Mural thrombi in mice with acute viral myocarditis

In an investigation of the relationship between mural thrombosis and congestive heart failure (CHF) in acute viral myocarditis, inbred BALB/c mice were inoculated intraperitoneally with the M variant of encephalomyocarditis (EMC) virus and sacrificed on days 7 (n = 33), 10 (n = 35) and 14 (n = 39). Myocarditis was found in 105 of 107 sacrificed mice (98.1%). Myocardial necrosis with cellular infiltration was evident after day 7 and was extensive in the ventricles and atria. In the 105 mice with myocarditis, 17 mice (16.2%) developed CHF after day 10, and 15 mice (14.3%) had thrombi as early as day 7. All thrombi were in the left and/or right atrium. The incidence of thrombi in mice with CHF was higher, but not significantly so, than that in mice without CHF (23.5% vs 12.5%). All 50 control mice had no myocardial lesions or thrombi. This study demonstrates that thrombus formation was not rare in the absence of CHF in the acute stage of viral myocarditis and suggests that clinically acute viral myocarditis has a risk of systemic and/or pulmonary embolism even when resting cardiac function is normal.     

Role of substance P in viral myocarditis in mice

Substance P (SP) is widely expressed in the central nervous system and in peripheral tissues such as myocardial nerves. We examined SP in viral myocarditis in mice induced by encephalomyocarditis virus (EMCV). Localization of SP in the hearts was examined immunohistochemically, and concentrations of SP in hearts and sera were measured by enzyme immunoassay. Substance P levels and density of SP-containing cells in murine hearts on day 6 after EMCV inoculation were decreased compared with those in normal controls. There was a negative correlation between SP levels in the hearts and ratio of heart weight to body weight of the mice at 6 days. Circulating SP levels were decreased in mice on day 6 after EMCV inoculation, and further decreased on day 14. Substance P in hearts and sera is decreased in viral myocarditis in mice, suggesting that SP may play a role in the pathogenesis of viral myocarditis, and that interaction of the neuropeptide nervous system and mast-cell immune system is important in the pathogenesis of viral myocarditis.     

小鼠病毒性心肌炎与心肌细胞凋亡的关系

实验通过给５周龄ＢＡＬＢ／Ｃ小鼠腹腔接种Ｃｏｘｓａｃｋｉｅ Ｂ３ｍ病毒（ＣＶＢ３ｍ），诱发小鼠急性病毒性心肌炎（ＶＭＣ），感染病毒９天后处死小鼠，利用光镜、电镜及原位末端标记法对心肌组织进行检测，结果显示：ＶＭＣ检出率为９３．３３％，心肌细胞凋亡阳性率为８０．００％，凋亡细胞多分布于心内膜下、心外膜下心肌组织，血管内皮细胞和坏死病灶周围，提示细胞凋亡可能是小鼠ＶＭＣ的发病机制之一。     

Virus receptor trap neutralizes coxsackievirus in experimental murine viral myocarditis

OBJECTIVE: The coxsackie and adenovirus receptor (CAR) and the decay-accelerating factor (DAF) are receptors for coxsackievirus B3 (CVB3), which is known as the major cause of human viral myocarditis. We investigated the potential for therapeutic use of soluble virus receptor fusion proteins. METHODS: We designed and generated a novel virus receptor trap (hCAR-hDAF:Fc) consisting of both CVB3 receptors and the Fc portion of human IgG1 and evaluated its antiviral effects in experimental CVB3 myocarditis. RESULTS: Among four soluble virus receptor fusion proteins (hCAR:Fc, hDAF:Fc, hCAR-hDAF:Fc and hDAF-hCAR:Fc), hCAR:Fc and hCAR-hDAF:Fc in the supernatant of transfected cells neutralized echovirus, adenovirus, and various serotypes of CVB in a dose-dependent manner. Both soluble viral receptor proteins bound to the VP0 and VP1 capsid proteins of CVB3. The in vivo efficacy of viral receptor proteins was evaluated by intramuscular injection of plasmid (hCAR:Fc or hCAR-hDAF:Fc) followed by electroporation in a murine model of CVB3 myocarditis. Serum levels of the virus receptor proteins increased relative to baseline values from day 3 and peaked on day 14 at 12.9-fold for hCAR:Fc and 7.1-fold for hCAR-hDAF:Fc. The 3-week survival rate was significantly higher in hCAR-hDAF:Fc-treated mice (61%) than in hCAR:Fc-treated mice (29%) and in controls (15%; p<0.05). Myocardial inflammation, fibrosis, and myocardial virus titers were all significantly reduced in the hCAR:Fc and hCAR-hDAF:Fc groups compared to the controls. CONCLUSION: Our soluble virus receptor trap, hCAR-hDAF:Fc, attenuated viral infection, myocardial inflammation, and fibrosis, resulting in higher survival rates in mice with coxsackieviral myocarditis. Furthermore, it consists exclusively of human components, and we demonstrated that this soluble virus receptor trap may be used as a potential candidate for a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.     

