Progression Of Experimental Autoimmune Myocarditis Research Articles

Sinomenine hydrochloride alleviates autoimmune myocarditis via suppressing Th1 cell induced-M1 macrophage pyroptosis

Abstract Background Myocarditis is typical of the complex inflammatory response in the heart with high morbidity and mortality, accompanied by tissue damage and cardiac fibrosis. Previous studies have demonstrated that CD4+ T cells are crucial to the cardiac autoimmunity of myocarditis, but the role of different CD4+ T cell subsets remains unclear. Recently, sinomenine hydrochloride, a natural compound from the traditional Chinese herb Sinomenium acutum, is reported to inhibit T cell proliferation in some systemic autoimmune diseases such as rheumatoid arthritis. Given that autoimmune myocarditis is an organ-specific autoimmune disease, the effect of sinomenine hydrochloride on autoimmune myocarditis demands further study. Purpose This study aims to elucidate the role of CD4+ T-helper 1 (Th1) cells in regulating inflammatory cell death in autoimmune myocarditis and investigate the pharmacological effect of sinomenine hydrochloride on CD4+ Th1 cells and autoimmune myocarditis. Methods Male Balb/c mice were immunized with myosin heavy chain-α peptides to establish the experimental autoimmune myocarditis (EAM) model, followed by the treatments with sinomenine hydrochloride by gavage. Tohoku Hospital Pediatrics-1 (THP-1) cell line and murine T cells were used for in vitro experiments. Results In the acute phase of EAM, we observed plenty of CD4+ T cells infiltrating the heart tissue. RNA sequencing revealed that among the genes encoding typical transcription factors of CD4+ T cell subset, only Tbx21 and Stat4, both representing Th1 cells, were upregulated, which consisted with immunofluorescence staining. Further studies indicated that Th1 cells not only mediated classical activated macrophage (M1 macrophage) polarization by secreting interferon-γ (IFN-γ) but also associated with pro-inflammatory macrophage pyroptosis in the EAM, with the cleavage of gasdermin D (GSDMD), caspase-1 and IL-1β. Subsequent in vitro experiments confirmed that IFN-γ could dramatically promote macrophage pyroptosis combined with ATP, leading to the release of IL-1β and IL-18(Figure 1). To find an effective method for Th1 cell suppression and myocarditis remission, we identified that sinomenine hydrochloride impeded Th1 cell activation, differentiation and proliferation in vitro. Furthermore, sinomenine hydrochloride inhibited Th1 cell infiltration and M1 macrophage pyroptosis in the EAM heart, In the chronic phase, cardiac remodeling and fibrosis were suppressed, and cardiac systolic function was significantly improved in the treatment group (Figure 2). Conclusion Our study demonstrates that Th1 cells-induced M1 macrophage pyroptosis is an essential pathogenic mechanism of EAM. Sinomenine hydrochloride can alleviate the severity and progression of EAM, which may become a novel therapeutic strategy for treating myocarditis.Figure 1Figure 2

Single cell RNA sequencing to dissect transcriptional signature of cardiac fibroblasts activation in experimental autoimmune myocarditis

