Myocarditis In Mice Research Articles

L‐arginine ameliorates experimental autoimmune myocarditis by maintaining extracellular matrix and reducing cytotoxic activity of lymphocytes

It was previously shown that administration of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) aggravated murine viral myocarditis by increasing myocardial virus titres. Experimental autoimmune myocarditis in mice and rats mimics human fulminant myocarditis. The effects of L-arginine, a precursor of nitric oxide, upon heart failure in experimental autoimmune myocarditis were evaluated. Dietary L-arginine (L-arginine group) and L-arginine plus N(G)-nitro-L-arginine methyl ester (L-arginine + l-NAME group) were administered to C57BL/6 mice immunized with porcine cardiac myosin over 3 weeks. An untreated myocarditis group was prepared. Cardiac damage was less in the L-arginine group compared with the other two groups, as was incidence of heart failure. In addition, extracellular matrix change was less prominent in the L-arginine group. Plasma concentrations of nitric oxide were elevated in the L-arginine group. Cytotoxic activities of lymphocytes were lower in L-arginine group than in other two groups. L-arginine treatment may be effective in preventing the development of heart failure in experimental myocarditis by maintaining extracellular matrix and reducing the cytotoxic activity of lymphocytes.

TIR-domain-containing adaptor protein inducing IFN-beta (TRIF) is essential in coxsackievirus B3-indused myocarditis in mice

TIR-domain-containing adaptor protein inducing IFN-beta (TRIF) is essential in coxsackievirus B3-indused myocarditis in mice

Interleukin-13 Protects Against Experimental Autoimmune Myocarditis by Regulating Macrophage Differentiation

We report here that interleukin (IL)-13 protects BALB/c mice from myocarditis, whether induced by peptide immunization or by viral infection. In contrast to mild disease in IL-4 knockout (KO) BALB/c mice, IL-13 KO BALB/c mice developed severe coxsackievirus B3 (CVB3)-induced autoimmune myocarditis and myocarditogenic peptide-induced experimental autoimmune myocarditis. Such severe disease was characterized by increased cardiac inflammation, increased total intracardiac CD45(+) leukocytes, elevated anti-cardiac myosin autoantibodies, and increased cardiac fibrosis. Echocardiography revealed that IL-13 KO mice developed severe dilated cardiomyopathy with impaired cardiac function and heart failure. Hearts of IL-13 KO mice had increased levels of the proinflammatory and profibrotic cytokines IL-1beta, IL-18, interferon-gamma, transforming growth factor-beta1, and IL-4 as well as histamine. The hallmark of the disease in IL-13 KO mice was the up-regulation of T-cell responses. CD4(+) T cells were increased in IL-13 KO hearts both proportionally and in absolute number. Splenic T cells from IL-13 KO mice were highly activated, and myosin stimulation additionally increased T-cell proliferation. CD4(+)CD25(+)Foxp3(+) regulatory T-cell numbers were decreased in the spleens of IL-13 KO mice. IL-13 deficiency led to decreased levels of alternatively activated CD206(+) and CD204(+) macrophages and increased levels of classically activated macrophages. IL-13 KO mice had increased caspase-1 activation, leading to increased production of both IL-1beta and IL-18. Therefore, IL-13 protects against myocarditis by modulating monocyte/macrophage populations and by regulating their function.

5′ terminal deletions in the genome of a coxsackievirus B2 strain occurred naturally in human heart

Enteroviruses can induce human myocarditis, which can be modeled in mice inoculated with group B coxsackieviruses (CVB) and in which CVB evolve to produce defective, terminally deleted genomes. The 5′ non-translated region (NTR) was enzymatically amplified from heart tissue of a fatal case of enterovirus-associated myocarditis in Japan in 2002. While no intact 5′ viral genomic termini were detected, 5′ terminal deletions ranged in size from 22 to 36 nucleotides. Sequence of the 5′ third of this viral genome is of a modern strain, closely related to CVB2 strains isolated in Japan in 2002. A CVB3 chimera containing the 5′ NTR with a 22 nt deletion produced progeny virus upon transfection of HeLa cells. When the 5′ 22 nucleotide deletion was repaired, the virus induced myocarditis in mice and replicated like wild type virus in murine heart cells. This is the first report of these naturally-occurring defective enteroviral genomes in human myocarditis.

