Experimental Autoimmune Myocarditis Model Research Articles

Acacetin alleviates autoimmune myocarditis by regulating CD4+ T cell mitochondrial respiration

BackgroundMyocarditis refers to an autoimmune inflammatory response of the myocardium with characterization of self-reactive CD4+ T cell activation, which lacks effective treatment and has a poor prognosis. Acacetin is a natural flavonoid product that has been reported to have anti-inflammatory effects. However, acacetin has not been investigated in myocarditis.MethodsOral acacetin treatment was administered in an experimental autoimmune myocarditis model established with myosin heavy chain-alpha peptide. Echocardiography, pathological staining, and RT-qPCR were used to detect cardiac function, myocardial injury, and inflammation levels. Flow cytometry was utilized to detect the effect of acacetin on CD4+ T cell function. RNA-seq, molecular docking, and microscale thermophoresis (MST) were employed to investigate potential mechanisms. Seahorse analysis, mitoSOX, JC-1, and mitotracker were utilized to detect the effect of acacetin on mitochondrial function.ResultsAcacetin attenuated cardiac injury and fibrosis as well as heart dysfunction, and reduced cardiac inflammatory cytokines and ratio of effector CD4+ T and Th17 cells. Acacetin inhibited CD4+ T cell activation, proliferation, and Th17 cell differentiation. Mechanistically, the effects of acacetin were related to reducing mitochondrial complex II activity thereby inhibiting mitochondrial respiration and mitochondrial reactive oxygen species in CD4+ T cells.ConclusionAcacetin may be a valuable therapeutic drug in treating CD4+ T cell-mediated myocarditis.Graphical

Identification of key TE associated with myocarditis based on RNA and single-cell sequencing data mining

Cardiomyopathy is a severe cardiac condition characterized by complex immune regulatory mechanisms. While the role of immune genes is recognized, the specifics of their regulation in cardiomyopathy are not fully understood. Recent studies highlight the significance of transposable elements (TEs) in various diseases, particularly their potential to modulate immune responses. This paper utilizes publicly available databases to explore the role of TEs in myocarditis: RNA Seq data and single-cell sequencing data were analyzed, with a focus on the mouse model of experimental autoimmune myocarditis (EAM). The RNA-Seq analysis revealed substantial upregulation of a range of immune genes in cardiac tissue. Further investigation using single-cell sequencing of cardiac immune cells identified specific expression of certain transposable elements (TEs) across different types of immune cells in the heart. Additionally, there was an overall increase in the expression of the ERVB7-1. LTR-MM transposon across various cells in the EAM model, suggesting a widespread impact of this transposon on the immune response in this disease context. The findings of this study highlight the intricate interplay between transposable elements and the immune system in cardiomyopathy, providing new insights into the molecular mechanisms underlying this condition. The discovery of specific TEs expression in cardiac immune cells and the overall increase in ERVB7-1. LTR-MM expression across the EAM model underscore the potential of these elements in modulating immune responses and contribute to our understanding of cardiomyopathy's pathogenesis. These observations open avenues for further research into the role of TEs in cardiac disases and may lead to novel therapeutic strategies.

Role of CCRL2 in the Pathogenesis of Experimental Autoimmune Myocarditis via P21-Activated Kinase 1/NOD-Like Receptor Protein 3 Pathway.

Myocarditis, a severe inflammatory disease, is becoming a worldwide public health concern. This study aims to elucidate the effect of Chemokine (C C motif) receptor-like 2 (CCRL2) in experimental autoimmune myocarditis (EAM) occurrence and its potential regulatory mechanisms.EAM was simulated in a mouse model injected with α-myosin-heavy chain. The changes on EAM were assessed through histological staining of heart tissues, including measuring cardiac troponin I (cTnI), proinflammatory cytokines, transferase-mediated dUTP nick end labeling (TUNEL) assay, and cardiac function. Then, the heart tissues from the EAM mouse model and control groups were analyzed through transcriptome sequencing to identify the differential expressed genes (DEGs) and hub genes related to pyroptosis. Downregulation of CCRL2 further verified the function of CCRL2 on EAM and p21-activated kinase 1/NOD-like receptor protein 3 (PAK/NLRP3) signaling pathways in vivo.The EAM model was constructed successfully, with the heart weight/body weight ratio, serum level of cTnI, and concentrations of proinflammatory cytokines elevation. Moreover, cell apoptosis was also significantly increased. Transcriptome sequencing revealed 696 and 120 upregulated and downregulated DEGs, respectively. After functional enrichment, CCRL2 was selected as a potential target. Then, we verified that CCRL2 knockdown improved cardiac function, alleviated EAM occurrence, and reduced PAK/NLRP3 protein expression.CCRL2 may act as a novel potential treatment target in EAM by regulating the PAK1/NLRP3 pathway.

