The Role And Underlying Mechanisms Of Atg7 In LPS-induced Septic Cardiomyopathy

Background:Sepsis is a life-threatening systemic inflammatory response syndrome,followed with severe complications and multiple organ damage,especially the heart.Septic cardiomyopathy,one of the most severe complications of sepsis,is a key pathological process leading to death.Therefore,It is of great scientific significance to find and explore treatment options and intervention targets for sepsis cardiomyopathy.In recent years,studies have found that there is a close relationship between autophagy and sepsis cardiomyopathy.Some evidences have shown that early autophagy can antagonize the inflammatory response of sepsis cardiomyopathy,but late autophagy damage leads to cardiomyocyte apoptosis and systolic dysfunction;others have shown that autophagy may be able to aggravates sepsis cardiomyopathy.The role of autophagy in sepsis cardiomyopathy has not yet been fully elucidated.The autophagy associated gene(autophagy associated gene,Atg)family is involved in the formation and regulation of autophagy.Among the all,Atg7,a key member of the Atg family,is a hotspot in autophagy research.Atg7 has El-like ubiquitination activating enzyme activity,which promotes the conversion of LC3I to LC3II and the formation of the E3-like ubiquitinase Atg12-Atg5 complex,which means Atg7 plays a crucial role in the formation of autophagic vesicles.So far,whether Atg7 can participate in the development of sepsis cardiomyopathy by regulating autophagy or other non-autophagy pathways has not been reported at home and abroad yet,thus it is worthy of further study.Objective:This study focused on(1)the expression changes of Atg7 in the heart and cardiomyocytes of LPS-induced septic cardiomyopathy mice;(2)the effects of specific Atg7’s overexpression or deletion on the survival rate and cardiac function of mice;(3)the potential mechanism of the regulation in septic cardiomyopathy by Atg7;(4)The effect of Atg7-regulated non-autophagy pathway on septic cardiomyopathy.Methods:Part 1:(1)To detect the expression changes of Atg7 and LC3I/II in wild type(C57)mice and primary rat cardiomyocytes(NRCM)under the stimulation of LPS.Animal experiments:The model was established by intraperitoneal injection of 5 mg/kg LPS.The experimental animals were randomly divided into control group(CON),LPS stimulated 3h group,6h group,12h group,24h group and 48h group;Cell experiment:Separated NRCM added with 1μg/mL LPS to establish a cell model.The experiment was divided into control group and LPS stimulated 3h group,6h group,12h group,24h group and 48h group.Tissue and cell proteins were obtained at corresponding time points,and the expression of target protein was detected by Western-blot.(2)To detect the effects of specific Atg7’s overexpression or deletion on the survival rate and cardiac function of mice.Animal experiments:Male C57 mice(8-10 weeks old,24.5-25.5 g),cardiac-specific Atg7 knockout mice(Atg7-CKO)and cardiac-specific Atg7 overexpressing mice(Atg7-TG)were administered.The mortality of mice was observed with 30 mg/kg LPS injected;the cardiac function of mice was detected after 9-12 h with5 mg/kg LPS injected;the tissue morphology changes were observed by HE staining,and the expression of autophagosomes and lysosomes was observed by electron microscopy.Colocalization of LC3 and LAMP1 was observed,TUNEL was used to detect cardiomyocyte apoptosis;total cardiac protein and RNA were extracted to detect autophagy,apoptosis and AMPKα/mTORC1 signaling pathway changes.Part 2:(1)The potential mechanism of Atg7 in LPS sepsis cardiomyopathy.Animal experiments:Atg7-TG mice were given(TG+Met)mice 1 week after metformin(Metformin,Met,100 mg/kg/d),and TG mice were hybridized with AMPKa knockout mice to obtain Atg7 overexpression and AMPKα knockdown;5 mg/kg LPS was intraperitoneally injected to construct a model of sepsis cardiomyopathy,and the heart function of mice was monitored by ultrasound 9-12 h after LPS injection.Mouse heart tissues were collected,and AMPKα2/mTORC1 signaling pathway,lysosome and apoptosis-related proteins were detected by Western-blot.(2)Detected the role of Atg7-regulated non-autophagy pathway in sepsis cardiomyopathy.The heart tissue of 5 mg/kg LPS-treated mice was collected,and the expression of Nrf2 pathway-related proteins was detected by western-blot when Atg7 was silenced or overexpressed.Cell experiment:NRCM was extracted and cultured,Nrf2-siRNA was transfected with lip6000 to silence Nrf2 in cardiomyocytes,and Atg7 was silenced with Ad-Atg7-KO,and then cell were collected after LPS stimulation for 9-12 h.And the oxidative stress level were detected,then the regulation of Nrf2 expression by Atg7 was further verified..Results:Part 1:(1)With the stimulation of LPS the expression of Atg7 and LC3Ⅱ/LC3Ⅰ in cardiac tissue and cardiomyocytes increased,The expression level was the highest at 12h.