Baricitinib protects ICIs-related myocarditis by targeting JAK1/STAT3 to regulate Macrophage polarization.

PURPOSE: The emergence of immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment, but these drugs can also cause severe immune-related adverse effects (irAEs), including myocarditis. Researchers have become interested in exploring ways to mitigate this side effect, and one promising avenue is the use of baricitinib, a Janus kinase inhibitor known to have anti-inflammatory properties. This study aimed to examine the potential mechanism by which baricitinib in ICIs-related myocarditis. METHODS: To establish an ICIs-related myocarditis model, BALB/c mice were administered murine cardiac troponin I (cTnI) peptide and anti-mouse programmed death 1 (PD-1) antibodies. Subsequently, baricitinib was administered to the mice via intragastric administration. Echocardiography, HE staining, and Masson staining were performed to evaluate myocardial functions, inflammation, and fibrosis. Immunofluorescence was used to detect macrophages in the cardiac tissue of the mice.In vitro experiments utilized raw264.7 cells to induce macrophage polarization using anti-PD-1 antibodies. Different concentrations of baricitinib were applied to assess cell viability, and the release of pro-inflammatory cytokines was measured. The activation of the JAK1/STAT3 signaling pathway was evaluated through western blot analysis. RESULTS: Baricitinib demonstrated its ability to improve cardiac function and reduce cardiac inflammation, as well as fibrosis induced by ICIs. Mechanistically, baricitinib treatment promoted the polarization of macrophages towards the M2 phenotype. In vitro and in vivo experiments showed that anti-PD-1 promoted the release of inflammatory factors. However, treatment with baricitinib significantly inhibited the phosphorylation of JAK1 and STAT3. Additionally, the use of RO8191 reversed the effects of baricitinib, further confirming our findings. CONCLUSION: Baricitinib demonstrated its potential as a protective agent against ICIs-related myocarditis by modulating macrophage polarization. These findings provide a solid theoretical foundation for the development of future treatments for ICIs-related myocarditis.

STAT1 regulates neutrophil gelatinase B-associated lipocalin induction in influenza-induced myocarditis.

Influenza is a significant public health and economic threat around the world. Epidemiological studies have demonstrated a close association between influenza pandemics and cardiovascular mortality. Moreover, it has been shown that there is a decrease in cardiovascular mortality in high-risk patients following vaccination with the influenza vaccine. Here, we have investigated the role of anti-viral STAT1 signaling in influenza-induced myocarditis. Wild-type mice (C57BL/6) were infected with either influenza A/PR/8/34 or control, and cellular response and gene expression analysis from the heart samples were assessed 7 days later. The expression of interferon response genes STAT1, STAT2, Mx1, OASL2, ISG15, chemokines CCL2, CCL3, CXCL9 and CXCL10, and the frequency of neutrophils (CD45+CD11b+Ly6G+) and CD4+ T cells (CD45+CD4+) were all significantly increased in influenza-infected mice when compared to vehicle controls. These data suggest that influenza infection induces interferons, inflammatory chemokines, and cellular recruitment during influenza infection. We further investigated the role of STAT1 in influenza-induced myocarditis. The frequency of neutrophils and the levels of lipocalin 2 were significantly increased in STAT1-/- mice when compared to WT controls. Finally, we investigated the role of Lcn2 in viral-induced myocarditis. We found that in the absence of Lcn2, there was preserved cardiac function in Lcn2-/- mice when compared to WT controls. These data suggest that the absence of Lcn2 is cardioprotective during viral-induced myocarditis.

Exosomal let-7a-5p derived from human umbilical cord mesenchymal stem cells alleviates coxsackievirus B3-induced cardiomyocyte ferroptosis via the SMAD2/ZFP36 signal axis. / 人脐带间å 质干细胞来源的外泌体let-7a-5p通过SMAD2/ZFP36ä¿¡å·è½´å‡è½»æŸ¯è¨å¥‡ç— æ¯’B3诱导的心肌细胞铁死亡.

