Coxsackievirus B3 infection induces autophagic flux, and autophagosomes are critical for efficient viral replication.

Autophagy is an intrinsic cellular process that can degrade cytoplasmic components. It has been reported that several pathogens hijack this process to facilitate their replication. Coxsackievirus B3 (CVB3), a member of the family Picornaviridae, induces autophagy upon infection. However, the details of CVB3-induced autophagy remain a subject of debate. This study applied a combination of multiple assays for the measurement of autophagy and demonstrated that CVB3 induces a complete autophagic flux. Experiments with infected HEK293A cells revealed that autophagosomes were induced upon CVB3 infection. Most of these autophagosomes were mCherry positive in mCherry-GFP-LC3 cells. Conversely, mCherry-positive autophagosomes were rescued to green positive when treated with the acidification inhibitors chloroquine (CQ) and bafilomycin A1 (BAF), suggesting that autophagosomes fused with late endosomes or lysosomes. The co-localization of LC3-positive puncta with lysosome-associated membrane protein 1 (LAMP1) or LysoTracker confirmed that the autophagosomes fused primarily with lysosomes. Interestingly, the disruption of autophagosome formation by 3-methyladenine (3-MA) or ATG5 siRNA treatment during viral infection significantly decreased CVB3 replication. However, inhibitors of lysosomal acidification, fusion, or degradation did not affect viral replication. Therefore, autolysosomes may not be critical for viral replication in vitro.

Innate immune interleukin-1 receptor-associated kinase 4 exacerbates viral myocarditis by reducing CCR5(+) CD11b(+) monocyte migration and impairing interferon production.

BACKGROUND: Viral myocarditis follows a fatal course in ≈30% of patients. Interleukin-1 receptor-associated kinase 4 (IRAK4), a major nodal signal transducer in innate immunity, can play a pivotal role in host inflammatory response. We sought to determine how IRAK4 modulates inflammation and outcome in a mouse model of viral myocarditis. METHODS AND RESULTS: Myocarditis was induced after intraperitoneal inoculation of coxsackievirus B3 into C57Bl/6 IRAK4-deficient mice and their littermate controls. Mortality and viral proliferation were markedly reduced in IRAK4(-/-) mice compared with their IRAK4(+/+) littermates. Disease resistance of IRAK4(-/-) mice paralleled increased amounts of protective heart-infiltrating CCR5(+) monocytes/macrophages and enhanced interferon-α and interferon-γ production 2 days after infection. Competitive bone marrow chimera demonstrated that intact IRAK4 function inhibited heart-specific migration of bone marrow-derived CCR5(+) cells. Mechanistically, lack of IRAK4 resulted in interferon regulatory factor 5 homodimerization via reduced melanoma differentiation-associated protein 5 degradation and enhanced Stat1 and Stat5 phosphorylation. Consequently, antiviral interferon-α and interferon-γ production, as well as CCR5(+) cell recruitment, increased, whereas the overall proinflammatory response was drastically reduced in the absence of IRAK4. CONCLUSIONS: Innate immunity signal transducer IRAK4 exacerbates viral myocarditis through inhibition of interferon production and reduced mobilization of protective CCR5(+) monocytes/macrophages to the heart. The combination of IRAK4 inhibitors and antiviral adjuvants may become an attractive therapeutic approach against viral myocarditis in the future.

Adiponectin promotes coxsackievirus B3 myocarditis by suppression of acute anti-viral immune responses.

Adiponectin (APN) is an immunomodulatory adipocytokine that improves outcome in patients with virus-negative inflammatory cardiomyopathy and mice with autoimmune myocarditis. Here, we investigated whether APN modulates cardiac inflammation and injury in coxsackievirus B3 (CVB3) myocarditis. Myocarditis was induced by CVB3 infection of APN-KO and WT mice. APN reconstitution was performed by adenoviral gene transfer. Expression analyses were performed by qRT-PCR and immunoblot. Cardiac histology was analyzed by H&E-stain and immunohistochemistry. APN-KO mice exhibited diminished subacute myocarditis with reduced viral load, attenuated inflammatory infiltrates determined by NKp46, F4/80 and CD3/CD4/CD8 expression and reduced IFNß, IFNγ, TNFα, IL-1ß and IL-12 levels. Moreover, myocardial injury assessed by necrotic lesions and troponin I release was attenuated resulting in preserved left ventricular function. Those changes were reversed by APN reconstitution. APN had no influence on adhesion, uptake or replication of CVB3 in cardiac myocytes. In acute CVB3 myocarditis, cardiac viral load did not differ between APN-KO and WT mice. However, APN-KO mice displayed an enhanced acute immune response, i.e. increased expression of myocardial CD14, IFNß, IFNγ, IL-12, and TNFα resulting in increased cardiac infiltration with pro-inflammatory M1 macrophages and activated NK cells. Up-regulation of cardiac CD14 expression, type I and II IFNs and inflammatory cell accumulation in APN-KO mice was inhibited by APN reconstitution. Our observations indicate that APN promotes CVB3 myocarditis by suppression of toll-like receptor-dependent innate immune responses, polarization of anti-inflammatory M2 macrophages and reduction of number and activation of NK cells resulting in attenuated acute anti-viral immune responses.