Baicalein alleviates cardiomyocyte death in EAM mice by inhibiting the JAK-STAT1/4 signalling pathway

BackgroundThe experimental autoimmune myocarditis (EAM) model is valuable for investigating myocarditis pathogenesis. M1-type macrophages and CD4+T cells exert key pathogenic effects on EAM initiation and progression. Baicalein (5,6,7-trihydroxyflavone, C15H10O5, BAI), which is derived from the Scutellaria baicalensis root, is a primary bioactive compound with potent anti-inflammatory and antioxidant properties. BAI exerts good therapeutic effects against various autoimmune diseases; however, its effect in EAM has not been thoroughly researched. PurposeThis study aimed to explore the possible inhibitory effect of BAI on M1 macrophage polarisation and CD4+T cell differentiation into Th1 cells via modulation of the JAK-STAT1/4 signalling pathway, which reduces the secretion of pro-inflammatory factors, namely, TNF-α and IFN-γ, and consequently inhibits TNF-α- and IFN-γ-triggered apoptosis in cardiomyocytes of the EAM model mice. Study design and methodsFlow cytometry, immunofluorescence, real-time quantitative polymerase chain reaction (q-PCR), and western blotting were performed to determine whether BAI alleviated M1/Th1-secreted TNF-α- and IFN-γ-induced myocyte death in the EAM model mice through the inhibition of the JAK-STAT1/4 signalling pathway. ResultsThese results indicate that BAI intervention in mice resulted in mild inflammatory infiltrates. BAI inhibited JAK-STAT1 signalling in macrophages both in vivo and in vitro, which attenuated macrophage polarisation to the M1 type and reduced TNF-α secretion. Additionally, BAI significantly inhibited the differentiation of CD4+T cells to Th1 cells and IFN-γ secretion both in vivo and in vitro by modulating the JAK-STAT1/4 signalling pathway. This ultimately led to decreased TNF-α and IFN-γ levels in cardiac tissues and reduced myocardial cell apoptosis. ConclusionThis study demonstrates that BAI alleviates M1/Th1-secreted TNF-α- and IFN-γ-induced cardiomyocyte death in EAM mice by inhibiting the JAK-STAT1/4 signalling pathway.

Activation of ETAR and ETBR in myocardial tissue characterizes heart failure induced by experimental autoimmune myocarditis

BackgroundEndothelial dysfunction is characterized by an imbalance between endothelium-derived vasodilatory and vasoconstrictive effects and may play an important role in the development of heart failure. An increasing number of studies have shown that endothelial-derived NO-mediated vasodilation is attenuated in heart failure patients. However, the role of endothelin-1 (ET-1) in heart failure remains controversial due to its different receptors including ET-1 receptor type A (ETAR) and ET-1 receptor type B (ETBR). The aim of this study was to determine whether ET-1 and its receptors are activated and to explore the role of ETAR and ETBR in heart failure induced by myocarditis.MethodsWe constructed an animal model of experimental autoimmune myocarditis (EAM) with porcine cardiac myosin. Twenty rats were randomized to the control group (3 weeks, n = 5), the extended control group (8 weeks, n = 5), the EAM group (3 weeks, n = 5), the extended EAM group (8 weeks, n = 5). HE staining was used to detect myocardial inflammatory infiltration and the myocarditis score, Masson’s trichrome staining was used to assess myocardial fibrosis, echocardiography was used to evaluate cardiac function, ELISA was used to detect serum NT-proBNP and ET-1 concentrations, and immunohistochemistry and western blotting were used to detect ETAR and ETBR expression in myocardial tissue of EAM-induced heart failure. Subsequently, a model of myocardial inflammatory injury in vitro was constructed to explore the role of ETAR and ETBR in EAM-induced heart failure.ResultsEAM rats tended to reach peak inflammation after 3 weeks of immunization and developed stable chronic heart failure at 8 weeks after immunization. LVEDd and LVEDs were significantly increased in the EAM group compared to the control group at 3 weeks and 8 weeks after immunization while EF and FS were significantly reduced. Serum NT-proBNP concentrations in EAM (both 3 weeks and 8 weeks) were elevated. Therefore, EAM can induce acute and chronic heart failure due to myocardial inflammatory injury. Serum ET-1 concentration and myocardial ETAR and ETBR protein were significantly increased in EAM-induced heart failure in vivo. Consistent with the results of the experiments in vivo, ETAR and ETBR protein expression levels were significantly increased in the myocardial inflammatory injury model in vitro. Moreover, ETAR gene silencing inhibited inflammatory cytokine TNF-α and IL-1β levels, while ETBR gene silencing improved TNF-α and IL-1β levels.ConclusionsET-1, ETAR, and ETBR were activated in both EAM-induced acute heart failure and chronic heart failure. ETAR may positively regulate EAM-induced heart failure by promoting myocardial inflammatory injury, whereas ETBR negatively regulates EAM-induced heart failure by alleviating myocardial inflammatory injury.

