Masao Hirasawa

Enterovirus RNA Replication in Cases of Dilated Cardiomyopathy: Light Microscopic In Situ Hybridization and Virological Analyses of Myocardial Specimens Obtained at Partial Left Ventriculectomy

Objective: Recently, attention has been focused on enteroviral infection of the heart in the genesis of dilated cardiomyopathy (DCM). To determine the location of enteroviral RNA in the myocardium, we performed light microscopic in situ hybridization (ISH) and virological analyses of myocardial specimens obtained at partial left ventriculectomy (PLV). Methods: Posterolateral walls of the left ventricle from 26 DCM patients were examined. Myocardial specimens were tested for the presence of enteroviral genomes by polymerase chain reaction (PCR). We selected two age‐matched groups (10 patients each) in which entoroviruses were either present (EV‐plus group) or not (EV‐minus group). For both groups, we examined in situ localization of enteroviral RNA in the myocardium by ISH, Results: In PCR studies, both sense and antisense enteroviral RNA were detected in the myocardium of seven patients in the EV‐plus group. The presence of this RNA indicates active viral replication in the myocardium. Five of seven patients who exhibited both sense and antisense enteroviral RNA died early after surgery. On ISH, three patients had evidence of active replication of enteroviral genomes. Viral genomes were present in myocardial lesions, especially in endocardial sites. Viral signals were found in degenerating myocardial cells, in‐terstitial inflammatory cells, and endothelial cells of small vessels. These positive signals were not detected in the myocardium of the EV‐negative group. Conclusions: We detected both sense and antisense enteroviral RNA in various myocardial lesions. This suggests that active enteroviral replication plays a role in the development of myocardial lesions in DCM patients. Active viral replication appears to be a prognostic factor for DCM after PLV. Further study of active viral replication in myocardial lesions will provide information useful for evaluating different therapeutic strategies for DCM.

Mechanism of Inflammation in Murine Eosinophilic Myocarditis Produced by Adoptive Transfer with Ovalbumin Challenge

Background: Interleukin (IL)-5, RANTES and CC chemokine receptor 3 (CCR3) are essential for induction of eosinophil recruitment in organs, but the precise pathogenesis of eosinophilic myocarditis is still unclear. We investigated the relationships between these cytokines and receptors in the development of inflammation in murine myocarditis produced by adoptive transfer, with reference to eosinophil infiltration and signal transduction. Methods: The splenocytes from male donor DBA/2 mice were separated after ovalbumin (OVA) sensitization. These cells had a CD4/CD8 ratio of approximately 3.0. Cells (2.0 × 107) were individually transfused to recipient adoptive male DBA/2 mice, and OVA challenge was performed serially. The heart and spleen of the recipient were analyzed to determine the kinetics of IL-5, RANTES, CCR3 and eosinophil production with simultaneous determination of Janus kinase 3 (JAK3) mRNA. Results: Approximately 85% of recipient mice developed myocarditis; 35% had recognizable cell infiltration in the left ventricular endocardium, an effect which was absent in control mice. Eosinophilic myocarditis was usually associated with animals having several degenerative changes in myocardial cells, and IL-5, RANTES and CCR3 expressions were usually present in these eosinophils (p < 0.05). CCR3 and JAK3 mRNAs were detected in the spleens and hearts of recipient animals providing histological evidence for kinetics related to eosinophil infiltration. Conclusion: The murine model of adoptive transferred myocarditis is suitable for studying the mechanism of eosinophilic myocarditis. A unique pathogenesis of this disorder may be controlled by the synergism of CD4 dominancy in the donor and JAK-STAT signaling in the recipient, which may cause recruitment of eosinophils into heart lesions.

Spontaneous myocarditis in DBA/2 mice. Light microscopic study with transmission and X-ray analytical electron microscopic studies.

Abstract DBA/2 inbred mice spontaneously develop myocarditis and a unique form of subepicardial inflammation of the right ventricle characterized by a prominent eosinophilic infiltrate with calcinosis. We studied this myocarditis using light microscopy and both transmission and analytical X-ray electron microscopy, paying particular attention to eosinophil-associated cardiocyte injury. At 5 weeks of age, many eosinophils and mononuclear cells (MNCs) were seen in the subepicardium of the right ventricle. Electron microscopy showed that cardiocytes underwent degenerative changes, including myofibrillar lysis, accumulation of Z-band material and mitochondrial inclusions, and rupture of plasma membranes. The infiltrating eosinophils appeared to be activated, and cells with cytoplasmic vacuoles, suggestive of degranulation, were noted. The myocardial injury was most severe in the 7th week and healed with myocardial fibrosis and calcinosis by the 8th week. Analytical X-ray electron microscopy showed that the calcinosis was initiated in mitochondrial inclusions of injured cardiocytes. The peripheral eosinophil count did not increase during the course of the disease, but there was a positive correlation between the ratio of eosinophils to infiltrated white blood cells (Eo/WBCs) in the right ventricle and the severity of myocardial damage. Eosinophils may play a significant part in subepicardial cardiocyte injury seen in DBA/2 mice.

Immunologic interaction between infiltrating eosinophils and T lymphocytes in murine spontaneous eosinophilic myocarditis

Background: Eosinophilic myocarditis often occurs spontaneously in DBA/2 mice. Relationships between infiltrating eosinophils (Eos), T lymphocytes and interleukin-5 (IL-5) in this disorder were investigated microscopically and immunohistochemically. Methods: Hearts from male DBA/2 mice were studied from 5 to 10 weeks of age. Anti-CD4 and anti-CD8 antibodies were used. Infiltrating Eos, white blood cells (WBC), and CD4+ and CD8+ T cells were counted. Interactions were assessed with multiple regression and forward stepwise regression tests. Additionally, IL-5 distribution in heart tissue was histologically observed with special reference to immunological findings. Results: Beginning at 5 weeks, several necrotic foci containing many damaged cardiocytes were seen in the epicardium of the right ventricle. Eos and lymphocytes were numerous within and around the necrotic foci. Eos were occasionally seen adjacent to degenerating cardiocytes. Three quarters of the mice exhibited such Eo-related myocardial damage. CD4+ lymphocytes were often seen infiltrating actively inflamed foci where Eos could also be observed. The lesional CD4+-to-CD8+ ratio (CD4/CD8) was 5.2 ± 3.3 at 6 weeks, 6.8 ± 3.7 at 7 weeks, and 1.5 ± 0.8 at 10 weeks of age. The lesional Eo/WBC ratio was directly proportional to the CD4/CD8 ratio (p < 0.05), and was also significantly related to lesional CD4+ T cell count (p < 0.05). IL-5 was also expressed in Eo-rich areas. Conclusions: In this mouse strain with susceptibility to eosinophilic myocarditis, Eo infiltration was related to increased lesional CD4+ cell count. We suspect that CD4+ T cells induce local eosinotaxis, mediated by IL-5, and participate in myocardiocyte injury via Eo induction.