Ablation of Matrix Metalloproteinase-9 Increases Severity of Viral Myocarditis in Mice

Coxsackievirus B3 (CVB3) causes human myocarditis, which can result in cardiac damage, maladaptive remodeling, and heart failure. Matrix metalloproteinases (MMP)-8 and -9 have been identified in virus-infected myocardium, but their particular roles and underlying mechanisms of effect are unknown. For the first time, we examine the severity of CVB3-induced myocarditis in MMP-8-and MMP-9-deficient mice. CVB3-infected MMP-8 and MMP-9 knockout (KO) mice and corresponding wild-type (WT) mice were euthanized and harvested at 9 days after infection. Expression of MMP-2, -8, -12, and -13 and tissue inhibitors of MMPs was assessed by zymography or immunoblotting on harvested hearts, and in situ hybridization was performed to detect active infection. Infected MMP-9 KO mice had greater myocardial injury and foci of infection than WT mice despite similar pancreatic infection. Increased fibrosis (10.6+/-2.7% versus 7.1+/-2.6%, P=0.04), viral titer, as well as decreased cardiac output, were evident in MMP-9 KO compared with WT mice as assessed by picrosirius red staining, plaque assay, and echocardiography, respectively. Immune infiltration was also greatly increased in MMP-9 KO compared with WT mice (15.2+/-12.6% versus 2.0+/-3.0%, P<0.002). Myocardial interferon-beta1, interferon-gamma, interleukin-6, interleukin-10, and macrophage inflammatory protein-1alpha expression was elevated in MMP-9 KO mice as measured by quantitative real-time polymerase chain reaction and ELISA. In contrast, MMP-8 KO mice had the same degree of cardiac injury, fibrosis, and viral infection as their WT counterparts. During acute CVB3 infection, MMP-9 appears necessary to halt virus propagation in the heart, promote proper immune infiltration and remodeling, and preserve cardiac output.

Imatinib mesylate attenuates fibrosis in coxsackievirus b3-induced chronic myocarditis

Coxsackievirus B3 (CVB3)-induced chronic myocarditis in mice is accompanied by severe fibrosis and by sustained elevation of platelet-derived growth factor (PDGF)-A, -B, and -C levels in the cardiac tissue. To test if PDGF stimulation of resident fibroblasts causally contributes to fibrosis, we employed inhibition of PDGF receptor signalling with the orally available kinase inhibitor Imatinib. Chronic myocarditis was induced by CVB3 infection of major histocompatibility complex (MHC) class II knockout (B6Aa(0)/Aa(0)) mice. The mice were treated with 100 mg/kg Imatinib or vehicle, respectively, twice daily for 34 days. Expression of PDGF-C and of inflammatory cytokines were analysed by semi-quantitative RT-PCR. PDGFalpha receptor phosphorylation was detected by immunoblotting of cardiac tissue extracts and in situ by immunohistochemistry. Fibrosis formation was analysed by Sirius-Red staining and hydroxyproline (HP) determination. Fibronectin, and tenascin expression was analysed by RT-PCR and immunohistochemistry. Matrix metalloproteinase (MMP) activity was assessed with collagen, synthetic peptides, and gelatine as substrates. Imatinib significantly inhibited the myocarditis-related PDGFalpha receptor activation in the heart tissue. The virus titres in the hearts, inflammatory infiltrations, and elevated PDGF levels were unaffected by the Imatinib treatment. A significant attenuation of fibrosis occurred in Imatinib-treated animals. The Sirius Red-stained fibrotic area was reduced from 5.30 +/- 0.50 to 3.21 +/- 0.35%, and the HP content was reduced from 362 +/- 43 to 238 +/- 32 microMol/10 mg dry weight vs. 190 +/- 27 in uninfected controls. The expression of fibronectin, EIIIA+ fibronectin, and tenascin C were likewise reduced. The diminished matrix protein deposition was not caused by elevated MMP activity, since MMP activity was not changed or even reduced under Imatinib. The data suggest a causal role for elevated PDGF expression and PDGF receptor activity in the pathogenesis of cardiac fibrosis.

