| Background:Acute myocardial infarction(AMI)is a serious ischemic heart disease with significant morbidity and mortality worldwide.Despite advances in the treatment of acute myocardial infarction(AMI),the early mortality in patients with acute myocardial infarction has decreased significantly,many patients still develop progressive heart failure in several years as a result of declining cardiac function.Therefore,it is important to explore new and viable strategies to reduce myocardial damage and aid cardiac repair following MI.Thyroid hormone can promote cardiac function recovery after cardiac injury,but the possible molecular mechanism is still unclear.IGF-1 is a growth-promoting peptide hormone,which can regulate the proliferation,differentiation and energy metabolism of various cells.IGF-1binding to igf-1 receptor can phosphorylate igf-1 receptor and activate the PI3K/AKT signaling pathway,thus regulating cell growth,proliferation,differentiation,apoptosis and metabolism,effectively inhibiting apoptosis of cardiomyocytes,reducing inflammatory response,and resisting myocardial remodeling.However,whether the IGF-1/PI3K/AKT signaling pathway mediates the protective effect of thyroid hormone on post-infarction myocardium remains to be further studied.Objective:In this study,a mouse model of myocardial infarction was established to investigate the effects of thyroid hormone T3 pretreatment on cardiac function and cardiac pathology in mice after myocardial infarction,and to explore the role of IGF-1mediated PI3K/AKT signaling pathway in T3 promoting cardiac repair and its molecular mechanism.Methods:Male C57BL/6J mice aged 8-10 weeks were randomly divided into four groups:Sham group,MI group,MI+T3 group,MI+T3+BMS-754807 group.Drugs or vehicle were administered intraperitoneally daily for 3 days before surgery.Then,the MI model was established by ligating the left anterior descending(LAD)coronary artery as described with minor modifications.Cardiac function of mice was detected at postoperative time points(3 days and l weeks after surgery),then serum and heart tissues of mice were collected respectively for the detection of related pathological changes after myocardial infarction,including myocardial infarction size,degree of myocardial fibrosis in the infarction scar area,level of inflammatory factors and angiogenesis in the area surrounding infarction.By comparing the protein expression levels of IGF-1,IGF-1R,p-PI3 K and p-AKT in cardiac tissues of each group,the possible mechanism of IGF-1 mediated PI3K/AKT signaling pathway in promoting cardiac function recovery after myocardial infarction in T3 was explained.Results:(1)In the sham group,none of the mice that underwent surgery died at the end of the observation period(100% survival).However,cardiac rupture after MI caused high mortality,and the survival rate through 7 days postoperation reached 68.5% in the MI group(P<0.05).T3 pretreatment increased the survival rate after MI(80.2%;P<0.05),but BMS-754807 suppressed this effect;the MI+T3+ BMS-754807 group had the lowest survival rate of 62.5%(P<0.05).(2)The results indicated that left ventricular function was impaired after MI.Its characteristics are: left ventricular ejection fraction(LVEF),left ventricular short axis(FS)and left ventricular posterior wall thickness(LVPW)reduction;Left ventricular end-systolic diameter(LVESd)and left ventricular end-diastolic diameter(LVEDd)increased(P<0.05).Compared with MI animals,T3 pretreatment animals exhibited preserved LVEF,FS and LVPW and reduced LVESd and LVEDd values(P<0.05),suggesting that T3 pretreatment can bring sustained functional and structural benefits.We further found that,compared with the Sham group,the heart rate of postoperative mice was significantly increased,but there was no significant difference in heart rate between the MI+T3 group and the MI group,suggesting that the T3 pretreatmentinduced increase in the values of LVEF in MI animals may not be associated with heart rate.