Research On The Role Of PI3K In Myosin Induced Autoimmune Myocarditis In Mice And The Underlying Mechanism

Part I Myosin induced autoimmune myocarditis Model in miceBackgroundCardiovascular disease is still a serious threat to human health due to their high prevalence, and considerable morbidity and mortality. More than 17.3 million people die of cardiovascular disease each year. Of these,2% are the result of inflammatory heart diseases. Myocarditis diverse clinical symptoms, many will develop dilated cardiomyopathy. A variety of infectious and non-infectious factors can induce myocarditis, but so far, the cause of myocarditis is not clear, on the clinical development and progression of myocarditis lack of effective interventions. Studies have shown that continuous immune response plays an important role in promoting progress in the process of myocarditis. Several animal models have been used to study myocarditis. Coxsackie B3 virus into mice model used to study infectious myocarditis. For autoimmune myocarditis (non-infectious), the most commonly used animal model are autoimmune myocarditis mice model induced by myosin or troponin.PurposeUse myosin produce an autoimmune myocarditis mice model.MethodsMice were injected subcutaneously rodent cardiac myosin mixture with complete Freund's adjuvant to induce autoimmune myocarditis. Mice were grouped:40 8-12-week-old BALB/C wild-type mice, the mice were randomly divided into four groups in principle, specific groups were as follows:control group (A):10 female BALB/C mice, without any treatment. Low concentrations of EAM model group (B): 10 female BALB/C mice, rodent cardiac myosin heavy chain amino acid fragment with a mixture of Freund's adjuvant injected subcutanous 25?g/200?l. The concentration of EAM model group (C):10 female BALB/C mice, rodent cardiac myosin heavy chain amino acid fragment with a mixture of Freund's adjuvant injected subcutanous 50?g/200?l. High concentrations of EAM model group (D):10 female BALB/C mice, rodent cardiac myosin heavy chain amino acid fragment with a mixture of Freund's adjuvant injected subcutanous 100?g/200?l. EAM mice injected amino acid-a mixture of Freund's adjuvant, and the control group was injected with PBS. Comparison with the control group of mice the incidence of myocarditis, change the ratio of heart weight/body weight, cardiac pathology changes and mortality in mice.ResultsMice were injected with myosin heavy chain amino acid fragment, severe myocarditis happened. Mouse heart weight/body weight and myocardial pathological changes in a concentration-dependent increase. In normal control group, low dose group, middle dose group, high concentration group, the incidence of myocarditis was 0/10,2/10,5/10,10/10, heart weight/body weight ratio (mg/g) were 5.53 ± 0.21,6.16 ± 0.32,7.04 ± 0.53,8.16 ± 0.87, morphology scores were 0,1.7 ± 0.3,2.2 ± 0.4,2.9 ± 0.6.ConclusionMyosin can induce autoimmune myocarditis in mice, so it can be used to explore mechanisms of autoimmune myocarditis. With 100?g/200?l concentration of MyHC-a, myocarditis incidence is high and the inflammations are typical, it is the most appropriate concentration.Part ? The role of PI3K in myosin-induced cardiac pathological changes in mice autoimmune myocarditisBackgroundBALB/C mice are of high sensitivity to the amino acid sequence of a-mysosin or myosin heavy chin source, and the use of ?-mysosin or myosin heavy chain amino acid derived immunized BALB/C mice can induce experimental autoimmune myocarditis, which has been confirmed in our previous study. Chironic autoimmune reponse is important molecular mechanism for the peogression of myocarditis. Currently, the molecular mechanism for the progeresion of myocarditis is still unknown, and it's still lack of effective clinically treatment strategies.Phosphatidylinositol 3-kinase (PI3K) is a very important protein kinase involved in inflammation regulation and immune response. In recent years, increasing evidence showed that pharcological inhibition of PI3K could inhibite a variety of autoimmune diseases. However, whether PI3K is also involved in immune-related heart diseases has not been informed.PurposeUse of pharmacological methods of inhibiting PI3K activity, we observed the myocardial injury in myosin-induced experimental autoimmune myocarditis mice, thus reveal the role of PI3K in experimental autoimmune myocarditis.MethodsTo exclude the influence of LY294002 under physiological conditions of mice, we first detected LY294002 toxicological effects. Mice were injected subcutaneously rodent cardiac myosin mixture with complete Freund's adjuvant to induced autoimmune myocarditis. Mice were divided into three groups, blank group, control group (EAM group) and experimental group (EAM+LY group). Experimental mice