Corrigendum to "The mitochondrial uncoupler 2,4-dinitrophenol modulates inflammatory and oxidative responses in Trypanosoma cruzi -induced acute myocarditis in mice" [Cardiovascular Pathology 72 (2024) 107653

We characterized the immune responses elicited by a DNA-prime/MVA-boost vaccine (TcVac3) constituted of antigenic candidates (TcG2 and TcG4), shown to be recognized by B and T cell responses in Trypanosoma cruzi (Tc) infected multiple hosts. C57BL/6 mice immunized with TcVac3 elicited a strong antigen-specific, high-avidity, trypanolytic antibody response (IgG2b>IgG1); and a robust antigen- and Tc-specific CD8+T cell response with type-1 cytokine (IFN-γ+TNF-α>IL-4+IL-10) and cytolytic effector (CD8+CD107a+IFN-γ+Perforin+) phenotype. The vaccine-induced effector T cells significantly expanded upon challenge infection and provided >92% control of T. cruzi. Co-delivery of IL-12 and GMCSF cytokine adjuvants didn’t enhance the TcVac3-induced resistance to T. cruzi. In chronic phase, vaccinated/infected mice exhibited a significant decline (up to 70%) in IFN-γ+CD8+T cells, a predominance of immunoregulatory IL-10+/CD4+T and IL10+/CD8+T cells, and presented undetectable tissue parasitism, inflammatory infiltrate, and fibrosis in vaccinated/infected mice. In comparison, control mice responded to challenge infection by a low antibody response, mixed cytokine profile, and consistent activation of pro-inflammatory CD8+T cells associated with parasite persistence and pathologic damage in the heart. We conclude that TcVac3 elicited type-1 effector T cell immunity that effectively controlled T. cruzi infection, and subsequently, predominance of anti-inflammatory responses prevented chronic inflammation and myocarditis in chagasic mice.

Viral myocarditis results from an adverse immune response to cardiotropic viruses, which causes irreversible myocyte destruction and heart failure in previously healthy people. The involvement of microRNAs and their usefulness as therapeutic targets in this process are unknown. To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. Cardiac microRNAs were profiled in both human myocarditis and in Coxsackievirus B3-injected mice, comparing myocarditis-susceptible with nonsusceptible mouse strains longitudinally. MicroRNA responses diverged depending on the susceptibility to myocarditis after viral infection in mice. MicroRNA-155, -146b, and -21 were consistently and strongly upregulated during acute myocarditis in both humans and susceptible mice. We found that microRNA-155 expression during myocarditis was localized primarily in infiltrating macrophages and T lymphocytes. Inhibition of microRNA-155 by a systemically delivered LNA-anti-miR attenuated cardiac infiltration by monocyte-macrophages, decreased T lymphocyte activation, and reduced myocardial damage during acute myocarditis in mice. These changes were accompanied by the derepression of the direct microRNA-155 target PU.1 in cardiac inflammatory cells. Beyond the acute phase, microRNA-155 inhibition reduced mortality and improved cardiac function during 7 weeks of follow-up. Our data show that cardiac microRNA dysregulation is a characteristic of both human and mouse viral myocarditis. The inflammatory microRNA-155 is upregulated during acute myocarditis, contributes to the adverse inflammatory response to viral infection of the heart, and is a potential therapeutic target for viral myocarditis.

BackgroundThe diagnosis of acute myocarditis typically requires either endomyocardial biopsy (which is invasive) or cardiovascular magnetic resonance imaging (which is not universally available). Additional approaches to diagnosis are desirable. We sought to identify a novel microRNA for the diagnosis of acute myocarditis.MethodsTo identify a microRNA specific for myocarditis, we performed microRNA microarray analyses and quantitative polymerase-chain-reaction (qPCR) assays in sorted CD4+ T cells and type 17 helper T (Th17) cells after inducing experimental autoimmune myocarditis or myocardial infarction in mice. We also performed qPCR in samples from coxsackievirus-induced myocarditis in mice. We then identified the human homologue for this microRNA and compared its expression in plasma obtained from patients with acute myocarditis with the expression in various controls.ResultsWe confirmed that Th17 cells, which are characterized by the production of interleukin-17, are a characteristic feature of myocardial injury in the acute phase of myocarditis. The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction. The human homologue, designated hsa-miR-Chr8:96, was identified in four independent cohorts of patients with myocarditis. The area under the receiver-operating-characteristic curve for this novel microRNA for distinguishing patients with acute myocarditis from those with myocardial infarction was 0.927 (95% confidence interval, 0.879 to 0.975). The microRNA retained its diagnostic value in models after adjustment for age, sex, ejection fraction, and serum troponin level.ConclusionsAfter identifying a novel microRNA in mice and humans with myocarditis, we found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. (Funded by the Spanish Ministry of Science and Innovation and others.)

