| Objectives:The adaptive changes of exercise to the heart have been shown to reduce the risk of cardiovascular disease and protect against myocardial ischemia-reperfusion(I/R)injury.The occurrence of most cardiovascular diseases is accompanied by sympathetic hyperactivity and parasympathetic depression.M2 acetylcholine receptor(M2AChR),as a large number of parasympathetic receptors,can produce parasympathetic excitation after binding with acetylcholine,which plays a regulatory role in myocardial mitochondrial autophagy and endoplasmic reticulum stress during I/R.Exercise can significantly increase the decrease of parasympathetic activity caused by cardiovascular disease,improve the levels of mitochondrial autophagy and endoplasmic reticulum stress and reduce cardiomyocyte apoptosis,but whether M2AChR inhibits the effects of mitochondrial autophagy and endoplasmic reticulum stress in exercise resistance to I/R injury has not been reported.This study is to explore the protective effects of aerobic exercise interventions in myocardial I/R injury,and the role of parasympathetic M2AChR expression in reducing myocardial endoplasmic reticulum stress and mitochondrial autophagy in I/R rats.Its molecular mechanism.Methods:In vivo animal experiment:60 3-month-old male SD rats were randomly divided into 5 groups:sham operation group(S),quiet control+ischemia-reperfusion group(I/R),aerobic exercise+ischemia-reperfusion group(EX+I/R),ischemia-reperfusion+choline group(I/R+CHO),aerobic exercise+ ischemia-reperfusion+M2 acetylcholine receptor inhibitor group(EX+I/R+ME).Among them,EX+I/R and EX+I/R+ME were given aerobic exercise for 8 weeks,and the other three groups were fed normally in cages for 8 weeks.After 8 weeks of intervention,they fasted for 12 hours.In short,3%pentobarbital sodium was used for intraperitoneal anesthesia,the breathing mask was connected to the small animal ventilator,and the whole animal was intubated through the right jugular vein.The chest cavity was opened,and a 6.0 silk thread was threaded in the left anterior descending coronary artery.The silk thread was tightened to simulate myocardial ischemia,and the loosening was reperfusion.Ischemic 30min was prepared in the model and then reperfused for 4 hours.I/R+CHO group and EX+I/R+ME group were injected with choline,a parasympathetic agonist,and Methoctramine,an inhibitor of M2AChR,into the right jugular vein before ischemia,respectively,and the same amount of normal saline was injected into the other groups.In group S,only thoracotomy was performed under the anterior descending branch of the left coronary artery without ischemia and perfusion.At the end of reperfusion,the hemodynamics was recorded by retrograde catheterization of the right common carotid artery into the left ventricle,and the left ventricular systolic blood pressure,diastolic blood pressure,maximum rising rate and maximum descending rate were measured,and the cardiac function was evaluated.Blood was taken from the abdominal aorta,and the heart was removed for later experiments.In vitro cell experiment:H9C2 cell model was established.In short,the cells were cultured in 6cm dishes,and when the cells grew to about 80%,the cells were starved in serum-free medium for 8 hours,and then the hypoxia/reoxygenation(H/R)model was established:In the anoxic stage,simulated blood deficiency and hypoxia in three-gas incubator(37?,94%N2+1%O2+5%CO2)were used for 12 hours,and in the reoxygenation stage,normal medium and normal O2 content(37?,5%CO2)were used for reoxygenation for 4 hours;AMPK agonist(AICAR,1mM),parasympathetic agonist(Cholin,0.5mM)and M2AChR inhibitor(Methoctramine,1?M)were used to interfere with H9C2 cells.The cell model group was divided into H9C2 control group,H/R group,H/R+AICAR group,H/R+Choline group and H/R+Methoctramine+AICAR group.Lab Chart 7.0 analyzes changes in parasympathetic nerves in HRV response;The changes of myocardial microstructure and the degree of myocardial fibrosis were measured by HE staining and Masson staining;the indexes of serum CK and LDH were detected to reflect the degree of cardiac injury,and the ultrastructural changes of myocardial mitochondria were observed by transmission electron microscope;Western blotting was used to detect the expression of parasympathetic nerve M2AChR protein,mitochondrial fusion mitotic protein MFN2,DRP1,mitochondrial autophagy protein PINK1/Parkin,BNIP3,LC3II,P62,endoplasmic reticulum stress protein GRP78,GRP94 and phosphorylation of p-PERK/PERK,P-eIF2a/eIF2?,ATF4,CHOP signal pathway in myocardial tissue and H9C2 cells;Myocardial apoptosis index was detected by TUNEL fluorescence staining.Results:(1)At the moment of myocardial ischemia 30min and reperfusion,aerobic exercise intervention or choline had no significant effect on HFn,LFn and LF/HF in the heart of I/R rats;After 30 min of reperfusion,aerobic exercise exerted a protective effect,gradually restored HFn in I/R rats,inhibited LFn and LF/HF,and this protective effect was significantly different after 1h of reperfusion,and had a similar effect to choline.The results showed that aerobic exercise significantly inhibited the decrease of parasympathetic nerve activity and increased HRV,to improve the autonomic nerve dysfunction induced by I/R.(2)Aerobic exercise intervention significantly reduced left ventricular LVEDP,increased left ventricular LVSP and ± dp/dt max in I/R rats,and had similar effects to choline.The results showed that the intervention of aerobic exercise improved the cardiac function of I/R rats.