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Viral myocarditis (VM) is an inflammatory pathology of the myocardium triggered by a viral infection that may cause sudden death or heart failure, especially in the younger population. Current treatments only stabilise and improve cardiac function without resolving the underlying inflammatory cause. The factors that induce VM to progress to heart failure are still uncertain, but neutrophils have been increasingly associated with the negative evolution of cardiac pathologies. The present study investigates the contribution of neutrophils to VM disease progression in different ways.In a Coxsackievirus B3- (CVB3) induced mouse model of VM, neutrophils and neutrophil extracellular traps (NETs) were prominent in the acute phase of VM as revealed by ELISA analysis and immunostaining. Anti-Ly6G-mediated neutrophil blockade starting at model induction decreased cardiac necrosis and leukocyte infiltration, preventing monocyte and Ly6CHigh pro-inflammatory macrophage recruitment. Furthermore, genetic peptidylarginine deiminase 4 (PAD4)-dependent NET blockade significantly reduced cardiac damage and leukocyte recruitment, significantly decreasing cardiac monocyte and macrophage presence. Depleting neutrophils with anti-Ly6G antibodies at 7 days post-infection, after the acute phase, did not decrease cardiac inflammation.Collectively, these results indicate that the repression of neutrophils and the related NET response in the acute phase of VM improves the pathological phenotype by reducing cardiac inflammation.Viral myocarditis (VM) is a form of acute heart failure prompted by viral infection that still lacks a specific therapy addressing the cardiac inflammation causing the disease. Increasing evidence suggests that neutrophils actively contribute to the severity of inflammatory and cardiovascular pathologies. Our study demonstrates that inhibition of neutrophil functions in the early phases of VM decreases cardiac damage and inflammation and, therefore, may be considered a very early therapeutic strategy in preventing disease progression.

Pathological innate and adaptive immune response upon viral infection may lead to cardiac injury and dysfunction. Stabilin-1 is a scavenger receptor that regulates several aspects of the innate immunity. Whether stabilin-1 affects the inflammatory response during viral myocarditis (VM) is entirely unknown. Here, we assess the role of stabilin-1 in the pathogenesis of VM and its suitability as a therapeutic target. Genetic loss of stabilin-1 increased mortality and cardiac necrosis in a mouse model of human Coxsackievirus B3 (CVB3)-induced myocarditis. Absence of stabilin-1 significantly reduced monocyte recruitment and strongly reduced the number of alternatively activated anti-inflammatory macrophages in the heart, enhancing a pro-inflammatory cardiac niche with a detrimental T lymphocyte response during VM. Yeast two-hybrid screening, confirmed by affinity chromatography, identified fibronectin as a stabilin-1 interacting partner. Absence of stabilin-1 specifically decreased monocyte adhesion on extracellular fibronectin in vitro. Loss of Type III repeats Extra Domain A (EDA) of fibronectin during VM also increased the mortality and cardiac necrosis as in stabilin-1 knockout mice, with reduced monocytic cardiac recruitment and increased T lymphocyte response. Collectively, stabilin-1 has an immune-suppressive role of limiting myocardial damage during VM, regulating anti-inflammatory monocyte-recruitment to the site of inflammation. ispartof: Journal Of Molecular And Cellular Cardiology vol:165 pages:31-39 ispartof: location:England status: published

Background Aging can be viewed as a status of chronic inflammation, in which neutrophils have a lower threshold for activation. The enzyme peptidylarginine deiminase 4 (PAD4), which catalyzes the conversion of arginine to citrulline, will be activated in a certain population of neutrophils. When this conversion takes place on the histones, neutrophils can form neutrophil extracellular traps (NETs), which are both prothrombotic and proinflammatory. Mice lacking this enzyme systemically were previously reported to be protected from age-related fibrosis. Purpose We aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, and in consequence NETs, are involved in cardiac fibrosis development, which in turn will result in impaired cardiac function. Methods We generated a mouse model of impaired NET release capability via deletion of PAD4, a NET-essential gene, under the neutrophil-specific promoter (PAD4fl/flMRP8Cre+). In order to study heart failure (HF) development, these specific deletion mice and their littermate controls were aged for a period of two years (coinciding with approximately 70 years of age in the human population; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated by echocardiography and histology, respectively. A separate set of young mice (12 weeks) were evaluated in parallel. Results We performed a comprehensive echocardiography analysis including both structural and functional parameters. As for systolic function, we could see that in old wild type (WT) mice, ejection fraction (EF) significantly decreased as compared to EF in young and healthy (YH) mice (YH - 67±6%, WT - 53±10%; p Conclusion Our data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac fibrosis, resulting in cardiac dysfunction. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds Wetenschappelijk Onderzoek (FWO) - Vlaanderen

