Mechanism Of Annexin A5 In Alleviating Sepsis-induced Myocardial Injury Through Regulating Microtubules Remodeling

Background:Septic cardiomyopathy(SCM)is one of the main causes of septic shock,affecting 20%to 65%of patients with sepsis.Severe SCM leads to refractory septic shock,with a mortality up to 70%.However,there are no evidence-based recommendations for the management of SCM.Therefore,it is urgent to find effective therapeutic methods for SCM in clinical practice.In recent years,microtubule(MT)remodeling has been proved to be an important mechanism of heart failure.Annexin A5(ANXA5),which is predominantly located on cytoskeletal,was reported to improve cardiac function in sepsis.However,the effects of ANXA5 on the function of cardiomyocytes in sepsis and whether the mechanism is related to MT remodeling remain unknown.Aims:To investigate the relationship between ANXA5 level and cardiac function in patients with septic shock.Then,the effects of ANXA5 on the function of cardiomyocytes in sepsis and the mechanisms were explored in LPS-induced AC 16 cardiomyocytes injury model.Method and results:1.A total of 31 patients with septic shock who were admitted to the Department of Intensive Care Unit,the Second Affiliated Hospital of Guangzhou Medical University from June 2017 to June 2018 were included and divided into afterload-related cardiac performance(ACP)>80%group and ACP≤80%group.The results showed that the plasma concentration of ANXA5 in ACP≤80%group were significantly higher than those in ACP>80%group.The plasma levels of ANXA5 were negatively correlated with ACP level.2.The cell model of SCM was established by lipopolysaccharide(LPS)-induced injury of AC 16 human cardiomyocytes.The ANXA5 interfering fragment si-ANXA5 was constructed to down-regulate the expression of ANXA5.ANXA5 overexpression plasmid was constructed to overexpress ANXA5.The expressional level of ANXA5 mRNA and protein were determined by real-time fluorescence quantitative polymerase chain reaction(real-time qPCR)and Western Blotting,respectively.Cell Counting Kit-8(CCK-8)assay was used to measure cell activity.The concentrations of inflammation-related cytokines[interleukin-1 beta(IL-1β),interleukin-6(IL-6),tumor necrosis factor-alpha(TNF-α),interleukin-10(IL-10),monocyte chemotactic protein-1(MCP-1)and matrixmetalloproteinase-9(MMP-9)]and biomarkers of myocardial injury[creatine kinase-MB(CK-MB)and cardiac troponin T(cTnT)]in the supernatant of cell culture medium were determined by Enzyme linked Immunosorbent Assay(ELISA).The video-based motion edge-detection system(IonOptix)was used to measure sarcomere length and the velocity of shortening/relengthening of cardiomyocytes(dl/dt).The changes of cytoskeleton were observed by immunofluorescence(IF)double-staining myosin and beta-tubulin(β-tubulin).Intracellular calcium concentration was measured by Fura-2-AM.The results indicated that LPS induced excessive secretion of inflammation-related factors,significantly increased concentration of biomarkers of myocardial injury in culture medium supernatant,decreased degree of sarcomere shortening and velocity of systole and diastole,decreased fluorescence intensity of myosin,disordered microtubule(MT)arrangement,loss of grid shape,intracellular calcium overload,and increased expression of ANXA5 mRNA and protein.Inhibition of ANXA5 expression aggravated LPS-induced damage.In contrast,overexpression of ANXA5 alleviated LPS-induced damage.3.MT depolymerizing agent colchicine and MT polymerizing agent epothilone B(EpoB)combined with LPS respectively were used to treat myocardial cells.The guanine nucleotide exchange factor(GEF)-H1 interfering fragment si-GEF-H1 was constructed to down-regulate the expression of GEF-H1.Then the sarcomere length and the velocity of systole and diastole were measured by the video-based motion edge-detection system(IonOptix).β-tubulin was stained by IF.The expression of protein tyrosinated tubulin(Tyr-tubulin),acetylated tubulin(Acet-tubulin),RhoA,GEF-H1 and microtubule associated protein Tau were measured by Western Blotting.The interaction between protein Tau and protein ANXA5 was determined by immunoprecipitation(CoIP).The results suggest that colchicine can aggravate LPS-induced MT instability and depolymerization,GEF-H1 release increase,Tau phosphorylation increase and myocardial contractile function worsen.EpoB has the opposite effect to colchicine.Down-regulation of GEF-H1 expression could inhibit RhoA activation and show similar effects to EpoB.The result of CoIP suggests that ANXA5 interacts with Tau.Overexpression of ANXA5 inhibited the release of GEF-H1 and the activation of RhoA/ROCK signaling pathway,showing similar effects to down-regulation of GEF-H1 expression.Conclusions:1.ANXA5 may be a biomarker for predicting the degree of cardiac dysfunction in patients with septic shock and may play a potential role in the pathogenesis of SCM.2.ANXA5 could inhibit inflammation-related factors release,cytoskeletal destruction and calcium overload in cardiomyocytes induced by LPS.ANXA5 also could alleviated cardiomyocytes injury and contractility decrease.3.MT depolymerization is the mechanism of LPS-induced myocardial dysfunction,and the RhoA/ROCK signaling pathway is involved.ANXA5 could interact with Tau to inhibit Tau phosphorylation,GEF-H1 release and RhoA/ROCK signaling pathway activation,reduce MT depolymerization and contractility decrease induced by LPS.