| BackgroundAcute myocardial infarction(AMI)is the leading threat to the human health which may lead to malignant ventricular arrhythmia,cardiac shock,and sudden death.Reperfusion therapy,taking thrombolytic therapy or primary percutaneous coronary intervention for example,is the most effective way for the treatment of AMI.Because it can reduce cardiac infarction area and improve the prognosis for patients by reopening the occluded coronary artery to restoration of blood flow.However,the restoration of blood flow also leads to the injury of the myocardium called myocardial ischemia reperfusion injury(MIRI)which is characterized as cardiac arrythmia,cardiac stunning,microvascular occlusion and most important of all,myocardial cell death.The programmed cell death during the MIRI is composed of apoptosis,necroptosis,pyroptosis and ferroptosis.Apoptosis and necroptosis are the most important ways for cell death during MIRI.It is proved that the infarction area is increased by a fold because of cardiomyocytes death during MIRI.There is no doubt that reducing the cardiomyocytes death is the key target for the treatment of MIRI.However,there is no effective way to reduce the cardiomyocytes death during MIRI.So,it has drawn a lot of attention to have a deeper understanding of the cardiomyocytes death mechanism and find an effective therapy.Adenosine kinase(ADK)is an important enzyme to metabolize adenosine.Adenosine mediates a protective role to reduce MIRI under ischemia,hypoxia and trauma condition.However,the adenosine has not applied to the clinical used in the MIRI treatment because it is degraded quickly in the body.It is of great importance to note that ADK is the most widely distributed enzymes metabolizing adenosine in the mammal cells and it can be found in the heart,liver and brain.Most of adenosine in the cardiomyocytes is transformed to adenosine monophosphate by ADK.So,the concentration of adenosine depends on the activity of ADK.Besides that,ADK can regulate the expression of inflammatory factors and is a potential phosphorylase kinase.A lot of studies have showed that ADK played an important role in the seizures and brain ischemia.The role of ADK in the MIRI and underlying mechanism need to be discussed.ObjectivesThis experiment aims to investigate:1.The potential protective role of ADK inhibitor in the MIRI.2.Whether ADK inhibitor regulates the programmed cell death in the MIRI.3.The underlying mechanisms of ADK in the regulation of MIRI.MethodsFor vivo study,we established mice MIRI model and studied the effect of ADK inhibitor or AAV9-shADK on the infarction size and cardiac function.We also tested the effect of ADK inhibitor on the programmed cell death during MIRI.For vitro study,H9c2 and rat neonatal cardiomyocytes were suffered to H/R to test the role of ADK in the regulation of programmed cell death during MIRI.(?)For vivo study1.Animal protocol:In protocol 1,the ADK inhibitor ABT702 was used.The mice were randomly assigned into 4 groups:sham group,ABT702 group,IR group and ABT702+IR group.For ABT702 group and ABT702+IR group,ABT702(2 mg/Kg)was intraperitoneally given 30 min before the surgery.For the sham and IR group,the same dose of DMSO was intraperitoneally given at the same time.In protocol 2,AAV9-shADK was used.The mice were randomly assigned into 3 groups:sham group,AAV9-GFP+IR group,AAV9-shADK+IR group.For the AAV9-shADK+IR group,the virus carrying AAV9-ADK-shRNA was injected via tail vein 3 weeks before the operation.For the AAV9-GFP+IR group,AAV9-scramble shRNA was introduced.2.Establishment of mice MIRI model:Mice were anaesthetized with isoflurane inhalation.At first,the LAD was ligated with 6-0 silk sutures threaded through a snare.The occlusion was maintained for 30 min,after then,the snare was released to achieve reperfusion for 4 h or 24 h.The sham group underwent the same procedures without ligating.After anesthetized with 0.8%pentobarbital sodium,the mice were harvested.3.Measurement of myocardial function:Cardiac function was assessed by animal heart ultrasonography under anesthetized using isoflurane