The Role Of Acetaldehyde Dehydrogenase 2 In Septic Shock Induced Myocardial Pyroptosis And The Underlying Mechanisms

BackgroundSepsis is a life-threatening condition characterized by organ dysfunction due to unregulated host immune response against infection,and is widely recognized as the ultimate cause of death from many diseases.Nearly 50 million cases of sepsis were reported worldwide in the year of 2017,and this number was considered significantly underestimated.More than 60%of patients with severe sepsis or septic shock were reported to have cardiac dysfunction,the presence of which is related to mortality as high as 70%-90%.Therefore,exploring novel therapeutic targets for septic shockinduced cardiac dysfunction is a key research imperative.Programmed death of cardiomyocytes is one of the critical mechanisms of impaired cardiac function.In the last two decades,several types of programmed cell death were newly recognized and interpreted,such as necroptosis,ferroptosis and pyroptosis.Of all the types of cell death,pyroptosis is most closely related to infection and inflammation,and it is usually induced after pathogens infect immune cells such as monocytes,macrophages and dendritic cells.Pyroptosis,known as Gasdermindependent inflammatory programmed cell death,is characterized by rapid rupture of cell membrane and the release of inflammatory cytokines and cellular contents.Thus,pyroptosis can be triggered by inflammation and further greatly amplifies the inflammation response,which may play key role in septic shock-induced cardiac dysfunction.NOD-like receptor protein 3(NLRP3)inflammasome/Caspase1/Gasdermin D(GSDMD)pathway is the well-known canonical regulatory pathway of pyroptosis.NRLP3 inflammasome is a multi-protein complex composed of intracellular receptor NLRP3,adaptor protein apoptosis-associated speck-like protein(ASC)and precursor pro-Caspase-1.The activation of this complex ultimately cleaves pro-Caspase-1 to its active form,which mediates the maturation and secretion of IL-1βand IL-18,and the cleavage of GSDMD,a key executor of pyroptosis.The existence of pyroptosis in cardiomyocytes induced by sepsis was proved by several studies,however its regulatory mechanism remains unclear and needs further research.Mitochondrial homeostasis is essential for myocardial survival.Mitochondrial fatty acid β-oxidation is the major pathway for fatty acid degradation and energy supply of cardiomyocytes,and its disturbance leads to energy shortage and mitochondrial instability.Disturbed fatty acid β-oxidation leads to inadequate energy supply as well as oxidative stress and impaired mitochondrial respiration due to accumulation of longchain saturated fatty acid acylcarnitines and triglycerides.It has been reported that mitochondrial instability is closely associated with NLRP3 inflammasome activation.However,whether fatty acid β-oxidation disorder is associated with NLRP3 inflammasome activation and cardiomyocyte pyroptosis remains unknown.The mitochondrial inner membrane protein hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex a subunit(HADHA)is a key protein for fatty acid β-oxidation,the enzymatic activity of which can be regulated by acetylation at Lysine 303.Loss of HADHA activity or mutations in its encoding gene can lead to cardiomyopathy and other disorders.Histone deacetylase 3(HDAC3)is a key deacetylase.Therefore,we hypothesized that HDAC3/HADHA might regulate NLRP3 inflammasome and pyroptosis by maintaining mitochondrial homeostasis in cardiomyocytes.Aldehyde dehydrogenase(ALDH2)is well-known for its role in maintaining mitochondrial homeostasis through aldehyde scavenging and antioxidant effects.Aldh2 rs671 genetic mutation is an independent risk factor for cardiovascular diseases,and about 30%to 50%of East Asian individuals carry this mutation,causing a 90%loss of its enzyme activity.However,as a protein mainly located in mitochondria,it is still unclear whether ALDH2 affects fatty acid β-oxidation and plays a role in myocardial pyroptosis.In a previous study by our group,it was found that ALDH2 could improve cardiac dysfunction by inhibiting endoplasmic reticulum stress and autophagy in mild sepsis.However,how ALDH2 functions in cardiac dysfunction in severe sepsis is unclear,and whether ALDH2 protects mitochondria through pathways other than scavenging toxic aldehydes is an interesting but poorly explored question.This study will demonstrate the role of ALDH2 as a therapeutic target for myocardial pyroptosis,and elucidate novel mechanisms focusing on the mitochondrion-NLRP3 inflammasome interaction.Objectives1.To clarify the role of ALDH2 in septic shock-induced cardiac dysfunction;2.To clarify the role of ALDH2 in improving cardiac dysfunction induced by