Fragestellung Nuclear factor kappa-B (NfκB) wird in präklinischen Studien eine wichtige Rolle bei kardialer Fibrose und Kardiomyopathien zugeschrieben. Die Aktivierung von NfκB, unter anderem durch geschädigte Mitochondrien, triggert inflammatorische Signalwege im Myokard. Es ist jedoch unklar, ob ein Zusammenhang von NfκB mit der Mitochondrienfunktion des Myokards bei nicht-ischämischer Diabetes-assoziierter Kardiomyopathie besteht. Wir prüften, ob die ventrikuläre myokardiale NfκB-Expression im Menschen (1) durch Typ 2 Diabetes Mellitus (T2DM) induziert wird und (2) mit einer eingeschränkten myokardialer Mitochondrienfunktion assoziiert ist.

Abstract Human herpesvirus 6A and 6B (HHV-6) can integrate into the germline, and as a result, ∼70 million people harbor the genome of one of these viruses in every cell of their body. Until now, it has been largely unknown if 1) these integrations are ancient, 2) if they still occur, and 3) whether circulating virus strains differ from integrated ones. Here, we used next-generation sequencing and mining of public human genome data sets to generate the largest and most diverse collection of circulating and integrated HHV-6 genomes studied to date. In genomes of geographically dispersed, only distantly related people, we identified clades of integrated viruses that originated from a single ancestral event, confirming this with fluorescent in situ hybridization to directly observe the integration locus. In contrast to HHV-6B, circulating and integrated HHV-6A sequences form distinct clades, arguing against ongoing integration of circulating HHV-6A or “reactivation” of integrated HHV-6A. Taken together, our study provides the first comprehensive picture of the evolution of HHV-6, and reveals that integration of heritable HHV-6 has occurred since the time of, if not before, human migrations out of Africa.

Nuclear factor kappa-B (NFkB) has been postulated to induce cardiac fibrosis and dysfunction in heart failure and triggers inflammatory pathways. NFkB can be induced by damaged mitochondria. Its association with myocardial mitochondrial respiratory function in non-ischemic diabetes-related heart failure in humans is yet unclear. We hypothesized that human ventricular myocardial NFkB expression (i) is increased by type 2 diabetes mellitus (T2DM), and (ii) relates to reduced myocardial mitochondrial respiration. Heart transplant recipients with or without T2DM (as determined by oral glucose tolerance test), about to undergo post-transplant surveillance endomyocardial biopsies, were included, if they had received hearts from donors without T2DM. Thus, time since transplantation equaled diabetes-exposure of the transplanted hearts (2.9±2.4 years). We assessed normalized NFkB p105 subunit (NFkB1) mRNA expression using real-time PCR and myocardial mitochondrial respiration using high-resolution respirometry. Study participants (T2DM: n=17, Non-DM: n=32) had no histological signs of allograft rejection, heart failure (left ventricular ejection fraction 66.2±6.5%) or coronary artery disease. Age and sex distribution were similar between Non-DM and T2DM (p=0.50; p=0.40, respectively). Myocardial NFkB1 expression was 60% higher in T2DM than in Non-DM (0.28 [0.21-0.44] vs. 0.45 [0.29-0.71] arbitrary units, p<0.05). Fasting blood glucose, but not fasting free fatty acids (p=0.55), related to myocardial NFkB1 mRNA expression (r=0.31; p<0.05). NFkB1 expression related inversely to state 3 respiration of fatty acids and glutamate (r=-0.33; p<0.05), suggesting impaired complex I-related respiration. Our findings support the concept of a key role of NFkB in diabetes-related heart failure via altered myocardial respiratory function in humans. NFkB1 might therefore serve as a future target for treating diabetes-related heart disease. Disclosure E. Zweck: None. D. Scheiber: None. T. Jelenik: Employee; Self; Boehringer Ingelheim Pharmaceuticals, Inc. P. Horn: None. D. Pesta: None. D. Lassner: None. H. Schultheiss: None. U. Boeken: None. P. AKhyari: None. A. Lichtenberg: None. M. Kelm: None. M. Roden: Advisory Panel; Self; Servier. Board Member; Self; Poxel SA. Consultant; Self; Eli Lilly and Company, Gilead Sciences, Inc., ProSciento, TARGET PharmaSolutions. Research Support; Self; Boehringer Ingelheim International GmbH, Novartis Pharma K.K., Sanofi US. Speaker’s Bureau; Self; Novo Nordisk A/S. R. Westenfeld: None. J. Szendroedi: None. Funding German Research Council (SFB1116); Heinrich-Heine University

