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Background and Aims: Viral infections are the leading cause of myocarditis. Besides acute cardiac complications, late-stage sequelae such as myocardial fibrosis may develop, importantly impacting the prognosis. Coxsackievirus B3 (CVB)-induced myocarditis in mice is the most commonly used translational model to study viral myocarditis and has provided the majority of our current understanding of the disease pathophysiology. Nevertheless, the late stages of disease, encompassing fibrogenesis and arrhythmogenesis, have been underappreciated in viral myocarditis research to date. The present study investigated the natural history of CVB-induced myocarditis in C57BL/6J mice, expanding the focus beyond the acute phase of disease. In addition, we studied the impact of sex and inoculation dose on the disease course., Methods and Results: C57BL/6J mice (12 weeks old; n=154) received a single intraperitoneal injection with CVB to induce viral myocarditis, or vehicle (PBS) as control. Male mice (n=92) were injected with 5 × 10 5 (regular dose) (RD) or 5 × 10 6 (high dose) (HD) plaque-forming units of CVB, whereas female mice received the RD only. Animals were sacrificed 1, 2, 4, 8, and 11 weeks after CVB or PBS injection. Virally inoculated mice developed viral disease with a temporary decline in general condition and weight loss, which was less pronounced in female animals (P<.001). In male CVB mice, premature mortality occurred between days 8 and 23 after inoculation (RD: 21%, HD: 20%), whereas all female animals survived. Over the course of disease, cardiac inflammation progressively subsided, with faster resolution in female mice. There were no substantial group differences in the composition of the inflammatory cell infiltrates: predominance of cytotoxic T cells at day 7 and 14, and a switch from arginase1-reactive macrophages to iNOS-reactive macrophages from day 7 to 14 were the main findings. There was concomitant development and maturation of different patterns of myocardial fibrosis, with enhanced fibrogenesis in male mice. Virus was almost completely cleared from the heart by day 14. Serum biomarkers of cardiac damage and cardiac expression of remodeling genes were temporarily elevated during the acute phase of disease. Cardiac CTGF gene upregulation was less prolonged in female CVB animals. In vivo electrophysiology studies at weeks 8 and 11 demonstrated that under baseline conditions (i.e. in the absence of proarrhythmogenic drugs), ventricular arrhythmias could only be induced in CVB animals. The cumulative arrhythmia burden throughout the entire stimulation protocol was not significantly different between CVB and control groups., Conclusion: CVB inoculation in C57BL/6J mice represents a model of acute self-limiting viral myocarditis, with progression to different patterns of myocardial fibrosis. Sex, but not inoculation dose, seems to modulate the course of disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Viral myocarditis (VMC) is an inflammatory disease of the myocardium caused by cardioviral infection, especially coxsackievirus B3 (CVB3), and is a major contributor to acute heart failure and sudden cardiac death in children and adolescents. LncRNA MALAT1 knockdown reportedly inhibits the differentiation of Th17 cells to attenuate CVB3-induced VMC in mice. Moreover, long non-coding RNAs (lncRNAs) interact with RNA-binding proteins (RBPs) to regulate UPF1-mediated mRNA decay. However, it remains unclear whether MALAT1 can bind to UPF1 to mediate the mRNA decay of its target genes in VMC. Herein, we aimed to explore the effect of lncRNA MALAT1 on UPF1-mediated SIRT6 mRNA decay in VMC using in vivo and in vitro experiments. CVB3-infected BABL/C mice were used as VMC models, and MALAT1 interfering adenovirus was injected to achieve MALAT1 knockdown. The heart function of the VMC mice was assessed using echocardiography. Pathological changes in myocardial tissues were assessed after hematoxylin-eosin staining. Myocardial injury and inflammation were evaluated by measuring creatine kinase isoenzyme B, cardiac troponin T, interleukin (IL)-1β, and IL-18. TUNEL staining was performed to assess apoptosis in myocardial tissues. In vitro experiments were performed using H9c2 cells after transfection and CVB3 infection. The lactic dehydrogenase release, caspase-1 activity, and IL-1β and IL-18 levels in the cellular supernatant were detected. Western blotting was performed to determine the expression of pyroptosis-related proteins (GSDMD-N, NLRP3, ASC, and Cleaved-Caspase-1) and Wnt/β-catenin signal pathway-related proteins (Wnt1, β-catenin, and p-GSK-3β). RNA immunoprecipitation and RNA stability assays assessed the relationship between MALAT1, UPF1, and SIRT6. CVB3-infected mice and H9c2 cells exhibited elevated MALAT1 and reduced SIRT6 expression. MALAT1 knockdown or SIRT6 overexpression suppressed inflammation and pyroptosis and inhibited the activation of the Wnt/β-catenin signal pathway in myocardial tissues and cells. MALAT1 enhanced the enrichment of SIRT6 mRNA by UPF1 and disturbed the stability of SIRT6 mRNA to promote the development of VMC. MALAT1 can bind UPF1 to mediate SIRT6 mRNA decay and activate the Wnt/β-catenin signal pathway in VMC., Competing Interests: Declarations Conflict of interest The authors declare that they have no competing interests. Ethical Approval and Consent to Participate The experimental design was approved by the Ethics Committee of Hunan Children’s Hospital (Approval no. KYSQ-2022–304). All experiments involving mice were conducted based on the instructions on the Guide for Care and Use of Laboratory Animals issued by the National Institutes of Health. Consent for Publication Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNβ as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis., (© 2023. The Author(s).)

Nonischaemic myocardial fibrosis is associated with cardiac dysfunction, malignant arrhythmias and sudden cardiac death. In the absence of a specific aetiology, its finding as late gadolinium enhancement (LGE) on cardiac magnetic resonance imaging is often attributed to preceding viral myocarditis. Athletes presenting with ventricular arrhythmias often have nonischaemic LGE. Previous studies have demonstrated an adverse effect of exercise on the course of acute viral myocarditis. In this study, we have investigated, for the first time, the impact of endurance training on longer-term outcomes such as myocardial fibrosis and arrhythmogenicity in a murine coxsackievirus B3 (CVB)-induced myocarditis model. Male C57BL/6J mice (n = 72) were randomly assigned to 8 weeks of forced treadmill running (EEX) or no exercise (SED). Myocarditis was induced 2 weeks later by a single intraperitoneal injection with CVB, versus vehicle in the controls (PBS). In a separate study, mice (n = 30) were subjected to pretraining for 13 weeks (preEEX), without continuation of exercise during myocarditis. Overall, continuation of exercise resulted in a milder clinical course of viral disease, with less weight loss and better preserved running capacity. CVB-EEX and preEEX-CVB mice tended to have a lower mortality rate. At sacrifice (i.e. 6 weeks after inoculation), the majority of virus was cleared from the heart. Histological assessment demonstrated prominent myocardial inflammatory infiltration and cardiomyocyte loss in both CVB groups. Inflammatory lesions in the CVB-EEX group contained higher numbers of pro-inflammatory cells (iNOS-reactive macrophages and CD8 + T lymphocytes) compared to these in CVB-SED. Treadmill running during myocarditis increased interstitial fibrosis [82.4% (CVB-EEX) vs. 56.3% (CVB-SED); P = 0.049]. Additionally, perivascular and/or interstitial fibrosis with extensive distribution was more likely to occur with exercise [64.7% and 64.7% (CVB-EEX) vs. 50% and 31.3% (CVB-SED); P = 0.048]. There was a numerical, but not significant, increase in the number of scars per cross-section (1.9 vs. 1.2; P = 0.195), with similar scar distribution and histological appearance in CVB-EEX and CVB-SED. In vivo electrophysiology studies did not induce sustained monomorphic ventricular tachycardia, only nonsustained (usually polymorphic) runs. Their cumulative beat count and duration paralleled the increased fibrosis between CVB-EEX and CVB-SED, but the difference was not significant (P = 0.084 for each). Interestingly, in mice that were subjected to pretraining only without continuation of exercise during myocarditis, no differences between pretrained and sedentary mice were observed at sacrifice (i.e. 6 weeks after inoculation and training cessation) with regard to myocardial inflammation, fibrosis, and ventricular arrhythmogenicity. In conclusion, endurance exercise during viral myocarditis modulates the inflammatory process with more pro-inflammatory cells and enhances perivascular and interstitial fibrosis development. The impact on ventricular arrhythmogenesis requires further exploration., (© 2024. The Author(s).)

