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

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.

Ethyl Pyruvate Ameliorates Experimental Autoimmune Myocarditis

Ethyl pyruvate (EP) has profound anti-inflammatory and immunomodulatory properties. Here, its effects were determined on experimental autoimmune myocarditis (EAM) induced in mice by heart-specific myosin-alpha heavy chain peptide immunization. EP was applied intraperitoneally, daily, starting with the immunization. Severity of EAM was determined by histological assessment of immune cell infiltrates into the heart. Cells were phenotypically characterized by flow cytometry. Concentration of cytokines in cell culture supernatants and sera was determined by ELISA. EP reduced the infiltration of immune cells into the heart and lessened heart inflammation. Smaller number of total immune cells, as well as of CD11b+ and CD11c+ cells were isolated from the hearts of EP-treated mice. A reduced number of antigen-presenting cells, detected by anti-CD11c, MHC class II and CD86 antibodies, as well as of T helper (Th)1 and Th17 cells, detected by anti-CD4, IFN-γ and IL-17 antibodies, was determined in mediastinal lymph nodes draining the heart, in parallel. In the spleen, only the number of CD11c+ cells were reduced, but not of the other examined populations, thus implying limited systemic effect of EP. Reduced production of IFN-γ and IL-17 by myosin-alpha heavy chain peptide-restimulated cells of the lymph nodes draining the site of immunization was observed in EP-treated mice. Our results clearly imply that EP restrains autoimmunity in EAM. Therapeutic application of EP in the treatment of myocarditis in humans should be addressed in the forthcoming studies.

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

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. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Science Centre (Poland)

Sacubitril/Valsartan Alleviates Experimental Autoimmune Myocarditis by Inhibiting Th17 Cell Differentiation Independently of the NLRP3 Inflammasome Pathway

Sacubitril/valsartan (Sac/Val) is a recently approved drug that is commonly used for treatment of heart failure. Several studies indicated that Sac/Val also regulated the secretion of inflammatory factors. However, the effect and mechanism of this drug modulation of inflammatory immune responses are uncertain. In this study, an experimental autoimmune myocarditis (EAM) mouse model was established by injection of α-myosin-heavy chain peptides. The effect of oral Sac/Val on EAM was evaluated by histological staining of heart tissues, measurements of cardiac troponin T and inflammatory markers (IL-6 and hsCRP). The effects of Sac/Val on NLRP3 inflammasome activation and Th1/Th17 cell differentiation were also determined. To further explore the signaling pathways, the expressions of cardiac soluble guanylyl cyclase (sGC) and NF-κB p65 were investigated. The results showed that Sac/Val downregulated the inflammatory response and attenuated the severity of EAM, but did not influence NLRP3 inflammasomes activation. Moreover, Sac/Val treatment inhibited cardiac Th17 cell differentiation, and this might be associated with sGC/NF-κB p65 signaling pathway. These findings indicate the potential use of Sac/Val for treatment of myocarditis.

RIP1/RIP3/MLKL Mediates Myocardial Function Through Necroptosis in Experimental Autoimmune Myocarditis

Cardiomyopathy often leads to dilated cardiomyopathy (DCM) when caused by viral myocarditis. Apoptosis is long considered as the principal process of cell death in cardiomyocytes, but programmed necrosis or necroptosis is recently believed to play an important role in cardiomyocyte cell death. We investigated the role of necroptosis and its interdependency with other processes of cell death, autophagy, and apoptosis in a rat system of experimental autoimmune myocarditis (EAM). We successfully created a rat model system of EAM by injecting porcine cardiac myosin (PCM) and showed that in EAM, all three forms of cell death increase considerably, resulting in the deterioration of cardiac conditions with an increase in inflammatory infiltration in cardiomyocytes. To explore whether necroptosis occurs in EAM rats independent of autophagy, we treated EAM rats with a RIP1/RIP3/MLKL kinase-mediated necroptosis inhibitor, Necrostatin-1 (Nec-1). In Nec-1 treated rats, cell death proceeds through apoptosis but has no significant effect on autophagy. In contrast, autophagy inhibitor 3-Methyl Adenine (3-MA) increases necroptosis, implying that blockage of autophagy must be compensated through necroptosis. Caspase 8 inhibitor zVAD-fmk blocks apoptosis but increases both necroptosis and autophagy. However, all necroptosis, apoptosis, and autophagy inhibitors independently reduce inflammatory infiltration in cardiomyocytes and improve cardiac conditions. Since apoptosis or autophagy is involved in many important cellular aspects, instead of suppressing these two major cell death processes, Nec1 can be developed as a potential therapeutic target for inflammatory myocarditis.

