LncRNA MAAMT facilitates macrophage recruitment and proinflammatory activation and exacerbates autoimmune myocarditis through the SRSF1/NF-κB axis.

Long non-coding RNAs (lncRNAs) have been implicated in dilated cardiomyopathy (DCM). However, the biological functions and regulatory mechanisms underlying DCM remain elusive. Using a mouse model of experimental autoimmune myocarditis (EAM) to mimic DCM, we successfully constructed a dynamic lncRNA expression library for EAM by lncRNA microarray and found that the expression of a macrophage-enriched lncRNA, MAAMT, was significantly increased in the myocardial tissue of mice at the acute stage of EAM. Functionally, MAAMT knockdown alleviated the recruitment and proinflammatory activation of macrophages of heart, spleen, and peripheral blood of mice at the acute stage of EAM, reduced myocardial inflammation and injury, and eventually reversed ventricular remodelling and improved cardiac function in chronic EAM model mice. Mechanistically, we identified serine/arginine-rich splicing factor 1 (SRSF1) as an MAAMT-interacting protein in macrophages using RNA pull-down assays coupled with mass spectrometry. MAAMT knockdown attenuated the ubiquitination-mediated degradation of SRSF1, increased the protein expression of SRSF1, and restrained the activation of the NF-κB pathway in macrophages, thereby inhibiting the proinflammatory activation of macrophages. Collectively, our results demonstrate that MAAMT is a key proinflammatory regulator of myocarditis that promotes macrophage activation through the SRSF1-NF-κB axis, providing a new insight into early effective treatment strategies for DCM.

TGF-ß Signalling Regulates Cytokine Production in Inflammatory Cardiac Macrophages during Experimental Autoimmune Myocarditis.

Myocarditis is characterized by an influx of inflammatory cells, predominantly of myeloid lineage. The progression of myocarditis to a dilated cardiomyopathy is markedly influenced by TGF-ß signalling. Here, we investigate the role of TGF-ß signalling in inflammatory cardiac macrophages in the development of myocarditis and post-inflammatory fibrosis. Experimental autoimmune myocarditis (EAM) was induced in the LysM-Cre × R26-stop-EYFP × Tgfbr2-fl/fl transgenic mice showing impaired TGF-ß signalling in the myeloid lineage and the LysM-Cre × R26-stop-EYFP control mice. In EAM, immunization led to acute myocarditis on day 21, followed by cardiac fibrosis on day 40. Both strains showed a similar severity of myocarditis and the extent of cardiac fibrosis. On day 21 of EAM, an increase in cardiac inflammatory macrophages was observed in both strains. These cells were sorted and analysed for differential gene expression using whole-genome transcriptomics. The analysis revealed activation and regulation of the inflammatory response, particularly the production of both pro-inflammatory and anti-inflammatory cytokines and cytokine receptors as TGF-ß-dependent processes. The analysis of selected cytokines produced by bone marrow-derived macrophages confirmed their suppressed secretion. In conclusion, our findings highlight the regulatory role of TGF-ß signalling in cytokine production within inflammatory cardiac macrophages during myocarditis.

Ivermectin ameliorates acute myocarditis via the inhibition of importin-mediated nuclear translocation of NF-κB/p65.

