Myocarditis In Mice Research Articles

Previous cardiovascular injury is a prerequisite for immune checkpoint inhibitor-associated lethal myocarditis in mice.

Immune checkpoint inhibitors (ICIs) are antineoplastic drugs designed to activate the immune system's response against cancer cells. Evidence suggests that they may lead to immune-related adverse events, particularly when combined (e.g., anti-CTLA-4 plus anti-PD-1), sometimes resulting in severe conditions such as myocarditis. We aimed to investigate whether a previously sustained cardiac injury, such as pathological remodelling due to hypertension, is a prerequisite for ICI therapy-induced myocarditis. We evaluated the cardiotoxicity of ICIs in a hypertension (HTN) mouse model (C57BL/6). Weekly doses were administered up to day 21 after the first administration. Our analysis encompassed the following parameters: (i) survival and cardiac pathological remodelling, (ii) cardiac function assessed using pressure-volume (PV)-loops, with brain natriuretic peptide (BNP) serving as a marker of haemodynamic dysfunction and (iii) cardiac inflammation (cytokine levels, infiltration, and cardiac antigen autoantibodies). After the first administration of ICI combined therapy, the treated HTN group showed a 30% increased mortality (P=0.0002) and earlier signs of hypertrophy and pathological remodelling compared with the untreated HTN group. BNP (P=0.01) and TNF-α (<0.0001) increased 2.5- and 1.7-fold, respectively, in the treated group, while IL-6 (P=0.8336) remained unchanged. Myocarditis only developed in the HTN group treated with ICIs on day 21 (score >3), characterised by T cell infiltration and increased cardiac antigen antibodies (86% showed a titre of 1:160). The control group treated with ICI was unaffected in any evaluated feature. Our findings indicate that pre-existing sustained cardiac damage is a necessary condition for ICI-induced myocarditis.

Modulation of myocardial inflammation, fibrosis development and ventricular arrhythmogenicity by endurance exercise in murine coxsackievirus B3 myocarditis

Abstract Aims Nonischaemic myocardial fibrosis is associated with progressive deterioration of myocardial function and forms the substrate for arrhythmias in atria and ventricles. In the absence of a specific aetiology, its origin is commonly attributed to preceding viral myocarditis. Athletes presenting with ventricular arrhythmias often demonstrate nonischaemic fibrosis on magnetic resonance imaging. Previous translational studies have shown an adverse effect of exercise on the course of acute viral myocarditis. Nevertheless, the effect on myocardial fibrosis development, a frequent and important consequence of viral myocarditis, has not yet been investigated. In the present study, we evaluate for the first time the impact of endurance exercise on longer-term myocardial inflammation, myocardial fibrosis and arrhythmogenicity using a murine viral myocarditis model. Methods and results Male C57BL/6J mice (11 weeks of age, n=72) were randomly assigned to 8 weeks of treadmill running (EEX) or without exercise (SED). Two weeks into the study (training or control), animals were injected intraperitoneally with either coxsackievirus B3 to induce acute viral myocarditis (CVB) or vehicle (PBS). Exercising myocarditis mice showed lower mortality (11% vs. 27%), albeit without statistical significance (P=0.23). At sacrifice (i.e. 6 weeks after inoculation), prominent myocardial inflammatory infiltration and cardiomyocyte loss was observed in both CVB groups, without difference on semiquantitative histological scoring. Nevertheless, the infiltrating cells in the CVB-EEX group were more often proinflammatory in nature (predominantly iNOS-reactive macrophages and positive CD8+/CD4+ ratio) compared to these in CVB-SED (predominantly arginase-1-reactive macrophages and higher CD4+/CD8+ ratio). In addition, the EEX group showed more haemosiderin-laden macrophages and spindled fibroblasts as signs of organisation. At that time, the majority of virus was cleared from the heart. Treadmill running during myocarditis enhanced development of interstitial fibrosis (with limited or extensive distribution)(82.4% in CVB-EEX vs. 56.3% in CVB-SED; P=0.049) and perivascular and/or interstitial fibrosis with extensive distribution (64.7% &amp; 64.7% in CVB-EEX vs. 50% &amp; 31.3% in CVB-SED; P=0.048). The highest scar counts were observed in CVB-EEX, but the mean count (1.9 vs. 1.2; P=0.19) did not significantly differ between the myocarditis groups. In CVB-EEX, the lesions contained denser and more organised collagen bundles. In vivo electrophysiology studies showed similar ventricular arrhythmia inducibility (P&amp;gt;0.20) and arrhythmia burden (P=0.49) in the myocarditis groups, although the longest arrhythmias and highest cumulative burden occurred in CVB-EEX. Conclusion Endurance exercise training during viral myocarditis modulates the longer-term inflammatory process and promotes perivascular and interstitial fibrosis development, potentially enhancing ventricular arrhythmogenicity.

