SUMO1 Modified Drp1 Promoting BNIP3-dependent Mitochondrial Iron-derived ROS Pathway Mediates Apelin-13-induced Cardiomyocyte Hypertrophy

Objective: APJ is a G protein-coupled receptor with homology to angiotensin receptors,which is also a endogenous receptor of Apelin.Several studies indicate that the Apelin/APJ system is involved in cardiovascular diseases such as heart failure and hypertension.Previous studies in our laboratory report that Apelin-13 promotes cardiomyocyte hypertrophy through ferritinophagy activation and SFXN1-dependent mitochondrial iron-overload pathway.Mitotophagy(Mitophagy)as a selective autophagy process that specifically cleaves damaged mitochondria,is involved in inducing mitochondrial iron metabolism disorders.This study aims to investigate the effect of Apelin-13 on mitophagy,as well as BNIP3-dependent mitophagy pathway on Apelin-13 induced mitochondrial iron-derived ROS.Additionally,based on previous studies,we explore a new mechanism by which SUMOylation of Drp1-BNIP3 dependent mitochondrial iron-derived ROS pathway mediates Apelin-13-induced cardiomyocyte hypertrophy.The research reveal a novel molecular mechanism that Apelin-13/APJ system induces cardiac hypertrophy.It may stand for a new experimental basis for exploring whether APJ receptors could be a new target for treating cardiac hypertrophy.Methods:1.Western Blot: detection of the expression of Beclin 1,LC3II/I,BNIP3,Drp1,SUMO1,BNP in H9c2 rat cardiomyocytes;2.RNA interference: Design RNA interference chain to interfere with the expression of SUMO1,Drp1,BNIP3 in H9c2 cardiomyocytes;3.Mito-SOX fluorescent probe was used to detect the change of mitochondrial ROS in H9c2 rat cardiomyocytes;4.RPA mitochondrial iron fluorescent probe was used to observe the content of mitochondrial chelated iron in H9c2 cardiomyocytes;5.The colocalization of SUMO1 and Drp1,BNIP3 and LC3 B,as well as BNIP3 and mitochondria were detected by immunofluorescenc in H9c2 cells.6.Transmission electron microscopy was utilized to observe the occurrence of mitophagy;7.ScepterTM Handheld cell counter test the diameter and volume of H9c2 cells.8.BCA protein quantitation kit is used for the concentration of proteins in H9c2 cells.9.Immunocytochemical analysis of BNP,SUMO1,Drp1,BNIP3 protein expression in human autopsy cardiac hypertrophy heart tissue specimens,bilateral renal double-clamp rat renal vascular hypertension model rat heart tissue and heart tissue from C57BL/6J mouse with exogenous injection Apelin-13;Results:1.Apelin-13 promotes the expression of mitophagy marker protein BNIP3 in H9c2 cardiomyocytes cells with a dose and time dependence;APJ antagonist F13 A inhibits the effect of Apelin-13 on BNIP3;2.Apelin-13 induces the occurrence of mitophagy in H9c2 cardiomyocytes under transmission electron microscope;3.Apelin-13 increases the expression of mitochondrial fission protein Drp1 in H9c2 cells in dose and time-dependent manners;APJ antagonist F13 A reverses the inducive effect of Apelin-13 on the effect of Drp1;4.Drp1 specific inhibitor,Mdivi-1,significantly suppresses Apelin-13 induced the expression of mitochondrial fission protein Drp1,mitophagy marker protein BNIP3 and autophagy marker proteins Beclin 1,LC3II/I;5.Drp1 specific inhibitor,Mdivi-1,significantly blocks Apelin-13 induced the colocalization of mitophagy marker protein BNIP3 and autophagy marker protein LC3II;Drp1 specific inhibitor,Mdivi-1,significantly blocks Apelin-13 induced the colocalization of mitophagy marker protein BNIP3 and mitochondria.6.Drp1-si RNA significantly reverses Apelin-13 induced the