Viral myocarditis (VMC) is one of the most common acquired heart diseases in children and teenagers. However, its pathogenesis is still unclear, and effective treatments are lacking. This study aimed to investigate the regulatory pathway by which exosomes alleviate ferroptosis in cardiomyocytes (CMCs) induced by coxsackievirus B3 (CVB3). CVB3 was utilized for inducing the VMC mouse model and cellular model. Cardiac echocardiography, left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS) were implemented to assess the cardiac function. In CVB3-induced VMC mice, cardiac insufficiency was observed, as well as the altered levels of ferroptosis-related indicators (glutathione peroxidase 4 (GPX4), glutathione (GSH), and malondialdehyde (MDA)). However, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSCs-exo) could restore the changes caused by CVB3 stimulation. Let-7a-5p was enriched in hucMSCs-exo, and the inhibitory effect of hucMSCs-exolet-7a-5p mimic on CVB3-induced ferroptosis was higher than that of hucMSCs-exomimic NC (NC: negative control). Mothers against decapentaplegic homolog 2 (SMAD2) increased in the VMC group, while the expression of zinc-finger protein 36 (ZFP36) decreased. Let-7a-5p was confirmed to interact with SMAD2 messenger RNA (mRNA), and the SMAD2 protein interacted directly with the ZFP36 protein. Silencing SMAD2 and overexpressing ZFP36 inhibited the expression of ferroptosis-related indicators. Meanwhile, the levels of GPX4, solute carrier family 7, member 11 (SLC7A11), and GSH were lower in the SMAD2 overexpression plasmid (oe-SMAD2)+let-7a-5p mimic group than in the oe-NC+let-7a-5p mimic group, while those of MDA, reactive oxygen species (ROS), and Fe2+ increased. In conclusion, these data showed that ferroptosis could be regulated by mediating SMAD2 expression. Exo-let-7a-5p derived from hucMSCs could mediate SMAD2 to promote the expression of ZFP36, which further inhibited the ferroptosis of CMCs to alleviate CVB3-induced VMC.

Loss of TRIM29 mitigates viral myocarditis by attenuating PERK-driven ER stress response in male mice.

Viral myocarditis, an inflammatory disease of the myocardium, is a significant cause of sudden death in children and young adults. The current coronavirus disease 19 pandemic emphasizes the need to understand the pathogenesis mechanisms and potential treatment strategies for viral myocarditis. Here, we found that TRIM29 was highly induced by cardiotropic viruses and promoted protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated endoplasmic reticulum (ER) stress, apoptosis, and reactive oxygen species (ROS) responses that promote 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 monocytic myeloid-derived suppressor cells (mMDSC) in vivo. Mechanistically, TRIM29 interacted with PERK to promote SUMOylation of PERK to maintain its stability, thereby promoting PERK-mediated signaling pathways. Finally, we demonstrated that the PERK inhibitor GSK2656157 mitigated viral myocarditis by disrupting the TRIM29-PERK connection, thereby bolstering cardiac function, enhancing cardiac antiviral responses, and curbing inflammation and immunosuppressive mMDSC in vivo. Our findings offer insight into how cardiotropic viruses exploit TRIM29-regulated PERK signaling pathways to instigate viral myocarditis, suggesting that targeting the TRIM29-PERK axis could mitigate disease severity.

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.

Mapping the interplay of immunoproteasome and autophagy in different heart failure phenotypes.

Proper protein degradation is required for cellular protein homeostasis and organ function. Particularly, in post-mitotic cells, such as cardiomyocytes, unbalanced proteolysis due to inflammatory stimuli and oxidative stress contributes to organ dysfunction. To ensure appropriate protein turnover, eukaryotic cells exert two main degradation systems, the ubiquitin-proteasome-system and the autophagy-lysosome-pathway. It has been shown that proteasome activity affects the development of cardiac dysfunction differently, depending on the type of heart failure. Studies analyzing the inducible subtype of the proteasome, the immunoproteasome (i20S), demonstrated that the i20S plays a double role in diseased hearts. While i20S subunits are increased in cardiac hypertrophy, atrial fibrillation and partly in myocarditis, the opposite applies to diabetic cardiomyopathy and ischemia/reperfusion injury. In addition, the i20S appears to play a role in autophagy modulation depending on heart failure phenotype. This review summarizes the current literature on the i20S in different heart failure phenotypes, emphasizing the two faces of i20S in injured hearts. A selection of established i20S inhibitors is introduced and signaling pathways linking the i20S to autophagy are highlighted. Mapping the interplay of the i20S and autophagy in different types of heart failure offers potential approaches for developing treatment strategies against heart failure.

