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Human herpesvirus 6 (HHV-6) A and B are two betaherpesviruses that are associated with many conditions including roseola, drug-induced hypersensitivity syndrome, limbic encephalitis, and myocarditis. HHV-6 is integrated in the germline [chromosomically integrated HHV-6 (ciHHV-6)] in ∼0.8% of the human population. To date, the prevalence, species distribution, and treatment responses of ciHHV-6 are unknown for cardiac patients.We determined the prevalence of HHV-6 and ciHHV-6 genotypes in 1656 endomyocardial biopsies of patients with persisting unexplained symptoms of heart failure. Infection of cardiac tissue was identified by nested PCR, electron microscopy, and immunohistochemistry. Virus load and mRNA levels were followed in ciHHV-6 patients treated with ganciclovir. HHV-6 was detected in 273 of 1656 cardiac tissues (16.5%; HHV-6B, 98.2%, HHV-6A, 1.8%) by PCR. Nineteen of the 1656 patients (1.1%) presented with persistently high HHV-6 copy numbers indicative of ciHHV-6. Sequencing confirmed ciHHV-6A in seven patients (36.8%) which was considerably higher than detected in non-ciHHV-6 patients. Inheritance was demonstrated in three selected families, confirming ciHHV-6 chromosomal integration by PCR and fluorescence in situ hybridization. HHV-6 reactivation and chromosomal integration were confirmed in peripheral blood mononuclear cells and heart tissue. Virus particles were identified in degenerating myocytes and interstitial cells. Antiviral treatment abolished viral mRNA and ameliorated cardiac symptoms.Virus replication in cardiac tissue of ciHHV-6 heart failure patients suggests that ciHHV-6 reactivation causes persistence of unexplained heart failure symptoms. We demonstrated that antiviral treatment, effective in decreasing viral transcripts and clinical complaints of cardiomyopathies, is a new therapeutic option for ciHHV-6-associated diseases.

Parvovirus B19 infections are acquired by majority in the childhood and persist lifelong within bone marrow precursor cells and the vascular endothelium in a high percentage of the general population. Because many infected patients remain asymptomatic, the clinical has remained doubtful. The study

Human herpesvirus 6 (HHV-6) A and B are lymphotropic viruses with life-long persistence, primarily associated with non-cardiac diseases, and discussed as a possible etiologic factor of myocarditis and cardiomyopathy.To analyze the long-term spontaneous course of cardiac patients suffering from suspected inflammatory cardiomyopathy (CMi) with persisting HHV-6 A and B infections by follow-up biopsies.We prospectively evaluated patients (n=73) with biopsy-proven viral HHV-6 A and B infection in endomyocardial biopsies (EMBs), followed up by reanalysis of EMBs and left ventricular ejection fraction (LV-EF) measurements after a median period of 8.8 months (range 4-73 months). Beyond, we studied HHV-6 prevalence in isolated peripheral blood cells (PBCs) and HHV-6 species in EMBs. HHV-6 species-specific cellular infection sites within the myocardium were identified by immunohistochemistry (IHC).We identified 73 patients with cardiac HHV-6 A and B persistence or newly detected in follow-up EMB (95.0% B). Proof of HHV-6 in PBCs was primarily associated with A. Persistence of cardiac HHV-6 B genome was significantly associated with cardiac dysfunction at follow-up (LV-EF deteriorated from 58.2±16.0 to 51.8±17.2%, p0.001), and LV improvement was observed when HHV-6 B persistence resolved (LV-EF increased from 54.9±15.4 to 60.7±13.1%, p0.001).Persistence of cardiac HHV-6 B genomes was significantly associated with cardiac dysfunction, and hemodynamic parameters improved in association with HHV-6 B clearance.