Erythroparvovirus (B19V) genomes have been detected in various organs of infected individuals including endothelial cells of the heart muscle. However, the role of B19V as a causative pathogen of myocardial damage is still unknown. The majority of reports focus on the presence of viral DNA ignoring proof of viral RNAs as important markers for viral activity. During this study, we established (RT-) qPCR to characterize expression of B19V RNAs (NS1 and VP1/2) in endomyocardial biopsies (EMBs) of 576 patients with unexplained heart failure. 403/576 (70%) EMBs were positive for B19V DNA. B19V mRNAs NS1 and/or VP1/2, indicating viral activity, could be detected in 38.5% of B19V DNA positive samples using the newly established B19V RT-PCRs. 22.1% of samples were characterized by only NS1 mRNA detection while 6.0% revealed only VP1/2 mRNA expression. Detection of both intermediates was successful in 10.4% of samples. Applying the molecular testing, our study revealed that a high proportion (38.5%) of B19V DNA positive EMBs was characterized by viral transcriptional activity. Further prospective studies will evaluate relevance of viral transcription intermediates as a diagnostic marker to differentiate between latent B19V infection and clinically relevant transcriptionally active B19V-infection of the heart muscle.

The lifetime risk of developing heart failure is approximately 20%, and survival rates remain poor. Myocardial mitochondrial function has been suggested to play a pivotal role in heart failure pathophysiology. Human studies on ex vivo mitochondrial function have mostly been limited to atrial tissue obtained during open heart surgery and have provided contradictory results. This study aimed at measuring myocardial mitochondrial function in transcatheter ventricular endomyocardial biopsies and assessing the relationship between oxidative capacity and heart function. We enrolled 40 heart failure patients undergoing ventricular assist device surgery or heart transplantation (34 males, age 57 ± 11 years, body mass index 26.6 ± 4.8 kg/m 2 ) and 29 heart transplant recipients of comparable age and body mass index with normal left ventricular function undergoing surveillance biopsies (23 males, 57 ± 12 years, body mass index 26.2 ± 4.1 kg/m 2 ). High-resolution respirometry was established in the myocardium to measure oxidative capacity ex vivo. The mitochondrial oxidative capacity was 90% higher in ventricular compared to atrial tissues ( n = 11, p < 0.01) of explanted hearts. Respiration rates were comparable in ventricular samples of heart failure patients obtained during open heart surgery by standard tissue preparation or ex vivo endomyocardial biopsy ( r = 0.9988, p < 0.0001, n = 8), and the mitochondrial oxidative capacity in samples from these patients remained stable for 8 h when stored in either of two common preservation buffers. The oxidative capacity was 44% lower in heart failure than in transplant recipients (67 ± 3 vs. 97 ± 5 pmol/[s mg], p < 0.0001) and correlated positively with heart function ( r = 0.49, p < 0.01). High-resolution respirometry of ventricular tissue is feasible in transcatheter biopsies, facilitating clinical studies on myocardial mitochondrial function in patients not undergoing heart surgery.

Aims: Human parvovirus B19 (B19V) infection directly induces apoptosis and modulates CXCR4 expression of infected marrow-derived circulating angiogenic cells (CACs). This leads to dysfunctional endogenous vascular repair. Treatment for B19V-associated disease is restricted to symptomatic treatment. Telbivudine, a thymidine analogue, established in antiviral treatment for chronic hepatitis B, modulates pathways that might influence induction of apoptosis. Therefore, we tested the hypothesis of whether telbivudine influences B19V-induced apoptosis of CAC. Methods and Results: Pretreatment of two CAC-lines, early outgrowth endothelial progenitor cells (eo-EPC) and endothelial colony-forming cells (ECFC) with telbivudine before in vitro infection with B19V significantly reduced active caspase-3 protein expression (−39% and −40%, both p < 0.005). Expression of Baculoviral Inhibitor of apoptosis Repeat-Containing protein 3 (BIRC3) was significantly downregulated by in vitro B19V infection in ECFC measured by qRT-PCR. BIRC3 downregulation was abrogated with telbivudine pretreatment (p < 0.001). This was confirmed by single gene PCR (p = 0.017) and Western blot analysis. In contrast, the missing effect of B19V on angiogenic gene expression postulates a post-transcriptional modulation of CXCR4. Conclusions: We for the first time show a treatment approach to reduce B19V-induced apoptosis. Telbivudine reverses B19V-induced dysregulation of BIRC3, thus, intervening in the apoptosis pathway and protecting susceptible cells from cell death. This approach could lead to an effective B19V treatment to reduce B19V-related disease.