Sacubitril/Valsartan inhibits M1 type macrophages polarization in acute myocarditis by targeting C-type natriuretic peptide.

Studies have shown that Sacubitril/valsartan (Sac/Val) can reduce myocardial inflammation in myocarditis mice, in addition to its the recommended treatment of heart failure. However, the underlying mechanisms of Sac/Val in myocarditis remain unclear. C-type natriuretic peptide (CNP), one of the targeting natriuretic peptides of Sac/Val, was recently reported to exert cardio-protective and anti-inflammatory effects in cardiovascular systems. Here, we focused on circulating levels of CNP in patients with acute myocarditis (AMC) and whether Sac/Val modulates inflammation by targeting CNP in experimental autoimmune myocarditis (EAM) mice as well as LPS-induced RAW 264.7 cells and bone marrow derived macrophages (BMDMs) models. Circulating CNP levels were higher in AMC patients compared to healthy controls, and these levels positively correlated with the elevated inflammatory cytokines IL-6 and monocyte count. In EAM mice, Sac/Val alleviated myocardial inflammation while augmenting circulating CNP levels rather than BNP and ANP, accompanied by reduction in intracardial M1 macrophage infiltration and expression of inflammatory cytokines IL-1ß, TNF-α, and IL-6. Furthermore, Sac/Val inhibited CNP degradation and directly blunted M1 macrophage polarization in LPS-induced RAW 264.7 cells and BMDMs. Mechanistically, the effects might be mediated by the NPR-C/cAMP/JNK/c-Jun signaling pathway apart from NPR-B/cGMP/NF-κB pathway. In conclusion, Sac/Val exerts a protective effect in myocarditis by increasing CNP concentration and inhibiting M1 macrophages polarization.

STING-Targeted PET Imaging for Specific Detection and Therapeutic Monitoring of Myocarditis.

Imaging strategies for the specific detection and therapeutic monitoring of myocarditis are still lacking. Stimulator of interferon genes (STING) is a signal transduction molecule involved in an innate immune response. Here, we evaluated the feasibility of the recently developed STING-targeted radiotracer [18F]FBTA for positron emission tomography (PET) imaging to detect myocardial inflammation and monitor treatment in myocarditis mice. [18F]FBTA-PET imaging was performed in myocarditis mice and normal mice to verify the specificity of [18F]FBTA for the diagnosis of myocarditis. We also performed PET imaging in mice with myocarditis treated to verify the ability of [18F]FBTA in therapeutic monitoring. The expression of STING and inflammatory cell types was confirmed by flow cytometry and immunohistochemistry. [18F]FDG-PET imaging of myocarditis was used as a contrast. [18F]FBTA-PET imaging showed that the average radioactive uptake was significantly higher in the hearts of the myocarditis group than in the control group. STING was highly overexpressed in cardiac inflammatory cells, including macrophages, dendritic cells (DCs), and T cells. However, there was no significant difference in cardiac radiotracer uptake of [18F]FDG between the myocarditis group and the control group. Moreover, cardiac uptake of [18F]FBTA was significantly reduced in cyclosporin A-treated myocarditis mice and myocardial STING expression was also significantly reduced after the treatment. Overall, we showed that a STING-targeted PET tracer [18F]FBTA can be used to monitor changes in the inflammatory microenvironment in myocarditis. Besides, [18F]FBTA-PET is also suitable for real-time monitoring of myocarditis treatment, representing a promising diagnostic and therapeutic monitoring approach for myocarditis.

Targeting NLRP3 signaling reduces myocarditis-induced arrhythmogenesis and cardiac remodeling.