inhalation.Transthoracic echocardiography was performed in M mode images,left ventricular ejection fraction(LVEF)and fraction shortening(LVFS)were measured.4.Measurement of infarction size and area at risk:After reperfusion for 4 h,mice were killed.The heart sections were stained with Evans blue dye(EBD)and 1%2,3,5-triphenyltetrazolium chloride(TTC)solution to indicate infarction size and area at risk.5.Measurement of serum lactate dehydrogenase(LDH)and S-adenosylhomocysteine(SAH):The blood was collected in the anticoagulant tube and centrifuged at 2500 g,4? for 15 min.Serum was collected to detect LDH,SAH by ELISA kit.6.Transmission electron microscope examination:1 ?m3 myocardium tissue from area at risk were submerged in 2.5%glutaraldehyde overnight.And after that,transmission electron microscope was used for examination.7.Measurement of myocardial apoptosis:In vivo experiment,myocardial specimen were submerged in 4%paraformaldehyde and fixed by paraffin.After serial sections,the sections were stained with Terminal deoxynucleotide nick-end labeling(TUNEL)to indicate the apoptosis cells.8.Measurement of myocardial necroptosis:In vivo experiment,Immunofluorescence staining of EBD-CaV3 was performed to indicate the necroptosis area.9.Protein expression detection:30 ?g protein was extracted from the myocardium tissue and separated by 10%-15%SDS-PAGE.After electro-transferred to PVDF membrane and incubated in the primary antibodies,secondary antibodies,the bands were detected.10.mRNA detection:The mRNA was extracted from myocardium tissue according to the instruction and detected by RT-qPCR.11.Statistical analysis:The data were presented as the Means ±SEM after passing normality or equal variance tests.Student's t test or one-way analysis of variance followed by Tukey's post hoc test was used to compare statistical significance.Statistical significance was considered at P<.05.(?)For vitro study1.Cell protocol:To investigate the role of ADK in regulating the programmed cell death in MIRI and underlying mechanism.H9c2 cells and neonatal rat cardiomyocytes were subjected to hypoxia and reoxygenation where the apoptosis,necroptosis and associated protein were detected.To recognise whether ADK regulates the cell programmed cell death via X-linked inhibitor of apoptosis protein(XIAP),XIAP was knocked down by siRNA.Then,apoptosis and necroptosis of cardiomyocytes were detected.To make it clear that which adenosine receptor had major effect on mediating the role of ADK during MIRI,we tested the mRNA of adenosine receptor by real-time quantitative PCR(RT qPCR).Then the A2B antagonist was used and the apoptosis and necroptosis of the cells were detected.At last,mitochondrial membrane potential,mitochondrial ROS,and the opening of mitochondrial permeability transition pore(mPTP)were detected.2.Hypoxia/Reoxygenation(H/R)Procedure:After starved for 12 h,the cells were transferred into hypoxic workstation for 12 h(94%N2+5%CO2+1%O2).And then culturing cells under normal conditions for 1-4 h before harvesting the cells.3.X-linked inhibitor of apoptosis protein(XIAP)knockdown by siRNA:XIAP was knocked down in H9c2 cells using siRNA.4.Measurement of Mitochondrial ROS:Mitochondrial superoxide was assessed using MitoSoxTM Red.5.Measurement of Mitochondrial Membrane potential(??m):??m was detected using JC-1 method.6.Measurement of Cardiomyocytes Necroptosis:In vitro experiments,cells were stained with Annexin V/PI and detected by flow cytometry.7.Measurement of Cardiomyocytes Apoptosis:In vitro experiments,the cardiomyocytes were suffered to H/R stimulation and stained with TUNEL immunofluorescence kit to indicate apoptosis cells.8.Measurement of ATP:ATP levels were determined by using an ATP Assay Kit(Beyotime)and were normalized to total protein content,which was evaluated by the bicinchoninic acid method.9.Measurement of opening of mPTP:Opening of the mPTP was monitored by loading cells with Calcein-AM and CoCl2.10.Protein Expression and RNA Detection:The process was the same with in vivo experiments.11.Statistical analysis:The process was the same with in vivo