白细胞介素-23在小鼠病毒性心肌炎中的表达

目的:研究小鼠病毒性心肌炎(VMC)模型中白细胞介素-23(IL-23)基因和蛋白的表达,探究其在VMC发病中的作用及其意义.方法:用柯萨奇病毒B3(CVB3)建立Balb/cVMC小鼠模型,实验组注射100 TCID 50病毒液0.1 ml;对照组注射等量磷酸盐缓冲液.在注射的第0、1、2、3、4和6周应用逆转录-聚...     

Effects of prednisolone on acute viral myocarditis in mice

The effect of prednisolone on viral myocarditis was studied in BALB/c mice with encephalomyocarditis virus myocarditis. Prednisolone was injected intramuscularly, 10 mg/kg once a day, on days 4 to 13 (experiment 1) and on days 8 to 17 (experiment 2). The control mice in each experiment received injections of distilled water. In experiment 1, myocardial virus titers were maximal but neutralizing antibodies were rarely present on day 4, and viral titers were still elevated and antibody titers were high on day 8. The survival rate of the prednisolone group was significantly lower (p less than 0.05) than that of the control group on days 21, 22 and 23. On day 10, the antibody titers of the prednisolone group were significantly lower (p less than 0.01) than those of the control group, and viral titers of the prednisolone group remained significantly elevated (p less than 0.01), whereas viruses were rarely isolated in the control group. In experiment 2, the survival rate and antibody titers were not significantly different in the prednisolone and control groups. In both experiments, no viruses were isolated on day 14. The present study suggests that corticosteroids given in the early stage aggravate the course of acute viral myocarditis, and that they may not have detrimental effects if given when neutralizing antibody titer levels are high, although they are not expected to have a beneficial effect.     

酮替芬对小鼠病毒性心肌炎的作用研究

目的研究肥大细胞膜稳定剂酮替芬对脑心肌炎病毒（EMCV）心肌炎的作用。方法4周龄DBA／2小鼠经腹腔接种EMCV制成病毒性心肌炎模型,评价灌胃给予酮替芬对小鼠病毒性心肌炎的作用。观察小鼠在接种EMCV后14d生存率的变化。小鼠心肌切片HE、Masson trichrome染色,观察心室肌坏死和炎症细胞浸润面积的变化。荧光定量RT—PCR检测小鼠心脏促炎细胞因子（IL-6、TNF—α）和肥大细胞蛋白酶类胰蛋白酶及糜蛋白酶（mMCP-4、mMCP-5、mMCP-6）mRNA表达。结果酮替芬治疗使小鼠的14d生存率明显改善（75％比25％,P〈0．01）;小鼠心脏／体重比值显著改善（P〈0．05）;小鼠左心室坏死和炎症细胞浸润面积明显减少（P〈0．05）;IL-6、TNF—α、mMCP－4、mMCP－5及mMCP－6mRNA的表达明显下调（P〈0．05）。结论酮替芬可改善EMCV心肌炎小鼠的心肌炎症和生存率,其作用机制可能与下词心肌促炎细胞因子和肥大细胞特异蛋白酶类胰蛋白酶和糜蛋白酶的表达有关。     

NO与小鼠病毒性心肌炎发生发展的关系

目的 探讨ＮＯ在小鼠病毒性心肌炎发生发展中的作用。方法 建立柯萨奇病毒Ｂ３（ＣＶＢ３）病毒性心肌炎ＢＡＬＢ／Ｃ小鼠模型及ＢＡＬＢ／Ｃ小鼠腹腔巨噬细胞（Ｍφ）培养体系。酶法测定小鼠心肌及Ｍφ分泌ＮＯ含量;免疫组化法检测心肌ｉＮＯＳ;微量滴定法测定心肌内感染性病毒;ＲＴ－ＰＣＲ法检测心肌病毒ＲＮＡ;ＴＵＮＥＬ法检测凋亡细胞;光镜、电镜检查心肌损伤状况。结果 （１）小鼠心肌内ＮＯ于ＣＶＢ３感染后７～３０     