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ethnopharmacological Relevance: Jingfang Granules (JFG) originate from the traditional herbal formula Jingfang Baidu powder. It has the effects of inducing sweating and dispelling wind. It is a classic medication used for treating external pathogenic factors and viral diseases. However, the therapeutic mechanism of JFG for viral myocarditis needs further clarification., Aim of the Study: This study aimed to explore the therapeutic efficacy of JFG on coxsackievirus B3-induced viral myocarditis (VMC), along with the elucidation of its underlying mechanisms., Materials and Methods: C57 BL/6JNifdc mice were divided randomly into several groups: control, model, Jingfang Granule groups (0.23, 0.46, and 0.69 g/20g, respectively), and a positive group (oseltamivir, 19.33 mg/kg). Following the establishment of the VMC model, the mice underwent an 8 -week treatment regimen. Pathological alterations in cardiac tissues and inflammatory protein expression were monitored. Differential gene analysis was conducted utilizing transcriptomic techniques. The differential gene mucolipin 1 (Mcoln1) was knocked down by transfection with siRNA in H9C2 cell, and investigative techniques such as immunoblotting, qRT-PCR, immunofluorescence, JC-1 staining, reactive oxygen species (ROS) detection, and mitochondrial stress testing were employed to examine its mechanism of action., Results: JFG significantly mitigates the pathological damage observed in the cardiac tissues of CVB3-induced VMC mice and attenuates the expression of inflammatory genes. Subsequently, differentially expressed genes are identified through transcriptomic analysis and validated via PCR. Among these, the upregulation of Mcoln1 promotes autophagy, facilitating the clearance of damaged mitochondria and excessive ROS. This has been substantiated through in vitro experiments. Excessive ROS precipitates a reduction in mitochondrial membrane potential, instigating cell apoptosis. In accordance with TUNEL staining results, JFG acts to inhibit cell apoptosis. To ascertain whether Mcoln1 is a crucial target for JFG in treating VMC, Mcoln1 was suppressed in H9C2 cells. The suppression of Mcoln1 hinders the elevation in autophagy levels post-JFG treatment, obstructs the enhancement of mitochondrial function, and impedes the clearance of ROS. Furthermore, the inhibitory effect of JFG on cell apoptosis is attenuated., Conclusion: The research findings indicate that JFG has a protective effect on CVB3-induced H9C2 cell injury. JFG may exert its effects in VMC treatment by enhancing autophagy to suppress cell apoptosis through the mitochondrial pathway, thereby counteracting cell damage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Viral myocarditis is an inflammatory disease of the myocardium, often leads to cardiac dysfunction and death. PARKIN (PRKN) and PINK1, well known as Parkinson's disease-associated genes, have been reported to be involved in innate immunity and mitochondrial damage control. Therefore, we investigated the role of parkin and PINK1 in coxsackievirus B3 (CVB3)-induced viral myocarditis because the etiology of myocarditis is related to abnormal immune response to viral infection and mitochondrial damage. After viral infection, the survival was significantly lower and myocardial damage was more severe in parkin knockout (KO) and PINK1 KO mice compared to wild-type (WT) mice. Parkin KO and PINK1 KO showed defective immune cell recruitment and impaired production of antiviral cytokines such as interferon-gamma, allowing increased viral replication. In addition, parkin KO and PINK1 KO mice were more susceptible to CVB3-induced mitochondrial damage than WT mice, resulting in susceptibility to viral-induced cardiac damage. Finally, using publicly available RNA-seq data, we found that pathogenic mutants of the PRKN gene are more common in patients with dilated cardiomyopathy and myocarditis than in controls or the general population. This study will help elucidate the molecular mechanism of CVB3-induced viral myocarditis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Increasing evidence has shown that extracellular vesicles or exosomes released from virus-infected cells contain viral particles, genomes, or other pathogenic factors that move to neighbor cells, contributing to virus dissemination and productive infection. Our recent study demonstrated that exosomes carrying CVB3 virions exhibited greater infection efficiency than free virions because they accessed various entry routes, overcoming restrictions to viral tropism. However, the pathogenicity of exosomes carried CVB3 and their effect on immunological properties have not yet been completely explained. In the current study, we sought to explore whether exosomes exert their effect on the CVB3-induced pathogenesis or evade the immune attack. Our results showed that exosomes-carried CVB3 could effectively infect viral receptor-negative immune cells in vivo, resulting in inducing immune system loss. Importantly, the exosomes-carried CVB3 had the ability to escape the neutralizing antibodies activity resulting in inducing the severe onset of myocarditis. Using the genetically engineered mouse with deficiency of exosomes, we observed that the exosomes-carried CVB3 reinforced an aggravated pathogenesis. By understanding how exosomes promote the course of viral disease, clinical applications of exosomes can be developed., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2023 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

The coxsackievirus and adenovirus receptor (CAR) is very well known as an epithelial tight junction and cardiac intercalated disc protein; it mediates attachment and infection via the coxsackievirus B3 (CVB3) and type 5 adenovirus. Macrophages play important roles in early immunity during viral infections. However, the role of CAR in macrophages is not well studied in relation to CVB3 infection. In this study, the function of CAR was observed in the Raw264.7 mouse macrophage cell line. CAR expression was stimulated by treatment with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α). In thioglycollate-induced peritonitis, the peritoneal macrophage was activated and CAR expression was increased. The macrophage-specific CAR conditional knockout mice (KO) were generated from lysozyme Cre mice. The expression of inflammatory cytokine (IL-1β and TNF-α) was attenuated in the KO mice's peritoneal macrophage after LPS treatment. In addition, the virus was not replicated in CAR-deleted macrophages. The organ virus replication was not significantly different in both wild-type (WT) and KO mice at days three and seven post-infection (p.i). However, the inflammatory M1 polarity genes (IL-1β, IL-6, TNF-α and MCP-1) were significantly increased, with increased rates of myocarditis in the heart of KO mice compared to those of WT mice. In contrast, type1 interferon (IFN-α and β) was significantly decreased in the heart of KO mice. Serum chemokine CXCL-11 was increased in the KO mice at day three p.i. compared to the WT mice. The attenuation of IFN-α and β in macrophage CAR deletion induced higher levels of CXCL-11 and more increased CD4 and CD8 T cells in KO mice hearts compared to those of WT mice at day seven p.i. These results demonstrate that macrophage-specific CAR deletion increased the macrophage M1 polarity and myocarditis in CVB3 infection. In addition, chemokine CXCL-11 expression was increased, and stimulated CD4 and CD8 T cell activity. Macrophage CAR may be important for the regulation of innate-immunity-induced local inflammation in CVB3 infection.

Background: Different serotypes of coxsackievirus B (CVB), which is the most common cause of viral myocarditis, target cardiomyocytes through Coxsackie and Adenovirus Receptor and Decay-Accelerating Factor. Both receptors are expressed in the fetal heart. We hypothesized that infection with different serotypes of CVB during early pregnancy plays a role in pathogenesis of congenital heart defect (CHD)., Methods: In this study, we use a murine model to infect with CVB1, CVB4, and combination of CVB3 + CVB4 during a critical period in gestation. We examined offspring of pregnant mice for fetal death and heart defects following viral infection., Result: Fetuses from uninfected control dams showed normal heart development, while maternal CVB infection precipitates CHD: majorly ventricular septal defects (VSD) and non-compaction of ventricular myocardium (NC), with some infrequent cases of double outlet right ventricle, left ventricle wall rupture, right ventricle hypertrophy, and thickened/dysplastic semilunar valves. Infection of pregnant dams with CVB1 leads to 44% VSD and 41.2% NC cases, while with CVB4 leads to 31.7% VSD and 13.3% NC cases. Co-infection with CVB3 + CVB4 increases fetal pathology to 51.3% VSD and 41% NC cases. Infection can also result in fetal death, with higher incidences with CVB3 + CVB4 with 46.2% cases, compared to 33.3% by CVB1 and 21.7% by CVB4. Male fetuses were more susceptible to all phenotypes., Conclusion: Our report shows that prenatal CVB infections can lead to pathogenesis of certain heart defects in mouse model, particularly exacerbated with co-infections. This data confirms a link between prenatal CVB infection and CHD development and highlights it is not unique to just one serotype of CVB., (© 2022 Wiley Periodicals LLC.)