Aims Viral myocarditis (VM) is severe cardiac inflammation that can result in sudden death or congestive heart failure in previously healthy adults, with no effective therapy. Liver X receptor (LXR) agonists have both anti-inflammatory and lipid-lowering properties. This study investigates whether LXR agonist T0901317 may modulate viral replication and cardiac inflammation during VM. Methods and results (i) Adult mice were administered T0901317 or vehicle with the onset of inflammation during CVB3 virus myocarditis or (ii) treated 2 days prior to CVB3 infection. Against what we expected, T0901317 treatment did not alter leucocyte infiltration after CVB3 infection; yet pre-administration with T0901317 resulted in increased mortality upon CVB3 infection, higher cardiac viral presence, and increased cardiomyocyte damage when compared with the vehicle. Furthermore, we show a correlation of fatty acid synthase (FAS) and sterol regulatory element-binding protein 1c (SREBP-1c) with CVB3 viral load in the heart and that T0901317 is able to enhance the cardiac expression of FAS and SREBP-1c. Finally, we show in vitro that T0901317 is able to exaggerate CVB3-mediated damage of Vero cells, whereas inhibitors of FAS and the SREBP-1c reduce the viral presence of CVB3 in neonatal cardiomyocytes. Conclusion LXR agonism does not modulate cardiac inflammation, but exacerbates virus-mediated myocardial damage during VM by stimulating lipid biosynthesis and enhancing CVB3 replication.

Rationale: To maintain cardiac mechanical and structural integrity after an ischemic insult, profound alterations occur within the extracellular matrix. Osteoglycin is a small leucine-rich proteoglycan previously described as a marker of cardiac hypertrophy. Objective: To establish whether osteoglycin may play a role in cardiac integrity and function after myocardial infarction (MI). Methods and Results: Osteoglycin expression is associated with collagen deposition and scar formation in mouse and human MI. Absence of osteoglycin in mice resulted in significantly increased rupture-related mortality with tissue disruption, intramyocardial bleeding, and increased cardiac dysfunction, despite equal infarct sizes. Surviving osteoglycin null mice had greater infarct expansion in comparison with wild-type mice because of impaired collagen fibrillogenesis and maturation in the infarcts as revealed by electron microscopy and collagen polarization. Absence of osteoglycin did not affect cardiomyocyte hypertrophy in the remodeling remote myocardium. In cultured fibroblasts, osteoglycin knockdown or supplementation did not alter transforming growth factor-β signaling. Adenoviral overexpression of osteoglycin in wild-type mice significantly improved collagen quality, thereby blunting cardiac dilatation and dysfunction after MI. In osteoglycin null mice, adenoviral overexpression of osteoglycin was unable to prevent rupture-related mortality because of insufficiently restoring osteoglycin protein levels in the heart. Finally, circulating osteoglycin levels in patients with heart failure were significantly increased in the patients with a previous history of MI compared with those with nonischemic heart failure and correlated with survival, left ventricular volumes, and other markers of fibrosis. Conclusions: Increased osteoglycin expression in the infarct scar promotes proper collagen maturation and protects against cardiac disruption and adverse remodeling after MI. In human heart failure, osteoglycin is a promising biomarker for ischemic heart failure.