Abstract Introduction Dilated cardiomyopathy (DCM) is a condition that evolves from inflammation of the myocardium, and it is often associated with a poor prognosis and fatal outcome. Experimental autoimmune myocarditis (EAM) represents a CD4+ T cell-dependent animal model of acute myocarditis followed by the development of post-inflammatory DCM. Purpose The aim of the study was to investigate the transcriptional changes in cardiac fibroblasts during disease progression in a mouse model of EAM at the single cell level. Methods 6–8-week-old BALB/c mice (or reporter mice expressing EGFP under the collagen type I promoter) were immunized with αMyHC peptide together with Complete Freunds’ Adjuvant. Live cardiac cells were sorted from healthy hearts (d0), at acute myocarditis (d19), during resolution of inflammation (d25), and at the DCM stage (d35) using the BD FACSAria II Cell Sorter. Single-cell cDNA libraries were prepared using 10x Genomics technology, and the sequencing was performed on an Illumina NovaSeq 6000 platform, targeting 50000 reads per cell. Single cell RNA sequencing data was analyzed using RStudio software and Seurat packages. At d0 and d19 of EAM, bulk RNAseq was performed on live cardiac EGFP+CD45-CD34- fibroblasts. Results Following a quality control check, we included in the analysis 25058 cells. We identified 13 distinct main cell populations, including fibroblasts, lymphoid and myeloid immune cells, endothelial cells, smooth muscle cells, and cardiomyocytes (Fig. 1A). Differential expression analysis revealed that fibroblasts in acute myocarditis (d19) showed activation of genes related to immune processes such as cytokine-cytokine receptor interaction, IL-17, and TNF-alpha signaling pathways. Subset analysis on 4901 cardiac fibroblasts resulted in 5 distinct fibroblast subsets, FB1–FB5 (Fig. 1B). A major fibroblast subset found in the heart (FB1) expanded during the course of EAM (Fig. 1C). Of note, cells expressing the myofibroblastic signature (FB4) were identified at d19 only (Fig. 1C). Fibroblast subset FB1 activated expression of proinflammatory genes, such as Ly6a, Ly6c1, Cxcl14, Ccl2, and Ccl7, particularly during the inflammatory phase of EAM (d19, Fig. 1D). Surprisingly, the expression of profibrotic genes, including Col1a1, Col3a1, and Postn in FB1, was downregulated during the progression of EAM (Fig. 1E). Bulk RNA sequencing analysis confirmed that cardiac fibroblasts in acute myocarditis (d19) activated immune processes (chemokine production), but also showed dysregulated expression of genes involved in cytoskeletal re-organization and extracellular matrix turnover. Conclusions Our data indicated that acute inflammation in the heart activated a pro-inflammatory transcriptional response in cardiac fibroblasts and induced a fibroblast-to-myofibroblast transition. Understanding changes in transcriptomic signatures on a single cell level can help to develop new therapies in inflammatory heart diseases.

Abstract 13808: Investigating Autophagy in Experimental Autoimmune Myocarditis Using Single Cell RNA Sequencing and Spatial Transcriptomics

Background: Autophagy has emerged as an important process in the context of heart failure. Experimental autoimmune myocarditis (EAM) represents an animal model of acute myocarditis (d19) and post-inflammatory dilated cardiomyopathy (d35). Aim: To explore autophagy dynamics in a mouse model of EAM at the single cell level. Methods: 6-8-week-old BALB/c mice were immunized with αMyHC peptide and complete Freund’s adjuvant. Single-cell RNA (scRNA) libraries were prepared using 10x Genomics technology and sequenced, targeting 50’000 reads/cell. For spatial transcriptomics, snap-frozen hearts embedded in OTC were sectioned into 10 μm-thick fragments and placed on 10x Genomics Visium Gene Expression Slide and processed using 10x Genomics Visium protocols. The data were analyzed using RStudio software. LC3B and Beclin-1 proteins were detected by IHC. Results: After quality control check, we included in the RNAseq analysis 25’058 cells clustering in 11 distinct cell populations (Fig. 1A). Expression of autophagic genes Atg7 and Becn1 was relatively consistent across all cell populations, whereas Atg3 and Map1lc3b (encoding LC3B) showed high expression specifically in monocytes. Analysis of monocytes indicated up-regulation of Atg3, Atg7 and Becn1 during the progression of EAM. Spatial transcriptomics analysis identified 5911 spots under the tissue and results showed an up-regulation of Atg3, Atg7, Becn1 and Map1lc3b in the hearts during the inflammatory phase of EAM. This increased expression was found throughout the entire heart section (Fig. 1B). Moreover, we detected the presence of LC3B and Beclin-1 proteins specifically localized within the inflammatory compartment of inflamed hearts (d19). Conclusions: In the EAM model, acute inflammation in the heart is associated with activated autophagy. Targeting this autophagic response could potentially improve disease outcomes. Financed by the National Science Centre (Poland), grant 2019/35/B/NZ5/00551

Linagliptin, A Xanthine-Based Dipeptidyl Peptidase-4 Inhibitor, Ameliorates Experimental Autoimmune Myocarditis

This study sought to show the mechanism of how to ameliorate experimental autoimmune myocarditis (EAM) by administering dipeptidyl peptidase (DPP)-4 inhibitor linagliptin. The number of RAR-related orphan nuclear receptor gamma-positive Th17 cells infiltrated to the EAM myocardium was significantly attenuated by linagliptin treatment. Tandem mass spectrometry-based analysis demonstrated that DPP-4 binds to cathepsin G in EAM hearts, thereby protecting cathepsin G activity through inhibiting SerpinA3N activity. Linagliptin suppresses oxidative stress in EAM hearts as well. Thus, we found that DPP-4 plays a detrimental role in the progression of EAM by interacting with cathepsin G, which, in turn, suppresses SerpinA3N activity.

