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Murine models of coxsackievirus B3 (CVB3)-induced myocarditis well represent the different outcomes of this inflammatory heart disease. Previously, we found that CVB3-infected A.BY/SnJ mice, susceptible for severe acute and chronic myocarditis, have lower natural killer (NK) cell levels than C57BL/6 mice, with mild acute myocarditis. There is evidence that myeloid-derived suppressor cells (MDSC) may inhibit NK cells, influencing the course of myocarditis. To investigate the MDSC/NK interrelationship in acute myocarditis, we used CVB3-infected A.BY/SnJ mice. Compared to non-infected mice, we found increased cell numbers of MDSC in the spleen and heart of CVB3-infected A.BY/SnJ mice. In parallel, S100A8 and S100A9 were increased in the heart, spleen, and especially in splenic MDSC cells compared to non-infected mice. In vitro experiments provided evidence that MDSC disrupt cytotoxic NK cell function upon co-culturing with MDSC. MDSC-specific depletion by an anti-Ly6G antibody led to a significant reduction in the virus load and injury in hearts of infected animals. The decreased cardiac damage in MDSC-depleted mice was associated with fewer Mac3+ macrophages and CD3+ T lymphocytes and a reduced cardiac expression of S100A8, S100A9, IL-1β, IL-6, and TNF-α. In conclusion, impairment of functional NK cells by MDSC promotes the development of chronic CVB3 myocarditis in A.BY/SnJ mice.

Aims Left ventricular (LV) dysfunction in viral myocarditis is attributed to myocardial inflammation and fibrosis, inducing acute and long‐time cardiac damage. Interventions are not established. On the basis of the link between inflammation, fibrosis, aldosterone, and extracellular matrix regulation, we aimed to investigate the effect of an early intervention with the mineralocorticoid receptor antagonist (MRA) eplerenone on cardiac remodelling in a murine model of persistent coxsackievirus B3 (CVB3)‐induced myocarditis. Methods and results SWR/J mice were infected with 5 × 104 plaque‐forming units of CVB3 (Nancy strain) and daily treated either with eplerenone (200 mg/kg body weight) or with placebo starting from Day 1. At Day 8 or 28 post infection, mice were haemodynamically characterized and subsequently sacrificed for immunohistological and molecular biology analyses. Eplerenone did not influence CVB3 load. Already at Day 8, 1.8‐fold (P

Aims The coxsackievirus B3 (CVB3) mouse myocarditis model is the standard model for investigation of virus-induced myocarditis but the pancreas, rather than the heart, is the most susceptible organ in mouse. The aim of this study was to develop a CVB3 mouse myocarditis model in which animals develop myocarditis while attenuating viral infection of the pancreas and the development of severe pancreatitis. Methods and results We developed the recombinant CVB3 variant H3N-375TS by inserting target sites (TS) of miR-375, which is specifically expressed in the pancreas, into the 3ʹUTR of the genome of the pancreo- and cardiotropic CVB3 variant H3. In vitro evaluation showed that H3N-375TS was suppressed in pancreatic miR-375-expressing EndoC-βH1 cells >5 log10, whereas its replication was not suppressed in isolated primary embryonic mouse cardiomyocytes. In vivo, intraperitoneal (i.p.) administration of H3N-375TS to NMRI mice did not result in pancreatic or cardiac infection. In contrast, intravenous (i.v.) administration of H3N-375TS to NMRI and Balb/C mice resulted in myocardial infection and acute and chronic myocarditis, whereas the virus was not detected in the pancreas and the pancreatic tissue was not damaged. Acute myocarditis was characterized by myocardial injury, inflammation with mononuclear cells, induction of proinflammatory cytokines, and detection of replicating H3N-375TS in the heart. Mice with chronic myocarditis showed myocardial fibrosis and persistence of H3N-375TS genomic RNA but no replicating virus in the heart. Moreover, H3N-375TS infected mice showed distinctly less suffering compared with mice that developed pancreatitis and myocarditis after i.p. or i.v application of control virus. Conclusion In this study, we demonstrate that by use of the miR-375-sensitive CVB3 variant H3N-375TS, CVB3 myocarditis can be established without the animals developing severe systemic infection and pancreatitis. As the H3N-375TS myocarditis model depends on pancreas-attenuated H3N-375TS, it can easily be used in different mouse strains and for various applications.