MicroRNA profiling identifies microRNA-155 as an adverse mediator of cardiac injury and dysfunction during acute viral myocarditis.

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.

Cardiac fibroblasts aggravate viral myocarditis: cell specific coxsackievirus B3 replication.

Myocarditis is an inflammatory disease caused by viral infection. Different subpopulations of leukocytes enter the cardiac tissue and lead to severe cardiac inflammation associated with myocyte loss and remodeling. Here, we study possible cell sources for viral replication using three compartments of the heart: fibroblasts, cardiomyocytes, and macrophages. We infected C57BL/6j mice with Coxsackievirus B3 (CVB3) and detected increased gene expression of anti-inflammatory and antiviral cytokines in the heart. Subsequently, we infected cardiac fibroblasts, cardiomyocytes, and macrophages with CVB3. Due to viral infection, the expression of TNF-α, IL-6, MCP-1, and IFN-ß was significantly increased in cardiac fibroblasts compared to cardiomyocytes or macrophages. We found that in addition to cardiomyocytes cardiac fibroblasts were infected by CVB3 and displayed a higher virus replication (132-fold increase) compared to cardiomyocytes (14-fold increase) between 6 and 24 hours after infection. At higher virus concentrations, macrophages are able to reduce the viral copy number. At low virus concentration a persistent virus infection was determined. Therefore, we suggest that cardiac fibroblasts play an important role in the pathology of CVB3-induced myocarditis and are another important contributor of virus replication aggravating myocarditis.

MicroRNA-21 and -146b are involved in the pathogenesis of murine viral myocarditis by regulating TH-17 differentiation.

Viral myocarditis (VMC) is a common cardiovascular disease, and microRNAs (miRNAs) have been postulated to be involved in its pathology. Using microarrays, we observed that miRNA-21 and -146b were upregulated in a murine model of VMC. We also found that miRNA-451 was downregulated. In vivo silencing of miRNA-21 and -146b resulted in less-severe VMC. Overexpression of miRNA-451 did not ameliorate the severity of VMC. Further work revealed that inhibition of miRNA-21 and -146b decreased the expression levels of Th17 and RORγt . Overexpression of miRNA-451 had no effect on IL-17 and RORγt expression. Inhibition of miRNA-21 and -146b might ameliorate myocardium inflammation by mediating downregulation of RORγt expression, indicating that these miRNAs are involved in the pathogenesis of murine VMC.

Testosterone and interleukin-1ß increase cardiac remodeling during coxsackievirus B3 myocarditis via serpin A 3n.

Myocarditis and dilated cardiomyopathy (DCM) are often caused by viral infections and occur more frequently in men than in women, but the reasons for the sex difference remain unclear. The aim of this study was to assess whether gene changes in the heart during coxsackievirus B3 (CVB3) myocarditis in male and female BALB/c mice predicted worse DCM in males. Although myocarditis (P = 4.2 × 10(-5)) and cardiac dilation (P = 0.008) were worse in males, there was no difference in viral replication in the heart. Fibrotic remodeling genes, such as tissue inhibitor of metalloproteinase (TIMP)-1 and serpin A 3n, were upregulated in males during myocarditis rather than during DCM. Using gonadectomy and testosterone replacement, we showed that testosterone increased cardiac TIMP-1 (P = 0.04), serpin A 3n (P = 0.007), and matrix metalloproteinase (MMP)-8 (P = 0.04) during myocarditis. Testosterone increased IL-1ß levels in the heart (P = 0.02), a cytokine known to regulate cardiovascular remodeling, and IL-1ß in turn increased cardiac serpin A 3n mRNA (P = 0.005). We found that 39 of 118 (33%) genes identified in acute DCM patients were significantly altered in the heart during CVB3 myocarditis in mice, including serpin A 3n (3.3-fold change, P = 0.0001). Recombinant serpin A 3n treatment induced cardiac fibrosis during CVB3 myocarditis (P = 0.0008) while decreasing MMP-3 (P = 0.04) and MMP-9 (P = 0.03) levels in the heart. Thus, serpin A 3n was identified as a gene associated with fibrotic cardiac remodeling and progression to DCM in male myocarditis patients and mice.

