The Effect And Mechanism Of ALOX12 In Myocardial Ischemia Reperfusion Injury

Background:Acute myocardial infarction(AMI)is a major cause of death and disability worldwide.In patients with AMI,timely and successful reperfusion of ischemic myocardium with the thrombolytic therapy or primary percutaneous coronary intervention(PPCI)as well as coronary artery bypass graft is the most effective treatment option for limiting the infarct size,improving cardiac function and clinical outcomes.However,the structure of ischemic myocardium will be damaged,resulting in cell death and the expansion of infarct area after ischemia-reperfusion,which is called ischemia-reperfusion injury.Occurrence of myocardial I/R injury might offset part of the beneficial effects of restoring blood flow,and has become the main cause of adverse cardiac events after recanalization of myocardial infarction.Though ischemic pre-conditioning is the only clinic practice showing benefits to reduce infarct size after reperfusion,its application is very limited as it is only applicable for selective coronary intervention.Therefore,it is a reasonable strategy and an urgent thing to explore the molecular and cellular mechanism of reperfusion injury and to find the core target.Myocardial reperfusion triggers a series of complex cellular biological processes from abnormal energy metabolism to oxidative stress,including oxidative stress,inflammation,microvascular occlusion and apoptosis.The rapid recovery of energy and oxygen during reperfusion will result in the metabolic activities changing rapidly.Metabolic disorder may be an important source of reperfusion injury.The previous results of metabonomics suggest that metabolic changes at the beginning of tissue reperfusion can cause a wide range of molecular,biochemical and cellular events related to organ damage and pathological remodeling.However,in the process of myocardial ischemia-reperfusion injury,the dynamic changes of key metabolites and how to regulate the reperfusion injury are not clear.Therefore,it is of great significance for us to explore the potential mechanism of myocardial reperfusion injury and it may have important guiding significance for clinical translation.Object:To clarify the changes of metabolic related pathways in the process of myocardial ischemia-reperfusion,identify the key regulatory factor and explore the potential molecular mechanism.Methods:Part one: We collected the serum from patients with myocardial infarction before and after revascularization,and established myocardial ischemia-reperfusion model in mice.The changes of each metabolic component in the stage of ischemia and reperfusion were detected by metabonomics.It was found that 12-HETE,an arachidonic acid metabolite regulated by ALOX12,was most sensitive to reperfusion injury.The expression of ALOX12 before and after reperfusion was determined by Western blot and immunofluorescence staining.And some molecular biological experiments were performed to explore the potential molecular mechanism of ALOX12 expression rising in the reperfusion stage.Part two: The myocardial ischemia-reperfusion injury model was established in both WT and Alox12-KO mice.The cardiac function was evaluated by echocardiography,the injury and necrosis of cardiomyocytes were evaluated by biochemical indexes such as myocardial enzyme and troponin,the inflammatory response of cardiomyocytes was evaluated by CD11 b and ly6 G by immunofluorescence staining,and the apoptosis of cardiomyocytes was evaluated by TUNEL staining and Western blot analysis.In addition,H&E staining was performed to evaluate the morphological changes of mouse myocardium,PSR staining was performed to detect the degree of myocardial fibrosis,TTC staining was performed to evaluate the myocardial necrosis.At the same time,the changes of myocardial inflammation,myocardial necrosis,cardiac function and other cell events were analyzed by RNA-seq analysis.Part three: The myocardial ischemia-reperfusion injury model was established in both WT and Alox12-KO mice to explore the potential molecular mechanism.The downstream target molecules were determined by detecting the changes of related signal pathways in myocardial ischemia-reperfusion by transcriptome.Western blot was performed to verify the regulation of Alox12 on downstream target molecules.In addition,the model of myocardial ischemia-reperfusion injury was established with WT,Alox12 knockout,downstream target knockout and Alox12 / downstream target double knockout mice.The changes of myocardial enzymes and troponin were detected to evaluate the injury of myocardial cells in each group.H&E staining was performed to evaluate the morphological changes of myocardial tissue.At the same time,we compared the changes of inflammation,apoptosis and cardiac function related genes between alox12 knockout group and double knockout group through the analysis of RNA-seq data.Part four: Hypoxia/Reoxygenation(H/R)model was established with primary cardiomyocytes,and the effect of 12-HETE,a catalytic metabolite of ALOX12,on MIRI was detected by RNA-seq data