| BackgroundMyocardial infarction(MI)is a serious cardiovascular disease that endangers the physical and mental health of human beings.The development and maturity of reperfusion therapy technology and the establishment and perfection of infrastructure have greatly reduced the mortality rate of MI and improved the prognosis of patients,but chronic heart failure caused by ischemic events affects more and more patients.Studies have shown that myocardial fibrosis after MI plays an important role in the progression of chronic heart failure,and the degree of myocardial fibrosis is closely related to the increased mortality and hospitalization rate of heart failure patients.Therefore,inhibition of myocardial fibrosis in the treatment of heart failure is of great significance.Excessive deposition of collagen in extracellular matrix is the main sign of myocardial fibrosis.It is important to study the involvement of collagen in myocardial fibrosis in the treatment of heart diseases.Collagen Ⅰ and Collagen Ⅲ are the major collagen proteins in fibrosis.At present,most of the studies focus on the transcription level of collagen mRNA,and few studies on post-transcription.Collagen biosynthesis is a complex process that requires multiple co-translational transport and post-translational modifications in the endoplasmic reticulum.Among them,lysine modification is a highly regulated sequential process that occurs inside and outside the cell,with specific hydroxyllysine residues in the helical domain being glycosylated with the addition of galactose or glucose.Collagen beta(1-O)galactosyltransferase 25 domain 1(GLT25D1)is the key collagen glycosyltransferase for collagen glycosylation,which is initiated during its post-translational modification by catalyzing the transfer of β-galactosyl groups to hydroxylysine(Hyl)residues of collagen.However,its role in myocardial fibrosis after myocardial infarction remains unclear,particularly in cardiac fibroblasts,the major collagen-producing cells associated with myocardial fibrosis.AimsThe aim of this study was to clarify the role of GLT25D1 in myocardial fibrosis after myocardial infarction and its possible mechanism,so as to find a drug target for improving or treating myocardial fibrosis after myocardial infarction.Methods1.The mouse model of MI was established.The survival curve was drawn to analyze the difference of survival time.2.The mouse model of MI was established.On the 14 th day after operation,the cardiac function of mice was evaluated by echocardiography.HE staining was used to observe the morphological changes of histiocytes.Masson staining was used to observe the distribution of collagen fibers in myocardial tissue to evaluate the degree of fibrosis.Immunohistochemical staining was used to detect the expression of CD31,the vascular endothelial marker,observe the microvessel density(MVD)and evaluate the neovascularization.The myocardial cell membrane was displayed by WGA fluorescence staining to judge the hypertrophy of myocardial cells.3.The proteins of the infarct border area were extracted,and the differentially expressed proteins(DEPs)14 days after myocardial infarction were detected and screened by 4D Label-free quantitative proteomics.The biological functions of the DEPs were analyzed by bioinformatics technology,and the fibrosis-related items in GO analysis were sorted out to screen target proteins.4.At the tissue level,collagen deposition was assessed by immunohistochemical staining of anti-Col 1 antibody and anti-Col 3 antibody.The expression of α-SMA,Col 1,Col 3 and FN were detected by Western Blot.And Western Blot was also used to verify the differential expression of GLT25D1 in the heart tissue of myocardial infarction mice.5.Primary cardiac fibroblasts were isolated from the hearts of C57BL/6 newborn mice at 1-3 days of age by collagenase Ⅰ digestion and differential attachment.Vimentin,the marker of fibroblast,was detected by immunofluorescence staining to determine its purity.6.Appropriate GLT25D1 small interfering RNA(si-GLT25D1)was designed and transfected into primary cardiac fibroblasts at a final concentration of 100 nM for 48 h.The silencing efficiency of siRNA was detected by qRT-PCR.And the inhibitory effect of siRNA on target gene expression was detected by Western Blot.7.The expressions of Col 1,Col 3,α-SMA,FN,TGF-β1,Smad 3 and p-Smad3 in primary cardiac fibroblasts transfected with si-GLT25D1 were used Western Blot to detect.8.Male C57BL/6 mice aged 4-6 weeks were injected via tail vein with an AAV9 viral vector overexpressing