septic shock through myocardial pyroptosis mediated NLRP3 inflammasome/Caspase1/GSDMD signaling pathway;3.Around the mitochondria-NLRP3 inflammasome,explore the mechanism of ALDH2 in septic shock-induced cardiacdysfunction through HDAC3/HADHA signaling pathway.MethodsWe used a septic shock model in mice to investigate the effects of enhanced ALDH2 activity or Aldh2 knockout on cardiac function and survival,myocardial pyroptosis and mitochondrial damage after septic shock.Rat cardiomyocyte cell line H9C2 was used to establish cell model of pyroptosis in vitro.The potential mechanism of ALDH2 was investigated around mitochondrial damage-NLRP3 inflammasome and pyroptosis.1.Establishment of mouse model of septic shock-induced cardiacdysfunction:Male mice aged 6-8 weeks were selected for experiments on the eve of the establishment of the model.After fasting,the mice were intraperitoneally injected with 15 mg/kg LPS for 12h,then anesthetized the mice to collect samples for use.The ALDH2 agonist,Alda-1,was injected intraperitoneally 30 minutes before LPS administration.Experimental groups:wild-type mice were randomly divided into 4 groups,including Control group(Control group),Alda-1 pretreatment group(Alda-1 group),septic shock model group(LPS group)and Alda-1 pretreatment and septic shock model group(LPS+Alda-1 group).C57BL/6J mice(WT type)and acetaldehyde dehydrogenase 2 knockout mice(Aldh2-/-type)were randomly divided into 4 groups with 8-10 mice in each group:Wild-type control group(WT group),aldehyde dehydrogenase 2 gene knockout type control group(Aldh2-/-group),wild-type and septic shock group(WT+LPS group),aldehyde dehydrogenase 2 gene knockout type and septic shock group(Aldh2-/-+LPS group).2.Establishment and experiment of cell model:To study whether ALDH2 has a protective effect on myocardial pyroptosis in spsis,we used the rat H9C2 cell line to establish a pyroptosis model of spsis in vitro,stimulated with 2 μg/mL LPS for 24 hours and then stimulated with 40 μmol/L ATP for 45 minutes.The ALDH2 smallmolecule agonist Alda-1(20 μmol/L)stimulated cells 30 min before LPS with or without administration.Experimental grouping:Control group,LPS+ATP group,Alda1 group,LPS+ATP+Alda-1 group;and detected the level of pyroptosis,the level of NLRP3 inflammasome,mitochondrial DNA/ox-mtDNA release,and mitochondrial respiratory function.In order to study the effect of ALDH2 on mitochondrial oxidative stress,the changes of mitochondrial ROS(mtROS)level and mitochondrial membrane potential were observed after H9C2 cells were stimulated with 4-HNE.In order to investigate whether ALDH2 has a protective role in myocardial pyroptosis in sepsis through HDAC3 and HADHA pathway,this study used siRNA to interfere with HDAC3 and HADHA to explore their effects.3.Measurement of myocardial function:Color Doppler ultrasound was used to detect cardiac LVFS,LVEF,HR and other indicators in mice to reflect the cardiac function of sepsis mice for 12h.4.Murine sepsis score:After mouse modeling,murine sepsis score was used to evaluate the severity of sepsis.The score includes 7 aspects,namely appearance,level of consciousness,activity,response to stimulus,eyes,respiration rate,and respiration quality.5.Measurement of ALDH2 activity:After extracting myocardial tissue mitochondrial protein and detecting the protein concentration,it was found that ALDH2 had aldehyde dehydrogenase activity,and the enzymatic activity of ALDH2 was quantitatively analyzed by adding the metabolic substrate NAD+to reduce to NADH,and the change of absorbance at 340 nm wavelength.6.Measurement of myocardial cell death:TUNEL and methylgreen staining were used to evaluate the myocardial cell death after the myocardial tissue was fixed and made into paraffin sections.7.Measurement of mitochondrial oxidative stress levels in tissues and cells:After staining with fluorescent probes DHE and MitoSOX Red,the levels of mitochondrial oxidative stress in tissues and cells were respectively evaluated through fluorescence intensity.8.Morphological imaging of pyroptosis:PI staining was used to evaluate the integrity of cell membrane,and fluorescence images of pyroptosis were captured by confocal microscopy,and the bright field in the same field of vision was captured to observe cell morphology.9.Measurement of mitochondrial respiratory function:Seahorse XFe96 cellular mitochondrial stress test kit was used to measure Oxygen consumption rate(OCR)to evaluate cellular mitochondrial respiratory function.10.Measurement of NLRP3 inflammasome activation:After the cells were fixed and permeable,fluorescent primary antibodies of NLRP3 and ASC were used for overnight incubation,and then fluorescent secondary antibodies were used for incubation and sealing.NLRP3/ASC speck