Virus-negative Inflammatory Cardiomyopathy Research Articles

FOXO3A acts as immune response modulator in human virus-negative inflammatory cardiomyopathy

ObjectiveInflammatory cardiomyopathy is characterised by inflammatory infiltrates leading to cardiac injury, left ventricular (LV) dilatation and reduced LV ejection fraction (LVEF). Several viral pathogens and autoimmune phenomena may cause cardiac...

Immunosuppressive therapy in virus-negative inflammatory cardiomyopathy: 20-year follow-up of the TIMIC trial.

AimsLong-term results of the Tailored IMmunosuppression in virus-negative Inflammatory Cardiomyopathy (TIMIC) trial protocol have been evaluated.Methods and resultsEighty-five patients with endomyocardial biopsy-proven virus-negative chronic inflammatory cardiomyopathy were enrolled in the randomized, double-blind, placebo-controlled TIMIC trial and received prednisone and azathioprine (n = 43) vs. placebo (n = 42) for 6 months. Immunosuppressive treatment promoted an improvement in cardiac function in 88% of the cases compared with none of the patients in the placebo group, which were switched to a 6-month immunosuppressive therapy at the end of the 6-month study period. Long-term (up to 20 years) clinical outcomes of the whole cohort of 85 patients originally enrolled in the TIMIC trial (Group A) were compared with those of a 1:2 propensity score-matched control cohort of patients untreated with the TIMIC protocol (Group B) and followed for a comparable period of time. The primary outcome was a composite of cardiovascular death and heart transplantation. At long-term follow-up, the risk of cardiovascular death [hazard ratio (HR) 6.77; 95% confidence interval (CI) 2.36–19.45] and heart transplantation (HR 7.92; 95% CI 1.80–34.88) was significantly higher in Group B patients. Group A showed a persistent improvement in the left ventricular ejection fraction compared with Group B (HR 7.24; 95% CI 3.05–17.18). A higher number of Group B patients underwent implantable cardioverter defibrillator implantation. The incidence of recurrent myocarditis was similar between groups, and patients with evidence of a recurrent cardiac inflammatory process promptly responded to a TIMIC protocol application.ConclusionVirus-negative inflammatory cardiomyopathy benefits from immunosuppressive therapy even after long-term follow-up. Recurrence appears to respond to a new TIMIC protocol application.

Myocardial Aldosterone Receptor and Aquaporin 1 Up-Regulation Is Associated with Cardiomyocyte Remodeling in Human Heart Failure.

Background: Abnormal aldosterone signaling is a recognized source of cardiovascular damage. Its influence on cardiomyocyte structure, function, and hormonal receptors when associated with heart failure is still unreported. Methods: Twenty-six consecutive patients with heart failure (LVEF < 40%) and normal coronaries and valves underwent left ventricular endomyocardial biopsy (EMB) for evaluation of myocardial substrate. Biopsy samples were processed for histology, electron microscopy, immunohistochemistry, and Western blot analysis of myocardial aldosterone receptor and aquaporin-1 correlated with plasma aldosterone (AD) and renin activity (PRA). Eight patients with virus-negative inflammatory cardiomyopathy (ICM) had a control EMB after 6 months of immunosuppressive therapy and recovery of cardiac function with re-evaluation of cardiomyocyte structure and receptor expression. Results: EMB in addition to the diagnosis of myocarditis (15 cases), dilated cardiomyopathy CM (6), alcohol CM (2), and diabetic CM (3) showed vacuolar degeneration and cloudy swelling of cardiomyocytes corresponding at electron microscopy to ions and water accumulation into cytosol, membrane-bound vesicles, nucleus, and other organelles, and was associated with an increased AD, PRA, and myocardial expression of aldosterone receptor (2.6 fold) and aquaporin 1 (2.7 fold). In the 8 patients recovered from ICM, cardiomyocyte diameter reduced with disappearance of intracellular vacuoles and normalization of cytosol, nucleus, and cell organelles’ electron-density, along with down-regulation of aldosterone receptor and aquaporin-1. Conclusion: Human heart failure is associated with overexpression of myocardial aldosterone receptor and aquaporin-1. These molecular changes are paralleled by intracellular water overloading and cardiomyocyte swelling and dysfunction. Cardiac recovery is accompanied by down-regulation of hormonal receptors and normalization of cell structure and composition.

