The Mechanism Of Cardioprotection Induced By Endogenous TIGAR And Exogenous NADPH

Objective:To investigate the effects of endogenous TIGAR and exogenous NADPH on cardioprotection.Methods:For in vivo cardiac ischemia/reperfusion(I/R),an open-chest,in situ heart model was used.The I/R injury was imposed by ligation of the left anterior descending(LAD)coronary artery with 6-0 suture threaded through a snare for ischemia followed by withdrawal of the snare for reperfusion.Endogenous TIGAR protein expression levels were determined by western blot at different time point.System TIGAR knockout mice were used to measure the change of heart NADPH by ELISA and P62,LC3 II/I,and GAPDH by western blot.Infarct size(IS)and ATP were assessed after 30 min of ischemia followed by 120min reperfusion using different dose of exogenous NADPH(NADPH4mg/kg,8mg/kg or 16mg/kg).Levels of LC3BII/I,P62,PINK1 and PARKIN were also analysed by western blot;and we detected ROS steady-state levels within the myocardium in-situ using DHE and histology of rats heart after I/R using Hematoxylin and Eosin(HE)Staining.Cardiac troponin I(cTn-I)and lactate dehydrogenase(LDH)levels in the plasma of rats were detected with ELISA kits after 30 min of ischemia followed by 240min.Results:At ischemia 30min,ischemia 60min or ischemia 30min plus reperfusion 60min,endogenous TIGAR protein expression levels increased.Compared with wild type mice,the concentration of NADPH in TIGAR knockout mice heart was lower;but the expression level of P62 and LC3 II/I increased.When delivered in the clinically relevant context of reperfusion,exogenous NADPH(8mg/kg or 16mg/kg)significantly reduced myocardial infarct size and the levels of LDH,cTn-I and reactive oxygen species(ROS).After NADPH treatment,the increased LC3BⅡ/Ⅰ,P62 and PARKIN during I/R decreased,suggesting that NADPH repaired the impaired autophagy flux.Conclusions:Elevation of endogenous TIGAR during I/R is a defense mechanism generating little but not enough NADPH.Giving additional exogenous NADPH(8mg/kg or 16mg/kg)protected against myocardial I/R injury through mediating autophagy flux and ROS.