expression of mitophagy marker protein BNIP3 and autophagy marker proteins Beclin 1,LC3II/I;7.Drp1-si RNA significantly blocks Apelin-13 induced the colocalization of mitophagy marker protein BNIP3 and autophagy marker protein LC3II;Drp1-si RNA significantly inhibits the colocalization of mitophagy marker protein BNIP3 and mitochondria;Drp1-si RNA significantly blocks Apelin-13 promoted the mitochondrial iron and ROS production in H9c2 cardiomyocytes;8.Apelin-13 increases the expression of ubiquitin-like protein SUMO1 in H9c2 cells in dose and time-dependent manners;APJ antagonist F13 A blocks the promotive effect of Apelin-13 on the SUMO1;9.Apelin-13 induces the colocalization of SUMO1 and Drp1;10.SUMO1-si RNA significantly blocks the expression of Apelin-13 induced mitochondrial fission protein Drp1,mitophagy marker protein BNIP3 and autophagy marker proteins Beclin 1,LC3II/I;11.SUMO1-si RNA significantly suppresses the Apelin-13 induced colocalization of mitophagy marker protein BNIP3 and autophagy marker protein LC3II;SUMO1-si RNA significantly inhibits Apelin-13 induced the colocalization of mitochondrial autophagy marker protein BNIP3 and mitochondria;SUMO1-si RNA significantly blocks Apelin-13 promoted the mitochondrial iron and ROS production in H9c2 cardiomyocytes;12.BNIP3-si RNA significantly blocks Apelin-13 promoted the mitochondrial iron and ROS production in H9c2 cardiomyocytes.13.Mitochondria-targeted antioxidant,Mito-TEMPO,significantly inhibits Apelin-13 induced the expression of ubiquitin-like protein SUMO1,mitochondrial fission protein Drp1,mitophagy marker protein BNIP3 and autophagy marker proteins Beclin 1,LC3II/I trigerred by Apelin-13;14.Mitochondria-targeted antioxidant,Mito-TEMPO,significantly blocks Apelin-13 stimulated the colocalization of SUMO1 and Drp1,as well as mitophagy marker protein BNIP3 and autophagy marker protein LC3II;Mitochondria-targeted antioxidant,Mito-TEMPO,significantly blocks Apelin-13 stimulated the colocalization of mitophagy marker protein BNIP3 and mitochondria15.SUMO1-si RNA blocks the increase of H9c2 cardiomyocyte diameter,volume,protein contents,as well as the expression of cardiac hypertrophic factor BNP trigerred by Apelin-13;Drp-si RNA inhibits the increase of H9c2 cardiomyocyte diameter,volume,protein contents,as well as the expression of cardiac hypertrophic factor BNP induced by Apelin-13;Drp1 specific inhibitor,Mdivi-1 blocks the increase of H9c2 cardiomyocyte diameter,volume,protein contents,as well as the expression of cardiac hypertrophic factor BNP induced by Apelin-13;16.BNIP3-si RNA blocks the increase of H9c2 cardiomyocyte diameter,volume,protein contents,as well as the expression of cardiac hypertrophic factor BNP induced by Apelin-13;Mitochondria-targeted iron chelator,DXZ,suppresses the increase of H9c2 cardiomyocyte diameter,volume,protein contents,as well as the expression of cardiac hypertrophic factor BNP induced by Apelin-13;Mitochondria-targeted antioxidant,Mito-TEMPO suppresses the increase of H9c2 cardiomyocyte diameter,volume,protein contents,as well as the expression of cardiac hypertrophic factor BNP induced by Apelin-13;17.BNP,SUMO1,Drp1 and BNIP3 are higher expressed in human autopsy cardiac hypertrophy heart tissue specimens,bilateral renal double-clamp rat renal vascular hypertension model rat heart tissue and the heart tissue of C57BL/6J mice with exogenous injection of Apelin-13,compared with control group;Conclusion: SUMO1 modified Drp1 promoting BNIP3-dependent mitochondrial iron-derived ROS pathway mediates Apelin-13-induced cardiomyocyte hypertrophy...