Treatment of virus negative chronic myocarditis: long term follow-up of TIMIC trial

Abstract Background The TIMIC trial demonstrated the short term efficacy of immunosuppression (IMS) in virus-negative inflammatory cardiomyopathy. However long term outcome of patients responsive to this treatment is still unclear. Purpose To assess the long term outcome of patients enrolled in the TIMIC trial. Methods The 85 (51 M, 34 F, 42.7±14.7 years) patients with endomyocardial biopsy proven virus-negative chronic inflammatory cardiomyopathy, enrolled in the TIMIC trial and treated either with prednisone and azathioprine (prednisone 1mg kg–1 day–1 for 4 weeks followed by 0.33 mg kg–1 day–1 for 5 months and azathioprine 2 mg kg–1 day–1 for 6 months) or placebo for 6 months, were evaluated in a long-term follow-up of up to 18 years (mean 15.2±2.0 SD, range 12–18 ys). Patients treated with placebo at the end of the 6-month enrollment because of the lack of improvement received IMS for further 6 months. Results At 6 months' follow-up 89% patients showed a significant improvement of left ventricular ejection fraction compared to baseline (LVEF rising from 27.0±5.7 to 46.2±6.4, % p&amp;lt;0.001). This improvement was maintained in the long term follow-up (LVEF 51.1±6.4%, p&amp;lt;0.001). Importantly, the 42 patients initially on placebo showed an improvement comparable to the branch on IMS either on short (EF from 27.7% to 46.8±6.2%) and on long term follow up (LVEF from 27.7% to 50.6±5.6%, p&amp;lt;0.001). Nine patients (11%) did not show a significant improvement of LVEF either at short and long term follow-up and among them 4 died and 2 had a cardiac transplantation. Five patients (5.8%) had a relapse of the cardiac inflammatory process that promptly responded to a new TIMIC dose treatment. Four of them (4F, 41.2±9.4 ys age) had concomitant autoimmune diseases and the relapse was within the first 2 years after IMS discontinuation. One patient (M, 56 ys) had a relapse after 10 years following a flu-like syndrome. Conclusion Immunosuppressive therapy of virus negative inflammatory cardiomyopathy warrants in susceptible patients a long term recovery of heart function with very low incidence of recurrences, that respond to a new TIMIC protocol dose treatment. Long term efficacy of TIMIC-IMS therapy Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): AIFA

Inflammation, iron and vitamin D metabolism in different cardiomyopathy aetiologies

Abstract Immune activation coincides with disturbances in iron and vitamin D metabolism in patients with cardiomyopathy. In this study, we investigated whether there are differences regarding immune activation, iron and vitamin D metabolism between the different cardiomyopathy aetiologies. Patients and methods: Parameters of iron metabolism (haemoglobin, iron, transferrin, transferrin saturation, ferritin, hepcidin), vitamin D metabolism (Ct-FGF23, parathormone, phosphate, vitamin D) and immune activation (C-reactive protein and neopterin) were determined in 149 patients (98 men, 51 women) with non-ischaemic cardiomyopathy. Results: Patients with amyloid cardiomyopathy presented with higher neopterin, ferritin and hepcidin levels than other cardiomyopathy aetiologies. Furthermore, they showed the highest rate of cardiovascular events. C-reactive protein levels were significantly higher in patients with inflammatory cardiomyopathy. Patients with virus positive cardiomyopathy presented with significantly higher ferritin and Ct-FGF23 levels compared to patients with virus negative inflammatory cardiomyopathy. Conclusion: This study indicates that there are some differences regarding the extent of immune activation and inflammation as well as alterations in iron metabolism disorders between different cardiomyopathy aetiologies. Further studies with larger patient cohorts are needed to investigate these findings more precisely.

P6450Plasminogen activator inhibitor type-1 (PAI-1) expression relates to the presence of myocardial inflammation in patients with nonischemic cardiomyopathy