Identification of molecules associated with disease progression of autoimmune myocarditis to dilated cardiomyopathy

Abstract Myocarditis is an inflammatory disease characterized by immune infiltrates into the myocardium. In many patients, the disease progresses to a chronic phase dilated cardiomyopathy (DCM) in which there is extensive fibrosis leading to ventricular dilation and impaired cardiac function. Less than 50% of these patients survive more than 5 years. Immunosuppressive treatments such as corticosteroids (CSs) are effective in managing the acute phase of the disease, but do not prevent progression to the DCM. Using the experimental autoimmune myocarditis (EAM) animal model, we previously demonstrated that the inflammatory cytokine macrophage migration inhibitory factor (MIF) plays a role in resistance to CS treatment. MIF knockout mice were treated with the corticosteroid Dexamethasone (Dex) and proved to be resistant to both EAM and DCM. We also observed that treatment with MIF inhibitors and Dex decreased the expression of chemokines and adhesion molecules and implicated these molecules in the progression of disease. Within these, osteopontin (OPN), a powerful chemoattractant, was downregulated. We hypothesized that OPN plays a role in the disease process and investigated its role in EAM and DCM using OPN knockout mice. Our results show that OPN knockout mice developed EAM, but that progression to DCM was impaired. Furthermore, we observed a decrease in CD45+CD133+ fibroblast progenitor cells infiltrating the myocardium in the absence of OPN, which could contribute to attenuated disease in KO mice. This study may provide insights into the mechanisms driving progression of EAM to DCM and point to potential therapeutic targets.

TLR4 Activation Promotes the Progression of Experimental Autoimmune Myocarditis to Dilated Cardiomyopathy by Inducing Mitochondrial Dynamic Imbalance.

Mitochondrial dynamic imbalance associates with several cardiovascular diseases. However, the role of mitochondrial dynamics in TLR4 activation-mediated dilated cardiomyopathy (DCM) progress remains unknown. A model of experimental autoimmune myocarditis (EAM) was established in BALB/c mice on which TLR4 activation by LPS-EB or TLR4 inhibition by LPS-RS was performed to induce chronic inflammation for 5 weeks. TLR4 activation promoted the transition of EAM to DCM as demonstrated by increased cardiomyocyte apoptosis, myocardial fibrosis, ventricular dilatation, and declined heart function. TLR4 inhibition mitigated the above DCM changes. Transmission electron microscope study showed that mitochondria became fragmented, also with damaged crista in ultrastructure in EAM mice. TLR4 activation aggravated the above mitochondrial aberration, and TLR4 inhibition alleviated it. The mitochondrial dynamic imbalance and damage in DCM development were mainly associated with OPA1 downregulation, which may be caused by elevated TNF-α level and ROS stress after TLR4 activation. Furthermore, OMA1/YME1L abnormal degradation was involved in the OPA1 dysfunction, and intervening OMA1/YME1L in H9C2 significantly alleviated mitochondrial fission, ultrastructure damage, and cell apoptosis induced by TNF-α and ROS. These data indicate that TLR4 activation resulted in OPA1 dysfunction, promoting mitochondrial dynamic imbalance and damage, which may involve in the progress of EAM to DCM.

Inhibition of MIF as a novel treatment for autoimmune myocarditis and dilated cardiomyopathy.

