肌球蛋白致自身免疫性心肌疾病的实验研究

目的　探索在没有病毒感染的情况下 ,自身免疫机制在心肌炎和心肌病发病中的作用。方法　 (1)猪心肌肌球蛋白致BALB/C鼠自身免疫性心肌疾病模型的复制 :实验组 (n =32 )于实验的第 0 ,7和 30天 3次注射肌球蛋白 ,对照组 (n =2 0 )与实验组同样的时间注射弗氏完全佐剂 ,于初次免疫后的第 15 ,2 1和 12 0天分别留取血清和心肌标本。 (2 )病毒性心肌炎模型的复制 :病毒接种组 (n =2 2 )给予 10 3 TCID50 / 0 1mlCVB3 溶液腹腔注射 ,对照组 (n =10 )给予Vero细胞冻融液 0 1ml腹腔注射 ,接种时间及采样时间同肌球蛋白免疫组。 (3)抗体检测 :ELISA法检测小鼠血清抗肌球蛋白抗体。结果　病理结果显示 ,肌球蛋白致心肌炎小鼠急性期 (15和 2 1d)以心肌纤维变性坏死和淋巴细胞浸润较明显 ,间质病变较轻 ,内膜和心瓣膜无明显改变 ;慢性期 (12 0d)仅见心肌纤维化 ,急慢性期均可检测到高滴度的抗肌球蛋白抗体 (P <0 0 0 1) ,对照组为正常心肌 ,抗体滴度低。病毒接种组慢性期亦显示成纤维细胞增生。结论　心肌肌球蛋白可以作为自身抗原刺激机体产生自身免疫反应 ,在没有病毒感染的情况下 ,单一的自身免疫机制即可导致心肌炎向心肌病的转化。     

Critical role of RAGE and HMGB1 in inflammatory heart disease

Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1–RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.Inflammatory cardiomyopathy is a relatively common cause of acute heart failure in the young, for which an efficient and specific therapy is lacking. Although most patients recover completely, some present a deteriorating course. Recent work from our laboratory and others has focused on the dysregulation of the immune system as an essential modulator of disease induction and progression in heart failure (1, 2). In this context, release of cardiac troponin I (TnI) from damaged cardiomyocytes into the circulation is believed to trigger an autoimmune response to TnI (3, 4).Our group has established an animal model in which immunization with murine cardiac TnI induces severe myocardial inflammation and fibrosis, followed by severe heart failure (1). However, the exact pathomechanism and immune modulators involved in this inflammatory process are not yet fully elucidated.Extensive work has cast light on the role of high-mobility group box 1 (HMGB1) in the pathogenesis of infectious and noninfectious inflammatory diseases. HMGB1, first described as a DNA binding protein, has subsequently been associated with various pathological conditions such as cardiovascular disease (5, 6), cancer (7), and ischemia/reperfusion (I/R) injury (5). It is a key modulator of innate immune responses and regulates in part adaptive immunity (8). In response to cellular stress, HMGB1 acts as a damage-associated molecular pattern (DAMP) signal after passive release into the extracellular milieu during cell death or active secretion by mononuclear and other cell types (7). It binds to receptors such as receptor for advanced glycation end products (RAGE) and Toll-like receptors (TLRs) such as TLR-2 and -4, leading to the expression of inflammatory cytokines, chemokines, and corresponding receptors (9).Some studies describe differential effects of HMGB1- or RAGE-dependent signaling with regard to their concentration and release in a particular model or mode of application (10, 11). In a rodent model of myocardial infarction, exogenously administered HMGB1 had a beneficial effect on postinfarct myocardial remodeling (10). Kitahara and colleagues demonstrated reduced necrosis and smaller infarct size after myocardial infarction in transgenic mice overexpressing HMGB1 (12). However, in a murine model of I/R injury, our group recently showed that treatment of wild-type (wt) mice with recombinant HMGB1 increased the infarct size (6). In the same model of I/R injury, RAGE-deficient mice demonstrated significantly reduced myocardial damage compared with wt mice (6).The effect of HMGB1 and RAGE on the pathogenesis of cardiac disorders is not only described in preclinical animal models but is also investigated in human cardiac disorders. Different studies have revealed an elevated HMGB1 level in patients with heart failure correlating with disease severity (13–17). In addition to this, some studies have identified RAGE as a prognostic factor in human heart failure (16, 18, 19).In the present study, we aimed to clarify the role of HMGB1 and RAGE in an experimental model of murine autoimmune myocarditis. This experimental approach enables a reproducible sterile cardiac inflammation, as described previously (1, 20). Furthermore, the clinical relevance of both proteins should be investigated.Hence, we first studied the expression kinetics of HMGB1 in the inflamed myocardium and serum of wt mice. Then, inhibition of HMGB1 by glycyrrhizin (GL) was performed and heart tissue was analyzed in TnI-induced experimental autoimmune myocarditis (EAM) of wt mice. Additionally, RAGE knockout (RAGE-ko) mice were immunized with TnI to further study the role of RAGE signaling in this EAM model. Finally, the role of the HMGB1–RAGE axis in our model was analyzed by an adeno-associated virus (AAV)9-mediated cardiac overexpression of HMGB1. In a last step, we studied the clinical relevance of HMGB1 and RAGE in patients with myocarditis.     

