MCU-dependent Mitochondrial Iron Overload Induces Mitophagy And Mediates Apelin-13-induced Proliferation Of Human Aortic Vascular Smooth Muscle Cells

Aim: Apelin is an endogenous ligand of seven transmembrane G protein-coupled receptor APJ,which plays an important role in the formation of cardiovascular system and the occurrence and development of related diseases.Our previous studies showed that apelin-13 promotes the proliferation of vascular smooth muscle cells(VSMC),and it was found that autophagy mediated the process of VSMC induced by apelin-13.Mitochondrial calcium uniporter(MCU)is also a kind of iron ion channel.Inhibition of MCU channel can significantly affect the transport of iron in mitochondria.Iron overload is the excessive deposition of iron ions in the organs of the whole body,including the heart,blood vessels,liver,and endocrine glands.Mitochondrial iron overload catalyzes Fenton reaction,which leads to the production of hydroxyl radicals and regulates cell proliferation.The purpose of this study was to investigate the effects of apelin-13 on MCU,mitochondrial iron overload,mitochondrial reactive oxygen species(ROS)and mitophagy.Based on previous studies to discuss the molecular mechanism of MCU dependent mitochondrial iron overload induced mitophagy mediated apelin-13 induced HA-VSMCs proliferation.Toreveal the new mechanism of HA-VSMCs proliferation induced by apelin-13/APJ system,and to provide a new experimental basis for the development of anti-atherosclerotic drugs targeting APJ receptor.Methods:1.The expression of MCU,Drp1,PINK1,Parkin,VDAC1,Tom20,PCNA in HA-VSMCs was detected by Western Blot.2.RNA interference: interference chain SiDrp1,SiPINK1 and SiParkin interfere with the expression of Drp1,PINK1 and Parkin in HA-VSMCs.3.The percentage of G1,S,G2/M cells in HA-VSMCs was detected by flow cytometry.4.The formation of mitophagy in HA-VSMCs was observed by transmission electron microscopy(TEM).5.The colocalization of mitochondria and LC3 was observed by immunofluorescence assay in HA-VSMCs6.The expression of MCU,Drp1 in patient arteriosclerosis,double kidney-double clip rats hypertensive specimen was observed by immunohistochemistry.7.RPA red fluorescent probe was used to dectect the mitochondrial chelated iron in HA-VSMCs.8.Mito-SOX red fluorescent probe was used to assess the mitochondrial ROS production in HA-VSMCs.9.The concentration of proteins in HA-VSMCs was tested by BCA proteinquantitation kit.Results:1.The expression of MCU,Drp1,and mitochondrial iron in patient arteriosclerosis coronary artery tissue was higher than normal coronary artery tissue.The expression of MCU,Drp1,and mitochondrial iron in double kidney-double clip rats hypertensive vascular coronary artery tissue was higher than normal rat aortic vascular smooth muscle tissue.2.Apelin-13 promoted the expression of MCU in concentration-(0-1 ?M,24h)and time(0-24h)dependent manner in HA-VSMCs;APJ receptor antagonist F13 A mediated apelin-13 promoted the expression of MCU in HA-VSMCs.3.Apelin-13 induced mitochondrial iron overload in HA-VSMCs;APJ receptor antagonist F13 A inhibited mitochondrial iron overload induced by apelin-13 in HA-VSMCs;MCU inhibitor Ru360 suppressed mitochondrial iron overload induced by apelin-13 in HA-VSMCs.4.Apelin-13 increased the production of mitochondrial ROS in HA-VSMCs;APJ receptor antagonist F13 A blocked the production of mitochondrial ROS induced by apelin-13 in HA-VSMCs;Ferric ammonium citrate(FAC)increased the production of mitochondrial ROS induced by apelin-13 in HA-VSMCs;F13A inhibited the production of mitochondrial ROS induced by apelin-13 in HA-VSMCs;MCU inhibitor Ru360 inhibited apelin-13-induced mitochondrial ROS production in HA-VSMCs.5.APJ receptor antagonist F13 A inhibited the expression of Drp1 induced by apelin-13 in HA-VSMCs;MCU inhibitor Ru360 inhibited the expression of Drp1 induced by apelin-13 in HA-VSMCs;Mitochondrial ROS scavenger Mito-TEMPO inhibited the expression of Drp1 induced by apelin-13 in HA-VSMCs.6.Transmission electron microscopy(TEM)showed that apelin-13 induced mitophagy in HA-VSMCs;Mitochondrial division inhibitor 1Mdivi-1 mediated mitophagy induced by apelin-13;immunofluorescence showed that apelin-13 induced mitochondria co-localization with LC3 in HA-VSMCs;Mdivi-1 and SiRNA-Drp1 inhibited the co-localization of mitochondria and LC3 induced by apelin-13 in HA-VSMCs.7.Mito-TEMPO inhibited the expression of PINK1,Parkin,VDAC1 and Tom20 induced by apelin-13,Mdivi-1 and SiRNA-Drp1 inhibited the expression of PINK1,Parkin,VDAC1,Tom20 induced by Apelin-13.8.Western blot showed that APJ receptor antagonist F13 A inhibited the proliferation of HA-VSMCs induced by apelin-13 in HA-VSMCs;Ru360inhibited the proliferation of HA-VSMCs induced by apelin-13;Mito-TEMPO inhibited the proliferation of HA-VSMCs induced by apelin-13;Mdivi-1 and SiRNA-Drp1 inhibited the proliferation of HA-VSMCs induced by apelin-13;SiRNA-PINK1 and SiRNA-Parkin inhibited the proliferation of HA-VSMCs induced by apelin-13.Conclusion:MCU-dependent mitochondrial iron overload induces mitophagy and mediates apelin-13-induced proliferation of HA-VSMCs.