Lupeol alleviates autoimmune myocarditis by suppressing macrophage pyroptosis and polarization via PPARα/LACC1/NF-κB signaling pathway

BackgroundAutoimmune myocarditis, with increasing incidence and limited therapeutic strategies, is in urgent need to explore its underlying mechanisms and effective drugs. Pyroptosis is a programmed cell death that may contribute to the pathogenesis of myocarditis. Nonetheless, no direct evidence validated the role of pyroptosis in autoimmune myocarditis. Lupeol (Lup), a pentacyclic triterpene, possesses various biological activities such as antidiabetic properties. However, the effects of Lup on autoimmune myocarditis and pyroptosis remain unelucidated. PurposeThis study aimed to reveal the role of pyroptosis in autoimmune myocarditis and explore the protective effects of Lup, and its engaged mechanisms. MethodsThe experimental autoimmune myocarditis (EAM) mouse model was established by immunization with a fragment of cardiac myosin in Balb/c mice. Lup and MCC950 were administered after EAM induction. The protective effects were assessed by inflammation score, cardiac injury, chronic fibrosis, and cardiac function. Mechanistically, the effects of Lup on the M1 polarization and pyroptosis of macrophages were evaluated. Transcriptome sequencing and molecular docking were subsequently employed, and the underlying mechanisms of Lup were further explored in vitro with small interfering RNA and adenovirus. ResultsAdministration of Lup and MCC950 alleviated EAM progression. Western blotting and immunofluorescence staining identified macrophages as the primary cells undergoing pyroptosis. Lup inhibited the expression of pyroptosis-associated proteins in macrophages during EAM in a dose-dependent manner. Furthermore, Lup suppressed pyroptosis in both bone marrow-derived macrophages (BMDMs) and THP-1-derived macrophages in vitro. In addition, Lup inhibited the M1 polarization of macrophages both in vivo and in vitro. Mechanistically, the protective effects of Lup were demonstrated via the suppression of the nuclear factor-κΒ (NF-κB) signaling pathway. Transcriptome sequencing and molecular docking revealed the potential involvement of peroxisome proliferator-associated receptor α (PPARα). Subsequently, we demonstrated that Lup activated PPARα to reduce the expression level of LACC1, thereby inhibiting the NF-κB pathway and pyroptosis. ConclusionOur findings indicated the crucial role of macrophage pyroptosis in the pathogenesis of EAM. Lup ameliorated EAM by inhibiting the M1 polarization and pyroptosis of macrophages through the PPARα/LACC1/NF-κB signaling pathway. Thus, our results provided a novel therapeutic target and agent for myocarditis.

Abstract 16577: Unraveling T-cell and Macrophage Chemokine Crosstalk in Immunotherapy-Induced Myocarditis

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that activate immunity against tumor cells but can cause immune related adverse events such as myocarditis, a rare but serious side effect with up to 40% mortality. Hypothesis: Macrophages contribute to recruitment of cytotoxic T-cell subsets to the heart in the setting of ICI, causing myocarditis. Methods: To study the role of macrophage depletion on ICI myocarditis, we treated MRL/Pdcd1-/- mice with liposomal clodronate. To identify timing of immune infiltration, we performed serial single cell T-cell receptor and RNA sequencing of CD45+ cells from the hearts/blood of MRL/Pdcd1-/- myocarditis mice at 1, 2, and 4 weeks of age. We treated MRL mice with anti-PD1/CTLA4 to induce myocarditis and assessed if anti-CXCR3 could decrease effector CD8+ T-cell cardiac infiltration and mitigate myocarditis. We studied the effects of in vitro CXCR3 blockade on CXCR3+ T-cell migration in a transwell assay. Results: Serial single-cell TCR sequencing/scRNA-seq of immune cells from MRL/ Pdcd1-/- vs. MRL control hearts showed infiltration of CXCL9/10+ macrophages ( Fig. 1, A ) along with progressive clonal expansion of CXCR3+ effector CD8+ T-cells in the heart starting at 2 weeks of age, prior to clinical onset of myocarditis. Macrophage depletion with liposomal clodronate reduced myocarditis and improved survival in MRL/ Pdcd1-/- mice ( Fig. 1, B-C) . CXCR3 blockade with anti-CXCR3 antibody reduced myocarditis, decreased CXCR3+ CD8+ T-cell infiltration, and improved survival in mice treated with anti-CTLA4/anti-PD1 antibodies (Fig. 1, D-E) . CXCR3 blockade in transwell assay also reduced migration of CD8+CXCR3+ T-cells towards the macrophage compartment (Fig. 1, F) . Conclusions: CXCL9/10+ macrophages likely play a critical role in development of ICI myocarditis through the recruitment of CXCR3+ effector T-cells into the heart, making this pathway an attractive therapeutic target.