BACKGROUND: Myocarditis substantially increases the risk of ventricular arrhythmia. Approximately 30% of all ventricular arrhythmia cases in patients with myocarditis originate from the right ventricular outflow tract (RVOT). However, the role of NLRP3 signaling in RVOT arrhythmogenesis remains unclear. METHODS: Rats with myosin peptide-induced myocarditis (experimental group) were treated with an NLRP3 inhibitor (MCC950; 10 mg/kg, daily for 14 days) or left untreated. Then, they were subjected to electrocardiography and echocardiography. Ventricular tissue samples were collected from each rat's RVOT, right ventricular apex (RVA), and left ventricle (LV) and examined through conventional microelectrode and histopathologic analyses. In addition, whole-cell patch-clamp recording, confocal fluorescence microscopy, and Western blotting were performed to evaluate ionic currents, intracellular Ca2+ transients, and Ca2+-modulated protein expression in individual myocytes isolated from the RVOTs. RESULTS: The LV ejection fraction was lower and premature ventricular contraction frequency was higher in the experimental group than in the control group (rats not exposed to myosin peptide). Myocarditis increased the infiltration of inflammatory cells into cardiac tissue and upregulated the expression of NLRP3; these observations were more prominent in the RVOT and RVA than in the LV. Furthermore, experimental rats treated with MCC950 (treatment group) improved their LV ejection fraction and reduced the frequency of premature ventricular contraction. Histopathological analysis revealed higher incidence of abnormal automaticity and pacing-induced ventricular tachycardia in the RVOTs of the experimental group than in those of the control and treatment groups. However, the incidences of these conditions in the RVA and LV were similar across the groups. The RVOT myocytes of the experimental group exhibited lower Ca2+ levels in the sarcoplasmic reticulum, smaller intracellular Ca2+ transients, lower L-type Ca2+ currents, larger late Na+ currents, larger Na+-Ca2+ exchanger currents, higher reactive oxygen species levels, and higher Ca2+/calmodulin-dependent protein kinase II levels than did those of the control and treatment groups. CONCLUSION: Myocarditis may increase the rate of RVOT arrhythmogenesis, possibly through electrical and structural remodeling. These changes may be mitigated by inhibiting NLRP3 signaling.

CXCL4:NLRP3-mediated pyroptosis product that regulates cardiac fibrosis.

Severe myocarditis is often accompanied by cardiac fibrosis, but the underlying mechanism has not been fully elucidated. NOD-like receptor protein 3 (NLRP3) inflammation is involved in the development of myocarditis and is closely related to the form of cell death. Inhibiting pyroptosis mediated by NLRP3 inflammasome can reduce cardiac fibrosis, although its exact mechanism remains unknown. In this study, we induced Viral myocarditis (VMC) via infection of CVB3 to explore the relationship between pyroptosis and fibrosis. Our results showed that intraperitoneal injection of an NLRP3 inhibitor MCC950 or use of NLRP3-/- mice inhibited cardiac pyroptosis mediated by NLRP3 inflammasome in VMC. CXCL4 is a chemokine that has been reported to have pro-inflammatory and pro-fibrotic functions. In VMC, we further found that pyroptosis of Mouse myocardial fibroblasts (MCF) promoted the secretion of CXCL4 by activating Wnt/ß-Catenin signaling. Subsequently, the transcriptome sequencing data showed that CXCL4 could promote cardiac fibrosis by activating PI3K/AKT pathway. In summary, infection of CVB3 induced host oxidative stress to further activate the NLRP3 inflammasome and ultimately lead to heart pyroptosis, in which MCF secreted CXCL4 by activating Wnt/ß-Catenin signaling and CXCL4 participated in cardiac fibrosis by activating PI3K/AKT pathway. Therefore, our findings revealed the role of CXCL4 in VMC and unveiled its underlying mechanism. CXCL4 appears to be a potential target for the treatment of VMC.

miR-29b-3p regulates cardiomyocytes pyroptosis in CVB3-induced myocarditis through targeting DNMT3A.

