TXNIP Knockout Improves Cardiac Function After Myocardial Infarction By Promoting Angiogenesis And Reducing Cardiomyocyte Apoptosis And Inflammation

Background:Myocardial infarction(MI)is a dominant cause of death.It has a major threat to human health.Although the clinical treatment methods have made great progress compared with the previous ones,the mortality rate of the disease remains high.It is urgent to find effective and non-invasive means to prevent and treat MI.On the one hand,the myocardium has compensatory angiogenesis and the establishment of collateral circulation after MI,which is of great significance for increasing myocardial blood flow and improving cardiac function.However,in the pathological condition of MI,compensatory angiogenesis in the myocardium is far from sufficient to restore cardiac blood flow,and external means are required to enhance the level of angiogenesis.On the other hand,preventing cardiomyocyte apoptosis and reducing the inflammatory response after MI both reduce the damage to the myocardium caused by hypoxia-ischemia.Thioredoxin interacting protein(TXNIP)belongs to the superfamily of proteins containing inhibin domains.It plays an important role as a scaffold protein.It is also an important member in cell signal transduction.The transcription and protein instability of TXNIP are regulated by various proteins.So,TXNIP is promising as a good therapeutic target.It is known that the expression of TXNIP increased after MI and it negatively regulated cardiac function after MI.However,the mechanism of the effect of TXNIP on cardiac function after MI is not fully understood.In addition,the effect and mechanism of TXNIP on angiogenesis after MI need to be explored.Objective:1 We used MI model to observe the effect of TXNIP on cardiac structure and function,myocardial fibrosis level,the ratio of heart weight to body weight,oxidative stress level,angiogenesis level,cardiomyocyte apoptosis level and the activation levels of the Nucleotide-binding oligomerization domain-like receptor family pyrin domain-like receptor containing pyrin domain 3(NLRP 3)inflammasome.2 This study aims to explore the specific mechanism of TXNIP gene knockout promoting angiogenesis after MI.Methods:1 The TXNIP knockout mouse(TXNIP-KO)and TXNIP knockin mouse(TXNIP-KI)were established.Eight-week-old male TXNIP knockout mice,TXNIP knockin mice and Wild type(WT)littermates were selected,and three kinds of mice were randomly divided into myocardial infarction group and sham operation group.Myocardial infarction model was established.2 4 days after the operation,some mice were sacrificed,and the heart tissue was collected.TUNEL(Td T-mediated d UTP nick end labeling)was used to detect cardiomyocytes apoptosis.Western blot was used to detect the expression of TXNIP,Hypoxia inducible factor-1α(HIF-1α),Phosphorylated protein kinase B(p-AKT),Vascular endothelial growth factor(VEGF),Phosphorylated adenosine5’-monophosphate activated protein kinase(p-AMPK),Nucleotide-binding oligomerization domain-like receptor family pyrin domain-like receptor containing pyrin domain 3(NLRP 3),Cleaved cysteinyl aspartate specific proteinase-1(cleaved caspase-1),Interleukin-1β(IL-1β)and Cleaved cysteinyl aspartate specific proteinase-3(cleaved caspase-3).Quantitative real-time PCR was performed to detect the expression of TXNIP,HIF-1α,VEGF,Prolyl hydroxylase 1(PHD 1),and Factor inhibiting hypoxia-inducible factor(FIH).The superoxide dismutase(SOD)activity and malondialdehyde(MDA)level in each group were also measured.3 At day 7 after MI,hearts of sacrificed animals were analyzed by immunohistochemistry to assess CD31 expression and determine the density of angiogenesis.4 In the remaining mice,one month after the operation,the cardiac function of the mice in each group was detected by a small animal echocardiography,and then the mice were sacrificed.The level of myocardial fibrosis was observed by Masson staining.Results:1 MI model based on TXNIP transgenic mice was successfully establishedThe genotypes of TXNIP-KO and TXNIP-KI homozygous mice were identified by PCR.Surgical ligation of left anterior descending coronary artery in mice included in MI group.After ligation,the apex of the heart became white,the heart beat was weakened,and the ST segment of the electrocardiogram was elevated.The above phenomena indicated that the MI model was successful established.2 TXNIP knockout improved left ventricular function after MICompared with the WT-MI group,left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)increased significantly in TXNIP-KO-MI group(P < 0.05),and left ventricular internal diameter