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.     

白细胞介素-10基因修饰的未成熟树突状细胞诱导自身免疫性心肌炎特异性耐受的实验研究

目的探讨白细胞介素(IL)-10基因修饰的未成熟树突状细胞(iDC)对实验性自身免疫性心肌炎的作用。方法从Lewis大鼠骨髓培养成熟树突状细胞(mDC)和iDC,pcDNA3-IL-10质粒转染iDC。分别将2×106 mDC、iDC、pcDNA3-iDC、pcDNA3-IL-10-iDC或PBS回输5天前以猪心肌肌球蛋白免疫的实验性自身免疫性心肌炎大鼠体内。3周后观察心肌炎症、超声心动图、Th1/Th2细胞因子、主要组织相容性Ⅱ(MHC-Ⅱ)类分子及共刺激分子表达。以T细胞增殖实验及过继转移实验检测pcDNA3-IL-10-iDC诱导耐受的抗原特异性。结果IL-10基因修饰的iDC显著抑制抗原特异性T细胞增殖,pcDNA3-IL-10-iDC治疗后实验性自身免疫性心肌炎大鼠心肌炎症显著减轻,心功能改善,MHC-Ⅱ、共刺激分子表达显著降低,脾细胞分泌细胞因子呈Th2型。结论IL-10基因修饰的iDC可诱导实验性自身免疫性心肌炎产生抗原特异性耐受,其机制与IL-10诱导的Th1/Th2偏离及MHC-Ⅱ、共刺激分子表达下调等有关。     

A CCR1 antagonist prevents the development of experimental autoimmune myocarditis in association with T cell inactivation

Chemokines play an important role in induction of chemotaxis of immune cells. CCR1 is a chemokine receptor expressed on neutrophils, monocytes, and T lymphocytes. The role of CCR1 in immunity is not well examined. We demonstrated the role of CCR1 on T lymphocytes and the effect of a CCR1 antagonist, BX471 in myocarditis. Lewis rats were immunized with cardiac myosin on day 0 to establish experimental autoimmune myocarditis. Rats were then administered BX471 subcutaneously every day (group BX0: n = 7) or from day 14 (group BX14: n = 7) and were killed on day 21. We confirmed expression of CCR1 in cells infiltrating the myocardium by immunohistochemistry and FACS analysis. The development of myocarditis was almost completely prevented in group BX0, and myocarditis-affected areas were significantly decreased in size in group BX14. Cardiac function was markedly improved. Ribonuclease protection assay showed that the CCR1 antagonist treatment suppressed mRNA expression for IL-6, IL-1beta, and TNF-alpha in the hearts. An antigen-specific T cell proliferation assay was performed with CD4-positive T cells isolated from control rats immunized with cardiac myosin. T cell proliferation was inhibited by the CCR1 antagonist. Additionally, we showed by Western blot that the CCR1 antagonist suppressed ERK1/2 and JNK activities in T cells stimulated with myosin and that IL-2 reversed this suppression. The CCR1 antagonist reduced the severity of EAM by inhibiting cytokine expression and inducing T cell inactivation. Thus, the CCR1 antagonist may provide a novel therapeutic strategy treatment of myocarditis.     

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.     

HMG-CoA reductase inhibitor attenuates experimental autoimmune myocarditis through inhibition of T cell activation

OBJECTIVE: This study tested the hypothesis that 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor affects T cell-mediated autoimmunity through inhibition of nuclear factor-kappaB (NFkappaB) and reduces the severity of experimental autoimmune myocarditis (EAM). METHODS: EAM was induced in Lewis rats by immunization with myosin. High-dose or low-dose fluvastatin or vehicle was administered orally for 3 weeks to rats with EAM. RESULTS: Fluvastatin reduced the pathophysiological severity of myocarditis. Fluvastatin inhibited expression of NFkappaB in the nuclei of myocardium in EAM. Fluvastatin reduced production of Th1-type cytokines, including interferon (IFN)-gamma and interleukin (IL)-2, and inhibited expression of inflammatory cytokine mRNAs in the myocardium. Infiltration of CD4-positive T cells into the myocardium and T cell proliferative responses were suppressed by fluvastatin. Plasma lipid levels did not differ between the groups. CONCLUSIONS: Fluvastatin ameliorates EAM by inhibiting T cell responses and suppressing Th1-type and inflammatory cytokines via inactivation of nuclear factor-kappaB, and this activity is independent of cholesterol reduction.     

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.     