Cell-mediated immune cardiocyte injury in viral myocarditis of mice and patients

The cellular immune mechanism of cardiocyte injury in viral myocarditis was investigated by observing and analyzing the interactions among cardiocytes, T cells, B cells, natural killer (NK) cells and macrophages in situ in the myocardium of our murine model (C3H/He mice) and of human patients. In murine coxsackie B3 virus myocarditis, lymphocyte subsets were identified by light and electron microscopic immunohistochemical techniques with antibodies against specific antigens of pan T (Thy 1.2), helper/inducer T (Th/i) (Lyt 1+), cytotoxic/suppressor T (Tc/s) (Lyt 2+), B (Ig+) and asialo GM1+ cells in the myocardium. In the acute phase of myocarditis, asialo GM1+ (mostly NK) cells predominated over pan T cells and peaked on day 9. Pan T cells then reached a peak on day 14. The T4/T8 (Lyt 1+/Lyt 2+) ratio was 1.3 +/- 0.5 on day 5 and reached a peak of 9.1 +/- 3.6 with an increase of Lyt 1+ cells on day 14. Thereafter, NK cells and T cells gradually decreased and could still be seen in fibrotic foci even 3 and 12 months later. B cells were so scarce that no quantitative evaluation could be made. Electron microscopy revealed that macrophages were in close contact with Th/i cells, target cardiocytes and less commonly, B cells; Tc/s and NK cells also occasionally conjugated with apparently viable or degenerated cardiocytes. Some lymphocytes were located in widened intercellular spaces of dissociated intercalated discs, and in intracytoplasmic widened confines of some cardiocytes (emperipolesis). These results suggest that in the acute phase of myocarditis, NK cells initiate the reaction, and then sensitized cytotoxic T cells and activated macrophages aggravate cell-mediated injury by their close contacts with target cardiocytes; close contacts among macrophages; Th/i cells and a few B cells, and the increased T4/T8 ratio may facilitate regulation of the complex immune network; in the chronic phase, residual but active NK and cytotoxic T cells may sustain cytotoxicity. In the endomyocardial biopsies obtained from 8 patients with viral or idiopathic myocarditis from 3 to 48 days after the clinical onset, conventional electron microscopy revealed actual contacts among cardiocytes, macrophages and lymphocytes. As in our murine model, some lymphocytes had emperipolesed in cardiocytes or were located in widened spaces of dissociated intercalated discs. In 4 of these 8 patients infiltration of Leu 2a+ Tc/s, Leu 3+ Th/i and Leu 7+ cells was identified immunohistochemically, and T4/T8 ratios varied widely from 0.1 to 3.8 in the endomyocardial biopsides.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased severity of viral myocarditis in mice lacking lymphocyte maturation

The role of lymphocytes in the pathogenesis of viral myocarditis is controversial. To better understand how lymphocyte maturation controls a virus-induced myocarditic process, a murine model of viral myocarditis was utilized. Encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene (RAG)-2 knockout 129/SV mice. The RAG2 participate in the maturity of T and B lymphocytes. Survival rate, heart weight (HW), HW to body weight (BW) ratio, viral genome, cardiac inflammation and myocardial necrosis were evaluated after EMCV (500 plaque forming unit/mouse) inoculation. On post-inoculation day 10, the survival rate of C57BL/6, 129/SV and RAG2 knockout mice were 42, 90 and 0%, respectively. Myocardial viral titer was significantly (P<0.05) higher in C57BL/6 and RAG2 knockout mice than in 129/SV mice. In situ hybridization demonstrated the EMCV genome in the myocardium of RAG2 knockout and C57BL/6 mice, but not in 129/SV mice. At day 8, HW and HW/BW ratios were elevated (P<0.05) in RAG2 knockout mice as well as C57BL/6 mice compared with 129/SV mice. Myocardial necroses were more severe in RAG2 knockout mice than in wild-type 129/SV mice. In conclusion, matured lymphocytes protect the development of viral myocarditis which includes viral replication and myocardial apoptosis.     