Th2 regulation of viral myocarditis in mice: different roles for TLR3 versus TRIF in progression to chronic disease.

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

[Outbreak of acute enterovirus intestinal infection in Sakhalin region in August 2010].

The investigation of cases of acute intestinal infections in the Sakhalin region of Russia in August, 2010 is described. Epidemiological and molecular biological studies were conducted. After initial PCR screening and determining the nucleotide sequences of the positive samples the following enteroviruses were found: Coxsackie A2 - 42 samples (45%), Coxsackie A4--31 sample (34%), Enterovirus 71--6 samples (6,5%), Coxsackievirus B5--6 samples (6,5%), Coxsackie B3--4 samples (4%) and Coxsackie B1--4 samples (4%). The phylogenetic analysis of sequences showed that the closest analogues for the nucleotide sequences of these genotypes were previously identified in Japan, Korea and China in 2000-2010.

Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo.

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.

Coxsackievirus B3 infection activates the unfolded protein response and induces apoptosis through downregulation of p58IPK and activation of CHOP and SREBP1.

Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used cardiomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a (activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-associated gene, p58(IPK), which often is upregulated during infections with other types of viruses, was downregulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections between the three UPR pathways, we found that the ATF6a-induced downregulation of p58(IPK) was associated with the activation of PKR (PERK) and the phosphorylation of eIF2alpha, suggesting that p58(IPK), a negative regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58(IPK) and induction/activation of CHOP, SREBP1, and caspase-12.

Bosentan enhances viral load via endothelin-1 receptor type-A-mediated p38 mitogen-activated protein kinase activation while improving cardiac function during coxsackievirus-induced myocarditis.

Reduced cardiac output is one of the consequences of myocarditis. Bosentan, an endothelin-1 receptor (ET1R) antagonist, could be useful to reduce cardiac afterload, preserving cardiac output. In this study, we investigated the potential therapeutic use of bosentan in an animal model of viral myocarditis. Using a mouse model of coxsackievirus B3 (CVB3)-induced myocarditis, we demonstrated preserved ejection fraction (EF) and fractional shortening (FS) by treatment with bosentan (68+/-5.8% EF and 40+/-3.7% FS for treated versus 48+/-2.2% EF and 25+/-2.6% FS for controls; P=0.028). However, bosentan enhanced cardiac viral load (10.4+/-6.7% in the bosentan group versus 5.0+/-5.5% in control group; P=0.02), likely through enhancement of p38 mitogen-activated protein kinase (MAPK) phosphorylation (0.77+/-0.40% ATF2 activation in the bosentan group versus 0.03+/-0.02% in controls; P=0.0002), mediated by endothelin receptor type-A. We further demonstrate that a water soluble inhibitor of p38 MAPK, SB203580 HCl, is a potent inhibitor of virus replication in the heart (0.28% antisense viral genome stained area for 3 mg/kg dose versus 2.9% stained area for controls; P=0.01), attenuates CVB3-induced myocardial damage (blinded cardiac histopathologic scores of 1.8+/-1.6 and 2.05+/-1.2 for the 3 mg/kg and 10 mg/kg doses, respectively, versus 3.25+/-1.2 for the controls), and preserves cardiac function (69+/-3.5% EF for 3 mg/kg dose and 71+/-6.7% EF for 10 mg/kg dose versus 60+/-1.5% EF control; P=0.038 and P=0.045, as compared to control, respectively). Bosentan, a prescribed vasodilator, improves cardiac function but enhances viral load and myocarditis severity through ETRA mediated p38 MAPK activation; p38 MAPK is a desirable antiviral target. Caution must be exercised during treatment of suspected infectious myocarditis with supportive vasoactive remedies.

Osteopontin: a fibrosis-related marker molecule in cardiac remodeling of enterovirus myocarditis in the susceptible host.