Abstract Myocarditis is an inflammatory disease of the myocardium and a major cause of sudden death in young adults. It is characterized by the presence of immune infiltrates and necrotic myocytes in the myocardium. Importantly, patients often progress to a more severe form of the disease termed dilated cardiomyopathy (DCM), which leads to ventricular dilation and impaired cardiac function. Despite the inflammatory and autoimmune nature of this disease condition, immunosuppressive treatments such as corticosteroids (CSs) have not been very effective in preventing myocarditis and its progression to DCM. We hypothesized that macrophage migration inhibitory factor (MIF) may play a role in resistance to CSs, as it is the only known pro-inflammatory cytokine to be induced by CSs. Importantly, MIF counter-regulates CS-mediated immunosuppression. Using the experimental autoimmune myocarditis (EAM) animal model, we observed that MIF−/− mice treated with Dexamethasone (Dex) were highly resistant to EAM and progression to DCM. Furthermore, using small molecule inhibitors of MIF combined with Dex treatment recapitulated this phenotype in wild type mice. Investigating the mechanism we found that treatment with MIF inhibitors and Dex decreased the expression of key chemokines and adhesion molecules and implicated these molecules in the progression of EAM to DCM. Our results suggest that MIF promotes the recruitment of inflammatory cells to the myocardium to promote DCM. Moreover, the results suggest therapeutic inhibition of MIF in combination with CSs as a novel treatment approach to prevent DCM. Last, our studies may provide new insights into the mechanisms driving DCM.

Dipeptidyl Peptidase-4 Suppression Leads to Attenuation of Cardiac Dysfunction Caused by Autoimmune Myocarditis Through Inhibiting Cathepsin-G Activity

We have shown previously that dipeptidyl peptidase-4 (DPP-4) plays a critical role in the development of experimental autoimmune myocarditis(EAM). Here we show that the mechanism how DPP-4 promotes the progression of EAM in mice. Mouse EAM model were given a diet mixed with linagliptin, a DPP-4 inhibitor (3mg/kg/day)(LINA-group), or normal diet (CONT-group). Left ventricular ejection fraction after 21 days of EAM induction was significantly higher in LINAgroup than in CONT group. Immunostaining showed that the number of Th17 cells infiltrated to the EAM heart was significantly smaller in LINA-group than in CONT-group. Consistent with this finding and the fact that Th17 cells express high level of active DPP-4, the activity of DPP-4 in the EAM heart was significantly lower in LINA-group than in CONT-group. Mass spectrometry and co-immunoprecipitation-western blot analyses revealed that DPP-4 physically interacts with cathepsin-G(CTSG) in inflamed myocardium. The CTSG activity in the EAM heart was significantly higher in EAM mice compared to those in untreated mice, and the activity of CTSG in inflamed myocardium was significantly lower in LINA-group than in CONT-group. The amount of angiotensin II, a product catalyzed by CTSG, in inflamed myocardium was also significantly lower in LINA-group than in CONT-group. In conclusion, suppression of DPP-4 causes a salutary effect in the cardiac function during EAM by inhibiting CTSG activity in the heart.

Mulberry Leaf Diet Protects Against Progression of Experimental Autoimmune Myocarditis to Dilated Cardiomyopathy Via Modulation of Oxidative Stress and MAPK‐Mediated Apoptosis

To examine the protective effects of dietary administration of Mulberry leaves (ML) on postmyocarditis dilated cardiomyopathy (DCM) focusing on oxidative and endoplasmic reticulum stresses and adverse myocardial remodeling. In this study, we used a rat model of cardiac myosin-induced experimental autoimmune myocarditis to test the effects of ML diet (MLD) (5%) on various markers of cardiac remodeling and function. After 4weeks of immunization, the rats were fed with 5% MLD for 4weeks. By the end of the study, echocardiography was performed to assess the myocardial dimensions. The heart tissue was used for histopathology and Western blotting analyses. Our study showed that the postmyocarditis rats exhibited increased oxidative stress when compared with the control rats. MLD supplementation suppressed this change, compared with vehicle treatment. In addition, postmyocarditis rats showed significant elevation of the endoplasmic reticulum stress markers, which were prevented by the MLD supplementation. Similarly the vehicle-treated rats suffered with the adverse myocardial remodeling in the form of fibrosis as evidenced by the Azan-Mallory staining and immunohistochemistry for collagen-III levels, compared with the control rats. However, MLD treatment not only markedly attenuated cardiac fibrosis, but also improved the left ventricular ejection fraction and fractional shortening. Interestingly, the myocardial levels of endothelin-1, activated members of mitogen-activated protein kinase (MAPK) pathway, and vascular endothelial growth factor (VEGF) were significantly attenuated by MLD, indicating that the antihypertrophic effects of MLD are partially mediated via endothelin-1, MAPK, and VEGF pathway. Collectively, these results suggest that supplementation of rats with 5% MLD has the ability to regulate cardiac remodeling and improves cardiac function and hence contributes to prevent the development of postmyocarditisdilated cardiomyopathy.

