| Background There are 290 million patients suffered with cardiovascular disease(CVD)in China,and 2 out of every 5 patients die from CVD.Among them,ischemic heart disease(IHD)can be regarded as the "No.1 killer",so the seeking of effective prevention and treatment measures for IHD is our country’s major medical needs.Myocardial ischemia can be caused by coronary artery contraction,vasospasm and thrombosis which lead to Ischemic injury.Therefore,recover heart reperfusion as soon as possible is the basic measure for the prevention and treatment of ischemic injury.However,with in-depth clinical observation and experimental studies,it has been found that reperfusion injury after tissue ischemia would further aggravate functional and metabolic disorders and structural damage caused by ischemia,which is known as ischemia/reperfusion injury(I/R injury).For cardiac tissue,since Jennings et al.,first proposed the concept of I/R injury in 1960,the prevention and treatment of Myocardial ischemia/reperfusion injury(MI/R)has always been an important problem during the clinical treatment of IHD and requiring prompt solution.In-depth and detailed analysis of the molecular basis and internal underlying signal mechanism of MI/R damage and in turn to develop new strategies for the prevention and treatment of MI/R are the main task of experimental research.Currently,the use of small molecule drugs for exogenous treatment and the activation of endogenous protection mechanism to prevent MI/R injury are two main research directions.In order to further explore the molecular basis and internal underlying signal mechanism of MI/R injury,this study adopted the mouse model of myocardial ischemia reperfusion respectively from in vivo and in vitro experiment to explore the new drug prevention and treatment methods of myocardial ischemia reperfusion injury and analyze the underlying signal mechanism.This thesis is divided into two parts.Part 1: Duet effects of Metformin: promoting autophagy through both AMPKα1 and α2 pathway in myocardial ischemia/reperfusion injuryAutophagy is a crucial self-clean process essential for sustaining intracellular protein and organelle quality.In the heart,autophagy remains basal level under normal condition which regulates energy salvage,nutrient homeostasis and old,malfunctional organelles degradation in myocardium.In contrast to these salutary function at basal level,delirious autophagy is a programmed cell death process which induce cellular damage attributed to many disorder and diseases such as ischemic heart disease,cardiac hypertrophy and heart failure.Autophagic flux,initiating form formation of autophagosome,autophagosome-lysosome fusion to final degradation,starts in ischemia period during MI/R.Autophagy induction in myocardial ischemia is generally regarded as protective mechanism referring to its compensation for the energy loss through regeneration of fatty acids and amino acids which can recycled through TCA cycle to generate ATP.However,uncontrolled and excessive induction of autophagy in MI/R injury is detrimental.It used to be deemed that autophagy is further enhanced during cardiac reperfusion combined with accumulation of autophagosomes.Therefore,to explore the key mechanism of the regulation of myocardial autophagy function and the effect of myocardial autophagy on myocardial ischemia reperfusion injury has become a research focus in recent years.Our previous studies and related reports have shown that both myocardial ischemia and reperfusion period can seriously affect the level of autophagy,but it is still controversial.It has been suggested that during reperfusion period,the myocardium may lead to excessive activation of autophagy which causing the destruction of myocardial cell structure and inducing apoptosis or necrosis.However,with the concept of "autophagy flux" had been proposed,the increased levels of autophagy related proteins in the myocardium may also regarded as the substrate accumulation caused by autophagy dysfunction.So it is necessary to differentiate the levels of myocardial autophagy between ischemia and reperfusion.AMP-activated protein kinase(AMPK),as an important myocardial "survival signal",is one of the most important myocardial survival kinases.In the myocardium,AMPK exists in the form of heterotrimeric,which includes three functional subunits: α,β,γ,and the Alpha