analysis.Western blot analysis was performed to detect the effect of 12-HETE on the phosphorylation of AMPK.The adenovirus of ALOX12-WT and its enzyme activity mutation(ALOX12(M))were constructed,and their effects on AMPK phosphorylation were detected by Western blot.The effects of ALOX12,12-HETE and ALOX12(M)on the downstream signaling pathways were compared by RNA-seq data analysis of cardiomyocytes from Alox12-WT,12-HETE and Alox12(M)and their control groups.Part five: In vitro cell experiments were performed to explore the specific molecular mechanisms by which ALOX12 inhibits the activation of AMPK.Western blot analysis was performed to detect the changes of upstream phosphokinases/kinases of AMPK.The interaction between ALOX12 and the upstream phosphokinases/kinases of AMPK was detected by immune-coprecipitation and GST precipitation.The immunoprecipitation mass spectrometry,ubiquitination and immuno-coprecipitation were used to detect how ALOX12 could regulate the upstream kinase or phosphatase of AMPK.At the same time,whether the mutation of the active site of Alox12 affects the regulation of AMPK signal pathway was also detected.Results:Part one: The results of metabonomics showed that the AA metabolic pathway was the most significant upregulated in the reperfusion stage,and 12-HETE,the specific catalytic product of Alox12,up-regulated significantly in the AA pathway.At the same time,Western blot analysis showed that the expression of Alox12 increased significantly in the reperfusion stage,similar to the results of metabonomics,the highest expression of Alox12 was also found at 6h of reperfusion.Immunofluorescence staining also showed that the expression of Alox12 was significantly higher than that of Sham group.And the results showed that the upregulation of USP10 in MIR may be the potential mechanism of Alox12 expression elevated in cardiomyocytes.Part two: The cardiac function in Alox12-KO mice was significantly preserved,and lowered serum markers of cardiac injury,including myocardial enzymes and troponin comparing to the WT cohort.Furthermore,TTC staining of cardiac tissue revealed a significantly smaller infarct size in the Alox12-KO hearts comparing to the WT,while histopathology and Western blot analysis showed less degrees of inflammatory cell infiltration,cell death and fibrosis.RNA-seq analyses suggest that the genes induced in the Alox12-KO hearts were significantly enriched for heart function and cellular survival,while the genes downregulated in the Alox12-KO hearts were significantly enriched for pro-inflammatory and cell death.Part three: RNA-seq data and immunoblotting analysis showed that AMPK activity was significantly increased by Alox12 deletion.Notably,AMPKα2 deficiency alone exacerbated reperfusion-induced cardiac injury and dysfunction.More importantly,the ameliorating effect of Alox12 knockout on heart dysfunction,inflammatory response,cell death and fibrosis after reperfusion were largely reversed by AMPKα2 ablation,as indicated by serum markers of cardiac injury and histopathology evidences.This effect was firmly validated by a transcriptome-based analysis where the Alox12-dependent gene expression changes were largely reversed by AMPKα2-KO.Part four: RNA-seq analysis showed that 12-HETE enhanced the I/R-induced inflammatory response and cell death related gene expression,and inhibited the function of cardiomyocytes.12-HETE treatment in cardiomyocytes showed a negligible impact on AMPK activity.We generated an ALOX12 enzyme-dead mutant ALOX12(M)by substituting the amino acid 403 from alanine to threonine which had no 12-HETE producing capacity.Interestingly,expression of the ALOX12(M)protein in the Alox12-knockdown cells significantly reversed the activity status of AMPK in the same fashion as the ALOX12-WT protein.Furthermore,RNA-seq analysis revealed an extensive alteration in gene expression in the cardiomyocytes following ALOX12(M)overexpression,affecting genes involved in cardiomyocyte function,cellular injury and inflammatory response in a similar fashion as the ALOX12-WT protein.Part five: Western blot showed that ALOX12 could down regulate the expression of TAK1.Immuno-coprecipitation and GST-pull down results showed that ALOX12 directly interacted with TAK1.Additional experiments showed that Alox12 promoted the degradation of TAK1 by regulating the ubiquitination of TAK1.IP-MS combined with molecular experiments showed that ALOX12 promoted the NEDD4-mediated TAK1 degradation,and this function had nothing to do with the catalytic activity of ALOX12.Conclusion:We identified the AA metabolic pathway was significantly activated in the reperfusion stage and a marked accumulation of ALOX12-dependent 12-HETE was also observed.The elevated ALOX12 can promote cardiomyocyte apoptosis and necrosis,myocarditis and myocardial fibrosis.ALOX12 mainly aggravates myocardial reperfusion injury by inhibiting AMPK activation.Moreover,independent from its capacity on 12-HETE production,ALOX12 suppressed AMPK activity via promoting the degradation of TAK1 by NEDD4,which beyond its enzymatic activity.