(3)HE staining and Masson’s trichrome staining at the was performed at the endpoint to evaluate infarct size,myocardial fibrosis and structural changes.The results showed that infarct size(31.3% VS 0%)and fibrosis(29.8% VS 0%)were dramatically increased in the MI group compared to the sham group(P<0.05),and that T3 infusion markedly reduced infarct size(22.5% VS 31.3%)and limited myocardial fibrosis spaces(21.9% VS 29.8%)and collagen volumes after MI(P<0.05).Conversely,BMS-754807 suppressed this effect,resulting in a larger infarct area(37.4%VS 22.5%)and more severe myocardial fibrosis(37.1%VS 21.9%)in the MI+T3+ BMS-754807 group(P<0.05).(4)The extent of apoptosis was detected through a TUNEL assay in peri-infarct tissues.Notably,TUNEL-positive nuclei were rarely observed in the sham group.However,the number of TUNEL-positive nuclei was significantly increased after MI.The ratio of TUNEL-positive nuclei was 28.6% in the MI group,and T3 pretreatment greatly reduced the ratio of TUNEL-positive nuclei to 14.1%(P<0.05),suggesting that T3 protects cardiomyocytes against MI-induced apoptosis.However,BMS-754807 inhibited the effect of T3,and the ratio of TUNEL-positive cells in the MI+T3+BMS-754807 group was 35.9%(P<0.05).We also examined the expression levels of apoptosis-related proteins,including pro-apoptotic factors(Bax and cleaved caspase 3)and anti-apoptotic factors(Bcl-2),to clarify the anti-apoptotic effect of T3.Western blot analyses revealed that the expression of Bax and cleaved caspase-3 was significantly increased,while the expression of Bcl-2 was decreased in the MI group compared with the sham group.Conversely,T3 infusion markedly suppressed Bax and cleaved caspase3 expression and preserved Bcl-2 expression compared with that in the MI group,and relative to animals treated with saline or T3 infusion,the MI+T3+ BMS-754807 group showed higher expression of Bax and cleaved caspase 3 and the lower expression of Bcl-2(all P<0.05).(5)Serum ELISA showed that compared with Sham group,serum inflammatory cytokines(IL-6 and TNF-a)were significantly increased in mice 3 days after acute myocardial infarction(P<0.05),while serum IL-6 and TNF-a levels were significantly decreased in mice after T3 pretreatment(P<0.05),suggesting that T3 has the benefit of reducing inflammatory response.However,this benefit disappeared with the addition of BMS-754807,and the levels of inflammatory factors in the MI+T3+ BMS-754807 group increased significantly(P<0.05).(6)The results of CD31 immunofluorescence staining indicated that,compared with Sham group,the neovascularization density in the periinfarction area after acute myocardial infarction decreased(42.3% VS 66.4%;P < 0.05);After T3 pretreatment,neovascularization density increased compared with MI group(58.1% VS 42.3%;P<0.05),suggesting that T3 has a potential role in promoting angiogenesis;However,BMS-754807 inhibited this effect of T3,and the density of new blood vessels in the MI+T3+ BMS-754807 group decreased(40.9%% VS 58.1%;P < 0.05).(7)Western blot showed that compared with Sham group,the expression levels of IGF-1,IGF-1r and p-PI3 K were increased in myocardial infarction surgery,but the expression levels of p-AKT were not significantly increased.Compared with the MI group,T3 pretreatment significantly increased the expression of IGF-1,IGF-1R,pPI3 K and p-AKT(p <0.05),that is,T3 increased the level of IGF-1,promoted the binding of IGF-1 and IGF-1R,and activated the PI3K/AKT signaling pathway.However,after the addition of BMS-754807,the effect of T3 was significantly inhibited,and the expression of p-PI3 K and p-AKT was significantly decreased(P<0.05).Conclusion:(1)T3 pretreatment promotes survival rates and improves cardiac function of mice after MI.(2)T3 pretreatment confers cardioprotection on mice subjected to MI.These protective effects are associated with decreased cardiomyocyte apoptosis and infarct size,limited LV remodeling,stimulated angiogenesis,and a blunted inflammatory response.(3)T3 pretreatment mediates post-MI cardioprotection via the activation of the IGF-1/PI3K/AKT signaling pathway. |
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