MAP4 Phosphorylation Aggravates Myocardial Hypoxic Damage Via Regulating Intercellular Mitophagy-NLRP3 Inflammasome Axis

BackgroundAt the early stage of severe burn,multiple organs,including the heart,are subjected to different degrees of hypoxic-ischemic damage because of the combined actions of various factors such as the increased permeability of vascular,the activation of renin angiotensin system and the vascular stenosis caused by endothelial cell damage and swelling.Mitochondria,as the main organelle providing energy for the heart,functions as the main effector organelle of ischemia-hypoxia injuried heart caused by severe burn.Damaged mitochondria induced by ischemia-hypoxia injury can be characterized by the release of reactive oxygen species（ROS）,mitochondrial DNA（mt DNA）,cytochrome c（Cytc）,Ca²⁺overload and other pathological molecules.These molecules can activate multiple pathological responses including mitophagy and NLR family pyrin domain containing 3（NLRP3）inflammasome.Up to now,the roles of mitophagy and NLRP3 inflammasome in hypoxic myocardial injury have been extensively studied and recognized.Recently,the investigations on mitophagy-NLRP3 inflammasome axis have been gradually increased,but the mutual regulation is still unclear.Especially,the function of mitophagy-NLRP3inflammasome axis in burn induced heart injury is little known.In addition,it has been reported that mitophagy-NLRP3 inflammasome axis exists between different cell types.In burned heart,myocardial cells and endothelial cells are the two main effector cells,so whether mitophagy-NLRP3 inflammasome axis exists in these two cells requires more efforts.Figuring this question out can not only clarify the interaction between mitophagy and NLRP3inflammasome,but also enrich the mechanism of burn induced myocardial injury.Besides,this study is able to encourage the establishment of new methods to alleviate severe burn induced hypoxic myocardial injury.Mitophagy,an essential part of selective autophagy,is a highly regulated process involved in the degradation of damaged or redundant mitochondria.To date,two mitophagy mechanisms have been proposed.One depends on PTEN-inducible putative kinase 1（Pink1）-Parkin pathway for the ubiquitination of mitochondrial proteins followed by interactions with the adaptor protein p62（SQSTM1）,which is responsible for connecting ubiquitin with microtubule-associated protein light chain 3（LC3,a marker of phagophore）.The other mechanism relies upon either proteins or lipids on the outer mitochondrial membrane as receptors for LC3.NIP3-like protein X（Nix/Bcl-2L）,Bcl-2,and FUN14 domain containing 1（FUNDC1）have been shown to act as LC3 receptors and are known as LC3-binding proteins.The LC3 interacting region（LIR）and ubiquitin-interacting motif（UIM）have been regarded as the functional domains in the LC3-binding proteins,which underly binding to the LC3LIR/AIM docking site（LDS）or UIM/AIM docking site（UDS）.LIR is a short linear motif that consists of up to 13 amino acids with a W/F/Y-XX-L/I/V core sequence（where X indicates sites that can be replaced by any amino acid）.The UIM domain consists of 20 amino acids with a principal sequence of XXXXX-L-XX-A-XXX-S-XXXXXXX,which is associated with the induction of ubiquitination and binding to ubiquitinated proteins.In mitophagy,the interaction between LIR or UIM-containing proteins and LC3 is the core step,which promotes the engulfment of mitochondria by autophagosomes.The process can be described as follows:after mitochondrial damage,the release of ROS,mt DNA and other molecules activates autophagy,and the damaged mitochondria are recoginized and engulfed by autophagosomes,and then are digested by lysosome.Within one cell,when mitophagy consumes damaged mitochondria,it also clears the stimulating factors of NLRP3inflammasome such as ROS,and results in inhibiting NLRP3 inflammasome activation.Moreover,mitophagy also directly clears NLRP3 inflammasome components including NLRP3,apoptosis-associated speck-like protein containing a CARD（ASC）and caspase1,and leads to NLRP3 inflammasome inhibition.However,studies have shown that mitophagy-NLRP3 inflammasome axis also exists between different cells,such as,mesenchymal stromal cells inhibit the NLRP3 inflammasome of macrophage by activating mitophagy.This