Intramyocardial inflammation is considered an adverse prognostic factor in inflammatory cardiomyopathy (CMi). However, the precise nature of immune system factors relevant for the prediction of long-term course remains elusive. The aim of this study was to analyse the prognostic relevance of perforin in a large cohort of patients with CMi.We investigated 495 consecutive patients with suspected CMi, undergoing endomyocardial biopsies (EMBs), and examined haemodynamic measurements after a long follow-up period (interquartile range 10.2-37.1 months). In EMBs, myocardial inflammation was assessed by histology and immunohistology. At follow-up, 388 patients (Group I) showed stable mild dysfunction or significant improvement, with LVEF rising from 46.2 ± 14.8% to 64.3 ± 12.3% (P 0.0001). Lack of improvement of LV function or significant deterioration of LVEF from 42.1 ± 14.2% to 32.3 ± 11.6% (P 0.0001) was observed in 107 patients (Group II). Multivariable statistical analysis of LVEF and immunohistochemical parameters in all patients revealed that the single most important predictor of LVEF development was detection of perforin in EMBs, with an odds ratio (OR) of 7.922 [95% confidence interval (CI) 4.380-14.326; P 0.001] for deteriorating LVEF. Importantly, baseline LVEF (OR 0.962), LV end-diastolic diameter (OR 1.847), and other immmunohistochemical parameters (CD3, Mac-1, CD45R0, LFA-1, HLA-1, and ICAM-1) made minor or insignificant contributions to LVEF course in these 495 patients.In this EMB-based analysis of the long-term course of CMi we identified, for the first time, that detection of perforin in the myocardium is a key predictor of LVEF course.

Most patients with giant cell myocarditis (GCM) present with acute fulminant heart failure due to rapidly progressive myocyte necrosis [1, 2]. Immunosuppressive therapies, including high-dose steroids and cyclosporine, have only modestly improved the prognosis in GCM [3, 4]. Morbidity and mortality remain high and many patients still require heart transplantation. GCM has been reported in association with autoimmune disorders. This supports an autoimmune etiologic role. Nevertheless, autoantigens in GCM remain poorly defined [5]. To gain insight into the pathogenesis of this mercurial disease, we studied patients with GCM with regard to histopathological appearances and gene expression pattern of inflammation in endomyocardial biopsies (EMB). Between January 2009 and November 2011 we verified eight patients with GCM in EMB (mean age 42 ± 8.6 years, 5 male/3 female) admitted to our hospital for evaluation of suspected myocarditis. EMBs were obtained from the right ventricular septum. Coronary artery disease and other possible causes of myocardial dysfunction (i.e. valvular heart disease) had been excluded by angiography prior to EMB in all patients. Other comorbidities such as uncontrolled hypertension and diabetes, increased alcohol or drug uptake as well as systemic or autoimmune diseases were also excluded. Histology was developed by hematoxylin eosin (HE) and Elastica-van-Gieson (EvG) staining in light microscopy. For immunohistological evaluation, antibodies were used as follows: CD3 lymphocytes (Dako, Glostrup, Denmark, dilution 1:25), CD11a/LFA-1 lymphocytes (Immuno Tools, Friesoythe, Germany, dilution 1:250), CD11b/Mac-1 macrophages (ImmunoTools, Friesoythe, Germany, dilution 1:500), HLA-1 (Dako, Glostrup, Denmark, dilution 1:2,000), ICAM-1/CD54 (ImmunoTools, Friesoythe, Germany, dilution 1:500), VCAM-1/CD106 (ImmunoTools, Friesoythe, Germany, dilution 1:1,000), and CD31 (Dako, Glostrup, Denmark, dilution 1:100). Nested PCR for cardiotropic viral genomes was performed and was negative for erythrovirus, entero and adenovirus, human herpes virus type 6 and Epstein–Barr virus. Differential gene expression for TLR5, TLR7, TLR8, CCL20 and CCR6 was determined by real-time PCR. 20 genderand age-matched patients (mean age 45.2 ± 9.2 years, 11 male/9 female) without evidence of inflammation in EMB served as controls. They were referred for evaluation of repeated chest discomfort but had no symptoms of heart failure. The diagnostic workup finally revealed that their complaints were non-cardiac in origin. The study was performed within the CRC Transregio 19 and was approved by the local ethics committees as well as by the committees of respective federal states. An informed written consent is obtained from each patient. Regarding the baseline characteristics of GCM patients, mean LVEF was significantly reduced, paralleled by an increase in LVEDD in contrast to controls (LVEF 37.5 ± 10.7 %, LVEDD 59.7 ± 3.1 mm vs. LVEF 67.6 ± 4.4 %, LVEDD 51.2 ± 2.1 mm, P \ 0.0001). All S. Elezkurtaj Institute of Pathology, Charite-University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany

Background. The diagnosis of acute myocarditis (AMC) and inflammatory cardiomyopathy (DCMi) can be difficult. Speckle tracking echocardiography with accurate assessments of regional contractility could have an outstanding importance for the diagnosis.Methods and Results.N=25patients with clinically diagnosed AMC who underwent endomyocardial biopsies (EMBs) were studied prospectively. Speckle tracking imaging was examined at the beginning and during a mean follow-up period of 6.2 months. In the acute phase patients had markedly decreased left ventricular (LV) systolic function (mean LV ejection fraction (LVEF)40.4±10.3%). At follow-up inn=8patients, inflammation persists, correlating with a significantly reduced fractional shortening (FS,21.5±6.0%) in contrast to those without inflammation in EMB (FS32.1±7.1%,P<0.05). All AMC patients showed a reduction in global systolic longitudinal strain (LS,−8.36±−3.47%) and strain rate (LSR,0.53±0.29 1/s). At follow-up, LS and LRS were significantly lower in patients with inflammation, in contrast to patients without inflammation (−9.4±1.4versus−16.8±2.0%,P<0.0001;0.78±0.4versus1.3±0.3 1/s). LSR and LS correlate significantly with lymphocytic infiltrates (for CD3r=0.7,P<0.0001, and LFA-1r=0.8,P<0.0001).Conclusion. Speckle tracking echocardiography is a useful adjunctive assisting tool for evaluation over the course of intramyocardial inflammation in patients with AMC and DCMi.

Parvovirus B19 is a frequent virus detected in endomyocardial biopsies of patients with clinically suspected myocarditis or dilated cardiomyopathy (DCM). Viruses often cause a more symptomatic disease with increased tissue injury if they become reactivated. A disease-specific differential expression of microRNAs (miRNAs) has been described in the regulation of replicating viruses. Analyzing patients with latent and reactivated B19V infection, we found 29 differentially regulated miRNAs and, in order to test whether predicted genes are differentially expressed, selected mRNAs were tested by TaqMan-QPCR.

Toll-like receptor 9 (TLR9) is a member of the innate immune system and has been shown to influence myocardial function, but its role in myocarditis is hitherto unknown. We therefore investigated whether or not TLR9 plays a role in this disease in coxsackievirus B3 (CVB3)-induced myocarditis in mice. Left ventricular (LV) function, cardiac immune cell infiltration, virus mRNA, and components of the TLR9 downstream pathway were investigated in TLR9-deficient [knockout (KO)] and wild-type (WT) mice after infection with CVB3. Murine cardiac TLR9 expression was significantly increased in WT mice with acute CVB3 infection but not in WT mice with chronic myocarditis. Furthermore, in the acute phase of CVB3-induced myocarditis, CVB3-infected KO mice displayed improved LV function associated with reduced cardiac inflammation indexed by reduced amounts of immune cells compared with CVB3-infected WT mice. In contrast, in the chronic phase, LV function and inflammation were not seen to differ among the infected groups. The cardioprotective effects due to TLR9 deficiency were associated with suppression of the TLR9 downstream pathway as indexed by reduced cardiac levels of the adapter protein myeloid differentiation factor (MyD)-88 and the proinflammatory cytokine TNF-α. In addition, TLR9 deficiency led to an activation of the antiviral cytokine interferon-β in the heart as a result from viral infection. In conclusion, the MyD88/TNF-α axis due to TLR9 activation in the heart contributes the development of acute myocarditis but not of chronic myocarditis.

Parvovirus B19 (B19V) has been identified as the most common virus in dilated cardiomyopathy (DCM) patients by PCR techniques. We investigated the detectability and the expression pattern of B19V proteins by immunohistology (IH) in endomyocardial biopsies (EMBs) from DCM patients, and its association with the standard proof of B19V genomes by nested PCR (nPCR.EMBs from 30 DCM patients were analyzed by nPCR for B19V genomes, and IH of B19V VP1/VP2 expression was carried out using the antibody clone R92F6. The specificity of this antibody was investigated on 293T cells transfected with pB19-M20. Positive anti-B19V IH staining (positive in n=14/46.6%) and nPCR proof of B19V genomes (positive in n=15/50%) were significantly associated (p=0.0003).Based on the B19V nPCR results, the sensitivity of anti-B19V-VP1/VP2 IH was 80.0%, and the specificity was 86.0%. B19V immunostaining was observed on interstitial cells in all IH positive cases, and was noted additionally on endothelial cells in 1, and on cardiomyocytes in 4 cases.IH detection of B19V VP1/VP2 expression provides a high association with the nPCR proof of B19V genomes in DCM patients. IH detection of B19V proteins enables a differentiation of B19V VP1/VP2 expressing cells within the myocardium.