formation was evaluated under fluorescence or confocal microscopy.11.Western Blot:After extracting 30μg tissue or cell protein,8%-15%gel was used to detect the expression levels of toxic aldehyde 4-HNE,ALDH2,NLRP3,N-GSDMD,Caspase-1 p20,HADHA,HDAC3 and other proteins by western blot.12.Measurement of HD AC3 organelle localization:Use the kit to separatethe mitochondria,nucleus and cytoplasm proteins of the cells,then add the corresponding volume of 4x protein loading buffer.After heating proteins,the process was carried out as the above protein detection step.13.Co-immunoprecipitation:This technology was used to detect the acetylation level of the mitochondrial protein HADHA.After extracting the protein through general steps,the protein was incubated with the primary antibody and magnetic beads in turn.After centrifugation,the supernatant was discarded,and the corresponding volume of 4x protein loading buffer was added.After heating proteins,the process was carried out as the above protein detection procedure.14.Statistical analysis:the continuous data were presented as mean±standard deviation.Group comparisons were performed by one-way analysis of variances(ANOVA)with Tukey’s post hoc test or Student’s t-test.Kaplan-Meier curve was used for survival rate,and log-rank test was used for comparing survival rate between groups.P<0.05 was considered statistically significant(2-tailed).Results1.In mouse model of septic shock-induced cardiacdysfunction,ALDH2 significantly improved cardiac dysfunction and mortalityCompared with the control group,LPS treatment of Wild type(WT)mice resulted in a very high murine spsis score and significantly impaired the cardiac function of mice,and was positively correlated with the mortality of mice.Aldh2 knockout(Aldh2/-type)further aggravated murine sepsis score,mortality and cardiac dysfunction in mice compared with LPS group;and these phenomena were improved after Alda-1 pretreatment.In addition,it was also observed that LPS can significantly reduce the enzymatic activity of ALDH2 in heart tissue of wild-type mice.2.ALDH2 attenuates myocardial pyroptosis through NLRP3 inflammasome/Caspase-1/GSDMD pathway in septic shockHE staining showed that LPS stimulation increased inflammatory cell infiltration and broken myocardial fibers compared with the control group,and TUNEL staining found that the number of died cardiomyocytes increased significantly,which was further aggravated and effectively relieved in Aldh2 knockout mice(Aldh2-/-)and Alda1 pretreated mice,respectively.Plasma LDH levels also indicate the protective role of ALDH2 in septic shock-induced cardiac dysfunction.After LPS stimulation,the transcription and expression levels of IL-1β,IL-6 and TNF-α were significantly increased(except TNF-α expression,P=0.052).Alda-1 pretreatment significantly decreased the transcription and expression levels of these cytokines.The plasma IL-18 level was also consistent with the above results.To elucidate the role of pyroptosis in septic shock-induced cardiac dysfunction,mice were pretreated with N ecrosulfonamide Necrosulfonamide(NS A,a recognized inhibitor of necroptosis),and it was found that,compared with the LPS group,the NSA pretreatmentandLPS group significantly improved the LPS-induced cardiac dysfunction.In addition,the protein expression of N-GSDMD,a marker of pyroptosis was detected in sepsis-stimulated tissues and typical morphological changes of pyroptosis were observed in vitro,and ALDH2 significantly improved the above phenomena.To further explore the signaling pathway,Western blot results showed that LPS stimulation triggered higher levels of NLRP3 and Caspase-1 p20 in Aldh2-/-mice compared with wild-type mice.In contrast,Alda-1 pretreatment significantly reduced LPS-induced upregulation of NLRP3 and Caspase-1 p20.Both immunohistochemical staining and immunofluorescence staining showed that the protein expression of Caspase-1 in LPS-stimulated heart tissue was increased,and the protein expression of Caspase-1 was further enhanced in Aldh2-/-mice,while Alda-1 pretreatment significantly reversed its expression.The same results were obtained by flow cytometry and immunofluorescence staining of Caspase-1.Fluorescence colocalization staining showed that LPS and ATP stimulation caused a significant increase in the number of NLRP3/ASC speck compared with Alda-1 pretreatment group.These results suggest that activated ALDH2 can reduce NLRP3 inflammasome activation.3.ALDH2 inhibited the expression of NLRP3 and the activation of NLRP3 inflammasome through reducing mtDNA and ox-mtDNATo explore the potential role of ALDH2 in regulating the mitochondria-NLRP3 inflammasome pathway,we focused on in vitro studies of mitochondrial DNA release.It was found that,compared