Mycophenolate mofetil prevents the development of experimental autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) is characterized by the appearance of multinucleated giant cells. EAM leads to severe myocardial damage and is a useful model of human giant cell myocarditis. We investigated whether mycophenolate mofetil (MMF), which is a potent immunosuppressant, prevents the development of myocarditis in a rat EAM model, and focused on the role of osteopontin (OPN) in the pathogenesis of this disorder. Adult Lewis rats were immunized with porcine cardiac myosin to establish EAM. The early MMF treatment completely prevented the development of EAM, and the late MMF treatment was also effective even against established EAM. Echocardiogram demonstrated that left ventricular function was also improved by the treatment with MMF. Real-time RT-PCR analysis showed that both early and late MMF treatments significantly inhibited myocarditis-induced OPN mRNA expression in the heart. Immunohistochemistry revealed that OPN expression was prominent in the myocardium on day 14, whereas expression was observed in the infiltrated macrophages on day 21. Mycophenolic acid (MPA) did inhibit agonist-induced OPN expression in cultured cardiomyocytes. These results show the therapeutic potential of MMF for autoimmune myocarditis and provide new insights into the pathogenesis of this disease.     

炙甘草汤对实验性自身免疫性心肌炎的治疗作用及机制研究

目的 探讨炙甘草汤对实验性自身免疫性心肌炎的疗效及可能的作用机制.方法 40只BALB/c鼠随机分成空白对照组、实验性自身免疫性心肌炎(EAM)组、炙甘草汤组及强的松组各10只.采用重组α肌球蛋白重链多肽(MyHC-α)诱导小鼠制备实验性自身免疫性心肌炎(EAM)模型.将弗氏佐剂与MyHC-α等体积混合,分别在第0 d...     

Suppression of myocardial inflammation using suramin, a growth factor blocker.

Autoimmune myocardial injuries are involved in the pathogenesis of myocarditis and dilated cardiomyopathy, but effective strategies for treating myocardial inflammation have not yet been established. The present study investigated the effects of suramin, a growth factor blocker, on experimental autoimmune myocarditis (EAM) in rats. Lewis rats were immunized with cardiac myosin and placed into one of 4 groups: every 72 h for 1 month the control group (C) was subcutaneously injected with saline; group L received 4mg/kg of suramin; group M, 10 mg/kg: group H, 40 mg/kg. The heart weight/body weight ratios of the M and H groups were significantly lower than that of the C group. Macroscopic and microscopic scores for myocarditis were reduced in the M and H groups. The expression of transforming growth factor (TGF)-beta mRNA in the heart was significantly decreased in the M and H groups compared with the C. In the next experiment, we investigated the effects of suramin on the cytokine milieu in EAM. The serum level of interleukin-10 on day 15 was significantly increased by suramin treatment. Furthermore, suramin increased the number of T cells with Th2 function in the popliteal lymph nodes. Suramin suppressed myocardial inflammation in EAM and was associated with modulation of the Th1/Th2 cytokine milieu and reduced TGF-beta1 expression in the heart.     

