Ad-A20 administration mediated protection against CVB3-induced myocarditis.

<p>Mice were intravenously injected with saline or 3×10⁹ pfu of either Ad-A20 or Ad-LacZ 2 days before 10³ TCID₅₀ dose of CVB3 infection. Mice without infection were as control group. (A and B) The body weight change (A) and survival rate (B) were respectively monitored daily until day 7 and day 10 post-infection. (C) Serological indices of myocarditis, the activity of CK, CK-MB and cTnI in mouse serum were detected on day 7 post-infection. (D<b>)</b> Paraffin sections of heart tissues were prepared on day 7 and cardiac inflammation was revealed by H&E staining (magnification:×200). The severity of myocarditis was scored by a standard 0–4 scale according to the foci of mononuclear infiltration and myocardial necrosis. Individual experiments were conducted 3 times with similar results, with 1 representative shown for each group. Data show the means±SEM of 6 mice per group. *, <i>P</i><0.05; **, <i>P</i><0.01; N.D., not detected.</p

Suppression of pro-inflammatory cytokines production in CVB3 infected mice with Ad-A20 administration.

<p>Mice were intravenously injected with saline or 3×10⁹ pfu of either Ad-A20 or Ad-LacZ 2 days before 10³ TCID₅₀ dose of CVB3 infection at day 0. Mice without infection were as control group. (A) Heart tissue homogenates prepared at indicated time points were subjected to western blot analysis with anti-A20 antibody. β-actin was used as loading control. Similar results were obtained in three independent experiments. (B) Heart tissue homogenates were prepared at the indicated time points. Protein levels of pro-inflammatory cytokines including TNF-α, IL-6, IL-1β and MCP-1 were determined by ELISA on day 0, 4, 7, 10 respectively post CVB3 infection. Data show the means±SEM of 6 mice per group. *, <i>P</i><0.05; **, <i>P</i><0.01; NS, no significance.</p

The effect of A20 on CVB3-induced NF-κB signaling in cardiac myocytes.

<p>Cardiac myocytes were pre-infected with adenovirus (Ad-LacZ or Ad-A20) to over-express A20 or lentivirus (LV-ctrl or LV-shA20) to knock down endogenous A20 for 48 h, then exposed to CVB3 (MOI = 10) for the indicated time. (A and D) Cell lysates were examined for phosphorylation of IκB-α, p65 and protein expression of total IκB-α, p65 and A20. β-actin was probed as the loading control. Left, representative western blots; Right, quantitative results, the band intensity was measured and the ratio of phospho-IκBα and phospho-p65 to β-actin was calculated. (B and E) NF-κB p65 DNA binding activity of nuclear extracts from cardiac myocytes was measured using the NF-κB p65 transcription factor assay kit. (C and F) Cell lysates were harvested at the indicated time, immunoprecipitated with anti-IKKγ antibody, incubated with GST-IκBα substrate, and analyzed by immunoblotting for phospho-GST-IκBα levels. Left, representative western blots; Right, quantitative results. Each assay was done 3 times. Values were presented as the means±SEM. *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001.</p

A20 (TNFAIP3) Alleviates CVB3-Induced Myocarditis via Inhibiting NF-κB Signaling

<div><h3>Background</h3><p>Viral myocarditis, which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. However, efficient therapies targeting inflammation are still lacking and much needed. A20, also known as tumor necrosis factor alpha induced protein 3 (TNFAIP3) is a key negative regulator of inflammation. But whether A20 may affect cardiac inflammation during acute viral myocarditis remains to be elucidated. The aim of this study was to investigate the potential protective effect of A20 on CVB3-induced myocarditis.</p> <h3>Methodology/Principal Findings</h3><p>Mice were intraperitoneally inoculated with CVB3 to establish acute viral myocarditis model. We found that the expression of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and monocyte chemotactic protein-1 (MCP-1) were markedly and persistently increased during the progression of CVB3-induced myocarditis, and positively correlated with the disease severity. Notably, intravenous injection <em>in vivo</em> with adenovirus expressed A20 (Ad-A20) remarkably reduced CVB3-induced pro-inflammatory cytokines production and alleviated the severity of myocarditis. Further, we observed that nuclear factor-kappaB (NF-κB) signaling which mediates inflammatory response was significantly inhibited in CVB3-infected mice with Ad-A20 treatment. Finally, we revealed that A20 was required to inhibit CVB3-induced NF-κB signaling by restricting TNF receptor associated factor 6 (TRAF6) ubiquitylation.</p> <h3>Conclusion/Significance</h3><p>This study demonstrates the protective role of A20 against CVB3-induced myocarditis, which may provide a new therapeutic strategy for the treatment of viral myocarditis.</p> </div

The effect of A20 on endogenous TRAF6 ubiquitylation in CVB3-infected cadiac myocytes.

