The Molecular Mechanisms Of Ubiquitin-like Protein FAT10 Regulates Cardiac Fibrosis After Myocardial Infarction

Background:Cardiac fibrosis is a pivotal pathological process of myocardial remodeling after myocardial infarction（MI）.Excessive cardiac fibrosis will damage left ventricular compliance,leading to heart failure and even cardiac sudden death（SCD）.Therefore,inhibiting cardiac fibrosis after MI becomes a critical treatment strategy to improve the prognosis of patients with MI.To date,numerous research has noted the activity of ubiquitin and ubiquitin-like proteins（UBLs）on MI-induced cardiac fibrosis.Human HLA-F adjuvant transcript 10（FAT10）,as a member of the ubiquitin like protein family,can directly mediate the degradation of target proteins through proteasome,and regulate various biological functions such as cell apoptosis.In our previous studies,FAT10 was firstly showed to be expressed in heart.In addition,FAT10 could repressed MI injury,and protect against MI-accompanied arrhythmia.However,whether FAT10 acts in the development of cardiac fibrosis after MI is unclear.Objective:The aim of this study is to explore the regulatory effect of FAT10 on cardiac fibrosis after MI and its specific molecular mechanism,and to provide a theoretical basis for finding a novel targets to treat cardiac fibrosis after MI.Methods:In the present study,we used the method of CRISPR/Cas9 to construct global FAT10 knockout(Fat10^-/-)mice.Then we established the MI mouse model and cardiac fibroblasts（CFs）fibrosis model,to explore the regulatory effect of FAT10 on cardiac fibrosis after MI and its molecular mechanism both in vivo and vitro.The details as follows:1.To explore the effect of FAT10 on cardiac fibrosis after MI in vivoMale Fat10^-/-mice were used at 8 weeks of age.Age and sex-matched wild-type（WT）littermates were used as controls.MI mouse model was established by Left anterior descending coronary artery（LAD）.The degree of cardiac fibrosis was detected by Masson staining;Western blotting,immunohistochemistry and biofluorescence were used to detect the expression changes of related fibrotic markers（a-SMA,Coll-1,Coll-3）;Doppler echocardiography was used to observe the changes of cardiac function.Besides,as a rescue experiment,Fat10 overexpressed adenovirus（ad-Fat10）was injected at the apical during the LAD operation process with the equivalent amount of vector virus injection（ad-con）.A similar cardiac function evaluation was done with doppler echocardiography and histology assays.2.Effect of FAT10 on proliferation and migration of CFs in vitroNRCFs were treated with 10 ng/m L TGF-β1 for 24h to establish fibrosis model.To determine whether FAT10 modulates cardiac fibrosis in vitro,cells were transfected with ad-Fat10 and sh-Fat10 viruses before TGF-β1 treatment.Ed U test was used to detect the effect of FAT10 on CFs proliferation;Transwell,would-healing and RTCA experiments were used to detect the effect of FAT10 on CFs migration.3.To find the potential protein that FAT10 affects the process of myocardial fibrosisImmunoprecipitation-mass spectrometry（IP-MS）analysis was carried out to screen the potential interacting proteins of FAT10.Then we used Western Blot to further verify.4.Identification of Smad3 as a key factor mediating the effect of FAT10 on cardiac fibrosis.In mouse model,Smad3-specific inhibitor SIS3 was selected for in vivo rescue experiments.The degree of cardiac fibrosis was detected by Masson staining;The expression changes of related fibrotic markers（a-SMA,Coll-1,Coll-3）were detected by Western blot,immunohistochemistry and biofluorescence;The cardiac function was detected by echocardiography.In vitro assays,Smad3 overexpression and interfering virus were constructed for cell rescue assay.Edu assay was performed to detect the effect of CFs proliferation;Transwell,would-healing,and RTCA assays were performed to detect the effect of migration.5.To determine the specific mechanism of FAT10 regulating Smad3We used the protein synthesis inhibitor（CHX）,and proteasome inhibitor（MG132）to determine whether FAT10 affects the degradation of Smad3.Further,RNA interference of the FAT10 specific E1-activated enzyme（UBA6）,the E2-transferase enzyme（UBE2Z）ubiquitin,and the interference of Ubiquitin were used to detect the specific degradation mechanism.Then,the interaction between FAT10and Smad3 protein was identified