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

Modulation of TLR4/NFκB Pathways in Autoimmune Myocarditis.

Myocarditis is an inflammatory and oxidative disorder characterized by immune cell recruitment in the damaged tissue and organ dysfunction. In this paper, we evaluated the molecular pathways involved in myocarditis using a natural compound, Coriolus versicolor, in an experimental model of autoimmune myocarditis (EAM). Animals were immunized with an emulsion of pig cardiac myosin and complete Freund's adjuvant supplemented with mycobacterium tuberculosis; thereafter, Coriolus versicolor (200 mg/Kg) was orally administered for 21 days. At the end of the experiment, blood pressure and heart rate measurements were recorded and the body and heart weights as well. From the molecular point of view, the Coriolus versicolor administration reduced the activation of the TLR4/NF-κB pathway and the levels of pro-inflammatory cytokines (INF-γ, TNF-α, IL-6, IL-17, and IL-2) and restored the levels of anti-inflammatory cytokines (IL-10). These anti-inflammatory effects were accompanied with a reduced lipid peroxidation and nitrite levels and restored the antioxidant enzyme activities (SOD and CAT) and GSH levels. Additionally, it reduced the histological injury and the immune cell recruitment (CD4+ and CD68+ cells). Moreover, we observed an antiapoptotic activity in both intrinsic (Fas/FasL/caspase-3) and extrinsic (Bax/Bcl-2) pathways. Overall, our data showed that Coriolus versicolor administration modulates the TLR4/NF-κB signaling in EAM.

Ursolic acid reduces oxidative stress injury to ameliorate experimental autoimmune myocarditis by activating Nrf2/HO-1 signaling pathway.

Background: Oxidative stress is crucial in experimental autoimmune myocarditis (EAM)-induced inflammatory myocardial injury. Ursolic acid (UA) is an antioxidant-enriched traditional Chinese medicine formula. The present study aimed to investigate whether UA could alleviate inflammatory cardiac injury and determine the underlying mechanisms. Methods: Six-week-old male BALB/c mice were randomly assigned to one of the three groups: Sham, EAM group, or UA intervention group (UA group) by gavage for 2weeks. An EAM model was developed by subcutaneous injection of α-myosin heavy chain derived polypeptide (α-MyHC peptide) into lymph nodes on days 0 and 7. Echocardiography was used to assess cardiac function on day 21. The inflammation level in the myocardial tissue of each group was compared using hematoxylin and eosin staining (HE) of heart sections and Interleukin-6 (IL-6) immunohistochemical staining. Masson staining revealed the degree of cardiac fibrosis. Furthermore, Dihydroethidium staining, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA) were used to determine the mechanism of cardioprotective effects of UA on EAM-induced cardiac injury, and the level of IL-6, Nrf2, and HO-1. Results: In EAM mice, UA intervention significantly reduced the degree of inflammatory infiltration and myocardial fibrosis while improving cardiac function. Mechanistically, UA reduced myocardial injury by inhibiting oxidative stress (as demonstrated by a decrease of superoxide and normalization of pro- and antioxidant enzyme levels). Interestingly, UA intervention upregulated the expression of antioxidant factors such as Nrf2 and HO-1. In vitro experiments, specific Nrf2 inhibitors reversed the antioxidant and antiapoptotic effects of ursolic acid, which further suggested that the amelioration of EAM by UA was in a Nrf2/HO-1 pathway-dependent manner. Conclusion: These findings indicate that UA is a cardioprotective traditional Chinese medicine formula that reduces EAM-induced cardiac injury by up-regulating Nrf2/HO-1 expression and suppressing oxidative stress, making it a promising therapeutic strategy for the treatment of EAM.