Increased expression of CCR5 in experimental autoimmune myocarditis and reduced severity induced by anti-CCR5 monoclonal antibody

Experimental autoimmune myocarditis (EAM) is a T-cell-mediated autoimmune disease. CCR5, which is expressed mostly on activated T cells and monocytes/macrophages, are potent chemotactic factors for autoimmune myocarditis. We investigated the role of CCR5 in the formation of experimental autoimmune myocarditis. Expression of CCR5 and its cognate ligands was assessed by RT-PCR and immunohistochemical analysis. Single-cell suspension of splenocytes and whole blood specimens from EAM mice were subjected to flow-cytometry analysis. We investigated the critical role of CCR5 in EAM mice by adoptively transferring CCR5-positive/negative T cells to mice and by neutralizing CCR5 with monoclonal antibody to observe the influence on the severity and prevalence of myocarditis. In this report, we found that CCR5-positive cells predominate in infiltrated inflammatory cells in cardiac tissue of EAM mice and CCR5-positive T cells in peripheral blood increased markedly in EAM mice compared with controls. Moreover, we demonstrated that the severity of myocarditis was significantly reduced when CCR5-negative T cells from EAM mice were adoptively transferred. When administrated with CCR5-positive T cells, the myocarditis was significantly aggravated. We also demonstrated that blockade of CCR5 with monoclonal antibodies significantly reduced severity of myocarditis in EAM mice. Overall, these findings indicate that CCR5 is important in the induction of EAM and inhibition of CCR5 with monoclonal antibody significantly reduces the severity of myocarditis. CCR5 may have the potential to become a new therapy target against autoimmune myocarditis.     

Allogeneic administration of fetal membrane-derived mesenchymal stem cells attenuates acute myocarditis in rats

We reported previously that the autologous administration of bone marrow-derived mesenchymal stem cells (BM-MSC) significantly attenuated myocardial dysfunction and injury in a rat model of acute myocarditis by stimulating angiogenesis and reducing inflammation. Because BM aspiration procedures are invasive and can yield low numbers of MSC after processing, we focused on fetal membranes (FMs) as an alternative source of MSC to provide a large number of cells. We investigated whether the allogeneic administration of FM-derived MSC (FM-MSC) attenuates myocardial injury and dysfunction in a rat myocarditis model. Experimental autoimmune myocarditis (EAM) was induced in male Lewis rats by injecting porcine cardiac myosin. Allogeneic FM-MSC obtained from major histocompatibility complex-mismatched ACI rats (5 × 10⁵ cells/animal) were injected intravenously into Lewis rats one week after myosin administration. At day 21, severe cardiac inflammation and deterioration of cardiac function were observed. The allogeneic administration of FM-MSC significantly attenuated inflammatory cell infiltration and monocyte chemoattractant protein 1 expression in the myocardium and improved cardiac function. In a T-lymphocyte proliferation assay, the proliferative response of splenic T lymphocytes was significantly lower in cells obtained from FM-MSC-treated EAM rats that reacted to myosin than in cells obtained from vehicle-treated rats with EAM. T-lymphocyte activation was significantly reduced by coculture with FM-MSC. The allogeneic administration of FM-MSC attenuated myocardial dysfunction and inflammation, and the host cell-mediated immune response was attenuated in a rat model of acute myocarditis. These results suggest that allogeneic administration of FM-MSC might provide a new therapeutic strategy for the treatment of acute myocarditis.     