The characteristics of dilated cardiomyopathy (DCM) resulting from chronic viral myocarditis are remodeling processes of the extracellular matrix. Based on our findings of enhanced osteopontin (OPN) expression in inflamed human hearts, we further investigated in the murine model of acute and chronic coxsackievirus (CV)B3-myocarditis the role of OPN regarding its involvement in resolution of cardiac virus infection and fibrosis. In hearts of A.BY/SnJ mice susceptible to chronic CVB3-myocarditis, a pronounced increase of OPN expression levels was detected by microarray analysis and quantitative RT-PCR during acute stages of myocarditis. Combined immunohistochemistry and in situ hybridization identified infiltrating macrophages as main OPN producers. In contrast to resistant C57BL/6 and OPN gene-deficient mice, transcription levels of matrix metalloproteinase-3, TIMP1 (tissue inhibitor of metalloproteinases-1), uPA (urokinase-type plasminogen activator), and transforming growth factor beta1 were elevated in susceptible mice, and as a consequence, procollagen-1alpha mRNA expression and fibrosis was considerably enhanced. Treatment of infected susceptible mice with the vitamin D analog ZK 191784 led to decreased myocardial expression levels of OPN, metalloproteinase-3, TIMP1, uPA, and procollagen-1alpha and subsequently to reduced fibrosis. Concurrently, the fibrosis-relevant signaling molecules pERK (phosphorylated extracellular signal-regulated kinase) and pAkt (phosphorylated Akt), increased in A.BY/SnJ mice, were diminished in ZK 191784-treated mice. Here, we show that high expression levels of OPN in acute myocarditis are associated with consecutive development of extensive fibrosis that can be reduced by treatment with a vitamin D analog. Thus, OPN may serve as a diagnostic tool as well as a potential therapeutic target to limit cardiac remodeling in chronic myocarditis.

Myocarditis and inflammatory cardiomyopathy: current evidence and future directions.

Inflammatory cardiomyopathy, characterized by inflammatory cell infiltration into the myocardium and a high risk of deteriorating cardiac function, has a heterogeneous aetiology. Inflammatory cardiomyopathy is predominantly mediated by viral infection, but can also be induced by bacterial, protozoal or fungal infections as well as a wide variety of toxic substances and drugs and systemic immune-mediated diseases. Despite extensive research, inflammatory cardiomyopathy complicated by left ventricular dysfunction, heart failure or arrhythmia is associated with a poor prognosis. At present, the reason why some patients recover without residual myocardial injury whereas others develop dilated cardiomyopathy is unclear. The relative roles of the pathogen, host genomics and environmental factors in disease progression and healing are still under discussion, including which viruses are active inducers and which are only bystanders. As a consequence, treatment strategies are not well established. In this Review, we summarize and evaluate the available evidence on the pathogenesis, diagnosis and treatment of myocarditis and inflammatory cardiomyopathy, with a special focus on virus-induced and virus-associated myocarditis. Furthermore, we identify knowledge gaps, appraise the available experimental models and propose future directions for the field. The current knowledge and open questions regarding the cardiovascular effects associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are also discussed. This Review is the result of scientific cooperation of members of the Heart Failure Association of the ESC, the Heart Failure Society of America and the Japanese Heart Failure Society.

Sequential changes in serum cytokines reflect viral RNA kinetics in target organs of a coxsackievirus B infection in mice.

INTRODUCTION: The pattern of cytokine responses related to viral replication during the course of the common human coxsackievirus B3 (CVB3) infection is not known. METHODS: Serum levels of 21 cytokines and chemokines were studied (Luminex technique) in CVB3-infected in mice on days 3, 6, and 9 post-infection (p.i.). CVB3 was measured quantitatively (reverse transcriptase polymerase chain reaction) in the liver and pancreas. RESULTS: Virus levels peaked on day 3 in both the liver and pancreas, but were 1,000-fold higher in the pancreas. IL-17alpha, IFN-gamma, KC, MCP-1, MIP1beta, and RANTES were detected on all days. On day 3 p.i., IL-6, IL-12(p40), KC, MCP-1, RANTES, and TNF-alpha were found to peak. On day 6 p.i., IL-1beta, IL-9, IL-12(p70), IL-13, IL-17alpha, and IFN-gamma peaked. On day 9 p.i., MIP1beta, IL-1beta, MCP-1, and TNF-alpha were still increased. These changes in cytokines may be used to monitor the progress of enteroviral infections in clinical settings.

Gonadectomy of male BALB/c mice increases Tim-3(+) alternatively activated M2 macrophages, Tim-3(+) T cells, Th2 cells and Treg in the heart during acute coxsackievirus-induced myocarditis.