Effect of hydrodynamics-based delivery of IL-18BP fusion gene on rat experimental autoimmune myocarditis

Interleukin-18 (IL-18) is a powerful and important cytokine in myocarditis. IL-18-binding protein (IL-18BP), a naturally occurring antagonist of IL-18, is presumed to play a vital regulatory function in IL-18-mediated immune responses. The purpose of this study was to evaluate the alterations of IL-18 and its related protein expressions and the effect of hydrodynamics-based delivery of the IL-18BP gene for treatment of rat experimental autoimmune myocarditis (EAM).Rats were immunized on Day 0 and killed on 2, 3 and 4 weeks to determine IL-18 and its related protein expression and target cells in EAM hearts. On Day 6, rats were injected with a recombinant plasmid encoding IL-18BP-Ig or SP-Ig. On Day 17, rats were detected with echocardiography and then be killed. IL-18BP gene therapy was effective in controlling EAM, as monitored by a decreased ratio of heart weight to body weight, reduced myocarditis areas, reduced expression of atrial natriuretic peptide, brain natriuretic peptide, IL-17, IFN-γ, IL-6 and IL-10. Furthermore, the effect of serum containing IL-18BP on the expression of immune-relevant genes in IL-1α-stimulated NC cells and splenocytes cultured from EAM rats was examined. The results showed that IL-18BP significantly suppressed the expression of IL-17 as well as other proinflammatory genes such as transforming growth factor-β, prostaglandin E2 synthase, cyclooxygenase-2 in IL-1α-stimulated NC cells, and IL-18BP also significantly suppressed the expression of IL-17, IL-17R, IL-21 and IL-17-related transcriptional factor retinoic acid-related orphan nuclear receptor, signal transducer and activator of transcription-3 and Foxp3 in IL-1α-stimulated splenocytes cultured from EAM rats. IL-18 and its related protein played an important role on the development of EAM. IL-18BP effectively prevented progression of EAM by blocking IL-17 and related inflammatory genes expression. This might be a possible mechanism of the amelioration of EAM by IL-18BP treatment.

161 : IL6 promotes the progression of experimental autoimmune myocarditis to dilated cardiomyopathy

We have recently reported that IL17A −/− animals are protected from progression of experimental autoimmune myocarditis (EAM) to dilated cardiomyopathy (DCM) as determined by echocardiography on day 54. IL17A −/− mice had lower IL6 levels in their heart compared with wildtype (WT) BALB/c animals. This was the only cytokine found to differ significantly between the two groups at both days 14 and 21. To investigate the IL6 requirement for DCM development we used two experimental approaches. First, we over-expressed IL6 in WT and IL17A −/− animals through hydrodynamic gene delivery (HGD) on days −1 or 7 of EAM. Mice over-expressing IL6 showed worsening of heart function compared to controls. Second, we blocked IL6R signaling using an anti-IL6R monoclonal antibody (mAb) in WT mice with EAM. Mice administered the anti-IL6R mAb on days 7 and 14 or 14 and 21 of EAM were protected from heart failure and DCM development by day 54 of disease. At day 14 of EAM there was reduced STAT3 phosphorylation in the heart after anti-IL6R mAb administration and increased STAT3 phosphorylation following HGD of IL6. In the mice receiving anti-IL6R mAb there was a significant decrease in the total number of CD45 + cells infiltrating the heart as measured by flow cytometry with similar composition in controls. Mice over-expressing IL6, both WT and IL17A −/− genotypes, had more CD11b + F4/80 + macrophages as well as CD11b + Ly6G + neutrophils in the heart. IL6R mAb treated mice had lower circulating complement C3 levels where over-expressing mice had higher C3 levels than controls. Our data suggest that in EAM IL6 promotes the development of DCM during early events around day 7.