experiments.Results1.ADK inhibition reduces myocardial injury and improves cardiac function after I/RADK was transiently increased from 2 to 4 h and returned to normal levels after 6 h of reperfusion.The ADK inhibitor ABT702 or AAV9-shADK significantly reduced myocardial infarct size compared with that of the I/R group.LDH release was also reduced by ADK inhibition.Both LVEF and LVFS were improved by pre-treatment with ADK inhibitor or ADK knockdown.ADK inhibition markedly reduced I/R-induced damage to muscle segments and mitochondria.ADK inhibition reduced the heart rates while did not affect blood pressures in mice.2.ADK inhibition prevents I/R-induced cell apoptosis and necroptosisTUNEL staining revealed that ADK inhibition decreased the apoptotic cells in the area at risk compared with that in the I/R group.ADK inhibition suppressed the increasement in the activation of caspase-9,caspase-8 and caspase-3 but not the increasement in the activation of caspase-12 in I/R-injured hearts.Bcl-2 associated X(Bax)and B-cell lymphoma 2(Bcl-2)were unchanged by I/R injury or by ADK inhibition in heart tissues.ADK inhibition suppressed the phosphorylation of P38 mitogen-activated protein kinases(P38 MAPK)in I/R-injured hearts.ADK inhibition decreased I/R-induced myocardial necrosis,as indicated by CaV3 and EBD staining.Receptor-interacting serine/threonine-protein kinase(RIP1)and the phosphorylation of RIP1 were both increased by I/R injury but were not changed by ADK inhibition.ADK inhibition prevented the I/R-induced increase in RIP3 and the phosphorylation of RIP3.The expression of mixed lineage kinase domain-like pseudokinase(MLKL),the phosphorylation of MLKL and the phosphorylation of calcium-calmodulin-dependent protein kinase ?(CaMKII)were increased after I/R injury,which was prevented by ADK inhibition.Moreover,neither I/R injury nor ADK inhibition affected the content of CaMKII in heart tissues.3.ADK inhibition reduces cell apoptosis and necroptosis in H/R-injured H9c2 cellsADK inhibition only suppressed the activation of caspase-9,caspase-8 and caspase-3 in H/R-treated H9c2 cells.ADK inhibition suppressed the phosphorylation of P38 MAPK but had no effect on the content of Bax,Bcl-2 and P38 MAPK.Necroptotic cells were identified by cell flow cytometry as Annexin V and PI double-positive cells.H/R promoted cell necroptosis,and ADK inhibition decreased H/R-induced necroptotic cells.RIP3 and the phosphorylation of RIP3 were effectively decreased by ADK inhibition.Further study indicated that ADK inhibition prevented H/R-induced increase in MLKL,and the phosphorylation of MLKL and CaMKII.4.XIAP mediates the protective role of ADK inhibition in cell apoptosis and necroptosisNeither cIAP1 nor cIAP2 levels were changed by H/R treatment with/without ADK inhibition.However,less XIAP and phosphor-XIAP were detected by immunoblotting after H/R stimulation compared with control cells;ADK inhibition reversed these changes.H/R injury decreased the phosphorylation of Akt at Ser473,which was restored by ADK inhibition.Using the Akt inhibitor MK-2206 abrogated the effect of ADK inhibition on XIAP and phosphorylation of XIAP.To further evaluate the role of XIAP in ADK-mediated cell death,we then knocked down XIAP using small interfering RNA.XIAP silencing prevented the protective effect of ADK inhibition on H/R-induced cell apoptosis and activation of caspase-9,caspase-8 and caspase-3.XIAP silencing also eliminated the effect of ADK inhibition on H/R-induced cell necroptosis.Moreover,the changes in RIP3,MLKL and the phosphorylation of RIP3 and CaMKII caused by ADK inhibition were also prevented by XIAP knockdown.5.Adenosine receptors contribute to the protective role of ADK inhibition in H/R-induced cell deathUsing an adenosine receptor antagonist,the increase in XIAP and the phosphorylation of XIAP induced by ADK inhibition were attenuated in H/R-treated H9c2 cells.To determine which adenosine receptor is critical for the effects of ADK inhibition on I/R-injured hearts,the mRNA expression of adenosine receptors was analysed in mouse hearts and H9c2 cells.A2B