| INTRODUCTIONCurrently cardiovascular disease is still a main cause of death worldwideespeciallyin the industrial countries. Althoughthe most effective way by thrombolytic drugs orprimary PCI and CABG decreasing mortality of acute myocardial infarction(AMI), thecomplication resulted from reperfusion, including myocardial stunning, ventriculararrhythmias, and microvascular dysfunction,are still confused and unsolved issues.Especially combined with diabetic mellitus (DM), the hospitalization mortality of AMIincreased to2-4fold compared with non-DM patients. So how to alleviating myocardialischemia/reperfusion injury (IRI) becomes an challenge for all the cardiologists andresearchers.The concept of remote ischemic conditioning (RIPC) was first described in1993by Przyklenk et al in dogs. Transient ischemia of one coronary artery territory was shownto reduce the effects of subsequent potentially lethal ischemia in the territory of anothercoronary artery. But the mechanism of cardioprotection invoked by remote limb ischemicpostcondtioning (RIPostC) is not documented, especially under the conditions combinedwith DM, the efficacy of RIPostC is still controversial.In the last decade, regulation of autophagy is giving arise to much interest ofscientists. Autophagy is an intracellular bulk process of self-digestion involving thelytisosomal degradation of cytoplasmic organelles and macromolecules. It plays anadaptive role in the heart or other organs at baseline to perform housekeeping functions andis dramatically augmented upon nutrient depletion or exposure to other stresses, thusmaintaining cellular homeostasis and energy balance and providing cytoprotectiveresponses to adverse conditions.Recent numerous studies have reported that the activation of macroautophagy (hereinafter referred to as autophagy) plays a key role in alleviating IRI, but thecontribution of autophagy to cardioprotection remains to be determined[1,2].MicroRNAs (miRNAs) are a class of short (19-25nt) noncoding RNAs andcapable of negatively regulating gene expression through complementary binding of the‘‘seed sequence’’ of target mRNAs and affect gene coding of30%of human gene.Numerous compelling evidences have demonstrated that miRNAs are involved in theregulation of cardiovascular diseases such as atherosclerosis, myocardial infarction andheart failure.Although many recent findings have revealed that under stress conditions, anumber of miRNAs are capable of modulating autophagic activity, it is not yet clearwhether miRNA related with RIPostC are involved in the regulation of autophagy in IRI.Based on the current research evidences, we sought to determine:(1) Whether RIPostC will offer potential benefits in ameliorating myocardialI/R injury in mice myocardial I/R injury model with or without DM.(2) If so,Is autophagy pathway involved in the cardioprotection activity ofRIPostC?(3) Whether RIPostC will induce miRNAs changes which may be associatedwith cardioprotection?(4) Whether and how miR27a, a miRNA related with RIPostC, regulateautophagy under HR conditions in H9c2cells.METHODSA Streptozocin (STZ) induced DM mice model and myocardial I/R model wereestablished. RIPostC was induced in normal and DM mice using three cycles of5min ofischemia and5min of reperfusion of the left hindlimb. ND animals and DM animalswere divided into four groups separately: shame group, IR group, IR+RIPostC group,and IR+RIPostC+3-MA group. Eject fraction of left ventricular (LVEF) was determinedby echocardiography, myocardial infarct size was calculated by Evans blue and TTCstaining. We evaluating the degree of autophagy using Immunohistochemistry Analysis,western blot (Beclin-1,LC3and p62)and the number of autophagosomes detected bytransmission electron microscopy (TEM). For the STZ-induced DM mice IRI model，we further divided into four groups:shame group, IR group, IR+Met treatment groups, and IR+Met+RIPostC group. Thenweinvestigated the efficacy ofMet plus RIPostCcompared with Met alonetherapy.Analyzing the infarct size and cardiac function using the above methods, detectingserum TnT by ELISA kit.Cell apoptosis was analyzed by terminal deoxynucleotidyl(TUNEL) staining and western blot.Cell autophagy was also evaluated by the abovemethods including Immunohistochemistry, TEM, and western blot. To further explore themechanism of autophagy regulation of RIPostC combined Met therapy for DM-IRI, wepretreated IR models using autophagy inhibitor wortmannin(Wort) and AMPK inhibitorcompound C(CC) to investigate the effects on the cardiac function and autophagypathway.Then in DM IRI model, we further investigated the efficacy of RIPostC plusMetformin (Met) combination therapy compared with Met alone therapy. We preparedRNAsamples