with the control group,LPS and ATP significantly increased the levels of mtDNA and ox-mtDNA,and the activation of ALDH2 significantly reduced the release of mtDNA and ox-mtDNA by sepsis.In addition,after transfection of mtDNA or ox-mtDNA into H9C2 cells in vitro,Western blot and fluorescence co-localization staining results showed that both mtDNA and ox-mtDNA transfection increased NLRP3 expression and activation of NLRP3 inflammasome.To elucidate the role of ALDH2 in the release of mitochondrial DNA in sepsis induced myocardial pyroptosis,we investigated the relationship between ALDH2 in oxidative stress,toxic aldehyde accumulation,and mitochondrial damage.We found that ALDH2 activation significantly reduced the accumulation of protein adduces of 4hydroxylenal(4-HNE)induced by LPS stimulation compared with the control group.DHE staining revealed that ALDH2 significantly ameliorated the LPS-induced excessive oxidative stress in mouse heart tissue.In addition,compared with the control group,LPS and ATP reduced the oxygen consumption rate(OCR),which could reflect the mitochondrial respiratory function.,Aldh2 knockdown aggravated the mitochondrial respiratory function damage,while Alda-1 pretreatment improved the mitochondrial respiratory function.To further verify the critical role of ROS in this model,endotoxin-induced mitochondrial damage was significantly ameliorated after application of the ROS scavenger,NAC.To verify the effects of toxic aldehydes and ALDH2 on mtDNA/ox-mtDNA release,it was found that 4-HNE significantly increased the release of mtDNA and ox-mtDNA,whereas ALDH2 decreased mtDNA and ox-mtDNA release compared to the control group.These results suggest that ALDH2 may rescue mitochondrial damage caused by oxidative stress and accumulation of toxic aldehydes.4.ALDH2 elevated acetylation level of HADHA through suppressing the translocation of HD AC3 from nuclei to mitochondriaTo further elucidate the mechanism by which ALDH2 regulates mtDNA and oxmtDNA,we studied mitochondrial membrane proteins except 4-HNE and ROS,while HADHA is a key protein in mitochondrial energy metabolism.Western blot results showed that sepsis significantly reduced the level of HADHA acetylation compared to the control group,although there was no change in total HADHA protein levels,and ALDH2 reversed this change.To further determine the role of HADHA in mitochondrial respiratory dysfunction,siRNA was used to silence hadha and it was found that hadha knockdown compromised the ALDH2-induced improvement in oxygen consumption.Similarly,knocking down hadha effectively impaired ALDH2induced improvement of pyroptosis.These results suggest that deacetylation of HADHA is associated with mitochondrial damage and pyroptosis of cardiomyocytes.Western blot results showed that knockdown of hdac3 could effectively rescue the deacetylation of HADHA.Although there were no significant differences in total HDAC3 protein levels among all groups,both HDAC3 fluorescence co-location staining and Western blot results showed that,compared with the control group,HDAC3 translocated to mitochondria after LPS and ATP stimulation,and ALDH2 inhibited its translocation.In addition,hdac3 knockdown effectively decreased the protein expression level of Caspase-1 p20 induced by LPS and ATP stimulation.Hadha knockdown increased the number of cells with NLRP3/ASC speck induced by ATP in LPS-primed cells,and Hdac3 knockdown attenuated this effect.When Hadha and Hdac3 were knocked down at the same time,Hadha knockdown effectively impaired the beneficial effect of Hdac3 knockdown for inhibiting NLRP3 inflammasome activation after LPS and ATP stimulation.Hdac3 knockdown also effectively inhibited the protein level of Caspase-1 p20.In addition,after interfering with both Hdac3 and Aldh2 by siRNA,it was found that Hdac3 interference remarkedly reversed the decreased acetylation level of HADHA induced by LPS and ATP with or without Aldh2 interference,it was found that Aldh2 knockdown significantly reduced HADHA acetylation after LPS and ATP stimulation,and Hdac3 knockdown remarkedly reversed the adverse consequences of Aldh2 deficiency.These results suggest that ALDH2 may play a protective role against myocardial pyroptosis in septic shock through the mitochondrial HDAC3/HADHA-NLRP3 inflammasome pathway.Conclusion1.Aldh2 knockout aggravates septic shock-induced cardiac dysfunction and mortality.2.ALDH2 attenuates myocardial pyroptosis through NLRP3 inflammasome/Caspase-1/GSDMD pathway in septic shock.3.ALDH2 inhibited the expression of NLRP3 and the activation of NLRP3 inflammasome through reducing mtDNA and ox-mtDNA.4.ALDH2 elevated acetylation level of HADHA through suppressing the translocation of HDAC3 from nuclei to mitochondria.