Background: Myocardial inflammation and apoptosis are key processes in coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). Accumulating evidence reveals the essential roles of long noncoding RNAs (lncRNAs) in the pathogenesis of AVMC. Here, we aimed to evaluate the biological functions of a novel lncRNA guanylate-binding protein 9 (lncGBP9) in AVMC progression and further explore its underlying mechanisms., Methods: Initially, mouse models of AVMC were constructed by CVB3 infection. The expression and localization of lncGBP9 in heart tissues were analyzed using RT-qPCR and FISH. Adeno-associated virus serotype 9 (AAV9)-mediated lncGBP9 knockdown was then employed to clarify its roles in survival, cardiac function, and myocardial inflammation and apoptosis. Moreover, the mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) were detected by RT-qPCR and ELISA, and the regulation of lncGBP9 knockdown on the NF-κB signaling pathway was investigated by Western blotting. Using an in vitro model of HL-1 cardiomyocytes exposed to CVB3 infection, the effects of lncGBP9 knockdown on cell viability, inflammation, and apoptosis were further evaluated in vitro., Results: Increased lncGBP9 expression was detected in the heart tissues of AVMC mice and CVB3-infected HL-1 cells, and was mainly located in the cytoplasm. Knockdown of lncGBP9 remarkably alleviated the severity of AVMC in CVB3-infected mice, as verified by improved cardiac function, and reduced myocardial inflammation and apoptosis. Additionally, lncGBP9 knockdown suppressed the NF-κB signaling pathway and consequently reduced productions of pro-inflammatory cytokines in vivo. In vitro functional assays further confirmed that lncGBP9 knockdown promoted cell viability, inhibited cell apoptosis, and reduced pro-inflammatory cytokines release in CVB3-infected HL-1 cells through suppressing NF-κB activation., Conclusions: Collectively, lncGBP9 knockdown exerts anti-inflammatory and anti-apoptotic effects in CVB3-induced AVMC, which may be mediated in part via NF-κB signaling pathway. These findings highlight lncGBP9 as an attractive target for therapeutic interventions., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Viral myocarditis (VMC), which is defined as inflammation of the myocardium with consequent myocardial injury, may develop chronic disease eventually leading to dilated cardiomyopathy (DCM). Molecular mechanisms underlying the progression from acute VMC (aVMC), to chronic VMC (cVMC) and finally to DCM, are still unclear. Here, we established mouse models of VMC and DCM with Coxsackievirus B3 infection and conducted NMR-based metabolomic analysis of aqueous metabolites extracted from cardiac tissues of three histologically classified groups including aVMC, cVMC and DCM. We showed that these three pathological groups were metabolically distinct from their normal counterparts and identified three impaired metabolic pathways shared by these pathological groups relative to normal controls, including nicotinate and nicotinamide metabolism; alanine, aspartate and glutamate metabolism; and D-glutamine and D-glutamate metabolism. We also identified two extra impaired metabolic pathways in the aVMC group, including glycine, serine and threonine metabolism; and taurine and hypotaurine metabolism Furthermore, we identified potential cardiac biomarkers for metabolically distinguishing these three pathological stages from normal controls. Our results indicate that the metabolomic analysis of cardiac tissues can provide valuable insights into the molecular mechanisms underlying the progression from acute VMC to DCM.

Atrial dysfunction is a relatively common complication of acute myocarditis, although its pathophysiology is unclear. There is limited information on myocarditis-associated histological changes in the atria and how they develop in time. The aim of this study therefore was to investigate inflammation, fibrosis and viral genome in the atria in time after mild CVB3-induced viral myocarditis (VM) in mice. C3H mice (n = 68) were infected with 10 5 PFU of Coxsackievirus B3 (CVB3) and were compared with uninfected mice (n = 10). Atrial tissue was obtained at days 4, 7, 10, 21, 35 or 49 post-infection. Cellular infiltration of CD45+ lymphocytes, MAC3+ macrophages, Ly6G+ neutrophils and mast cells was quantified by (immuno)histochemical staining. The CVB3 RNA was determined by in situ hybridization, and fibrosis was evaluated by elastic van Gieson (EvG) staining. In the atria of VM mice, the numbers of lymphocytes on days 4 and 7 (p < .05) and days 10 (p < .01); macrophages on days 7 (p < .01) and 10 (p < .05); neutrophils on days 4 (p < .05); and mast cells on days 4 and 7 (p < .05) increased significantly compared with control mice and decreased thereafter to basal levels. No cardiomyocyte death was observed, and the CVB3 genome was detected in only one infected mouse on Day 4 post-infection. No significant changes in the amount of atrial fibrosis were found between VM and control mice. A temporary increase in inflammation is induced in the atria in the acute phase of CVB3-induced mild VM, which may facilitate the development of atrial arrhythmia and contractile dysfunction., (© 2022 The Authors. International Journal of Experimental Pathology published by John Wiley & Sons Ltd on behalf of Company of the International Journal of Experimental Pathology (CIJEP).)

Cardiomyocyte apoptosis is critical for the development of viral myocarditis (VMC), which is one of the leading causes of cardiac sudden death in young adults. Our previous studies have demonstrated that elevated calpain activity is involved in the pathogenesis of VMC. This study aimed to further explore the underlying mechanisms. Neonatal rat cardiomyocytes (NRCMs) and transgenic mice overexpressing calpastatin were infected with coxsackievirus B3 (CVB3) to establish a VMC model. Apoptosis was detected with flow cytometry, TUNEL staining, and western blotting. Cardiac function was measured using echocardiography. Mitochondrial function was measured using ATP assays, JC-1, and MitoSOX. Mitochondrial morphology was observed using MitoTracker staining and transmission electron microscopy. Colocalization of dynamin-related protein 1 (Drp-1) in mitochondria was examined using immunofluorescence. Phosphorylation levels of Drp-1 at Ser637 site were determined using western blotting analysis. We found that CVB3 infection impaired mitochondrial function as evidenced by increased mitochondrial ROS production, decreased ATP production and mitochondrial membrane potential, induced myocardial apoptosis and damage, and decreased myocardial function. These effects of CVB3 infection were attenuated by inhibition of calpain both by PD150606 treatment and calpastatin overexpression. Furthermore, CVB3-induced mitochondrial dysfunction was associated with the accumulation of Drp-1 in the outer membrane of mitochondria and subsequent increase in mitochondrial fission. Mechanistically, calpain cleaved and activated calcineurin A, which dephosphorylated Drp-1 at Ser637 site and promoted its accumulation in the mitochondria, leading to mitochondrial fission and dysfunction. In summary, calpain inhibition attenuated CVB3-induced myocarditis by reducing mitochondrial fission, thereby inhibiting cardiomyocyte apoptosis. Calpain is activated by CVB3 infection. Activated calpain cleaves calcineurin A and converts it to active form which could dephosphorylate Drp-1 at Ser637 site. Then, the active Drp-1 translocates from the cytoplasm to mitochondria and triggers excessive mitochondrial fission. Eventually, the balance of mitochondrial dynamics is broken, and apoptosis occurs., (© 2021. The Author(s).)

Viral myocarditis (VMC) is an inflammatory heart condition which can induce dilated cardiomyopathy (DCM). However, molecular mechanisms underlying the progression of VMC into DCM remain exclusive. Here, we established mouse models of VMC and DCM by infecting male BALB/c mice with Coxsackievirus B3 (CVB3), and performed NMR-based metabonomic analyses of mouse sera. The mouse models covered three pathological stages including: acute VMC (aVMC), chronic VMC (cVMC) and DCM. We recorded 1 D 1 H-NMR spectra on serum samples and conducted multivariate statistical analysis on the NMR data. We found that metabolic profiles of these three pathological stages were distinct from their normal controls (CON), and identified significant metabolites primarily responsible for the metabolic distinctions. We identified significantly disturbed metabolic pathways in the aVMC, cVMC and DCM stages relative to CON, including: taurine and hypotaurine metabolism; pyruvate metabolism; glycine, serine and threonine metabolism; glycerolipid metabolism. Additionally, we identified potential biomarkers for discriminating a VMC, cVMC and DCM from CON including: taurine, valine and acetate for aVMC; glycerol, valine and leucine for cVMC; citrate, glycine and isoleucine for DCM. This work lays the basis for mechanistically understanding the progression from acute VMC to DCM, and is beneficial to exploitation of potential biomarkers for prognosis and diagnosis of heart diseases.