receptor was significantly increased in both I/R-injured mouse hearts and H/R-injured H9c2 cells showed by RT-qPCR.A1 receptor was also changed.Blockade of the A2B receptor using an A2B receptor antagonist effectively prevented the effects of ADK inhibition on XIAP and the phosphorylation of Akt and XIAP.Blockade of the A2B receptor destroyed the protective effects of ADK inhibition on H/R-induced cell apoptosis and necroptosis.Moreover,changes induced by ADK inhibition in caspase-9,caspase-8,caspase-3,RIP3,MLKL and the phosphorylation of RIP3,MLKL and CaMKII were reversed by blockade of the A2B receptor.Using neonatal rat cardiomyocytes,the critical role of the A2B receptor in the regulation of H/R injury by ADK inhibition in cardiomyocytes was further confirmed.6.ADK inhibition improves mitochondrial function and prevents the production of ROSH/R injury induced depolarization of the mitochondrial membrane as indicated by??m,whereas ADK inhibition notably improved it in H9c2 cells.As the decline in??m is due to the opening of the mPTP,Calcein-AM was used to detect mPTP opening.H/R injury promoted the opening of the mPTP,while this change was prevented by ADK inhibition in H9c2 cells.Mitochondrial superoxide production was also reduced by ADK inhibition.Moreover,ADK inhibition increased ATP production in H/R-injured H9c2 cells.ADK inhibition alleviated H/R-induced changes in mitochondria.Conclusion1.ADK inhibition limited myocardial infarct size and improved cardiac function after MIRI.2.ADK inhibition reduced apoptosis and necroptosis during MIRI.3.XIAP was phosphorylated and up-regulated via the A2B adenosine receptor/Akt pathway after ADK inhibition.4.ADK might be a potential therapeutic target for reperfusion-induced injury.BackgroundAs what is mentioned above,reperfusion therapy is the most effective way for the treatment of acute myocardial infarction(AMI).However,the restoration of blood flow also leads to the injury of the myocardium called myocardial ischemia reperfusion inj ury(MIRI).MIRI is an important target in the treatment of AMI as it can improve the prognosis for patients and reduce the risk of heart failure.Programmed cell death plays an important part in the process of MIRI.And necroptosis is one of the most important types of programmed cell death.4-hydroxy-2-nonenal(4-HNE),as one of the most toxic aldehydes,is the major secondary product of lipid peroxidation.It is regarded as one of the most formidable aldehydes,and can attack nuclear acid,protein,phospholipids to regulate their function as soon as it is produced.The concentration of 4-HNE under normal condition is 0.3-5?M and plays physiological roles as a signaling molecule while 4-HNE is increased by 10-100 times under oxidative stress and plays cytotoxic roles by inhibiting gene expression or modifying proteins.During MI/R injury,4-HNE is increased by 6-fold in reperfusion-injured hearts.It is well known that 4-HNE has ability to regulate apoptosis and autophagy during MIRI,the role of 4-HNE on the necroptosis and underlying mechanism need to be discussed.ObjectivesThe main objectives of this experiment are to investigate:1.Changes in necroptosis and 4-HNE during MIRI.2.Whether 4-HNE regulates the necroptosis in the MIRI.3.The underlying mechanisms of 4-HNE in the regulation of necroptosis in MIRI.MethodsFor vivo study,we established mice MIRI model and langendorff-perfused isolated hearts to study the effect of 4-HNE on the necroptosis during MIRI.For vitro study,H9c2 were suffered to 4-HNE stimulation to test the underlying mechanism about how 4-HNE regulates necroptosis during MIRI.