evidence opens up a novel path to investigate the mutual regulation of mitophagy-NLRP3inflammasome axis.Inflammasomes,the well-known upstream process of pyroptosis and the principal initiator of the innate immune response,can be divided into various categories.The NLRP3inflammasome is one of the most familiar and consists of NLRP3,ASC,and caspase-1.Along with the activation of the NLRP3 inflammasome,pro-caspase-1 is catalyzed into its active form,then cleaves gasdermin（GSDM）family proteins,and leads to pyroptosis.Among the GSDM family,gasdermin D（GSDMD）is regarded as the primary effector molecule and is constitutively autoinhibited by the binding of its C-terminal（GSDMD-C）repressor domain to its N-terminal（GSDMD-N）pore-forming domain.During the induction of pyroptosis,inflammatory caspases,mainly caspase-1,have been reported to ease this autoinhibition by catalyzing the proteolytic cleavage of the interdomain loop,causing the liberation of the GSDMD-N pyroptotic inducer.And GSDMD-N could form pores on the cell membrane to induce pyroptosis and release a large number of pro-inflammatory factors such as interleukin-1β（IL-1β）and interleukin-18（IL-18）.Previous studies have showed that pro-inflammatory molecule tumor necrosis factorα（TNF-α）released by retinal Muller cells activates mitophagy of retinal epithelial cells.Moreover,IL-1βdirectly injures mitochondria and stimulates the release of mt DNA,resulting in mitophagy activation.These data indicate NLRP3inflammasome possesses the capability to act as the upstream signal of mitophagy activation because of the release of pro-inflammatory cytokines.Therefore,we assume that NLRP3inflammasome in cardiac endothelial cells activates mitophagy in cardiomyocytes in burned models.Microtubule-associated proteins（MAPs）are well-known cytosolic skeleton proteins that promote microtubule（MT）polymerization under physiological conditions.As one of the most intensively studied MAPs,microtubule-associated protein 4（MAP4）is ubiquitously expressed in nonneural cells,and its function is affected by phosphorylation.It is reported that the S696,S768 and S787 sites in the proline-rich region of the microtubule binding domain of human MAP4 are the key sites for phosphorylation.The phosphorylation of the above sites can regulate the depolymerization of MAP4 and MT,resulting in the imbalance of microtubule homeostasis.In hypoxic cardiomyocytes,MAP4 translocates to mitochondria and impaires it and induces mitochondrial apoptosis after its phosphorylation.And,in current study,after sequence alignment,LIR domain which directly interacts with LC3 has been discovered in MAP4.Therefore,MAP4 contains the basic conditions to function as a mitophagy receptor,indicating it possesses the potential to regulate mitophagy.In addition,our previous results have clarified that MAP4 phosphorylation is involved in the inflammatory response induced by lipopolysaccharide（LPS）and TNF-αin pulmonary endothelial cells.Considering LPS is a classical stimulating factor of NLRP3 inflammasome,so the relationship between MAP4 phosphorylation and NLRP3 inflammasome in endothelial cells exists in theory and needs to be clarified.Based on these evidences,MAP4 is likely to be a molecule that simultaneously activates endothelial cell NLRP3 inflammasome and cardiomyocytes mitophagy,so it is a useful tool to clarify the relationship of intercellular mitophagy-NLRP3 inflammasome axis.In burned models,the injury of endothelial cells is thought to be earlier than the hypoxic damage of cardiomyocytes,and tissue inflammation is the basis of other pathological reactions,and MAP4 phosphorylation has the potential regulatory capacities both in the NLRP3 inflammasome of endothelial cell and the mitophagy of cardiomyocytes.Therefore,we assume that in severe burn induced hypoxic cardiac injury,MAP4 phosphorylation regulates the mitophagy of cardiomyocytes by activating the NLRP3 inflammasome of endothelial cell.We found that（1）In cardiac endothelial cells,MAP4 phosphorylation activates NLRP3 inflammasome to injury its morphology and function;（2）In cardiomyocytes,MAP4 phosphorylation regulates mitophagy and hypoxic myocardial damage through its LIR domain;（3）In burned heart,NLRP3 inflammasome in endothelial cells may be the main cause of mitophagy activation in