Abstract 13301: TRIM29 Causes Pathogenesis of Viral Myocarditis by Inducing PERK-Mediated Endoplasmic Reticulum Stress and Reactive Oxygen Species Responses

Introduction: Viral myocarditis is an inflammatory disease of the myocardium which contributes to a significant fraction of sudden death in children and young adults. The ongoing coronavirus disease 19 pandemic emphasizes the need for understanding the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Methods: To understand the role of the novel E3 ligase TRIM29 in controlling viral myocarditis, wild-type and TRIM29 knockout mice were infected with coxsackievirus B3 (CVB3) for inducing viral myocarditis. Mice survival and heart function were monitored by transthoracic echocardiography, and hearts were harvested for histology and immunohistochemistry analysis. Real-time PCR, western blotting, co-immunoprecipitation, enzyme-linked immunoassay, flow cytometry, over-expression and knockdown techniques were used to understand the pathogenic mechanisms of TRIM29 in regulating endoplasmic reticulum stress response after virus infection in this study. Results: We found that TRIM29 was highly induced by cardiotropic viruses, including CVB3 and encephalomyocarditis (EMCV), and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK) mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that restrict viral replication in cardiomyocytes in vitro . TRIM29 deficiency protected mice from viral myocarditis by promoting cardiac antiviral functions and reducing PERK-mediated inflammation and immunosuppressive cells in vivo . Mechanistically, TRIM29 interacted with PERK to catalyze SUMOylation of PERK for maintaining its stability, thereby promoting PERK-mediated signaling pathways. Conclusions: Our data provide the first evidence that TRIM29-mediated PERK signaling pathways cause pathogenesis of viral myocarditis and identify the TRIM29-PERK axis as a potential therapeutic target for treating viral induced cardiovascular diseases.

The role of blood podoplanin in patients with viral myocarditis

Podoplanin (PDPN), a small mucin-like glycoprotein, was recently found to promote the generation of cardiac ectopic lymphoid follicles and anti-heart autoantibodies (AHA) in viral myocarditis (VMC) mice. Herein, we investigated the blood PDPN expression and its potential clinical value in VMC patients. Overall, 40 VMC patients were enrolled among 112 hospitalized patients with suspected myocarditis. Their serum PDPN levels were higher than those in controlled acute myocardial infarction (AMI) patients (n = 40) and healthy individuals (n = 30) (both p < 0.01) and positively correlated with CRP, IL-17, and IL-4 (all p < 0.01). Elevation of serum PDPN discriminated VMC from AMI (OR = 4.061, p < 0.01) and PDPN addition to the basic model (age, CRP, and peak cTNI) increased AUC values (from 0.822 to 0.933, p = 0.04). Additionally, the serum levels of PDPN ligand CCL-21 were also increased and correlated with PDPN (R = 0.59, p < 0.01) in VMC patients, accompanied by AHA production. Moreover, the anti-MHC antibody was closely related to PDPN levels (R = 0.53, p < 0.01), and anti-MHC-positive patients with VMC displayed higher percentages of CD4+IL-17A+PDPN+T cells and CD19+CCR7+B cells (both p < 0.05). Noticeably, VMC patients complicated by ventricular arrhythmias (27.50%) presented with AHA production and higher PDPN levels (p < 0.05). Finally, we screened out and verified that miR-182-5p directly targeted PDPN and negatively regulated its expression (all p < 0.01). These data suggested that blood PDPN might be a novel inflammation-associated biomarker for the early diagnosis of VMC and may contribute to AHA production by binding CCL-21 to recruit Th17 and B cells, which were regulated by miR-182-5p.