The incidence of cardiovascular disease, including inflammatory heart diseases like myocarditis, is increased in men. Similarly, male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe acute inflammation in the heart compared to females. To better understand the effect of male sex hormones on cardiac inflammation, we gonadectomized (Gdx) male BALB/c mice and examined acute CVB3-induced myocarditis compared to sham controls. Viral replication in the heart was not significantly altered between Gdx and sham mice. However, gonadectomy significantly reduced testosterone levels and inflammation in the heart. FACS analysis of cell populations isolated from the heart revealed that CD11b(+) cells were significantly reduced in Gdx males. However, a GR1(+)F4/80(+) subset of CD11b(+) cells was significantly increased. Because this subset also expressed the interleukin (IL)-4R and IL-10, we refer to these cells as "alternatively activated" or M2 macrophages. A greater percentage of M2 macrophages in Gdx males expressed the inhibitory receptor Tim-3, while fewer expressed IL-1beta and IL-10. Only M2 macrophages upregulated TLR4 and Tim-3, whereas GR1(-)IL-4R(lo) macrophages did not. Additionally, IL-4(+)CD4(+) Th2 cells, Foxp3(+) regulatory T (Treg) cells and Tim-3(+)CD4(+) T cells were significantly increased in the heart following Gdx. Thus, we report for the first time that the inhibitory receptor Tim-3 is expressed on M2 macrophages. Our findings show that sex hormones and/or other mediators released from the testes inhibit anti-inflammatory populations in the heart including Tim-3(+) M2, Tim-3(+)CD4(+) T cells, Th2 and Treg resulting in more severe acute cardiac inflammation in males following CVB3 infection.

Coxsackievirus-B4 Infection of Human Primary Pancreatic Ductal Cell Cultures Results in Impairment of Differentiation into Insulin-Producing Cells.

Coxsackievirus-B4 (CV-B4) E2 can persist in the pancreatic ductal-like cells (Panc-1 cell line), which results in an impaired differentiation of these cells into islet-like cell aggregates (ICA). In this study, primary pancreatic ductal cells obtained as a by-product of islet isolation from the pancreas of seven brain-dead adults were inoculated with CV-B4 E2, followed-up for 29 days, and the impact was investigated. Viral titers in culture supernatants were analyzed throughout the culture. Intracellular viral RNA was detected by RT-PCR. Levels of ductal cell marker CK19 mRNA and of insulin mRNA were evaluated by qRT-PCR. The concentration of c-peptide in supernatants was determined by ELISA. Ductal cells exposed to trypsin and serum-free medium formed ICA and resulted in an increased insulin secretion. Ductal cells from five brain-dead donors were severely damaged by CV-B4 E2, whereas the virus persisted in cultures of cells obtained from the other two. The ICAs whose formation was induced on day 14 post-inoculation were scarce and appeared tiny in infected cultures. Also, insulin mRNA expression and c-peptide levels were strongly reduced compared to the controls. In conclusion, CV-B4 E2 lysed human primary pancreatic ductal cells or persisted in these cells, which resulted in the impairment of differentiation into insulin-producing cells.

Astragaloside IV reduces cardiomyocyte apoptosis in a murine model of coxsackievirus B3-induced viral myocarditis.

Apoptosis plays a crucial role in regulating cardiomyopathy and injuries of coxsackievirus B3 (CVB3)-induced viral myocarditis (VM). It has been reported that Astragaloside IV (AST-IV) from Astragalus membranaceus could inhibit apoptosis under a variety of pathological conditions in vivo or in vitro. However, the functional roles of AST-IV in CVB3-induced VM still remain unknown. Here, we found that AST-IV significantly enhanced survival for CVB3-induced mice. AST-IV protected the mice against CVB3-induced virus myocarditis characterized by the increased body weight, decreased serum level of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), supressed expression of Ifn-γ, Il-6 in heart, enhanced systolic and diastolic function of left ventricle. At the pathological level, AST-IV ameliorated the mice against CVB3-induced myocardial damage and myocardial fibrosis. In vitro, the results from flow cytometry showed that AST-IV significantly suppressed CVB3-induced cardiomyocytes apoptosis, which also were verified in vivo. Moreover, an increased expression of pro-apoptotic genes including FAS, FASL, cleaved caspase-8 and cleaved caspase-3 was found in CVB3-induced cardiomyocytes, while those was inhibited in cardiomyocytes treated with AST-IV. Taken together, the data suggest that AST-IV protected against CVB3-induced myocardial damage and fibrosis, which may partly attribute to supress activation of FAS/FASL signaling pathway.