BACKGROUND: Viral myocarditis (VMC) is a disease resulting from viral infection, which manifests as inflammation of myocardial cells. Until now, the treatment of VMC is still a great challenge for clinicians. Increasing studies indicate the participation of miR-29b-3p in various diseases. According to the transcriptome sequencing analysis, miR-29b-3p was markedly upregulated in the viral myocarditis model. The purpose of this study was to investigate the role of miR-29b-3p in the progression of VMC. METHODS: We used CVB3 to induce primary cardiomyocytes and mice to establish a model of viral myocarditis. The purity of primary cardiomyocytes was identified by immunofluorescence. The cardiac function of mice was detected by Vevo770 imaging system. The area of inflammatory infiltration in heart tissue was shown by hematoxylin and eosin (H&E) staining. The expression of miR-29b-3p and DNMT3A was detected by quantitative real time polymerase chain reaction (qRT-PCR). The expression of a series of pyroptosis-related proteins was detected by western blot. The role of miR-29b-3p/DNMT3A in CVB3-induced pyroptosis of cardiomyocytes was studied in this research. RESULTS: Our data showed that the expression of miR-29b-3p was upregulated in CVB3-induced cardiomyocytes and heart tissues in mice. To explore the function of miR-29b-3p in CVB3-induced VMC, we conducted in vivo experiments by knocking down the expression of miR-29b-3p using antagomir. We then assessed the effects on mice body weight, histopathology changes, myocardial function, and cell pyroptosis in heart tissues. Additionally, we performed gain/loss-of-function experiments in vitro to measure the levels of pyroptosis in primary cardiomyocytes. Through bioinformatic analysis, we identified DNA methyltransferases 3A (DNMT3A) as a potential target gene of miR-29b-3p. Furthermore, we found that the expression of DNMT3A can be modulated by miR-29b-3p during CVB3 infection. CONCLUSIONS: Our results demonstrate a correlation between the expression of DNMT3A and CVB3-induced pyroptosis in cardiomyocytes. These findings unveil a previously unidentified mechanism by which CVB3 induces cardiac injury through the regulation of miR-29b-3p/DNMT3A-mediated pyroptosis.

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.

Exploring the Role of Platelets in Virus-Induced Inflammatory Demyelinating Disease and Myocarditis.

Theiler's murine encephalomyelitis virus (TMEV) infection has been used as a mouse model for two virus-induced organ-specific immune-mediated diseases. TMEV-induced demyelinating disease (TMEV-IDD) in the central nervous system (CNS) is a chronic inflammatory disease with viral persistence and an animal model of multiple sclerosis (MS) in humans. TMEV infection can also cause acute myocarditis with viral replication and immune cell infiltration in the heart, leading to cardiac fibrosis. Since platelets have been reported to modulate immune responses, we aimed to determine the role of platelets in TMEV infection. In transcriptome analyses of platelets, distinct sets of immune-related genes, including major histocompatibility complex (MHC) class I, were up- or downregulated in TMEV-infected mice at different time points. We depleted platelets from TMEV-infected mice by injecting them with platelet-specific antibodies. The platelet-depleted mice had significantly fewer viral antigen-positive cells in the CNS. Platelet depletion reduced the severities of TMEV-IDD and myocarditis, although the pathology scores did not reach statistical significance. Immunologically, the platelet-depleted mice had an increase in interferon (IFN)-γ production with a higher anti-TMEV IgG2a/IgG1 ratio. Thus, platelets may play roles in TMEV infection, such as gene expression, viral clearance, and anti-viral antibody isotype responses.

The central role of natural killer cells in mediating acute myocarditis after mRNA COVID-19 vaccination.

BACKGROUND: Vaccine-related acute myocarditis is recognized as a rare and specific vaccine complication following mRNA-based COVID-19 vaccinations. The precise mechanisms remain unclear. We hypothesized that natural killer (NK) cells play a central role in its pathogenesis. METHODS: Samples from 60 adolescents with vaccine-related myocarditis were analyzed, including pro-inflammatory cytokines, cardiac troponin T, genotyping, and immunophenotyping of the corresponding activation subsets of NK cells, monocytes, and T cells. Results were compared with samples from 10 vaccinated individuals without myocarditis and 10 healthy controls. FINDINGS: Phenotypically, high levels of serum cytokines pivotal for NK cells, including interleukin-1ß (IL-1ß), interferon α2 (IFN-α2), IL-12, and IFN-γ, were observed in post-vaccination patients with myocarditis, who also had high percentage of CD57+ NK cells in blood, which in turn correlated positively with elevated levels of cardiac troponin T. Abundance of the CD57+ NK subset was particularly prominent in males and in those after the second dose of vaccination. Genotypically, killer cell immunoglobulin-like receptor (KIR) KIR2DL5B(-)/KIR2DS3(+)/KIR2DS5(-)/KIR2DS4del(+) was a risk haplotype, in addition to single-nucleotide polymorphisms related to the NK cell-specific expression quantitative trait loci DNAM-1 and FuT11, which also correlated with cardiac troponin T levels in post-vaccination patients with myocarditis. CONCLUSION: Collectively, these data suggest that NK cell activation by mRNA COVID-19 vaccine contributed to the pathogenesis of acute myocarditis in genetically and epidemiologically vulnerable subjects. FUNDING: This work was funded by the Hong Kong Collaborative Research Fund (CRF) 2020/21 and the CRF Coronavirus and Novel Infectious Diseases Research Exercises (reference no. C7149-20G).