Attenuation of autoimmune myocarditis in rats by mesenchymal stem cell transplantation through enhanced expression of hepatocyte growth factor

Mesenchymal stem cells (MSCs) have various effects, including angiogenic, myogenic, and paracrine actions. In this study, we determined whether MSC transplantation attenuates experimental autoimmune myocarditis (EAM). The mechanisms involved were also investigated. Male Lewis rats were immunized with myosin to establish EAM on day 0. MSCs, isolated from isogenic rats, were injected directly into the myocardium on day 14 (group MSC-2W), day 21 (group MSC-3W), or day 28 (group MSC-4W). In the MSC transplantation groups, cardiac systolic function detected by echocardiography was significantly improved, the EAM affected area determined by histological examination was significantly decreased, and capillary density was increased compared to that in the control groups. Expression of hepatocyte growth factor protein was enhanced by MSC transplantation. MSC transplantation inhibited myocardial expression of interleukin-2, -6, and -10 mRNAs. MSC transplantation reduces the severity of EAM by inducing neovascularization and inhibiting inflammatory cytokine production. Enhanced expression of hepatocyte growth factor was associated with these effects. Autoimmune myocarditis may be a good clinical target for MSC transplantation.     

Tea catechins improve left ventricular dysfunction, suppress myocardial inflammation and fibrosis, and alter cytokine expression in rat autoimmune myocarditis

BACKGROUND: Myocarditis is a clinically serious disease. Tea catechins have been shown to reduce inflammation; however the effects of catechins on the development of myocarditis have not been well studied. AIMS: To clarify the role of catechins, using an experimental autoimmune myocarditis (EAM) model. METHODS AND RESULTS: Lewis rats were immunized with porcine cardiac myosin to establish EAM. Tea catechins were administered orally from day 0 to day 21 (Group A, n=12), from day 14 to day 21 (Group B, n=8), or saline (Group C, n=9) daily. Rats were killed on day 21. Echocardiograms indicated that Group A showed significantly improved cardiac function compared to Group C. Pathologically, non-treated EAM hearts showed severe myocardial cell infiltration and fibrosis; however Group A showed significantly less area. Immunohistochemistry revealed enhanced expression of NF-kappaB and ICAM-1 in non-treated EAM hearts, which was suppressed by catechin administration in Group A. mRNA levels of TNF-alpha were decreased and Th2 cytokines were markedly enhanced in Group A compared with the control group. Late catechin administration (Group B) showed limited effects on EAM. CONCLUSION: The catechins suppressed ventricular remodelling in EAM; thus catechin treatment might be a promising option for the prevention of EAM myocarditis.     

Structural and electrical ventricular remodeling in rat acute myocarditis and subsequent heart failure

OBJECTIVE: We reported that experimental autoimmune myocarditis (EAM) rats showed dramatic changes in ventricular action potential and enhanced arrhythmogenicity in the acute phase, but mechanisms for this are still unclear. To investigate the mechanisms of cardiac remodeling in acute myocarditis and subsequent heart failure, physiological and molecular changes were evaluated along the time course of EAM. METHODS: Six-week-old Lewis rats were immunized with porcine cardiac myosin. On days 14, 21, 35 and 60 after immunization, histology, hemodynamics and electrophysiological parameters (i.e., effective refractory period (ERP), monophasic action potential duration (MAPD) and PVC inducibility) were evaluated and compared with control rats. After these studies, the expression levels of Kv(+) and L-Ca(2+) channels, ion transporters and BNP expressions in the left ventricle were examined by quantitative real time RT-PCR and Western blot analysis. RESULTS: EAM rats showed acute myocarditis with massive infiltration of the mononuclear cells on days 14 and 21. Subsequently, a chronic dilated cardiomyopathy (DCM)-like structural change was observed on day 60. Hemodynamic parameters were worse in EAM than controls. ERP and MAPD were longer in EAM than controls, with a peak on day 21, which was parallel to PVC inducibility. mRNA levels of Kv4.2, Kv1.5, KChIP2, frequenin and SERCA2a, and the protein levels of Kv4.2 and Kv1.5, were reduced, especially in the acute phase. CONCLUSIONS: The initial reduction of Ito-related molecules, such as the expression levels of Kv4.2, 1.5, frequenin and KChIP2, and the prolongation of MAPD are considered to be a key mechanism of ventricular remodeling and cause the characteristic clinical findings in EAM in the acute inflammatory phase and chronic DCM phase.     

