Inhibition of RNA Helicase Activity Prevents Coxsackievirus B3-Induced Myocarditis in Human iPS Cardiomyocytes.

AIMS: Coxsackievirus B3 (CVB3) is known to be an important cause of myocarditis and dilated cardiomyopathy. Enterovirus-2C (E2C) is a viral RNA helicase. It inhibits host protein synthesis. Based on these facts, we hypothesize that the inhibition of 2C may suppress virus replication and prevent enterovirus-mediated cardiomyopathy. METHODS AND RESULTS: We generated a chemically modified enterovirus-2C inhibitor (E2CI). From the in vitro assay, E2CI was showed strong antiviral effects. For in vivo testing, mice were treated with E2CI intraperitoneally injected daily for three consecutive days at a dose of 8mg/kg per day, after CVB3 post-infection (p.i) (CVB3 + E2CI, n = 33). For the infected controls (CVB3 only, n = 35), mice were injected with PBS (phosphate buffered saline) in a DBA/2 strain to establish chronic myocarditis. The four-week survival rate of E2CI-treated mice was significantly higher than that of controls (92% vs. 71%; p < 0.05). Virus titers and myocardial damage were significantly reduced in the E2CI treated group. In addition, echocardiography indicated that E2CI administration dramatically maintained mouse heart function compared to control at day 28 p.i chronic stage (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%; EF, 67. 9 ± 2.9% vs. 54.7 ± 4.7%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Moreover, E2CI is effectively worked in human iPS (induced pluripotent stem cell) derived cardiomyocytes. CONCLUSION: Enterovirus-2C inhibitor (E2CI) was significantly reduced viral replication, chronic myocardium damage, and CVB3-induced mortality in DBA/2 mice. These results suggested that E2CI is a novel therapeutic agent for the treatment of enterovirus-mediated diseases.

Differentiated pattern of complement system activation between MOG-IgG-associated disease and AQP4-IgG-positive neuromyelitis optica spectrum disorder.

Background: Myelin oligodendrocyte glycoprotein antibody (MOG) immunoglobulin G (IgG)-associated disease (MOGAD) has clinical and pathophysiological features that are similar to but distinct from those of aquaporin-4 antibody (AQP4-IgG)-positive neuromyelitis optica spectrum disorders (AQP4-NMOSD). MOG-IgG and AQP4-IgG, mostly of the IgG1 subtype, can both activate the complement system. Therefore, we investigated whether the levels of serum complement components, regulators, and activation products differ between MOGAD and AQP4-NMOSD, and if complement analytes can be utilized to differentiate between these diseases. Methods: The sera of patients with MOGAD (from during an attack and remission; N=19 and N=9, respectively) and AQP4-NMOSD (N=35 and N=17), and healthy controls (N=38) were analyzed for C1q-binding circulating immune complex (CIC-C1q), C1 inhibitor (C1-INH), factor H (FH), C3, iC3b, and soluble terminal complement complex (sC5b-9). Results: In attack samples, the levels of C1-INH, FH, and iC3b were higher in the MOGAD group than in the NMOSD group (all, p<0.001), while the level of sC5b-9 was increased only in the NMOSD group. In MOGAD, there were no differences in the concentrations of complement analytes based on disease status. However, within AQP4-NMOSD, remission samples indicated a higher C1-INH level than attack samples (p=0.003). Notably, AQP4-NMOSD patients on medications during attack showed lower levels of iC3b (p<0.001) and higher levels of C3 (p=0.008), C1-INH (p=0.004), and sC5b-9 (p<0.001) compared to those not on medication. Among patients not on medication at the time of attack sampling, serum MOG-IgG cell-based assay (CBA) score had a positive correlation with iC3b and C1-INH levels (rho=0.764 and p=0.010, and rho=0.629 and p=0.049, respectively), and AQP4-IgG CBA score had a positive correlation with C1-INH level (rho=0.836, p=0.003). Conclusions: This study indicates a higher prominence of complement pathway activation and subsequent C3 degradation in MOGAD compared to AQP4-NMOSD. On the other hand, the production of terminal complement complexes (TCC) was found to be more substantial in AQP4-NMOSD than in MOGAD. These findings suggest a strong regulation of the complement system, implying its potential involvement in the pathogenesis of MOGAD through mechanisms that extend beyond TCC formation.

