The Underlying Mechanism Of Ubiquitin C-terminal Hydrolase L1 On Cardiac Fibrosis Following Myocardial Infarction

Background Abnormal cardiac fibrosis indicates cardiac dysfunction and poor prognosis in myocardial infarction(MI)patients.Many studies have demonstrated that the ubiquitin proteasome system(UPS)plays a significant role in the pathogenesis of fibrosis.Ubiquitin Cterminal hydrolase L1(UCHL1)is a member of the UPS,which is associated with fibrosis of various heart diseases.However,whether UCHL1 regulates cardiac fibrosis following MI has yet to be determined.Methods To determine if UCHL1 regulates cardiac fibrosis,the MI model of C57 mice was established by permanent ligation of left anterior descending coronary artery.The expression of UCHL1 was detect in sham and MI hearts at 1,7 and 14 days after operation using immunohistochemistry and the protein levels of UCHL1,Col1 and ?-SMA was detect in sham and MI hearts at 1,7 and 14 days after operation using western blot.To assess whether UCHL1 mediates post-MI cardiac fibrosis in vivo,MI mice were intraperitoneally injected with UCHL1 inhibitor LDN57444(LDN)for 14 days after operation.The cardiac fibrosis and infarct size of MI was detected by western blot and Masson's trichrome staining,cardiac function was examined by echocardiography.Primary cardiac fibroblasts(CFs)were isolated from the heart of normal C57 mice,and TGF-?1 was used to induce the activation of CFs.The the protein levels of UCHL1 and Col1 and ?-SMA was detect in CFs treated with and without TGF-?1 for 12 h,24h and 36 h.We next suppressed activity of UCHL1 in TGF-?1 induced CFs with LDN,and detected the protein level of Col1 and ?-SMA by western blot.Afterwards,TGF-?1 induced CFs were treated with UCHL1 siRNA,the protein levels of Col1 and ?-SMA was determined by western blot,and the protein level of ?-SMA was examined by immunofluorescence.To identify the underlying mechanism of UCHL1 on post-MI fibrosis,we performed immunoprecipitation-mass spectrometer(IP-MS)in TGF-?1-induced CFs to find UCHL1 interactors.Co-immunoprecipitation was performed to identify whether there exists an interaction between UCHL1 and glucose-regulated protein of 78 k Da(GRP78)in CFs induced by TGF-?1.Subsequently,the co-localization between UCHL1 and GRP78 in sham-operated heart / MI heart and CFs with / without TGF-?1 treatment was detected by immunofluorescence.To find out whether GRP78 is regulated by UCHL1,we determined the protein level of GRP78 in CFs transfected with scrambled siRNA / UCHL1 siRNA.Cycloheximide chase assay was performed to determine the protein level of GRP78 in CFs transfected with scrambled siRNA / UCHL1 siRNA treated with CHX at 0h,1h,2h,3h,4h,and 5h.Protein degradation was inhibited using MG132 in TGF-?1 stimulated CFs transfected with scrambled siRNA / UCHL1 siRNA,then the immunoprecipitation was performed using GRP78 antibody in the harvested protein samples,and the ubiquitination level of GRP78 protein was determined by Western blot.To deternine if the effect of UCHL1 on fibrosis was related to GRP78,we examined the protein levels of GRP78 in CFs with / without UCHL1 knockdown with / without TGF-?1 stimulation.TGF-?1 stimulated CFs with / without UCHL1 knockdown treated with / without GRP78 inhibitor HA15 and the protein levels of Col1 and ?-SMA were detected.Results In the study,we found that the protein levels of UCHL1 and Col1 and ?-SMA was increased at 7 days and 14 days post MI.LDN significantly reduced the protein levels of Col1 and ?-SMA in MI hearts at 14 days,dramatically prevented MI-associated infarct size and cardiac fibrosis and improved cardiac function.And the expression of UCHL1 and Col1 and ?-SMA in CFs was significantly increased at 24 and 36 hours after TGF-?1 stimulation.LDN also significantly inhibited the protein levels of Col1 and ?-SMA in CFs induced by TGF-?1.Meanwhile,we found that UCHL1 siRNA treatment downregulated the protein levels of Col1 and ?-SMA in TGF-?1 induced CFs.Subsequently,we screened the interactor of UCHL1 using IP-MS with number of PSMs > 20 as the threshold,and 6 candidates were selected as following: Endoplasmic reticulum chaperone Bi P(also known as GRP78),Annexin A6,Carnitine Opalmitoyltransferase 1 muscle isoform,Glycogen phosphorylase brain form,Elongation factor Tu mitochondrial and Cysteine and glycine-rich protein 3.We further confirmed the interaction between UCHL1 and GRP78 by immunoprecipitation in TGF-?1 induced CFs.Immunofluorescence staining showed that UCHL1 colocalised with GRP78 in fibrotic areas of MI hearts as well as in CFs with/without TGF-?1 treatment.We found that knockdown of UCHL1 in CFs elevates the GRP78 protein levels.Cycloheximide chase assay found that the rate of GRP78 degradation was significantly decreased with UCHL1 knock down.Ubiquitination assay found downregulation of UCHL1 by UCHL1 siRNA decreased polyubiquitination of GRP78.We found that GRP78 protein levels were upregulated by TGF-?1 and knockdown of UCHL1 led to greater increases in GRP78 levels.The GRP78 inhibitor HA15 abolished the downregulation of Col1 and ?-SMA in response to UCHL1 siRNA in CFs with TGF-?1 stimulation.Conclusions UCHL1 is upregulated in post MI mouse hearts.Inhibition of UCHL1 by LDN alleviated post-MI fibrosis and improved cardiac function.UCHL1 is also upregulated in CFs treated with TGF-?1.Both inhibition and knockdown of UCHL1 downregulated TGF-?1-mediated activation of CFs.UCHL1 interacts with GRP78.UCHL1 prompted GRP78 degradation by interacting with and ubiquitinated it.The effect of UCHL1 on cardiac fibrosis is due to its control of GRP78.This work identifies that the UCHL1-GRP78 axis is involved in cardiac fibrosis after MI and the newly found mechanism may provide a pivotal direction to alleviate the maladaptive cardiac fibrosis following MI.