<p>(A) Cardiac myocytes were pre-infected with adenovirus Ad-LacZ or Ad-A20 for 48 h, then they were treated with CVB3 (MOI = 10) for the indicated time, lysed in RIPA buffer, immunoprecipitated with RIP1, RIP2 and TRAF6 antibody. Immunoprecipitated protein complex was then analyzed by immunoblotting for ubiquitin. Immunoblotting for RIP1, RIP2 and TRAF6 on immunoprecipitates was shown below as a control. The whole cell lysates (WCLs) were subjected to immunoblot analysis with specific antibodies as indicated. (B) Cardiac myocytes were pre-infected with lentivirus LV-ctrl or LV-shA20 for 48 h, then they were treated with CVB3 (MOI = 10), lysed in RIPA buffer, immunoprecipitated with TRAF6 antibody as described above. Data are representative of three independent experiments.</p

Additional file 1 of Integrative and quantitive evaluation of the efficacy of his bundle related pacing in comparison with conventional right ventricular pacing: a meta-analysis

Figure S1. QRS duration of HBRP compared to the one at baseline: this figure showed the difference between post long-term HBRP QRS duration and intrinsic QRS duration at baseline (WMD = weight mean difference, and CI = confidence interval). Figure S2. Fluoroscopic time and lead impedence of HBRP compared to RVAP’s: this figure showed fluoroscopic time and lead impedence of HBRP compared to RVAP’s. A showed higher dose of radiation in HBRP during the procedure; B showed lower lead impedence in HBRP measured during the procedure. Figure S3. Pacing threshold and pacing R wave amplitude of HBRP compared to ones of RVAP: this figure showed pacing threshold and pacing R wave amplitude of HBRP compared to ones of RVP. A showed pacing threshold of HBRP different from the one of RVP during procedure; B showed difference of amplitude of R wave between 2 different pacing patterns. Figure S4: this figure presented horizontal funnel plots for test of publication bias: A. LVEF; B. LVEDV; C. LVESV; D. inter-ventricular asynchrony; E. NYHA class; F. mitral regurgitation; G. myocardial performance index (Tei index); H. 6MWT; I. PASP; J. QRS duration (HBRP versus baseline); K. QRS duration (HBRP versus RVP); L. fluoroscopy exposure time; M. lead impedence; N. pacing threshold; O. amplitude of R wave. Table S1. Sensitivity analysis of different group of studies for each parameter: this table showed sensitivity analyses by the way of exclusion of 1 study at a time to determine the stability of the overall treatment effects (LVEF = left ventricular ejection fraction, LVEDV = left ventricular end diastolic volume, LVESV = left ventricular end systolic volume, NYHA = New York Heart Association, MPI = myocardial performance index, 6MWT = 6 min walk test, PASP = pulmonary artery systolic pressure, Pth = pacing threshold, and RWA = R wave amplitude) (DOCX 355 kb

In Vivo Delivery of Adenoviral Vector Containing Interleukin-17 Receptor A Reduces Cardiac Remodeling and Improves Myocardial Function in Viral Myocarditis Leading to Dilated Cardiomyopathy

<div><p>Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM.</p></div

The levels of IL-6 and TNF-α in the supernatants of cardiac fibroblast cultures treated with IL-17.

<p>The IL-17 (10 ng/ml)-treated group produced higher levels of IL-6 and TNF-α cytokines than the control- and IL-17 (5 ng/ml)-treated groups. Moreover, IL-6 in the IL-17 (5 ng/ml)-treated group was highly expressed in the supernatants of the heart homogenates (ELISA). The values in the IL-17-treated CFs were normalized to the values in the control cells (n = 5 per group). The data represent the mean ± SEM. **<i>P</i><0.01 vs control group, ^##<i>P</i><0.01 vs control group.</p

Degradation of CFs related to the MMPs/TIMPs expression following neutralization of IL-17.

<p>(<i>A</i>) Representative western blots showing the expression of the cardiac ADAMTS-1 protein in each group. (<i>B</i>) Representative western blots showing the expression of the cardiac TIMP-1 protein in each group. (<i>C</i>) Representative western blots showing the expression of the cardiac MMP-2 protein in each group. The expression of the cardiac ADAMTS-1 and MMP-2/TIMP-1 proteins in each group is expressed as a ratio of the GAPDH expression for the same sample. On day 90, 3 mice per group were analyzed. *<i>P</i><0.05 vs control group.</p

The number of Th17 cells in the spleen and cardiac tissue is decreased by Ad-IL-17AR:Fc.

<p>(<i>A</i>) Representative flow cytometry images of Th17 (CD4⁺ IL-17⁺) cells from each group gated on CD4⁺ T cells. Numbers in the upper right quadrants and lower right quadrants indicate the separate percentages of Th17 cells and CD4⁺ T cells, respectively. PE = phycoerythrin; FITC = fluorescein isothiocyanate. (<i>B</i>) The percentage of Th17 (CD4⁺ IL-17⁺) cells in each group was analyzed using CellQuest software. (<i>C</i>) The levels of IL-6 and TNF-α in the supernatants of heart homogenates detected using ELISA. The data represent the mean ± SEM for 6 mice per group. **<i>P</i><0.01 vs control group, *<i>p</i><0.05 vs control group.</p