Haruo Hanawa

Rat dilated cardiomyopathy after autoimmune giant cell myocarditis.

One of the possible causes of dilated cardiomyopathy is considered to be a sequel to myocarditis. Two mechanisms have been proposed in the process of progression of myocarditis into dilated cardiomyopathy: one is a persistent viral infection, and the other is an autoimmune myocardial injury. To clarify the possible part played by the autoimmune mechanism in the process, using an animal model, we investigated whether autoimmune myocarditis, exclusively not related to viral infection, might develop into dilated cardiomyopathy. Experimental autoimmune myocarditis was elicited in Lewis rats by immunization with cardiac myosin fraction. Rats of the control group were immunized with ovalbumin. The clinical course was observed over 4 months. Six rats from the myosin-immunized group died during the acute phase and the healing phase, and all those rats had severe myocarditis. All rats that survived until the end of the study showed enlarged and discolored hearts. Aneurysmal changes were observed in the right ventricle during thoracotomy. The ratio of heart weight to body weight of the myosin-immunized group was significantly higher than that of the control group (3.36 +/- 0.49 versus 2.69 +/- 0.06 g/kg, respectively; P < .005). The lengths of the anterior interventricular fissure and the posterior interventricular fissure of the hearts of the myosin-immunized group were significantly longer than those of the control group. The external diameter of the left ventricle of the myosin-immunized group was also significantly larger than that of the control group. Diffuse myocardial muscle loss and replacement fibrosis were the prominent histological findings of the rats of the myosin-immunized group.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrodynamic-Based Delivery of an Interleukin-22-Ig Fusion Gene Ameliorates Experimental Autoimmune Myocarditis in Rats

IL-22 is one of several cytokines with limited homology to IL-10. However, the biological activities of IL-22 are mostly unknown. The purpose of this study was to evaluate the effect of IL-22 on rat experimental autoimmune myocarditis (EAM) and elucidate an aspect of the biological activities of IL-22. Rats were immunized on day 0; IL-22-Ig-treated rats were injected with pCAGGS-IL-22-Ig and control rats with pCAGGS-Ig using hydrodynamics-based gene delivery on day 1 or day 6. IL-22-Ig gene therapy administered on day 1 or day 6 after immunization was effective in controlling EAM as monitored by the heart weight to body weight ratio, and the myocarditis area in rats was sacrificed on day 17. Examination of the expression of IL-22-related genes in purified cells from EAM hearts suggested that IL-22-Ig acting target cells were noncardiomyocytic (NC) noninflammatory cells such as fibroblasts, smooth muscle cells, and endothelial cells. Therefore, we examined the effect of rIL-22 or serum containing IL-22-Ig on the expression of immune-relevant genes in IL-1-stimulated NC cells cultured from EAM hearts. Results showed that the expression of immunologic molecules (PGE synthase, cyclooxygenase-2, MIP-2, MCP-1, IL-6, and cytokine-induced neutrophil chemoattractant-2) in IL-1-stimulated NC cells was significantly decreased by rIL-22 or serum containing IL-22-Ig. EAM was suppressed by hydrodynamics-based delivery of plasmid DNA encoding IL-22-Ig, and the reason for this effectiveness may be that IL-22 suppressed gene expression of PG synthases, IL-6, and chemokines in activated NC noninflammatory cells.

High-level expression of naked DNA delivered to rat liver via tail vein injection.

High levels of foreign gene expression in mouse hepatocytes can be achieved by rapid tail vein injection of a large volume of a naked DNA solution, the ‘hydrodynamics‐based procedure’. Rats are more tolerant of the frequent phlebotomies required for monitoring blood parameters than mice, and thus are better for some biomedical research.

Recombinant murine interleukin-12 facilitates induction of cardiac myosin-specific type 1 helper T cells in rats.