Abstract Background Plasminogen activator inhibitor type-1 (PAI-1) is an important inhibitor of the fibrinolytic pathway and an acute phase reactant in response to inflammatory cytokines, resulting in thrombosis, arteriosclerosis, and tissue fibrosis. It has been identified as a potential biomarker for coronary artery disease and metabolic syndrom. However, so far, nothing is known about its importance in nonischemic cardiomyopathy. Aims To analyzed PAI-1 expression in patients with different forms of cardiomyopathies and evaluate possible influence of PAI-1-dependent pathomechanisms in patients with intramyocardial inflammation. Methods and results In this study, endomyocardial biopsies (EMBs) from 309 patients (mean age 48.0±13.9 years) with different forms of cardiomyopathies were enrolled, including 123 patients with dilated cardiomyopathy (DCM) (mean LVEF 28.01±9.06%) and 186 patients with EMB-proven virus-negative inflammatory cardiomyopathy (DCMi) (mean LVEF 31.76±14.14%). 10 patients (mean LVEF 60.50±4.76%) without viral or inflammation in EMB served as controls. Hemodynamic parameters were measured by catheterization and echocardiography. EMBs were performed at first admission after exclusion of ischemic of valvular heart disease. In EMBs PAI-1 was assessed by immunohistology including digital imaging analysis. PAI-1 expression was significantly higher in patients with DCMi in contrast to DCM patients and controls (0.517±2.20 vs. 0.187±0.598 vs. 0.023±0.032% Area Fraction; p=0.0002). PAI-expression correlates significantly with lymphocytic infiltrates (for CD3 r=0.56, p&lt;0.0001, and LFA-1 r=0.59, p&lt;0.0001). This was found to be independent of hemodynamic parameters, and age. Conclusion Myocardial inflammation is associated with a significant increase in PAI-1 expression in DCMi independently of the hemodynamic conditions. This new pathophysiological axis could be a potiential therapeutic target in future treatment stategies in DCMi.

P2574A 10 years observation study in patients with virus-negative inflammatory cardiomyopathy after immunosuppressive treatment

P2574A 10 years observation study in patients with virus-negative inflammatory cardiomyopathy after immunosuppressive treatment

Myocardial expression of Toll-like receptor 4 predicts the response to immunosuppressive therapy in patients with virus-negative chronic inflammatory cardiomyopathy.

We sought to determine whether myocardial expression of Toll-like receptor 4 (TLR4) may predict the response to immunosuppression. Endomyocardial biopsies from 237 patients with virus-negative inflammatory cardiomyopathy treated with immunosuppression were retrospectively examined for the expression of TLR4, differentiating those patients responding to immunosuppression (n = 193) from non-responder patients (n = 44). A semiquantitative evaluation of the immunoreactivity (grading from 0 to 4) for TLR4 and human leucocyte antigen (HLA)-DR was performed together with real-time PCR and western blot for TLR4. Cardiomyocyte apoptosis and necrosis was evaluated by in situ ligation with hairpin probes. A focal intense positive cytoplasmic immunostaining for TLR4 was observed in cardiomyocytes of all responders (P < 0.001 vs. non-responders). A grading 2 or above (2+) at baseline showed a sensitivity of 100% and 90.9% specificity with a positive predictive value of 98% as a predictor of an immunosuppression-positive response. Real-time PCR and western blot analysis for TLR4 were 4.3-fold and 4.6-fold higher, respectively, in responders vs. non-responders. Correlation between TLR4 grading and TLR4 mRNA molecular and protein expression was highly significant. HLA-DR did not discriminate between the two groups. Cardiomyocyte death by apoptosis was 3.7-fold higher in responders vs. non-responders and significantly correlated with TLR4 expression, while necrosis was comparable. Intensity of baseline TLR4 expression correlated with the variation in ejection fraction after 6 months of immunosuppression. TLR4 is highly expressed in human myocarditis responding to immunosuppression. It can be considered as a new sensitive marker in patient selection predicting a good response to immunosuppressive therapy.

THE ROLE OF IMMUNITY SUPPRESSION IN TREATMENT OF MYOCARDITIS

Regardless the common consensus on the benefits of immunity suppression in eosinophilic, granulomatous, giant cell and lymphocyte myocarditis, associated with systemic connective tissue diseases, and in rejection of transplanted heart, role of immune suppression therapy (IST) in treatment of lymphocyte inflammatory cardiomyopathy remains controversial. Previous retrospective studies revealed clinical improvement in 90% of patients with virus-negative inflammatory cardiomyopathy and absence of response or worsening of heart function in 85% of virus-positive inflammatory cardiomyopathy after immunity suppression. Other studies identified the enhanced expression of HLA in cardiomyocytes as an additional indicator of sensitivity of inflammatory cardiomyopathy to IST. Recently, the singlecenter randomized study was performed with double blind application of prednisone and azatioprin as addition to maintenance therapy in 85 patients with virus-negative inflammatory cardiomyopathy. The results of the study showed significant improvement of the left ventricle ejection fraction and decrease of the left ventricle dimensions in 88% patients among 43 of treated comparing to 42 patients on placebo demonstrated worsening of cardiac functioning in 83% cases (the TIMIC study). This data confirms the efficacy of immunity suppression in virus-negative inflammatory cardiomyopathy. Insufficiency of response in 12% cases undermines existence of non-revealed viruses and mechanisms of damage and inflammation, non-sensitive to immunity suppression. Restoring of cardiac function in patients actively responded to immunity suppression, was determined by inhibition of cardiomyocytes death and increase of cells proliferation with ad novo synthesized contractile material.

