Tissue-nonspecific Alkaline Phosphatase Overexpression Enhancing Myocardial Fibrosis After Myocardial Infarction In Mouse

PART Ⅰ CORRELATION BETWEEN TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE AND MYOCARDIAL FIBROSIS AFTER MYOCARDIAL INFARCTIONObjective : To investigate the relationship of tissue-nonspecific alkaline phosphatase(TNAP)with myocardial fibrosis after myocardial infarction(MI).Methods:(1)Forty-six acute MI patients admitted to our hospital from March to November 2019 were enrolled in this study and assigned as the MI group,and another 41 patients with negative findings in coronary angiograms during the same period were collected as the control group.The serum TNAP activity was detected by alkaline phosphatase kit.The serum contents of procollagen type Ⅰ C-peptide(PⅠCP)and procollagen Ⅲ N-terminal peptide(PⅢNP)were detected by enzyme-linked immunosorbent assay(ELISA).(2)Myocardial tissues from 5 patients whodied of MI were collected.Collagen fiber area of heart was detected by Sirius red staining and the expression of TNAP was detected by immunohistochemistry.(3)Ten 8-week-old male C57BL/6J mice were randomly divided into sham group and MI group(n=5).In 2 weeks after MI,all mice were sacrificed,and collagen fiber area of heart was detected by Masson staining and the expression of TNAP was detected by immunohistochemistry.Results :(1)Compared with the control group,the MI group had significantly increased serum TNAP activity(8.49±3.30 king U/100 ml)and the concentration of serum TNAP(67.22±12.83)U/L(both P<0.01).(2)Compared with the control group,the serum contents of PⅠCP(42.07-87.68)mg/L and PⅢNP(1.35-2.27)mg/L in patients with MI increased significantly(both P<0.01).(3)The serum TNAP activity was significantly correlated with the serum contents of PICP and PⅢNP(r=0.38,P<0.01,r=0.40,P<0.01).(4)There was more collagen deposition and high TNAP level in the infarct border area of remote MI lesion than in the distal infarct(both P<0.01).(5)Compared with the mice of the sham group,the expression of TNAP and Collagen fiber area in MI group increased significantly(both P<0.01),and the expression was consistent with the distribution of fibers in the MI group.Conclusion:TNAP is involved in the pathophysiological process of myocardial fibrosis after MI.PART Ⅱ EFFECT OF TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE OVEREXPRESSION ON MYOCARDIAL FIBROSIS AFTER MYOCARDIAL INFARCTION IN MOUSEObjective : To investigate the effect of tissue-nonspecific alkaline phosphatase overexpression on myocardial fibrosis(MF)after myocardial infarction(MI)in mouse.Methods:(1)Adv-TNAP adenovirus liquid was injected into the heart of mice by direct myocardial injection.Western blotting was used to detect the expression level of TNAP protein at different transfection time(1,3,5,7,14 and 21d),and RT-PCR was used to detect its m RNA level.(2)Forty-five 8-week-old male C57BL/6J mice were randomly divided into sham group(sham),MI group(MI+Adv-EGFP)and TNAP overexpression group(MI+Adv-TNAP)with 15 mice in each group.In 2weeks after MI,cardiac function of mice was detected by Echocardiography,pathological changes was detected by HE staining.The fibrotic area of heart was detected by Masson staining and the expression of TNAP was detected by immunohistochemistry.The collagen volume fraction(CVF)and the ratio of Col Ⅰ/Col Ⅲ were measured by polarizing method with Sirius red staining.(3)Western blotting was used to detect the expression of TNAP,α-SMA,TGF-β1,Smad2,p-Smad2,collagen Ⅰ and collagen Ⅲ.Results:(1)RT-PCR showed that the expression of TNAP mRNA peaked at 5 days,lasting for 3 W,and Western blotting showed that TNAP protein began to increase at 3 days,lasting for 3 W.(2)Compared with MI group,the Left ventricular ejection fraction(LVEF)(%)and Left ventricular fraction shortenin(LVFS)(%)of the heart in MI+Adv-TNAP group were significantly reduced(P<0.05).(3)HE:MI+Adv-TNAP group showed myocardial fiber broken,disordered and fibrous scars was obvious in myocardial infarction area.(4)Compared with the sham group,the area of MF and the expression of TNAP in the MI group were significantly increased(both P<0.01).Compared with MI group,the area of cardiac fibrosis and the expression of TNAP in the MI+Adv-TNAP group were increased(both P<0.05),and the expression of TNAP was consistent with the distribution of fibers.(5)Compared with MI group,the CVF and the proportion of the ratio of Col Ⅰ/Col Ⅲ in the MI+Adv-TNAP group were significantly increased(P<0.05).(6)Compared with the sham group,the expression of α-SMA,TGF-β1,p-Smad2,collagen Ⅰ,collagen Ⅲ protein and the ratio of Col Ⅰ/Col Ⅲ in the MI group were increased(P<0.05).compared with the MI group,the expression of α-SMA,TGF-β1,p-Smad2 and collagen I protein and the ratio of Col Ⅰ/Col Ⅲ in the MI+Adv-TNAP group were increased significantly(P<0.05),but the expression level of collagen Ⅲ protein in the MI+Adv-TNAP group was not significantly different.Conclusion : TNAP overexpression can promote the myocardial fibrosis after myocardial infarction in mouse.