(?)For vivo study1.Animal Protocol:In vivo study,the wide type(WT)mice and ALDH2-transgenic(ALDH2-Tg)mice(6-8 weeks)were randomly assigned into 3 groups:sham group,I/R group and ALDH2-Tg+I/R group.In Langendorff model,the wide type C57BL/6 mice were randomly assigned into 2 groups:sham group and 4-HNE group.2.Establishment of Mice MIRI Model:The mice MIRI and sham models were established as mentioned before.The occlusion was maintained for 30 min,after then,the snare was released to achieve reperfusion for 4-24 h and the mice were anesthetized with 0.8%pentobarbital sodium.3.Langendorff model:The Langendorff model was used to examine the direct effects of 4-HNE on the isolated mouse hearts.After being anesthetized and heparinized,the mouse hearts were harvested quickly and installed on the langendorff apparatus.The isolated hearts were perfused with Krebs-Henseleit buffer(KH buffer)and stabilized at 37? with 95%O2 and 5%CO2 for 20 min and then perfused the heart with KH buffer containing 60 uM 4-HNE or vehicles for 1 h.During that period,left ventricular developed pressure(LVDP)and left ventricular pressure rising rate(dp/dt)were recorded to measure the left ventricular function.4.Measurement of Myocardial Function:Cardiac function was assessed by animal heart ultrasonography and left ventricular ejection fraction(LVEF)and fraction shortening(LVFS)were measured.5.Measurement of Infarction Size and Area at Risk:After MIRI,the heart section was stained with Evans blue dye(EBD)and 1%2,3,5-triphenyltetrazolium chloride(TTC)solution to indicate infarction size and area at risk.6.Measurement of Serum lactate dehydrogenase(LDH)and S-adenosylhomocysteine(SAH):The blood was collected in the anticoagulant tube and centrifuged at 2500 g,4? for 15 min.Serum was collected to detect LDH,SAH by ELISA kit.7.Measurement of Myocardial Necroptosis:In vivo experiments,immunofluorescence staining of EBD-CaV3 was performed to indicate where the necroptosis is.8.Immunohistochemical staining:The sections were incubated with anti-4-HNE antibody overnight at 4? followed by washing and staining with secondary antibodies.After that,DAB was used as chromogenic substrate.And the slices were counterstained with hematoxylin.9.Protein Expression Detection:30 ?g protein was extracted from the myocardium tissue and separated by 10%-15%SDS-PAGE.After electro-transferred to PVDF membrane and incubation in the primary antibodies,secondary antibodies,the bands were detected.10.Co-immunoprecipitation:The formation of necrosome and ubiquitination of receptor interacting protein 1(RIP 1)were assessed by immunoprecipitation method.H9c2 cells were lysed in NP-40.Then,protein was incubated with 2 ?g RIP1 antibody or IgG overnight and then was added with 15 ?L protein A/G agarose for 2 h.After washing,the beads were added 20 ?l loading buffer and boiled.Supernatants were subjected to SDS-PAGE and analyzed.11.mRNA detection:The mRNA was extracted from myocardium tissue or H9c2 cells according to the instruction and detected by RT-qPCR.12.Statistical analysis:The data were presented as the means ± SEM after passing normality or equal variance tests.Student's t test or one-way analysis of variance followed by Tukey's post hoc test was used to compare statistical significance.Statistical significance was considered at P<.05.(?)For vitro study1.Cell Protocol:To investigate the role of 4-HNE in regulating the necroptosis in MIRI and underlying mechanism.H9c2 cells were subjected to 4-HNE stimulation with different concentration for different period.To recognise whether 4-HNE regulate the cell programmed cell death via RIP1,the expression of RIP 1 was knocked down by siRNA.Then,the necroptosis and necroptosis pathway associated protein in cardiomyocytes were detected.To make it clear that how 4-HNE regulates the amount of RIP 1,the mRNA,degradation rate and ubiquitination of RIP1 were detected.2.4-HNE stimulation:For 4-HNE stimulation,the cells were cultured in regular culture medium with vehicle or diverse concentration of 4-HNE(20 ?M,40 ?M,60?M,80 ?M)and kept in incubator for various hours(1 h,2 h,4 h,6 h).3.Hypoxia/Reoxygenation(H/R)Procedure:After starved for 12 h,the cells were transferred into hypoxic workstation for 12 h(94%N2+5%CO2+1%O2).And then culturing cells under normal conditions for 1-4 h before harvesting the cells.4.Measurement of Cardiomyocytes Necroptosis:In vitro experiments,cells were stained with Annexin V/PI and detected by flow cytometry.5.RIP1 knockdown by siRNA:RIP1 was knocked down in H9c2 cells using siRNA.6.Protein Expression Detection and