hypoxic cardiomyocytes.Thus,MAP4 phosphorylation plays an important role in regulating intercellular mitophagy-NLRP3 inflammasome axis,which not only enriches the relationship between mitophagy and NLRP3 inflammasome,but also provides a new treatment strategy for hypoxic myocardial injury at the early stage of severe burn.Materials and Methods1.To explore the influences of MAP4 phosphorylation on mitophagy,angiogenesis and NLRP3 inflammasome in myocardium,the MAP4 knock in（KI）mice were used.To achieve our purposes,the myocardium samples of MAP4 KI and wild-type（WT）mice were collected to detect the expressions of mitochondrial proteins including voltage-dependent anion channel1（VDAC1）,TOM20 and TIM23,autophagy-related protein LC3,angiogenic signaling pathway-associated proteins including angiopoietin 2（Ang2）,TIE2,vascular endothelial growth factor A（VEGFA）,vascular endothelial growth factor receptor 2（VEGFR2）,platelet endothelial cell adhesion molecule（PECAM-1/CD31）and CD34,and NLRP3 inflammasome associated proteins including NLRP3,ASC and caspase1 by Western blot（WB）,visualize myocardial mitophagosomes and endothelium morphology through transmission electron microscope（TEM）,and measure microvascular density through immunofluorescence（IF）.2.To elucidate the impacts of MAP4 phosphorylation on mitophagy activation in vitro,the primary cardiomyocytes of MAP4 KI and WT mice were isolated.Collect the whole protein of primary cardiomyocytes after a certain period of culture,and the proteins were used for detecting the expressions of autophagy-related protein LC3 and mitochondrial proteins including VDAC1,TOM20 and TIM23 through WB,measuring the number of mitophagosomes by TEM and assaying the condition of mitophagy by IF.In addition,to further assess the effects of MAP4 phosphorylation on mitophagy in vitro,overexpression adenoviruses encoding mouse MAP4（alanine,Ala）and MAP4（Glutamate,Glu）were constructed,and then primary cardiomyocytes were transfected with these adenoviruses to mimic intracellular MAP4 dephosphorylation or hyperphosphorylation form of MAP4.And then,the expression of autophagy-related protein LC3,the contents of mitochondrial proteins（VDAC1,TOM20 and TIM23）,the number of mitophagosomes and the condition of mitophagy were once again measured.3.The hypoxia model of mice was established to measure the impacts of MAP4phosphorylation on hypoxia-induced mitophagy in vivo.After hypoxia exposure（7.5%O₂ for7 days）,the myocardium samples were collected to test the variations of autophagy related proteins and the number of mitophagosomes through WB or TEM.Next,to identify the effects of MAP4 phosphorylation on hypoxia-induced mitophagy in vitro,the primary cardiomyocytes isolated from MAP4 KI mice or transfected with MAP4（Glu）adenovirus were used as effective MAP4 phosphorylation models.And then,after hypoxia（1%O₂ for 9hours）,the cardiomyocytes were collected for analyzing the expression of autophagy related molecules using WB,and detecting the number of mitophagosomes by TEM and assaying the condition of mitophagy via IF.Additionally,to mimic intracellular dephosphorylation of MAP4,the MAP4（Ala）adenovirus or microtubule-affinity regulating kinase 4（MARK4）small interfering RNA（si RNA）was transfected to cardiomyocytes.After that,the samples were used for analyzing autophagy related proteins and detecting mitophagosomes.4.In order to clarify the interaction between MAP4 and LC3,we firstly collected the whole protein of primary cardiomyocytes for co-immunoprecipitation（IP）experiments.On this basis,MAP4 and LC3 were expressed in vitro,and glutathione S-transferase（GST）-pull down experiment was then carried out to clarify the direct interaction between them.Subsequently,the amino acids at MAP4 34-37 and 47-50 were knocked out to determine the direct interaction sites between MAP4 and LC3.5.To investigate the effects of MAP4 phosphorylation on angiogenesis,the tube formation assay was conducted.Human umbilical vein endothelial cells（HUVECs）were pre-treated with specific adenovirus（MAP4 Glu and MAP4 Ala）or NLRP3 si RNA for 24 h or 48h.Next,HUVECs were collected and counted strictly,and 10000 cells were inoculated on the surface of matrix gel for 24 h,and then the cells were observed and photographed using a microscope.Then,the total length of tubes was calculated using Image J software.6.To explore the influences of MAP4 phosphorylation on NLRP3 inflammasome in vitro,the HUVECs were incubated with MAP4（Glu）and MAP4（Ala）adenoviruses for 48hours.Then,the protein samples were collected for detecting NLRP3 inflammasome proteins（NLRP3,ASC,caspase1）expressions through WB,and the cells were acquired to operate propidium iodide（PI）staining and NLRP3/ASC costaining.The medium samples were obtained for measuring IL-18 concentration and lactate dehydrogenase（LDH）release.Results1.MAP4 phosphorylation activated myocardial mitophagy under normoxic condition.