腺病毒载体介导的ICOSIg融合基因对实验性自身免疫性心肌炎作用的研究

目的探讨腺病毒载体介导的ICOSIg基因治疗对实验性自身免疫性心肌炎（EAM）的作用。方法将人ICOS胞外域与免疫球蛋白IgGFc段融合,构建ICOSIg融合基因的腺病毒表达载体P—Adeno—ICOSIg。PoeⅠ消化腺病毒载体后转染HEK293细胞,生产表达ICOSIg融合蛋白的腺病毒;构建增强型绿色荧光蛋白腺病毒作为对照。皮下注射猪心肌肌球蛋白诱导Lewis大鼠EAM模型,随机分为4组,分别于免疫当天（第0天）（A组,n=15）和第14天（B组,n=15）通过股静脉注射ICOSIg重组腺病毒,观察共刺激分子融合蛋白对T细胞激活阶段和炎症阶段的作用,C组（n=10）和D组（n=10）分别在第0天和第14天注射表达增强型绿色荧光蛋白（EGFP）的重组腺病毒。另设E组（n=10）未接受免疫的正常对照组。免疫第28天,超声心动图检测后处死动物。HE染色检测心肌炎症浸润程度。Westernblot检测心肌ICOS、ICOSL、B7—1及B7-2蛋白表达。实时定量RT—PCR定量心肌IL-2、IL-4和IFN-γ mRNA水平。结果第28天B组大鼠心功能指标（短轴缩短率：40．6±6．2比26．2±4．6,20．8±5．6,21．7±9．6）、心肌炎症程度较对照组显著改善。Western blot显示B组ICOS、ICOSL及B7—1蛋白表达显著下调,各组间B7-2蛋白表达差异未见统计学意义。B组大鼠心肌组织IFN-γ mRNA表达降低（19．8±7．9比66．6±4．5,79．6±5．9,80．9±5．1）,IL-4 mRNA表达增加（42．3±8．6比19．7±3．8,22．3±9．0,28．3±2．2）,IL-2表达各组间无显著差异,因此B组IFN-γ／IL-4比值显著低于A、C及D组（0．47±0．36比2．91±0．22,1．89±0．42,2．32±0．83）,表明Th细胞因子平衡向Th2方向偏离。结论炎症高峰期以ICOSIg阻断共刺激分子通路能够减轻EAM心肌组织炎症,改善大鼠心脏功能。其机制可能是下调心肌组织局部ICOS、ICOSL和B7—1表达及对炎症性细胞因子的抑制作用。     

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.     

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.     

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.     

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...     

Pioglitazone, a peroxisome proliferator-activated receptor gamma activator, ameliorates experimental autoimmune myocarditis by modulating Th1/Th2 balance

OBJECTIVES: The aim of this study is to investigate the effect of Peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand on experimental autoimmune myocarditis (EAM). BACKGROUND: Rat EAM model resembles the giant cell myocarditis in human. Recent studies have suggested that Th1 cytokines are involved in the initiation and progression of EAM, whereas Th2 cytokines are associated with the remission. PPAR gamma, which is a member of nuclear hormone receptor superfamily, has been known to affect not only glucose homeostasis but also immune responses, by regulating the Th1/Th2 balance. METHODS: Lewis rats were immunized with cardiac myosin and divided into three groups: Group N, normal control rats (n = 16); Group E, EAM rats (n = 17); and Group P, EAM rats treated with a PPAR gamma activator pioglitazone (n = 20). RESULTS: Cardiac dysfunction and remodeling were inhibited in Group P compared to Group E. Heart weight/body weight ratio and the degree of inflammation and fibrosis were significantly lower in Group P than in Group E. The mRNA levels of macrophage inflammatory protein-1alpha (MIP-1alpha), which plays an important role in the recruitment of inflammatory cells in the early stage of EAM, were upregulated in the heart of Group E, but not in the heart of Group P. Furthermore, the treatment with pioglitazone decreased the expression levels of proinflamatory cytokine (TNF alpha and IL-1beta) genes and Th1 cytokine (IFN-gamma) genes, and increased the expression levels of Th2 cytokine (IL-4) gene. CONCLUSIONS: PPAR gamma ligands may have beneficial effects on myocarditis by inhibiting MIP-1alpha expression and modulating the Th1/Th2 balance.     