Reconstituted Extracellular Vesicles from Human Platelets Decrease Viral Myocarditis in Mice.

Patients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease-specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human-platelet-derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)-like module-containing mucin-like hormone receptor-like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll-like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL-1β), transforming growth factor-beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7-9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T-cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

Abstract P1049: The Impact Of Endurance Exercise On Longer-term Myocardial Fibrosis Development And Ventricular Arrhythmogenicity In Murine Coxsackievirus B3 Myocarditis

Aims: Diagnostic work-up of athletes often reveals nonischaemic myocardial fibrosis, which is associated with cardiac dysfunction and malignant arrhythmias. Its isolated finding on magnetic resonance imaging is commonly attributed to preceding viral myocarditis. Previous murine studies have shown a negative effect of exercise early in the course of viral myocarditis. In this study, we investigate, for the first time, the impact of endurance exercise on myocardial fibrosis development and arrhythmogenicity in a murine viral myocarditis model. Methods &amp; Results: Eleven-week-old male C57BL/6J mice (n=72) were randomised to 8 weeks of treadmill running (EEX) or without exercise (SED). After 2 weeks, animals received a single injection with either coxsackievirus B3 to induce acute viral myocarditis (CVB) or vehicle as control (PBS). Exercise resulted in a milder clinical disease course, with less weight loss and better preserved running capacity. In addition, mortality was lower in exercising myocarditis mice (11 vs. 27%), however without statistical significance (P=0.23). Exercise during myocarditis increased the occurrence of interstitial fibrosis (limited or extensive distribution) at sacrifice ( i.e. 6 weeks after inoculation)(82.4% in CVB-EEX vs. 56.3% in CVB-SED; P=0.049). Additionally, exercise enhanced development of perivascular and/or interstitial fibrosis with extensive distribution (64.7% &amp; 64.7% in CVB-EEX vs. 50% &amp; 31.3% in CVB-SED; P=0.048). CVB-EEX animals demonstrated more myocardial scars on average, albeit not significantly (1.9 vs. 1.2; P=0.19), with similar scar distribution. Despite manifest fibrotic remodelling, collagen and fibrogenetic genes were not upregulated in the myocarditis groups at the time of sacrifice. In vivo right ventricular programmed electrical stimulation showed comparable ventricular arrhythmia inducibility in the myocarditis groups (P&gt;0.20). The longest arrhythmias and highest cumulative arrhythmia burden occurred in CVB-EEX, however, without reaching statistical significance when compared to CVB-SED (P=0.49). Conclusion: Endurance exercise during viral myocarditis enhances perivascular and interstitial fibrosis development, which may promote ventricular arrhythmogenicity.

S1PR1-dependent migration of ILC3s from intestinal tissue to the heart in a mouse model of viral myocarditis.

Type 3 innate lymphocytes (ILC3s) have recently been reported as key factors in inflammatory diseases, however, their role in viral myocarditis is unclear. By flow cytometry, CVB3 (Coxsachievirus B3)-induced myocarditis mice were detected to increase the number of ILC3s, and their main type was NKp46 + ILC3. In contrast, application of CD90.2 neutralizing antibody in T-cell-deficient mice reduced the number of ILCs and improved myocarditis. ILCs from CD45.1 mouse intestinal lamina propria lymphocytes were adoptively transferred into recipient mice, and a comparable proportion of CD45.1+ cells were observed in the hearts of CVB3-infected recipient mice. The upregulation of S1PR1 (Recombinant Sphingosine 1 Phosphate Receptor 1), KLF2 (Kruppel-like factor 2), CXCR6, and CXCL16 in the hearts of CVB3-infected mice, as well as the greatly reduced numbers of ILCs infiltrating the hearts after S1PR1 inhibition, suggest that intestinal ILCs may migrate to the hearts via the CXCL16/CXCR6 axis. Taken together, our results demonstrate that increased ILC3 in the heart during viral myocarditis may contribute to inflammatory progression, and that this increased population of ILC3 likely originates from the intestine.