Autoimmunity after viral myocarditis is considered to be one of the causes of dilated cardiomyopathy. Cytokines are assumed to play an important role in the pathogenesis. We recently reported that interleukin (IL)-2 and interferon (IFN)-gamma mRNA are expressed in the myocardium of rats with experimental autoimmune myocarditis (EAM). However, the role of cytokines in autoimmune myocardial injury in detail is still not clear. Reverse transcription-polymerase chain reaction identified IL-12 (p40) mRNA in antigen-presenting cells in the initial phase of EAM. Cardiac myosin-specific T lymphocytes (MSTLs) were cultured with cardiac myosin peptide (CMP) in the presence of IL-2 and/or IL-12 and were transferred to other naive rats. The results showed that EAM could be effectively induced by transfer of MSTLs cultured with IL-12, whereas transfer of MSTLs cultured with IL-2 was less effective. However, IL-2 acts synergistically with IL-12, and MSTLs cultured with both cytokines most efficiently induce EAM. In vitro experiments showed that MSTLs cultured with both IL-12 and IL-2 produced a much greater amount of IFN-gamma than did MSTLs cultured with either IL-12 or IL-2 alone. The amount of IFN-gamma production was correlated with pathogenicity of MSTLs. Transfer experiments after sorting further demonstrated that the transfer was affected by CD4+ helper T (Th) cells but not by CD8+ cytotoxic T lymphocytes. IL-12 and IL-2 synergistically enhance the pathogenicity of MSTLs. Furthermore, a type 1 Th (Th1) cytokine, IFN-gamma, which is a potent regulatory cytokine of autoimmunity, is produced by MSTLs. IL-12 and IL-2 potentiate the expansion of cardiac myosin-specific Th1 cells and play an important role in the development of autoimmune myocardial injury.

Predictors of Disease Course in Patients With Acute Myocarditis

Background—Clinical manifestations of acute myocarditis, with distinct onset, vary from asymptomatic to fatal. The predictors of the course of the disease in patients with acute myocarditis at initial presentation have not yet been established. In this study, we examined the predictive values of various parameters in the disease course of patients with myocarditis. Methods and Results—Twenty-one consecutive patients who had been diagnosed as having acute myocarditis by histological examinations were analyzed. The patients with myocarditis were divided into the survival group (n=13) and the fatal group (n=8). We examined the parameters of the clinical state, hemodynamic variables, required therapies, biochemical laboratory data, and cytokines. The control groups were composed of 23 patients with old myocardial infarction and 20 healthy volunteers. The fatal group had lower blood pressure and higher pulmonary capillary wedge pressure compared with those values in the survival group. Mechanical ventilation support was more frequently required in the fatal group. Serum levels of soluble Fas (sFas) and soluble Fas ligand (sFasL) were significantly higher in the myocarditis group than in the 2 control groups. Furthermore, levels were significantly higher in the fatal group than in the survival group for sFas (13.93±4.77 versus 3.77±0.52 ng/mL, respectively;P <0.001) and sFasL (611.4±127.7 versus 269.5±37.3 pg/mL, respectively;P <0.05). Other clinical states, hemodynamic variables, required therapies, and biochemical laboratory parameters were not different between the 2 groups. Conclusions—Elevation of sFas and sFasL levels at initial presentation appear to be a good serological marker to predict the prognosis of acute myocarditis.

Low dose carvedilol inhibits progression of heart failure in rats with dilated cardiomyopathy