Fenofibrate,a peroxisome proliferator-activated receptor alpha activator,suppresses experimental autoimmune myocarditis by stimulating the interleukin-10 pathway in rats

Experimental autoimmune myocarditis (EAM) in rats is an animal model of human giant cell myocarditis and postmyocarditis dilated cardiomyopathy. As the heart consumes large amounts of energy, heart diseases such as myocarditis and dilated cardiomyopathy are associated with abnormal fatty acid metabolism. Peroxisome proliferator-activated receptor alpha (PPARalpha) is a regulator of the oxidative degradation of fatty acids. To investigate the role of PPARalpha in EAM, fenofibrate (a PPARalpha activator) was administered to rats with EAM for 4 weeks. Reductions in the ratios of both ventricular weight to body weight and the area of inflammatory lesions to the total area of heart sections were observed in fenofibrate-treated rats when compared with controls. Fenofibrate ameliorated changes in serum albumin and sialic acid, which are markers of inflammation. Cardiac expression of interleukin-10 (IL-10) mRNA was more pronounced in the fenofibrate group than in the control group (1.3 +/- 0.2 vs 0.7 +/- 0.1; p < 0.01), and the area of intact myocardium correlated with the IL-10 mRNA level (p = 0.0297, r = 0.620). We suggest that PPARalpha activators may prevent the progression of myocarditis through increased expression of the gene encoding the anti-inflammatory cytokine IL-10, although the mechanisms involved remain to be determined.     

Alteration of IL-17 related protein expressions in experimental autoimmune myocarditis and inhibition of IL-17 by IL-10-Ig fusion gene transfer.

BACKGROUND: T-helper (Th)1/Th2 cytokine balance plays an important role in the pathogenesis of myocarditis. Recently, some studies indicate that interleukin (IL)-17, known as a T cell (Th17)-derived proinflammatory cytokine, is the major mediator of tissue inflammation in inflammatory and autoimmune diseases. Experimental autoimmune myocarditis (EAM) is a T cell-mediated autoimmune disease; however, the pathogenic role of IL-17 in the development of rat EAM remains largely unknown. METHODS AND RESULTS: In the present study, alterations of IL-17-related protein expressions were investigated and then the effect of hydrodynamic-based delivery of plasmid DNA encoding the IL-10-Ig gene on rat EAM and the effect of IL-10-Ig on IL-17 was evaluated. The results showed that IL-17 was expressed more highly than IFN-gamma expressed by Th1 cells in alphabetaT cells and the peaks of IL-17 related protein expression in the heart were the early phase of EAM. Moreover, we observed that IL-10-Ig gene therapy was effective in controlling EAM and that IL-10-Ig significantly suppressed the expression of IL-17 as well as other proinflammatory cytokines, IL-1beta and TNF-alpha, in IL-1-stimulated splenocytes cultured from EAM rats. CONCLUSIONS: IL-17 is highly produced by alphabetaT cells in the early phase of EAM hearts and IL-17 inhibition might be a possible mechanism of the amelioration of EAM by IL-10-Ig treatment. These data suggest that IL-17 produced by Th17 plays an important role in the pathogenesis of rat EAM.     

基质金属蛋白酶-9抑制剂治疗自身免疫性心肌炎大鼠模型的实验研究

目的 观察基质金属蛋白酶(MMP)-9抑制剂米诺环素对实验性自身免疫性心肌炎(EAM)Lewis大鼠模型的治疗效果,并探讨其机制.方法 Lewis大鼠60只,6～8周龄,体重150～170 g.双足底注射心肌C蛋白片段和完全弗氏辅佐剂的油状混合物,腹腔注射百日咳毒素建立自身免疫性心肌炎大鼠模型.根据药物干预的时期不同将大鼠分为早期干预组、中期干预组和晚期于预组3组,每组20只,不同的干预组又分为米诺环素治疗组(治疗组)和对照组两个亚组,每个亚组10只.早期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第1～21天.中期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第8～28天.晚期干预组治疗组为腹腔注射50 mg/kg的米诺环素,1次/d,连续21 d,免疫注射后第15～35天.各对照组均为在相同时间给予与治疗组相同体积的生理盐水腹腔注射.干预结束后,处死动物,心脏取材,进行系列检测.组织病理学石蜡切片苏木素-伊红(HE)染色检测心肌炎症分级,天狼猩红染色检测心肌胶原纤维含量,免疫组织化学染色检测心肌巨噬细胞和T细胞浸润,实时定量PCR检测MMP-2、MMP-9的mRNA表达水平,冰冻切片用于原位明胶酶谱法检测明胶酶活性.结果 早期干预组和中期干预组的治疗组大鼠心肌组织石蜡切片显示心肌组织炎症积分均显著低于其相应对照组[分别为1.51±0.36比3.03±1.35(P＜0.05)和2.11±0.82比3.75±0.29(P＜0.01)],免疫组织化学显示心肌巨噬细胞和T淋巴细胞浸润数目均显著低于其相应对照组,天狼猩红染色显示心肌间质纤维积分和纤维含量均明显低于其相应对照组[分别为1.51±0.35比2.75±0.29(P＜0.01)和1.61±0.42比2.50±0.41(P＜0.05)],实时定量PCR检测心肌MMP-2和MMP-9 mRNA表达低于其相应对照组,原位明胶酶法显示心肌明胶酶活性显著低于其相应对照组[分别为62 366±2131比162 367±5095(P＜0.01)和113 197±4809比184 256±5427(P＜0.01)].晚期干预组治疗组与对照组相比较心肌组织炎症积分、心肌巨噬细胞和T淋巴细胞浸润数目、心肌间质纤维化积分和纤维含量、心肌组织MMP-2和MMP-9 mRNA表达及心肌明胶酶活性差异均无统计学意义.结论 MMP-9抑制剂抑制EAM的早期病理发展,其机制可能与降低心肌炎症细胞浸润,延缓心肌间质纤维化,从转录水平降低明胶酶mRNA的表达,同时从蛋白水平降低明胶酶活性表达有关.