A Case of Autoimmune Myocarditis Treated with IL-17 Inhibition

Myocarditis is defined as inflammation targeting the heart muscle. Autoimmune myocarditis is a subtype of myocarditis that can occur as an isolated entity in which the primary targeted organ is the heart or as part of a systemic autoimmune disease. The pathogenesis of autoimmune myocarditis has been studied using experimental autoimmune myocarditis mouse models. These studies have established the role of TH17 and IL-17 in the development and progression of myocarditis. While IL-17 inhibition has been tested in these mouse models, there are no reported cases of IL-17 inhibitors being utilized in humans to treat autoimmune myocarditis. We report a patient diagnosed with autoimmune myocarditis in the setting of psoriasis treated initially with prednisone and methotrexate without improvement. The patient was then started on an IL-17 inhibitor secukinumab, directly targeting the immune mechanisms found to be responsible for autoimmune myocarditis and its progression as evidenced by the experimental autoimmune myocarditis mouse models. We provide nuclear scan imaging pre- and post-treatment with secukinumab documenting resolution of our patient’s autoimmune myocarditis. This novel case highlighting the success of IL17 inhibition in treating autoimmune myocarditis, a disease with no truly effective treatment and with potentially devastating consequences, provides an exciting avenue for future research in larger patient populations to further assess the efficacy of this treatment modality.

IL-17 Upregulates MCP-1 Expression via Act1 / TRAF6 / TAK1 in Experimental Autoimmune Myocarditis

Myocarditis is a myocardial inflammatory infiltration heterogeneous disease. At present, various interventions are not effective in the treatment of myocarditis. IL-17, an important pro‐inflammatory factor secreted mainly by Th17 cells, can promote the expression of multiple cytokines. MCP-1 is an important cytokine that mediates mononuclear cell infiltration. Studies have found that IL-17 could stimulate the expression of MCP-1 to mediate inflammatory infiltration. But the mechanism by which IL-17 induces MCP-1 expression in experimental autoimmune myocarditis (EAM) remains unclear. The purpose of this study is to establish an EAM model to explore the role of Act1/TRAF6/TAK1 cascade in the induction of MCP-1 by IL-17. In the present study, we found that in EAM, IL-17 could stimulate the expression of MCP-1 by activating Act1/TRAF6/TAK1 cascade. After interfering TAK1 with si-TAK1, myocardial tissue inflammation was greatly alleviated, and both MCP-1 mRNA and protein expression were downregulated. In conclusion, IL-17 can activate AP-1, NF-κB via Act1/TRAF6/TAK1 upregulation of MCP-1 expression in EAM.

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.

Bazedoxifene Regulates Th17 Immune Response to Ameliorate Experimental Autoimmune myocarditis via Inhibition of STAT3 Activation

Myocarditis is a type of inflammatory cardiomyopathy that has no specific treatment. Accumulating evidence suggests that Th17 cells play a prominent role in the pathogenesis of myocarditis. Interleukin-(IL)-6-mediated signal transducer and activation of transcription 3 (STAT3) signaling is essential for Th17 cell differentiation and secretion of inflammatory cytokines. Bazedoxifene inhibits IL-6/STAT3 signaling in cancer cells, but its effect on the Th17 immune response induced by myocarditis remains unknown. Here we explore the effect of Bazedoxifene on Th17 immune response and cardiac inflammation in a mouse model of experimental autoimmune myocarditis, which has been used to mimic human inflammatory heart disease. After eliciting an immune response, we found Bazedoxifene ameliorated cardiac inflammatory injury and dysfunction. Th17 cells and related inflammatory factors in splenic CD4+ T cells at day 14 and in the heart at day 21 were increased, which were reduced by Bazedoxifene. Furthermore, Bazedoxifene could regulate autophagy induction in polarized Th17 cells. In conclusion, Bazedoxifene affected STAT3 signaling and prevented cardiac inflammation deterioration, so may provide a promising therapeutic strategy for the treatment of experimental autoimmune myocarditis (EAM).