Mao-to Prolongs the Survival of and Reduces TNF-α Expression in Mice with Viral Myocarditis

Goal of this study was to evaluate effects of Mao-to on development of myocarditis induced by encephalomyocarditis (EMC) virus in mice. Mice were randomly divided into five groups. Group N included uninfected controls (n = 18), while group A, B and C underwent intraperitoneal injection of EMC virus. Group A was administered oral saline from day 0 to day 4. Group B was administered oral Mao-to (500 mg−1 kg−1 day−1) from day 0 to day 4. Group C was administered Mao-to from day 2 to day 6. Group D was administered Mao-to from day 5 to day 10. Treated mice were followed for survival rates during 2 weeks after infection. Body weight (BW) and organ weights including heart (HW), lungs, thymus and spleen were examined on days 4, 6 and 14. Survival rate of group C (36.4%) was significantly improved compared with group A, B or D (0% of each, P < 0.05). HW and HW/BW ratio in group C was significantly (P < 0.05) lower than those in group A, B or D. Viral titers of hearts were significantly different among groups A, B and C. Cardiac expression in tumor necrosis factor-α (TNF-α) was significantly reduced in group C in comparison with group A, B or D on day 6 by immunohistochemical study. Administration of Mao-to starting on day 2 improves mortality resulting from viral myocarditis in mice with reduced expression of cardiac TNF-α. These findings suggest that timing of Mao-to is crucial for preventing cardiac damage in mice with viral myocarditis.

DNA Mismanagement Leads to Immune System Oversight

Trex1, a major 3' DNA exonuclease in mammalian cells, has been thought to act primarily in DNA replication or repair. Surprisingly, the major phenotype resulting from Trex1 deficiency in humans and mice is a chronic inflammatory disease. In this issue, Yang et al. (2007) report that Trex1 deficiency causes chronic activation of the ATM-dependent DNA-damage checkpoint and accumulation of a discrete single-stranded DNA species in the cytoplasm, either of which could contribute to chronic inflammation.

French Maritime Pine Bark Extract Inhibits Viral Replication and Prevents Development of Viral Myocarditis

Background French maritime pine bark extract (Pycnogenol) revealed diverse anti-inflammatory actions by an inhibition of NF-κB–dependent gene expression. The aim of this study was to determine whether Pycnogenol had a beneficial effect on viral myocarditis in mice. Methods and Materials Four-week-old inbred male DBA/2 mice were inoculated intraperitoneally with 10 plaque-forming units (pfu) of the encephalomyocarditis (EMC) virus. Pycnogenol was administered orally at a dose of 1 or 10 mg/kg per day for the histologic study, and 10 or 100 mg/kg for the gene expression study, beginning on the day of viral inoculation. Results The area of myocardial infiltration and necrosis on day 7 was significantly smaller in the hearts of mice treated with Pycnogenol 10 mg/kg (16.2 ± 8.9% and 19.2 ± 9.7%, respectively, n = 10, mean ± SEM) compared with controls (27.6 ± 15.0% and 30.1 ± 15.7%, respectively, n = 10, P < .05). There was a nonsignificant trend for less myocardial infiltration in the Pycnogenol 1 mg/kg group. Myocardial virus concentration on day 7 was 8.4 ± 0.3 × 10 3 pfu/mg in mice treated with 1 mg/kg of Pycnogenol, and 2.7 ± 0.6 × 10 4 pfu/mg in control mice, and the difference was statistically significant ( P < .05). Gene expressions of tumor necrosis factor, type-I procollagen, stem cell factor, and mast cell tryptase were significantly suppressed in the hearts of mice treated with Pycnogenol 100 mg/kg. Conclusions These results suggest that Pycnogenol exerts its beneficial effects on viral myocarditis by decreasing virus replication, and by suppressing expression of pro-inflammatory cytokines, genes related to cardiac remodeling, and mast cell–related genes in the hearts of mice.

Th1-type immune responses by Toll-like receptor 4 signaling are required for the development of myocarditis in mice with BCG-induced myocarditis

The immunological aspects of autoimmune myocarditis are difficult to understand because of the existence of many infectious agents and animal models suggesting different mechanisms in autoimmune myocarditis. To overcome these difficulties, two strains of mice, C3H/HeN and C3H/HeJ, showing different immune responses to mycobacteria, were immunized with myosin mixed with BCG. The C3H/HeN mice with a wild-type Toll-like receptor 4 (TLR4) showed severe myocarditis, whereas the C3H/HeJ mice with nonfunctional mutated TLR4 did not. CD4 + cells from both strains of mice exhibited appreciable proliferative responses following myosin stimulation; however, the cytokines from these cells differed between these two strains. The C3H/HeN mice showed T helper (Th)1-type cytokine responses, whereas the expressions of mRNA in C3H/HeJ mice were Th2-type cytokine. When both of these strains of immunized mice were inoculated with a plasmid encoding cDNA of interleukin (IL)-4 or agonistic IL-4, the development of myocarditis was inhibited in C3H/HeN mice. Moreover, C3H/HeJ mice, in which development of myocarditis was not induced by immunization of myosin mixed with BCG, showed myocarditis after injection of IL-4 antagonistic mutant DNA for the induction of Th1-type immune responses. The results suggested that the induction of autoimmune myocarditis by myosin is affected by Th1-type immune responses.

