AMPK Alleviated Myocardial Ischemia-Reperfusion Injury By Regulating Drp1 Mediated Mitochondrial Dynamics

Myocardial ischemia/reperfusion injury refers to the phenomenon that myocardial tissue function,metabolic disorders and structural damage become worse after the restoration of blood flow in ischemic tissue.Mitochondrial dysfunction is a salient feature of MIRI.Activation of AMP-activated protein kinase(AMPK)can attenuate MIRI,while the potential mechanism is still unclear.This study sought to explore whether AMPK activation could alleviate MIRI by regulating GTPase dynamin-related protein 1(Drp1)mediated mitochondrial dynamics.Methods : Animal level: Forty-eight clean grade male C57BL/6J mice were randomly divided into four groups,Control group,Control + AICAR group,I/R group,I/R+AICAR group,12 mice in each group.Control + AICAR group,I/R+AICAR group was injected with AICAR(30mg/kg)in caudal vein 30 min before surgery,Control group and I/R group were injected with the same amount of normal saline,followed by langendorff perfusion system for isolated mouse heart ischemia/reperfusion(I/R)treatment.Left ventricular pressure was measured by BL-420 N biological function experiment system.Through the biochemical assay kit each perfusion fluid changes of creatine kinase(CK)and lactic dehydrogenase(LDH);using 2,3,5-triphenyltetrazoliumchloride(TTC)staining method determination of myocardial infarction area;The levels and activities of malondialdehyde(MDA)and total superoxide dismutase(SOD)in myocardial tissue were determined by biochemical assay kit.Western blot was used to detect AMPK/Drp1 total protein and its phosphorylation level in myocardial tissue of mice in each group.The expression of mitochondrial fission factors(Fis1,Mff)and fusion factors(Mfn1,Mfn2)were detected by q PCR.Cellular level: H9C2 cells,the cells were divided into the Control group,Control + AICAR group,H/R group,H/R + AICAR group,H/R + Mdivi-1 group,H/R+ Compound C group H/R + Compound C + Mdivi-1 group.Pretreated with drugs for 1h before in vitro hypoxia/reoxygenation(H/R)treatment,and then placed in anoxic tank for 12 h and then reoxygenated for 12 h.The levels of mitochondrial membrane potential(MMP)and reactive oxygen species(ROS)were determined by flow cytometry.The morphology of mitochondria was observed by transmission electron microscopy(TEM).Molecular biological techniques were used to detect the total AMPK/Drp1 protein and its phosphorylation level,as well as the expression of Fis1,Mff,Mfn1 and Mfn2.Result The results of Animal experiments showed that AICAR,the AMPK activator,could significantly improve left ventricular function,decrease arrhythmia incidence and myocardial infarction area.Meanwhile,AICAR caused an increase of superoxide dismutase(SOD)and a decrease of malondialdehyde(MDA)content in myocardial homogenate.Molecular biological results showed that AICAR decreased the expression of mitochondrial fission genes(Mff,Fis1)and increased the expression of mitochondrial fusion genes(Mfn1,Mfn2).Furthermore,AICAR inhibited phosphorylation of Drp1(Ser 616)and enhanced phosphorylation of Drp1(Ser 637).In the cell experiment,trends were similar to those seen in animal experiments.AICAR improved mitochondrial membrane potential(MMP),reduced reactive oxygen species(ROS)production and inhibited mitochondrial damage.Mitochondrial fission inhibitor Mdivi-1 has similar results to AICAR,significantly improved MMP,inhibited ROS production,reduced the expression of Fis1 and Mff,and improved the expression of Mfn1 and Mfn2.Further research confirmed that AMPK inhibitor Compound C couldn’t reverse the myocardial protection effect of Mdivi-1.Conclusion This study confirmed that activation of AMPK exerted the protective effects of MIRI,its protective effect is related to inhibition of Drp1 mediated mitochondrial overfission.