Noninvasive ultrasound stimulation to treat myocarditis through splenic neuro-immune regulation

BackgroundThe cholinergic anti-inflammatory pathway (CAP) has been widely studied to modulate the immune response. Current stimulating strategies are invasive or imprecise. Noninvasive low-intensity pulsed ultrasound (LIPUS) has become increasingly appreciated for targeted neuronal modulation. However, its mechanisms and physiological role on myocarditis remain poorly defined.MethodsThe mouse model of experimental autoimmune myocarditis was established. Low-intensity pulsed ultrasound was targeted at the spleen to stimulate the spleen nerve. Under different ultrasound parameters, histological tests and molecular biology were performed to observe inflammatory lesions and changes in immune cell subsets in the spleen and heart. In addition, we evaluated the dependence of the spleen nerve and cholinergic anti-inflammatory pathway of low-intensity pulsed ultrasound in treating autoimmune myocarditis in mice through different control groups.ResultsThe echocardiography and flow cytometry of splenic or heart infiltrating immune cells revealed that splenic ultrasound could alleviate the immune response, regulate the proportion and function of CD4+ Treg and macrophages by activating cholinergic anti-inflammatory pathway, and finally reduce heart inflammatory injury and improve cardiac remodeling, which is as effective as an acetylcholine receptor agonists GTS-21. Transcriptome sequencing showed significant differential expressed genes due to ultrasound modulation.ConclusionsIt is worth noting that the ultrasound therapeutic efficacy depends greatly on acoustic pressure and exposure duration, and the effective targeting organ was the spleen but not the heart. This study provides novel insight into the therapeutic potentials of LIPUS, which are essential for its future application.Graphical

Thalidomide and a Dipeptidyl Peptidase 4 Inhibitor in a Rat Model of Experimental Autoimmune Myocarditis.

Myocarditis is a potentially fatal disease, but curative treatments have not yet been established. Myocardial inflammation is an important pathogenesis of this disease, and immunosuppressants such as methylprednisolone and immunoglobulin have been used for treatment; however, the effectiveness needs to be improved. Thalidomide and dipeptidyl peptidase (DPP) 4 inhibitors were recently investigated regarding their immunomodulatory properties. This study aimed to test whether thalidomide or a DPP4 inhibitor (evogliptin) can improve the effectiveness of myocarditis treatment using a rat model of experimental autoimmune myocarditis (EAM). Rats with or without myocarditis were administered thalidomide at 100 mg/kg/day and DPP4 inhibitor at 10 mg/kg/day orally. Measurement of echocardiography, serum inflammatory cytokines, myocardial histopathological examination, and immunohistochemical staining for leukocytes, macrophages, CD4+ T cells, and cytoskeleton were performed after 3 weeks, and the fibrosis area was measured after 3 and 6 weeks. Thalidomide and DPP4 inhibitor did not reduce the severity of myocarditis compared with the EAM without treatment rats by comparing the echocardiographic data, myocardial CD4+, macrophages, neutrophil infiltrations, and the heart weight/body weight ratio in 3 weeks. The levels of inflammatory cytokines were not lower in the thalidomide and DPP4 inhibitor-treated group than in the untreated group in 3 weeks. In 6 weeks, thalidomide and DPP4 inhibitors did not reduce the fibrosis area compared to untreated groups. Although thalidomide and the DPP4 inhibitor had an immunomodulatory effect and are used against inflammatory diseases, they did not ameliorate myocardial inflammation and fibrosis in this rat model of EAM.

Activation of cardiac macrophages, endothelial cells and fibroblasts in experimental autoimmune myocarditis

