Myosin-induced Myocarditis Research Articles

Role of T Cells in Viral and Immune-mediated Myocarditis

Myocarditis is characterized by inflammatory cell infiltration into the myocardium and a high risk of deteriorating cardiac function with a heterogeneous etiology. Both viral- and myosin-induced myocarditis experimental models are used to mimic myocarditis in humans. Here, coxsackie virus B3-induced and non-virus-induced myocarditis models and data obtained in clinical studies were reviewed. Experimental murine myocarditis following immunization with α-myosin together with complete Freund adjuvant represents the classical immune-mediated model. T helper 1 (Th1) and Th2 pathways and important cytokines are involved in the autoimmunity of myocarditis, and the dynamic balance between Th17 and regulatory T cell seems to have an important role in the process of myocarditis. The purpose of this review is to summarize the existing understanding of the immunological mechanisms underlying myocarditis and exploring gaps in knowledge in both animal and human studies, since these mechanistic insights are a critical requirement for the development of novel therapeutic and vaccination strategies.

Cardioprotective effects of omega 3 fatty acids from fish oil and it enhances autoimmunity in porcine cardiac myosin-induced myocarditis in the rat model.

This experiment proposed to investigate the efficiency of omega 3 fatty acids from fish that improves autoimmune against myocarditis in the rat. Fish oil was extracted from fresh Tuna fish and performed FAME analysis and mice bioassay. The autoimmune myocarditis was induced by subcutaneous injection of porcine cardiac myosin (PCM) into the footpads of rats on the first and seventh day. Rats were dissected on the 21st day to analyze the histopathological, hemodynamic, echocardiographic factors, and immunohistochemistry expressions. In the study, 73.90% of total fatty acids were recorded. Histological analysis revealed that omega 3 fatty acids administrated groups showed tremendous development in the multifocal myocardia hyaline degeneration and necrosis with inflammatory changes. Moreover, omega 3 fatty acids inhabited the expressions of inflammatory cells (CD4, CD8 and CD11b) and suppressed the level of NF-κB. The echocardiographic factors such as heartbeat, SBP, DBP, levels of LVDs, LVDd, LVPW percentage of LVFS, EF, expression levels of inflammatory cytokines (TNF, IL-1β, IFN-ɤ, IL-2, and IL-6) also significantly suppressed by omega 3 fatty acids. Hence, the present study proved that consuming fatty acid-enriched fish might be a successful therapy for improving the inflammatory profile, regenerates the heart tissues, and controlled the production of inflammatory cells.

Phosphoinositide 3-kinase inhibitor LY294002 ameliorates the severity of myosin-induced myocarditis in mice

BackgroundMyocarditis, characterized by myocyte necrosis, fibrosis, and degeneration with mononuclear cell infiltration, always causes heart failure in patients. Phosphoinositide 3-kinase (PI3K) is a pivotal kinase known to regulate inflammatory responses in cardiac diseases. Although previous research has suggested that PI3K was involved in cardiac diseases such as myocardial infarction, it is still unclear whether the inhibition of PI3K is essential for the treatment of myosin-induced myocarditis. The aim of this study was to explore whether pharmacological blockade of PI3K is able to protect mice against experimental autoimmune myocarditis (EAM). Materials and methodsWe used the cardiac myosin-induced murine EAM model to investigate the therapeutic effect of PI3K inhibitor LY294002 on autoimmune myocarditis in mice. ResultsLY294002 significantly alleviated EAM injury in mice, as indicated by the reduction of cardiac necrosis, inflammatory infiltrates, and CD3+ T cells. LY294002 also decreased the expression of p-Akt upon cardiac myosin treatment in the cardiac tissue of the mice. In the present study, LY294002 resulted in a moderate reduction in absolute CD4+ cell numbers and a significant decrease in the absolute numbers of CD8+ cells. Consequently, LY294002 increased the CD4+/CD8+ ratio compared with peptide treatment alone. ConclusionThis report provides evidence that PI3K inhibitor LY294002 has potent effects against cardiac injury caused by EAM, suggesting that it has therapeutic value for the treatment of myocarditis.