were injected intraperitoneally PI3K inhibitor LY294002.comparison the incidence of myocarditis, the ratio of heart weight/body weight, changes in cardiac pathology changes and mortality with the control group of in mice.ResultsLY294002 toxicological test results showed that, under physiological conditions, we can't find visible pathological injury in heart, liver, kidneys, lungs and other organs in mice with the given LY294002 dose.Mice were injected with myosin heavy chain amino acid fragment, severe myocarditis cell infiltration and myocardial necrosis happened.The heart weight/ body weight ratio increased compared with normal mice (8.16 ± 0.87 vs5.53 ± 0.21, p <0.01), and the experimental mouse had a high mortality (8/10). After giving PI3K inhibitor LY294002 intervention, the heart weight/body weight ratio of the experimental group of mice was significantly reduced compared with the control group (6.04 ± 1.03 vs 8.16 ± 0.87, p<0.05). Myocarditis injury in mice significantly reduced (morphological lesion score 1.7+0.6 vs 2.9 ± 0.6, p<0.01). Mice 7-day mortality was significantly lower than the control group (3/10 vs 8/10).ConclusionInhibition of PI3K can led a protective effect on myocardial injury in experimental autoimmune myocarditis, suggesting that PI3K signaling pathway may be a potential therapeutic target for autoimmune myocarditis.Part ? The effects of PI3K on the progression of inflammatory and immune response in experimental autoimmunoe myocarditis in miceBackgroundThe release of pro-inflamamtory cytokines is very important regulating mechanism of inflammatory action. A lot of studies has showed that pro-inflamamtiry-cytokines (such as TNF-?, IL-1? IFN-?) were involved in the pathogenesis and progression of autoimmune heart diseases. Evidence has demonstrated that the rectuitment of inflammatory cells by these chemokines requires the activation of PI3K/AKT signaling.This suggesting there exist a link between PI3K and inflammation. The AKT belong to the serine/threonine protein kinase and is the most important signaling transduction tatrget downstream of PI3K. Previously, in the first part, we have demonstrated the protective effects of pharmacological inhibition of PI3K in experimental autoimmune myocarditis in mice, which suggested the role of PI3K signaling in the pathogenesis of myocardist. However, the mechanism by which PI3K regulate this process is still unrevealed.PurposeInvestigate the relationship between PI3K signal pathway and inflammation in the EAM, revealing the molecular mechanism of how PI3K influence the development and progress of autoimmune myocarditis.MethodsImmunohistochemistry was used to detect the number of CD3+T cells in mice cardiac muscle cells in the control group, EAM, EAM+LY294002 group. Using western blotting to analysis PI3K downstream signal transduction proteins AKT phosphorylation levels in mice myocardium of the above-mentioned groups Lymphocytes taken from the normal mouse, divided into control group, EAM Group, EAM+low concentrations of LY294002 group and EAM+high concentration LY294002 group.Using flow cytometry to check lymphocytes CD4+and CD8+T cells of the above groups and the absolute value of the ratio between the two. ELISA techniques were used to detect TNF-?, IL-1?, and IFN-? in the serum in each group and each cultured lymphocytes group.ResultsBy immunohistochemistry results we observed in EAM mice heart tissue slices CD3 staining was strongly positive, while in the control group and the EAM+ LY294002 group, CD3 staining not obvious. Flow cytometry showed that the application of myosin made CD4+and CD8+T cells increase in the absolute value (24.3% vs 23.22% and 11.95% vs 9.36%) compared with the control group. And the application of 1?M LY pretreated cells, CD4+and CD8+T cells in absolute value were decreased compared with EAM group (24.05% vs24.3% and 7.44% vs 11.95%); 15?M LY application after pretreatment of cells, CD4+and CD8+T cells in absolute value had a more significant decline compared with EAM model group (21.37% vs24.3% and 7.19% vs 11.95%). Application of myosin causes CD4+/CD8+T cells reduced compared with the control group (2.03 vs 2.48), the use of 1?M and 15?M LY294002, made the CD4+/CD8+T cells value reached 3.23 and 2.97 respectively. Western blotting analysis showed that the mice by intraperitoneal injection of LY294002, the myosin-induced AKT phosphorylation levels decreased (0.71 ± 0.12 vs 1.14 ± 0.21). ELISA analysis showed that the mouse serum pro-inflammatory cytokines expression levels were significantly increased compared to the normal mice (TNF-a, IL-1?. and IFN-y3 kinds of inflammatory factors were compared with the control group: 174 ± 24.21 vs 48.2 ± 1.52; 18.2 ± 1.87 vs 4.07 ± 0.74; 220 ± 29.4 vs 40.43 ± 2.34 respectively), but