subunit is a catalytic subunit that contains phosphorylation sites with kinase activity.Alpha subunits include alpha 1 and alpha 2 subunits.Alpha 1 is mainly located in the cytoplasm,and alpha 2 is highly expressed in the nucleus of liver,myocardium,skeletal muscle and other tissues.Although the myocardial protective effect of AMPK activation and its regulation of myocardial autophagy have been widely reported,while the previous reports have not clearly distinguished whether there is a difference between AMPKα1 and AMPKα 2 in the regulation of myocardial autophagy during myocardial ischemia reperfusion.In recent years,more and more studies have shown that metformin,a classic first-line therapeutic medicine for the treatment towards type 2 diabetes,has a variety of biological effects.Experimental studies have found that metformin can alleviate myocardial ischemia reperfusion injury and effectively activate myocardial AMPK.Further more,metformin can promote autophagy,reduce oxidative stress and improve the ability of cells to resist stress by activating AMPK to inhibit m TOR signal.Therefore,metformin is closely related to myocardial AMPK,myocardial autophagy and myocardial protection,but the relevant mechanisms are still under discussion.Objective The aim of this study is to identify the state of autophagic flux in ischemia phase and reperfusion phase;to find out the role of cytoplasmic AMPKα1-m TOR and nuclear AMPKα2-CARM1 pathway within autophagic flux during MI/R injury and whether metformin has duet effects on these both pathways.Methods Adult male C57BL/6 mice aged 6-8 months were used to construct the myocardial ischemia reperfusion injury model in vivo.Myocardial tissues of ischemic area were collected after 30 minutes of ischemia and 4 hours after reperfusion,protein expression levels were detected by western blotting method,and protein interactions were determined by immunoprecipitation method.According to the experimental protocol,after four weeks of intraperitoneal injection of Metformin(125 g/kg,i.p.),myocardial ischemia reperfusion injury model was established to observe the level of myocardial autophagy and extent of myocardial injury.In part of the study,Adult male AMPKα2 KO mice aged 6-8 months mice with C57 background were used investigate the biological effects of AMPKα1 and α2,wild-type C57 mice of the same age were used as controls.Results 1.Myocardial ischemia reperfusion leads to impaired autophagy flux.To investigate the discrepancy of autophagic flux in respond to ischemia and reperfusion phase separately in myocardium,we established myocardial ischemia-reperfusion model(ischemia for 30 min followed with reperfusion for 4 h).Compared with sham group,autophagosome abundance Atg5 and LC3-II were seen to increase in ischemia group and markedly weaken in reperfusion group.Lysosome membrane protein LAMP2 reduced obviously in reperfusion group,indicated dysfunctional autophagic flux with the accumulation of p62.These results shed light on the different state of autophagic flux during ischemia and reperfusion phase with apparent impairment of autophagy during reperfusion.2.The cytoplasmic and nuclear components of myocardial tissue were isolated in order to find out the underlying mechanism of impaired autophagy in reperfusion phase,we detected AMPKα1 signaling in cytoplasm.The expression of AMPKα1 after 30 min ischemia do not change noticeably.While,the phosphorylation level of AMPK in myocardium was significantly increased,and the phosphorylation level of downstream m TOR was subsequently decreased,suggesting that ischemia could stimulate AMPK in myocardium.But after 4 h of reperfusion,AMPKα1 expression decreased companied with abased level of phosphor-AMPK,which led to augmented m TOR phosphorylation.The above results suggest that myocardial autophagy dysfunction during reperfusion period may be caused by decreased AMPKα1 in myocardial cytoplasm,which weakens AMPK activity and inhibits myocardial autophagy through m TOR signaling.3.Our previous study indicated that,once TFEB shuttles to nucleus,TFEB and CARM1 format co-activated complex recruiting autophagic genes then.The nuclear TFEB-CARM1 complex was markedly impaired during reperfusion phase which is consistent with blunt autophagy,paralleled with the repression of nuclear TFEB.Besides,CARM1 expression also decreased during reperfusion.S-phase