Interleukin-17A is dispensable for myocarditis but essential for the progression to dilated cardiomyopathy (135.25)

Abstract A third of myocarditis cases progress to dilated cardiomyopathy (DCM); however, the mechanisms controlling this process are largely unknown. Immunization of BALB/c mice with myocarditogenic peptide in CFA adjuvant induced the infiltration of pathogenic IL-17A-secreting T helper type-17 cells into the inflamed heart during the acute stage; and mild-to-severe DCM in the chronic stage of the disease. However, IL-17A deficiency did not abrogate the incidence or severity of myosin-induced myocarditis, nor did it ameliorate the severe disease of IFNγ-deficient mice, suggesting that IL-17A plays a minimal role during acute myocarditis. In contrast, during the chronic stage, IL-17A-deficient mice but not WT littermates were fully protected from the progression to DCM. Flow cytometry and cytokine analysis revealed an important role for IL-17A in the upregulation of IL-6, TNFα, and IL-1β in the heart tissue, and the recruitment of CD11b+ monocytes and Gr1+ granulocytes into the heart. These differences were associated with a significant decrease in myocardial fibrosis and dysregulation of matrix metalloproteinases. Furthermore, neutralization of endogenous IL-17A after the onset of myocarditis decreased myocardial fibrosis and improved systolic function. Our findings reveal a critical role for IL-17A in adverse cardiac remodeling leading to end-stage DCM, and suggest that targeting IL-17A may be an attractive therapeutic strategy for patients with inflammatory DCM.

Eosinophils are required for the development of myosin-induced myocarditis and dilated cardiomyopathy (135.28)

Abstract Eosinophilic infiltration has been associated with severe myocarditis and depressed cardiac function; however, demonstrating a causal relationship between eosinophils and myocarditis and dilated cardiomyopathy (DCM) has long remained elusive. We show here that Interleukin-5 (IL-5) is dispensable for the recruitment of eosinophils into the inflamed heart, the development of myosin-induced myocarditis (EAM) and the progression of EAM to DCM. However, IL-5-overexpressing transgenic mice developed peripheral eosinophilia, severe eosinophilic myocarditis, increased myocardial fibrosis and accelerated progression to DCM. In addition, eosinophil-deficient (dblGATA1) mice developed attenuated EAM, decreased myocardial fibrosis and greatly improved left ventricular function. Treatment with resorcinol, a specific inhibitor of eosinophilic peroxidase, significantly decreased myocarditis and myosin-induced heart hypertrophy. We conclude that eosinophils are required for the development of EAM, and the progression to DCM, at least in part through their secretion of eosinophilic peroxidase.

Genetic Differences in Bone Marrow-Derived Lymphoid Lineages Control Susceptibility to Experimental Autoimmune Myocarditis

Bone marrow (BM) transplantation has been used to study the cellular basis of genetic control of autoimmune diseases, but conclusions remain elusive due to the contradictory findings in different animal models. In the current study, we found that BM cells from myocarditis-susceptible A.SW mice can render irradiated, myocarditis-resistant B10.S recipient mice susceptible to myosin-induced myocarditis, indicating that hematopoietic cells express the genetic differences controlling susceptibility to autoimmune myocarditis. We then sought to differentiate the role of lymphoid vs nonlymphoid components of BM in the pathogenesis of myocarditis by comparing mixed chimeras receiving BM from A.SW wild-type or RAG(-/-) mice mixed with BM from B10.S wild-type mice. This experiment clearly demonstrated that T and B lymphocytes were indispensable for transferring the susceptible phenotype to disease-resistant recipients. Our findings significantly narrow the cellular expression of genetic polymorphisms controlling the EAM phenotype.

Interleukin-13 Protects Against Experimental Autoimmune Myocarditis by Regulating Macrophage Differentiation

We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45(+) leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1beta, IL-18, interferon-gamma, transforming growth factor-beta1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4(+) T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4(+)CD25(+)Foxp3(+) regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206(+) and CD204(+) macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1beta and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function.