Viral diseases are a major threat to modern society and the global health system. It is therefore of utter relevance to understand the way viruses affect the host as a basis to find new treatment solutions. The understanding of viral myocarditis (VMC) is incomplete and effective treatment options are lacking. This review will discuss the mechanism, effects, and treatment options of the most frequent myocarditis-causing viruses namely enteroviruses such as Coxsackievirus B3 (CVB3) and Parvovirus B19 (PVB19) on the human heart. Thereby, we focus on: 1. Viral entry: CVB3 use Coxsackievirus-Adenovirus-Receptor (CAR) and Decay Accelerating Factor (DAF) to enter cardiac myocytes while PVB19 use the receptor globoside (Gb4) to enter cardiac endothelial cells. 2. Immune system responses: The innate immune system mediated by activated cardiac toll-like receptors (TLRs) worsen inflammation in CVB3-infected mouse hearts. Different types of cells of the adaptive immune system are recruited to the site of inflammation that have either protective or adverse effects during VMC. 3. Autophagy: CVB3 evades autophagosomal degradation and misuses the autophasomal pathway for viral replication and release. 4. Viral replication sites: CVB3 promotes the formation of double membrane vesicles (DMVs), which it uses as replication sites. PVB19 uses the host cell nucleus as the replication site and uses the host cell DNA replication system. 5. Cell cycle manipulation: CVB3 attenuates the cell cycle at the G1/S phase, which promotes viral transcription and replication. PVB19 exerts cell cycle arrest in the S phase using its viral endonuclease activity. 6. Regulation of apoptosis: Enteroviruses prevent apoptosis during early stages of infection and promote cell death during later stages by using the viral proteases 2A and 3C, and viroporin 2B. PVB19 promotes apoptosis using the non-structural proteins NS1 and the 11 kDa protein. 7. Energy metabolism: Dysregulation of respiratory chain complex expression, activity and ROS production may be altered in CVB3- and PVB19-mediated myocarditis. 8. Ion channel modulation: CVB3-expression was indicated to alter calcium and potassium currents in Xenopus laevis oocytes and rodent cardiomyocytes. The phospholipase 2-like activity of PVB19 may alter several calcium, potassium and sodium channels. By understanding the general pathophysiological mechanisms of well-studied myocarditis-linked viruses, we might be provided with a guideline to handle other less-studied human viruses., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Viral myocarditis (VMC) is a cardiac disease associated with myocardial inflammation and injury induced by virus infection. Cardiomyocytes have recently been regarded as key players in eliciting and modulating inflammation within the myocardium. Kruppel-like factor 10 (KLF10) is a crucial regulator of various pathological processes and plays different roles in a variety of diseases. However, its role in VMC induced by coxsackievirus B3 (CVB3) infection remains unknown. In this study, we report that cardiac KLF10 confers enhanced protection against viral myocarditis. We found that KLF10 expression was downregulated upon CVB3 infection. KLF10 deficiency enhanced cardiac viral replication and aggravated VMC progress. Bone marrow chimera experiments indicated that KLF10 expression in nonhematopoietic cells was involved in the pathogenesis of VMC. We further identified MCP-1 as a novel target of KLF10 in cardiomyocytes, and KLF10 cooperated with histone deacetylase 1 (HDAC1) to negatively regulate MCP-1 expression by binding its promoter, leading to activation of MCP-1 transcription and recruitment of Ly6C high monocytes/macrophages into the myocardium. This novel mechanism of MCP-1 regulation by KLF10 might provide new insights into the pathogenesis of VMC and a potential therapeutic target for VMC., (© 2020. CSI and USTC.)

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 3'UTR 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., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2019. For permissions, please email: journals.permissions@oup.com.)

Viral myocarditis (VMC) is a widely studied but poorly understood inflammatory cardiomyopathy which mainly affects children and young adults and results in adverse outcomes. Cardiomyocyte apoptosis was reported important in the progress of coxsackievirus B3 (CVB3)-induced VMC and the blocking of this process may contribute to the therapeutic effect towards VMC. Therefore, this study was designed to explore whether survivin, one of the strongest antiapoptotic proteins, can protect cardiomyocytes from apoptosis in VMC and to discover its related mechanisms. Here, the cultured neonatal mouse cardiomyocytes (NMCs) were exposed to CVB3 to establish the cell model of VMC and the results of Western Blot showed that the protein expression of survivin in CVB3-infected NMCs varied at different post-infection time. Lentivirus was next used to examine the function of survivin in CVB3-infected NMCs. TUNEL assay demonstrated that the overexpression of survivin interrupted CVB3-induced apoptosis. It was next examined whether caspase-3 and -9 were involved in the antiapoptotic pathway initiated by survivin via Western Blot. The results showed a reverse relationship between the protein expression of survivin and that of cleaved caspase-3 and cleaved caspase-9, suggesting that survivin may attenuate apoptosis through restraining the activity of caspase-3 and -9. Moreover, the supernatant fluid of cultured NMCs was extracted to detect the quantitation of released lactate dehydrogenase (LDH) and a sharp decrease was discovered in the survivin-overexpressed group compared to the CVB3-infected group, indicating a protective role of survivin in the cell model of CVB3-induced myocarditis. This study demonstrated that survivin was triggered by CVB3 infection in NMCs and survivin executed its antiapoptotic effects via caspase-3- and caspase-9-dependent signaling pathway.

Calpains are calcium-activated neutral cysteine proteases. The dysregulation of calpain activity has been found to be related to cardiovascular diseases, for which calpain inhibition is used as a treatment. Viral myocarditis (VMC) is primarily caused by Coxsackievirus group B3 virus infection (CVB3). CVB3 virus infection induces autophagy and hijacks this process to facilitate its replication. In this study, we found that calpain was significantly activated in hearts affected by VMC. However, pharmacologically inhibiting calpain aggravated VMC symptoms in mice due to myocardial inflammation and cardiac dysfunction. The inhibition of calpain activity in vitro led to the accumulation of LC3-II and increased levels of p62/SQSTM1 protein expression, suggesting that autophagic flux was impaired by calpain inhibition. These effects of calpain inhibition were also observed in capn4-specific myocardial knockout mice in vivo. Furthermore, our results provided evidence that calpain inhibition in VMC, unlike other cardiovascular diseases, exacerbated the disease symptom by impairing CVB3-induced autophagic flux, which may subsequently reduce virus autolysosome degradation. Our findings indicated that calpain inhibition may not be a good treatment for VMC disease in a clinical setting., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: Coxsackie-B-viruses (CVB) are frequent causes of acute myocarditis and dilated cardiomyopathy, but an effective antiviral therapy is still not available. Previously, we and others have demonstrated that treatment with an engineered sCAR-Fc (soluble coxsackievirus-adenovirus receptor fused to the carboxyl-terminus of human IgG) efficiently neutralizes CVB3 and inhibits the development of cardiac dysfunction in mice with acute CVB3-induced myocarditis. In this study, we analyzed the potential of sCAR-Fc for treatment of chronic CVB3-induced myocarditis in an outbred NMRI mouse model., Methods: NMRI mice were infected with the CVB3 strain 31-1-93 and treated with a sCAR-Fc expressing adeno-associated virus 9 vector 1, 3, and 7 days after CVB3 infection. Chronic myocarditis was analyzed on day 28 after infection., Results: Initial investigations showed that NMRI mice develop pronounced chronic myocarditis between day 18 and day 28 after infection with the CVB3 strain 31-1-93. Chronic cardiac infection was characterized by inflammation and fibrosis as well as persistence of viral genomes in the heart tissue and by cardiac dysfunction. Treatment of NMRI mice resulted in a distinct reduction of cardiac inflammation and fibrosis and almost complete elimination of virus RNA from the heart by day 28 after infection. Moreover, hemodynamic measurement revealed improved cardiac contractility and diastolic relaxation in treated mice compared with mice treated with a control vector (mean±SD; maximal pressure, 81.9±9.2 versus 69.4±8.6 mm Hg, P =0.02; left ventricular ejection fraction, 68.9±8.5 versus 54.2±11.5%, P =0.02; dP/dt max , 7275.2±1674 versus 4432.6±1107 mm Hg/s, P =0.004; dP/dt min , -4046.9±776 versus -3146.3±642 mm Hg/s, P =0.046). The therapeutic potential of sCAR-Fc is limited, however, since postponed start of sCAR-Fc treatment either 3 or 7 days after infection could not attenuate myocardial injury., Conclusions: Early therapeutic employment of sCAR-Fc, initiated at the beginning of the primary viremia, inhibits the development of chronic CVB3-induced myocarditis and improves the cardiac function to a level equivalent to that of uninfected animals.

