The Protected Effects And Mechanisms Caused By Ischemic Postconditioning On Hypertrophic Myocardium Induced By Pressure-overloaded Suffering From Reperfusion Injury In Mice

Objective:The research is aimed to evaluate effects caused by ischemia postconditioning (IPost) on mouse hypertrophic heart in vivosuffering from ischemia/reperfusion (I/R) injury, and further exploring cardioprotective mechanisms induced by reperfusion injurysalvage kinase (RISK)signal pathways in isolated mouse hypertrophic hearts. The presentstudy focuses on the following issues: 1) Through microsurgery to establish the transverse aortic constriction (TAC) mouse model and to explore the changed tendency of heartstructure and function. 2) To establish the advanced Langendorff model of mouse hypertrophic heart and to determine the effect of ischemic IPost protection in hypertrophic myocardiumsubjected to I/R injure and tostudy the role of RISKsignal pathways in mediatingsuch protection. 3) To explore the cardioprotective molecular mechanisms of extracellularsignal-regulated kinase 1/2 (ERK1/2) as the upstream target enzyme of Bcl-2 and Bax that decrease cell apoptosis.Methods:Part one:C57/BL wild mice, aged 12wk old, weresubjected tosham-operation (SH) or transversing aortic constriction to establish left ventricular hypertrophy. Echocardiographic assessments, hemodynamic determination, organ weight measurement, morphological and histological examination were performed at 1, 4, 8, 12 and 16 wks aftersurgery. At the meanwhile mRNA levels of atrial natriuretic peptide (ANP), Brain natriuretic peptide (BNP),α-myosin heavy chain (α-MHC),β-myosin heavy chain (β-MHC), Transforming growth factor (TGF-β1),sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) mRNA were determined by reverse transcription polymerase chain reaction (RT-PCR) technique. Part two: TAC was induced in aged 12 wks old C57/BL mice to establish left ventricular hypertrophy for 4 wks. Hypertrophic myocardium I/R injure was induced by 30min globe ischemia, followed by 15min or 120min reperfusion in Langendorff model. Hearts were randomly divided into 4 groups. 1) I/R group:30min global ischemia and 120min reperfusion; 2) IPost group:IPost with 3 episodes of 10s of ischemia and 10s reperfusion after 30min global ischemia and 15min or 2h reperfusion; 3) I/R+ inhibitor (ERK1/2 inhibitor PD98059 or Akt inhibitor wortmannin) group:after 30min global ischemia and then give 15min reperfusion by KH fuild with inhibitor PD98059 or wortmannin, then as I/R group; 4) IPost+inhibitor group:IPost with 3 episodes of 10s of ischemia and 10s reperfusion after 30min global ischemia and then with I/R+ inhibitor group. at the end of reperfusion the effects of IPost on infarctsize, hemodynamics bymini-balloon were measured;Protein levels of ERK1/2?P70S6K?Akt?GSK-3βwere determined by Western blot at the 15min or end of reperfuaion. Part three: TAC was induced in aged 12 wks old C57/BL mice to establish left ventricular hypertrophy for 4 wks. Hypertrophic myocardium I/R injure was induced by 30min globe ischemia, followed by 120min reperfusion in Langendorff model. Hearts were randomly divided into 4 groups:I/R group;IPos group (three cycles of 10s reperfusion interspersed by 10s of no–flow ischemia) ;I/R+PD98059 group; IPost+PD98059 group. At the end Hemodynamic determination, infarctionsize (IS) measurement were performed. Protein levels of ERK1/2, Bcl-2, Bax, mitochondrial and cytosolic Cyt.C were determined by Weatern blot. Apoptosis was measured by terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL)staining.Results:Part one: (1) Compared withsH group, LVESd? LVEDd?Awsth?Awdth?Pwsth ? Pwdth ? progressive increased after TAC. Meanwhile, left ventricular fractionalshortening (LVFS%)significantly decreased at 16wk (P<0.05). LVSP ?dp/dtmax?dp/dtmin in TAC group were progressive increased after 4 wk. From 8~12 wk these parameters maintainedstabilization and thensharply decreased at 16 wk (all P<0.05). However LVEDP was increased at 8 wk and there wasstatistically difference (P<0.05). These echocardiographic and hemodynamic changes indicated a development of LVH and eventually progressing towards to heart failure. (2) Histologically, cardiac collagen measured by percentage ofsirius Red positivestained area and apoptosis indexshowed a progressive increase from 4 to 16 wk. (3) Compared withsH group, mRNA levels of ANP?BNP?β-MHC?TGF-β1was time-dependently increased whileα-MHC andsERCA2a were time-dependently decreased. (all P<0.05). Part two and three: 1)The Langendorff model of mouse hypertrophic heart wassuccessfully established,;2)Infarctsize wassignificantly reduced in postconditioning group (23.6±2.8)% compared with I/R group hearts [(40.2±4.6)%, P<0.05], IPost group had higher Lvsp, dp/dtmax(all P<0.05); Myocardial function was equally improved compared with I/R group (all P<0.05); and apoptosis index decreased(AI%) [(16±2) /100 vs (37±5) /100, P<0.05] .3)ERK1/2?P70S6Kphosphorylation of myocardial wassignificantly increased in IPost group at 15min and 2 h reperfusion (all P<0.05).Compared with I/R, PI3K-Akt?GSK-3βphosphorylation of myocardial was nosignificantly changes in IPost group at 15min and 2h reperfusion (all P>0.05). 4)Compared with I/R group, increased protein levels of phosphorylated ERK1/2, Bcl-2, mitochondrial Cyt.C;decreased protein levels of Bax, cytosolic Cyt.C. I/R+PD98059 group had no effects on above-mentioned parameters. However, in IPost+PD98059 group, addition of ERK1/2 inhibitor PD98059, at the first 15min of reperfusion, reversed all changes observed in IPost group and eliminated IPost protection by increasing IS to a levelsimilar as in I/R group. The histopathological changes in mitochondria and myocardium destroyed by ischemia repefusion were alleviated markedly by ischemic postconditioning.Conclusions:The conclusion of the research can be drawn from the following four aspects, 1) Pressure-overload induced by TAC resulted in a development of LVH from early concentric hypertrophy to late eccentric hypertrophy. And eventually toward cardiac dysfunction or heart failure. 2) IPost plays a pivotal role in reducing ischemia/reperfusion injury by decrease AI and mitochondria injury in hypertrophic myocardium in vitro.ERK1/2signaling pathway involved in the protection of IPost, but PI3K-Aktsignaling pathway did not participate it. 3) ERK1/2signaling pathway involved in the protection of IPost by regulating the myocyte apoptosis proteins of Bcl-2?Bax and Cyt.C.IPost attenuated ischemia/reperfusion induced apoptosis via increasing the ratio of Bcl-2/Bax in mitochondrial, inhibiting mitochondrial Cyt.C release.