Background Heart failure with preserved ejection fraction (HFpEF) is associated with cardiac inflammatory responses, indicating a potential role of the immune system in the pathology of diastolic dysfunction. The cytoplasmatic pattern recognition receptor, nucleotide binding oligomerization domain 2 (NOD2) belongs to the innate immune system and induces among others the NLRP3 inflammasome, known to be involved in myocarditis and coronary heart disease. Purpose The aim of this study was to explore the role of NOD2 in Angiotensin II (AngII)-induced diastolic heart failure. Methods In NOD2−/− knock down and C57Bl6/j-wild type (WT) mice, diastolic dysfunction was induced by subcutaneous administration of 1.4mg/kg*day–1 AngII. Twenty-one days after first AngII administration, left ventricular (LV) function was evaluated by pressure tip catheter. Cardiac fibrosis, inflammation, and the expression of NOD2 and the NLRP3 component Apoptosis-associated speck like protein containing a caspase recruitment domain (ASC) were determined via immunohistochemistry, real-time PCR or Western Blot. Results LV NOD2 mRNA expression was 2.3-fold (p Conclusion NOD2−/− deteriorates LV diastolic dysfunction and worsens pathophysiological key mechanisms in mice with AngII-induced diastolic heart failure.

Inflammation in myocarditis induces cardiac injury and triggers disease progression to heart failure. NLRP3 inflammasome activation is a newly identified amplifying step in the pathogenesis of myocarditis. We previously have demonstrated that mesenchymal stromal cells (MSC) are cardioprotective in Coxsackievirus B3 (CVB3)-induced myocarditis. In this study, MSC markedly inhibited left ventricular (LV) NOD2, NLRP3, ASC, caspase-1, IL-1β, and IL-18 mRNA expression in CVB3-infected mice. ASC protein expression, essential for NLRP3 inflammasome assembly, increased upon CVB3 infection and was abrogated in MSC-treated mice. Concomitantly, CVB3 infection in vitro induced NOD2 expression, NLRP3 inflammasome activation and IL-1β secretion in HL-1 cells, which was abolished after MSC supplementation. The inhibitory effect of MSC on NLRP3 inflammasome activity in HL-1 cells was partly mediated via secretion of the anti-oxidative protein stanniocalcin-1. Furthermore, MSC application in CVB3-infected mice reduced the percentage of NOD2-, ASC-, p10- and/or IL-1β-positive splenic macrophages, natural killer cells, and dendritic cells. The suppressive effect of MSC on inflammasome activation was associated with normalized expression of prominent regulators of myocardial contractility and fibrosis to levels comparable to control mice. In conclusion, MSC treatment in myocarditis could be a promising strategy limiting the adverse consequences of cardiac and systemic NLRP3 inflammasome activation.