BACKGROUND: Myocarditis is an important clinical issue which lacks specific treatment by now. Ivermectin (IVM) is an inhibitor of importin α/ß-mediated nuclear translocation. This study aimed to explore the therapeutic effects of IVM on acute myocarditis. METHODS: Mouse models of coxsackie B3 virus (CVB3) infection-induced myocarditis and experimental autoimmune myocarditis (EAM) were established to evaluate the effects of IVM. Cardiac functions were evaluated by echocardiography and Millar catheter. Cardiac inflammatory infiltration was assessed by histological staining. Cytometric bead array and quantitative real-time PCR were used to detect the levels of pro-inflammatory cytokines. The macrophages and their M1/M2 polarization were analyzed via flow cytometry. Protein expression and binding were detected by co-immunoprecipitation, Western blotting and histological staining. The underlying mechanism was verified in vitro using CVB3-infected RAW264.7 macrophages. Cyclic polypeptide (cTN50) was synthesized to selectively inhibit the nuclear translocation of NF-κB/p65, and CVB3-infected RAW264.7 cells were treated with cTN50. RESULTS: Increased expression of importin ß was observed in both models. IVM treatment improved cardiac functions and reduced the cardiac inflammation associated with CVB3-myocarditis and EAM. Furthermore, the pro-inflammatory cytokine (IL-1ß/IL-6/TNF-α) levels were downregulated via the inhibition of the nuclear translocation of NF-κB/p65 in macrophages. IVM and cTN50 treatment also inhibited the nuclear translocation of NF-κB/p65 and downregulated the expression of pro-inflammatory cytokines in RAW264.7 macrophages. CONCLUSIONS: Ivermectin inhibits the nuclear translocation of NF-κB/p65 and the expression of major pro-inflammatory cytokines in myocarditis. The therapeutic effects of IVM on viral and non-viral myocarditis models suggest its potential application in the treatment of acute myocarditis.

Rho kinase inhibitor Y-27632 downregulates IL-1ß expression in mice with experimental autoimmune myocarditis.

Autoimmune myocarditis is the limited or diffuse inflammation of the myocardium due to dysfunctional cellular and humoral immunity mechanisms. We constructed mouse models of experimental autoimmune myocarditis (EAM) using peptide MyHC-α614-629. On the day after secondary immunization, the mice were intraperitoneally injected with Rho kinase (ROCK) inhibitor Y-27632. On day 21, the cardiac tissues were harvested and weighed. The hearts of EAM mice were significantly enlarged and whitened. Furthermore, body weight (BW) slowly increased during the treatment period, the heart weight (HW) and the ratio of HW/eventual BW were increased, and inflammatory infiltration and fibrosis were aggravated in the myocardial tissue. Y-27632 treatment improved the aforementioned phenotypic and pathological features of EAM mice. Mechanistic analysis revealed a significant increase in Notch1, Hes1, Jag2, Dil1, Toll-like receptor (Tlr) 2, and interleukin (IL)-1ß expression in the myocardial tissue of EAM mice. Notably, IL-1ß expression was correlated with that of Notch1 and Tlr2. Following Y-27632 treatment, the expression of key target genes of the Notch signaling pathway (Notch1, Hes1, Dil1, and Jag2) and Tlr2 were obviously decreased. Y-27632 treatment also decreased the number of monocytes in the spleen of EAM mice. Thus, ROCK inhibitor Y-27632 exerted a protective effect in EAM mice by downregulating IL-1ß expression. This study aimed to provide a reference point for the future treatment of myocarditis in clinical settings.

TNF-α protects from exacerbated myocarditis and cardiac death by suppressing expansion of activated heart-reactive CD4+ T cells.

AIMS: Tumour necrosis factor α (TNF-α) represents a classical pro-inflammatory cytokine, and its increased levels positively correlate with the severity of many cardiovascular diseases. Surprisingly, some heart failure patients receiving high doses of anti-TNF-α antibodies showed serious health worsening. This work aimed to examine the role of TNF-α signalling on the development and progression of myocarditis and heart-specific autoimmunity. METHODS AND RESULTS: Mice with genetic deletion of TNF-α (Tnf+/- and Tnf-/-) and littermate controls (Tnf+/+) were used to study myocarditis in the inducible and the transgenic T cell receptor (TCRM) models. Tnf+/- and Tnf-/- mice immunized with α-myosin heavy chain peptide (αMyHC) showed reduced myocarditis incidence, but the susceptible animals developed extensive inflammation in the heart. In the TCRM model, defective TNF-α production was associated with increased mortality at a young age due to cardiomyopathy and cardiac fibrosis. We could confirm that TNF-α as well as the secretome of antigen-activated heart-reactive effector CD4+ T (Teff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to Teff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4+ T lymphocytes from both models of myocarditis showed a strongly increased fraction of Teff cells in hearts, spleens, and in the blood of Tnf+/- and Tnf-/- mice. Indeed, antigen-activated Tnf-/- Teff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive Teff. CONCLUSION: TNF-α signalling promotes myocarditis development by activating cardiac endothelial cells. However, in the case of established disease, TNF-α protects from exacerbating cardiac inflammation by inducing activation-induced cell death of heart-reactive Teff. These data might explain the lack of success of standard anti-TNF-α therapy in heart failure patients and open perspectives for T cell-targeted approaches.