IL17A and IFNγ jointly control rapidly fatal eosinophilic myocarditis in mice (P4120)

Abstract Deviation to autoaggressive T helper 17 (Th17) responses has been reported to account for the protective effect of interferon-gamma (IFNγ) in certain animal models of autoimmune disease. Building on our previous findings that interleukin-17 (IL17) signaling is critical for the progression of experimental autoimmune myocarditis (EAM) to dilative heart failure, we sought to address the question of whether IL17 mediated the severe form of EAM observed in IFNγ-deficient mice. To our surprise, we found that IFNγ-/-IL17A-/- double-knockout (DKO) mice had greater mortality compared to IFNγ-/- mice, starting at day 14 and reaching 50% by day 21 of EAM. DKO hearts were severely infiltrated, indicating acute inflammatory heart failure as the mode of death in these mice. We found pronounced eosinophilic infiltration in DKO mice, implicating eosinophils as mediators of the enhanced morbidity. Cardiac production of CCL11/eotaxin was increased, alongside Th2 deviation among heart-infiltrating CD4+ T cells. Intriguingly, blockade of IL4 in IFNγ-/-IL17A-/-DKO mice inhibited Th2 deviation, but failed to resolve eosinophilia or morbidity. However, developmental ablation of eosinophils partly rescued the survival of DKO mice. These data point to a novel collaboration between IFNγ and IL17A in controlling eosinophil recruitment.

IL6 is necessary for the progression of experimental autoimmune myocarditis to dilated cardiomyopathy (171.11)

Abstract Previous findings in our lab have showed that IL17A deficient animals are protected from the progression of experimental autoimmune myocarditis (EAM) to dilated cardiomyopathy (DCM). These findings showed an increase of systemic IL6 in the WT animals that progressed to heart failure, whereas the protected IL17A deficient animals had lower IL6 levels. This was the only cytokine shown to differ consistently between the two groups at both day 14 and 21. We sought to address the question of whether systemic IL6 levels can mediate disease and is therefore critical for DCM development. To investigate the IL17A requirement for DCM development through its induction of IL6, we used two experimental approaches. First, we over-expressed IL6 in WT animals with EAM through hydrodynamic gene delivery on day -1 prior EAM induction. WT mice, which over-expressed an IL6 insert, showed worsening of heart function by echo on day 54 compared to controls with empty plasmid. Second, we blocked IL6R signaling using an anti-IL6R monoclonal antibody (mAb) in WT BALB/c mice with EAM. In mice administered the anti-IL6R mAb on days 6 and 13 of EAM, the mice were protected from heart failure and DCM development by day 60 of disease compared to animals receiving isotype control antibodies. Our data suggest that IL6 early during EAM is necessary for the development of DCM.

Involvement of AMPK and MAPK signaling during the progression of experimental autoimmune myocarditis in rats and its blockade using a novel antioxidant

There are various reports suggesting the role of angiotensin (Ang) receptor blockers, Ang converting enzyme inhibitors, calcium channel blockers, diuretics and antioxidants against the progression of experimental autoimmune myocarditis (EAM) to dilated cardiomyopathy (DCM). Most of them were reported to be effective during this adverse cardiac remodeling. Recently much attention has been paid to studying the involvement of AMP-activated protein kinase (AMPK) and mitogen activated protein kinase (MAPK) in various cardiovascular ailments. AMPK acts as a master sensor of cellular energy balance via maintenance of lipid and glucose metabolism. Evidences also suggest the relation between AMPK and oxidative stress during physiological and pathological myocardial cellular function. Since, it is of interest to identify the roles of AMPK and MAPK during the progression of EAM to DCM and also the effect of edaravone, a novel free radical scavenger, against its progression. For this, we have carried out western blotting, histopathological staining and immunohistochemical analyses to measure the myocardial expressions of AMPK signaling and oxidative stress related parameters in normal and vehicle or edaravone-treated EAM rats, respectively. We identified the myocardial levels of phospho Akt and phosphoinositide 3-kinase, which are the upstream proteins of AMPK and MAPK activation and both were up-regulated in the vehicle-treated rats, whereas candesartan treatment significantly reversed these changes. We have also measured the myocardial levels of p-AMPKα, different isoforms of protein kinase C and MAPK signaling proteins. All of these protein levels were significantly elevated in the hearts of DCM rats whereas edaravone treatment significantly reversed these changes. In viewing these results, we can suggest that along with MAPK, AMPK signaling also plays a crucial role in the progression of EAM and it can be effectively blocked by the treatment with a novel antioxidant, edaravone.