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans.

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid-protein-specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti-VP2 and anti-VP1 sera detected the viral proteins extracted from CVB3-infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti-CVB3 antibodies in virus-infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3-induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti-CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus-induced myocarditis.

Antiviral activity of coxsackievirus B3 3C protease inhibitor in experimental murine myocarditis.

BACKGROUND: We investigated the efficacy of a 3C protease inhibitor (3CPI) in a murine coxsackievirus B3 (CVB3) myocarditis model. CVB3 is a primary cause of viral myocarditis. The CVB3 genome encodes a single polyprotein that undergoes a series of proteolytic events to produce several viral proteins. Most of this proteolysis is catalyzed by the 3C protease (3CP). METHODS AND RESULTS: By way of a micro-osmotic pump, each mouse received 50 mM 3CPI in 100 µL of 100% dimethyl sulfoxide (DMSO) during a 72-hour period. On the day of pump implantation, mice (n = 40) were infected intraperitoneally with 10(6) plaque-forming units of CVB3. For the infected controls (n = 50), the pump was filled with 100% DMSO without 3CPI. The 3-week survival rate of 3CPI-treated mice was significantly higher than that of controls (90% vs 22%; P < .01). Myocardial inflammation, viral titers, and viral RNA levels were also reduced significantly in the 3CPI-treated group compared with these measures in the controls. CONCLUSIONS: The protein-based drug 3CPI inhibited the activity of 3CP of CVB3, significantly inhibited viral proliferation, and attenuated myocardial inflammations, subsequent fibrosis, and CVB3-induced mortality in vivo. Thus, this CVB3 3CPI has the potential to be a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.

Antiviral effects of small interfering RNA simultaneously inducing RNA interference and type 1 interferon in coxsackievirus myocarditis.

Antiviral therapeutics are currently unavailable for treatment of coxsackievirus B3, which can cause life-threatening myocarditis. A modified small interfering RNA (siRNA) containing 5'-triphosphate, 3p-siRNA, was shown to induce RNA interference and interferon activation. We aimed to develop a potent antiviral treatment using CVB3-specific 3p-siRNA and to understand its underlying mechanisms. Virus-specific 3p-siRNA was superior to both conventional virus-specific siRNA with an empty hydroxyl group at the 5' end (OH-siRNA) and nonspecific 3p-siRNA in decreasing viral replication and subsequent cytotoxicity. A single administration of 3p-siRNA dramatically attenuated virus-associated pathological symptoms in mice with no signs of toxicity, and their body weights eventually reached the normal range. Myocardial inflammation and fibrosis were rare, and virus production was greatly reduced. A nonspecific 3p-siRNA showed relatively less protective effect under identical conditions, and a virus-specific OH-siRNA showed no protective effects. We confirmed that virus-specific 3p-siRNA simultaneously activated target-specific gene silencing and type I interferon signaling. We provide a clear proof of concept that coxsackievirus B3-specific 3p-siRNA has 2 distinct modes of action, which significantly enhance antiviral activities with minimal organ damage. This is the first direct demonstration of improved antiviral effects with an immunostimulatory virus-specific siRNA in coxsackievirus myocarditis, and this method could be applied to many virus-related diseases.

Foreign gene transfer to cardiomyocyte using a replication-defective recombinant coxsackievirus B3 without cytotoxicity.