Co-immunoprecipitation:The process was the same with in vivo study.7.Statistical analysis:The process was the same with in vivo study.Results1.Reperfusion injury induces cell necroptosis and increases 4-HNE production in mouse heartsAs shown in Figure 1A and B,MI/R injury obviously induced myocardial necrosis.And we found that RIP1,p-RIP1,RIP3,p-RIP3,MLKL,p-MLKL,and p-CaMKII were all upregulated in reperfusion-injured hearts.And H9c2 cells were also exposed to H/R stimulation.In line with the results in vivo,RIP1,RIP3 and the phosphorylation of them were all upregulated in H/R treated cells.We then detected the change of 4-HNE and found more 4-HNE was immunoblotted or immunochemically stained in MI/R injured hearts.Identically,4-HNE was also increased in H/R treated H9c2 cells.2.Detoxifying 4-HNE protects against MI/R injury by reducing necroptosisLess 4-HNE was detected in I/R injured heart from ALDH2-Tg group as compared with sham group.Cardiac function after I/R injury was improved by overexpression of ALDH2 as indicated by LVEF and LVFS.The myocardial infarct size was limited in ALDH2-Tg group compared to the I/R group.LDH was also decreased in ALDH2-Tg group.EBD-CaV3 staining showed that necrotic area was significantly reduced in ALDH2-Tg group.Moreover,MI/R-induced upregulation of RIP1,p-RIP1,p-RIP3,MLKL,p-MLKL,and p-CaMKII were prevented by ALDH2 overexpression.RIP1 was immunoprecipitated to detect the formation of necrosome and found the binding of RIP1 to RIP3 was reduced in ALDH2-Tg group.3.4-HNE induces myocardial necroptosis in Langendorff-perfused heartsTo explore the role of 4-HNE in cell necroptosis,the Langendorff-perfused mouse heart model was used.After stabilized for 20 min,the hearts were perfused with 60 ?M 4-HNE or vehicle for 1 h.4-HNE adducts were significantly increased in 4-HNE perfused hearts as indicated by both western blots and immunohistochemical staining.The cardiac function was impaired by 4-HNE perfusion as indicated by the decreased LVDP and dp/dt.Importantly,the perfusion of 4-HNE increased RIP1,p-RIP1,p-RIP3,MLKL,p-MLKL,and p-CaMKII.Furthermore,more RIP3 was coimmunoprecipitated with RIP1 in 4-HNE perfused hearts.4.4-HNE induces cell necroptosis in H9c2 cellsTo confirm the effect of 4-HNE on necroptosis,H9c2 cells were pretreated with Z-VAD-FMK(the pan-caspase inhibitor and was used to inhibit apoptosis)and then were stimulated with 4-HNE.Necroptotic cells were measured using flow cytometry and were defined as Annexin V+/PI+cells.4-HNE promoted cell necroptosis.Moreover,RIP1,p-RIP1,and p-RIP3 were all upregulated by 4-HNE in a time dependent manner.Meanwhile,RIP1,p-RIP1,p-RIP3,MLKL,p-MLKL,and p-CaMKII were also upregulated by 4-HNE in a dose-dependent manner.5.RIP1 mediates the effect of 4-HNE on cardiomyocytes necroptosisTo determine the critical role of RIP1 in the regulation of cardiomyocytes necroptosis by 4-HNE,the H9c2 cells were transfected with scramble or RIP1 siRNA.The effect of 4-HNE on cell necroptosis was mitigated by knockdown of RIP1.Moreover,the 4-HNE-induced changes of p-RIP1,p-RIP3,MLKL,p-MLKL and p-CaMKII were prevented by RIP1 deficiency.6.4-HNE reduces the ubiquitin-dependent degradation of RIP1The mRNA level of RIP1 wasn't changed by 4-HNE stimulation.To assess the protein degradation of RIP1,CHX was used to inhibit the gene transcription and we found that less protein was degraded in 4-HNE group.The K-48 linked polyubiquitination of RIP 1 was detected and we found that less ubiquitination was co-immunoprecipitated with RIP1 after 4-HNE stimulation.cIAP1 and cIAP2 were coimmunoprecipitated with RIP1 and we found the binding of cIAP1 and cIAP2 to RIP1 wasn't changed by 4-HNE stimulation.To further underlie the mechanism of reduced ubiquitination of RIP1 by 4-HNE,the carbonylation of RIP 1 was examined and more 4-HNE was coimmunoprecipitated with RIP1.Conclusion1.4-HNE increased necroptosis during MIRI.2.4-HNE enhanced necroptosis by increasing necroptosis during MIRI.3.4-HNE inhibited the K-48 degradation of RIP1 and increased necroptosis. |
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