Comparison of angiotensin converting enzyme inhibition and angiotensin II receptor blockade for the prevention of experimental autoimmune myocarditis

The angiotensin converting enzyme inhibitor captopril prevents myosin-induced experimental autoimmune myocarditis. Captopril inhibits production of angiotensin II and increases bradykinin signaling, among other actions. To test whether captopril inhibits disease through blockade of angiotensin signaling, we tested the ability of losartan, an angiotensin II receptor blocker, to prevent myosin-induced myocarditis. A/J mice immunized with the heavy chain of cardiac myosin in complete Freund's adjuvant develop acute myocarditis by day 21 post-immunization, consisting of severe focal inflammation, necrosis and fibrosis. Administration of losartan (250 mg/L in the drinking water) or captopril (75 mg/L in the drinking water) significantly reduced inflammation, necrosis and fibrosis in myosin-immunized mice. The heart weights and the heart weight-to-body weight ratios were also significantly reduced in both treatment groups. However, whereas captopril reduced myosin-specific delayed-type hypersensitivity, losartan did not. Both captopril-treated mice and losartan-treated mice showed a decrease in myosin-specific autoantibody production. Because losartan treatment significantly reduced myocarditis, fibrosis and autoantibody production in EAM, it is likely that prevention of angiotensin II receptor stimulation is a major mechanism underlying the inhibition of myosin-induced myocarditis by captopril.

CD4+/CD8+ macrophages infiltrating at inflammatory sites: a population of monocytes/macrophages with a cytotoxic phenotype

We found a population of nonlymphoid cells expressing both CD4 and CD8 in peripheral blood mononuclear cells (PBMCs) of human T-cell leukemia virus type-I pX transgenic rats with autoimmune diseases. These cells, which showed a monocytic phenotype, were also found in wild-type rats, and their number increased by adjuvant-assisted immunization. GM-CSF increased the number of these double-positive (DP) monocytes in PBMCs. Consistent with the idea that DP monocytes differentiate into DP macrophages at sites of inflammation, we found infiltration of DP macrophages at the site of myosin-induced myocarditis in wild-type rats; these cells exhibited a T-helper 1 (Th1)-type cytokine/chemokine profile and expressed high levels of Fas ligand, perforin, granzyme B, and NKR-P2 (rat orthologue of human NKG2D). Adoptive transfer of GFP-positive spleen cells confirmed hematogenous origin of DP macrophages. DP monocytes had a cytotoxic phenotype similar to DP macrophages, indicating that this phenotypic specialization occurred before entry into a tissue. In line with this, DP monocytes killed tumor cells in vitro. Combined evidence indicates that certain inflammatory stimuli that induce GM-CSF trigger the expansion of a population of DP monocytes with a cytotoxic phenotype and that these cells differentiate into macrophages at inflammatory sites. Interestingly, human PBMCs also contain DP monocytes.

Immunomodulatory and Immunoadsorptive Treatments

Patients with myocarditis and dilated cardiomyopathy (DCM) demonstrate abnormalities of the cellular and humoral immune system. Data from experimental and clinical sources indicate a causal relationship between myocarditis and DCM. It has not yet been possible, however, to establish the relative contributions of cellular and humoral immune disturbances to the pathogenesis and progression of myocarditis and of DCM. An experimental model of autoimmune myocarditis induced by myosin has revealed that activated autoreactive T lymphocytes may mediate myocardial damage [1]. Further, T-cell depletion can modify or abort myosin-induced myocarditis. The transfer of peripheral blood leucocytes can transmit impairment in left ventricular function in patients suffering from myocarditis to mice with severe combined immune deficiency [2]. Publications have indicated that indices of T-cell activation, such as soluble interleukin-2 receptor or b2-microglobulin, are elevated in a substantial proportion of DCM cases [3,4]. Other studies, in contrast, have identified various cardiac autoantibodies against cardiac cellular proteins in patients with myocarditis and DCM. These antibodies include those against mitochondrial proteins, contractile proteins, cardiac–b1-adrenergic receptors, and muscarinergic receptors [5–9]. Elucidation remains to be provided, however, of the functional role of cardiac autoantibodies in the development of DCM and possibly myocarditis. Further clarification is required as to whether these autoantibodies are directly pathogenic, or whether they reflect an in-