BACKGROUND: Replication-competent coxsackievirus B3 (CVB3) has been used as a gene transfer vector for cultured cardiomyocytes and hearts in vivo. However, CVB3 induces cell lysis when it replicates in infected cells. In this study, we investigated whether a replication-defective rCVB3 vector could be generated and used as a noncytotoxic gene transfer vector for cardiomyocytes. METHODS: We generated a replication-defective luciferase-expressing CVB3 plasmid. This recombinant cDNA and pCMV-P1 plasmids were amplified and cotransfected into Hek293 cells using transfection reagents. Replication-defective rLuCVB3 virus was recovered from the cells and cell culture supernatants for 3 days after transfection. The generated rLuCVB3 viruses were concentrated on a 30% sucrose cushion and semiquantified using a luciferase assay. In addition, foreign gene delivery by the rLuCVB3 was tested in cultured cardiomyocytes and intact mouse hearts after rLuCVB3 infection. RESULTS: Luciferase was expressed in Hek293, HeLa cells and cardiomyocytes after rLuCVB3 infection. In addition, these cells did not show a significant cytopathic effect after 72 h. Luciferase protein expression or activity were detected for 3 days in the myocardium of rLuCVB3-infected mouse hearts without producing cytotoxicity or inflammation. CONCLUSION: As a proof-of-concept, these data indicate that a replication-defective rCVB3 vector can be generated and used as a novel gene transfer system to transfect exogenous genes into cardiomyocytes without generating cytotoxicity.

Role of the myristoylation site in expressing exogenous functional proteins in coxsackieviral vector.

We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.

PICOT attenuates cardiac hypertrophy by disrupting calcineurin-NFAT signaling.

PICOT (protein kinase C-interacting cousin of thioredoxin) was previously shown to inhibit pressure overload-induced cardiac hypertrophy, concomitant with an increase in ventricular function and cardiomyocyte contractility. The combined analyses of glutathione S-transferase pull-down experiments and mass spectrometry enabled us to determine that PICOT directly interacts with muscle LIM protein (MLP) via its carboxyl-terminal half (PICOT-C). It was also shown that PICOT colocalizes with MLP in the Z-disc. MLP is known to play a role in anchoring calcineurin to the Z-disc in the sarcomere, which is critical for calcineurin-NFAT (nuclear factor of activated T cells) signaling. We, therefore, suggested that PICOT may affect calcineurin-NFAT signaling through its interaction with MLP. Consistent with this hypothesis, PICOT, or more specifically PICOT-C, abrogated phenylephrine-induced increases in calcineurin phosphatase activity, NFAT dephosphorylation/nuclear translocation, and NFAT-dependent transcriptional activation in neonatal cardiomyocytes. In addition, pressure overload-induced upregulation of NFAT target genes was significantly diminished in the hearts of PICOT-overexpressing transgenic mice. PICOT interfered with MLP-calcineurin interactions in a dose-dependent manner. Moreover, calcineurin was displaced from the Z-disc, concomitant with an abrogated interaction between calcineurin and MLP, in the hearts of PICOT transgenic mice. Replenishment of MLP restored the hypertrophic responses and the increase in calcineurin phosphatase activity that was inhibited by PICOT in phenylephrine-treated cardiomyocytes. Finally, PICOT-C inhibited cardiac hypertrophy to an extent that was comparable to that of full-length PICOT. Taken together, these data suggest that PICOT inhibits cardiac hypertrophy largely by negatively regulating calcineurin-NFAT signaling via disruption of the MLP-calcineurin interaction.

Prognostic values of novel biomarkers in patients with AL amyloidosis.

As cardiac involvement is the most important prognostic marker in light-chain amyloidosis (AL), revised Mayo staging for AL incorporated N-terminal pro-brain natriuretic peptide (NTproBNP) and troponin T (TnT). However, prognostic value of novel biomarkers, such as soluble suppression of tumorigenicity 2 (sST2), growth differentiation factor 15 (GDF15), or osteopontin (OPN) is unknown in AL amyloidosis. We aimed to investigate additive predictive effects of novel biomarkers for overall mortality rates of AL amyloidosis patients. Levels of sST2, GDF15, and OPN were quantified at diagnosis in a total of 73 AL amyloidosis patients at Samsung Medical Center from 2010 to 2016. The median follow-up duration of the censored cases was 18.0 (12.4-28.1) months. A total of 25 deaths occurred during the follow-up period. Two novel biomarkers, sST2 and GDF-15 showed satisfactory predictive performances for both one-year and overall survival from ROC analysis. Best cut-off values for predicting one-year mortality were selected. Elevated sST2 and GDF-15 levels showed significant incremental prognostic values in addition to NT-ProBNP and TnT for overall mortality. Patients were assigned 1 point for elevated sST2 or GDF-15. The mean values of NT-proBNP, TnT, mean LV wall thickness, and septal e' velocity differed significantly according to the scores. Patients with higher scores showed significantly worse prognosis even in patients with advanced revised Mayo staging. Two novel biomarkers, sST2 and GDF-15, showed satisfactory prognostic value for overall survival of AL amyloidosis patients. Furthermore, sST2 and GDF-15 showed additive incremental values over conventional biomarkers and further discriminated prognosis of patients in advanced stages.