The mitochondrial uncoupler 2,4-dinitrophenol modulates inflammatory and oxidative responses in Trypanosoma cruzi-induced acute 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.

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.

Genetic susceptibility and autoimmunity triggered by microbial infections are factors implicated in the pathogenesis of dilated cardiomyopathy, the most common cause of heart failure in young patients. Here we show that dendritic cells (DCs) loaded with a heart-specific self peptide induce CD4+ T-cell-mediated myocarditis in nontransgenic mice. Toll-like receptor (TLR) stimulation, in concert with CD40 triggering of self peptide-loaded dendritic cells, was shown to be required for disease induction. After resolution of acute myocarditis, DC-immunized mice developed heart failure, and TLR stimulation of these mice resulted in relapse of inflammatory infiltrates. Injection of damaged, syngeneic cardiomyocytes also induced myocarditis in mice if TLRs were activated in vivo. DC-induced myocarditis provides a unifying theory as to how tissue damage and activation of TLRs during infection can induce autoimmunity, relapses and cardiomyopathy.

Objective To investigate IL-17 and IL-23 levels change in Coxsackie virus-induced myocarditis in mice,aiming at effect of IL-17 in viral myocarditis.Methods A total of 100 male BALB/c mice,4 weeks old,including 80 in the viral myocarditis (VMC)group and 20 in the control group,were used.Mice in VMC group were peritoneally injected (PI)with Coxsackie virus B3(CVB3/Nancy).Mice were sacrificed at day 14,28,42 and 56 after PI.For different stages post infection,pathological changes of the mouse were observed,and IL-17 of heart by immunohistochemistry.Splenocytes were collected from the mice and restimulated with phytohemagglutinin (PHA)for 2 days in vitro.IL-17 mRNA and IL-23 mRNA of splenocytes were measured by RT-PCR and compared with those in the control.Results (1)IL-17 mRNA extracted from splenocytes increased at day 28 after PI as compared with that in the control and 14 days (both P＜0.01),and decreased at day 42 and 56.(2)All of the VMC group and control mice expressed IL-23 mRNA,but IL-23 mRNA level,which up-regulated at day 14,increased earlier than IL-17 mRNA,and continued to rise to a high level after that.(3)Heart IL-17 level which examined by integrate optical density also increased at day 28 of viral myocarditis in mouse as compared with that in the control and 14 days ( both P＜0.01).And the protein levels decreased at day 42 and 56 then.Conclusion The mRNA and protein levels of IL-17 were promoted in Coxsackie virus-induced myocarditis mice after 28 days.IL-23 mRNA expression was up-regulated also.This result suggested that Th17 subset is differentiated in chronic stage of viral myocarditis. Key words: Interleukin; Coxsackie virus; Myocarditis; Mice

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

The capacity for different reovirus reassortant viruses to induce acute myocarditis in mice correlates with cytopathogenic effect in primary cultures of murine cardiac myocytes. Multiple viral genes encoding proteins involved in viral RNA synthesis are determinants of this disease. We therefore evaluated the role of viral RNA synthesis in induction of acute myocarditis by infecting primary cultures of cardiac myocytes with a panel of myocarditic and nonmyocarditic viruses and quantitating RNA synthesis. RNA synthesis correlated with induction of myocarditis and with the S1 and M1 reovirus genes. Since one consequence of viral RNA synthesis is generation of infectious virus, we looked next at viral yield from cardiac myocyte cultures. Yield of infectious virus at an early time postinfection or as a final yield from primary infections did not correlate with myocarditis, but instead both correlated with the S1 gene. The S1 gene also determined the fraction of cells infected during primary infections in the culture, which varied dramatically between viruses. Viral yields per infected cell were similar for most myocarditic and nonmyocarditic reoviruses and did not correlate with induction of myocarditis or any reovirus gene. Together, the data provide two insights into reovirus-induced acute myocarditis in mice. First, while the S1 gene. which encodes the viral attachment protein sigma1 (as well as a nonstructural protein, sigma1s, of unknown function) does not determine the myocarditic potential of these viruses, it does determine the efficiency with which they infect cardiac myocytes. Second, while viral RNA synthesis is a determinant of acute myocarditis, this is not due to generation of infectious virus. This finding suggests that some other consequence of viral RNA synthesis, for example, induction of interferon, may determine reovirus-induced acute myocarditis.