kinase-associated protein 2(SKP2),acting as E3 ubiquitin ligase,was reported to regulate CARM1 degradation in nucleus.Recent study also revealed that SKP2 up-stream transcriptional repressor Fox O3 a was reported to be regulated by nucleus AMPKα2.So in order to explore the modulation of CARM1 during MI/R injury,we tested CARM1 upstream signaling AMPKα2-Fox O3a-SKP2.According to our results,AMPKα2 and p-Fox O3 a decreased during reperfusion,which led to SKP2 out of inhibition and then degradation of CARM1 increased.These data collaborated with TFEB nucleus translocation inhibited by AMPKα1-m TOR signaling in cytoplasm incurred TFEB-CARM1 interaction recession and autophagic dysfunction.4.Metformin has been reported to have cardioprotective effect through stimulation of AMPK catalytic activity,while the influence of metformin on AMPKα1 and α2 isoforms is blurred.To determine the effect of metformin of AMPKα1 and α2,vehicle and metformin group of mice with or without metformin treatment for 4 weeks(125 μg/kg,i.p.)were subjected to MI/R surgery in vivo.Of note,after metformin treatment,in cytoplasm,AMPKα1 elevated concomitant with AMPK phosphorylation sustained during both ischemia and reperfusion period.AMPK activation regulated m TOR inhibition,which led to permission of TFEB nucleus translocation.We also verified that,compared with vehicle group,AMPKα2 expression increased after metformin treatment during reperfusion.AMPKα2 dependent Fox O3 a phosphorylation also increased in nucleus,which induced reduction of SKP2 and upregulation of CARM1.Accordingly,metformin treatment improved the nucleus TFEB-CARM1 complex.5.We next evaluated the state of autophagic flux,the data implied that Atg5 LC3 II and LAMP2 increased,p62 accumulation alleviated in reperfusion after metformin treatment compared with vehicle group.Mice hearts were transfected with AAV9-m RFP-GFP-LC3 4 weeks before MI/R surgery.m RFP-GFP-LC3 was dyed and imaged by immunofluorescence.Autophagosomes marked by m RFP-GFP-LC3 showed both m RFP and GFP signals,whereas GFP signals weakened and only m RFP signals could been observed after the fusion of autophagosomes with lysosomes.It showed that autophagosome fusion with lysosome and lysosome degradation was inhibited during reperfusion,however,metformin treatment could restore autophagic flux.Taken together,these results indicated that impairment of cytoplasmic AMPKα1-m TOR and nuclear AMPK-SKP2-CARM1 signaling during reperfusion phase was activated by metformin treatment,which rejuvenated autophagic flux during MI/R injury..6.To examine the importance of AMPK-mediated signalings in metformin cardioprotective effect,AMPKα2 KO mice were subjected to MI/R surgery.Compared with WT mice,AMPKα1-m TOR pathway was partly influenced by AMPKα2 deficiency,indicated by decreased AMPK phosphorylation,slight elevation of m TOR phosphorylation,and,in consequence,further decrease of TFEB in nucleus during reperfusion.In nucleus,AMPKα2 deficiency also induced p-Fox O3 a being further inhibited during reperfusion phase,therefore led to SKP2 upregulated and CARM1 decreased.Then,we gave KO mice metformin treatment and tested its autophagic flux.It showed that autophagic flux in reperfusion was partly blocked in KO+Met group,evidenced by decreased Atg5,LC3 II,LAMP2 and increased p62,compared with WT+Met group.RFP-GFP-LC3 dyed and imaged by immunofluorescence also showed that metformin enhanced autophagic flux was suppressed in KO+Met group.These results indicated that deficiency of AMPKα2 resulted in inhibition of nuclear Fox O3a-SKP2-CARM1 and also influenced cytoplasmic AMPKα1-m TOR-TFEB signaling.These in turn made metformin enhanced autophagy dysfunctional.The data above shed light on AMPK dependent cardioprotective effect of metformin treatment.7.Our above results have shown that autophagic flux was partly impaired and autophagosome clearance was dramatically inhibited in reperfusion phase.Corresponding to the above results,metformin treatment could enhance autophagic flux and clearance of autophagosome.We also observed that metformin treatment markedly minimized MI/R injury induced myocardial infarct size compared with vehicle group mice.Reduction of LDH,also evidenced that metformin alleviated MI/R injury.Furthermore,MI/R induce cardiac contractile dysfunction and left ventricular dilation were restored in metformin