Attenuation of myocardial inflammation by celacade™ (Immune modulation therapy) in a murine model of myosin-induced myocarditis

Attenuation of myocardial inflammation by celacade™ (Immune modulation therapy) in a murine model of myosin-induced myocarditis

Cryptic epitope identified in rat and human cardiac myosin S2 region induces myocarditis in the Lewis rat.

Myocarditis is a common cause of dilated cardiomyopathy leading to heart failure. Chronic stages of myocarditis may be initiated by autoimmune responses to exposed cardiac Ags after myocyte damage. Cardiac myosin, a heart autoantigen, induced experimental autoimmune myocarditis (EAM) in susceptible animals. Although cardiac myosin-induced myocarditis has been reported in Lewis rats, the main pathogenic epitope has not been identified. Using overlapping synthetic peptides of the S2 region of human cardiac myosin, we identified an amino acid sequence, S2-16 (residues 1052-1076), that induced severe myocarditis in Lewis rats. The myocarditic epitope was localized to a truncated S2-16 peptide (residues 1052-1073), which contained a sequence identical in human and rat cardiac myosin. The S2-16 peptide was not myocarditic for three other strains of rats, in which the lack of myocarditis was accompanied by the absence of strong S2-16-specific lymphocyte responses in vitro. For Lewis rats, S2-16 was characterized as a cryptic epitope of cardiac myosin because it did not recall lymphocyte and Ab responses after immunization with cardiac myosin. Lymphocytes from S2-16 immunized rats recognized not only S2-16, but also peptides in the S2-28 region. Furthermore, peptide S2-28 was the dominant epitope recognized by T cells from cardiac myosin immunized rats. S2-16 was presented by Lewis rat MHC class II molecules, and myocarditis induction was associated with an up-regulation of inflammatory cytokine production. S2-16-induced EAM provides a defined animal model to investigate mechanisms of EAM and modulation of immune responses to prevent autoimmune myocarditis.

Prevention of myosin-induced autoimmune myocarditis in mice by anti-L3T4 monoclonal antibody.

This study was aimed at studying the effect of the induction of immune tolerance to swine cardiac myosin from anti-L3T4 monoclonal antibody injection and whether the immune tolerance could protect mice with myosin-induced myocarditis from myocardial injury. Twenty-four Balb/c mice were divided into two groups at random. All of the mice were immunized with swine cardiac myosin on the 1st day, 14th, 28th, 42nd, and 52nd day. Immune tolerance was induced by triplicate injections of 400 microg anti-L3T4 McAb on the 0 day (intravenous), 1st day, and 2nd day (intraperitoneal) in McAb-treated group. In the saline-treated group, saline of the same volume as anti-L3T4 monoclonal antibody was used as a control. The sera and hearts biopsies of all mice were collected on the 58th day. The anti-cardiac myosin antibody was examined with ELISA, and pathological changes of heart were observed by light microscope. It was shown that mice immunized with swine cardiac myosin could produce anti-myosin antibody and the anti-cardiac myosin antibody was positive in most of the saline-treated group but negative in the McAb-treated group. Morphologically, myocardial degeneration, necrosis, and infiltration of inflammatory cells were found in the saline-treated group but not in the McAb-treated group. In conclusion, this study indicated that the immune tolerance to cardiac myosin was induced by the anti-L3T4 monoclonal antibody, and accordingly myocardial injury could be prevented by induction of immune tolerance.