B-arrestin deletion abrogates the innate immune response in murine CVB3 myocarditis

β-arrestins (βarrs), initially discovered as desensitizers of GPCR signaling, are now known to function as intracellular scaffolds and signaling transducers mediating a wide array of signaling cascades including immune responses to inflammatory stimuli. An important cause of immune cell infiltration and cardiac inflammation are viral pathogens such as enterovirus coxsackievirus B3 (CVB3) induced myocarditis. While viral myocarditis is a leading cause of dilated cardiomyopathy, the precise molecular mechanisms for the acute immune response to CVB3 infection are not well understood. Given the important role for βarrs in the acute immune response, we tested the hypothesis that βarrs are necessary for the activation of the acute immune response and mediate the progression and severity of cardiotropic CVB3-induced myocarditis in mice. Five to 7-week-old mice devoid of βarr1 (βarr1 KO) or βarr2 (βarr2 KO) were infected intraperitoneally with 1x103-1x104 plaque forming units (PFU) of CVB3 per animal. Wild type (WT) C57BL/6 mice infected with CVB3 were used as controls. The survival rate 7 days post infection (dpi) was significantly improved in male βarr1 KOs (80%) and βarr2 KOs (75%) compared to male WT control mice (35%, p=0.013). Female WT, βarr1 KO, and βarr2 KO mice showed similar survival rates 7 dpi., ranging from 70% to 80%. Early mortality among all groups before 5 dpi was associated with high viral titers (~6.0 ± 0.5 log10PFU/mg) in the liver compared to mortality after 5 dpi (~2.4 ± 1.7 log10PFU/mg) as determined by plaque assay. Viral titers in the heart (WT=3.7 ± 0.9 log10PFU/mg, βarr1KO=3.6 ± 1.3 log10PFU/mg, βarr2KO=3.5 ± 1.2log10PFU/mg) were similar among all groups suggesting that βarrs are not involved in viral replication. Morphometric analysis showed similar weight loss (~18%) across all groups at 7 dpi corroborated by viral cardiac PFU. Immunofluorescent staining of the heart with the pan-inflammatory marker, CD45, revealed high immune cell infiltration in infected WT mice (34.2% ± 4.5%), whereas immune cell infiltration was significantly blunted in infected βarr1 KO (6.0% ± 1.1%; p&lt;0.0001) and βarr2 KO (8.5% ± 0.9%; p&lt;0.0001) mice. The degree of immune cell infiltration in hearts of infected WT mice was directly correlated with an increase in heart viral titer, whereas immune cell infiltration remained blunted across a broad range of heart viral titers in both βarr1 and 2 KO mice. The blunted immune response in both βarr KOs compared to WT infected controls was consistent across multiple immune cell markers including CD4 for helper T cells and CD11b for innate immune cells. In conclusion, we demonstrate that βarrs are necessary for immune cell recruitment to the heart in CVB3 myocarditis and deletion of βarrs in male mice may be cardioprotective as indicated by an increase in survival rate. These data suggest that βarrs may serve as potential therapeutic targets in the treatment of acute viral myocarditis. This work was supported by the National Institutes of Health Grants HL056687 (to H.A.R.). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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

Inhibition of phosphoglycerate kinase 1 attenuates autoimmune myocarditis by reprogramming CD4+ T cell metabolism.

CD4+ T cells are the major drivers of cardiac-specific autoimmunity in myocarditis, specifically Th1, Treg, and most significant Th17 cells. But the molecular mechanisms of their activation remain unclear. We aimed to elucidate the regulatory role of phosphoglycerate kinase 1 (PGK1) in CD4+ T cells and experimental autoimmune myocarditis (EAM). EAM was induced in BALB/c mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Single-cell sequencing analysis found that glycolysis and PGK1 expression were elevated in cardiac CD4+ T and Th17 cells from myocarditis mice. Mice treated with PGK1 inhibitor NG52 showed less cardiac inflammation and fibrosis and better contractile function, as well as reduced cardiac infiltrating Th17 and Th1 cells and increased proportion of Treg. NG52 suppressed CD4+ T cell activation and differentiation of mice and myocarditis patients in vitro. Mechanistically, inhibition of PGK1 suppressed glycolytic activity and decreased pyruvate dehydrogenase kinase 1 (PDHK1) phosphorylation, thereby increasing reactive oxygen species (ROS) production in mitochondria and thus preventing Th17 cell differentiation. PGK1 may act as a key metabolic regulator of CD4+ T cell differentiation and regulates Th17 cell differentiation by regulating glycolysis and the PDHK1-ROS axis. Targeting PGK1 might be a promising strategy for the treatment of myocarditis.

