin,TOM70 and Bnip3,mito and cyto Bnip3 approaching to these levels in group EE.Group LEP+EE showed increases in the mito Parkin,in the Parkin translocation and in the fluor.intensity of TOM70,it showed all Bnip3-related data to have significant decreases.Group W+EEP+EE showed significant increases in all Parkin-related data,but in which Parkin translocation is significantly lower than in group EEP+EE.,and group W+EEP+EE showed significant increases in the cyto Bnip3 and in the TOM20 fluor.intensity.Group W+LEP+EE is associated with the increased translocation and fluor.intensity of Parkin.Compared with group EEP+EE,group W+EEP+EE showed significant increases in the fluor.intensity of Parkin and significant decreases in the fluor.intensities of Bnip3 and TOM20.Compared with group LEP+EE,group W+LEP+EE showed significant decreases in the Parkin translocation,but in which,the Bnip3 translocation,and the fluor.intensities of Bnip3 and TOM20 are significantly increased.Conclusions:(1)single bout exhaustive exercise results in significant myocardial injury,hypoxia-ischemia,ultrastructural injury,and oxidative stress injury,but it is without excessive mitochondrial damage and the induction of apoptosis.Mechanisms by exhaustive exercise the suppression of m PTP opening and the Bnip3-dependent mitophagy induced by the enhanced mitochondrial fission,perceptibly participating in mitochondrial protection.(2)EP is a non-injury type of exercise,inducing the activity of SOD,decreasing H202 levels,providing the adaptation to the myocardium.During the early phase of EP-induced cardioprotection,m PTP was inhibited,the levels of repairing mitophagy was elevated by the induction of H2O2,involving the participations of Parkin and Bnip3,but in which Bnip3 may has more significant effect.During late phase of EP-induced cardioprotection,Parkin-mediated manner plays leading role in repairing mitophagy.(3)Autophagic inhibitor wortmannin has negative effect on the mitochondria to EP-induced cardioprotection,but it dose not lead exacerbated injury to EP itself.During early phase of EP-induced cardioprotection,inhibited cellular autophagy resulted in the elevation of type 1 pre-apoptosis,further the mitochondrial protection was lost.During late phase of EP-induced cardioprotection,wortmannin reversely induced apoptotic autophagy,the cardioprotection was lost consequently.