Abstract：

Objective To investigate the effects of matrix metalloproteinase-9 (MMP-9) inhibitor minocyclin hydrochloride in Lewis rats with experimental autoimmune myocarditis (EAM). Methods EAM was induced by injection of cardiac C protein emulsified in completed Freund adjuvant in double footpad and intra peritoneal injection of pertussis toxin on 6- to 8-week old Lewis rats. Sixty EAM Lewis rats were dividedinto 3 groups (early, middle and late intervention groups, n =20 each: 10 minocyclin treated and 10 control rats). In early intervention group, rats in treatment group received intraperitoneal injection of minocyclin hydrochloride from 1st to 21st day after immunization; in middle intervention group, rats were treated from 8th to 28th day after immunization and in late intervention group, rats were treated from 15th to 35th day after immunization (50 mg/kg body weight, once daily). Control rats received intraperitoneal injection of same volumetric physiological saline at corresponding time periods. At the end of intervention, rats were euthanatized and hearts were harvested. Paraffin sections were used for hematoxylin and eosin stain to determine the inflammatory score, for picrosirius stain to determine fibrosis score and collagen content, and for immunohistological stain to determine macrophages and T lymphocytes. Real time PCR was used to detect mRNA expression of myocardial MMP-2 and MMP-9. Cryostat sections were used for in situ zymography to detect protein activity of gelatinase. Results Inflammatory score in cardiac paraffin slides, number of cardiac macrophages and T lymphocytes, cardiac interstitial fibrosis score and content, expression of MMP-2, 9 mRNA and activity of gelatinase in treatment group were all significantly lower than in control group for early and middle intervention groups ( inflammatory score: early control group vs. treatment group: 3.03 ± 1.35 vs. 1.51 ±0. 36,P ＜0. 05, middle control group vs. treatment group: 3.75 ±0. 29 vs. 2. 11 ±0. 82,P ＜0. 01; cardiac interstitial fibrosis score, early control group vs. treatment group: 2. 75 ±0. 29 vs. 1.51 ± 0.35, P＜0.01, middle control group vs. treatment group: 2.50 ±0.41 vs. 1.61 ±0.42, P＜0.05;gelatinase, early control group vs. treatment group: 162 367 ±5095 vs. 62 366 ±2131, P ＜0. 01, middle control group vs. treatment group: 184 256 ±5427 vs. 113 197 ±4809, P ＜0. 01 ) while these parameters were similar between minocyclin-treated and control rats in late intervention group ( all P ＞ 0. 05 ).Conclusions MMP-9 plays an important role in the pathogenesis of autoimmune myocarditis. Inhibition of MMP-9 in early and middle stage could significantly attenuate inflammatory responses and myocardial fibrosis in this experimental EAM model.     

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.     