Background: The alarmins S100A8 and S100A9 are damage-associated molecular patterns, which play a pivotal role in cardiovascular diseases, inflammation, and viral infections. We aimed to investigate their role in Coxsackievirus B3 (CVB3)–induced myocarditis. Methods and Results: S100A8 and S100A9 mRNA expression was 13.0-fold ( P =0.012) and 5.1-fold ( P =0.038) higher in endomyocardial biopsies from patients with CVB3-positive myocarditis compared with controls, respectively. Elimination of CVB3 led to a downregulation of these alarmins. CVB3-infected mice developed an impaired left ventricular function and displayed an increased left ventricular S100A8 and S100A9 protein expression versus controls. In contrast, CVB3-infected S100A9 knockout mice, which are also a complete knockout for S100A8 on protein level, showed an improved left ventricular function, which was associated with a reduced cardiac inflammatory and oxidative response, and lower CVB3 copy number compared with wild-type CVB3 mice. Exogenous application of S100A8 to S100A9 knockout CVB3 mice induced a severe myocarditis similar to wild-type CVB3 mice. In CVB3-infected HL-1 cells, S100A8 and S100A9 enhanced oxidative stress and CVB3 copy number compared with unstimulated infected cells. In CVB3-infected RAW macrophages, both alarmins increased MIP-2 (macrophage inflammatory protein-2) chemokine expression, which was reduced in CVB3 S100A8 knockdown versus scrambled siRNA CVB3 cells. Conclusions: S100A8 and S100A9 aggravate CVB3-induced myocarditis and might serve as therapeutic targets in inflammatory cardiomyopathies.

Background: The cytoplasmatic pattern recognition receptor, NOD2 (nucleotide-binding oligomerization domain 2), belongs to the innate immune system and is among others responsible for the recognition of single-stranded RNA. With Coxsackievirus B3 (CVB3) being a single-stranded RNA virus, and the recent evidence that the NOD2 target, NLRP3 (NOD-like receptor family, pyrin domain containing 3) is of importance in the pathogenesis of CVB3-induced myocarditis, we aimed to unravel the role of NOD2 in CVB3-induced myocarditis. Methods and Results: Endomyocardial biopsy NOD2 mRNA expression was higher in CVB3-positive patients compared with patients with myocarditis but without evidence of persistent CVB3 infection. Left ventricular NOD2 mRNA expression was also induced in CVB3-induced myocarditis versus healthy control mice. NOD2 knockdown (−/− ) mice were rescued from the detrimental CVB3-mediated effects as shown by a reduced cardiac inflammation (less cardiac infiltrates and suppression of proinflammatory cytokines), cardiac fibrosis, apoptosis, lower CAR (Coxsackievirus and adenovirus receptor) expression and CVB3 copy number, and an improved left ventricular function in NOD2 −/− CVB3 mice compared with wild-type CVB3 mice. In agreement, NOD2 −/− decreased the CVB3-induced inflammatory response, CVB3 copy number, and apoptosis in vitro. NOD2 −/− was further associated with a reduction in CVB3-induced NLRP3 expression and activity as evidenced by lower ASC (apoptosis-associated speck-like protein containing a CARD) expression, caspase 1 activity, or IL-1β (interleukin-1β) protein expression under in vivo and in vitro CVB3 conditions. Conclusions: NOD2 is an important mediator in the viral uptake and inflammatory response during the pathogenesis of CVB3 myocarditis.

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro- inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.

Increased levels of interleukin-6 (IL-6) have been observed in patients with acute myocarditis and are associated with poor prognosis. This study was designed to examine whether treatment with anti-IL-6 receptor antibody improves cardiac dysfunction and left ventricular (LV) remodeling in experimental Coxsackie virus B3 (CVB3)-induced myocarditis. C57BL6/J mice were subjected to acute CVB3 infection. One day after viral infection mice were treated with a single injection of an anti-IL-6 receptor antibody (MR16-1, tocilizumab) or control IgG. Seven days after viral infection, LV function was examined by conductance catheter technique, cardiac remodeling assessed by estimation of titin phosphorylation, cardiac fibrosis, and inflammatory and antiviral response by immunohistochemistry, RT-PCR and cell culture experiments. Compared to controls, infected mice displayed an impaired systolic and diastolic LV function associated with an increase in cardiac inflammation, fibrosis and impaired titin phosphorylation. IL-6 receptor blockade led to a shift of the immune response to a Th1 direction and significant reduction of viral load. In addition, cardiac immune response, extracellular matrix regulation and titin function improved, resulting in a preserved LV function. IL-6 receptor blockade exerts cardiac beneficial effects by antiviral and immunomodulatory actions after induction of an acute murine CVB3 virus myocarditis.

Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.