BACKGROUND: Diagnosing lymphocytic myocarditis (LM) is challenging because of the large variation in clinical presentation and the limitations inherent in current diagnostic tools. The objective of this study was to analyze infiltration of inflammatory cells in quadriceps skeletal muscle of LM patients and investigate the potential diagnostic value of assaying infiltrating inflammatory cells.METHODS: Quadriceps muscle tissue, obtained at autopsy from control patients (n = 9) and LM patients (n = 21), was analyzed using immunohistochemistry for infiltration of lymphocytes (CD45), macrophages (CD68), neutrophilic granulocytes (myeloperoxidase), and several lymphocyte subtypes (CD3, CD4, CD8, CD20) and using polymerase chain reaction for a panel of myocarditis-associated viruses. Additionally, quadriceps muscle from mice with acute coxsackievirus B3-induced myocarditis and control mice was analyzed for presence of lymphocytes and virus.RESULTS: In quadriceps muscle of LM patients the number of infiltrating lymphocytes were significantly increased and LM was diagnosed with specificity of 100% and sensitivity of 71%. Parvovirus B19 was the primary virus found in our patient groups, found in quadriceps tissue of 3 LM patients (although it was also found in 1 control patient). In the mice, enteroviral RNA was present in the quadriceps muscle, although enteroviral capsid proteins and lymphocyte infiltration were found primarily in the adipose tissue within and directly adjacent to the myocyte tissue, rather than in the myocyte tissue itself.CONCLUSIONS: LM is associated with lymphocyte infiltration and viral presence in quadriceps muscle. This indicates that skeletal muscle biopsy/lymphocyte quantification might be a potential diagnostic tool for LM patients.

Optimal healing after myocardial infarction requires not only the induction of inflammation, but also its timely resolution. In patients, 30 days post myocardial infarction, circulating monocytes have increased expression of Semaphorin3A (Sema3A) as compared to directly after admission. This increased expression coincides with increased expression of Cx3CR1—a marker of non-classical monocytes that are important for immune resolution hence proper wound healing. In mice, the expression of Sema3A also increases in response to myocardial ischemia being expressed by infiltrating leukocytes. Comparing Sema3A heterozygote (HZ) and wild type (WT) mice post myocardial infarction, revealed increased presence of leukocytes in the cardiac tissues of HZ mice as compared to WT, with no differences in capillary density, collagen deposition, cardiomyocyte surface area, chemokine—or adhesion molecules expression. Whilst infarct sizes were similar 14 days after myocardial infarction in both genotypes, Sema3A HZ mice had thinner infarcts and reduced cardiac function as compared to their WT littermates. In vitro experiments were conducted to study the role of Sema3A in inflammation and resolution of inflammation as a potential explanation for the differences in leukocyte recruitment and cardiac function observed in our in vivo experiments. Here, recombinant Sema3A protein was able to affect the pro-inflammatory state of cultured bone marrow derived macrophages. First, the pro-inflammatory state was altered by the induced apoptosis of classical macrophages in the presence of Sema3A. Second, Sema3A promoted the polarization of classical macrophages to resolution-phase macrophages and enhanced their efferocytotic ability, findings that were reflected in the infarcted cardiac tissue of the Sema3A HZ mice. Finally, we demonstrated that besides promoting resolution of inflammation, Sema3A was also able to retard the migration of monocytes to the myocardium. Collectively our data demonstrate that Sema3A reduces cardiac inflammation and improves cardiac function after myocardial infarction by promoting the resolution of inflammation. Electronic supplementary material The online version of this article (doi:10.1007/s00395-017-0630-5) contains supplementary material, which is available to authorized users.

Background: Cardiovascular diseases remain the predominant cause of death worldwide, with the prevalence of heart failure continuing to increase. Despite increased knowledge of the metabolic alterations that occur in heart failure, novel therapies to treat the observed metabolic disturbances are still lacking. Methods: Mice were subjected to pressure overload by means of angiotensin-II infusion or transversal aortic constriction. MicroRNA-146a was either genetically or pharmacologically knocked out or genetically overexpressed in cardiomyocytes. Furthermore, overexpression of dihydrolipoyl succinyltransferase (DLST) in the murine heart was performed by means of an adeno-associated virus. Results: MicroRNA-146a was upregulated in whole heart tissue in multiple murine pressure overload models. Also, microRNA-146a levels were moderately increased in left ventricular biopsies of patients with aortic stenosis. Overexpression of microRNA-146a in cardiomyocytes provoked cardiac hypertrophy and left ventricular dysfunction in vivo, whereas genetic knockdown or pharmacological blockade of microRNA-146a blunted the hypertrophic response and attenuated cardiac dysfunction in vivo. Mechanistically, microRNA-146a reduced its target DLST—the E2 subcomponent of the α-ketoglutarate dehydrogenase complex, a rate-controlling tricarboxylic acid cycle enzyme. DLST protein levels significantly decreased on pressure overload in wild-type mice, paralleling a decreased oxidative metabolism, whereas DLST protein levels and hence oxidative metabolism were partially maintained in microRNA-146a knockout mice. Moreover, overexpression of DLST in wild-type mice protected against cardiac hypertrophy and dysfunction in vivo. Conclusions: Altogether we show that the microRNA-146a and its target DLST are important metabolic players in left ventricular dysfunction.