GLT25D1(AAV9-GLT25D1).After 4 weeks,echocardiography was performed and frozen sections were made.The expression of green fluorescent protein(GFP)in heart tissue was observed under an upright fluorescence microscope,and the effect of GLT25D1 overexpression was detected by Western Blot.9.Mouse model of myocardial infarction was established 4 weeks after tail vein injection of AAV9-GLT25D1.On the 14 th day after operation,the mice were examined by echocardiography and the hearts were sampled.HE staining was used to observe the morphological changes of histiocytes.Masson staining was used to observe the distribution of collagen fibers in myocardial tissue to evaluate the degree of fibrosis.In addition,Western Blot was used to detect expression levels of Col 1 and Col 3.Results1.InMI group,5 mice died on the 1st,3rd and 5th day after operation.The main cause of death were cardiac rupture,respiratory obstruction and hemothorax.After 42 days of continuous follow-up,10 mice in Sham group and 7 mice inMI group survived,P = 0.0251 by Log-Rank test,and the difference was statistically significant.2.Compared with Sham group,left ventricular ejection fraction(EF)and fractional shortening(FS)were decreased,left ventricular end-systolic diameter(LVIDs)and end-diastolic diameter(LVIDd)were significantly increased,and left ventricular anterior wall thickness was significantly thinner inMI group.HE staining showed that the myocardial cells in Sham group were arranged regularly,while inMI group,a large number of myocardial cells were necrotic,inflammatory cells were infiltrated,myocardial interstitial fibers were proliferated,and the orientation of myocardial fibers was disordered.Masson staining showed that collagen fibrils were deposited and the degree of fibrosis was increased inMI group.Moreover,CD31 immunohistochemical staining showed that the number of newly formed capillaries increased inMI group compared with Sham group.WGA staining results showed that the cross-sectional area of myocardial cells in non-infarcted zone was increased inMI group compared with Sham group.3.4D Label-free quantitative proteomics analysis identified 1053 DEPs in the infarct border region of the heart,of which 609 were up-regulated.77 fibrosis-related proteins were obtained by screening fibrosis-related items from GO enrichment analysis.GLT25D1 is a protein that has not been studied and may be involved in myocardial fibrosis.4.Compared with Sham group,the expression of Col Ⅰ and Col Ⅲ inMI group was significantly increased detected by immunohistochemical study on pathological section of mouse heart.Western Blot results showed that the levels of fibrosis markers inMI group were significantly higher than those in Sham group.Furthermore,the expression of GLT25D1 was significantly increased in the infarct border region of MI mice.5.qRT-PCR and Western Blot results showed that the mRNA and protein expression of GLT25D1 were significantly decreased in si-GLT25D1 treated primary cardiac fibroblasts 48 h after transfection.6.After 48 h of transfection,the expression levels of α-SMA,Col 1,Col 3 and FN in si-GLT25D1 group were significantly lower than those in si-NC group.Furthermore,the expression levels of TGF-β1 and p-Smad 3 / Smad 3 were significantly decreased after GLT25D1 silencing.7.Echocardiography was performed 4 weeks after tail vein injection of AAV9-GLT25D1.The results showed that the overexpression of GLT25D1 had no significant effect on the cardiac function of mice in physiological condition.Western Blot analysis showed that the expression of GLT25D1 in AAV9-GLT25D1 group was significantly higher than that in saline group and AAV9-vector group,suggesting that the overexpression of GLT25D1 in vivo was successful.8.Myocardial infarction model was established after overexpressing GLT25D1 in vivo.After 14 days,echocardiography results showed that compared with MI+AAV9-vector group,MI+AAV9-GLT25D1 group further aggravated the cardiac function damage,EF and FS were significantly reduced.Compared with MI+AAV9-vector group,GLT25D1-overexpressing group had significantly increased Col 1 and Col 3 expression in cardiac tissue.HE staining and Masson staining confirmed that myocardial fibrosis was significantly increased inMI+AAV9-GLT25D1 group.ConclusionGLT25D1 expression is increased in myocardial fibrosis after myocardial infarction,and inhibition of GLT25D1 can affect the activation of cardiac fibroblasts.The mechanism may be that GLT25D1 is involved in myocardial fibrosis after myocardial infarction through TGF-β1/Smad 3 signaling pathway. |