Background— The hallmarks of diabetic cardiomyopathy are cardiac oxidative stress, intramyocardial inflammation, cardiac fibrosis, and cardiac apoptosis. Given the antioxidative, antiinflammatory, and antiapoptotic potential of high-density lipoprotein (HDL), we evaluated the hypothesis that increased HDL via gene transfer (GT) with human apolipoprotein (apo) A-I, the principal apolipoprotein of HDL, may reduce the development of diabetic cardiomyopathy. Methods and Results— Intravenous GT with 3×10 12 particles/kg of the E1E3E4-deleted vector Ad.hapoA-I , expressing human apoA-I, or Ad.Null , containing no expression cassette, was performed 5 days after streptozotocin (STZ) injection. Six weeks after apoA-I GT, HDL cholesterol levels were increased by 1.6-fold ( P Ad.Null vector (STZ- Ad.Null ). ApoA-I GT and HDL improved LV contractility in vivo and cardiomyocyte contractility ex vivo, respectively. Moreover, apoA-I GT was associated with decreased cardiac oxidative stress and reduced intramyocardial inflammation. In addition, compared with STZ- Ad.Null rats, cardiac fibrosis and glycogen accumulation were reduced by 1.7-fold and 3.1-fold, respectively ( P P P P P Ad.Null rats. HDL supplementation ex vivo reduced hyperglycemia-induced cardiomyocyte apoptosis by 3.4-fold ( P P Conclusion— ApoA-I GT reduced the development of streptozotocin-induced diabetic cardiomyopathy.

Angiotensin-(1–7) is associated with beneficial effects in cardiovascular diseases. In this study, we determined the effect of AVE0991, a nonpeptide angiotensin-(1–7) receptor agonist, on cardiac function in an animal model of diabetes mellitus type I. Diabetes was induced in Sprague–Dawley rats by a single injection of streptozotocin (70 mg/kg). Diabetic and non-diabetic animals were fed with AVE0991 (20 mg/kg per day) or control chow. Normoglycemic control chow- or AVE0991-fed rats served as controls ( n = 10/group). After five weeks, metabolic cage experiments were performed to assess metabolic parameters. Six weeks after induction of diabetes, cardiac function was monitored using a Millar-tip catheter system. AVE0991 had no effect on any of the investigated hemodynamic parameters under normoglycemic conditions. Hyperglycemia was comparable in diabetic animals with or without AVE0991 treatment. Diabetic control rats suffered from severe systolic dysfunction, indicated by a significant decrease in heart rate, left ventricular systolic pressure, systolic blood pressure and an impairment of left ventricular contractility. Administration of AVE0991 clearly rescued cardiac function under diabetic conditions as indicated by a normalisation of blood pressure and contractility parameters. Our data demonstrates a dominant beneficial impact of AVE0991 on the diabetic heart, implying a cardioprotective role for angiotensin-(1–7) under hyperglycemic conditions and thus pointing to new therapeutic strategies using angiotensin-(1–7) agonists to treat cardiovascular complications in diabetes mellitus.

Background: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity. Methods and results: Five days after a single injection of Dox (20 mg/kg; i.p.), left ventricular pressure–volume loops were measured in wild-type and TLR4-deficient mice (TLR4−/−) Dox-treated and control mice. Analyses of possible pathophysiological mechanisms were performed in left ventricular tissue and isolated myocytes, respectively. Dox injection resulted in an impairment of left ventricular function and neurohumoral activation, indexed by increased ET-1 expression. This was further associated with an increase in cardiac oxidative stress, inflammation and apoptosis, as indicated by an up-regulation of cardiac lipid peroxidation, TNF-α expression and enhanced content of TUNEL-positive cells. In contrast, TLR4−/−Dox mice showed improved left ventricular function with reduced oxidative and inflammatory stress response including reduced cardiac apoptosis. These results were found to be associated with an increase of GATA-4 expression. Conclusions: TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in Dox-induced cardiomyopathy.

The mechanisms contributing to diabetic cardiomyopathy, as well as the protective pathways of the kallikrein-kinin-system (KKS), are incompletely understood. In a kallikrein-overexpressing rat model of streptozotocin (STZ)-induced diabetic cardiomyopathy, we investigated the involvement of inflammatory pathways, endothelial dysfunction, and oxidative stress. Six weeks after STZ injection, impairment of left ventricular (LV) function parameters measured by a Millar-tip catheter (peak LV systolic pressure; dP/dtmax; dP/dtmin) was accompanied by a significant increment of ICAM-1 and VCAM-1 (CAMs) expression, as well as of beta2-leukocyte-integrins+ (CD18+, CD11a+, CD11b+) and cytokine (TNF-alpha and IL-1beta)-expressing infiltrates in male Sprague-Dawley (SD-STZ) rats compared with normoglycemic littermates. Furthermore, SD-STZ rats demonstrated a significant impairment of endothelium-dependent relaxation evoked by acetylcholine and significantly increased plasma TBARS (plasma thiobarbituric acid reactive substances) levels as a measure of oxidative stress. These diabetic cardiomyopathy-associated alterations were significantly attenuated (P

