The Effect Of PEDF/PEDF-R Induced Acute Ischemic Myocardial Cells Transforming Into "Hibernation" And The Underlying Mechanism

BackgroudAcute myocardial infarction(AMI)occurred in coronary artery occlusion,blood flow interruption,so that the affected area due to severe persistent ischemia.After AMI occur,the majority of myocardial cell death will be inevitable the fate of death in myocardial infarction if myocardial ischemia persists,only part of the edge region can be sustained in chronic ischemia,and gradually transform into"hibernation",formed hibernating myocardium and maintain survival.However,recent studies have shown that the number of this part of the hibernating myocardium is closely related to the prognosis after revascularization treatment after AMI.Pigment epithelium-derived factor(PEDF),a non-inhibitory member of the serine protease inhibitor superfamily(SERPINS),is a glycoprotein with a molecular weight of 50 kD identified in various human tissues,such as eye,brain,muscle,adipose tissue,liver and bone.PEDF is characterized as a multifunctional protein possessing antiangiogenic,antitumorigenic,antioxidant,anti-inflammatory,antithrombotic,neurotrophic and neuroprotective properties.In the previous studies,we found that the expression PEDF was significantly decreased in the ischemia zone;Over-expression of PEDF reduced vascular permeability and infarct size,protected myocardial cells in an ischemic heart and improved functional recovery in an AMI rat model.In cultured myocardial cells;PEDF attenuates hypoxia-induced apoptosis and necrosis in H9c2 cells by inhibiting p53 mitochondrial translocation via PEDF-R.During these studies,we found that a survival myocardial cell protected by PEDF in hypoxic culture conditions is different with normal myocardial cells.Therefore,we hypothesis that PEDF is able to regulate metabolism and function of myocardial cells in acute ischemia,inducing ischemic myocardial cells transform into hibernation,so as to effectively save more myocardial cells,which could be the cell foundation of revascularization treatment.PEDF is a secreted protein with various biological effects and deposits in the cell membrane.Up to now,a plurality of membrane receptors has been identified in different cell types.Current evidence demonstrates that the 80-kDa adipose triglyceride lipase(ATGL)is a receptor for PEDF in retinal epithelial cells and myocardial cells.The extracellular PEDF shows retina survival activity by interacting with this 80-kDa receptor protein on cell-surfaces.Our previous studies have demonstrated that PEDF-R mediates PEDF attenuates hypoxia-induced apoptosis and necrosis in cultured myocardial cells.This receptor has intracellular and extracellular regions,and a phospholipase domain.Interestingly,PEDF-R(ATGL)has been described,in adipose cells,as a member of the new calcium-independent PLA2/nutrin/patatin-like phospholipase domain-containing 2(PNPLA2)family that possesses phospholipase A2 and triglyceride lipase activities.The two enzyme activities are known to release bioactive fatty acids,lysophospholipids and diacylglycerol that operate as second messengers that mediate signal transduction.We speculate that PEDF binds to PEDF-R so as to regulate metabolism and function of ischemic myocardial cells,promotes their transform into"hibernation" and exerts cardioprotective effects.The effects of FFAs,LPA as an intracellular signaling molecule exert anti-oxidation,regulation of blood sugar and other aspects of the cell protection has been recognized;DAG play a broader role by activating the protein kinase C(PKC)in the cells.In summary,we hypothesize when AMI,PEDF will bind and activate the two enzymes activity of PEDF-R,releasing FFAs,DAQ PLA involved in cell metabolism and function of myocardial cells to promote them transform into similar"hibernation",so as to temporarily reduce the consumption of myocardial cells and maintain them survival.These hibernating myocardium will be the cell foundation of revascularization treatment.Part I:A modified closed-chest rat model of myocardial ischemia/reperfusion permits pre-transfection of target gene.Objectives:To design a modified closed-chest rat model of myocardial ischemia/reperfusion.Here we design a modified closed-chest rat model of MI/R that can pre-transfect target genes and evaluate its feasibility and effectiveness.Methods:Thoracotomy was performed through the 4th intercostal space in rats and ligatures and loose sutures were preset on the left descending coronary artery(LAD).The the transfected group were injected GPF-lentiviruses on the site of ligatures,and the sham group were injected enhanced-infection solution(ENIS).GPF,1L-1,TNF-? in the left ventricle,C-reactive protein(CRP),angiotensin ?(Ang ?)and antidiuretic hormone(ADH)in the serum and echocardiographic parameters were examined on 1,3,5,7 days after surgery.Then,MI/R was performed,and the ratio of infarction area to area at risk was calculated using TTC/Evan's blue staining method.Result:GFP expression gradually increased and stabilized five days later after transfection.IL-1,TNF-? increased in the transfected group were stronger than that in the sham group 1-3 days after surgery(P<0.05),and both of them normalized on the 7th day in the two groups.CRP,Ang ? and ADH increased 1-3 days after surgery and normalized on the 5th day;HR increased on the 1st day and normalized 5 days later,CO,EF%and FS%decreased on the 1st day and normalized 5(CO)or 7(EF%,FS%)days later,no statistical difference between the two groups.The ratio of infarction area to area at risk did not show an obvious difference between the transfected group and