Experimental Research On The Effect And Molecular Mechanism Of CTRP9 In Inhibiting The Activity Of Cardiac Fibroblasts And Ameliorating Myocardial Fibrosis After Myocardial Infarction

Background:With the implementation of the early reperfusion strategies and the introduction of new pharmacological methods,the 30-day mortality rate of acute myocardial infarction(AMI)has been reduced by 60%,and the prognosis has been greatly improved during acute cardiac events,but the increased survival rate has led to a significant increase in patients with chronic heart failure after myocardial infarction.Chronic heart failure seriously affects patients’ cardiac function and quality of life,and also significantly increases the risk of death.Fibrosis after myocardial infarction is an important cause of the development of chronic heart failure.The expression of TGF-β in myocardial tissue is significantly increased in animal experimental models of myocardial infarction and heart failure,and plays a key role in the process of myocardial fibrosis.TGF-β signal can induce the transformation of fibroblasts to myofibroblasts,and myofibroblasts are the main cellular source of extracellular matrix(ECM)protein deposition in the fibrotic region.Fibroblast TGF-β signaling pathway has been explored for a long time as a target for the treatment of fibrosis,however,no effective therapeutic targets and methods have been developed.Complement 1q/tumor necrosis factor-related proteins(CTRPs)and adiponectin have similar structural and biochemical characteristics,both of which belong to the complement 1q protein family;CTRP9 has the highest degree of amino acid identity to adiponectin in its globular Clq domain in the CTRP paralogs,which is highly expressed in the heart as a secreted glycoprotein;CTRP9 participates in the occurrence and development of various cardiovascular metabolic disorders and related risk factors,and plays an important role in the protection of cardiovascular disease,the regulation of inflammation and the regulation of glucose and lipid metabolism.Our previous research has shown that CTRP9 significantly inhibits cardiomyocyte apoptosis via PKA pathway,and play a significant role in myocardial protection,thereby ameliorating pathological remodeling after myocardial infarction;CTRP9’s protective effect on cardiovascular diseases,especially the protection of late cardiac contraction function is related to its anti-cardiac fibrosis.The molecular mechanism has not been fully clarified,which has become an obstacle to its further research.Objective:To investigate the effect and molecular mechanism of CTRP9 on reducing the activity of cardiac fibroblasts and myocardial fibrosis after AMI in vivo and in vitro,and to clarify the following questions:1)Whether CTRP9 alleviates myocardial fibrosis after AMI,improves cardiac contractile function?2)Whether CTRP9 affects the role of TGF-β1 in promoting fibrosis after AMI by PKA?3)Whether CTRP9 inhibits the proliferation,the transformation to myofibroblasts and the secretion of fibrosis-related proteins of CFs induced by TGF-β1?What is the molecular mechanism?Part Ⅰ CTRP9 improves cardiac function and ameliorates cardiac fibrosis in myocardial infarction mice.Methods:1)Construction of AMI-injured mouse model:Male adult C57BL/6 mice and KO-CTRP9 mice were subject to AMI by left anterior descending coronary artery ligation or sham surgery;divided into 6 groups:MI+Vehicle group,MI+CTRP9 group,MI+CTRP9+H89 group,sham operation+Vehicle group,KO-MI+Vehicle group,KO-sham operation+Vehicle group.2)The left ventricular ejection fraction(LVEF)was measured to assess cardiac function of mouse using Vevo 2100 high-resolution small animal ultrasound machine.3)Detection of myofibroblasts in cardiac tissue:α-SMA and vimentin double staining positive cells were detected by immunofluorescence staining.4)Detection of CTRP9 in myocardium:The expression of CTRP9 in different regions of myocardium was detected by immunofluorescence staining.5)Detection of serum CTRP9 and TGF-β1:The content of serum CTRP9 and TGF-β1 in mice was detected by ELISA kit.6)Detection of myocardial fibrosis:The myocardial fibrosis of mice was detected by Masson staining,and the expression of Collagen Ⅰ and TGF-1 in myocardial tissue was detected by Western Blotting.7)PKA Activity Assay:PKA and p-PKA were detected by Western blotting.Results:1)On the fourth day after myocardial infarction,serum CTRP9 levels in mice were significantly decreased(519.7±17.77 ng/ml in MI+Vehicle group,907.0±40.19 ng/ml in Sham+vehicle group,P<0.001),serum TGF-β1 levels were significantly increased(118.5±6.872 pg/ml in the MI+Vehicle group,66.79±2.690 pg/ml in the Sham+Vehicle group,P<0.001).2)On the 28th day after myocardial infarction,the left ventricular ejection fraction(LVEF)decreased significantly(Sham+Vehicle