| Objective:Sepsis induced cardiac dysfunction（SICD）is a partial reversible decrease of myocardial contractility,fluid load response and catecholamine response caused by sepsis.The mortality of patients with SICD is significantly increased.The pathogenesis of myocardial depression in sepsis is still unclear.Some studies suggest that mitochondrial damage and reactive oxygen species（ROS）overproduction play an important role in myocardial depression in sepsis.Therefore,it is of great significance to remove damaged mitochondria,maintain healthy mitochondrial network and reduce ROS overproduction in the treatment of sepsis.Mitophagy can specifically degrade damaged mitochondria.Mitophagy is regulated by precise molecular signal,PINK1/Parkin is one of the most important ubiquitin dependent regulatory pathways of mitochondrial autophagy.Parkin ubiquitinates mitochondrial substrates.The ubiquitinated mitochondrial protein is recognized by cargo protein and then wrapped by autophagy bilayer membrane structure,which mediates the process of mitophagy.Therefore,the specific ubiquitination of protein and the total amount of ubiquitinated protein may be the key to affect the clearance of mitophagy.Ubiquitin specific proteases 30（USP30）is a deubiquitinase located in the outer membrane of mitochondria.Cell experiments show that overexpression of USP30 can reduce ubiquitin protein and inhibit mitophagy.However,the role and regulatory mechanism of USP30 in sepsis induced myocardial injury are still unclear.Therefore,the purpose of this study is to investigate whether USP30 can regulate mitophagy in sepsis induced myocardial injury and its possible mechanisms.Methods:PartⅠ.The role of mitophagy in LPS induced myocardial injury.1.E establishing of myocardial injury model in rats induced by LPS:after 6 hours,12hours,24 hours and 48 hours of LPS exposure,the hemodynamics of rats were monitored,and the heart samples were harvested for histopathological detection,mitophagy related protein detection:mitochondrial USP30,PINK1,Parkin,ubiquitin mitochondrial protein,SQSTM1/p62 and LC3BⅡand colocation of mitochondrial outer membrane protein TOMM20 and autophagy membrane protein LC3B by immunofluorescence imaging to evaluate the mitophagy.After pretreatment with cyclosporin A（CSA）,a mitophagy inhibitor,mitophagy associated proteins was detected at 6 hours and 12 hours after LPS exposure.2.Rat cardiac fibroblast H9c2（2-1）cell line was cultured in vitro.The sepsis-like cell model was established by adding LPS into the culture medium.Apoptosis,intracellular total ROS,adenosine triphosphate（ATP）content and mitochondrial membrane potential（MTP）were detected.PartⅡ.The effect of USP30 on mitophagy in LPS induced myocardial injury.1.Lentivirus was used to transfect H9c2（2-1）cells to produce stable transfection cell lines that overexpressed and knockdown of USP30 2.The total amount of ROS and ATP were detected after LPS treatment in cells described above,the expression of USP30,PINK1,Parkin,p62,LC3BⅡand mitochondrial ubiquitinated proteins were detected by Western blotting;the colocalization of TOMM20 and LC3B was detected by immunofluorescence imaging to evaluate the level of mitophagy.PartⅢ.The mechanism of USP30 regulating mitophagy in LPS induced myocardial injury.Overexpression of parkin gene of USP30 overexpression stable strain,knockdown of parkin gene of USP30 knockdown stable strain,overexpression and knockdown of parkin gene of normal cultured H9c2（2-1）cell line,respectively.The above cells were treated with LPS to detect USP30,p62,LC3BⅡand mitochondrial ubiquitinated proteins.The colocalization level of TOMM20 and LC3BⅡwas detected by fluorescence imaging.Results:1.Myocardial injury was observed 6 hours after LPS intraperitoneal injection,and was most significant 12 hours after LPS injection,with the decrease of±dp/dtmax and the increase of LVEDP.In the LPS injury model of H9c2（2-1）cells,MTP and ROS production increased,ATP synthesis decreased.Apoptosis was rare.Mitophagy detection showed that the level of mitophagy was low at 6 hours after LPS intraperitoneal injection,increased at 12 hours,and then decreased gradually.PINK1/Parkin increased significantly 6 hours after LPS treatment,and Parkin was recruited from cytoplasm to mitochondria,which was significantly earlier than the peak of mitophagy.After 6 hours of LPS treatment,the expression level of USP30 in mitochondria increased significantly,and the expression of mitochondrial ubiquitinated protein decreased significantly.After 12 hours of LPS treatment,the expression of USP30 in mitochondria decreased gradually,while the expression of mitochondrial ubiquitinated protein increased gradually.2.In the LPS injury model of USP30 knockdown cell line transfected by lentivirus,the increase of mitochondrial ubiquitinated protein and mitophagy,the decrease of ROS production and the increase of ATP synthesis were observed.In the LPS injury model of USP30 overexpression cell line,mitochondrial ubiquitinated protein decreased,mitophagy was inhibited,ROS production increased and ATP decreased.3.Knockdown of USP30 gene could not reverse the inhibition of mitophagy induced by Parkin gene knockdown,but overexpression of USP30 could partially counteract the enhancement of mitophagy induced by parkin overexpression.Conclusion:In LPS induced myocardial injury model,mitophagy plays a protective role;USP30 regulates mitophagy in LPS induced myocardial injury by regulating the ubiquitination substrates of Parkin.Decreased USP30 increases mitophagy and promotes the recovery of LPS induced myocardial injury. |