Background— Cardiac hypertrophy and subsequent heart failure triggered by chronic hypertension represent major challenges for cardiovascular research. Beyond neurohormonal and myocyte signaling pathways, growing evidence suggests inflammatory signaling pathways as therapeutically targetable contributors to this process. We recently reported that microRNA-155 is a key mediator of cardiac inflammation and injury in infectious myocarditis. Here, we investigated the impact of microRNA-155 manipulation in hypertensive heart disease. Methods and Results— Genetic loss or pharmacological inhibition of the leukocyte-expressed microRNA-155 in mice markedly reduced cardiac inflammation, hypertrophy, and dysfunction on pressure overload. These alterations were macrophage dependent because in vivo cardiomyocyte-specific microRNA-155 manipulation did not affect cardiac hypertrophy or dysfunction, whereas bone marrow transplantation from wild-type mice into microRNA-155 knockout animals rescued the hypertrophic response of the cardiomyocytes and vice versa. In vitro, media from microRNA-155 knockout macrophages blocked the hypertrophic growth of stimulated cardiomyocytes, confirming that macrophages influence myocyte growth in a microRNA-155 -dependent paracrine manner. These effects were at least partly mediated by the direct microRNA-155 target suppressor of cytokine signaling 1 (Socs1) because Socs1 knockdown in microRNA-155 knockout macrophages largely restored their hypertrophy-stimulating potency. Conclusions— Our findings reveal that microRNA-155 expression in macrophages promotes cardiac inflammation, hypertrophy, and failure in response to pressure overload. These data support the causative significance of inflammatory signaling in hypertrophic heart disease and demonstrate the feasibility of therapeutic microRNA targeting of inflammation in heart failure.

Rationale: 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. Objective: To identify microRNAs involved in viral myocarditis pathogenesis and susceptibility. Methods and Results: 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. Conclusions: 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.

Clinical use of the antineoplastic agent doxorubicin (DOX) is limited by its cardiomyocyte toxicity. Attempts to decrease cardiomyocyte injury showed promising results in vitro, but failed to reduce the adverse effects of DOX in vivo, suggesting that other mechanisms contribute to its cardiotoxicity as well. Evidence that DOX also induces cardiac injury by compromising extracellular matrix integrity is lacking. The matricellular protein thrombospondin-2 (TSP-2) is known for its matrix-preserving function, and for modulating cellular function. Here, we investigated whether TSP-2 modulates the process of doxorubicin-induced cardiomyopathy (DOX-CMP). TSP-2-knockout (TSP-2-KO) and wild-type (WT) mice were treated with DOX (2 mg/kg/week) for 12 weeks to induce DOX-CMP. Mortality was significantly increased in TSP-2-KO compared to WT mice. Surviving DOX-treated TSP-2-KO mice had depressed cardiac function compared to WT animals, accompanied by increased cardiomyocyte apoptosis and matrix damage. Enhanced myocyte damage in the absence of TSP-2 was associated with impaired activation of the Akt signaling pathway in TSP-2-KO compared to WT. The absence of TSP-2, in vivo and in vitro, reduced Akt activation both under non-treated conditions and after DOX. Importantly, inhibition of Akt phosphorylation in cardiomyocytes significantly reduced TSP-2 expression, unveiling a unique feedback loop between Akt and TSP-2. Finally, enhanced matrix disruption in DOX-treated TSP-2-KO hearts went along with increased matrix metalloproteinase-2 levels. Taken together, this study is the first to provide evidence for the implication of the matrix element TSP-2 in protecting against DOX-induced cardiac injury and dysfunction.