FN14 Signaling Plays a Pathogenic Role in a Mouse Model of Experimental Autoimmune Myocarditis

BackgroundThe pathogenesis of inflammatory cardiomyopathy is affected by the activation of autoimmune-mediated cascades. To study these cascades, we developed an experimental model of troponin I (TnI)-induced autoimmune myocarditis (EAM). One factor playing a pivotal role in the context of autoimmune disorders is the receptor fibroblast growth factor-inducible 14 (FN14). Thus, the impact of FN14 in the development of autoimmune myocarditis was investigated.Methods and ResultsTnI-immunization led to a significantly increased myocardial FN14 mRNA and protein expression in wild-type (wt) mice. To investigate the precise role of FN14 in EAM, FN14 knockout (ko) and wt littermates were immunized with TnI or control buffer. The animals were evaluated for cardiac parameters and indicators of myocardial injury. FN14 deficiency resulted in better cardiac performance, less myocardial inflammation, fibrosis, and cardiac damage. A lower myocardial mRNA expression of inflammatory cytokines and chemokines as well as their receptors could be demonstrated in TnI-immunized FN14ko compared to wt mice also immunized with TnI. Western blot analysis revealed a contribution of nuclear factor kappa-light-chain-enhancer of activated B cells to FN14-induced signaling cascades.ConclusionsIn the pathogenesis of autoimmune myocarditis, the inflammatory response to cardiac injury is attenuated in FN14ko mice. Thus, inhibition of FN14 in patients might represent a novel therapeutic strategy in the treatment of inflammatory cardiomyopathy.     

卡维地洛减轻自身免疫性心肌炎-抗氧化相关的抗炎作用

目的卡维地洛在实验性心脏损伤模型中具有显著的心脏保护作用。本研究探讨是否卡维地洛能减轻自身免疫性心肌炎,并探讨其可能的主要机制。方法 6周龄Lewis大鼠诱导自身免疫性心肌炎,随机分为正常对照、阳性对照、不同剂量的卡维地洛、美多洛尔、普萘洛尔、以及卡维地洛消旋体治疗组。观察其对急性心肌炎症程度,及对炎症心肌内多种炎性细胞因子mRNA表达及蛋白表达、心肌细胞内蛋白氧化产物和细胞膜脂质过氧化产物TBARS含量的影响。结果尽管两种剂量的卡维地洛、美多洛尔和普萘洛尔显示出同等的β受体阻滞作用,只有卡维地洛治疗使心肌炎症得到减轻,心重/体重、炎症分级严重程度明显减轻;卡维地洛消旋体虽无β受体阻滞作用,但心肌炎严重程度也明显减轻;与阳性对照组比较,卡维地洛治疗显著降低蛋白羧基含量和TBARS产物,而美多洛尔和普萘洛尔治疗组则无明显变化;也只有卡维地洛治疗明显降低心肌炎性细胞因子mRNA表达和IL-1β表达。体外实验证实卡维地洛和卡维地洛消旋体保护离体心肌细胞膜的脂质过氧化、并以剂量依赖方式抑制培养的U937细胞LPS刺激后IL-1β释放。结论卡维地洛治疗减轻自身免疫性心肌炎,其心脏保护作用可能与其抗氧化和抑制炎症细胞因子...     

Therapy with granulocyte colony-stimulating factor in the chronic stage, but not in the acute stage, improves experimental autoimmune myocarditis in rats via nitric oxide

We systematically investigated serial efficacy of granulocyte colony-stimulating factor (G-CSF) therapy upon experimental autoimmune myocarditis (EAM) in rats treated with and without the inhibition of nitric oxide (NO) with the analyses of tissue regeneration. G-CSF could mobilize multipotent progenitor cells of bone marrow into the peripheral blood and may improve ventricular function. A rat model of porcine myosin-induced EAM was used. After the immunization of myosin, G-CSF (10 μg/kg/day) or saline was injected intraperitoneally on days 0-21 in experiment 1 and on days 21-42 in experiment 2. Additional myosin-immunized rats were orally given 25 mg/kg/day of N^G-nitro-l-arginine methylester (l-NAME), an inhibitor of nitric oxide synthase (NOS), in each experiment (each group; n = 8-21). Serum cytokines and peripheral blood cell counts were measured in each group. In experiment 1, G-CSF treatment aggravated cardiac pathology associated with increased macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) levels and enhanced superoxide production. In experiment 2, G-CSF treatment reduced the severity of myocarditis with increased capillary density and improved left ventricular ejection fraction. In the rats with EAM treated with G-CSF associated with oral l-NAME treatment in experiment 2, the severity of myocarditis was not reduced. Myocardial c-kit⁺ cells were demonstrated only in G-CSF-treated group in experiment 2 but not in other groups. G-CSF has differential effects on EAM in rats associated with the modulation of cytokine network. The overwhelming superoxide production by G-CSF administration in the acute stage may worsen the disease. G-CSF therapy improved cardiac function via NO system in a rat model of myocarditis in the chronic stage, but not in the acute stage, possibly through the myocardial regeneration and acceleration of healing process.