Vascular adhesion protein-1-targeted PET imaging in autoimmune myocarditis.

BACKGROUND: Vascular adhesion protein-1 (VAP-1) is an adhesion molecule and primary amine oxidase, and Gallium-68-labeled 1,4,7,10-tetraazacyclododecane-N,N',N″,N‴-tetra-acetic acid conjugated sialic acid-binding immunoglobulin-like lectin 9 motif containing peptide ([68Ga]Ga-DOTA-Siglec-9) is a positron emission tomography (PET) tracer targeting VAP-1. We evaluated the feasibility of PET imaging with [68Ga]Ga-DOTA-Siglec-9 for the detection of myocardial lesions in rats with autoimmune myocarditis. METHODS: Rats (n = 9) were immunized twice with porcine cardiac myosin in complete Freund's adjuvant. Control rats (n = 6) were injected with Freund's adjuvant alone. On day 21, in vivo PET/computed tomography (CT) imaging with [68Ga]Ga-DOTA-Siglec-9 was performed, followed by ex vivo autoradiography, histology, and immunohistochemistry of tissue sections. In addition, myocardial samples from three patients with cardiac sarcoidosis were studied. RESULTS: [68Ga]Ga-DOTA-Siglec-9 PET/CT images of immunized rats showed higher uptake in myocardial lesions than in myocardium outside lesions (SUVmean, 0.5 ± 0.1 vs 0.3 ± 0.1; P = .003) or control rats (SUVmean, 0.2 ± 0.03; P < .0001), which was confirmed by ex vivo autoradiography of tissue sections. Immunohistochemistry showed VAP-1-positive staining in lesions of rats with myocarditis and in patients with cardiac sarcoidosis. CONCLUSION: VAP-1-targeted [68Ga]Ga-DOTA-Siglec-9 PET is a potential novel technique for the detection of myocardial lesions.

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 2 weeks. 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.

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: It 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.

The role of non-coding RNAs in myocarditis: a narrative review.

Background and Objective: Myocarditis is a heterogeneous disease that can lead to acute heart failure, dilated cardiomyopathy (DCM), and sudden death. However, the knowledge of the precise molecular mechanisms of myocarditis is fairly limited. In recent years, non-coding RNAs (ncRNAs) have been demonstrated to be involved in many physiological and pathological processes in myocarditis and to have the potential to be used as novel diagnostic and therapeutic strategies for myocarditis. This review summarizes the role of ncRNAs in myocarditis and discusses their potential as noninvasive biomarkers and therapeutic targets for myocarditis. Methods: Literature on ncRNAs and myocarditis published in PubMed was extensively reviewed for analysis and discussion. Key Content and Findings: This review describes the roles of different ncRNAs in myocarditis and summarizes their potential in diagnosing and treating myocarditis. Multiple functions and mechanisms of ncRNAs in myocarditis have been uncovered. Conclusions: Current studies show that ncRNAs are widely involved in the occurrence and development of myocarditis caused by infection, autoimmunity, and the use of immune checkpoint inhibitors (ICIs) through their regulation of cell apoptosis, immune response, viral replication, and other aspects. Small-sample clinical studies have assessed the diagnostic value of ncRNAs. These results provide a new theoretical basis for diagnosing and treating myocarditis.

Specialized Proresolving Mediators Protect Against Experimental Autoimmune Myocarditis by Modulating Ca2+ Handling and NRF2 Activation.

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum-adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

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-ß1, 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-ß1/Smad-2/3/Ets-1 signaling pathway in EAM mice.

Protein Kinase C-Mediated Hyperphosphorylation and Lateralization of Connexin 43 Are Involved in Autoimmune Myocarditis-Induced Prolongation of QRS Complex.