group(74.46±1.668)%,MI+Vehicle group(25.19±1.488)%,P<0.001),myocardial fibrosis was significantly increased((0.4950±0.08382)%in Sham+Vehicle group(left ventricular free wall),(25.41 ± 1.041)%in MI+Vehicle group(border region),P<0.001),TGF-β1 level in myocardium was significantly increased(P=0.0025),the number of myofibroblasts increased significantly(0.2500±0.2500 in the Sham+Vehicle group(left ventricular free wall)and 7.500±0.6455 in the MI+Vehicle group(border region)P<0.001),the PKA activity increased(P=0.0023),and the expression of collagen Ⅰ increased significantly(P=0.0238).3)Compared with WT-MI+Vehicle group,the mortality rate of KO-MI+Vehicle group increased(10/22 in KO-MI+Vehicle group,10/27 in WT-MI+Vehicle group),the cardiac contractile function was further reduced(P=0.0010),myocardial fibrosis was more severe(P=0.0395),PKA activity was partially inhibited(P=0.0063),TGF-β1 expression was more(P=0.0126),and collagen I expression increased more(P=0.0090);4)After rCTRP9 supplementation,the death of myocardial infarction mice was significantly reduced(10/27 in MI+Vehicle mice group and 4/26 in MI+CTRP9 group);the cardiac contractile function was significantly improved(P<0.001);myocardial fibrosis was significantly reduced(MI+Vehicle group fibrosis was(25.41±1.041)%,MI+CTRP9 group fibrosis was(17.51 ± 0.8927)%,P<0.001);PKA activity increased(P<0.001),and TGF-β1 expression decreased(P<0.01),collagen I expression decreased(P<0.01),and the myocardial protective effect of CTRP9 was blocked after being blocked with PKA inhibitor H-89.Part II The molecular mechanism of CTRP9 inhibiting the activaty and migration of cardiac fibroblasts.Methods:1)Isolation and culture of primary cardiac fibroblasts:Primary cardiac fibroblast cells were isolated from the left ventricular of neonatal SD rat using the method of enzymatic digestion and differential adhesion.2)Detection of proliferation activity of Cells:The cell proliferation activity of CFs were detected by MTS kit.3)Detection of fibroblast-to-myofibroblast Transformation:The expression of α-SMA was detected by immunofluorescence staining and Western blotting to evaluate CFs-to-myofibroblast transformation.4)Migration ability test:Wound healing assay and Transwell test were used to test the migration ability of CFS.5)Detection of fibrosis-related protein expression in cardiac fibroblasts:Western blotting was used to detect collagen Ⅰ,Ⅲ and CTGF secreted by CFS.Results:1)TGF-β1 increased the proliferation of cardiac fibroblasts(TGF-β1 group vs Contral group P<0.001);CTRP9 significantly inhibited the effect of TGF-β1(TGF-β1+CTRP9 group vs TGF-β1 group P<0.001);The inhibitory effect of CTRP9 was blocked by PKA inhibitor H-89(TGF-β1+CTRP9+H-89 group vs TGF-β1+CTRP9 group P<0.001).2)TGF-β1 induced the expression of α-SMA in cardiac fibroblasts and the transformation to myofibroblasts(TGF-β1 group vs Contral group P<0.001);CTRP9 significantly inhibited the effect of TGF-β1,increased expression of α-SMA was significantly inhibited(TGF-β1+CTRP9 group vs TGF-β1 group P<0.01);the inhibitory effect of CTRP9 was blocked by the PKA inhibitor H-89(TGF-β1+CTRP9+H-89 group vs TGF-β1+CTRP9 group P<0.05).3)TGF-β11 enhanced the migration ability of cardiac fibroblasts(TGF-β1 group vs Contral group P<0.001);CTRP9 significantly inhibited the effect of TGF-β1(TGF-β1+CTRP9 group vs TGF-β1 group P<0.001);the inhibitory effect of CTRP9 was blocked by the PKA inhibitor H-89(TGF-β1+CTRP9+H-89 group vs TGF-β1+CTRP9 group P<0.001).4)TGF-1 induced the increase of Smad3 phosphorylation and translocation into the nucleus of cardiac fibroblasts(TGF-β1 group vs Contral group P<0.001);CTRP9 inhibited the effect of TGF-β1(TGF-β1+CTRP9 group vs TGF-β1 group P≤0.05);H-89 blocked the inhibitory effect of CTRP9(TGF-β1+CTRP9+H-89 group vs TGF-β1+CTRP9 group P<0.01).5)TGF-β1 induced the increase of expression of collagen Ⅰ,collagen Ⅲ and CTGF in CFs(TGF-β1 group vs Contral group P<0.001);CTRP9 inhibited the effect of TGF-β1(TGF-β1+CTRP9 group vs TGF-β1 group P<0.001);the inhibitory effect of CTRP9 was blocked by the PKA inhibitor H-89(TGF-β1+CTRP9+H-89 group vs TGF-β1+CTRP9 group P<0.001).Conclusions:1.Studies have confirmed that CTRP9 significantly ameliorates myocardial fibrosis and improve cardiac function in mice with myocardial infarction via PKA.2.It was found Firstly,that CTRP9 reduces TGF-β1-induced proliferation and activaty,transformation to myofibroblasts and migration of cardiac fibroblasts via PKA pathway;CTRP9 reduces TGF-β1-induced phosphorylation and intranuclear translocation of Smad3 in myocardial fibroblasts via PKA pathway,thereby reducing the expression of fibrosis-related proteins and exerting anti-fibrosis effects.It is of great significance to screen targets for the treatment of fibrosis after myocardial infarction.