细胞凋亡与小鼠病毒性心肌炎发生发展的关系

通过对ＢＡＬＢ／Ｃ小鼠腹腔接种ＣＶＢ３ｍ后３，６，９，１２，１５，３０天批随机处死小鼠，取心脏进行检测发现，感染后９－１５天，病毒性心肌炎检出率可达９２．３０％。感染后３－９天，心肌中可分离出病毒，而病毒核酸可持续存在于感染后３个。应用电镜，原位末端标记法及免疫组化法检测发现：ＶＭＣ鼠心肌中可见心肌细胞和血管内皮细胞凋主ＴＧＦ－β１和Ｃ－ｍｙｃ蛋白的表达。     

Significance of pericardial cytokines in giant cell myocarditis in rats--pathological comparison to viral myocarditis in mice

To investigate the precise disease progression in myocarditis, Lewis rats were injected with porcine cardiac myosin, and C3H/He mice were inoculated with coxsackievirus B3. Both were killed serially, and the hearts were stained with hematoxylin-eosin to compare their pathological characteristics. In viral myocarditis, viral replication in the myocardium resulted in myocardial necrosis with inflammation, and the lesions were distributed transmurally, as previously reported. On the other hand, in giant cell myocarditis, inflammatory lesions appeared at first around the capillaries in the epicardium, and thereafter spread transmurally. Pericardial effusion was noticed in all the rats with myocarditis in the fulminant stage. Levels of interleukin (IL) -1beta and IL-6 in the pericardial effusion were elevated compared with the serum cytokines at the peak of inflammation. However, interferon-gamma in both the pericardial effusion and serum was not elevated. The cause of the myocardial lesions that developed in rats with giant cell myocarditis may be some active inflammatory process via the pericardial effusion.     

抗柯萨奇病毒B病毒性心肌炎胶囊复方提取物对病毒性心肌炎小鼠心肌的保护作用

目的探讨抗柯萨奇病毒B病毒性心肌炎胶囊(K-Cox B-JN)复方提取物对于阿霉素(ADM)诱导小鼠心肌损伤模型的保护作用。方法将复方提取物分为不同组别,观察其对心肌组织病理、小鼠心脾指数、小鼠血清肌酸激酶(CK)、磷酸肌酸激酶同工酶(CK-MB)、心肌肌钙蛋白I(c Tn I)的影响。结果 K-Cox B-JN复方总黄酮低、中、高三组、黄酮+皂苷及水提物组能够改善炎性和坏死病理改变,对于CK、CK-MB及c Tn I的活性具有明显抑制作用。结论 K-Cox B-JN复方提取物对病毒性心肌炎小鼠心肌具有保护作用。     

Impaired expression of cardiac adiponectin in leptin-deficient mice with viral myocarditis

A mouse model of encephalomyocarditis (EMC) virus-induced myocarditis was used to investigate the expression of adiponectin in damaged cardiomyocytes. We intraperitoneally injected EMC virus into leptin-deficient ob/ob (OB) mice and wild-type (WT) mice. OB mice were divided into two subgroups consisting of mice with no intervention and mice receiving leptin replacement starting simultaneously with viral inoculation. We determined differences in heart weight, cardiac histological score, numbers of infiltrating and apoptotic cells in the myocardium, expression levels of adiponectin and TNF-alpha mRNA in the heart, adiponectin immunoreactivity in myocytes, adiponectin and TNF-alpha concentrations in the heart, and immunoreactivity of adiponectin receptors in myocytes between OB mice and WT mice. There was significantly decreased adiponectin mRNA expression, immunoreactivity, and protein level in the heart, and reduced immunoreactivity of adiponectin receptor 1 in myocytes from OB mice on days 4 and 8 after viral inoculation as compared with those in WT mice, together with increased cardiac weight, severe inflammatory myocardial damage, and increased levels of cardiac TNF-alpha mRNA and protein. Replacement of leptin in OB mice inhibited the development of severe myocarditis through augmentation of adiponectin mRNA, immunoreactivity, and protein level, increased adiponectin receptor 1 immunoreactivity in myocytes, and suppressed levels of TNF-alpha mRNA and protein. These results suggest that impaired expression of cardiac adiponectin may contribute to the progression of viral myocarditis through enhanced expression of TNF-alpha under a leptin-deficient condition.     

病毒性心肌炎诊断现状与策略

病毒性心肌炎（VMC）患者早期的临床表现、体征及实验室检查结果均缺乏特异性,因而确诊相当困难,国际上尚无统一的标准。VMC的过度诊断难以避免,但因漏诊导致的失治误治,临床上也屡见不鲜。通过复习文献和结合临床研究,提出了以下诊断策略：①临床诊断：严格执行1999年的诊断标准,根据临床资料综合分析,在排除其他心肌疾病的基础上做出诊断;②疑似诊断：补充心脏核磁检查和抗心肌抗体的检测作为筛查手段之一,弥补根据病史、临床表现和实验室检查诊断的不足,避免对一些非典型VMC患者的漏诊或误诊;③病原学诊断：在当前不能普及进行心肌活检的情况下,慎用心肌活检,同时重视对心肌肌钙蛋白的分析。     