Myocarditis is a serious and potentially life-threatening disease, which leads to cardiac dysfunction and sudden cardiac death. An increasing number of evidence suggests that myocarditis is also a malignant complication of coronavirus pneumonia, associated with heart failure and sudden cardiac death. Prolonged QRS complexes that are related to malignant arrhythmias caused by myocarditis significantly increase the risk of sudden cardiac death in patients. However, the molecular mechanisms are not fully known at present. In this study, we identify protein kinase C (PKC) as a new regulator of the QRS complex. In isolated hearts of normal rats, the PKC agonist, phorbol-12-myristate-13-acetate (PMA), induced prolongation of the QRS complex. Mechanistically, hyperphosphorylation and lateralization of connexin 43 (Cx43) by PKC induced depolymerization and internalization of Cx43 gap junction channels and prolongation of the QRS duration. Conversely, administration of the PKC inhibitor, Ro-32-0432, in experimental autoimmune myocarditis (EAM) rats after the most severe inflammation period still significantly rescued the stability of the Cx43 gap junction and alleviated prolongation of the QRS complex. Ro-32-0432 reduced phosphorylation and blocked translocation of Cx43 in EAM rat heart but did not regulate the mRNA expression level of ventricular ion channels and the other regulatory proteins, which indicates that the inhibition of PKC might have no protective effect on ion channels that generate ventricular action potential in EAM rats. These results suggest that the pharmacological inhibition of PKC ameliorates the prolongation of the QRS complex via suppression of Cx43 hyperphosphorylation, lateralization, and depolymerization of Cx43 gap junction channels in EAM rats, which provides a potential therapeutic strategy for myocarditis-induced arrhythmias.

IL-17 upregulates MCP-1 expression via Act1 / TRAF6 / TAK1 in experimental autoimmune myocarditis.

Myocarditis is a kind of myocardial inflammatory infiltration disease. Many interventions are not effective in the treatment of myocarditis because the mechanism of myocarditis has not been elucidated. Previous studies have found that interleukin-17 (IL-17) could stimulate the expression of monocyte chemokine protein 1 (MCP-1) and mediate myocardial inflammatory infiltration. This study aimed to explore the role of Act1/TRAF6/TAK1 cascade in IL-17-induced MCP-1 expression based on a well-designed experimental autoimmune myocarditis (EAM) model. It was found that IL-17 could stimulate the expression of MCP-1 by activating Act1/TRAF6/TAK1 cascade in EAM. The expression of Act1, TRAF6 and TAK1 followed downregulation by the application of IL-17 antibody. Additionally, myocardial inflammatory cell infiltration was observably alleviated by interfering TAK1 with TAK1 siRNA, and both MCP-1 mRNA and protein expression followed downregulation. This study suggested that IL-17 could activate the Act1/TRAF6/TAK1 pathway to upregulate MCP-1 expression in the EAM, and will offer a new perspective for the study on the mechanism of myocarditis.

Angiotensin II receptor 1 controls profibrotic Wnt/ß-catenin signalling in experimental autoimmune myocarditis.

AIMS: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis. METHODS AND RESULTS: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-ß)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-ß-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-ß induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-ß failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)ß and nuclear ß-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, ß-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts. CONCLUSION: Ang II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-ß downstream signalling response including activation of profibrotic Wnt/ß-catenin pathway.

LNA oligonucleotide mediates an anti-inflammatory effect in autoimmune myocarditis via targeting lactate dehydrogenase B.