Role of Soluble ST2 as a Marker for Rejection after Heart Transplant.

BACKGROUND AND OBJECTIVES: Endomyocardial biopsy is obligatory during the first year after heart transplant (HTx) for the surveillance of acute rejection. Previous attempts using cardiac biomarkers for the detection of rejection failed to show enough evidence to substitute endomyocardial biopsy. Therefore, this study sought the possibility of using soluble ST2 (sST2), a novel cardiovascular marker, as a surrogate marker for acute allograft rejection after HTx. SUBJECTS AND METHODS: A total of 494 blood samples acquired at the time of endomyocardial biopsy were analyzed in 67 HTx cases from September 2006 to August 2014. Significant rejection was defined as International Society of Heart and Lung Transplant (ISHLT) score ≥2R and humoral rejection accompanied by hemodynamic instability. RESULTS: Twenty cases of HTx with 22 blood samples showed significant rejection in endomyocardial biopsy at 4.0 (2.0-9.0) months after HTx. The level of sST2 showed positive correlation with cardiac troponin I, and N-terminal pro-B-type natriuretic peptide (all p<0.001), and negative correlation with post-HTx months (p<0.001). The levels of sST2 according to the ISHLT scores were 36 (19-98), 28 (18-62), 15 (16-37), and 191 (85-343) ng/mL, consecutively 0R, 1R, 2R, and 3R+ (3R plus hemodynamically-unstable humoral rejection) (p=0.003). However, when we studied within-subject effects of sST2 using a mixed model, the sST2 level according to the predefined time point was not different according to the presence of significant rejection (p for interaction=0.94). CONCLUSION: Although sST2 is known as a promising predictor for cardiovascular events, its role in HTx patients to predict acute allograft rejection seems to be limited.

2,3,4-Trihydroxybenzyl-hydrazide analogues as novel potent coxsackievirus B3 3C protease inhibitors.

Human coxsackievirus B3 (CVB3) 3C protease plays an essential role in the viral replication of CVB3, which is a non-enveloped and positive single-stranded RNA virus belonging to Picornaviridae family, causing acute viral myocarditis mainly in children. During optimization based on SAR studies of benserazide (3), which was reported as a novel anti-CVB3 3C(pro) agent from a screening of compound libraries, the 2,3,4-trihydroxybenzyl moiety of 3 was identified as a key pharmacophore for inhibitory activity against CVB3 3C(pro). Further optimization was performed by the introduction of various aryl-alkyl substituted hydrazide moieties instead of the serine moiety of 3. Among the optimized compounds, 11Q, a 4-hydroxyphenylpentanehydrazide derivative, showed the most potent inhibitory activity (IC50 = 0.07 µM). Enzyme kinetics studies indicated that 11Q exhibited a mixed inhibitory mechanism of action. The antiviral activity against CVB3 was confirmed using the further derived analogue (14b) with more cell permeable valeryl ester group at the 2,3,4-trihydroxy moiety.

Soluble coxsackievirus B3 3C protease inhibitor prevents cardiomyopathy in an experimental chronic myocarditis murine model.

BACKGROUND: Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. CVB3 3C protease (3CP) cleaves the viral polyprotein during replication. We tested whether a water soluble 3CP inhibitor (3CPI) had antiviral effects in a chronic myocarditis model. METHODS: Chronic myocarditis was established using DBA/2 strain mice. Starting on post-infection (p.i) day 3, CVB3-infected mice (n=41) were treated with 3CPI by daily intraperitoneal (i.p.) injection at a concentration of 50 µM (1.7 mg/kg/day) per day for 3 consecutive days. Additional mice (n=49) were injected with PBS as a control. RESULTS: The 5-week survival rate was significantly higher with 3CPI treatment (82.3% versus 47.9%; P<0.05). Organ virus titers at day 3 and 7 and myocardial damage were significantly lower in 3CPI-treated mice. Echocardiography at day 31 indicated strong protection of heart function by 3CPI (FS, 51.2±1.5 versus 26.1±1.5%; P<0.001). Hemodynamic measurements indicated that 3CPI treatment markedly reduced CVB3-induced LV dysfunction on day 31 (dP/dTmax, 5302±352 versus 4103±408 mmHg/s, P<0.05; dP/dTmin, -3798±212 versus -2814±206 mmHg/s, P<0.01). CONCLUSIONS: Water soluble 3CPI was delivered through i.p. injection after CVB3 infection. This agent preserved heart function and decreased organ viral titers and myocardial damage. Soluble 3CPI may be beneficial in the treatment of cardiomyopathy associated with enterovirus infection.