Th cell differentiation in chronic stage of viral myocarditis in mice and interference of Qingxin-Ⅱ Recipe

To investigate part mechanisms of Th cell differentiation, and to observe the interference effect of Qingxin-II Recipe in the chronic stage of viral myocarditis (VMC), so as to provide some experimental evidences for illuminating the pathogenesis of VMC and treatment mechanisms of Qingxin-II Recipe. According to 20%-40% death rate of experiment in advance, 100 BALB/c male mice (4 weeks old and weighing 12-15 g) were used. Twenty mice were randomly assigned to normal control group, and the other 80 mice were intraperitoneally injected with 0.1 ml normal saline containing coxsackie virus B3 at the 1st, 4th and 28th day (the virus densities were 1:2000, 1:1000 and 1:500 respectively) to induce chronic VMC. At the 42nd day, the surviving mice were randomly divided into untreated group and treatment group, with 20 mice in each group. Mice in the treatment group were orally administered with 0.2 ml Qingxin-II Recipe every day, while mice in the normal control group and the untreated group were administered with 0.2 ml normal saline. All the mice were sacrificed after 45 days, and the sera, heart and spleen cells were collected. Then the myocardial pathological changes were observed by using a light microscope, and the levels of IL-4, IL-10 and IFN-gamma in serum were detected by enzyme linked immunosorbent assay (ELISA). And the Th cell differentiation was observed by flow cytometry. No obvious myocardial pathological changes were observed in mice of the normal control group. Myocardial pathological changes in the treatment group were slighter than those in the untreated group. The difference of serum IL-10 level between the normal control group and the untreated group showed no significance (P>0.05), and the levels of IFN-gamma and IL-4 of the untreated group were higher than those of the normal control group (P<0.05 or P<0.01). There was no statistical difference in IL-10 level between the treatment group and the untreated group (P>0.05), while the serum levels of IL-4 and IFN-gamma of the treatment group were lower than those of the untreated group (P<0.05 or P<0.01). There was no significant difference of the Th1 cell responder between the treatment group and the untreated group (P>0.05), while the Th2 cell responder was inhibited significantly in the treatment group (P<0.05). Qingxin-II Recipe can restore the balance of Thl and Th2 cells through inhibiting the reaction of Th2.

Oral Administration of Candesartan Improves the Survival of Mice with Viral Myocarditis through Modification of Cardiac Adiponectin Expression

We examined the effects of the angiotensin II receptor type 1 blocker candesartan on myocarditis injury in a murine model of acute myocarditis. We hypothesized that candesartan improves cardiac damage by inducing cardiac expression of adiponectin. We examined changes in heart failure caused by myocarditis in mice by candesartan based on induction of cardiac adiponectin expression. We intraperitoneally injected encephalomyocarditis virus in C3H mice, then orally administered candesartan (10 mg/kg/day) or vehicle (control). The 7 day survival rate was 18% in the control group, but 60% in the candesartan group. The heart weight/body weight ratio in the candesartan group was significantly lower than in the control group. Circulating adiponectin concentrations on day 7 were significantly higher in the candesartan group compared with the control group (7.91 +/- 0.61 vs. 6.04 +/- 2.26 microg/ml, P < 0.05). Comparative expression of cardiac adiponectin mRNA in the candesartan group was significantly higher than in the control group on day 7 (55.4 +/- 41.3 vs. 5.3 +/- 7.7, P < 0.05). Immunohistochemical staining and in situ hybridization showed that cardiac expression of adiponectin protein and mRNA was present in the candesartan group on day 7. Oral administration of candesartan improves survival and decreases myocardial damage in mice with viral myocarditis and induces expression of cardiac adiponectin. The induction of adiponectin might provide cardioprotective effects against acute heart failure due to viral myocarditis.