Cardiac migration of endogenous mesenchymal stromal cells in patients with inflammatory cardiomyopathy.

Introduction. Mesenchymal stromal cells (MSC) have immunomodulatory features. The aim of this study was to investigate the migration and homing potential of endogenous circulating MSC in virus negative inflammatory cardiomyopathy (CMi). Methods. In 29 patients with (n = 23) or without (n = 6) CMi undergoing endomyocardial biopsies (EMB), transcardiac gradients (TCGs) of circulating MSC were measured by flow cytometry from blood simultaneously sampled from aorta and coronary sinus. The presence of MSC in EMB, cardiac inflammation, and SDF-1α mRNA expression were detected via immunohistochemistry and real-time PCR. Results. MSC defined as CD45−CD34−CD11b−CD73+CD90+ cells accounted for 0.010 [0.0025–0.048]%/peripheral mononuclear cell (PMNC) and as CD45−CD34−CD11b−CD73+CD105+ cells for 0.019 [0.0026–0.067]%/PMNC, both with similar counts in patients with or without cardiac inflammation. There was a 29.9% (P < 0.01) transcardiac reduction of circulating MSC in patients with CMi, correlating with the extent of cardiac inflammation (P < 0.05, multivariate analysis). A strong correlation was found between the TCG of circulating MSC and numbers of MSC (CD45−CD34−CD90+CD105+) in EMB (r = −0.73, P < 0.005). SDF-1α was the strongest predictor for increased MSC in EMB (P < 0.005, multivariate analysis). Conclusions. Endogenous MSC continuously migrate to the heart in patients with CMi triggered by cardiac inflammation.

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 IFNβ, IFNγ, TNFα, IL-1β 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, IFNβ, 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.

Immunosuppressive therapy in myocarditis.

Although there is general agreement on the favorable effect of immunosuppression in eosinophilic, granulomatous, giant-cell myocarditis and in lymphocytic myocarditis associated with connective tissue disorders and with rejection of a transplanted heart, its therapeutic role in lymphocytic inflammatory cardiomyopathy (ICM) is still debated. Previous retrospective studies reported a relevant clinical benefit in 90% of patients with virus-negative ICM and no response or cardiac impairment in 85% of those with virus-positive ICM following immunosuppression. Other studies identified cardiomyocyte HLA upregulation as an additional indicator of ICM susceptibility to immunosuppressive therapy. Recently in a single-center randomized prospective double-blind trial using a combination of prednisone and azathioprine in addition to supportive treatment in 85 virus-negative ICM patients, a significant improvement in left ventricular (LV) ejection fraction and a significant reduction in LV dimensions in 88% of 43 treated patients compared with 42 patients receiving placebo who showed a cardiac impairment in 83% of cases (TIMIC study) was reported. These data confirm the efficacy of immunosuppression in virus-negative ICM. Lack of response in 12% of cases suggests the presence of unscreened viruses or mechanisms of damage and inflammation not susceptible to immunosuppression. Recovery of cardiac function in responders to immunosuppression was associated with inhibition of cardiomyocyte death, increased cell proliferation and with newly synthesized contractile material.

Toll-like receptor 4 is a marker of immune-mediated damage in patients with inflammatory cardiomyopathy

Purpose: Toll-like receptor 4 has been involved in the development of experimental autoimmune myocarditis. The aim of the study is to determine whether Toll-like receptor 4 plays a role in human myocarditis responsive to immunosuppression. Methods: Left ventricular endomyocardial biopsies from a subgroup of patients from the TIMIC trial were analyzed for the immunohistochemical expression of Toll-like receptor 4. In particular 18 patients (8 males,16 females, age 48.6±12.1 ys) with chronic (>6 months), virus negative (negative PCR for EV, AV, EBV, PVB19, HCV, Inf A/B, HSV1/2) inflammatory cardiomyopathy (LVEF 29.4±7.3%), responders to a 6 months treatment with prednisone and azathioprine were studied (group A). Controls were 10 left ventricular biopsies of patients with virus-negative inflammatory cardiomyopathy not responders to immunosuppression (group B) and 10 normal left ventricular surgical samples (group C). A semiquantitative evaluation of the immunoreactivity (grading from 0 to 4) was applied. Results: A focal intense positive cytoplasmic immunoreactivity was observed in cardiac myocytes of all patients in group A (grading 3.5±0.5) (panel A), with a statistically significant difference (p<0.001) compared with group B (grading 0.8±0.4) (panel B) and controls (0.2±0.4) (panel C). ![Figure][1] Conclusion: Toll-like receptor 4 is highly expressed in human immune-mediated myocarditis. It can be considered as a new marker in patient selection for immunosuppressive/immunomodulatory therapy. [1]: pending:yes