Quercetin offers cardioprotection against progression of experimental autoimmune myocarditis by suppression of oxidative and endoplasmic reticulum stress via endothelin-1/MAPK signalling

In order to test the hypothesis that treatment with quercetin at a dose of 10 mg/kg protects from the progression of experimental autoimmune myocarditis (EAM) to dilated cardiomyopathy (DCM), we have used the rat model of EAM induced by porcine cardiac myosin. Our results identified that the post-myocarditis rats suffered from elevated endoplasmic reticulum (ER) stress and adverse cardiac remodelling in the form of myocardial fibrosis, whereas the rats treated with quercetin have been protected from these changes as evidenced by the decreased myocardial levels of ER stress and fibrosis markers when compared with the vehicle-treated DCM rats. In addition, the myocardial dimensions and cardiac function were preserved significantly in the quercetin-treated rats in comparison with the DCM rats treated with vehicle alone. Interestingly, the rats treated with quercetin showed significant suppression of the myocardial endothelin-1 and also the mitogen activated protein kinases (MAPK) suggesting that the protection offered by quercetin treatment against progression of EAM involves the modulation of MAPK signalling cascade. Collectively, the present study provides data to support the role of quercetin in protecting the hearts of the rats with post myocarditis DCM.

IL17A switches from a pathogenic to a protective function in experimental autoimmune myocarditis in the absence of IFNγ. (115.14)

Abstract Deviation to autoaggressive Th17 responses has been reported to account for the protective effect of IFNγ in certain animal models of autoimmune disease. Building on our previous findings that IL17 signaling was critical for the progression of experimental autoimmune myocarditis (EAM) to dilative heart failure, we sought to address the question of whether IL17 mediated the severe form of EAM observed in IFNγ-deficient mice. To our surprise, we found that IFNγ-/-IL17A-/- double-knockout (DKO) mice had greater mortality compared to IFNγ-/- mice, starting at day 14 and reaching 50% by day 21 of EAM. DKO hearts were severely infiltrated, indicating acute inflammatory heart failure as the mode of death in these mice. We have found evidence of aberrant eosinophilic Th2-deviated infiltration in DKO mice, implicating eosinophils in mediating the enhanced morbidity. Production of IL5 by CD4+ cells was increased in DKO hearts, as well as cardiac production of CCL11/eotaxin. Heart-infiltrating DKO CD4+ cells showed evidence of Th2 deviation. These data point to a novel collaboration between IFNγ and IL17A in controlling eosinophil recruitment by autoaggressive Th2 cells. Moreover, they indicate that myocarditis is not restricted to a single CD4+ subset - but instead reflects redundancy among Th1, Th2, and Th17 populations to elicit qualititative and quantitative variations in disease phenotype and progression.

The Anti-Inflammatory Mechanism of Prostaglandin E2 Receptor 4 Activation in Rat Experimental Autoimmune Myocarditis

Prostaglandins (PG) and their specific receptors for E type PG (EP) play an important role in inflammatory diseases. Although myocarditis results in inflammation of the heart, roles of PG and EP in its pathophysiology is still controversial. To clarify the role of PG and EP on the progression of myocarditis, we used an experimental autoimmune myocarditis model. A selective EP4 (EP4RAG) agonist was administered into both early (Day 0 to 21) and late (Day 14 to 21) -treated groups and the animals were killed on Day 21. We found that improved cardiac function was detected in the EP4RAG-treated groups in comparison to the untreated group. The infiltration area ratio in the early-treated (16.6% ± 4.6%) group was lower than those in the untreated group (32.1% ± 3.5%) (P < 0.05). The fibrosis area ratios in the early-treated (19.2% ± 6.3%) and the late-treated groups (24.4% ± 5.1%) were lower than those in the untreated group (37.4% ± 2.6%), respectively (P < 0.05). Moreover, we found that EP4RAG decreased T-cell proliferation and monocyte chemoattractant protein-1 production in vitro. We concluded that a selective EP4 agonist inactivates T-cells, which turns out to moderate the progression of experimental autoimmune myocarditis. Therefore, EP4 can be an effective target for myocarditis treatment.