Myricetin ameliorates experimental autoimmune myocarditis in mice by modulating immune response and inhibiting MCP-1 expression

Myocarditis is defined as an inflammatory disease of the myocardium, and the autoimmune response specific to myocardium plays an important role in chronic myocarditis. Inhibiting myocardial-specific autoimmune response and inflammation is crucial to treat myocarditis. Myricetin is a plant-derived flavonoid in nature which has potent anti-inflammatory and cardiovascular protective properties. However, the pharmacological effect of myricetin in autoimmune myocarditis is undefined. It is necessary to investigate the role and potential mechanisms of myricetin in autoimmune myocarditis. Therefore, purified cardiac myosin was subcutaneously injected to mice to establish the experimental autoimmune myocarditis (EAM) model. Myricetin was solubilized in normal saline and administered everyday by gavage from the day of immunization. After 21 days of treatment, it was found that myricetin significantly alleviated myocardial injury in EAM mice. The serum anti-cardiac myosin antibody, immunoglobulin (Ig) G, IgM levels and the proportion of T helper 17 (Th17) cells were decreased and the proportion of regulatory T (Treg) cells was increased with the treatment of myricetin in EAM mice. The myosin-specific T cell proliferation was inhibited by myricetin. Meanwhile, myricetin suppressed the expressions of monocyte chemoattractant protein-1 (MCP-1), phospho (p)-p65, p-c-Jun and Act1/TRAF6/TAK1 in H9C2 cells and myocardial tissues of EAM mice. These results revealed that myricetin inhibited the autoimmune response specific to myocardium and the expression of MCP-1 in cardiomyocytes, which suggested that myricetin ameliorated autoimmune myocarditis by modulating immune response and the expression of MCP-1. Therefore, myricetin may be a promising therapeutic strategy for autoimmune myocarditis.

Genetic Ablation of Ankrd1 Mitigates Cardiac Damage during Experimental Autoimmune Myocarditis in Mice.

Myocarditis (MC) is an inflammatory disease of the myocardium that can cause sudden death in the acute phase, and dilated cardiomyopathy (DCM) with chronic heart failure as its major long-term outcome. However, the molecular mechanisms beyond the acute MC phase remain poorly understood. The ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic stress/stretch-inducible protein, which can modulate cardiac stress response during various forms of pathological stimuli; however, its involvement in post-MC cardiac remodeling leading to DCM is not known. To address this, we induced experimental autoimmune myocarditis (EAM) in ANKRD1-deficient mice, and evaluated post-MC consequences at the DCM stage mice hearts. We demonstrated that ANKRD1 does not significantly modulate heart failure; nevertheless, the genetic ablation of Ankrd1 blunted the cardiac damage/remodeling and preserved heart function during post-MC DCM.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Clusterin is increased in morphologically normal cardiomyocytes in lymphocytic myocarditis.

Hormone therapy ameliorates ICI-related myocarditis in mice.

Hormone therapy ameliorates ICI-related myocarditis in mice.

Abstract 9345: Midline1 is a Critical Regulator of Viral Myocarditis

Introduction: Infections by viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could cause viral myocarditis. Innate immunity to viral nucleic acids mediates antiviral immunity as well as inflammatory organ injury. However, the innate immune mechanisms that control the pathogenesis of viral myocarditis are unclear. Methods: To understand the role of the E3 ligase Midline1 in controlling viral myocarditis, wild-type and Midline1 knockout mice were infected with coxsackievirus B3 (CVB3) or encephalomyocarditis (EMCV) for inducing viral myocarditis. Mice survivals and heart functions were monitored by transthoracic echocardiography, and hearts were harvested for histology analysis. Real-time PCR, western blotting, co-immunoprecipitation, enzyme-linked immunoassay, luciferase assay, flow cytometry, over-expression and knockdown techniques were used to understand the molecular mechanisms of Midline1 in regulating type I interferon production after virus infection in this study. Results: We find that Midline1 is induced by RNA viruses with heart tropisms CVB3 and EMCV. Knockdown or deletion of Midline1 in human or mouse macrophages enhances production of type I interferon (IFN) in response to double strand (ds) RNA, RNA viruses CVB3 or EMCV. Importantly, deletion of Midline1 protects mice from viral myocarditis induced by CVB3 or EMCV due to enhanced type I IFN production in vivo. Mechanistically, we show that Midline1 recruits protein phosphatase 1A (PPM1A) to dephosphorylate TANK binding kinase 1 (TBK1), which inactivates TBK1 to block TBK1 from interacting with its upstream adaptor mitochondrial antiviral signaling (MAVS), thereby dampening antiviral signaling during viral infections. Moreover, Midline1 stabilizes PPM1A by inducing K63-linked ubiquitination of PPM1A. Conclusions: Our results indicate that Midline1 serves as a negative regulator of viral myocarditis by downregulating innate immune activation induced by RNA viruses CVB3 and EMCV. Our data reveal that Midline1 is a critical regulator of innate immunity in viral myocarditis, thereby identifying a potential therapeutic target for treating viral induced cardiovascular diseases.