Abstract Background/Introduction Inflammation of cardiac tissue, termed myocarditis, is a common cause of dilated cardiomyopathy associated with abnormal tissue remodeling, stiffening of ventricles and heart failure. Experimental autoimmune myocarditis (EAM) represents CD4+ T cell-dependent animal model of acute myocarditis followed by development of cardiomyopathy, cardiac fibrosis and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac macrophages, endothelial cells and fibroblasts in myocarditis and post-inflammatory cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with alpha myosin heavy chain and complete Freund's adjuvant. Reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP), transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts and Rosa26-YFP/LysM-Cre and Rosa26-YFP/LysM-Cre/Tgfbr2 fl/fl with YFP expression and Tgfbr2 deletion in myeloid cell population were used in this study. Cardiac fibroblasts and macrophages were sorted using BD FACSAria™ II Cell Sorter and analyzed for the genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was measured as percentage of fibrotic area using Trichrome Massons's staining and by hydroxyproline assay. Cardiac hypertrophy was analyzed as means of cross-sectional cardiomyocyte area. Monocytes and endobcells were analyzed using BD FACSCanto™ II flow cytometry. Results Cardiac fibroblasts in response to acute myocarditis (d21 of EAM) showed activation of immune processes (mainly chemokine production such as Ccl6, Ccl9, Cxcl2, Cxcl3, Cxcl5, Cxcl9, Cxcl13), cytoskeletal re-organization (Cxadr, F11r, Gdpd2, Krt8, Krt19, Ptk2b, Rac2, Rhov, Rnd1, S100a9, Spire2, Was) and upregulation of genes involved in extracellular matrix turnover (Bmp7, Kng2, Lgals3, Cthrc1, Cela1, Spn) including collagens. Ablation of myofibroblasts (between d21–40 of EAM) resulted in markedly reduced heart weight and cardiomyocyte hypertrophy, attenuated expression of genes related to hypertrophy (Acta1, Actc1, Bnp, Cfl2, Pdlim5), improved stroke volume, ejection fraction and cardiac output but did not prevent development of post-inflammatory cardiac fibrosis measured at d40 of EAM. Analysis of monocytes and endothelial cells indicated excessive production of type I collagen by these cells at d21. Analysis of cardiac macrophages pointed out TGF-β-dependent expression of cytokines (Ifn, Il23a, Il10, Il12b, Cxcl1, Tnf) and theirs receptors (Cxcr1, Ccr4) at d21 of EAM. Conclusions Acute myocarditis activates proinflammatory and profibrotic responses in cardiac resident cells. Our data suggest that cardiac myofibroblasts play a particularly important role in development of post-inflammatory cardiomyopathy and heart failure. Targeting cardiac myofibroblasts might therefore represent a novel therapeutic strategy in inflammatory heart disease. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

Combination of Sophora flavescens alkaloids and Panax quinquefolium saponins modulates different stages of experimental autoimmune myocarditis via the NF-κB and TGF-β1 pathways.

Chronic cardiac inflammation and fibrosis can progress into severe forms of cardiomyopathy. Sophora flavescens alkaloids (KuShen) have been previously reported to exert anti-inflammatory effects, whereas Panax quinquefolium saponins (XiYangShen) has been shown to alleviate cardiac fibrosis. Therefore, the potential effects of their combination (KX) on different stages of autoimmune myocarditis were investigated in the present study. Mice were randomly divided into the following four groups: Control; experimental autoimmune myocarditis (EAM); KX-High (275 mg/kg); and KX-Low (138 mg/kg). A 21-day and a 60-day EAM model was established through multi-site subcutaneous injections of cardiac myosin mixed with complete Freund's adjuvant on days 0, 7, 21 and 42. Mice in the High and Low KX groups were treated by gavage (10 ml/kg) daily from day 0 (1 day before treatment) until sacrifice (day 21 or 60). Mice in the control and EAM groups received an equivalent volume of distilled water. The levels of lactate dehydrogenase (LDH), creatine kinase-myocardial band (CK-MB), cardiac troponin I (cTn-I), IL-1β, IL-6, TNF-α, TGF-β1, collagen type I (Col Ⅰ) and collagen type III (Col Ⅲ) were measured by ELISA in the mouse myocardial tissues or serum. Myocardial tissue structure and extent of fibrosis were visualized using H&E and Masson's staining. Western blotting and immunohistochemistry were used to measure the expression levels NF-κB and TGF-β1 pathway proteins in the myocardial tissues. The degree of inflammation in the 21-day EAM model was found to be significantly higher compared with that in the 60-day EAM model. KX significantly reduced the inflammatory response at 21 days by decreasing the expression levels of CK-MB, LDH, cTn-I, IL-1β, IL-6, TNF-α and TGF-β-activated kinase 1-binding protein 1/NF-κB pathway proteins. Myocardial fibrosis in the 60-day EAM model was also significantly worse compared with that in the 21-day EAM model. However, fibrosis was significantly delayed by treatment with KX. In addition, KX significantly decreased the expression levels of TGF-β1, Smad2, Smad4, Col I and Col III. Therefore, these data suggest that KX is beneficial for treating myocarditis by targeting multiple pathways.