MG53 protects against Coxsackievirus B3-induced acute viral myocarditis in mice by inhibiting NLRP3 inflammasome-mediated pyroptosis via the NF-κB signaling pathway.

Pyroptosis, a novel programmed cell death mediated by NOD-like receptor protein 3 (NLRP3) inflammasome, is a critical pathogenic process in acute viral myocarditis (AVMC). Mitsugumin 53 (MG53) is predominantly expressed in myocardial tissues and has been reported to exert cardioprotective effects through multiple pathways. Herein, we aimed to investigate the biological function of MG53 in AVMC and its underlying regulatory mechanism in pyroptosis. BALB/c mice and HL-1 cells were infected with Coxsackievirus B3 (CVB3) to establish animal and cellular models of AVMC. As inflammation progressed in the myocardium, we found a progressive decrease in myocardial MG53 expression, accompanied by a significant enhancement of cardiomyocyte pyroptosis. MG53 overexpression significantly alleviated myocardial inflammation, apoptosis, fibrosis, and mitochondrial damage, thereby improving cardiac dysfunction in AVMC mice. Moreover, MG53 overexpression inhibited NLRP3 inflammasome-mediated pyroptosis, reduced pro-inflammatory cytokines (IL-1ß/18) release, and suppressed NF-κB signaling pathway activation both in vivo and in vitro. Conversely, MG53 knockdown reduced cell viability, facilitated cell pyroptosis, and increased pro-inflammatory cytokines release in CVB3-infected HL-1 cells by promoting NF-κB activation. These effects were partially reversed by applying the NF-κB inhibitor BAY 11-7082. In conclusion, our results suggest that MG53 acts as a negative regulator of NLRP3 inflammasome-mediated pyroptosis in CVB3-induced AVMC, partially by inhibiting the NF-κB signaling pathway. MG53 is a promising candidate for clinical applications in AVMC treatment.

CD73/adenosine axis exerts cardioprotection against hypobaric hypoxia-induced metabolic shift and myocarditis in a sex-dependent manner.

BACKGROUND: Clinical and experimental studies have shown that the myocardial inflammatory response during pathological events varies between males and females. However, the cellular and molecular mechanisms of these sex differences remain elusive. CD73/adenosine axis has been linked to anti-inflammatory responses, but its sex-specific cardioprotective role is unclear. The present study aimed to investigate whether the CD73/adenosine axis elicits sex-dependent cardioprotection during metabolic changes and myocarditis induced by hypobaric hypoxia. METHODS: For 7 days, male and female mice received daily injections of the CD73 inhibitor adenosine 5'- (α, ß-methylene) diphosphate (APCP) 10 mg/kg/day while they were kept under normobaric normoxic and hypobaric hypoxic conditions. We evaluated the effects of hypobaric hypoxia on the CD73/adenosine axis, myocardial hypertrophy, and cardiac electrical activity and function. In addition, metabolic homeostasis and immunoregulation were investigated to clarify the sex-dependent cardioprotection of the CD73/adenosine axis. RESULTS: Hypobaric hypoxia-induced cardiac dysfunction and adverse remodeling were more pronounced in male mice. Also, male mice had hyperactivity of the CD73/adenosine axis, which aggravated myocarditis and metabolic shift compared to female mice. In addition, CD73 inhibition triggered prostatic acid phosphatase ectonucleotidase enzymatic activity to sustain adenosine overproduction in male mice but not in female mice. Moreover, dual inhibition prostatic acid phosphatase and CD73 enzymatic activities in male mice moderated adenosine content, alleviating glycolytic shift and proinflammatory response. CONCLUSION: The CD73/adenosine axis confers a sex-dependent cardioprotection. In addition, extracellular adenosine production in the hearts of male mice is influenced by prostatic acid phosphatase and tissue nonspecific alkaline phosphatase.

