The Function And Mechanism Of IPLA2β During Myocardial Ischemia-reperfusion Injury

Coronary heart disease is the result of atherosclerotic lesions in the coronary arteries that narrow or block the lumen of the vessels,resulting in myocardial ischemia,hypoxia,or necrosis.The key facts published on the World Health Organization’s website in 2017 showed that cardiovascular disease(CVDS)is the leading cause of death globally,killing more people each year than any other cause.According to statistics,cardiovascular diseases caused more than 17 million deaths in 2016,accounting for nearly 30%of all deaths worldwide,85%of which were caused by heart disease and stroke,especially ischemic heart disease.With the popularization and development of PCI technology at present,we reduce the myocardial infarction area effectively,improve clinical outcomes.However,we still failed to achieve the mortality rate of patients with myocardial infarction that should be theoretically reduced by using this technique.Later the researchers found that even restoring of blood flow to the ischemia myocardium,but the area of infarction myocardium did not significantly reduce,instead,there is further damage,namely heart ischemia-reperfusion injury.A large number of studies have confirmed that the accumulation of oxygen free radicals,calcium ion overload,inflammatory cell infiltration,mitochondrial dysfunction,excessive cell swelling and other pathological factors are involved in the pathological mechanism of myocardial ischemia/reperfusion injury.Heart ischemia-reperfusion injury is a complex and multi-factorial pathological process,which still needs further study.Years of research have confirmed that ischemia-reperfusion injury is a result of multiple factors,such as oxidative stress,intracellular Ca2+overload,inflammation,and metabolic derangements.Increasing evidence has shown that the above pathogenesis perturbs normal function of the endoplasmic reticulum,leading to ER stress.The ER,a major subcellular organelle,takes part in the synthesis,maturation and transport of various proteins.To rescue the unbalanced endoplasmic reticulum protein folding ability and rebuild endoplasmic reticulum homeostasis,cells conduct unfolded protein reaction(UPR)for self-preservation cells,which is evolutionarily conserved.Through three discrete signaling branches:PERK,IRE1/XBP1 and ATF6,UPR attenuated translation,increased the production of proteins involved in the functions of UPR.However,UPR promotes apoptosis if these targets are not met or the destruction time is prolonged within a certain time frame.Lysophosphatidylcholine(LPC),also called lysolecithin,is a class of lipid biomolecules derived from the cleavage of phosphatidylcholine(PC)by phospholipase A2(PLA2).LPC accumulates in the heart during ischemia and reperfusion,and some LPC species can be diagnostic markers for myocardial infarction.Phospholipase A2 is generally classified into three types:secretory(sPLA2),cytoplasmic(cPLA2)and Ca2+independent(iPLA2).The enzyme,intracellular membrane-associated calcium-independent phospholipase A2 beta(iPLA2β),catalyzes phospholipids reaction to produce fatty acids and LPC.iPLA2β participated in many physiological phenomena,calcium homeostasis and apoptosis,for instance.This enzyme,originally isolated from myocardial tissue,exhibits several specific characteristics due to its activation during ischemia,including calcium independence,a favor for plasmalogen phospholipids containing arachidonate at the sn-2 position,and inhibition by calmodulin(CaM)and an interaction with ATP in the existence of Ca2+.The cellular localization of iPLA2β is tissue-specific and dynamically variable.iPLA2β translocations to different organelles,including the plasma membrane,endoplasmic reticulum,mitochondria,and nuclear membrane,perform different functions.Previous studies have reported that inhibition of iPLA2β activity can reduce cell apoptosis.However,the mechanism by which iPLA2βworks in different signaling cascades and its function in disease remains unclear.Therefore,we hypothesize that the increased expression of iPLA2β in cardiac ischemia reperfusion injury leads to the imbalance of phospholipid metabolism of cardiac membrane,which activates endoplasmic reticulum stress and leads to apoptosis