A cyclooxygenase-2 inhibitor alters Th1/Th2 cytokine balance and suppresses autoimmune myocarditis in rats

Acute myocarditis is a clinically serious disease; however, no effective treatment has been elucidated. Cyclooxygenase (COX)-2 is a key factor for progression of inflammation. Although inflammation is an essential pathological feature of acute myocarditis, the role of COX-2 in this process remains unclear. Thus, the purpose of this study was to clarify the role of COX-2 in acute myocarditis. We used a rat experimental autoimmune myocarditis (EAM) model and a specific COX-2 inhibitor in this study. Lewis rats were immunized on day 0 with porcine cardiac myosin to establish EAM. We administered the COX-2 inhibitor (meloxicam, 0.1 mg/kg per day) daily; the rats were killed on day 21. Echocardiograms, body and heart weight, heart rate, blood pressure, and histological and molecular examinations were performed. Cytokine expression in the hearts and cell proliferation against cardiac myosin were also analyzed. The COX-2 inhibition during the immune response (late) phase attenuated EAM development; however, the inhibition during the antigen priming (early) phase did not attenuate EAM. The COX-2 inhibitor altered Th1/Th2 cytokine balance and inhibited cell proliferation in vitro. The COX-2 inhibitor suppresses the development of EAM. COX-2 regulation is promising for treating acute myocarditis.     

Attenuation of experimental autoimmune myocarditis by blocking activated T cells through inducible costimulatory molecule pathway

OBJECTIVE: Inducible costimulator (ICOS) is a member of the CD28 family. Although inflammation is an essential pathological feature of myocarditis, the role of ICOS in myocarditis remains unclear. METHODS AND RESULTS: Lewis rats were immunized on day 0 with purified porcine cardiac myosin to establish experimental autoimmune myocarditis (EAM). Flow cytometry was used to examine expression of ICOS on myocardial infiltrating cells. Anti-ICOS antibody or ICOS-immunoglobulin (ICOSIg) was administered intravenously, and rats were killed on day 14 or 21 to study effects of ICOS/ICOS-ligand (ICOSL) pathway blockade during the antigen priming phase (days 0-14) or immune response phase (days 14-21), respectively. The heart weight to body weight ratio was determined, and histological examination and echocardiogram were performed to evaluate the severity of the disease. Cytokine expression in the heart and T cell proliferation against cardiac myosin were analyzed. Flow cytometry revealed that the majority of infiltrating cells, especially CD4-positive cells, expressed ICOS. Blockade of the ICOS/ICOSL pathway during the immune response phase attenuated EAM development. However, blockade of the ICOS/ICOSL pathway during the antigen priming phase did not attenuate and exacerbate EAM. Blockade of T cell activation through ICOS suppressed expression of cytokines including INF-gamma, IL-4, IL-6, IL-10, IL-1 beta, and TNF-alpha and inhibited T cell proliferation in vitro. CONCLUSIONS: Blockade of T cell activation through ICOS during the immune response phase regulates development of EAM, and therefore, ICOS may be an effective target for treating myocarditis.     

Resveratrol ameliorates experimental autoimmune myocarditis.