Viral myocarditis is potentially fatal and lacking a specific treatment. Exosomes secreted by cardiac progenitor cells (CPCs) have emerged as a promising tool for cardioprotection and repair. In this study, we investigated whether CPCs-derived exosomes (CPCs-Ex) could utilize the mTOR signal pathway to reduce the apoptosis in viral myocarditis. In vitro, exosomes were, respectively, added to H9C2 cells after CVB3 infection to detect the anti-apoptosis effect of CPCs-Ex. Compared with the controls, the apoptosis rate was reduced, accompanied with the depressed expression of viral capsid protein 1 (VP1) and pro-apoptosis factors of Bim/caspase families. Meanwhile, the phosphorylation of Akt, mTOR, and p70S6K were promoted, but that of 4EBP1 was suppressed. In vivo, the results of apoptosis, expression of CVB3 and pro-apoptosis factors, and phosphorylation of Akt/mTOR factors of CVB3-infected cardiomyocytes were consistent with that of vitro. Following that, we use Rapamycin and MK-2206 to inhibit the Akt/mTOR signaling pathway, meanwhile, Rattus 4EBP1, p70S6K, Akt1 and Akt2 were transfected to H9C2 cells to establish the stably transfected cell lines. In the group with Rapamycin or MK-2206 pretreatment, CPCs-Ex also could decrease the apoptosis of H9C2 cells and expression of CVB3 mRNA, followed by decreased expression of apoptosis factors. In Akt2, p70S6K and 4EBP1 overexpression groups, CPCs-Ex promoted CVB3-induced apoptosis, VP1 expression and cleavage of caspase-3. Our results therefore identify CPCs-Ex exerts an anti-apoptosis effect in CVB3-infected cells by abrogating the proliferation of CVB3 and modulating the mTOR signaling pathways as well as the expression of Bcl-2 and caspase families. Viral myocarditis, mainly caused by CVB3 infection, is lacking a specific treatment. Our study identified an anti-apoptosis role of CPCs-Ex in CVB3-infected cells and rats, which shown that CPCs-Ex may be an effective tool to treat viral myocarditis. We believe that with more in-depth research on the functionality of CPCs-Ex, there will be a breakthrough in the treatment of viral myocarditis.

Fingolimod (FTY720) after phosphorylation, as the ligand of sphingosine 1-phosphate receptors (S1PRs), plays an important role in cell proliferation and differentiation. In this article, FTY720 in the treatment of coxsackievirus B3 (CVB3)-induced viral myocarditis was closely related to apoptosis and AKT/caspase-3 apoptotic pathways. We found that CVB3 inhibited myocardial apoptosis at the early stage with upregulating p-AKT level and downregulating activated caspase-3 level for replication of virus progeny, whereas it promoted apoptosis at a late stage with downregulating p-AKT and upregulating activated caspase-3 for releasing the newly synthesized virus to spread. Interestingly, FTY720 could reverse this trend; it promoted apoptosis at an early stage and inhibited apoptosis at the late stage in vivo and vitro, which proved the antiviral effect. We also found that S1PR1, S1PR4, and S1PR5, rather than S1PR2 and S1PR3, were regulated by FTY720 in this process. The results confirmed that FTY720 alleviates CVB3-induced myocarditis and inhibits viral replication through regulating S1PRs and AKT/caspase-3 pathways with a bidirectional regulation of apoptosis., (© 2019 Wiley Periodicals, Inc.)

Myocarditis, inflammation of the heart muscle, affects all demographics and is a major cause of sudden and unexpected death in young people. It is most commonly caused by viral infections of the heart, with coxsackievirus B3 (CVB3) being among the most prevalent pathogens. To understand the molecular pathogenesis of CVB3 infection and provide strategies for developing treatments, we examined the role of a key nuclear pore protein 98 (NUP98) in the setting of viral myocarditis. NUP98 was cleaved as early as 2 h post-CVB3 infection. This cleavage was further verified through both the ectopic expression of viral proteases and in vitro using purified recombinant CVB3 proteases (2A and 3C), which demonstrated that CVB3 2A but not 3C is responsible for this cleavage. By immunostaining and confocal imaging, we observed that cleavage resulted in the redistribution of NUP98 to punctate structures in the cytoplasm. Targeted siRNA knockdown of NUP98 during infection further increased viral protein expression and viral titer, and reduced cell viability, suggesting a potential antiviral role of NUP98. Moreover, we discovered that expression levels of neuregulin-1 (NRG1), a cardioprotective gene, and presenilin-1 (PSEN1), a cellular protease processing the tyrosine kinase receptor ERBB4 of NRG1, were reliant upon NUP98 and were downregulated during CVB3 infection. In addition, expression of these NUP98 target genes in myocardium tissue not only occurred at an earlier phase of infection, but also appeared in areas away from the initial inflammatory regions. Collectively, CVB3-induced cleavage of NUP98 and subsequent impairment of the cardioprotective NRG1-ERBB4/PSEN1 signaling cascade may contribute to increased myocardial damage in the context of CVB3-induced myocarditis. To our knowledge, this is the first study to demonstrate the link between NUP98 and the NRG1 signaling pathway in viral myocarditis.

The role of B cells in viral myocarditis (VMC) remains controversial. In order to establish a role and mechanism of action for B cells in acute VMC, we established an acute VMC mouse model by intraperitoneal injection of Coxsackie virus group B type 3 (CVB3). At day 7, mice were analyzed using myocardial histopathology, and the presence of M2 macrophages in spleen and heart. Mice were divided into four groups, all having a C57BL/6 background: control group; wild-type (WT) VMC; mMt/mMt (-/-) VMC (BKO), and BKO + B cell VMC. A role for B cells was demonstrated by a significant reduction in myocardial pathological score and an increase in the frequency of M2 macrophages in the BKO group, when compared to the WT group. Once BKO mice underwent B cell reconstitution with isolated WT B cells, the myocardial pathological score was increased significantly, while the frequency of M2 macrophages decrease. Our findings demonstrate that B cells increase myocardial inflammation by suppressing M2 polarization in acute VMC in vivo.

Latency associated peptide (LAP) is a protein expressed on the membrane of some regulatory T cells (Treg). LAP + Treg have a greater immunomodulatory effect than that of their negative counterparts. In this study, we presented the data on the proportion of LAP + Treg out of CD4 + cells in mice with viral myocarditis, which we believed was more sensitive and specific than that of the ratio of total Treg in CD4 + cells. Comparing with the previously recognized total Treg, LAP + Treg was a better biomarker on myocardial inflammation., (© 2018 Wiley Periodicals, Inc.)

Overwhelming cardiac inflammation has been reported to be the pathogenic mechanism of Coxsackievirus B3 (CVB3)-induced viral myocarditis (VMC), while the detailed molecular mechanisms remain unknown. Membrane-bound dipeptidases (MBD, also known as Dpep) have been shown to be involved in inflammatory diseases. However, the clear and direct evidence of their impacts on inflammation is still lacking. In this study, our results revealed that Dpep2 expression was remarkably increased during CVB3 infection, and primarily produced by the cardiac tissue-infiltrating macrophages instead of constitutive cardiomyocytes. Macrophages have been reported to play an important pathological role in driving VMC. Interestingly, macrophage-specific Dpep2 deletion robustly aggravated CVB3-induced cardiac inflammation, evidenced by augmented expression of TNF-α, IL-6, and MCP-1 in heart tissue. In addition, Dpep2-deficient bone-marrow derived macrophages (BMDMs) generated more TNF-α, IL-6, and MCP-1 after CVB3 stimulation compared with the control BMDMs. Moreover, this suppressive effect of Dpep2 on macrophages relied on its repression on NF-κB signaling pathway, but not on its conventional hydrolysate LTE4. Taken together, this study revealed that Dpep2 could protect against CVB3-induced VMC by acting as a suppressor of macrophage inflammation. Better understanding how macrophage Dpep2 dampened the cardiac inflammation would provide us with insights for the efficient control of CVB3-induced VMC.