The protective effect of LCZ696 in coxsackievirus B3-induced acute viral myocarditis mice.

Acute viral myocarditis (AVMC) is the aetiology of heart failure (HF) with few specific treatments. The improvement of left ventricular ejection fraction (LVEF) is a critical predictor for the prognosis of AVMC. LCZ696 is a drug used in HF to improve LVEF, with a few research on AVMC. In this research, we evaluated the effects and mechanism of LCZ696 in improving LVEF in AVMC. Eighty 4-week-old male BALB/c mice were randomly divided into four groups of 20: Sham; Sham+LCZ696 (60mg/kg/d); AVMC; AVMC+LCZ696. The above experiments were repeated by CVB3-infected HL-1 and Mdivi-1 to down-regulated dynamin-related protein 1(Drp1). Adeno-associated virus 9 (AAV9) with enhanced green fluorescent proteins (GFP) was injected to produce Drp1-overexpression mice and set up four groups: AVMC group, AVMC+AAV group, AVMC+LCZ696 group, and AVMC+LCZ696+AAV group (n=20 in each group). LVEF was evaluated by echocardiography at a similar heart rate (HR) at d7, Drp1 (p-Drp1), inflammation and apoptosis by histology and Western blot (WB), and mitochondrial by electron microscopy. Cardiac function were injured in AVMC that LCZ696 reversed (LVEF, %: Sham: 68.99±9.67; Sham+LCZ696: 71.96±6.20; AVMC: 30.95±6.40*; AVMC+LCZ696: 68.99±9.67*#, *P<0.05 vs. Sham, #P<0.05 vs. AVMC). LCZ696 attenuated p-Drp1 expression, inflammation, apoptosis, and mitochondrial fission (p-Drp1/Drp1: Sham: 1; Sham+LCZ696: 1.37±0.22; AVMC: 2.29±0.36*; AVMC+LCZ696: 1.43±0.08*#, *P<0.05 vs. Sham, #P<0.05 vs. AVMC). Some of the above results were repeated in CVB3-infected HL-1 cells and Mdivi-1. AAV increased Drp1 expression and mitochondrial fission, inflammatory, and apoptosis. Compared with the AVMC+AAV group, the LVEF increased from 24.44±0.03% to 32.33±0.05% in the AVMC+LCZ696+AAV group(P<0.05), p-Drp1/Drp1 decreased from 0.54±0.12 to 0.42±0.09*, and IL-6, c-IL-1β, and c-caspase-3/caspase-3 decreased from 1.07±0.22 to 0.72±0.08*, from 1.03±0.14 to 0.79±0.09*, and from 4.69±0.29 to 0.92±0.13*, respectively (*P<0.05). LCZ696 has a protective effect on AVMC by improving LVEF and reducing inflammation and apoptosis, which may be due to the inhibition of Drp1-mediated mitochondrial fission.

Critical roles of Parkin and PINK1 in antiviral immunity and protection from cell death in Enterovirus-induced viral myocarditis