Spironolactone alleviates myocardial fibrosis via inhibition of Ets-1 in mice with experimental autoimmune myocarditis.

Spironolactone improves cardiac structure, function and prognosis in patients with heart failure and delays the progression of cardiac fibrosis. However, the exact underlying mechanism of this process remains to be elucidated. The present study therefore aimed to explore the protective effect and underlying mechanism of the aldosterone receptor antagonist, spironolactone, on myocardial fibrosis in mice with experimental autoimmune myocarditis (EAM). The EAM model was induced in BALB/c mice via immunization with murine cardiac α-myosin heavy chain sequence polypeptides. The cardiac function of the mice was assessed using echocardiography and the levels of inflammatory cytokines were quantified using ELISA. E26 transformation-specific sequence-1 (Ets-1) expression was knocked down using lentivirus-mediated small interference RNA. Total collagen deposition was assessed using Masson's trichrome and Ets-1, TGF-β1, Smad2/3, collagen I and III protein expression levels were detected using immunohistochemistry and western blotting. MMP-2 and MMP-9 mRNA expression levels and activity was determined using reverse transcription-quantitative PCR and gelatin zymography, respectively. The results of the present study demonstrated that spironolactone significantly improved myocardium hypertrophy, diastolic cardiac function and decreased myocardial inflammation and collagen deposition induced by EAM. Spironolactone treatment significantly inhibited Ets-1 and smad2/3 phosphorylation. In addition, inhibition of Ets-1 reduced the expression and activity of MMP-2 and MMP-9 and decreased cardiac fibrosis in EAM mice. The results indicated that the improvement of myocardial fibrosis by spironolactone may be associated with the TGF-β1/Smad-2/3/Ets-1 signaling pathway in EAM mice.

Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model.

Simple SummaryFulminant myocarditis (FM) is a serious inflammatory lesion of the myocardium accompanied by cardiac dysfunction, transitioning to end-stage heart failure. Due to such a difficult pathology, a therapeutic strategy that exerts a steadfast effect has yet to be developed. Blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. The aim of this study was to investigate whether transplantation therapy with hybrid cell sheets of fibroblasts and QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM) induced by purified porcine cardiac myosin. The transplanted hybrid cell sheet exerts cardioprotective effects against EAM, resulting in limited left ventricular remodeling and partially improved cardiac functions due to revascularization, anti-inflammation, and anti-fibrosis. Thus, tissue engineering using hybrid cell sheets of fibroblasts constructed with QQMNCs is expected to provide an effective therapeutic option for patients with severe FM.Fulminant myocarditis causes impaired cardiac function, leading to poor prognosis and heart failure. Cell sheet engineering is an effective therapeutic option for improving cardiac function. Naïve blood mononuclear cells (MNCs) have been previously shown to enhance the quality and quantity of cellular fractions (QQMNCs) with anti-inflammatory and vasculogenic potential using the one culture system. Herein, we investigated whether autologous cell sheet transplant with QQMNCs improves cardiac function in a rat model with experimental autoimmune myocarditis (EAM). Fibroblast sheets (F-sheet), prepared from EAM rats, were co-cultured with or without QQMNCs (QQ+F sheet) on temperature-responsive dishes. QQ+F sheet induced higher expression of anti-inflammatory and vasculogenic genes (Vegf-b, Hgf, Il-10, and Mrc1/Cd206) than the F sheet. EAM rats were transplanted with either QQ+F sheet or F-sheet, and the left ventricular (LV) hemodynamic analysis was performed using cardiac catheterization. Among the three groups (QQ+F sheet, F-sheet, operation control), the QQ+F sheet transplant group showed alleviation of end-diastolic pressure–volume relationship on a volume load to the same level as that in the healthy group. Histological analysis revealed that QQ+F sheet transplantation promoted revascularization and mitigated fibrosis by limiting LV remodeling. Therefore, autologous QQMNC-modified F-sheets may be a beneficial therapeutic option for EAM.