Susceptibility to autoimmune myocarditis in mice: analysis of the Eam1 susceptibility locus (129.18)

Abstract H-2sA.SW and B10.S mice are susceptible and resistant to experimental autoimmune myocarditis (EAM), respectively. Our previous studies suggested that Eam1 on chromosome 1 is a non-MHC locus conferring susceptibility to cardiac myosin-induced (EAM) in A.SW mice. B10.A Eam1 congenic mice, carrying the Eam1 locus on the EAM-resistant B10.S background, develop EAM comparable to A.SW mice, suggesting that Eam1 plays a key role in the development of disease. We have previously reported that lymph node cells (LNC) of A.SW mice are more resistant to cyslophosphamide (Cy)-induced apoptosis in comparison to B10.S mice, and a similar apoptosis-resistant phenotype is found in NOD mice has been linked to the Idd5 locus overlapping with Eam1. Therefore, we hypothesized that the mechanisms by which Eam1 mediates the susceptibility to EAM could be due to its regulatory role in apoptosis. In the current study, we found that LNC from B10.A Eam1 congenic mice present an intermediate susceptibility to Cy-induced apoptosis, i.e. the percentage of apoptotic LNC with activated caspase 3, 8, 9 in congenic mice falls in between A.SW and B10.S mice. Interestingly, data suggest that A.SW mice have significantly lower levels of apoptotic LNC following immunization with cardiac myosin in CFA than B10.S mice. Our results therefore suggest that Eam1 partially down regulates LNC apoptosis and delays the removal of self-activated T cells, thereby providing an explanation for the role of Eam1 in the susceptibility to EAM. Supported in part by NIH grant HL077611

Complex genetic control of host susceptibility to coxsackievirus B3-induced myocarditis

The pathogenesis of viral myocarditis is a multifactorial process involving host genetics, viral genetics and the environment in which they interact. We have used a model of infection with coxsackievirus B3 (CVB3) to characterize the contribution of host genetics to viral myocarditis in mice of different genetic backgrounds but with a common H2 haplotype: A/J and B10.A-H2(a). Here we have used Evans blue dye as a quantitative biomarker for susceptibility to CVB3-induced myocarditis in addition to histopathological semiquantitative measures. We have found evidence of linkage between susceptibility to viral myocarditis and three loci. A locus on chromosome 1 centered on D1Mit200 was linked to sarcolemmal disruption in males (P=0.00005), a second locus on chromosome 4 centered on D4Mit81 was also linked to sarcolemmal disruption in males (P=0.0022). A third locus on distal chromosome 3 centered on D3Mit19 was linked to myocardial infiltration, with a logarithm of odds (LOD) score of 4.7 (P=0.0045), as well as sarcolemmal disruption in females (P=0.0015). These results provide strong evidence for the presence of loci contributing to the susceptibility of mice to viral myocarditis.

Coxsackievirus-induced myocarditis in mice: A model of autoimmune disease for studying immunotoxicity

Excellent animal models are available for virus-induced and autoimmune heart disease that are remarkably similar to human disease. Developing good animal models for heart disease is crucial because cardiovascular disease is now the leading cause of death in the United States and is estimated to be the leading cause of death in the world by the year 2020. A significant proportion of heart disease in Western populations is associated with inflammation. Myocarditis, or inflammation of the heart muscle, is the major cause of sudden death in young adults. Although most individuals recover from acute myocarditis, genetically susceptible individuals may go on to develop chronic myocarditis and dilated cardiomyopathy (DCM) resulting in congestive heart failure. In this article, we describe a model of autoimmune myocarditis and DCM induced by inoculation with heart-passaged coxsackievirus B3 (CVB3). Intraperitoneal inoculation of susceptible mice with CVB3 induces acute cardiac inflammation from days 7 to 14 postinfection (pi) that progresses to chronic myocarditis and DCM from day 28 to at least 56 pi. The model of CVB3-induced myocarditis presented here allows dissection of the contribution of viral infection and xenobiotics on immune dysregulation and inflammation in the heart. An improved understanding of the interaction between environmental exposures and the development of heart disease represents a clear challenge for immunotoxicologists.