Transforming growth factor ß1-mediated atrial fibrotic activity and the recovery of atrial mechanical contraction after surgical maze procedure.

BACKGROUND: The maze procedure for atrial fibrillation (AF) is effective in restoring sinus rhythm; however, a significant proportion of patients failed to recover atrial mechanical contraction (AMC). We hypothesized that preoperative atrial fibrotic activity would be related to the AMC recovery. METHODS: In atrial tissues of 128 consecutive patients who underwent mitral valve and combined maze surgery using cryoablation for persistent AF, preoperative mRNA levels of various biomarkers were measured including transforming growth factor-ß1 (TGF-ß1), atrial natriuretic peptide, brain natriuretic peptide (BNP), C-reactive protein, connective tissue growth factor, matrix metalloproteinase, N-terminal prohormone BNP, and tissue inhibitor of metalloproteinase. Presence of AMC was assessed using Doppler echocardiographic measurement of the transmitral A-wave velocity. RESULTS: At 1-year follow-up, patients without AMC (n=62) showed higher preoperative TGF-ß1 mRNA expression (0.42 versus 0.28, P=0.01) than those with AMC (n=66). There was no significant difference between the two groups regarding the other biomarkers. Recovery rate of AMC and the increment of A-wave velocity were reduced with the increase of preoperative TGF-ß1 mRNA level. Multiple logistic regression analysis revealed that TGF-ß1 mRNA levels were independently associated with the absence of AMC (odds ratio 7.47, 95% CI 1.63 to 34.4, P=0.01). CONCLUSIONS: TGF-ß1-mediated atrial fibrotic activity might exert a detrimental effect on the reversibility of atrial mechanical contraction after the maze and combined mitral valve surgery for long-standing persistent AF. Earlier surgical intervention might be needed to preserve atrial mechanical function.

Thioredoxin, adiponectin and clinical course of acute fulminant myocarditis.

BACKGROUND: In an animal model of viral myocarditis, plasma levels of thioredoxin and adiponectin have been reported to be associated with the severity of inflammation and recovery of ventricular dysfunction, respectively. However, there have been few reports about the clinical significance of these cytokine levels in human myocarditis. OBJECTIVES: To examine the hypothesis that cytokine levels correlate with clinical courses of patients with acute fulminant myocarditis (FM). METHODS: A total of 33 consecutive patients with biopsy-proven acute myocarditis were evaluated. Twenty patients were ascribed to an FM group and the other 13 patients were grouped as a non-fulminant group (NFM). Plasma cytokine levels at the time of admission and after 2 weeks were evaluated and correlated with the duration of mechanical circulatory support application. RESULTS: Plasma thioredoxin level at admission was raised in the FM group (3.08±2.15 ng/ml) compared with the NFM group (1.63±0.45 ng/ml, p=0.011) and reduced after an initial unstable period. However there was no significant difference in plasma adiponectin level between the two groups. In a multivariable regression model, increased plasma thioredoxin level (OR=5.79, 95% CI 1.67 to 20.1, p=0.006) and reduced plasma adiponectin level (OR=0.16, 95% CI 0.055 to 0.49, p=0.001) were associated with longer duration of mechanical circulatory support application in the patients with FM, which in turn was significantly related to death or cardiac transplantation. CONCLUSION: In patients with acute myocarditis, the plasma thioredoxin level was increased in the more severe form, and a reduced level of adiponectin was closely correlated with worse short-term outcome in patients with FM.