Objective To investigate the effect of myeloid differentiation factor88 (MyD88)on the experimental autoimmune myocarditis (EAM) in Balb/c mice.Methods Totally 90 Balb/c mice were randomly divided into three groups:MyD88 ( n =30),myocarditis ( n =30),and control ( n =30 )groups.An injection of MyD88 high expression adenovirus was carried out through vena caudalis at day 3.The mice of myocarditis group received porcine cardiac myosin at the same time and position.The mice of control group received PBS only.Serum and myocardium samples were gained at day 21.The inflammatory infiltration and the serum levels of cTnI,IgG,and IgM were examined.Results 21 days later,the inflammatory infiltration score of the myocarditis group was higher than the control group (2.39 ± 1.23 vs 0.68 ±0.65,P ＜0.05).The MyD88 group was significantly higher than the other 2 groups ( F =94.194,P ＜0.01 ),the myocarditis group ( 3.56 ± 1.34 vs 2.39 ± 1.23,P ＜ 0.05 ),and the control group ( 3.56 ±1.34 vs 0.68 ±0.65,P ＜0.01).The serum levels of cTnI,IgG,and IgM of the MyD88 group were significantly higher than the myocarditis group and the control group( P ＜0.01,P ＜0.05).Conclusions MyD88 participates in the pathogenesis of experimental autoimmune myocarditis in mice. Key words: Myeloid differentiation factor 88/metabolism; Autoimmune diseases/physiopathology/blood; Myocarditis/physiopathology/blood; Cardiac myosins; Mice

Described by the Brazilian scientist Carlos Chagas more than a century ago, Chagas disease (ChD) currently affects 8 to 10 million persons and causes more than 10 000 deaths each year.[1][1] Originally confined to Latin American countries, ChD had been considered an exotic disease and received less

There is an unmet need for accurate and practical screening to detect myocarditis. We sought to test the hypothesis that the extent of acute myocarditis, measured by late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging (CMR), can be estimated based on routine blood markers. A total of 44 patients were diagnosed with acute myocarditis and included in this study. There was strong correlation between myoglobin and LGE (rs = 0.73 [95% CI 0.51; 0.87], p < 0.001), while correlation was weak between LGE and TnT-hs (rs = 0.37 [95% CI 0.09; 0.61], p = 0.01). Receiver operating curve (ROC) analysis determined myoglobin ≥ 87 μg/L as cutoff to identify myocarditis (92% sensitivity, 80% specificity). The data were reproduced in an established model of coxsackievirus B3 myocarditis in mice (n = 26). These data suggest that myoglobin is an accurate marker of acute myocarditis.Graphical Receiver operating curve analysis determined myoglobin ≥ 87 μg/L as cutoff to identify myocarditis and these data were reproduced in an established model of coxsackievirus B3 myocarditis in mice: CMRI, cardiac magnetic resonance imaging; Mb, myoglobin; LGE, late gadolinium enhancement; ROC, receiver operating curve analysis.

T cell activation is assumed to play a crucial role in many viral infections. An important marker for the activation of T cells is the interleukin-2 receptor (IL-2R); resting T lymphocytes do not bear detectable amounts of IL-2R. AMT13, a rat monoclonal antibody against mouse IL-2R, inhibits interleukin-2-dependent cell growth both in vitro and in vivo. Therefore, to clarify the effects of anti-IL-2R antibody treatment upon coxsackievirus B3 (CB3)-infected C3H/He mice, AMT13, 1 microg/mouse per day, was administered, subcutaneously, starting on day 0 (group 2) in experiment I or on day 7 (group 4) in Experiment II for 7 days, respectively. Groups 1 and 3 were examined as infected controls. In both experiments, there was no significant difference in mortality or in the severity of myocarditis between the treated and the untreated groups. Also, myocardial CB3 titers on day 7 did not differ significantly between groups 1 and 2. In addition, the distribution of activated T cell subsets in the inflamed myocardium was not changed by the treatment, and the paucity of myocardial IL-2R-positive cells was confirmed in all groups. Effects of the antibody treatment were confirmed by a decrease in delayed type hypersensitivity. Although some reports have shown that anti-IL-2R antibody has been successfully applied to ameliorate acute renal graft-versus-host disease, to enhance survival of skin allografts, and to suppress diabetic insulitis, it did not exert a beneficial effect on acute CB3 myocarditis in mice.

Viral infections are able to induce autoimmune inflammation in the heart. Here, we investigated the role of virus-activated Toll-like receptor (TLR)3 and its adaptor TRIF on the development of autoimmune coxsackievirus B3 (CVB3) myocarditis in mice. Although TLR3- or TRIF-deficient mice developed similarly worse acute CVB3 myocarditis and viral replication compared to control mice, disease was significantly worse in TRIF compared to TLR3-deficient mice. Interestingly, TLR3-deficient mice developed an interleukin (IL)-4-dominant T helper (Th)2 response during acute CVB3 myocarditis with elevated markers of alternative activation, while TRIF-deficient mice elevated the Th2-associated cytokine IL-33. Treatment of TLR3-deficient mice with recombinant IL-33 improved heart function indicating that elevated IL-33 in the context of a classic Th2-driven response protects against autoimmune heart disease. We show for the first time that TLR3 versus TRIF deficiency results in different Th2 responses that uniquely influence the progression to chronic myocarditis.