Coxsackieviruses of group B (CVB) are well-known causes of acute and chronic myocarditis. Chronic myocarditis can evolve into dilated cardiomyopathy (DCM) characterized by fibrosis and cardiac remodeling. Interleukin-1β (IL-1β) plays a decisive role in the induction of the inflammatory response as a consequence of viral replication. In this study, we analyzed the effects of IL-1β neutralization on the transition of acute to chronic myocarditis in a mouse model of CVB3 myocarditis. Mice were treated with an anti-murine IL-1β antibody as a surrogate for Canakinumab at different time points post CVB3 infection. Treatment was performed in the early phase (day 1-14 pi, day 3-14 pi) or at a later stage of myocarditis (day 14-28 pi). Subsequently, the hearts were examined histologically, immunohistochemically and by molecular biology. A significant reduction of viral replication, cardiac damage and inflammation was found after administration of the antibody in the early phase and in the later phase of infection. Furthermore, less collagen I deposition and a considerable reduction of fibrosis were found in antibody-treated mice. Using microarray analysis, a significant upregulation of various extracellular matrix and fibrosis-associated molecules was found in CVB3-infected mice, including TGF-β, TIMP-1 and MMP12, as well as diverse matricellular proteins, whereas, these molecules were significantly downregulated in all IL-1β antibody-treated infected mice. Neutralization of IL-1β at different stages of enteroviral infection prevents the development of chronic viral myocarditis by reducing inflammation, interstitial fibrosis and adverse cardiac remodeling. These findings are relevant for the treatment of patients with acute and chronic myocarditis.

Adenosine deaminases acting on RNA (ADAR) are enzymes that regulate RNA metabolism through post-transcriptional mechanisms. ADAR1 is involved in a variety of pathological conditions including inflammation, cancer, and the host defense against viral infections. However, the role of ADAR1p150 in vascular disease remains unclear. In this study, we examined the expression of ADAR1p150 and its role in viral myocarditis (VMC) in a mouse model. VMC mouse cardiomyocytes showed significantly higher expression of ADAR1p150 compared to the control samples. Coimmunoprecipitation verified that ADAR1p150 forms a complex with Dicer in VMC. miRNA-222, which is involved in many cardiac diseases, is highly expressed in cardiomyocytes in VMC. In addition, the expression of miRNA-222 was promoted by ADAR1p150/Dicer. Among the target genes of miRNA-222, the expression of phosphatase-and-tensin (PTEN) protein was significantly reduced in VMC. By using a bioinformatics tool, we found a potential binding site of miRNA-222 on the PTEN gene's 3'-UTR, suggesting that miRNA-222 might play a regulatory role. In cultured cells, miR-222 suppressed PTEN expression. Our findings suggest that ADAR1p150 plays a key role in complexing with Dicer and promoting the expression of miRNA-222, the latter of which suppresses the expression of the target gene PTEN during VMC. Our work reveals a previously unknown role of ADAR1p150 in gene expression in VMC.

Aims: To investigate whether there exists a cardio-protective effect of Fasudil, a selective Rho kinase (ROCK) inhibitor, in an experimental murine model of acute viral myocarditis., Methods: Male BALB/c mice were randomly assigned to three groups: control, myocarditis treated with placebo and myocarditis treated with Fasudil (n = 40 animals per group). Myocarditis was established by intraperitoneal injection with coxsackievirus B3 (CVB3). Twenty-four hours after infection, Fasudil was intraperitoneally administered for 14 consecutive days. Twenty mice were randomly selected from each group to monitor a 14-day survival rate. On day 7 and day 14, eight surviving mice from each group were sacrificed and their hearts and blood were obtained to perform serological and histological examinations. Expression of ROCKs, IL-17, IL-1b, TNFα, RORgt, and Foxp3 were quantified with RT-PCR. Plasma levels of TNF alpha, IL-1 beta, and IL-17 were measured by ELISA. In addition, protein levels of IL-17 and ROCK2 in cardiac tissues were analyzed with Western blot., Results: Fasudil treatment significantly increased survival, attenuated myocardial necrotic lesions, reduced CVB3 replication and expression of ROCK2 and IL-17 in the infected hearts. This treatment also imposed a T-cell subpopulation shift, from Th17 to Treg, in cardiac tissues., Conclusions: ROCK pathway inhibition was cardio-protective in viral myocarditis with increased survival, decreased viral replication, and inflammatory response. These findings suggest that Fasudil might be a potential therapeutic agent for patients with viral myocarditis., (© 2018 John Wiley & Sons Ltd.)

Acute viral myocarditis (AVMC) is typically caused by cardiotropic viral infection. There is a paucity of specific treatment options available with proven efficacy. Chinese patented pharmaceutical product Shenfu injection (SFI) has potent efficacy on treating AVMC in clinical practice. However, the molecular mechanism is still unknown. We employed cross-platform metabolomics combined with computational systems analysis, based on reversed-phase liquid chromatography-mass spectrometry (RPLC-MS), hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) and gas chromatography-mass spectrometry (GC-MS), to deciphering the targeted metabolic pathways of SFI against AVMC induced by coxsackievirus B3 (CVB3). Quantitative real-time PCR (qRT-PCR) technique was further applied to determining the expressions of the key genes associated with the SFI-targeted metabolic pathways. We have identified 48 significantly changed metabolites related to CVB3-induced AVMC, and SFI can significantly regulate the abnormalities of 33 metabolites and 9 relevant enzymes. Combined metabolic pathway enrichment and topology analyses revealed that the mechanisms of SFI against CVB3-induced AVMC may be attributed to modulating the disordered homeostasis of sphingolipid metabolism, glycerophospholipid metabolism, arachidonic acid metabolism, tryptophan metabolism, and TCA cycle. It provides new experimental information on the pathogenesis of AVMC, unravels the potential targeted metabolic pathways of SFI against AVMC on the whole metabolic network and highlights the importance of metabolomics combined with computational systems analysis as a potential tool for deciphering drug-targeted metabolic pathways., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms.

Acute viral myocarditis (VM), characterised by leukocyte infiltration and dysfunction of the heart, is an important cause of sudden cardiac death in young adults. Unfortunately, to date, the pathological mechanisms underlying cardiac failure in VM remain incompletely understood. In the current study, we investigated if acute VM leads to cardiac metabolic rewiring and if this process is driven by local inflammation. Transcriptomic analysis of cardiac biopsies from myocarditis patients and a mouse model of VM revealed prominent reductions in the expression of a multitude of genes involved in mitochondrial oxidative energy metabolism. In mice, this coincided with reductions in high-energy phosphate and NAD levels, as determined by Imaging Mass Spectrometry, as well as marked decreases in the activity, protein abundance and mRNA levels of various enzymes and key regulators of cardiac oxidative metabolism. Indicative of fulminant cardiac inflammation, NF-κB signalling and inflammatory cytokine expression were potently induced in the heart during human and mouse VM. In cultured cardiomyocytes, cytokine-mediated NF-κB activation impaired cardiomyocyte oxidative gene expression, likely by interfering with the PGC-1 (peroxisome proliferator-activated receptor (PPAR)-γ co-activator) signalling network, the key regulatory pathway controlling cardiomyocyte oxidative metabolism. In conclusion, we provide evidence that acute VM is associated with extensive cardiac metabolic remodelling and our data support a mechanism whereby cytokines secreted primarily from infiltrating leukocytes activate NF-κB signalling in cardiomyocytes thereby inhibiting the transcriptional activity of the PGC-1 network and consequently modulating myocardial energy metabolism., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. An early and abundant neutrophil accumulation in the myocardium is a hallmark of early CVB3 infection. Yet the relative contribution of neutrophils to host susceptibility to CVB3 myocarditis remains largely unknown. Herein, peripheral neutrophil depletion was implemented in a BALB/c mouse model of acute CVB3 myocarditis using the specific 1A-8 (anti-Ly6G) or a RB6-8C5 (anti-Gr-1) mAb covering a wide range. Anti-Ly6G treatment led to systemic neutropenia throughout the disease, but did not alter virus replication, disease susceptibility and histopathological changes in the heart and pancreas of mice. In contrast, depletion of both neutrophils and monocytes/macrophages by anti-Gr-1 mAb prior to and after infection significantly promoted susceptibility of mice to CVB3 infection which was associated with exacerbated cardiac and pancreatic viral load. However, depletion of Gr1+ cells significantly suppressed acute myocarditis and pancreatic acini destruction at day 7 post infection via reducing Ly6C high monocyte population in the circulation. Additionally, cardiac interstitial fibrosis was not affected by neutrophil depletion, whereas Gr-1+ cells other than neutrophils increased cardiac fibrosis at day 21 p.i. by increasing cardiac expression of profibrotic cytokine TNF-α and TGF-β. Thus, Neutrophil function is most likely not essential for CVB3 control and peripheral neutrophils play dispensable role in the pathogenesis of acute myocarditis and pancreatitis during CVB3 infection. Whereas Gr-1+ cells other than neutrophils play a major role in limiting viral replication while promoting myocardial and pancreatic inflammatory injury and fibrosis.