（1）Firstly,we collected the myocardium of WT and MAP4 KI mice to detect the expressions of autophay related proteins,the contents of mitochondrial proteins and the number of mitophagosome.And the results indicated that when compared with WT mice,the LC3 expression was increased by 1 fold,and the expressions of mitochondrial proteins（VDAC1,TOM20,TIM23）were decreased significantly,and the number of mitophagosome was incremented in MAP4 KI mice.These data demonstrated that MAP4 phsophorylation activated myocardial mitophagy under normoxia in vivo.（2）Then,we collected the primary cardiomyocytes of WT and MAP4 KI mice to illustrate the influence of MAP4 phosphorylation on mitophagy in vitro.When compared with the primary cardiomyocytes from WT mice,the expressions of mitochondrial proteins（VDAC1,TOM20 and TIM23）were decreased by 40%,the ratio of LC3-II/LC3-I was increased to about 1.5 and the number of mitophagosome was augmented to 4-5/cells in the primary cardiomyocytes of MAP4 KI mcie.Together,these results indicated that MAP4phosphorylation activated myocardial mitophagy under normoxia in vitro.2.MAP4 phosphorylation aggragated mitophagy induced by hypoxia.（1）To elucidate the effect of MAP4 phosphorylation on mitophagy under hypoxia in vivo,the WT and MAP4 KI mice were exposed to hypoxia（7.5%O₂ for 7 days）.And their myocardium samples were collelected to detect the number of mitophagosome,the ratio of LC3-II/LC3-I,and the expressions of mitochondrial proteins.The results suggested that under hypoxia,MAP4 phosphorylation further improved the ratio of LC3-II/LC3-I by about 45%,increased the number of mitophagosomes to 3-4/cells,and decreased the contents of mitochondrial proteins including VDAC1,TOM20 and TIM23 by about 30%.Therefore,MAP4 phosphorylation strengthened the activation of myocardial mitophagy induced by hypoxia in vivo.（2）To illustrate the influence of MAP4 phosphorylation on mitophagy under hypoxia in vitro,the primary cardiomyocytes of WT and MAP4 KI mice were exposed to hypoxia（1%O₂ for 9 hours）.After hypoxia treatment,the cardiomyocytes were collected to exam the number of mitophagosome,measure the condition of mitophagy,detect the ratio of LC3-II/LC3-I and assess the expressions of mitochondrial proteins.The results suggested that under hypoxia,MAP4 phosphorylation further improved the ratio of LC3-II/LC3-I by about45%,increased the number of mitophagosomes to 5-6/cells,and decreased mitochondrial proteins including VDAC1,TOM20 and TIM23 contents by about 50%.Therefore,MAP4phosphorylation accelerated the activation of mitophagy induced by hypoxia in vitro.3.MAP4 dephosphorylation alleviated mitophagy activated by hypoxia.（1）The MAP4（Ala）adenovirus was a common way to induce intracellular MAP4dephosphorylation,so we employed it as the first way to mimic MAP4 dephosphorylation in primary cardiomyocytes.After 48 hours incubation with MAP4（Ala）adenovirus,the cardiomyocytes were obtained to measure the indicators of mitophagy.The results showed that when compared with control group,MAP4（Ala）reduced the ratio of LC3-II/LC3-I by about 50%,decreased the number of mitophagosomes to 2-3/cells,and increased the expressions of mitochondrial proteins（VDAC1,TOM20 and TIM23）by nearly 60%.It indicated that MAP4 dephosphorylation induced by MAP4（Ala）inhibited hypoxia-activated mitophagy.