Hypertension and/or aging impose stress on the heart with consecutive cardiac hypertrophy, inflammation and fibrosis, processes that lead to heart failure. Our group has previously shown that non-structural matrix proteins such as TSP-2 and SPARC are critical during hypertension, fibrosis, and age-related cardiac dysfunction. Osteoglycin (OGN) is a class III member of the small leucine rich proteoglycans, and was recently identified as a marker of increased left ventricular mass in a study using genomics in recombinant inbred hypertensive rat strains. Its precise biological role in cardiac remodeling, however, remains unclear. We hypothesize that OGN will influence cardiac hypertrophy and fibrosis and hence is critical in the cardiac response during hypertension and aging. We show in young adult mice (3 months old) that hypertension-induced pressure overload of the heart causes two phases of cardiac fibrosis, following 28 days of angiotensin-II (AngII) infusion. The first phase develops between days 3 to 7 after the AngII-treatment and consists of diffuse interstitially deposited loose fibers. During the second wave of fibrosis, which starts at 14 days, these loose fibers transition into more localized scars of denser collagens. Interestingly, the expression of OGN mRNA also shows a biphasic response, significantly decreasing prior to each phase of fibrosis, at day 1 (96% decrease, p=0.002) and day 14 (84% decrease, p=0.004) respectively. Moreover, hypertension causes exaggerated cardiac fibrosis (7.54±1.58 vs. 3.65±0.62 %, p=0.04) and increased heart weight to body weight ratios (6.39±0.31 vs. 5.42±0.17 mg/g, p=0.01) in young adult OGN null mice when compared to WT littermates, but no differences in the amount of infiltrating leukocytes (31.48±9.82 vs. 36.32±18.75 cells/mm2), after 28 days of AngII infusion. In addition, aging results in decreased cardiac function, as measured by ejection fraction, in 18 months versus 3 months old OGN null mice (42.65±2.74 vs. 58.05±1.37 %, p=0.0001), which is absent in WT mice (51.01±3.11 vs. 53.01±1.67 %, p=0.62). Furthermore, a follow-up study in WT and OGN null mice showed a decrease in stroke volume over time in the OGN null mice (39.30±2.90 μl at 6 months, 35.54±1.95 μl at 12 months and 31.13±1.29 μl at 18 months, p=0.04 between 6 and 18 months) but not in the WT mice (41.61±2.02 μl at 6 months, 39.04±1.95 μl at 12 months and 41.04±3.05 μl at 18 months). Collectively, these data suggest that OGN regulates cardiac fibrosis during pressure overload and is therefore important in the remodeling response to hypertension and aging.

Emerging evidence indicates that inflammatory activation is a crucial pathway in disease progression during HF. The co-stimulatory dyad CD40-CD40L is involved in pro-inflammatory activation of a wide range of immune and non-immune cells and plasma levels of soluble CD40L in HF patients are raised. Here we investigated whether CD40-CD40L interactions attenuate cardiac hypertrophy in hypertensive heart disease. CD40 knockout and wild type C57Bl6-J mice were treated with angiotensin II (AngII, 2.5 mg/kg/day) for 4 weeks. AngII-induced hypertension led to a significant increase in cardiac mass in Wt mice (+31%, P