BACKGROUND: Myosin-induced autoimmune myocarditis of rats is a model of human dilated cardiomyopathy. Resveratrol is a natural polyphenol found in grapes and wine that is reported to have cardioprotective and immunomodulatory effects. METHODS AND RESULTS: To examine the effect of resveratrol on myocarditis, vehicle or resveratrol (50 mg/kg per day) was administered to cardiac myosin immunized rats 1 day before the immunization. At 14 days after immunization, resveratrol had preserved cardiac function of myosin-immunized rats according to echocardiographic analysis. The heart weight/tibial length ratio of vehicle-treated myosin-immunized rats was increased by 1.8-fold compared with unimmunized rats, and resveratrol attenuated the heart weight increase. Resveratrol significantly decreased cellular infiltration, fibrosis, and expression of inflammatory cytokines in the myocardium. Expressions of antioxidant genes were increased in myosin-immunized hearts, and resveratrol decreased those expressions. Resveratrol also attenuated myocarditis 21 days after immunization. SIRT1, a potential effector of resveratrol, was increased in the myocardium of myosin-immunized rats compared with unimmunized rats. The SIRT1 protein was localized mainly in infiltrating mononuclear cells. CONCLUSIONS: Resveratrol significantly ameliorated myocardial injury and preserved cardiac function in a rat model of autoimmune myocarditis. Resveratrol may be a therapeutic modality for myocarditis.     

Effects of HMG-CoA Reductase Inhibitor on Experimental Autoimmune Myocarditis

Purpose  

Myocarditis is an acute inflammatory disease of the heart and is often a precursor of dilated cardiomyopathy. Experimental autoimmune myocarditis (EAM) has been used as a model for human myocarditis. The purpose of this study was to investigate the therapeutic role of 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor, rosuvastatin, on the development of EAM.     

Hepatocyte growth factor ameliorates the progression of experimental autoimmune myocarditis: a potential role for induction of T helper 2 cytokines

Hepatocyte growth factor (HGF) plays a role in cell protection, antiapoptosis, antifibrosis, and angiogenesis. However, the role of HGF in the immune system is not well defined. We examined the influence of HGF on T cells and the effects of HGF therapy in acute myocarditis. Lewis rats were immunized on day 0 with cardiac myosin to establish experimental autoimmune myocarditis (EAM). Human HGF gene with hemagglutinating virus of the Japan-envelope vector was injected directly into the myocardium on day 0 or on day 14 (two groups of treated rats). Rats were killed on day 21. Expression of c-Met/HGF receptor in splenocytes and myocardial infiltrating cells was confirmed by immunohistochemical staining or FACS analysis. Myocarditis-affected areas were smaller in the treated rats than in control rats. Cardiac function in the treated rats was markedly improved. An antigen-specific T cell proliferation assay was done with CD4-positive T cells isolated from control rats stimulated with cardiac myosin. HGF suppressed T cell proliferation and production of IFN-gamma and increased production of IL-4 and IL-10 secreted from CD4-positive T cells in vitro. Additionally, TUNEL assay revealed that HGF reduced apoptosis in cardiomyocytes. HGF reduced the severity of EAM by inducing T helper 2 cytokines and suppressing apoptosis of cardiomyocytes. HGF has potential as a new therapy for myocarditis.     

14<Superscript>th</Superscript> INTERNATIONAL CONGRESS ON CARDIOVASCULAR PHARMACOTHERAPY