from the non-DM IRI (3h time point) and RIPostC group, then detected bymiRCURY LNA miRNAs assays and further confirmation by real-time PCR.Selective miRNA, miR-27a, then were synthesized and transfected into H9C2cardiocyte. Using CCK-8and lactate dehydrogenase(LDHto evaluate function of cell,Cell apoptosis was analyzed by flow cytometry (FCM) and western blot. Using confocalfluorescent microscopic images, western blot and Transmission ElectronMicroscopy(TEM) to investigate autophagy. To further clarify the mechanism ofautophagy regulation of miR-27a, we a lentiviral vector express mir-27a and SMAD5hnRNA (high expression)were transferred into H9C2cell. Using a dual luciferasereporter gene to determined target gene of miR-27a. Finally we explored the possiblesignaling pathway, Akt-mTOR pathway by western blot.RESULTS1.IRI significantly activated autophagy in both ND and DM groups. RIPostC led toreduced infarct size(32.6±3.0%in ND-RIPostC compared with50.6±2.4%in ND-IR, p<0.05) and improved cardiac ejection fraction (49.70±3.46%in ND-RIPostC comparedwith31.30±3.95%in ND-IR, p <0.05) in normal mice, however RIPostC failed affordcardioprotection in DM mice. Western blot analysis demonstrated that within non-DMmodel RIPostC upreguated autophagy markers, Beclin-1and the ratio of LC3-II/LC3-I, downregulated SQSTM1/p62expression level during myocardial I/R injury. The extentof autophagy activation showed discrepancy at3h time point after reperfusion betweenND and DM animals, RIPostC failed to upregulate the level of Beclin-1and the ratio ofLC3-II/LC3-I in DM model. Further findings indicated baseline phosphorylation level ofAMPK in DM myocardial was significantly lower as compared with ND.2． For the STZ-induced DM mice IRI model, as compared with Control, bothRIPostC plus metformin and metformin treatments significantly decreased infarct size(54.93±6.46%in Control,34.25±3.36%in RIPostC plus Met, and24.69±4.10%inMet, both p <0.05). Interestingly, as compared with Met alone, RIPostC plusMet furtherdecreased infarct size (24.69±6.02%vs.34.25±3.36%, p <0.05). Similarly, bothRIPostC plus Met and Met alone treatment significantly increased ejection fraction of LV(LVEF)(45.61±5.43%,32.26±4.17%in Met, both p <0.05vs. Control). Moreover,LVEF in RIPostC plus Met was higher than Met alone treatment (45.61±5.43%vs.32.26±4.17%, p <0.05). Western blot indicated that RIPostC plus Metenhanced the ratioof LC3-Ⅱ/LC3-Ⅰand Beclin-1level(both, p<0.05), TEM showed RIPostC plusMetincreased the number of AVs compared with Met alone treatment（10.2±1.3vs4.6±0.6,p<0.05）; Furthermore, the autophagy induction of RIPostC plus Met was accompaniedby decreased apoptosis. As compared with Met alone treatment,RIPostC plus Metincreased Bcl-2level and cleaved Caspase3expression (P<0.05), decreased apoptosispercentage(P<0.05). Autophagic inhibitors, wortmannin (15μg/kg) and AMPK inhibitor,CC(20mg/kg), completely abolishedRIPostC plus Met and Met induced cardioprotection,and offset the induction of autophagy.3． In the normal mice IRI model, RIPostC led to obvious change of103miRNAs(vs IR,1.5fold up regulated or down regulated) by miRNAs assays detection. Weselected total miRNA to further detection by real-time PCR. As a result, we foundRIPostC induced upregualtion of miR-27a and miR-20a.4． HR induced the time-dependent changes of LC3and miR-27a, peaking athypoxia12h followed by reoxygenation4h. Pre-mir-27a (50nM) induced H9C2cellautophagy shown by increasing ratio of LC3-II/LC3-I, decreasing p62level,enhancingLC3B dots, and enhancing formation of autophagosomes after HR, whereas the autophagy was attenuated with mir-27a inhibito（r50nM）. The induction of autophagywas accompanied by enhanced cell survival and decreased apoptosis. Furthermore,treatment with lenti-V-mir27a and SMAD5siRNA attenuated the lenti-V-miR27ainduced autophagy. A dual luciferase reporter gene indicated SMAD5is a target gene ofmiR-27a. miR-27a negatively regulated SMAD5expression. In addition, mir-27adownregulated SMAD5downstream Akt-mTOR expression level.CONCLUSIONS1. Cardioprotectioninvoked by RIPostC in normal mice might be mediated in part viaup-regulation of autophagy. The loss of cardioprotective effect induced by RIPostC onmyocardial I/R in diabetes mice might be association with the under-induction ofmyocardium autophagy.2. RIPostC plus metformin therapy induced myocardial autophagy, inhibitedapoptosis, alleviated IRI compared with Met alone treatment.3. MiR-27a and miR-20a is probably miRNAs related with RIPostC inducedcardioprotection.4. MiR-27a protect H9C2cardiocyte from HR injury by inducing autophagy andinhibiting apoptosis via SMAD5-Akt-mTOR pathway. |
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