Abstract Background Myocarditis is an inflammatory disease of the heart muscle often resulting in cardiac dysfunction and death. One of the most common causes is a cardiac infection with viruses, especially with enteroviruses of the Coxsackievirus B (CVB) family. CVB3-induced viral myocarditis is a well-established model to elucidate viral myocarditis in mice. It is assumed that mitochondrial damage control and mitochondria-associated innate immunity is essential for defence against viral infection of the heart muscle. And Parkinson's disease genes Parkin and PINK1 are well known for their critical function in mitochondrial homeostasis and damage control. We investigated the roles of Parkin and PINK1 in CVB3-induced viral myocarditis mouse model. Material and methods Male C57BL/6J WT, Parkin KO and PINK1 KO mice were used in an age of 8 to 12 weeks. The animals were injected intraperitoneally with 1x104 plaque forming units of CVB3 diluted in phosphate-buffered saline. Control and survived virus-infected mice were sacrificed for tissue sampling at day 7 to investigate imaging and molecular workup. Result After virus inoculation, the Parkin KO and PINK1 KO groups showed a significantly lower 28-day survival rate compared with the WT group (p&amp;lt;0.001 in Parkin KO; p&amp;lt;0.001 in PINK1 KO). Evans-Blue Dye (EBD) staining image of day 7 after virus-infection showed that the hearts of Parkin KO and PINK1 KO mice were more severely damaged than the hearts of WT mice (3.2-fold, p&amp;lt;0.01 in Parkin KO; 2.6-fold, p&amp;lt;0.05 in PINK1 KO). H&amp;E staining showed that severe inflammatory reaction occurred in the hearts of WT mice, although the inflammatory reaction was much lower compared to WT in the hearts of Parkin KO and PINK1 KO mice (p&amp;lt;0.001 in Parkin KO; p&amp;lt;0.001 in PINK1 KO). In immunohistochemistry, EBD staining positive area colocalized well with CVB3 in Parkin KO and PINK1 KO mice hearts, although EBD staining positive area was rare and didn't colocalized well CVB3 in WT mice hearts. mRNA levels of cell markers (CD3, natural cytotoxicity receptors, F4/80), inflammatory cytokines (IL-1β, IL-6, TNF-α), type II interferon, macrophage-derived protective factors, and CXCL10 family cytokines were lower in virus-infected Parkin/PINK1 KO mice than WT mice. Western blots showed that phospho-p65 increased less than WT in the heart of Parkin KO (0.7-fold, p&amp;lt;0.05) and PINK1 KO mice (0.58-fold, p&amp;lt;0.05). Conclusion The Parkin KO and PINK1 KO mice showed a higher mortality rate and more severe cardiomyocyte damage than the WT mice in the viral myocarditis mouse model. Parkin and PINK1 is critical to the type II IFN pathway that inhibits virus replication. And Parkin and PINK1 play an important role in the recruitment of IFN-γ-producing lymphocytes through NF-κB pathway and CXCL10 family chemokines. Funding Acknowledgement Type of funding sources: None.

Abstract P3074: Immune Checkpoint Inhibition Reshapes Cardiac Immune Landscape Through Crosstalk Between Macrophage And T-cell

Immune checkpoint inhibitors (ICI) targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) or programmed cell death protein 1(PD-1)/ programmed death-ligand 1(PD-L1) have revolutionized cancer management since the approval. However, they are associated with immune-related adverse events (irAEs), including myocarditis. ICI-associated myocarditis is often fulminant and fatal. The associated major risk factor is the combination of CTLA-4 and PD-1 inhibition. The mechanistic basis of ICI-associated myocarditis is unclear and mechanism-based treatment strategies are needed. Crosstalk between T-cells and macrophages may represent one pathogenic mechanism underlying ICI-associated myocarditis. We employed an established mouse model Ctla4 +/- Pdcd1 -/- to study the crosstalk between T-cells and macrophage in the heart and its impact on reshaping cardiac immune landscape using unbiased single cell-RNA-seq technology. We discovered a more inflammatory cardiac microenvironment in ICI-associated myocarditis mouse, characterized by CD68 + cells infiltration, robust CCR2 signal, enhanced T-cells activation with interferon gamma (IFN-γ) secretion and increased frequency of Cxcl9 + Cxcl10 + macrophages with an activated phenotype enriched in response to IFN-γ. Importantly, the existence of Cxcl9 + Cxcl10 + macrophages (characterized by CD16α and CXCL9/CXCL10 expression) was confirmed in human ICI-associated myocarditis specimens which establishes a connection between mouse model and human disease. Analysis of cell communication between myeloid cells and T-cells predicted that Cxcl9 + Cxcl10 + macrophages communicate with T-cells via IFN-γ-IFN-γR and Cxcl9/Cxcl10-CXCR3 signals. Pseudotime time analysis suggests Cxcl9 + Cxcl10 + macrophages were derived from monocytes. Blocking IFN-γ signal prolonged the survival time of ICI-associated myocarditis mouse and reduced emergence of Cxcl9 + Cxcl10 + macrophages in the heart suggesting specific blocking the mediator of crosstalk between T-cells and macrophages attenuated ICI-associated myocarditis. This study will provide support to the usage of IFN-γ as a potential target to prevent ICI associated myocarditis during or at the end of ICI therapy in cancer patients.