Experimental Study Of Sestrin2 As An Endogenous Antioxidant To Attenuate Myocardial Ischemia-reperfusion Injury

Background:Acute myocardial infarction is the leading cause of death and disability worldwide.Although early reperfusion is the most effective method to save ischemic myocardium,blood flow recovery can induce pathological events,resulting in abnormal ischemia/reperfusion(I/R)injury.Although there are a large number of basic and clinical studies to reduce the adverse consequences of I/R injury,the results are not satisfactory.Existing studies have shown that the increase of oxidative stress during myocardial I/R plays a central role in the severity of I/R injury.Oxidative stress is usually accompanied by the increase of reactive oxygen species(ROS),which not only directly leads to myocardial oxidative damage(such as lipid peroxidation and DNA/RNA base oxidation),but also adversely affects cardiac systolic function and cardiomyocyte survival by affecting calcium(Ca²⁺)signal and kinase signal.At the same time,endoplasmic reticulum is the main place for intracellular protein processing and the most important Ca²⁺reservoir in cells.The production of excessive ROS during I/R destroys the special redox environment and Ca²⁺homeostasis in the endoplasmic reticulum,and aggravates endoplasmic reticulum stress and dysfunction.In addition,during I/R,excessive ROS promotes the production of various inflammatory mediators and exacerbates I/R injury by stimulating tissue inflammation.What's more,as a signal molecule,the overproduction of ROS can activate a series of intracellular signaling pathways.Elevated levels of ROS lead to the activation of mitogen-activated protein kinases(MAPKs),including p38 MAPK,extracellular signal-regulated kinase(ERK),and c-Jun N-terminal kinase(JNK),as well as their downstream signaling cascades,which aggravate myocardial I/R injury through a variety of effects.Although oxidative stress is the key mechanism leading to myocardial I/R injury,the results of antioxidant therapy for cardiovascular diseases are disappointing,and some preclinical therapies with significantly beneficial effects do not show obvious benefits in clinical transformation.It is unclear why these studies failed to prove beneficial effects;however,current strategies involving the endogenous inhibition of ROS production or the supplementation of exogenous antioxidants to manage oxidative stress may be ineffective.Thus,the enhancement of endogenous antioxidant capacity may be a more effective intervention method.Therefore,the identification of effective endogenous antioxidant targets is a focus of current research.Sestrin2(Sesn2)is a stress-inducible protein and a key factor in regulating ROS production.In a variety of stress conditions including ischemia and reperfusion injury,Sesn2 plays an important role in cell and tissue protection by activating AMP-activated protein kinase(AMPK)and nuclear factor erythroid 2 related factor 2,and inhibiting mammalian target of rapamycin complex 1.The previous studies of our research group found that Sesn2,as a scaffold protein,promoted liver kinase B1-mediated AMPK activation in ischemic heart.At the same time,by regulating AMPK/PGC-1?signaling pathway,Sesn2 protected mitochondrial function and inhibited cardiomyocyte apoptosis induced by myocardial ischemia.During myocardial I/R,Sesn2 regulated cardiac substrate metabolism and increases myocardial tolerance to I/R injury by promoting AMPK activation.Sesn2 has been reported to attenuate neuronal I/R injury by inhibiting oxidative stress,but it remains unclear whether oxidative stress is involved in the protective effect of Sesn2 on myocardial I/R injury;the specific mechanism is also unknown.Objects:(1)To determine whether Sesn2 acts as an endogenous antioxidant to reduce myocardial I/R injury and improve systolic function during cardiac I/R.(2)To investigate whether Sesn2 can reduce the endoplasmic reticulum stress and inflammatory response related to oxidative stress,and the activation of oxidative stress related signaling pathways in the process of myocardial I/R.Methods:N-acetylcysteine(NAC),a recognized antioxidant with obvious protective effect on myocardial I/R injury in mice,but not satisfactory in clinical transformation,was used as the positive control.WT and Sesn2 KO mice randomly assigned to the operation and sham groups were further randomly divided into the following six groups:WT,WT+NAC,Sesn2 KO,Sesn2 KO+adeno-associated virus 9(AAV9)-Sesn2,Sesn2KO+NAC,and Sesn2 KO+AAV9-Sesn2+NAC.Mice were subjected to ischemia for 45 minutes and reperfusion for 24 hours to construct the in vivo cardiac I/R model;or the hearts of mice were perfused in the Langendorff mode for 25 minutes of global ischemia and 30 minutes of reperfusion to construct an ex vivo myocardial I/R model to measure myocardial infarction area and cardiac function,and analyze the changes of related protein expression.Cardiomyocytes isolated from mice in each group were reoxygenated for 1 hour after 20 minutes of hypoxia to construct a cell hypoxia/reoxygenation(H/R)model to determine the changes of cardiomyocyte contractility,Ca²⁺signal,mitochondrial superoxide and oxidative stress-related signaling pathways.Results:No matter in vivo or ex vivo,the myocardial infarct size and the damage of cardiac function in Sesn2 KO hearts were significantly greater than those of WT hearts.Sesn2transfection mediated by AAV9 restored the protein level of Sesn2 in the hearts of Sesn2 KO mice,significantly reduced the myocardial injury and cardiac function damage of Sesn2 KO mice after I/R,and improved the contractile function and Ca²⁺signal of cardiomyocytes during H/R stress.These improvements may be because that Sesn2 acted as an endogenous antioxidant,inhibiting the production of superoxide during H/R and reducing oxidative stress injury.Regardless of the expression of Sesn2,NAC treatment significantly reduced the myocardial injury and cardiac function damage caused by I/R(or H/R),while the combination of AAV9-Sesn2 and NAC did not further improve the myocardial I/R(or H/R)injury.At the same time,the deletion of Sesn2 exacerbated the up regulation of endoplasmic reticulum stress-related proteins GRP78,ATF4 and CHOP,promoted the expression of inflammatory mediators COX-2,IL-6 and TNF-?,and over activated MAPK signaling pathway during I/R(or H/R).Restoring the expression of Sesn2 or NAC pretreatment in cardiomyocytes improved these changes caused by I/R(or H/R)in Sesn2 KO hearts(or cardiomyocytes),while the combination of AAV9-Sesn2 and NAC did not further reduce the endoplasmic reticulum stress,inflammatory response and MAPK pathway activation induced by I/R(or H/R)compared with NAC treatment alone.Conclusions:(1)As an endogenous antioxidant,Sesn2 can significantly reduce the oxidative stress injury caused by myocardial I/R and improve the cardiac systolic function during I/R.(2)Sesn2 inhibits the endoplasmic reticulum stress and inflammation related to oxidative stress in the process of myocardial I/R.(3)As an endogenous adaptive response,Sesn2 inhibits the over activation of redox sensitive MAPK cascade signals during myocardial I/R.