Immune mediators in inflammatory heart disease: insights from a mouse model

Cardiac myosin-induced murine myocarditis provides a useful model for assessing the role of immune mediators in the development of inflammatory heart disease. Myocarditis is characterized by leucocyte infiltration, fibrosis, and cardiomyocyte death, which collectively lead to deterioration of both systolic and diastolic function as assessed by pressure-volume relations. In severe cases, disease progresses to dilated cardiomyopathy and heart failure. Key factors that promote disease include complement, tumour necrosis factor-alpha, interleukin-4, and interleukin-12. Factors found to suppress myocarditis include interferon-gamma, interleukin-10, and cyotoxic T lymphocyte antigen-4. Final disease outcome is determined by the interplay of these immune mediators.

Cardiac myosin autoimmunity in acute Chagas' heart disease.

Infection with Trypanosoma cruzi, the agent of Chagas' disease, may induce antibodies and T cells reactive with self antigens (autoimmunity). Because autoimmunity is generally thought to develop during the chronic phase of infection, one hypothesis is that autoimmunity develops only after long-term, low-level stimulation of self-reactive cells. However, preliminary reports suggest that autoimmunity may begin during acute T. cruzi infection. The goal of the present study was to investigate whether cardiac autoimmunity could be observed during acute T. cruzi infection. A/J mice infected with the Brazil strain of T. cruzi for 21 days developed severe myocarditis, accompanied by humoral and cellular autoimmunity. Specifically, T. cruzi infection induced immunoglobulin G (IgG) autoantibodies and delayed type hypersensitivity (DTH) to cardiac myosin. This autoimmunity resembles that which develops in A/J mice immunized with myosin in complete Freund's adjuvant in that myosin-specific antibodies and DTH responses both develop by 21 days postinfection or postimmunization. While the levels of myosin IgG in T. cruzi-infected mice were slightly lower than those in myosin-immunized mice, the magnitude of myosin DTH in the two groups was statistically equivalent. In contrast, C57BL/6 mice, which are resistant to myosin-induced myocarditis and its associated autoimmunity, developed undetectable or low levels of myosin IgG and did not exhibit myosin DTH or myocarditis upon T. cruzi infection. Therefore, humoral and cellular cardiac autoimmunity can develop during acute T. cruzi infection in the genetically susceptible host.

Prevention of Autoimmune Myocarditis Through the Induction of Antigen-Specific Peripheral Immune Tolerance

Autoimmunity to cardiac antigens, in particular cardiac myosin, has been observed in humans with myocarditis and in animals with experimental inflammatory heart disease. Current treatments for myocarditis are in many cases immunosuppressive and might lead to increased cardiac damage by reducing host defenses against infectious agents. Therefore, we sought to develop an antigen-specific approach to inhibit autoimmunity in mice with myosin-induced experimental autoimmune myocarditis. Syngeneic splenocytes, coupled with cardiac myosin by use of ethylene carbodiimide, were administered intravenously before disease induction, and the effects of this peripheral tolerization on myosin-induced myocarditis were assessed. This antigen-specific immunotherapy significantly reduced both the incidence and severity of myocarditis, with the prevention of myocyte necrosis, mononuclear cell infiltration, and fibrosis. Myosin-specific delayed-type hypersensitivity and antibody production were significantly reduced, demonstrating that peripheral tolerance affected both T- and B-cell responsiveness to the autoantigen. These results suggest that the induction of antigen-specific peripheral immune tolerance may be an effective approach for the treatment of myocarditides with autoimmune involvement.

T-Cell-dependent antibody response to the dominant epitope of streptococcal polysaccharide, N-acetyl-glucosamine, is cross-reactive with cardiac myosin.