Treatment of myocarditis is often limited to symptomatic treatment due to unknown pathomechanisms. In order to identify new therapeutic approaches, the contribution of locked nucleic acid antisense oligonucleotides (LNA ASOs) in autoimmune myocarditis was investigated. Hence, A/J mice were immunized with cardiac troponin I (TnI) to induce experimental autoimmune myocarditis (EAM) and treated with LNA ASOs. The results showed an unexpected anti-inflammatory effect for one administered LNA ASO MB_1114 by reducing cardiac inflammation and fibrosis. The target sequence of MB_1114 was identified as lactate dehydrogenase B (mLDHB). For further analysis, mice received mLdhb-specific GapmeR during induction of EAM. Here, mice receiving the mLdhb-specific GapmeR showed increased protein levels of cardiac mLDHB and a reduced cardiac inflammation and fibrosis. The effect of increased cardiac mLDHB protein level was associated with a downregulation of genes of reactive oxygen species (ROS)-associated proteins, indicating a reduction in ROS. Here, the suppression of murine pro-apoptotic Bcl-2-associated X protein (mBax) was also observed. In our study, an unexpected anti-inflammatory effect of LNA ASO MB_1114 and mLdhb-specific GapmeR during induction of EAM could be demonstrated in vivo. This effect was associated with increased protein levels of cardiac mLDHB, mBax suppression and reduced ROS activation. Thus, LDHB and LNA ASOs may be considered as a promising target for directed therapy of myocarditis. Nevertheless, further investigations are necessary to clarify the mechanism of action of anti-inflammatory LDHB-triggered effects.

Electrical, structural, and autonomic atrial remodeling underlies atrial fibrillation in inflammatory atrial cardiomyopathy.

Background: There is growing evidence indicating a close relationship between inflammation and atrial fibrillation (AF). Although underlying inflammatory atrial cardiomyopathy may contribute to the development of AF, the arrhythmogenic remodeling caused by atrial inflammation has not been elucidated in detail. Herein, we examined electrical, structural, and autonomic changes in the atria in a mouse model of autoimmune myocarditis. Methods: BALB/c mice were immunized with cardiac myosin peptide (MyHC-α614-629) conjugated with complete Freund's adjuvant on days 0 and 7. Susceptibility to AF was assessed using right-atrial burst pacing. Results: The mice immunized with MyHC-α614-629 showed an inflammatory atrial cardiomyopathy phenotype, with enlarged atria; a high degree of inflammatory cell infiltration primarily consisting of CD4+ T cells, CD8+ T cells, Ly6GlowCD11b+ macrophages, and CD11c+ dendritic cells; and severe interstitial fibrosis with collagen deposition. These mice demonstrated significantly enhanced susceptibility to AF, as indicated by their increased AF induction rate and duration. In addition, the expression of potassium channels (Kcnh2, Kcnd3, and Kcnj2) and calcium handling-associated genes (Cacna1c, Camk2, Ryr2, and Atp2a2) was downregulated. Connexin 40 expression was significantly downregulated, leading to frequent lateralization to the inflamed atrium. Sympathetic and parasympathetic innervation and neurotrophin expression (nerve growth factor and brain-derived neurotrophic factor) were upregulated in the inflamed atria. Conclusion: Inflammatory atrial cardiomyopathy promotes susceptibility to AF via arrhythmogenic electrical, structural, and autonomic remodeling of the atria.

HMG-CoA Reductase Inhibitor, Simvastatin Is Effective in Decreasing Degree of Myocarditis by Inhibiting Metalloproteinases Activation.

BACKGROUND: Acute myocarditis often progresses to heart failure because there is no effective, etiology-targeted therapy of this disease. Simvastatin has been shown to be cardioprotective by decreasing matrix metalloproteinases' (MMPs) activity. The study was designed to determine whether simvastatin inhibits MMPs activity, decreases the severity of inflammation and contractile dysfunction of the heart in experimental autoimmune myocarditis (EAM). METHODS: Simvastatin (3 or 30 mg/kg/day) was given to experimental rats with EAM by gastric gavage for 21 days. Then transthoracic echocardiography was performed, MMPs activity and troponin I level were determined and tissue samples were assessed under a light and transmission electron microscope. RESULTS: Hearts treated with simvastatin did not show left ventricular enlargement. As a result of EAM, there was an enhanced activation of MMP-9, which was significantly reduced in the high-dose simvastatin group compared to the low-dose group. It was accompanied by prevention of myofilaments degradation and reduction of severity of inflammation. CONCLUSIONS: The cardioprotective effects of simvastatin in the acute phase of EAM are, at least in part, due to its ability to decrease MMP-9 activity and subsequent decline in myofilaments degradation and suppression of inflammation. These effects were achieved in doses equivalent to therapeutic doses in humans.

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.

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.