The cardioprotective properties of carvedilol (a vasodilating β‐adrenoceptor blocking agent) were studied in a rat model of dilated cardiomyopathy induced by autoimmune myocarditis. Twenty‐eight days after immunization, surviving Lewis rats (32/43=74%) were divided into three groups to be given 2 mg kg−1 day−1 (Group‐C2, n=10) or 20 mg kg−1 day−1 (Group‐C20, n=10) of carvedilol, or vehicle (0.5% methylcellulose, Group‐V, n=12). After oral administration for 2 months, body weight, heart weight (HW), heart rate (HR), rat α‐atrial natriuretic peptide (r‐ANP) in blood, central venous pressure (CVP), mean blood pressure (mean BP), peak left ventricular pressure (LVP), left ventricular end‐diastolic pressure (LVEDP), ±dP dt−1 and area of myocardial fibrosis were measured. Values were compared with those for normal Lewis rats (Group‐N, n=10). Two out of 12 (17%) rats in Group‐V died from day 28 to day 42 after immunization. No rat died in Groups‐C2, ‐C20 and ‐N. Although the CVP, mean BP, LVP and ±dP dt−1 did not differ among the three groups, the HW, HR and r‐ANP in Group‐C2 (1.14±0.03, 339±16 and 135±31) and Group‐C20 (1.23±0.04, 305±8 and 156±24) were significantly lower than those in Group‐V (1.36±0.04 g, 389±9 beats min−1 and 375±31 pg ml−1, respectively). The LVEDP in Group‐C2 was significantly lower than that in Group‐V (7.4±1.4 and 12.2±1.2 mmHg, respectively, P<0.05). The area of myocardial fibrosis in Group‐C2 was smaller than that in Group‐V (12±1 and 31±2%, P<0.01). These results indicate that a low dose of carvedilol has beneficial effects on dilated cardiomyopathy.

Characteristics of giant cells and factors related to the formation of giant cells in myocarditis.

Giant cell myocarditis is a serious and frequently fatal inflammatory heart disease of which the etiology remains unknown. In the present study, we investigated the origin of multinucleated giant cells in myocarditis with the use of an experimental model. We also examined the factors relating to the formation of giant cells in myocarditis. Severe myocarditis characterized by the appearance of multinucleated giant cells was induced in Lewis rats by immunization with cardiac myosin in complete Freunds adjuvant. Two types of giant cells, foreign body giant cell-like and myocytelike, were observed in this myocarditis. Immunohistochemical studies revealed that both types of multinucleated giant cells were stained with OX42 and ED1 (macrophage markers) and were not stained with anti-desmin antibody and HHF35 (markers for muscle fibers). Therefore, it is likely that multinucleated giant cells in this myocarditis are derived from macrophages. During the course of the disease, the appearance of multinucleated giant cells was restricted to a period corresponding with the fulminant phase of inflammation. When the severity of the disease was modulated by immunization with various doses of the antigen, multinucleated giant cells appeared only in severe myocarditis after inoculation of a large dose of the antigen. Administration of immunoadjuvants also affected the formation of giant cells. Most of the rats injected with cardiac myosin in complete Freunds adjuvant developed giant cell myocarditis.(ABSTRACT TRUNCATED AT 250 WORDS)

High Expression of IL-22 Suppresses Antigen-Induced Immune Responses and Eosinophilic Airway Inflammation via an IL-10–Associated Mechanism

Allergic inflammation in the airway is generally considered a Th2-type immune response. However, Th17-type immune responses also play important roles in this process, especially in the pathogenesis of severe asthma. IL-22 is a Th17-type cytokine and thus might play roles in the development of allergic airway inflammation. There is increasing evidence that IL-22 can act as a proinflammatory or anti-inflammatory cytokine depending on the inflammatory context. However, its role in Ag-induced immune responses is not well understood. This study examined whether IL-22 could suppress allergic airway inflammation and its mechanism of action. BALB/c mice were sensitized and challenged with OVA-Ag to induce airway inflammation. An IL-22–producing plasmid vector was delivered before the systemic sensitization or immediately before the airway challenge. Delivery of the IL-22 gene before sensitization, but not immediately before challenge, suppressed eosinophilic airway inflammation. IL-22 gene delivery suppressed Ag-induced proliferation and overall cytokine production in CD4+ T cells, indicating that it could suppress Ag-induced T cell priming. Antagonism of IL-22 by IL-22–binding protein abolished IL-22–induced immune suppression, suggesting that IL-22 protein itself played an essential role. IL-22 gene delivery neither increased regulatory T cells nor suppressed dendritic cell functions. The suppression by IL-22 was abolished by deletion of the IL-10 gene or neutralization of the IL-10 protein. Finally, IL-22 gene delivery increased IL-10 production in draining lymph nodes. These findings suggested that IL-22 could have an immunosuppressive effect during the early stage of an immune response. Furthermore, IL-10 plays an important role in the immune suppression by IL-22.