In this study, we investigated the roles of RIP1/RIP3 mediated cardiomyocyte necroptosis in CVB3-induced acute myocarditis. Serum concentrations of creatinine kinase (CK), CK-MB, and cardiac troponin I were detected using a Hitachi Automatic Biochemical Analyzer in a mouse model of acute VMC. Histological changes in cardiac tissue were observed by light microscope and expression levels of RIP1/RIP3 in the cardiac tissue were detected via Western blot and immunohistochemistry. The data showed that RIP1/RIP3 was highly expressed in cardiomyocytes in the acute VMC mouse model and that the necroptosis pathway specific blocker, Nec-1, dramatically reduced the myocardial damage by downregulating the expression of RIP1/RIP3. These findings provide evidence that necroptosis plays a significant role in cardiomyocyte death and it is a major pathway for cell death in acute VMC. Blocking the necroptosis pathway may serve as a new therapeutic option for the treatment of acute viral myocarditis.

Cathepsin B (CatB) is a cysteine proteolytic enzyme widely expressed in various cells and mainly located in the lysosomes. It contributes to the pathogenesis and development of many diseases. However, the role of CatB in viral myocarditis (VMC) has never been elucidated. Here we generated the VMC model by intraperitoneal injection of coxsackievirus B3 (CVB3) into mice. At day 7 and day 28, we found CatB was significantly activated in hearts from VMC mice. Compared with the wild-type mice receiving equal amount of CVB3, genetic ablation of CatB (Ctsb-/-) significantly improved survival, reduced inflammatory cell infiltration, decreased serum level of cardiac troponin I, and ameliorated cardiac dysfunction, without altering virus titers in hearts. Conversely, genetic deletion of cystatin C (Cstc-/-), which markedly enhanced CatB levels in hearts, distinctly increased the severity of VMC. Furthermore, compared with the control, we found the inflammasome was activated in the hearts of wild-type mice with VMC, which was attenuated in the hearts of Ctsb-/- mice but was further enhanced in Cstc-/- mice. Consistently, the inflammasome-initiated pyroptosis was reduced in Ctsb-/- mice hearts and further increased in Cstc-/- mice. These results suggest that CatB aggravates CVB3-induced VMC probably through activating the inflammasome and promoting pyroptosis. This finding might provide a novel strategy for VMC treatment.

Globally, coxsackievirus B4 (CV-B4) has been continuously isolated and evidence suggests an association with the development of pancreatitis and type I diabetes. In addition, CV-B4 is also associated with myocarditis and severe central nervous system (CNS) complications, which remain poorly studied and understood. In the present study, we established an Institute for Cancer Research (ICR) mouse model of CV-B4 infection and examined whether CV-B4 infection resulted in a predisposition to myocarditis and CNS infection. We found high survival in both the treatment and control group, with no significant differences in clinical outcomes observed. However, pathological lesions were evident in both brain and heart tissue of the CV-B4-infected mice. In addition, high viral loads were found in the neural and cardiac tissues as early as 2 days post infection. Expressions of IFN-γ and IL-6 in sera were significantly higher in CV-B4-infected mice compared to uninfected negative controls, suggesting the involvement of these cytokines in the development of histopathological lesions. Our murine model successfully reproduced the acute myocarditis and cerebral cortical neuron edema induced by CV-B4, and may be useful for the evaluation of vaccine candidates and potential antivirals against CV-B4 infection.

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.

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.

Viral myocarditis (VMC) is closely related to apoptosis, oxidative stress, innate immunity, and energy metabolism, which are all linked to mitochondrial dysfunction. A close nexus between mitochondrial dynamics and cardiovascular disease with mitochondrial dysfunction has been deeply researched, but there is still no relevant report in viral myocarditis. In this study, we aimed to explore the role of Dynamin-related protein 1 (Drp1)-linked mitochondrial fission in VMC. Mice were inoculated with the Coxsackievirus B3 (CVB3) and treated with mdivi1 (a Drp1 inhibitor). Protein expression of Drp1 was increased in mitochondria while decreased in cytoplasm and accompanied by excessive mitochondrial fission in VMC mice. In addition, midivi1 treatment attenuate inflammatory cells infiltration in myocardium of the mice, serum Cardiac troponin I (CTnI) and Creatine kinase-MB (CK-MB) level. Mdivi1 also could improved the survival rate of mice and mitochondrial dysfunction reflected as the up-regulated mitochondrial marker enzymatic activities of succinate dehydrogenase (SDH), cytochrome c oxidase (COX) and mitochondrial membrane potential (MMP). At the same time, mdivi1 rescued the body weight loss, myocardial injury and apoptosis of cardiomyocyte. Furthermore, decease in LVEDs and increase in EF and FS were detected by echocardiogram, which indicated the improved myocardial function. Thus, Drp1-linked excessive mitochondrial fission contributed to VMC and midivi1 may be a potential therapeutic approach., (Copyright © 2017 Elsevier Inc. All rights reserved.)

CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect., Competing Interests: The authors declare no competing financial interests.

Background: Viral myocarditis (VMC) treatment has long been lacking of effective methods. Our former studies indicated roles of calpain in VMC pathogenesis. This study aimed at verifying the potential of calpain in Coxsackievirus B3 (CVB3)-induced myocarditis treatment., Methods: A transgenic mouse overexpressing the endogenous calpain inhibitor, calpastatin, was introduced in the study. VMC mouse model was established via intraperitoneal injection of CVB3 in transgenic and wild mouse respectively. Myocardial injury was assayed histologically (HE staining and pathology grading) and serologically (myocardial damage markers of CK-MB and cTnI). CVB3 replication was observed in vivo and in vitro via the capsid protein VP1 detection or virus titration. Inflammation/fibrotic factors of MPO, perforin, IFNγ, IL17, Smad3 and MMP2 were evaluated using western blot or immunohistology stain. Role of calpain in regulating fibroblast migration was studied in scratch assays., Results: Calpastatin overexpression ameliorated myocardial injury induced by CVB3 infection significantly in transgenic mouse indicated by reduced peripheral CK-MB and cTnI levels and improved histology injury. Comparing with CVB3-infected wild type mouse, the transgenic mouse heart tissue carried lower virus load. The inflammation factors of MPO, perforin, IFNγ and IL17 were down-regulated accompanied with fibrotic agents of Smad3 and MMP2 inhibition. And calpain participated in the migration of fibroblasts in vitro, which further proves its role in regulating fibrosis., Conclusion: Calpain plays dual roles of facilitating CVB3 replication and inflammation promotion. Calpain inhibition in CVB3-induced myocarditis showed significant treatment effect. Calpain might be a novel target for VMC treatment in clinical practices., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Background: Viral myocarditis, which is often caused by coxsackievirus B3 (CVB3), is a serious clinical disorder characterized by excessive myocardial inflammation. Valproic acid (VPA) is described as a histone deacetylase inhibitor that has anti-inflammatory effects in several inflammatory diseases. However, the role and the detailed mechanism of VPA in viral myocarditis remain unclear., Methods: Experimental CVB3-induced myocarditis was induced in mice by intraperitoneally (i.p.) infected with CVB3. VPA was i.p. administered from day 0 to day 7. The survival, body weight loss, and myocarditis severity of mice were recorded. Th17 and Treg cells in spleen were analyzed by flow cytometry. Th17/Treg cell-related cytokine expressions were quantified by ELISA. The effect of VPA on Th17 and Treg cells differentiation was examined in vitro and in vivo., Results: Administration of VPA significantly attenuated the clinical severity of myocarditis, and the overall mortality from CVB3-induced myocarditis. The infiltration of Th17 and Treg cells, as well as the serum level of related cytokines (IL-17A and IL-10), were increased in CVB3 infected mice. In addition, VPA decreased the percentage of splenic Th17 cells while increased the percentage of Treg cells. Moreover, VPA downregulated the expression of IL-17A and upregulated IL-10 in serum and heart tissues of CVB3 infected mice. Additionally, VPA directly inhibited the differentiation of Th17 cells and promoted both the differentiation and suppressive function of Treg cells in vitro and in vivo., Conclusions: Our results suggest that VPA may thus be a promising strategy in the therapy of viral myocarditis.

