Sestrin2 Prevents Age-related Intolerance To Ischemia And Reperfusion Injury By Modulating Substrate Metabolism

With aging,individuals become less tolerant to a variety of stresses.In particular,the ability of the myocardium to tolerate ischemia becomes significantly compromised with aging,as demonstrated by clinical findings and animal models of myocardial infarction.Current revascularization for myocardial infarction is critical for the salvage of ischemic myocardium but may also trigger a series of undesirable events that can exacerbate cell death and increase the extent of myocardial infarction,a process known as ischemia/reperfusion(I/R)injury.Furthermore,multiple clinical trials have demonstrated that,despite reperfusion,patients age >70 yr who suffer from acute myocardial infarction have a significantly higher mortality rate compared with younger age groups.Our group and others have provided extensive evidence that suggests that activation of the AMPK signaling pathway is highly advantageous to the heart and is cardioprotective under I/R conditions.Recently,a novel group of proteins that lack kinase activity,known as the sestrin2(Sesn2),have been shown to increase the activation of AMPK in vitro and in vivo.Like AMPK,Sestrin2 has been shown to be a positive regulator of autophagy as evidenced by the finding that Sesn2-deficient cells exhibit a significant decline in autophagy when subjected to nutrient depletion.We postulate that Sestrin2 may represent particularly important survival mechanism in the heart during I/R,in part by amplifying AMPK activation during ischemia.The induction of Sestrin2 has been shown to be primarily mediated by the tumor-suppressor p53 and by hypoxia-inducible factor 1?(HIF-1?).Intriguingly,the stress-induced stabilization and activity of both p53 and HIF-1? are dampened with aging.Thus,in the event of myocardial ischemia in the aged heart,Sestrin2 induction and/or function is likely to be blunted which may alter AMPK activation.Moreover,Sestrins are indeed important in the aging process as Sestrin gene deletion in Drosophila results in hallmark age-related pathologies such as increased lipid accumulation,increased oxidative stress,poor cardiac performance,and defective autophagy.We have demonstrated that AMPK activation stimulates heart glucose transport both in vitro and in vivo and that AMPK has an essential role in the activation of glucose uptake in the ischemic heart.AMPK also activates 6-phosphofructo-2-kinase(PFK-2),which leads to the production of fructose 2,6-bisphosphate,further promoting the glucose utilization in the ischemic heart.Glucose metabolism has an important role in protecting the ischemic heart against hypoxic injury and apoptosis.Activation of glucose transport mediates the entry of glucose into the cell and is required to increase glucose metabolism in heart and skeletal muscle,whether in response to insulin stimulation,exercise,hypoxia or ischemia.Ischemia stimulates glucose uptake by translocating GLUT4(and to some extent GLUT1)from intracellular storage membranes to the sarcolemma.Pharmacologic stimulation of AMPK leads to the translocation of GLUT4 in the heart,based on membrane fractionation,immunofluorescence with confocal microscopy,and novel surface GLUT4 labeling techniques defining the subcellular GLUT4 localization.The downstream mechanisms through which AMPK triggers GLUT4 translocation remain more elusive.AMPK may mediate its effects by interacting with additional intracellular signaling pathways or acting directly on proteins involved in GLUT4 vesicular trafficking.Therefore,the present study,To determine whether ischemic AMPK activation—modulated by the Sesn2-AMPK complex in the heart—is impaired in aging that sensitizes the heart to ischemic insults,young C57BL/6mice(age3–4mo),middle-aged mice(age10–12mo),aged mice(age 24–26 mo)and Sestrin2 KO mice(Sestrin2 –knockout:age3–4mo)were subjected to I/ R injury(in vivo and ex vivo),discussed the less of Sesn2 on myocardial ischemic injury,and analysis of relevant signaling mechanisms.Aim:1.To characterize the critical role of Sestrin2 in cardiac-aging AMPK signaling regulation during ischemia and reperfusion stress.2.To determine the mechanisms by which Sestrin2 modulates glucose transport in the aged ischemic heart.Method: C57BL/6 young mice(3-4 months),middle age mice(10-12 months),aged mice(24-26 months)and Sestrin2 KO mice(Sestrin2 –knockout:3–4months)were subjected to left anterior descending coronary artery occlusion for in vivo regional ischemia.The ex vivo working heart system was used for measuring substrate metabolism.Result: The protein level of Sestrin2 in the hearts were gradually decreased with aging.Intriguingly,ischemic AMPK activation was blunted in the aged hearts as compared with young hearts(p<0.05),the AMPK downstream glucose uptake and the rate of glucose oxidation were significantly impaired in the aged hearts during ischemia and reperfusion(I/R)(p<0.05 vs.young hearts).The myocardial infarction size was larger in the aged hearts(p<0.05 vs.young hearts).The immunoprecipitation with Sestrin2 antibody revealed that cardiac Sestrin2 forms a complex with AMPK and upstream LKB1 during ischemia,intriguingly,the binding affinity between Sestrin2 and AMPK upstream LKB1 is impaired in the aged hearts during ischemia(p<0.05 vs.young hearts).Furthermore,Sestrin2 knock out hearts demonstrate a similar cardiac phenotype and response to ischemic stress as the wild type aged hearts,i.e.impaired ischemic AMPK activation and higher sensitivity to I/R-induced injury.Adeno-associated virus(AAV)-Sestrin2 were delivered into the aged hearts via a coronary delivery approach significantly rescued the protein level of Sestrin2 and the ischemic tolerance of aged hearts.Conclusion: Sestrin2 is a scaffold protein that mediates AMPK activation in the ischemic myocardium via an interaction with AMPK upstream LKB1.The decreased Sestrin2 levels in aging lead to a blunted ischemic AMPK activation,alterations in substrate metabolism and an increased sensitivity to ischemic insults.