P物质抗血清对小鼠病毒性心肌炎的作用研究

目的:通过观察P物质抗血清对脑心肌炎病毒(EMCV)心肌炎的作用,探讨P物质在病毒性心肌炎发病机制中的作用.方法:4周龄DBA/2小鼠经腹腔接种EMCV制成病毒性心肌炎模型,腹腔注射P物质抗血清观察其对小鼠病毒性心肌炎的作用.观察小鼠在接种EMCV后14 d生存率的变化.小鼠心肌切片苏木精-伊红染色、Masson trichrome染色,观察心室肌坏死和炎症细胞浸润面积的变化.甲苯胺蓝(TB)染色显示心室肌组织内肥大细胞,观察肥大细胞密度的变化.荧光定量RT-PCR检测小鼠心脏促炎细胞因子[白细胞介素-6(IL-6),肿瘤坏死因子-α(TNF-α)]、基质金属蛋白酶(MMP-9)和肥大细胞蛋白酶类胰蛋白酶及糜蛋白酶(mMCP-4, mMCP-5, mMCP-6) mRNA表达.ELISA法检测小鼠心脏IL-6、TNF-α及MMP-9的表达.结果:P物质抗血清治疗使小鼠的14 d生存率明显改善(80%: 30%, P<0.05);小鼠心脏/体重比值显著改善(P<0.01);小鼠左心室坏死和炎症细胞浸润面积明显减少(P<0.01);左心室肥大细胞密度明显降低(P<0.01);IL-6、TNF-α、MMP-9、mMCP-4、mMCP-5及mMCP-6 mRNA的表达明显下调(P<0.05);小鼠心脏IL-6, TNF-α及MMP-9蛋白质表达减少(P<0.05).结论:P物质抗血清可改善EMCV小鼠病毒性心肌炎的心肌炎症和生存率,下调心肌促炎细胞因子和基质金属蛋白酶的表达,降低肥大细胞密度及其特异蛋白酶类胰蛋白酶和糜蛋白酶的表达,表明P物质可能在病毒性心肌炎急性炎症和心室重构中起重要作用.     

Apoptosis in congestive heart failure induced by viral myocarditis in mice

Summary It has recently been speculated that progressive deterioration of left ventricular function in chronic heart failure is due to the ongoing loss of viable cardiac myocytes. However, as there is little direct evidence of significant apoptosis contributing to the pathogenesis in cardiac myocytes in vivo, the significance of apoptosis in heart failure remains to be clarified. We investigated the role of apoptosis in heart failure induced by encephalomyocarditis virus myocarditis. DBA/2 mice were inoculated with the virus (day 0), then killed, and their hearts were extracted 3 to 28 days later. Internucleosomal DNA fragmentation, chromatin binding dye staining, and in situ terminal transferase deoxyuridine triphosphate (dUTP) endlabeling were used to detect apoptosis. Internucleosomal DNA fragmentation (DNA ladder) was clearly demonstrated on days 5 to 14 in the virusinfected hearts when myocardial necrosis and infiltration of mononuclear cells were prominent in the hearts. Apoptotic cells demonstrated morphological changes typical of apoptosis (condensation of chromatin and nuclear fragmentation). Both Fas antigen and Fas ligand immunoreactivity were detected in the infiltrating mononuclear cells. The in situ terminal transferase dUTP end-labeling method demonstrated condensed nuclei of infiltrating mononuclear cells on day 7. However, nuclei of cardiac myocytes surrounded by the cellular infiltration were absent. The main source of apoptotic cells in the heart in mice with viral myocarditis appeared to be the infiltrating mononuclear cells. This work was supported in part by a grant-in-aid for General Scientific Research, and a research grant from the Ministry of Health and Welfare, Japan.     

The effect of cyclosporine on the immunopathogenesis of viral myocarditis in mice.

The effect of cyclosporine on the immunopathogenesis of viral myocarditis was studied using a murine viral myocarditis model. Mice in the treated group had a higher mortality rate compared with those of the infected control group before day 15 (47/67 vs. 14/31, p less than 0.05). On day 7, treated mice showed higher titers of anti-heart autoantibody than the control group (12 +/- 7 vs 4 +/- 2, p less than 0.05), but no significant difference was seen on day 14 (28 +/- 15 vs. 39 +/- 34). Histologic lesions, lymphocyte subsets in the peripheral blood and heart in situ, the neutralizing antibody, and virus concentrations in the heart showed no significant differences between these groups. This study suggests that with the use of cyclosporine the production of anti-heart autoantibody was enhanced in the early stages of viral myocarditis in mice, and was associated with higher mortality rate.