Alox12 Aggravates Myocardial Ischemia-reperfusion Injury By Inhibiting AMPK Activation

Aims Myocardial ischemia-reperfusion(MIR)injury is one of major causes of poor prognosis for revascularization after myocardial infarction,leading to additional cardiomyocyte death,permanent myocardium damage,and even heart failure.However,there is currently no clinically effective drug for MIR therapy.Arachidonate 12-lipoxygenase(ALOX12)is a key enzyme in arachidonic acid metabolic pathway and has potent capacity in regulating cell death and inflammation,whereas the role of ALOX12 in MIR remains unknown.In this study,we aimed to clarify the function and underlying mechanisms of ALOX12 on MIR.The therapeutic potential of ALOX12 inhibitor,ML355,for MIR therapy was also evaluated based on mouse MIR model.Methods 1.To investigate the regulatory role of ALOX12 in MIR injury,immunoblot technology and confocal technology were used to detect changes in protein expression of ALOX12 in the hypoxia-reoxygenation(H/R)neonatal rat cardiomyocytes(NRCMs).2.The cardiomyocyte-specific ALOX12 transgenic mice were generated and subjected to MIR surgery.We detected cardiac function of mice using small animal ultrasound detectors(echocardiography),and performed Hematoxylin-eosin(H&E),2,3,5-triphenyltetrazolium chloride(TTC),picric acid-Sirius red(PSR)and other pathological staining to evaluate myocardial cell infarct size and myocardial fibrosis.The systematic influence of ALOX12 on cardiac function,inflammation,cell death and other cellular evens were investigated by RNA-seq assay using ischemic heart tissue of ALOX12-CTG and NTG mice.3.The regulatory effect of ALOX12 on cardiomyocyte during MIR injury was examined based on in vitro H/R model of NRCMs those were transfected with corresponding adenoviral vectors to knockdown or overexpress ALOX12.RNA-seq assay was carried out to detect and compare the expression profiles of inflammation and cell death-related factors of cardiomyocytes in ALOX12-knockdown,ALOX12-overexpression and their corresponding control groups.4.We explored the underlying mechanisms of ALOX12-mediated myocardial injury based on RNA-seq assay and multivariate analysis,which were further verified immunoblotting methods.5.To evaluate the therapeutic potential on MIR by targeting on ALOX12,we applied ML355,a specific ALOX12 inhibitor,to treat mice in parallel with vehicle controls.The protective effects of ML355 were determined by cardiac function ultrasound and pathological tests.Results 1.The expression of ALOX12 protein was significantly up-regulated in primary myocardial cells treated with hypoxia.2.Cardiac-specific ALOX12 transgenic mice significantly exacerbated MIR-induced cardiac dysfunction,myocardium damage,inflammatory cell infiltration,cardiomyocyte death,and cardiac fibrosis.3.In primary cardiomyocytes,ALOX12 interference significantly reduced the H/Rtriggered cell damage and inflammatory response.4.Bioinformatics and western blot results show that ALOX12 aggravates myocardial ischemia-reperfusion injury by inhibiting AMPK phosphorylation and activation.5.ML355 significantly improved cardiac function,ameliorated myocardial necrosis,and inhibited inflammatory response in mouse MIR model.Conclusion In this study,we demonstrated that ALOX12 is a key regulator of MIR injury.Mice with cardiomyocyte ALOX12 overexpression significantly exacerbated cardiac dysfunction,tissue necrosis,inflammatory response,and heart fibrosis compared to non-transgenic controls after MIR insult.Our further investigations into molecular mechanisms clarified that the inhibited phosphorylation and activation of AMPK underlines ALOX12 function.Finally,we demonstrated that inhibiting ALOX12 by ML355 protected against MIR-induced heart injury.