Sulfated Polysaccharide Fucoidan Ameliorates Experimental Autoimmune Myocarditis in Rats

Homing of cardiac myosin-specific CD4-positive T cells into the myocardium is the initial pathologic event of experimental autoimmune myocarditis (EAM). Subsequently, various bystander inflammatory cells are recruited into the myocardium crossing vascular endothelial cell walls. Sulfated polysaccharide fucoidan binds selectin nonselectively and blocks its function. Therefore, this study was designed to evaluate whether in vivo fucoidan treatment can improve EAM. A 21-day infusion of physiological saline or fucoidan was administrated intraperitoneally to the rats with sham operation (sham-saline, n = 5; sham-fucoidan, n = 6) or those with cardiac myosin injection (EAM-saline, n = 10; EAM-fucoidan, n = 10). After 3 weeks, fucoidan treatment improved left ventricular ejection fraction (79.04 ± 2.81 vs 65.94% ± 3.22%; P < .01 vs EAM-saline) with a reduced ratio of heart weight to body weight (4.016 ± 0.239 vs 4.975 ± 0.252 mg/g; P < .05 vs EAM-saline) in EAM. Furthermore, fucoidan treatment decreased serum levels of BNP (292.0 ± 53.4 vs 507.4 ± 89.2 ng/mL; P < .05 vs EAM-saline) and the myocarditis area (31.66 ± 1.53 vs 42.51% ± 3.24%; P < .01 vs EAM-saline) in EAM. These beneficial effects of fucoidan were accompanied by inhibition of both macrophage and CD4-positive T-cell infiltration into the myocardium. Fucoidan, a nonselective selectin blocker, attenuates the progression of EAM. This observation may be explained, at least in part, by blocking the extravasation of inflammatory cells into the myocardium.

The antioxidant edaravone attenuates ER-stress-mediated cardiac apoptosis and dysfunction in rats with autoimmune myocarditis

Experimental autoimmune myocarditis (EAM) is mediated by myocardial infiltration by myosin-specific T-cells secreting inflammatory cytokines. In this study, rat models of EAM were prepared by injection with porcine cardiac myosin. One week after immunization, edaravone was administered intraperitoneally at 3 or 10 mg/kg/day to rats for 2 weeks. Cardiac function was measured by haemodynamic and echocardiographic studies and TUNEL assay was performed. Left ventricular (LV) expression of NADPH oxidase sub-units (p47phox and p67phox), pro-inflammatory cytokines (TNF-α), endoplasmic reticulum (ER) stress signalling proteins (GRP78, caspase-12 and GADD153) and mitogen-activated protein kinase (MAPK) family proteins (phospho-p38 MAPK and phospho-JNK) were measured by western blotting. Edaravone improved LV function in a dose-dependent manner. Central venous pressure was significantly low and LV ejection fraction and fractional shortening was significantly high in edaravone groups compared with those in the vehicle group. In addition, edaravone treatment down-regulated LV expressions of p47phox, TNF-α, GADD153, phospho-p38 MAPK and phospho-JNK. Furthermore, the LV expressions of p67phox, GRP78, caspase-12 and TUNEL-positive cells of rats with EAM treated with edaravone were significantly low compared with those of the vehicle group. These findings suggest that edaravone ameliorated the progression of EAM by inhibiting oxidative and ER stress and, subsequently, cardiac apoptosis.

The Ras antagonist farnesylthiosalicylic acid ameliorates experimental myocarditis in the rat

Myocarditis is an inflammatory disorder of the heart in which T lymphocytes have a central role. No effective treatment is currently at hand for management of the myocarditis. Lymphocyte function requires the active signal transducer Ras. We thus hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane and induces its rapid degradation, will attenuate experimental autoimmune myocarditis (EAM). Two groups of Lewis rats were induced to develop EAM by immunization with porcine cardiac myosin. Group A received 5 mg/kg of FTS, and group B received phosphate-buffered saline (PBS) according to two protocols: FTS or PBS was given 2 days before myosin immunization in protocol 1 and FTS or PBS was given 14 days after myosin immunization in protocol 2. FTS significantly suppressed myocarditis, and this effect was accompanied by a reduction in myosin-specific cellular and humoral immune responses. In the longer regimen, FTS treatment for 6 weeks was associated with preservation of myocardial function made evident by echocardiography. In vitro, FTS significantly attenuated the proliferation of lymphocytes from untreated myocarditic rats to myosin. FTS is effective in suppressing the progression of EAM and its consequent functional myocardial dysfunction. The effect may be mediated by suppression of the cellular and humoral responses to myosin.