of cardiac myocytes.Part 1:The level of iPLA2P and the regulation of ER stress in myocardial ischemia reperfusion injury in vivo and in vitroAimTo observe the concentrations of LPC and iPLA2β in myocardial ischemia reperfusion injury.To investigate the activation of ER stress during ischemia/reperfusion injury,and to detect the expression of the important downstream chaperone GRP78 and the spliced XBP1 in ER stress.Method and ResultThe serum collected from acute myocardial infarction(AMI)patients were detected the levels of LPC and iPLA2β through ELISA.Compared with non-AMI patients,the concentration of LPC was nearly 5 times higher(P<0.05),and the concentration of iPLA2β was 4 times higher(P<0.05).To establish the myocardial ischemia reperfusion mouse model,we ligated the left anterior descending branch for 45 min,and after 6,24,72 hours reperfusion the expression of iPLA2β and LPC concentration were detected.The mRNA and protein levels were found to be about twice as high as those in the sham group.Myocardial ischemia reperfusion model was established to simulate ischemia reperfusion injury(sI/R)in vitro.About 20%of cells died after 3 hours of ischemia and 3 hours of reperfusion,which was 4 times of that in the normal control group(P<0.05).Significant changes in cell morphology can be observed under the microscope.The concentration of LPC in cells after sI/R was twice as high as that in the control group,and the concentration of LPC in cell supernatant was also significantly increased(P<0.05).In addition,myocardial ischemia/reperfusion injury can activate the endoplasmic reticulum stress pathway,and the expression of molecular chaperone GRP78 is up-regulated,and the splicing of XBP1 is significantly increased.ConclusionMyocardial ischemia-reperfusion injury increased expression of iPLA2β and activated endoplasmic reticulum stress in vivo and vitro.Part 2:The effects of iPLA2β in myocardial I/R injuryAimIn order to clarify the role of iPLA2β in myocardial ischemia reperfusion,we constructed iPLA2β knockout mice to observe the effect of iPLA2β in cardiac ischemia reperfusion injury.And knock down or use iPLA2β inhibitor in vitro to observe the effect of iPLA2β during myocardial sI/R.Method and ResultWe created a Pla2g6 knockout mouse model(C57BL/6N)by CRISPR/Cas9-mediated genome engineering and used the littermate wild type mice as the control group.All mice were treated with ischemia reperfusion for 45 min ligation and 24 hours reperfusion.Heart tissue was isolated and stained with 2,3,5-triphenyltetrazole chloride(TTC).Compared with the control group,the area of myocardial infarction was reduced in the knockout group(P<0.05)and the TUNEL positive rate was decreased.The death of myocardial cells and TUNEL positive cells were significantly reduced after transfection with iPLA2β siRNA,or iPLA2β inhibitor BEL in primary neonatal rat cardiomyocytes caused by sI/R.ConclusionPla2g6 knockout mice reduced myocardial infarction area after myocardial ischemia reperfusion injury with decreased TUNEL positive rate.Knockdown or inhibition of iPLA2β in vitro reduced the mortality and TUNEL positive rate of myocardial cells during sI/R.Part 3:The effects and mechanisms of iPLA2β on apoptosis induced by ER stress during myocardial ischemia reperfusion injuryAimTo assess the effect of knockdown iPLA2β on apoptosis in sI/R model,and to further detect the organelle distribution of iPLA2β during sI/R,as well as the promoting effect of iPLA2β on ER stress induced apoptosis.Method and ResultiPLA2β was knocked down or inhibited by small interfering RNA or chemical inhibitor BEL in primary neonatal rat ventricular myocyte(NRVM),and apoptosis was detected by flow cytometry after sI/R.In addition,cell membrane protein pull-down assay showed that I/R injury resulted in decreased distribution of iPLA2β on the cell membrane.In addition,iPLA2β co-localization with the endoplasmic reticulum was increased after sI/R by immunofluorescence,and knockdown or inhibition of iPLA2βreduced apoptosis and intracellular calcium concentration through the ATF4-PERKeIF2a-CHOP pathway.ConclusioniPLA2β may participate in myocardial ischemia reperfusion injury through the apoptosis pathway induced by ER stress and the regulation of intracellular calcium concentration.