the sham group.Part ?:PEDF induced myocardial cells transforming into,'hibernation”in acute myocardial infarctionObjectives:In the previous studies,we found that PEDF attenuates hypoxia-induced apoptosis and necrosis in myocardial cells by inhibiting p53 mitochondrial translocation via PEDF-R.During these studies,we found that survival myocardial cells protected by PEDF in hypoxic culture conditions is different with normal myocardial cells.Therefore,we hypothesis that PEDF is able to regulate metabolism and function of myocardial cells in acute ischemia,inducing ischemic myocardial cells transform into hibernation,so as to effectively save more myocardial cells.These hibernating myocardium could improve the prognosis of acute myocardial infarction,and will be the cell foundation of revascularization treatment.In this section,we intend to clarify whether PEDF could induce acute ischemic myocardial cells transforming into "hibernation" in acute ischemia.Methods:Intramyocardial injection of lentiviras carnying PEDF and preset ligatures and loose sutures on the left descending coronary artery.Seven days after operation,ligatures sutures were tighten to establish AMI model.Small animal echocardiography was used to detect cardiac function at 1,2,4,6,8 weeks after AMI.Dopamine stress test performed 8 weeks after AMI,echocardiography cardiac reserve function was detected in each group.Result:The mortality was no different between PEDF-transfected group and GFP-transfected group.One and two weeks after acute myocardial infarction model established,EF%,FS%in GFP-transfected group were decrease stronger than PEDF-transfected group(P<0.05),but the proportion of animal deaths was no different between these two groups.EF%,FS%in PEDF-transfected group were greater improvement than with GFP-transfected group 6-8 weeks after acute myocardial infarction model established(P<0.05).Eight weeks after acute myocardial infarction model established,dopamine tress test showed that AEF%,?FS%in PEDF-transfected group was significantly higher than GFP-transfected group(P<0.05).Part ?:The mechanism of PEDF/PEDF-R induced myocardial ischemia cells transforming into "hibernation"Objectives:We have proved that PEDF induces myocardial cells transforming into "hibernation" in acute myocardial infarction.The PEDF can bind PEDF-R on the myocardial cell membrane and activate its enzyme activity,prompting lipolysis.The Lipolysis products as an intracellular signaling molecules play an important role in cellular signal transduction.In this part of the study,we explored the effects of PEDF on mitochondrial density,autophagy,Ca2+ levels and myocardial contractility in vitro culture of H9c2 cells and primary neonatal rat cardiomyocytes under hypoxia.Methods:We used D-Hanks'solution and hypoxia simulated myocardial ischemia.Recombinant PEDF protein was added into the medium,and siRNA assay was employed knockdown PEDF-R.Mito-Tracker(?)Red mitochondrial probes was used to labeled mitochondria,then using fluorescence microscopy and flow cytometry to detect mitochondrial density of each group.Western blot was used to detect the expression of mitochondrial autophagy-related protein Atg5.TEM was employed to observe autophagy bodies density of each group.Gas chromatography-mass spectrometry(GC-MS)analysis was used to detect intracellular free fatty acids(FFAs)level,and ELISA was employed to detect intracellular diacylglycerol(DAG)levels.Fura2-AM calcium ion probe was used to detect Ca2+ levels of the primary cardiomyocytes.Video monitoring system was used to observe myocardial contractility changes over time.Result:PEDF led to decrease the average density of the mitochondria under hypoxic conditions compared to hypoxia group(P<0.05),this effect was abolished after siPEDF-R,(P<0.05).Autophagic bodies increased under hypoxic conditions in H9c2 cells compared to normal cultured cells(P<0.05),autophagic bodies in PEDF group was further increased than hypoxia group(P<0.05),but this effect was significantly decreased after siPEDF-R(P<0.05).The Atg5 in PEDF group was all expressed higher than group hypoxia at different time points(6h,12h,18h,24h)after hypoxia(P<0.05).Compared with normal cultured cells,intracellular palmitic acid(PA)and diacylglycerol(DAG)were reduced under hypoxic conditions in the H9c2 cells(P<0.05);the intracellular PA and DAG in PEDF group were significantly further reduction compared to hypoxia group,and higher than normal cultured cells(P<0.05).But this effect was significantly decreased after the siPEDF-R(P<0.05).Intracellular Ca2+ levels increased after hypoxia compared to normal cultured cells(P<0.05),Ca2+ levels in PEDF group was decreased compared to hypoxia group(P<0.05),but this effect was abolished after siPEDF-R(P<0.05).Under hypoxic conditions,the contractility of myocardial cells in each group were reducing over time.PEDF induce the contractility of myocardial cells decreased faster compared to hypoxia group(P<0.05),the effect of PEDF inhibiting the contractility of myocardial cells was significantly attenuated(P<0.05).Conclusions:1.A modified closed-chest rat model of MI/R permits pre-transfection of the target gene and avoids the confounding factors of gene transfection and second open thoracic surgery.It could be a feasible and efficient method for research on MI/R.2.PEDF binds to PEDF-R and activates its enzyme activity,accordingly increases intracellular FFAs and DAG levels in H9c2 cells under hypoxic conditions,,FFAs induces autophagy advance and enhanced mitophagy,DAG mediates PKC reduce intracellular Ca2+ levels,thus inhibiting the contractility of myocardial cells in hypoxic conditions.