Relevance of inflammatory cell infiltrates for complete atrioventricular block in experimental murine myocarditis

There are few systemic pathologic studies on myocarditis. This study aimed to clarify the pathologic characteristics of murine myocarditis. We recorded serial electrocardiograms in experimental viral myocarditis in mice and then examined their cardiac pathology. After taking baseline electrocardiograms, we inoculated the mice intraperitoneally with the encephalomyocarditis virus. Electrocardiograms were serially recorded until 220 days after the virus inoculation. Serial electrocardiograms revealed ectopic beats, low voltage of the QRS complex, and the appearance of complete atrioventricular (AV) block. Corresponding myocardial lesions were found in the hearts of mice with these ectopic beats. Mononuclear cell infiltrations into the His bundle were most frequently found in mice with complete AV block. Inflammatory change with cellular infiltrations was the most common pathologic finding in mice with complete AV block. In clinical settings, anti-inflammatory therapy might be recommended for patients with myocarditis complicated with conduction disturbances.

Coronary flow reserve and heart failure in experimental coxsackievirus myocarditis. A transthoracic Doppler echocardiography study

The objective of this study was to apply transthoracic Doppler echocardiography (TTDE) in mice to study coronary flow reserve (CFR), an index of coronary microvascular function, in mild and severe forms of experimental viral myocarditis. Regarding methodology, BALB/c mice were infected with cardiotropic coxsackieviruses causing either a mild (Nancy strain) or a severe (Woodruff strain) myocarditis. Left ventricular dimensions, fractional shortening, and CFR (ratio of left coronary artery flow velocity during maximal adenosine-induced vasodilatation to rest) were measured by TTDE before infection and again 1 or 2 wk after infection. As a result, the resting flow velocity did not change after infection. In contrast, CFR reduced significantly 1 wk after infection with either virus variant [from 2.5 (SD 0.3) to 1.4 (SD 0.1) in severe and from 2.4 (SD 0.4) to 2.1 (SD 0.3) in mild myocarditis], being significantly lower in the severe than mild myocarditis. CFR remained low in severe myocarditis 2 wk after infection. Fractional shortening decreased to the same levels 1 wk after infection with either virus variant [from 0.54 (SD 0.02) to 0.43 (SD 0.03) in severe and from 0.51 (SD 0.03) to 0.44 (SD 0.02) in mild myocarditis, P < 0.05]. However, 2 wk after infection, mice with severe myocarditis had enlarged left ventricles and lower fractional shortening [0.31 (SD 0.03)] than mice with mild myocarditis [0.47 (SD 0.02), P < 0.01]. In conclusion, CFR measured with TTDE is reduced in coxsackievirus myocarditis in mice. Low CFR is associated with progressive heart failure, indicating that dysfunction of coronary microcirculation is a determinant of poor outcome in viral myocarditis.

Cardiac Protective Effect of Astragalus on Viral Myocarditis Mice: Comparison with Perindopril

In clinical practice, Astragali Radix (Astragalus), the root of Astragalus membranaceus Bunge, has been widely applied to treat patients with viral diseases, including viral myocarditis in China. The present study was designed to evaluate the protective effects of Astragalus on the function of sarcoplasmic reticulum calcium ATPase (SERCA2) activity and endothelin system at acute and chronic periods of myocarditis mice induced by CVB(3) infection. Astragalus feeding (2.2 mg/kg/day) could significantly increase the survival rate, alleviate pathological alterations and serum cardiac troponin I (cTnI), as well as restore impaired SERCA activity at the acute stage. Low affinity and capacity of ETR were reversed with Astragalus after the first CVB(3) inoculation up to 7 days and after the second virus inoculation up to 150 days. In the meantime, the contents of cardiac ET-1 and ANP were reduced. Comparison the myocarditis mice treated with Perindopril (0.44 mg/kg/day), an ACE inhibitor, shows that Astragalus achieved a similar effect on survival rate, SERCA2 and ET system. These results indicated that the beneficial effects of Astragalus and Perindopril for treating viral myocarditis might be partly mediated by preserving the functions of SERCA 2 activity and ET system.

351 Transthoracic Doppler echocardiography in coxsackievirus myocarditis in mice: Reduced coronary flow velocity reserve is associated with progressive heart failure

351 Transthoracic Doppler echocardiography in coxsackievirus myocarditis in mice: Reduced coronary flow velocity reserve is associated with progressive heart failure

Inhibition of Coxsackie B Virus Infection by Soluble Forms of Its Receptors: Binding Affinities, Altered Particle Formation, and Competition with Cellular Receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.