Immunosupressive and antiviral therapy of virus-positive and virus-negative inflammatory cardiomyopathy: short-term results and outcomes

Immunosupressive and antiviral therapy of virus-positive and virus-negative inflammatory cardiomyopathy: short-term results and outcomes

ADIPONECTIN PROMOTES COXSACKIEVIRUS B3 MYOCARDITIS BY SUPPRESSING THE ACUTE ANTIVIRAL IMMUNE RESPONSE

Adiponectin (APN) is an immunomodulatory adipocytokine that modulates outcome in patients with virus negative inflammatory cardiomyopathy and mice with autoimmune myocarditis. Moreover, APN controls antigen specific T cell responses and viral replication in hepatitis. Here, we investigated whether

Immunosuppressive therapy in virus-negative inflammatory cardiomyopathy

While there is general agreement on the favorable impact of immunosuppression in eosinophilic, granulomatous, giant cell and lymphocytic myocarditis and with inflammatory myocardial disease associated with connective tissue disorders or with rejection of a transplanted heart, its therapeutic role in the treatment of lymphocytic inflammatory cardiomyopathy (ICM) is still debated. Previous retrospective studies reported a relevant (≥ 10% left ventricular ejection fraction) clinical benefit in 90% of patients with virus-negative ICM and no cardiac impairment in 85% of patients with virus-positive ICM following immunosuppression. Some studies identified cardiomyocyte HLA up-regulation as an additional indicator of ICM susceptibility to immunosuppressive therapy. Recently in a single-center randomized prospective double-blind trial using a combination of prednisone (1mg/kg per day for 4 weeks followed by 0.33mg/kg per day for 5 months) and azathioprine (2mg/kg per day for 6 months) in addition to supportive treatment in 85 virus-negative ICM patients, a significant improvement of left ventricular ejection fraction and a significant reduction of left ventricular dimensions in 88% of 43 treated patients was reported when compared to 42 patients receiving placebo who showed a cardiac impairment initially in 83% of cases (TIMIC study). These data confirm the efficacy of immunosuppression in virus-negative ICM. The lack of response in 12% of cases suggests that the missing response might be due to the presence of viruses which were not screened for or to mechanisms of damage and inflammation not susceptible to immunosuppression. The recovery of cardiac function in responders to immunosuppression was associated with the inhibition of cardiomyocyte death, an increase of cell proliferation and with newly synthesized contractile material.

Randomized study on the efficacy of immunosuppressive therapy in patients with virus-negative inflammatory cardiomyopathy: the TIMIC study

Randomized study on the efficacy of immunosuppressive therapy in patients with virus-negative inflammatory cardiomyopathy: the TIMIC study

Randomized study on the efficacy of immunosuppressive therapy in patients with virus-negative inflammatory cardiomyopathy: the TIMIC study

To evaluate the efficacy of immunosuppression in virus-negative inflammatory cardiomyopathy. This randomized, double-blind, placebo-controlled study included 85 patients with myocarditis and chronic (>6 months) heart failure unresponsive to conventional therapy, with no evidence of myocardial viral genomes. Patients received either prednisone 1 mg kg(-1) day(-1) for 4 weeks followed by 0.33 mg kg(-1) day(-1) for 5 months and azathioprine 2 mg kg(-1) day(-1) for 6 months (43 patients, Group 1) or placebo (42 patients, Group 2) in addition to conventional therapy for heart failure. Primary outcome was the 6 month improvement in left-ventricular function. Group 1 showed a significant improvement of left-ventricular ejection fraction and a significant decrease in left-ventricular dimensions and volumes compared with baseline. None of Group 2 patients showed improvement of ejection fraction, that significantly worsened compared with baseline. No major adverse reaction was registered as a result of immunosuppression. These data confirm the efficacy of immunosuppression in virus-negative inflammatory cardiomyopathy. Lack of response in 12% of cases suggests the presence of not screened viruses or mechanisms of damage and inflammation not susceptible to immunosuppression.