Function, subcellular localization and assembly of a novel mutation of KCNJ2 in Andersen's syndrome.

Andersens syndrome (AS) (which is characterized by periodic paralysis, cardiac arrhythmias and dysmorphic features), a hereditary disease, and missense mutations of KCNJ2 (which encodes an inward rectifying potassium channel) have been reported recently. We performed clinical and molecular analyses of a patient with AS, and found a novel mutation (G215D) of KCNJ2. Twelve-lead electrocardiography revealed a long QT interval and frequent premature ventricular contractions, and polymorphic ventricular tachycardia was induced by programmed electrical stimulation. Use of a conventional whole-cell patch-clamp system with COS7 cells demonstrated that the G215D mutant was non-functional, and that co-expression of wild type (WT)- and mutant-KCNJ2 shows a dominant negative effect on both inward and outward currents. We performed confocal laser scanning microscopy to assess the cellular trafficking of WT- and mutant-KCNJ2 subunits tagged with yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), respectively. Tagging with the YFP did not affect the channel function of WT-KCNJ2 and both proteins showed similar plasma membrane fluorescence patterns. Furthermore, the result of fluorescence resonance energy transfer (FRET) studies at the plasma membrane region suggested that both YFP-tagged WT- and CFP-tagged mutant-KCNJ2 combine to construct a hetero-multimer of the potassium channel. In conclusion, the G215D mutant of KCNJ2 is distributed normally in the plasma membrane, but exhibits a dominant-negative effect and reduces the Kir2.1 current, presumably due to hetero-multimer construction.

Effects of cyclosporine, prednisolone and aspirin on rat autoimmune giant cell myocarditis

OBJECTIVES Preventive effects of cyclosporine, prednisolone and aspirin on autoimmune giant cell myocarditis in rats were investigated. BACKGROUND The therapeutic efficacy of immunosuppressants for human myocarditis is controversial. Although harmful effects of immunosuppressive therapy on experimental viral myocarditis have been reported, the effects on autoimmune myocarditis have not been investigated. Recently, a novel experimental autoimmune myocarditis model characterized by congestive heart failure and multinucleated giant cell has been established. Using this model, the preventive effects of cyclosporine, prednisolone and aspirin on autoimmune myocarditis were investigated. METHODS Lewis rats were immunized with cardiac myosin in complete Freunds adjuvant on days 0 and 7. In experiment 1, four groups of seven rats each were established. Rats in each group received for 21 days intraperitoneal injections of either 1) phosphate-buffered saline solution, 1 ml/day (control); 2) cyclosporine, 20 mg/kg body weight per day (cyclosporine 20); 3) prednisolone, 4 mg/kg per day; or 4) aspirin, 15 mg/kg per day. In experiment 2, two additional groups (five rats each) received for 21 days an injection of cyclosporine, 1 or 5 mg/kg per day (cyclosporine 1 and cyclosporine 5, respectively). All rats were killed on day 21, when histopathologic studies were performed and the titers of antimyosin antibodies were measured. RESULTS The rats in the control, prednisolone and aspirin groups became ill and immobile in week 3. In comparison, rats in the cyclosporine 5 and 20 groups were still active until death was induced. Heart weight/body weight, lung weight/body weight and liver weight/body weight ratios in the rats in the cyclosporine 5 and cyclosporine 20 groups were significantly lower than those in the control group, and no differences were detectable among rats in the control, prednisolone and aspirin groups. The rats in the latter three groups and the cyclosporine 1 groups showed severe myocarditis with multinucleated giant cells. However, myocarditis was effectively prevented in the rats in the cyclosporine 5 and 20 groups. The histologic scores in each group were 2.91 in the control group, 2.14 in the prednisolone group, 2.91 in the aspirin group and 0.02, 2.58 and 0.07, respectively, in the cyclosporine 20, 1 and 5 groups. Production of antimyosin antibodies was remarkably suppressed in rats in the cyclosporine 5 and 20 groups in comparison with values in all other groups. CONCLUSIONS Autoimmune myocarditis is preventable by cyclosporine but not by prednisolone or aspirin in usual dosages.