at end systole(LVIDs)decreased significantly in TXNIP-KO-MI group(P < 0.05).By contrast,LVEF,LVFS and left ventricular anterior wall thickness at end systole(LVAWs)in the TXNIP-KI-MI group were lower,but LVIDs was higher,and the difference was statistically significant(P <0.05).These results suggested that TXNIP knockout improved cardiac function after MI.3 TXNIP knockout reduced the myocardial fibrosis area after MIMasson staining was used to detect the percentage of cardiac fibrosis in each group.Compared with the WT-MI group,the percentage of the fibrosis area to the total area of the layer in the TXNIP-KO-MI group decreased,while significantly increased in the TXNIP-KI-MI group(P < 0.05).4 TXNIP knockout reduced the ratio of heart weight to body weightThe heart was harvested 30 days after operation,and the weight of the mouse and the heart was detected.The results showed that the heart weight to body weight ratio of the TXNIP-KO-MI group was significantly lower than that of the WT-MI group,while this ratio of the TXNIP-KI-MI group was significantly higher than that of the WT-MI group(P < 0.05).5 The expression of TXNIP increased after MIThe TXNIP m RNA and protein expressions of WT-MI group and TXNIP-KI-MI group were respectively and significantly higher than those in their corresponding Sham operation group(P < 0.05).And the expression of TXNIP in the TXNIP-KI-MI group was significantly higher than that in the WT-MI group(P < 0.05).TXNIP-KO mice were TXNIP knockout mice,so the m RNA or protein expression of TXNIP in either the Sham group or the MI group was almost close to zero.6 TXNIP knockout increased SOD activity and decreased MDA levels in myocardium after MITissues around MI were taken to detect SOD activity and MDA levels.Compared with the WT-MI group,the SOD activity in the TXNIP-KO-MI group was significantly increased,while that in the TXNIP-KI-MI group was significantly decreased(P < 0.05).Compared with the WT-MI group,the MDA level in the TXNIP-KO-MI group was significantly decreased,while that in the TXNIP-KI-MI group was significantly increased(P < 0.05).7 TXNIP knockout increased angiogenesis after MI by promoting HIF-1αexpressionThe blood vessel density of each group was detected by immunohistochemistry.Compared with the WT-MI group,the angiogenesis and the vessel density near the infarct area in the TXNIP-KI-MI group were significantly reduced.However,the angiogenesis and the micro-vessel density in the TXNIP-KO-MI group were significantly increased(P < 0.05).Western blot results showed that compared with WT-MI group,the protein expression levels of VEGF,HIF-1α,p-AKT and p-AMPK in TXNIP-KO-MI group were significantly increased(P < 0.05).The protein expression levels of VEGF,HIF-1α,p-AKT and p-AMPK in TXNIP-KI-MI group were significantly decreased(P <0.05).Real-time PCR results showed that compared with WT-MI group,the m RNA expression levels of VEGF and HIF-1α in TXNIP-KO-MI group were increased,and the m RNA expression levels of PHD 1 and FIH were decreased(P < 0.05).The m RNA expression levels of VEGF and HIF-1α in TXNIP-KI-MI group were decreased,while the m RNA expression levels of PHD 1 and FIH were increased(P < 0.05).The above results suggested that TXNIP knockout promoted the protein expression of HIF-1α and VEGF,and increased the angiogenesis after MI.On the one hand,TXNIP knockout increased the expression of HIF-1α by promoting the activation of AKT and AMPK,on the other hand,TXNIP knockout also maintain the stability of HIF-1α by inhibiting the expression of PHD 1 and FIH.8 TXNIP knockout reduced cardiomyocyte apoptosis after MICompared with WT-MI group,myocardial cell apoptosis index(AI %)in TXNIP-KO-MI group was significantly decreased(P < 0.05),and the expression of cleaved caspase-3 was decreased(P < 0.05).However,the myocardial cell apoptosis index(AI %)in TXNIP-KI-MI group was significantly increased(P < 0.05),and the expression of cleaved caspase-3 was increased(P < 0.05).9 TXNIP knockout inhibited the activation of NLRP3 inflammasomeWestern blot results showed that compared with WT-MI group,the protein expression of NLRP 3,cleaved caspase-1/caspase-1 and IL-1β in TXNIP-KO-MI group were all decreased,and the difference was statistically significant(P < 0.05).Conclusion:After MI,TXNIP knockout up-regulated the level of HIF-1α and VEGF,increased the number of angiogenesis,decreased cardiomyocyte apoptosis,inhibited the activation of NLRP 3 inflammasomes and ultimately improved the prognosis of ischemic myocardium.TXNIP was a protein with negative effects after MI and was expected to be a target for prevention and treatment of MI.