Objective Erythropoietin (EPO) has been shown to not only have cardioprotective effects but also attenuate autoimmune diseases. In the present study, we investigated the effect of EPO on cardiac inflammation and function, inflammatory cell infiltration, and cytokine expression in a rat model of experimental autoimmune myocarditis (EAM). Methods and results Male Lewis rats (6–8 weeks old) were immunized on day 0 with porcine cardiac myosin to establish EAM. The rats were subcutaneously administered either vehicle (saline) or human recombinant EPO (6,000 U/kg, 3 days/week) from day 0 to 20, and they were evaluated on day 21. In the EPO group, the inflammation area and heart weight/body weight ratio were significantly attenuated as compared with those in the vehicle group. Blood pressure and cardiac function were also improved in the EPO group. Immunohistochemistry revealed that EPO decreased the infiltration of macrophages and CD4 T cells, and degranulated mast cells in the myocardium. Real-time RT-PCR analysis demonstrated that inflammatory cytokine expression in the myocardium and lymphocytes was suppressed in the EPO group. However, in vitro experiments showed that EPO had no effect on antigen-induced proliferation and cytokine expression in lymphocytes. Conclusion EPO attenuates inflammatory cell infiltration and cytokine expression, and it improves cardiac function and reduces cardiac inflammation in EAM. This beneficial effect of EPO is unlikely to arise from a direct anti-inflammatory action on lymphocytes. These findings suggest the therapeutic potential of EPO for the treatment of myocarditis.     

Impaired up-regulation of CD25 on CD4+ T cells in IFN-gamma knockout mice is associated with progression of myocarditis to heart failure

Inflammation has been recognized increasingly as a critical pathologic component of a number of heart diseases. A mouse model of autoimmune myocarditis was developed to study the role of immune mediators in the development of cardiac dysfunction. We have found previously that IFN-gamma deficiency promotes inflammation in murine myocarditis. It has been unclear, however, how IFN-gamma deficiency in myocarditis affects cardiac function and what underlying immune mechanisms are responsible for these effects. In this work, we show that IFN-gamma knockout (KO) mice have more pronounced systolic and diastolic dysfunction and greater frequency of progression to dilated cardiomyopathy and heart failure compared with WT mice. Cardiac dysfunction in the KO mice is associated with the expansion of activated (CD44(high)) CD3+ T cells due to reduced apoptosis of CD4+, but not CD8+, T cells. CD4+ T cells in the KO mice show impaired up-regulation of CD25 upon activation, resulting in the expansion of CD4+CD44+CD25- T cells and their infiltration into the heart. CD4+CD25- T cells are less apoptosis-prone compared with the CD25+ population, and their infiltration into the heart is associated with greater severity of myocarditis. We conclude that IFN-gamma deficiency in autoimmune myocarditis is associated with preferential expansion of CD4+CD44+CD25- T cells resulting in increased cardiac inflammation. An exaggerated inflammatory response in IFN-gamma KO mice causes cardiac dysfunction, leading to dilated cardiomyopathy and heart failure.     

自身免疫性心肌炎心肌基质金属蛋白酶的其抑制因子表达改变的意义

目的:以人工合成多肽诱导的实验性自身免疫性心肌炎(experimental autoimmune myocar-ditis,EAM)小鼠为实验模型,分析心肌内基质金属蛋白酶(matrix metalloproteinases,MMPs)及组织基质金属蛋白酶抑制因子(tissue inhibitors of matrix mentalloproteinases,TIMPs)表达变化,探讨病理性自身免疫可能的致病机制。方法:人工合成多肽诱导易感品系(BALB/C)小鼠构建EAM模型。超声评价2组小鼠血流动力学变化情况。分别以免疫组化法、逆转录多聚酶链反应(RT-PCR)法,比较2组小鼠心肌组织中MMP-3、MMP-9及TIMP-1在蛋白质水平与基因水平的表达变化。Masson三色染色法分析2组心肌胶原含量情况。结果:病理切片观察模型组小鼠心肌炎症反应明显。超声检查结果显示,模型组较对照组心功能明显恶化。免疫组化与RT-PCR结果分析显示,模型组MMP-3、MMP-9蛋白质及基因表达水平明显高于对照组,而TIMP-1的蛋白质及基因表达水平均相对下降。Masson染色切片分析显示,2组心肌胶原含量差异无统计学意义。结论:EAM模型小鼠心肌组织MMPs表达水平增高,而TIMPs表达水平下降,2者表达比例失衡。该失衡状态可能影响心肌胞外基质的正常功能,并进一步影响心功能。