Objectives: To address the question as to whether echocardiographic and/or microcomputed tomography (microCT) analysis can be utilized to assess the extent of Coxsackie B virus (CVB) induced myocarditis in the absence of left ventricular dysfunction in the mouse., Background: Viral myocarditis is a significant clinical problem with associated inflammation of the myocardium and myocardial injury. Murine models of myocarditis are commonly used to study the pathophysiology of the disease, but methods for imaging the mouse myocardium have been limited to echocardiographic assessment of ventricular dysfunction and, to a lesser extent, MRI imaging., Methods: Using a murine model of myocarditis, we used both echocardiography and microCT to assess the extent of myocardial involvement in murine myocarditis using both wild-type mice and CVB cleavage-resistant dystrophin knock-in mice., Results: Areas of increased echogenicity were only observed in the myocardium of Coxsackie B virus infected mice. These echocardiographic abnormalities correlated with the extent of von Kossa staining (a marker of membrane permeability), inflammation, and fibrosis. Given that calcium phosphate uptake as imaged by von Kossa staining might also be visualized using microCT, we utilized microCT imaging which allowed for high-resolution, 3-dimensional images of radiodensities that likely represent calcium phosphate uptake. As with echocardiography, only mice infected with Coxsackie B virus displayed abnormal accumulation of calcium within individual myocytes indicating increased membrane permeability only upon exposure to virus., Conclusions: These studies demonstrate new, quantitative, and semi-quantitative imaging approaches for the assessment of myocardial involvement in the setting of viral myocarditis in the commonly utilized mouse model of viral myocarditis.

MicroRNAs (miRNAs) are a group of short, noncoding, regulatory RNA molecules the dysregulation of which contributes to the pathogenesis of myocarditis. Argonaute proteins are essential components of miRNA-induced silencing complex and play important roles during miRNA biogenesis and function. However, the expression pattern of four AGO family members has not yet been detected in the coxsackievirus B3 (CVB3)-induced myocarditis tissue samples. In this study, we detected the expression of four AGOs in the CVB3-infected mouse heart tissues and found that AGO1 and AGO3 up-regulated significantly at 4 and 8h after CVB3 infection. Further in vitro research indicated that up-regulated AGO1 and AGO3 are related to the down-regulated TNFAIP3, which is a negative regulator of NF-κB pathway. Subsequently, we confirmed that TNFAIP3 is a direct target of miR-19a/b, and during CVB3 infection, the expression of miR-19a/b and miR-125a/b is not significantly changed. TNFAIP3 level is mainly reduced by up-regulated AGO1 and AGO3. This research sheds light on the relationship between overexpressed AGO proteins and CVB3-induced myocarditis, and this provides potential therapeutic target for viral myocarditis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To investigate the innate immune injury and repair mechanism during recovery from Coxsackievirus B3 (CVB3) induced myocarditis, we established an acute viral myocarditis recovery model by infecting BALB/c mice with CVB3. Histopathological examination of cardiac tissues after infection showed a gradual increase of myocardial injury to the maximum degree at 8 dpi (days post infection), followed by a recovery process with reduced viral replication. We also measured expression changes of innate immune genes in heart after 4, 8 and 12 days of infection using innate immune real-time PCR array. The results showed expression alterations in many Pattern Recognition Receptors (PRRs) genes upon CVB3 infection, which activated multiple important signaling pathways during recovery process. The expression of TLRs, RLRs, PKR and cytokines were strongly induced and reached the peak at 4 dpi in early myocarditis stage, followed by a gradual reduction in recovery stage, during which the levels were even lower than normal at 12 dpi. The strong correlation between cardiac histopathology score and chemokine expression level suggested that the chemokines might play a role in pathological changes during early myocarditis stage. In addition, we also found that both cell survival signaling pathways (AKT1, p38MAPK) and antiviral signaling pathways (IKKα/β/ε) were activated and promoted the recovery during late myocarditis stage. Altogether, our observations improved the understanding of formation and progression of the pathological lesions, as well as the repair mechanism for acute viral myocarditis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Unlabelled: Viral myocarditis, which is most prevalently caused by coxsackievirus B3 (CVB3), is a serious clinical condition characterized by excessive myocardial inflammation. Recent studies suggest that regulation of protein acetylation levels by inhibiting histone deacetylase (HDAC) activity modulates inflammatory response and shows promise as a therapy for several inflammatory diseases. However, the role of HDAC activity in viral myocarditis is still not fully understood. Here, we aim to investigate the role of HDAC activity in viral myocarditis and its underlying mechanism. CVB3-infected BALB/c mice were treated with the HDAC inhibitor (HDACI) suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA). We found inhibition of HDAC activity aggravated rather than ameliorated the severity of CVB3-induced myocarditis, which was contrary to our expectations. The aggravated myocarditis by HDACI treatment seemed not to be caused by an elevated inflammatory response but by the increased CVB3 replication. Further, it was revealed that the increased CVB3 replication was closely associated with the HDACI-enhanced autophagosome formation. Inhibition of autophagosome formation by wortmannin or ATG5 short hairpin RNA dramatically suppressed the HDACI-increased CVB3 replication. The increased viral replication subsequently elevated CVB3-induced myocardial apoptosis. Conversely, inhibition of CVB3 replication and ensuing myocardial apoptosis by the antiviral drug ribavirin significantly reversed the HDACI-aggravated viral myocarditis. In conclusion, we elucidate that the inhibition of HDAC activity increases CVB3 replication and ensuing myocardial apoptosis, resulting in aggravated viral myocarditis. Possible adverse consequences of administering HDACI should be considered in patients infected (or coinfected) with CVB3., Importance: Viral myocarditis, which is most prevalently caused by CVB3, is characterized by excessive myocardial inflammation. Inhibition of HDAC activity was originally identified as a powerful anti-cancer therapeutic strategy and was recently found to be implicated in the regulation of inflammatory response. HDACI has been demonstrated to be efficacious in animal models of several inflammatory diseases. Thus, we hypothesize that inhibition of HDAC activity also protects against CVB3-induced viral myocarditis. Surprisingly, we found inhibition of HDAC activity enhanced myocardial autophagosome formation, which led to the elevated CVB3 viral replication and ensuing increased myocardial apoptosis. Viral myocarditis was eventually aggravated rather than ameliorated by HDAC inhibition. In conclusion, we elucidate the role of HDAC activity in viral myocarditis. Moreover, given the importance of HDACI in preclinical and clinical treatments, the possible unfavorable effect of HDACI should be carefully evaluated in patients infected with viruses, including CVB3., (Copyright © 2015, Zhou et al.)

Coxsackievirus infection can lead to viral myocarditis and its sequela, dilated cardiomyopathy, which represent major causes of cardiovascular mortality worldwide in children. Yet, the host genetic susceptible factors and the underlying mechanisms by which viral infection damages cardiac function remain to be fully resolved. Dysferlin is a transmembrane protein highly expressed in skeletal and cardiac muscles. In humans, mutations in the dysferlin gene can cause limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Dysferlin deficiency has also been linked to cardiomyopathy. Defective muscle membrane repair has been suggested to be an important mechanism responsible for muscle degeneration in dysferlin-deficient patients and animals. Using both naturally occurring and genetically engineered dysferlin-deficient mice, we demonstrated that loss of dysferlin confers increased susceptibility to coxsackievirus infection and myocardial damage. More interestingly, we found that dysferlin is cleaved following coxsackieviral infection through the proteolytic activity of virally encoded proteinases, suggesting an important mechanism underlying virus-induced cardiac dysfunction. Our results in this study not only identify dysferlin deficiency as a novel host risk factor for viral myocarditis but also reveal a key mechanism by which coxsackievirus infection impairs cardiac function, leading to the development of dilated cardiomyopathy., (© 2015 John Wiley & Sons Ltd.)

Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.