Diabetic cardiomyopathy includes fibrosis. Kallikrein (KLK) can inhibit collagen synthesis and promote collagen breakdown. We investigated cardiac fibrosis and left ventricular (LV) function in transgenic rats (TGR) expressing the human kallikrein 1 (hKLK1) gene in streptozotocin (STZ) - induced diabetic conditions. Six weeks after STZ injection, LV function was determined in male Sprague-Dawley (SD) rats and TGR(hKLK1) (n = 10/group) by a Millar tip catheter. Total collagen content ( Sirius Red staining) and expression of types I, III, and VI collagen were quantified by digital image analysis. SD-STZ hearts demonstrated significantly higher total collagen amounts than normoglycemic controls, reflected by the concomitant increment of collagen types I, III, and VI. This correlated with a significant reduction of LV function vs. normoglycemic controls. in contrast, surface-specific content of the extracellular matrix, including collagen types I, III, and VI expression, was significantly lower in TGR( hKLK1)- STZ, not exceeding the content of SD and TGR( hKLK1) controls. This was paralleled by a preserved LV function in TGR( hKLK1)- STZ animals. the kallikrein inhibitor aprotinin and the bradykinin (BK) B2 receptor antagonist icatibant reduced the beneficial effects on LV function and collagen content in TGR( hKLK1)- STZ animals. Transgenic expression of hKLK1 counteracts the progression of LV contractile dysfunction and extracellular matrix remodeling in STZ-induced diabetic cardiomyopathy via a BK B2 receptor-dependent pathway. Free Univ Berlin, Charite Univ Med, Dept Cardiol & Pneumonol, D-12220 Berlin, Germany Universidade Federal de São Paulo, Dept Biophys, São Paulo, Brazil Max Delbruck Ctr Mol Med, Berlin, Germany Universidade Federal de São Paulo, Dept Biophys, São Paulo, Brazil Web of Science

We attempted to develop a digital image analysis (DIA) system for endomyocardial biopsies (EMBs) to reliably quantify a) biopsy quality, b) immunohistochemically-marked infiltrates, and c) cell adhesion molecules (CAMs) in relation to net heart area (HA) for the semi-automated diagnosis of inflammatory cardiomyopathy (InfCM).140 EMBs from dilated cardiomyopathy (DCM) patients and 14 autopsy heart samples (controls) were immunostained for T-lymphocytes (CD2, CD3, CD4, CD8), beta(2)-integrin+ infiltrates (CD18, LFA-1, Mac-1) and CAMs (immunoglobulin superfamily: ICAM-1, HLA class I, HLA DR, VCAM-1, CD58; selectins: CD62E and CD62P; and the beta(1)-integrin chain CD29). EMB quality was assessed visually on a three-point scale. Infiltrates were quantified visually (per hpf) and by DIA (per mm2 HA). CAM expression was evaluated semiquantitatively and by DIA (area fraction [AF]: stained area relative to HA).DIA-evaluated HA correlated significantly with the visual assessment of EMB quality. The visual evaluation of both infiltrates and CAMs correlated significantly with the respective DIA-based quantification. DIA-quantified CAM-AF and infiltrates were discriminated by the CAM classification (CAMs+: n=87; 62%) compared to controls. DIA-quantified CAM immunoreactivity correlated significantly with the DIA-quantified counter-receptor+ infiltrates. DIA evaluation of biopsy quality, infiltrates, and CAMs was devoid of inter- and intraobserver variability.The DIA system presented here enables standardized and observer-independent assessment of EMB quality and intramyocardial inflammation (density of infiltrates and CAM expression) in DCM biopsies related to HA. Our data confirm that endothelial CAM count and counter-receptor+ immunocompetent infiltration are interdependent pathogenic and diagnostic hallmarks of InfCM.

The protective effects of high-density lipoprotein (HDL) under lipopolysaccharide (LPS) conditions have been well documented. Here, we investigated whether an effect of HDL on Toll-like receptor 4 (TLR4) expression and signalling may contribute to its endothelial-protective effects and to improved survival in a mouse model of LPS-induced inflammation and lethality. HDL cholesterol increased 1.7-fold (p