Rationale: Acute viral myocarditis (VM) is one of the leading causes of heart failure in young adults, lacking specific therapy. Osteonection (SPARC) is a non-structural matrix protein modulating cell-matrix interaction in the heart, and has been implicated in cardiac remodeling. Whether SPARC may regulate cardiac inflammation and necrosis in VM through its matri-cellular function is unknown. Methods and results: To address the implication of SPARC in VM, a human coxsackie B3 (CVB3) murine model of VM was performed in SPARC-null and WT animals. SPARC transcript- and protein levels significantly increased by 100% percent 1 week after CVB3 injection compared to control injection. Furthermore, absence of SPARC increased the mortality (4 out of 6 in null vs. 3 out of 7 in WT at 15 days), which was associated with increased cardiac inflammation and necrosis (9% ± 3.03 in KO vs. 1.3% ± 0.24 in WT). Concordantly, Adenoviral SPARC (adSPARC) over-expression, prior to CVB3 injection, improved the survival in both SPARC-null and WT mice, and reduced cardiac inflammation and necrosis by 55 percent in the SPARC null mice. Most importantly, adSPARC over-expression in WT animals resulted in improved cardiac function after viral injury as compared to control vector (fractional shortening was 28,2% ± 0,6 vs. 21,2% ± 0,8 respectively). Intra-vital microscopy of the cremaster muscle allowed us to study leukocyte behavior and permeability in vivo. This demonstrated significantly increased permeability with 450% and decreased leukocyte velocities in SPARC null mice upon TNF-α stimulation, in line with increased leukocyte extravasation. Moreover we performed in vivo measurement of the endothelial glycocalyx, a gel like structure at the luminal side of endothelial cells which is important in endothelial barrier properties. We found glycocalyx quality to be significantly diminished in SPARC null mice. VCAM-1 was further identified as a possible mediator of proper leukocyte recruitment through increased SPARC, since VCAM-1 staining clearly co-localized with SPARC at the endothelial cell level. Conclusion: Here, we demonstrated a protective role of SPARC in VM by improving cardiac function and reducing cardiac inflammation, necrosis and overall mortality. This protective role can be attributed to SPARC by improving endothelial barrier properties and thereby prohibiting exaggerated leukocyte recruitment. Clinical Relevance: As therapy for acute myocarditis is lacking and mainly supportive, we believe SPARC may be a novel target for the treatment of VM.

Background: Viral myocarditis is an important cause of heart failure and sudden cardiac death in young adults. Our previous studies have shown that many microRNAs are differentially expressed in the heart during viral myocarditis, including microRNA-221 and -222. Therefore we aimed at studying the biological role of these miRs during viral myocarditis in mice, induced by human Coxsackie B3 virus. Materials and methods: We measured cardiac microRNA levels during VM with microarrays and qRT-PCR. Wild type C3H mice were intraperitoneally infected with CVB3. Mice were sacrificed at day 4 and day 7 post infection. The hearts, and other organs, were weighted and collected for histological analyses, immunoblotting and qRT-PCR. In vitro experiments were performed in neonatal rat cardiomyocytes (nRCMs). Results: Levels of miR-221 and miR-222 were significantly elevated during acute viral myocarditis in mice, and depressed in biopsies from viral myocarditis patients. In situ hybridization and in vitro experiments showed that both miRs are expressed by cardiomyocytes and cardiac endothelial cells, and are up regulated in neonatal rat cardiomyocytes (nRCMs) in response to enteroviral infection. In vivo inhibition of miR-221 and miRNA-222 by systemically delivered antagomirs (day +1, +3 and +6) aggravated cardiac injury and inflammation at day 7 post-infection, related to a threefold increase of viral loads at day +7 as compared to scrambled antagomiRs. Likewise, inhibition of miR-221 and -222 significantly increased the number of viral particles in nRCMs, whereas overexpression of miR-221 and -222 in nRCMs inhibited enteroviral replication by 30 percent at 48h post-infection. We identified and confirmed a set of conserved miR-221/-222 targets, including ETS1 and ETS2, the chemokine CXCL12, the type I interferon inhibitor IRF2, the T cell development factor TOX and the anti-apoptotic proteins BCL2L11 and BMF. Furthermore, the Coxsackie and adenovirus receptor (CXADR), up regulated in biopsies of patients with dilated cardiomyopathy, is also targeted by miR-221/-222 in mice and rats. De-repression of CXADR upon inhibition of miR-221/-222 may explain the higher viral load observed in vivo and in vitro. Higher CXADR levels contribute to increased viral entry in the cardiomyocyte. Conclusions: Systemic inhibition of miR-221 and miR-222 increases initial cardiac viral loads and resulting inflammation and necrosis during VM in mice. Both targets involved in increased viral replication and enhanced inflammation with inhibition of the miR-221/-222 family are involved. This miR cluster thus represents a master switch in VM.