Complement components regulates ferroptosis in CVB3 viral myocarditis by interatction with TFRC.

BACKGROUND: Dysregulated cell death machinery and an excessive inflammatory response in Coxsackievirus B3(CVB3)-infected myocarditis are hallmarks of an abnormal host response. Complement C4 and C3 are considered the central components of the classical activation pathway and often participate in the response process in the early stages of virus infection. METHODS: In our study, we constructed a mouse model of CVB3-related viral myocarditis via intraperitoneal injection of Fer-1 and detected myocarditis and ferroptosis markers in the mouse myocardium. Then, we performed co-IP and protein mass spectrometry analyses to explore which components interact with the ferroptosis gene transferrin receptor (TFRC). Finally, functional experiments were conducted to verify the role of complement components in regulating ferroptosis in CVB3 infection. RESULTS: It showed that the ferroptosis inhibitor Fer-1 could alleviate the inflammation in viral myocarditis as well as ferroptosis. Mechanistically, during CVB3 infection, the key factor TFRC was activated and inhibited by Fer-1. Fer-1 effectively prevented the consumption of complement C3 and overload of the complement product C4b. Interestingly, we found that TFRC directly interacts with complement C4, leading to an increase in the product of C4b and a decrease in the downstream complement C3. Functional experiments have also confirmed that regulating the complement C4/C3 pathway can effectively rescue cell ferroptosis caused by CVB3 infection. CONCLUSIONS: In this study, we found that ferroptosis occurs through crosstalk with complement C4 in viral myocarditis through interaction with TFRC and that regulating the complement C4/C3 pathway may rescue ferroptosis in CVB3-infected cardiomyocytes.

Ivabradine Alleviates Experimental Autoimmune Myocarditis-Mediated Myocardial Injury.

Ivabradine (IVA) reduces heart rate by inhibiting hyperpolarization-activated cyclic nucleotide-gated channels (HCNs), which play a role in the promotion of pacemaker activity in cardiac sinoatrial node cells. HCNs are highly expressed in neural and myocardial tissues and are involved in the modulation of inflammatory neuropathic pain. However, whether IVA exerts any effect on myocardial inflammation in the pathogenesis of heart failure is unclear. We employed single-cell RNA sequencing (scRNA-seq) in porcine cardiac myosin-induced experimental autoimmune myocarditis rat model to determine the effects and mechanisms of IVA. Lewis rats (n = 32) were randomly divided into the normal, control, high-dose-IVA, and low-dose-IVA groups. Heart rate and blood pressure were measured on days 0 and 21, respectively. Echocardiography was performed on day 22, and inflammation of the myocardium was evaluated via histopathological examination. Western blot was employed to detect the expression of HCN1-4, MinK-related protein 1 (MiRP1), matrix metalloproteinase 2 (MMP-2), MMP-9, and transforming growth factor-ß (TGF-ß). Furthermore, enzyme-linked immunosorbent assay was performed to measure serum IL-1, IL-6, and TNF-α. The relative mRNA levels of collagen I, collagen III, and α-smooth muscle actin (α-SMA) were determined via qRT-PCR. We found that IVA reduced the total number of cells infiltrated into the myocardium, particularly in the subset of fibroblasts, endocardia, and monocytes. IVA administration ameliorated cardiac inflammation and reduced collagen production. Results of the echocardiography indicated that left ventricular internal diameter at end-systole LVIDs increased whereas left ventricular ejection fraction and left ventricular fractional shortening decreased in the control group. IVA improved cardiac performance. The expression of HCN4 and MiRP1 protein and the level of serum IL-1, IL-6, and TNF-α were decreased by IVA treatment. In conclusion, HCNs and the helper proteins were increased in the profile of myocardial inflammation. HCNs may be involved in the regulation of myocardial inflammation by inhibiting immune cell infiltration. Our findings can contribute to the development of IVA-based combination therapies for the future treatment of cardiac inflammation and heart failure.

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.

Expansion of Pathogenic Cardiac Macrophages in Immune Checkpoint Inhibitor Myocarditis.

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte-associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model (Ctla4+/-Pdcd1-/- mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2)+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2+ monocytes. It is important that a similar macrophage population expressing CXCL9, CXCL10, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8+ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9+Cxcl10+ macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ-induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.