Autoantibodies against myosin are associated with myocarditis and rheumatic heart disease. In this study, the antigenic cross-reactivity of myosin and N-acetyl-glucosamine (GlcNAc), the dominant epitope of Group A streptococcal polysaccharide, was examined. Six antimyosin monoclonal antibodies (MAbs) derived from mice with cardiac myosin-induced myocarditis were characterized. All MAbs cross-reacted with GlcNAc, mimicking a subset of MAbs derived from rheumatic carditis patients that bind both myosin and streptococcal polysaccharide. Variable (V) region gene usage was diverse, with five of six MAb heavy-chain V regions encoded by distinct members of the J558 family and the sixth encoded by a member of the VGAM3.8 family. Light-chain V-region segments were derived from the Vk1, Vk4/5, Vk10, and Vk21 families. These antimyosin, anti-GlcNac MAbs demonstrated several T-cell-dependent features: they were predominantly immunoglobulin G, were encoded by V-region genes expressed late in development, and displayed somatic mutation. A direct correlation between the extent of somatic mutation and the affinity for myosin was observed. Affinity for GlcNAc also increased with the frequency of mutation, demonstrating that affinity maturation can occur simultaneously for both self antigen and foreign antigen. Based on these observations, we immunized mice with GlcNAc coupled to bovine serum albumin and demonstrated that a T-cell-dependent response to GlcNAc leads to antimyosin reactivity. We speculate that the pathogenic antibody response in rheumatic carditis may reflect the conversion of a T-cell-independent response to GlcNAc to a T-cell-dependent cross-reactive response to GlcNAc and myosin.

Like frogs disguised as leaves

It has long been a controversial issue whether infection by Chlamydia trachomatis directly contributes to heart disease or is a bystander simply finding a place to live on atherosclerotic plaques. Molecular mimicry is an important factor in the exacerbation of various infectious diseases, and this appears to be a possible mechanism operating in the case of the C. trachomatis–heart disease link. Kurt Bachmaier and colleagues1xChlamydia infections and heart disease linked through antigenic mimicry. Bachmaier, K. et al. Science. 1999; 283: 1335–1339Crossref | PubMed | Scopus (335)See all References1 in Toronto have discovered that a peptide extracted from mouse heart muscle has sequence homology to outer membrane proteins of several Chlamydia spp. In mice, cardiac myosin-induced myocarditis is linked to CD4+ T-cell recognition of major histocompatibility complex (MHC) class II-associated peptides derived from α-myosin heavy chain. These peptides are known to be conserved between mouse and human heart muscle. All the Chlamydia-derived mimics of these myosin peptides induce inflammatory heart disease in mice, characterized by perivascular and pericardial infiltration of mononuclear cells and fibrotic changes, which looks like human myocarditis. Inflammation is restricted to heart muscle and is not seen in skeletal muscle. The authors suggest that the presence of bacterial DNA also acts as a powerful adjuvant for autoaggressive T cells. This work has shown that Chlamydia infection, which is of high prevalence in western societies, superimposed on a permissive genetic background and combined with associated risk factors, such as smoking, high cholesterol levels, stress and obesity, can indeed be a significant threat to the precipitation of inflammatory heart disease.

Cellular and molecular mechanisms of murine autoimmune myocarditis.

Dilated cardiomyopathy is a prevalent cause of progressive heart disease and sudden death, and most patients with cardiomyopathy have a history of viral myocarditis. Coxsackie B3 (CB3) picornaviruses can be detected in as many as 50% of these patients and CB3 infections have been epidemiologically linked to chronic heart disease. Several clinical and experimental studies suggest that chronic stages of disease are mediated by an autoimmune response against heart muscle myosin. Human heart disease can be mimicked in mice using cardiac myosin as autoantigen. Murine cardiac myosin-induced myocarditis is an organ-specific autoimmune disease and mediated by CD4+ T cells that recognize a myosin-specific peptide in association with MHC class II molecules. Here, the recent discovery of autoimmune epitopes derived from the alpha isoform of cardiac myosin, the functional roles of surface receptor and signal transduction molecules, and the molecular mechanisms of target organ susceptibility will be discussed.

Cellular immune mechanisms in myosin-induced myocarditis

Cardiac myosin-induced myocarditis proved to be a valuable virus-free murine model with which to investigate autoimmunological mechanisms in inflammatory heart disease. The disease was shown to be T cell-mediated. In this contribution the functional role of CD4 and CD8 molecules and the conditions that are required to make the cardiac tissue susceptible to an autoimmune attack are discussed.