(2)Compared with the C57+LPS group,the cardiac function of the TG+LPS group was significantly worsened,while the cardiac function of the CKO+LPS mice was improved.High concentration of LPS increased the mortality of mice,and the mortality of mice in TG+LPS group was 100%within 24h.There was no significant difference in mortality between CKO+LPS group and C57+LPS group.At the tissue level,the TG+LPS group had more disordered myocardial alignment than C57+LPS group,the inflammatory cell infiltration,myocardial cell vacuoles and cardiomyocyte apoptosis were significantly increased,but in the.There was no significant difference between CKO+LPS group and the C57+LPS group.Transmission electron microscopy showed that autophagosomes and lysosomes in C57+LPS group were less and increased synchronously.Autophagosomes in TG+LPS group increased significantly but lysosomes were much less.By contrast,in CKO+LPS group The corpuscles are significantly reduced while the lysosomes are increased;Immunofluorescence assay further confirmed that LPS promoted the enhancement of autophagic flow in cardiac tissue.In the TG+LPS group,LC3II was significantly aggregated in mouse heart tissue,the autophagosomes and the expression of lysosomal-associated protein LAMP1 enhanced,but the autophagic flow did not;However,a reverse trend found in CKO+LPS group;TUNEL staining showed that LPS promoted cardiomyocyte apoptosis,and the positive rate of cardiomyocyte apoptosis was significantly increased in TG+LPS group compared with C57+LPS group,while cardiomyocyte apoptosis was less in CKO+LPS group.The detection of related signal pathways showed that the expression of Atg7 and Bax,the ratio of LC3II/I and the degree of phosphorylation of mTORCl in the myocardial tissue of the TG+Met group was increased,while the expression of LAMP 1,LAMP2 and p-AMPKa was decreased.However,in C57+LPS group,a reverse trend can be seen.Part 2:With LPS stimulation,the cardiac function of the TG+AMPKα-KO group was significantly worse than that of the TG group,while the cardiac function of the TG+Met group was significantly improved.Compared with the TG group,the expression of Atg7 and Bax in the myocardial tissue of the TG+Met group,the ratio of LC3Ⅱ/Ⅰ and the degree of phosphorylation of mTORC1 was decreased,while the expression of LAMP1,LAMP2 and p-AMPKα was increased.However,a reverse trend can also be seen in the TG+AMPKα-KO group.The western blot analysis of NRF2 pathway-related proteins revealed that the expression of Nrf2,HO-1,SOD 1,SOD2 and P62 was significantly increased in CK+LPS group compared with C57+LPS group,but the expression of these protein molecules in TG+LPS group has no significant difference.In addition,the results of SOD and GPx activity in mouse heart tissue showed that compared with CON,LPS significantly decreased the expression of SOD and GPX in CON and TG groups.Compared with CON+LPS group,the expression of SOD and GPX in TG+LPS group Significantly reduced;the expression of SOD and GPX in the KO+LPS group was significantly increased compared with the TG+LPS group.The results of NRCM showed that compared with CON,LPS significantly decreased the expression of SOD and GPX in the CON group,the si-Nrf2 group,and the CKO+si-Nrf2 group.Compared with the CON+LPS group,the expression of SOD and GPX in the si-Nrf2+LPS group and the CKO+si-Nrf2+LPS group was significantly reduced.Conclusion:1.Cardiac tissue-specific knockout of Atg7 attenuates LPS-induced cardiac function deterioration,improves the inflammation,oxidative stress and apoptosis caused by LPS;Cardiac tissue-specific overexpression of Atg7 aggravates the deterioration of cardiac function caused by LPS,aggravates inflammatory response,oxidative stress and cardiomyocyte apoptosis induced by septic cardiomyopathy.2.Atg7-specific overexpression cannot improve LPS-induced cardiac dysfunction and decrease myocardial cell apoptosis.The mechanism may be that AMPKα2 activity is down-regulated in septic heart,mTROCl activity is up-regulated to inhibit lysosomal pathway activity,autophagic flow Not enhanced,the accumulation of autophagosomes in cardiomyocytes promoted cardiomyocyte apoptosis and deterioration of cardiac function.3.Atg7-specific overexpression protects LPS-induced sepsis cardiomyopathy.The mechanism is that Atg7-dependent autophagy loss leads to the accumulation of P62 in cardiomyocytes.Keapl in P62-degraded cells promotes the release of Nrf2 and enhances the anti oxidation of cardiomyocytes,thereby protecting cardiomyocyte from damage by LPS-induced sepsis cardiomyopathy.4.Atg7 may be a potential therapeutic target in LPS-induced septic cardiomyopathy.