（2）Then,MARK4 si RNA had been utilized as another way to mimic intracellular MAP4phosphorylation for MARK4 was a clarified upstream signal of MAP4 phsophaorylation.After incubated with MARK4 si RNA for 24 hours,the cardiomyocytes were acquired to measure mitophagy related indicators including mitophagosome number,mitochondrial proteins expressions and autophagy associated proteins.The results showed that when compared with control group,MARK4 si RNA reduced the ratio of LC3-II/LC3-I by about40%,decreased the number of mitophagosomes to 3-4/cells,and increased the expressions of mitochondrial proteins（VDAC1,TOM20 and TIM23）by nearly 50%.It indicated that MAP4dephosphorylation induced by MARK4 si RNA down-regulated hypoxia-activated mitophagy.4.To elucidate the mechanism of MAP4 phosphorylation regulating mitophagy,we focused on LIR domain.After detailed sequence alignment,two LIR domain（34-37,47-50）were discovered in MAP4.Next,IP was used to identify whether interaction existed in MAP4/LC3,and the result was positive.Then,GST-pull down assay was conducted,and the results indicated that direct interaction lied in MAP4/LC3.Futhermore,MAP4 LIR^△47,50 was confirmed to be the interactive sites of MAP4/LC3.Then,the LIR（47,50）-mutant MAP4（Glu）adenovirus was constructed,and it recovered the decreased expressions of mitochondrial proteins（VDAC1,TOM20 and TIM23）induced by hypoxia to control level,and reduced the myocardial cell toxicity increased by hypoxia to about 40%.In summary,MAP4 mediated hypoxia-induced mitophagy and myocardial cell injury through utilizing its LIR（47-50aa）.5.MAP4 phosphorylation damaged the morphology and function of cardiac endothelial cells.（1）In order to clarify the effect of MAP4 phosphorylation on the morphology and function of cardiac endothelial cells,we detected the expressions of endothelial cell markers,measured the contents of angiogenesis signaling pathway related proteins and visualized cardiac microvascular density.The results suggestd that MAP4 phosphorylation could seriously damage the endothelial cell morphology,reduce the number of capillaries by about30%（CD31 and Lectin）staining,and decrease the expressions of angiogenesis signaling pathway including CD31,CD34,Ang2,TIE2,VEGFA and VEGFR2.These data demonstrated MAP4 phosphorylation destroyed cardiac endothelial cells.（2）To investigate the influence of MAP4 phosphorylation on the function of endothelial cells,we conducted in vitro tube formation experiment.The results suggested that MAP4phosphorylation could reduce the length of the tube by about 50%.The result showed that MAP4 phosphorylation could damage the function of endothelial cells.6.MAP4 phosphorylation damaged endothelial cells through NLRP3 inflammasome.（1）To measure the influence of MAP4 phosphorylation on NLRP3 inflammasome in heart,we acquired the myocardium of WT and MAP4 KI mice for corresponding experiments.By detecting the expressions of NLRP3 inflammasome-related proteins such as NLRP3,ASC and caspase-1 p10 in the myocardium of WT and MAP4 KI mice,it was found that MAP4phosphorylation could increase the expressions of NLRP3 inflammasome-related proteins,indicating that it could activate NLRP3 inflammasome in myocardium.（2）.To measure the influence of MAP4 phosphorylation on NLRP3 inflammasome in endothelial cells,we acquired the cells treated by MAP4（Ala）or MAP4（Glu）adenovirus for48 hours.It was found that the expressions of NLRP3 inflammasome-related proteins（NLRP3,ASC and caspase1）increased nearly 1-fold and the co-localization of NLRP3/ASC increased about 3-fold after MAP4 Glu treatment.These data indicated that MAP4phosphorylation could activate NLRP3 inflammasome in endothelial cells.（3）.To clarify the role of NLRP3 inflammasome in the impaired effects of MAP4phosphorylation on endothelial cells,NLRP3 si RNA was used to inhibit NLRP3inflammasome.Through tube formation experiment,it was clear that after NLRP3inflammasome was inhibited,the inhibitory effect of MAP4 phosphorylation on the length of the tube was restored by 40%,indicating that MAP4 phosphorylation damaged endothelial cell function by activating NLRP3 inflammasome.Conclusions1.In endothelial cells,MAP4 phosphorylation damages the cells and their functions through activating NLRP3 inflammasome.2.In cardiomyocytes,MAP4 phosphorylation strengthens mitophagy induced by hypoxia through its LIR（47-50aa）domain,making MAP4 a potential mitophagy receptor.3.MAP4 phosphorylation regulates hypoxic myocardial injury through mediating mitophagy.4.The NLRP3 inflammasome in cardiac endothelial cells may be the main trigger of mitophagy activation in burned induced myocardial hypoxic injury.