Ischemic Postconditioning-induced Cardioprotection Is Attenuated In Enho Gene Deficient Mice

Objective: Ischemic postconditioning(IpostC)is one of the essential endogenous cardioprotection mechanisms that reduces myocardial ischemia-reperfusion injury(MIRI).The protection disappears when metabolismis disturbed,however its molecular mechanism is still unclear.Energy Homeostasis-associated gene(Enho)is able toimprove insulin resistance,regulate glucose,lipid metabolism and participate in heart protection.Our study aims to investigate effects ofcardioprotection by postconditioningin Enho gene deficient mice.Methods:24-weekEnho-/-mice and brood wild-type C57BL/6J mice(WT mice),built as invivomyocardial Ischemia/Reperfusion(I/R)model,were randomly divided into 7 groups: all groups receiving 45-min ischemia and then 24-h or 7-min reperfusion(Enho-/-mice n=3,WT mice n=6 for both)except Sham group: 1)Sham group: without ischemia treatment;2)Ischemia/Reperfusion group(Control group): given ischemia for 45 mins followed by persisting reperfusion;3)Ischemicpostconditioning group(IpostC Group): three cycles of 10 s I and 10 s R before persisting reperfusion;4)Adropin group:additionalinjection of Adropin(0.2mg/kg)slowly into external jugular vein at 5 mins before reperfusion;5)Adropin+Ischemicpostconditioninggroup(A+IpostC group): same as Adropin plus IpostC group;6)Adropin+Ischemicpostconditioning+LY294002 group(A+IpostC+LY group):additional injection ofPI3K-specific inhibitor LY294002(40mg/kg)i.p.15 mins before reperfusion plus the same treatment as A+IpostCgroup;7)Adropin+Ischemic post-conditioning+PD98059 group(A+IpostC+PD group):additional injection of MEK specific inhibitor PD98059(1mg/kg)i.p.15 mins before reperfusion plus the same treatment as A+IpostCgroup.After 24-hour Reperfusion,several mice(n=3-6)were sacrificed to contribute hearts.Infact size was eveluated by triphenyltetrazolium chloride(TTC),activated Caspase-3 protein by immunohistochemical stain;The rests(n=3-6)(after I 45min/R 7min)were used to detected the level of signal pathway proteins of Akt,Erk1/2 and GSK-3? by Western blot and the level of Bcl-2 and Bax gene by Real-time PCR.Results: WT mice: Compared with Control group,the infarct size and activated Caspase-3 proteins of the IpostC group were significantly reduced(48.65±4.02% vs.32.67±4.45%,P<0.05;0.386±0.022 vs.0.143± 0.016,P<0.05),while Bcl-2/Bax gene ratio increased(0.56±0.12 vs.1.54±0.24,P<0.05).We found additional exogenous recombinant Adropin only still exerted the same effect.However,Adropin combined with IpostC failed to further reduce the myocardial infarct size,which was blocked by LY294002 or PD98059 respectively.Enho-/-mice: Compared with Control group,the infarct size decreasedby exogenous recombinant Adropin(50.08±4.62% vs.41.29±3.04%,P<0.05)but not IpostC group,and further more in A+IpostC group(33.88±3.58%).Contrast against Control group in WT mice,the myocardial infarct size did not increase in the Enho gene deficienct mice,nor further decrease in IpostC group.Additional exogenous recombinant Adropin could restore cardioprotective effectsin the IpostC group,increasephosphorylation of Akt,Erk1/2 and GSK-3? protein,the ratio of Bcl-2/Bax(0.48±0.22 vs.1.55±0.16,P<0.05)and down-regulate the activation of Caspase-3 protein(0.378±0.024 vs.0.145±0.019,P<0.05),which could be blocked by LY294002 or PD98059.Conclusions:The lack of Enho gene leads to the weakeness of the cardioprotectionof Ischemic Postconditioning by down-regulation of Akt,Erk1/2 and GSK-3? protein phosphorylation.