Bioinspired macrophage-targeted anti-inflammatory nanomedicine: A therapeutic option for the treatment of myocarditis

Myocarditis is a disease characterized by inflammation of the heart muscle, which increases the risk of dilated cardiomyopathy and heart failure. Macrophage migration is a major histopathological hallmark of myocarditis, making macrophages a potential therapeutic target for the management of this disease. In the present study, we synthesized a bioinspired anti-inflammatory nanomedicine conjugated with protein G (PSL-G) that could target macrophages and induce macrophage polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Notably, PSL-G exhibited a higher affinity for macrophages than non-macrophage cells. The addition of PSL-G decreased the levels of pro-inflammatory cytokines (e.g., IL-1α, IL-6, and TNF-α), but increased the level of the anti-inflammatory cytokine IL-10 in macrophages treated with lipopolysaccharide and/or interferon-γ. Furthermore, the lifetime of PSL-G in murine blood circulation was found to be significantly higher than that of PSL. Systemic injection of PSL-G into a mouse model of experimental autoimmune myocarditis remarkably reduced macrophage migration in the myocardium (16-fold compared with the positive control group) and myocardial fibrosis (8-fold). Based on these results and the fact that macrophages play a critical role in the pathogenesis of various diseases, we believe that bioinspired macrophage-targeted anti-inflammatory nanomedicines may be effective therapeutic options for the treatment of autoimmune and autoinflammatory diseases, especially myocarditis.

Specialized pro-resolving mediators prevents cardiac dysfunction by modulating Ca2+ handling and NRF2 axis

Abstract Background and purpose Myocarditis is a severe inflammatory heart disease and a leading cause of sudden death in young adults; but currently no specific treatment is available. Lipoxins and their derivatives promote the resolution of inflammation contributing to recover tissue homeostasis; but their role in cardiac inflammation is poorly understood. Methods and results BML-111, a stable lipoxin A4 receptor agonist, protects against cardiac dysfunction in a murine model of experimental autoimmune myocarditis (EAM) by preventing Ca2+ mishandling. Cardiac proteomic analysis revealed an enhanced cardiac oxidative profile in EAM-induced mice with reduced activation of NRF2, a master antioxidant transcription factor. In vitro analysis showed that 15-epi-lipoxin A4 increased systolic Ca2+ release and sarcoplasmic reticulum (SR)-Ca2+ load in cardiomyocytes isolated from wild-type mice and augmented the rate of SR-Ca2+ uptake by SERCA2a, but failed to induce any functional change in cells from Nrf2−/− mice. BML-111 increased SERCA2a cardiac expression in wild-type mice, and the transcriptional activity of Nrf2 determined SERCA2a expression in human ventricular cells. Human myocarditis-positive myocardium showed a reduced expression of both ATP2A2 (SERCA2a) and NF2L2 (NRF2). Conclusions Our results demonstrated new cardioprotective mechanisms of pro-resolving lipid mediators that may emerge as innovative treatments for myocarditis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ISCIII [PI17/01344]), Sociedad Española de Cardiología: Proyecto Traslacional 2019Fondo Europeo de Desarrollo Regional (FEDER), FSE, and CIBER-CV, a network funded by ISCIII

Activated myofibroblasts promote cardiac hypertrophy and systolic dysfunction independently of cardiac fibrosis in experimental autoimmune myocarditis