treatment group.Chronic MI/R induced mortality also lower in metformin treatment group than vehicle control.Conclusions 1.Our data shows that the myocardial cytoplasmic AMPKα1-m TOR signal regulates TFEB nucleus translocation and the nuclear AMPKα2-foxo3a-skp2 signal regulates CARM1 level and interacts with TFEB to regulate autophagy.2.Both myocardial AMPKα1 and AMPKα2 were significantly decrease during reperfusion period,leading to autophagy dysfunction and blocked autophagy flux3.We highlight the duet effects of metformin through AMPKα1 and AMPKα2 pathway,improves the nucleus TFEB-CARM1 interaction and autophagy flux in reperfusion period,which reduces cardiomyocytes death and finally proves cardiac function4.The difference between activation of AMPKα1 and AMPKα2 pathway in the regulation of myocardial autophagy is a new mechanism for Metformin inhibiting myocardial ischemia reperfusion injury.Due to the long research cycle of animal experiments,our research group is constructing induced cardiomyocardial specific AMPKα1 and AMPKα2 knockout mice,which will further refine and clarify the difference and synergistic mechanism of AMPKα1 and AMPKα2 pathway in Metformin’s autophagy regulation.Part 2: Melatonin enhanced the cardioprotection of HTK solution against myocardial ischemia–reperfusion injuryCardiac surgery requires cardiac arrest for surgical needs.Heart is in an ischemic state when perforing cardiac arrest and ischemia reperfusion injury is inevitable as the aorta is blocked and opened.How to inhibit cardiac ischemia/reperfusion injury has been an eternal topic in cardiac surgery.It is of great significance for cardiac surgery to improve cardiac preservation during cardiac arrest,prolong cardiac preservation time and promote cardiac function recovery after reperfusion.Histidine-tryptophan-ketoglutarate,UW(University of Wisconsin)and Celsior are commonly used in clinical cardiac preservation.HTK is a kind of organ protection liquid.It contains Histidine,Tryptophane and Ketoglutarate,so using the initial letter of the three components is called HTK liquid.It is also called "CUSTODIOL liquid" because its registered trademark is "CUSTODIOL".It can be used not only for myocardial protection in cardiac surgery,but also for donor protection of transplanted heart,kidney,liver and pancreas.HTK solution is an electrolyte mixture containing sodium,potassium and calcium similar to the intracellular level.In myocardial protection,it belongs to the intracellular liquid type of cardiac paralysis solution,and is one of the most commonly used cardiac preservation solutions,which is used to reduce the I/R injury during surgery.However,its inhibitory effect on I/R injury still has much room for improvement.Melatonin,an amine hormone produced by human pineal gland,has been proved to be a powerful antioxidant.Melatonin participates in ROS clearance by ○1 scavenging free radicals,○2preventing oxidation and reduce the content of peroxides ○3 inhibiting lipid peroxidation to protect cell structure and prevent DNA damage.In recent years,melatonin has been proved to be a very important myocardial protective factor,but whether it can improve the myocardial protective effect of HTK solution and what is the potential mechanism is still unknown.Objective This study was designed to investigate: Whether Melatonin would enhance the myocardial protective effect of HTK solution to inhibit ischemia reperfusion injury;if so,to clarify possible underlying mechanisms.Methods Cardioprotection by histidine-tryptophan-ketoglutarate(HTK)solution with and without melatonin was compared using an in vitro perfused mice heart model of I/R.Four groups of in vitro C57BL/6 mice(aged 3-4 months)hearts endured different treatments with I/R time course: Control;I/R;Ischemia,HTK solution cardiac arrest and reperfusion(I/R+HTK);Ischemia,HTK solution + melatonin cardiac arrest and reperfusion(I/R+HTK+mel).The cardiac function of each group was measured quantitatively and the expression levels of lactate dehydrogenase and c Tnl in myocardium of each groups were quantitatively detected.After reperfusion,myocardial tissue was extracted to detect the expression of relevant myocardial injury markers and signal proteins.The levels of endoplasmic reticulum stress and mitochondrial damage were measured.Results 1.Ischemia reperfusion leads to severe cardiac impairment.Cardiac