by immunoprecipitation,immunofluorescence and GST Pull down.The Smad3 fragment and mutant plasmid were constructed to find the specific binding fragment and site of FAT10 and Smad3.Furthermore,Smad3mutant virus was constructed for functional experiment to determine whether the inhibition of FAT10 on cardiac fibrosis after MI was related to the direct targeting of Smad3.Results:1.FAT10 deficiency aggravated ischemic-induced cardiac fibrosis in vivoCompared with control group,MI-Fat10^-/-showed aggravated cardiac fibrosis,a higher expressions of typical fibrosis-related proteins（α-SMA,collagen I and collagen III）and deteriorated cardiac function.2.Overexpression of FAT10 in mice heart alleviated the ischemic-induced cardiac fibrosisRecombinant adenovirus expressing FAT10 were used in Fat10^-/-mice heart tissue by vector infection.Overexpression of FAT10 was associated with decreased cardiac fibrosis,diminished expressions of fibrotic markers and improved cardiac function.3.FAT10 reversed TGF-β1-induced fibrotic response in vitroOverexpression of FAT10 significantly inhibited TGF-β1-induced proliferation,migration and transformation of CFs.Conversely,inhibition of FAT10 significantly promoted TGF-β1-induced CFs value addition,migration and transformation.4.Smad3 plays a critical role of FAT10 regulating cardiac fibrosis after MISmad3 was first identified as a substrate binding protein of FAT10 by IP-MS technique,and Western Blot assay showed that FAT10 negatively regulates the protein expression of Smad3.In vivo rescue assay indicated that Smad3-specific inhibitor-SIS3 could block the exacerbation of myocardial fibrosis after MI induced by FAT10 deficiency.Additionally,in vitro experiments further verified that the inhibitory effect of overexpressed FAT10 on cellular fibrosis response disappears when Smad3 is disrupted.5.FAT10 promoted Smad3 de gradation through FAT10-proteasome system（FPS）The degradation rate experiment showed that after overexpression of FAT10,the expression of Smad3 protein decreased significantly compared with the control group with the prolongation of CHX treatment time;However,after the FAT10 is interfered,the decline rate is gentle.It is suggested that FAT10 can promote the degradation of Smad3,but this effect is blocked by the virus that interferes with FAT10 specific E1activating enzyme（Uba6）and the virus that interferes with FAT10 specific E2transferase（UBE2Z）.The above results confirm that FAT10 promotes Smad3degradation via FAT10 mediated proteasome system（FPS）.6.FAT10 binding to K378 lysine residue on Smad3 directlyTo explore the specific mechanism of FAT10 regulation of Smad3.We divided the Smad3 protein into 3 functional fragments according to its structure and constructed the corresponding Smad3 fragment plasmids.GST-Pull down experiments revealed that FAT10 interacted with Smad3-D3.Further,we mutated the four lysine（Lys,K）sites of the Smad3-D3 fragment to arginine（Arg,R）（K333R,K341R,K378R,K409R）,and immunoprecipitation experiments revealed that the interaction between Smad3 and FAT10 was lost after mutating the K378R site,and the above results confirmed that FAT10 binds to Smad3-D3 through and K378 site in the Smad3-C terminus was directly bound.7.FAT10 exerts its regulatory effect on fibrosis by targeting the Smad3-K378sitesTo further explore the K378 lysine binding sites of FAT10 and Smad3 in the regulation of cardiac fibrotic.NRCFs were simultaneously transiently infected with Smad3-K378R and ad-con or ad-Fat10 virus.The results revealed that when co-transfected with Smad3-K378R-wt and ad-con or ad-Fat10 virus,overexpression of FAT10 inhibited the function of Smad3,fibrotic marker protein,cell proliferation and migration.However,when Smad3 was mutated,the repression functions of FAT10 on Smad3 and fibrotic phenotype were abolished.In summary,our results show that FAT10 directly binds to Smad3 through the K378 site.Conclusion:1.Knockdown of FAT10 was found to aggravate myocardial fibrosis after infarction in mice for the first time,and overexpression of FAT10 improved the above phenotype.2.Overexpression of FAT10 inhibited the TGF-β1-induced cellular fibrosis response3.Smad3 is a key effector of FAT10 to inhibit post-infarction myocardial fibrosis4.FAT10 promotes the degradation of Smad3 via FPS by directly binding to the Smad3-K378 site,which in turn inhibits its expression.