Abstract Background/Introduction Heart-specific inflammation – myocarditis is a common cause dilated cardiomyopathy which is characterized by pathological tissue remodeling, ventricular stiffening, cardiomyopathy and heart failure. In experimental autoimmune myocarditis (EAM) susceptible mice immunized with alpha myosin heavy chain (αMyHC) and complete Freund's adjuvant (CFA) develop acute myocarditis driven by autoreactive CD4+ T cells that is followed by progressive fibrosis, cardiomyopathy and systolic dysfunction. Purpose The aim of the study was to investigate the role of cardiac fibroblasts and myofibroblasts in myocarditis and post-inflammatory dilated cardiomyopathy in mouse model of EAM. Methods EAM was induced in BALB/c mice by immunization with αMyHC/CFA. We used reporter mice expressing EGFP under collagen type I promoter (Coll-EGFP) and RFP under a control of α-smooth muscle actin (αSMA) promoter (αSMA-RFP) and transgenic αSMA-TK mice with ganciclovir-inducible ablation of proliferating myofibroblasts. Cardiac cells were quantified using flow cytometry. Cardiac fibroblasts (CD45-CD31-EGFP+) were sorted from healthy and myocarditis-positive (day 21) mice using BD FACSAria™ II Cell Sorter and analyzed for the whole genome transcriptomics by RNA sequencing. Echocardiography was performed on Vevo 2100 Imaging System. Cardiac fibrosis was assessed by Trichrome Massons's staining and hydroxyproline assay, whereas cardiac hypertrophy by analysing cross-sectional cardiomyocyte area. Profibrotic gene expression was assessed by qRT-PCR. Results The total number of cardiac fibroblasts (CD45-CD31-EGFP+) and the subset of myofibroblasts (CD45-CD31-EGFP+RFP+) remained unchanged at inflammatory (d21) and fibrotic stages (d40). Analysis of differentially expressed genes (min. 2x fold change, p value <0.05) pointed out activation of immune processes (mainly chemokine production), response to stress, cytoskeletal and extracellular matrix re-organization in cardiac fibroblasts in response to myocarditis. αSMA-TK mice treated with ganciclovir (from day 21) showed comparable percent of fibrotic area, but significantly reduced heart weight, decreased cardiomyocyte hypertrophy and improved ejection fraction and cardiac output at day 40 comparing to PBS-treated mice. Ganciclovir-treated mice showed also attenuated cardiac Acta2 and Srf but markedly enhanced Mmp2 expression. Conclusions In EAM model cardiac fibroblasts actively participate in proinflammatory and profibrotic responses, while activated myofibroblasts contribute to dilated cardiomyopathy development independently of cardiac fibrosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

Sex-Specific Differences of the Inflammatory State in Experimental Autoimmune Myocarditis.

Increasing evidence suggests male sex as a potential risk factor for a higher incidence of cardiac fibrosis, stronger cardiac inflammation, and dilated cardiomyopathy (DCM) in human myocarditis. Chronic activation of the immune response in myocarditis may trigger autoimmunity. The experimental autoimmune myocarditis (EAM) model has been well established for the study of autoimmune myocarditis, however the role of sex in this pathology has not been fully explored. In this study, we investigated sex differences in the inflammatory response in the EAM model. We analyzed the cardiac function, as well as the inflammatory stage and fibrosis formation in the heart of EAM male and female rats. 21 days after induction of EAM, male EAM rats showed a decreased ejection fraction, stroke volume and cardiac output, while females did not. A significantly elevated number of infiltrates was detected in myocardium in both sexes, indicating the activation of macrophages following EAM induction. The level of anti-inflammatory macrophages (CD68+ ArgI+) was only significantly increased in female hearts. The expression of Col3A1 and fibrosis formation were more prominent in males. Furthermore, prominent pro-inflammatory factors were increased only in male rats. These findings indicate sex-specific alterations in the inflammatory stage of EAM, with a pro-inflammatory phenotype appearing in males and an anti-inflammatory phenotype in females, which both significantly affect cardiac function in autoimmune myocarditis.

Androgen receptor inhibition alleviated inflammation in experimental autoimmune myocarditis by increasing autophagy in macrophages.

Experimental autoimmune myocarditis (EAM) is characterized by pronounced macrophage infiltration, cardiac necrosis, and cardiac fibrosis. Our previous studies have demonstrated that suppressed androgen receptor (AR) enables anti-inflammation to promote tissue repair by decreasing M1 macrophages and increasing M2 macrophages in an EAM model. Given that autophagy mediates inflammatory response in macrophages, we investigated whether AR inhibition executes its protective role in inflammation through the autophagy pathway in EAM. To determine whether AR inhibition can perform its anti-inflammatory effects by upregulating autophagy, we pre-treated mice with 3-methyl adenine (3-MA), a pharmacological inhibitor of autophagy. Immunofluorescence assay and Western blot were used to detect autophagy levels and autophagy activity in five different groups. Immunofluorescence marked F4/80 and LC3 to illustrate the autophagy level in macrophages. TUNEL assays were used to detect the apoptosis level in heart tissue of five different groups. We demonstrated that AR inhibition resolves injury with sustained inhibition of inflammatory cytokines associated with enhanced autophagy, especially in macrophages. Increased LC3II/I expression corroborated complete autolysosome formation detected by electron microscopy and correlated with degradation of SQSTM1/p62 in the AR inhibition group by Western blot. These effects could be reversed within 3-MA, a pharmacological inhibitor of autophagy. Specifically, pharmacological inhibition of autophagy increased apoptosis and inflammation, which could be attenuated by AR inhibition. AR inhibition alleviates the inflammatory response and tissue apoptosis by enhancing autophagy, especially in macrophages.