injury can be partially alleviated by reperfusion after HTK solution cardiac arrest.Different concentrations of melatonin were used to detect the optimal concentration,it was found that,compared with the I/R+HTK group,adding melatonin could enhance the protection of cardiac systolic and diastolic functions of HTK myocardial protective solution,significantly reduced the release of c Tnl and LDH in the perfusion fluid,and the 50 μmol concentration of melatonin was found to improve the heart rate systolic blood pressure product of isolated perfusion heart,so we used this concentration in all subsequent experiments.2.After the reperfusion period,the hearts of mice in each group were taken for TTC staining,and it was found that myocardial infarction was apparently found in the I/R group.While,compared with the I/R group,reperfusion after HTK solution cardiac arrest could reduce the myocardial infarction area,and the addition of melatonin could further reduce the myocardial infarction area after the ischemia reperfusion.3.The energy metabolism and mitochondrial status of myocardial cells in each group were detected.The detection of ATP synthesis activity and citrate synthesis activity showed that the addition of melatonin further improved the energy metabolism of myocardial cells in isolated perfusion heart compared with the ischemia reperfusion +HTK solution cardiac arrest group.The mitochondrial morphology and number of myocardial cells were observed under electron microscope.It was found that compared with the ischemia/reperfusion +HTK solution group,the mitochondrial morphology of myocardial cells added with melatonin was more complete,and the mitochondrial damage was further reduced.4.We detected endoplasmic reticulum stress related protein expression of each groups after I/R and found that,compared with the control group,the expressions of p-PERK,p-e IF2α and CHOP in myocardium of I/R group were significantly up-regulated,HTK solution failed to reduce the phosphorylation of e IF2α and PERK and CHOP,ATF4 experssion,while adding melatonin to HTK solution significantly reduced the phosphorylation of e IF2α and PERK and CHOP,ATF4 expression compared with the ischemia reperfusion group only with HTK solution cardiac arrest.HTK solution failed to reduce the endoplasmic reticulum stress but adding melatonin to HTK could reduce the endoplasmic reticulum stress.5.Were detected the levels of lipid peroxidation and carbonyl stress in myocardium of each group.It was found that the I/R+HTK group reduced the contents of 4-HNE and MDA in myocardium after I/R,reduced the degree of carbonylation of myocardial proteins,and improve the activity of ALDH2 in myocardium.Adding melatonin to HTK solution further enhanced the above protective effects and effectively inhibited myocardial carbonyl stress.6.AMPK phosphorylation activation level were Detected in each group,it was found that I/R+HTK group could effectively increase AMPK phosphorylation in myocardium during ischemia and reperfusion,and adding melatonin to HTK solution further up-regulated AMPK phosphorylation in myocardium during myocardial ischemia and reperfusion.Parallel experiments with AMPKα2 KO mice revealed that both the I/R+HTK group and I/R+HTK+meletonin group were unable to reduce the myocardial infarction area,and the myocardial protective effects were lost.Conclusions 1.it was clearly demonstrated that adding melatonin would enhance the protective effect of HTK solution on isolated cardiac arrest and reperfusion,reduce mitochondrial damage and improve energy metabolism,and inhibit myocardial apoptosis and infarction.2.Our findings have also suggested new clues: HTK solution failed to inhibit ER stress while adding melatonin to HTK could effectively reduce ER stress,melatonin alleviates carbonyl stress in myocardium during ischemia reperfusion;the protective effects of melatonin are closely related to AMPK in myocardium.These results provide a new experimental basis for the development of perioperative cardiac protection strategies in vitro.3.At present,the research group is conducting myocardial proteomics analysis in each treatment group to further explore the intrinsic molecular network mechanism of melatonin’s protective effect on myocardium after in vitro cardiac reperfusion in subsequent experiments. |
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