(3)The intervention of aerobic exercise can improve the dissolution and fracture of myocardial fiber structure and inhibit the excessive production of collagen fibers in I/R rats,which is similar to that of choline.The results showed that aerobic exercise intervention could resist the damage of myocardial microstructure and reduce the percentage of myocardial collagen volume.(4)Aerobic exercise intervention significantly inhibited the serum enzymes CK and LDH activity in I/R rats,and had similar effects to choline.The results showed that the intervention of aerobic exercise significantly reduced the degree of myocardial injury.(5)Methoctramine,a M2AChR inhibitor,attenuates cardiac function and myocardial microstructure of aerobic exercise-improved I/R rats,aggravates myocardial collagen volume percentage,and myocardial injury.It is suggested that M2AChR plays an important role in the intervention of aerobic exercise in reducing myocardial injury in rats.(6)The expression of M2AChR in the myocardium of I/R rats was significantly higher than that of the control group.Aerobic exercise intervention can significantly increase the expression of M2AChR in I/R rats,and has a similar effect to choline.It is speculated that M2AChR is compensated in the myocardium of I/R rats,and aerobic exercise intervention can promote the expression of M2AChR.(7)Aerobic exercise intervention significantly reduced the expression of GRP78 and GRP94 proteins in myocardium of I/R rats,inhibited the phosphorylation level of PERK/eIF2?/ATF4 pathway,and had similar effects to choline.The results showed that the intervention of aerobic exercise inhibited the level of myocardial endoplasmic reticulum stress induced by I/R rats.(8)Aerobic exercise intervention significantly up-regulated the expression of MFN2 protein,inhibited the expression of DRP1 protein and retained the structure and number of mitochondria in the myocardium of I/R rats,which was similar to that of choline.The results showed that aerobic exercise intervention could promote myocardial mitochondrial fusion,inhibit division and improve myocardial mitochondrial dynamics disorder induced by I/R.(9)Aerobic exercise intervention down-regulates myocardial PINK1/Parkin protein expression in I/R rats,significantly reduces BNIP3 and LC3II protein expression,promotes P62 protein expression,and has similar effects to choline.The results showed that the intervention of aerobic exercise inhibited the high level of myocardial mitochondrial autophagy induced by I/R rats.(10)Aerobic exercise intervention reduced the number of myocardial TUNEL-positive cells and the expression of CHOP protein,which had similar effects to choline.It is suggested that the intervention of aerobic exercise inhibits the high level of cardiomyocyte apoptosis induced by I/R.(11)Methoctramine,a M2AChR inhibitor,attenuates myocardial endoplasmic reticulum stress and mitochondrial autophagy in I/R rats,and up-regulates myocardial apoptosis.It is shown that aerobic exercise can control the parasympathetic nerve M2AChR to inhibit the endoplasmic reticulum stress and mitochondrial autophagy in I/R rats,and reduce the myocardial cell apoptosis in I/R rats.(12)With the extension of H/R time,the expression of HIF1-? protein in H9C2 cells gradually increased after H/R4h,and there was a significant difference after H/R12h;the expression of M2AChR protein gradually decreased after H/R4h,And there was a significant difference after H/R6h;GRP78,p-PERK,p-eIF2? protein expression decreased slightly after H/R4h,then gradually increased after H/R6h,and it was significant at H/R12h difference.It shows that with the increase of H/R time,the endoplasmic reticulum stress level increases,the degree of cell damage increases,and the damage is more significant at H/R12h.(13)AICAR can significantly inhibit the H/R12h-induced H9C2 cells GRP78,GRP94 Protein expression and PERK/eIF2?/ATF4 phosphorylation levels,while M2AChR inhibitors attenuate AICAR reduce H/R-induced endoplasmic reticulum stress levels in H9C2 cells.It is suggested that AICAR can inhibit H9C2 cell endoplasmic reticulum stress induced by H/R through M2AChR.(14)H/R12h could decrease the level of mitochondrial autophagy,while AICAR of 1mM and Methoctramine of 1?M could regulate some proteins of mitochondrial autophagy induced by H/R.It is shown that the effects of AICAR and M2AChR and H/R models on mitochondrial autophagy in H9C2 cells need to be further studied.Conclusions:(1)Aerobic exercise intervention can regulate parasympathetic nerve M2AChR to inhibit the phosphorylation of PERK/eIF2?/ATF4 pathway,reduce the stress level of endoplasmic reticulum and inhibit the myocardial injury induced by I/R.(2)Aerobic exercise intervention can regulate parasympathetic nerve M2AChR to inhibit PINK 1/Parkin pathway induced by I/R,improve the state of mitochondrial fusion and division,down-regulate the level of mitochondrial autophagy,and reduce myocardial injury induced by I/R.(3)AICAR simulated exercise can inhibit endoplasmic reticulum stress induced by H/R12h in H9C2 cells through M2AChR,only part of mitochondrial autophagy can be regulated,which can not be completely proved that AICAR can regulate H/R 12h,induced mitochondrial autophagy through M2AChR.To sum up:At animal level,aerobic exercise can activate parasympathetic nerve,inhibit mitochondrial autophagy and endoplasmic reticulum stress through M2AChR receptor,and down-regulate cardiomyocyte apoptosis,It plays an important role in reducing myocardial injury induced by I/R and improving cardiac function in rats;At the cellular level,AICAR can inhibit endoplasmic reticulum stress induced by H/R12h through M2AChR,but the regulation of mitochondrial autophagy needs to be further studied. |
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