the serum proinflammatory cytokine levels of LY294002 pretreatment are not elevated significantly (three kinds of inflammatory factors and EAM were compared:119 ± 4.03 vs 174 ± 24.21; 11.5 ± 1.56 vs 18.2 ± 1.87; 170 ± 6.43 vs 220 ± 29.4 respectively). In cell experiments, cultured lymphocytes stimulated by MyHC-a 3h, the expression of pro-inflammatory cytokines TNFa, IL-1? and IFNy was significantly higher (three kinds of stimulation of inflammatory cytokines were compared with the control group:78 ± 11.73 vs 19.8 ± 2.23; 47.8 ± 5.17 vs 12.3 ± 1.36; 92.8 ± 5.27 vs 8.23 ± 1.44). However, if given LY294002 pretreatment cells before adding MyHC-a stimulation, the expression of pro-inflammatory cytokines dependently inhibited (maximum dose intervention group were:30 ± 1.78 vs 78 ± 11.73; 17.6 ± 1.46 vs 47.8 ± 5.17; 22 ± 1.77 vs 92.8 ± 5.27).ConclusionPI3K inhibition can significantly improve cardiac inflammation and the adaptive immune cell imbalance in myocardial tissue in myosin induced experimental autoimmune mice.This indicating that PI3K may play a myocardial damage role by promoting inflammation and immunity reaction in mice experimental autoimmune myocarditis.Part IV The effects of PI3K on the progress of myocardial cell apoptosis in experimental autoimmune myocarditis in miceBackgroundStudies have shown that apoptosis of cardiomyocytes played an independent role in the pathological process of myocarditis. Bax, Bim and Bcl-2 belong to Bcl-2 protein family members, Bax and Bim play a major role in promoting apoptosis, and Bcl-2 play an anti-apoptotic role. Bax had been shown to be directly activated by Bim long before, and can trigger the release of cytochrome C. Activation of caspase promote waterfall cascade can induce apoptosis. Caspase belong to cysteine proteases family.caspase-3 is apoptosis execution protein and caspase-9 is induced starting proteins of apoptosis.Our previous studies have shown that PI3K play an important role in myocardial damage of experimental autoimmune myocarditis mice by promoting inflammation and immune responses, but whether the PI3K/AKT/mTOR pathway is involved in cell apoptosis in cardiac autoimmune myocarditis process is not clear.PurposeAfter inhibiting PI3K, observe the situation of cardiomyocyte apoptosis in autoimmune myocarditis mice, and further explore the role of PI3K signaling pathway on myocardial cells apoptosis in myosin induced mice autoimmune myocarditis.MethodsUsing western blotting techniques to detect heart PI3K/AKT/mTOR signaling pathway p85 (PI3K subunit), phosphorylation of AKT and mTOR protein in the control group, EAM Group, EAM+LY294002 group. Use Western blot and EMSA to detected the P65 (NF?B subunit) expression in the three group myocardial cells. Use TUNEL staining to observe the apoptosis of mice cardiomyocytes in the three groups. Using western blotting and RT-PCR techniques analysis the expression of Bax, Bim, caspase3 and Bcl-2.ResultsAfter intraperitoneal injection of PI3K inhibitor LY294002, compared with the same type of test mice, the activity of PI3K/AKT/mTOR signaling pathway significantly decreased, NF-?B activity decreased. EAM+LY294002 group compared with EAM group, activation of PI3K/AKT/mTOR signaling pathway is significantly decreased (p85, AKT and mTOR protein phosphorylation levels of three kinds of indicators were:3.7 ± 0.23 vs 6.2 ± 0.18; 2.18 ± 3.50 vs 5.93 ± 1.26; 0.69 ± 0.18 vs 2.32 ± 0.34). Myocardial nuclear protein western blotting and EMSA results show, in LY294002+EAM group,NF-?B activity is decreased more than 55% as compared to EAM group (nuclear P65 levels were:0.42 ± 0.10 vs 1.2 ± 0.16). TUNEL technique shows, in LY294002+EAM group, the number of cardiac myocyte apoptosis surge compared with the EAM group, western blotting technical analysis shows, in LY294002 +EAM group mice myocardium Bax, Bim, caspase3 and expression of Bcl-2 were:2.1 ± 0.20 vs 1.25 ± 0.24; 2.2 ± 0.05 vs 0.7 ± 0.06; 1.9 ± 0.29 vs 1.25 ± 0.20; 0.30 ± 0.03 vs 0.92 ± 0.21 compared with the EAM group. RT-PCR showed:LY294002+EAM group Bax, Bim, caspase3 and expression of Bcl-2 mRNA were:1.0 ± 0.21 vs 0.56 ±0.01; 1.75 ± 0.27 vs 0.8 ± 0.22; 2.4 ± 0.26 vs 1.2 ± 0.01; 0.6 ± 0.21 vs 0.9 ± 0.09 compared with EAM group. The results shows after inhibiting PI3K, the mRNA of apoptosis related protein Bax, Bim, caspase3 was significantly increased, while anti-apoptotic proteins Bcl-2 mRNA expression is suppressed.ConclusionInhibition of PI3K pathway can increase the expression of pro-apoptotic related proteins and inhibit the expression of anti-apoptosis-related proteins and eventually induce apoptosis in subacute and chronic myocarditis mouse cardiomyocytes. It showed that PI3K pathway plays a negative role in the regulation of cardiac cells apoptosis in EAM mouse.