Aims Heart failure with preserved ejection fraction (HFpEF) and pathological cardiac aging share a complex pathophysiology, including extracellular matrix remodelling (EMR). Protease-activated receptor 2 (PAR2) deficiency is associated with EMR. The roles of PAR1 and PAR2 have not been studied in HFpEF, age-dependent cardiac fibrosis, or diastolic dysfunction (DD). Methods and results Evaluation of endomyocardial biopsies from patients with HFpEF (n = 14) revealed that a reduced cardiac PAR2 expression was associated with aggravated DD and increased myocardial fibrosis (r = -0.7336, P = 0.0028). In line, 1-year-old PAR2-knockout (PAR2ko) mice suffered from DD with preserved systolic function, associated with an increased age-dependent alpha-smooth muscle actin expression, collagen deposition (1.7-fold increase, P = 0.0003), lysyl oxidase activity, collagen cross-linking (2.2-fold increase, P = 0.0008), endothelial activation, and inflammation. In the absence of PAR2, the receptor-regulating protein caveolin-1 was down-regulated, contributing to an augmented profibrotic PAR1 and transforming growth factor beta (TGF-beta)-dependent signalling. This enhanced TGF-beta/PAR1 signalling caused N-proteinase (ADAMTS3) and C-proteinase (BMP1)-related increased collagen I production from cardiac fibroblasts (CFs). PAR2 overexpression in PAR2ko CFs reversed these effects. The treatment with the PAR1 antagonist, vorapaxar, reduced cardiac fibrosis by 44% (P = 0.03) and reduced inflammation in a metabolic disease model (apolipoprotein E-ko mice). Patients with HFpEF with upstream PAR inhibition via FXa inhibitors (n = 40) also exhibited reduced circulating markers of fibrosis and DD compared with patients treated with vitamin K antagonists (n = 20). Conclusions Protease-activated receptor 2 is an important regulator of profibrotic PAR1 and TGF-beta signalling in the heart. Modulation of the FXa/FIIa-PAR1/PAR2/TGF-beta-axis might be a promising therapeutic approach to reduce HFpEF.

Aims The coxsackievirus B3 (CVB3) mouse myocarditis model is the standard model for investigation of virus-induced myocarditis but the pancreas, rather than the heart, is the most susceptible organ in mouse. The aim of this study was to develop a CVB3 mouse myocarditis model in which animals develop myocarditis while attenuating viral infection of the pancreas and the development of severe pancreatitis. Methods and results We developed the recombinant CVB3 variant H3N-375TS by inserting target sites (TS) of miR-375, which is specifically expressed in the pancreas, into the 30UTR of the genome of the pancreo- and cardiotropic CVB3 variant H3. In vitro evaluation showed that H3N-375TS was suppressed in pancreatic miR-375-expressing EndoC-beta H1 cells >5 log10, whereas its replication was not suppressed in isolated primary embryonic mouse cardiomyocytes. In vivo, intraperitoneal (i.p.) administration of H3N-375TS to NMRI mice did not result in pancreatic or cardiac infection. In contrast, intravenous (i.v.) administration of H3N-375TS to NMRI and Balb/C mice resulted in myocardial infection and acute and chronic myocarditis, whereas the virus was not detected in the pancreas and the pancreatic tissue was not damaged. Acute myocarditis was characterized by myocardial injury, inflammation with mononuclear cells, induction of proinflammatory cytokines, and detection of replicating H3N-375TS in the heart. Mice with chronic myocarditis showed myocardial fibrosis and persistence of H3N-375TS genomic RNA but no replicating virus in the heart. Moreover, H3N-375TS infected mice showed distinctly less suffering compared with mice that developed pancreatitis and myocarditis after i.p. or i.v application of control virus. Conclusion In this study, we demonstrate that by use of the miR-375-sensitive CVB3 variant H3N-375TS, CVB3 myocarditis can be established without the animals developing severe systemic infection and pancreatitis. As the H3N-375TS myocarditis model depends on pancreas-attenuated H3N-375TS, it can easily be used in different mouse strains and for various applications.

Abstract Background Myocarditis is defined by inflammatory involvement of the myocardium, either histologically by evidence of myocardial necrosis and cellular infiltration on endomyocardial biopsy (EMB), or non-invasively by presence of myocardial oedema using tissue mapping with cardiovascular magnetic resonance (CMR). Objective: to undertake intra-individual comparisons of EMB vs. CMR diagnostic algorithms of myocardial inflammation, as well as against an independent gold-standard of myocardial injury, high-sensitive troponin (hs-TropT). Methods Prospective multicentre study of consecutive patients (n=109) with clinical diagnosis of myocarditis. EMBs were analysed by 2 reference centres using the ESC diagnostic and their local algorithms. The CMR criteria used sequence-specific cut-offs for native T1 and T2 (standard deviation, SD); myocardial inflammation T1 ≥2SD, T2 ≥2SD and no inflammation: T1 and T2<2SD, with subcategories for acute/high-grade: T1 ≥5SD, T2 ≥2SD; chronic/low-grade: T1 ≥2SD, T2 ≥2SD; healed: T1 <2SD, T2 <2SD but myocardial impairment and non-inflammatory cardiomyopathy: T1 ≥2SD, T2 <2SD. Results The agreement between ESC criteria and CMR criteria (AUC: 0.56, p=0.381) was poor. There was a significant agreement between myocardial injury (hs-TropT ≥13.9 ng/L) and CMR criteria (AUC: 0.84, p<0.001), but not ESC algorithm. hs-TropT levels had significant associations with native T1 and T2 (r=0.37 and 0.35, p<0.001), but not with immunohistochemical inflammatory markers. Viral presence was similarly proportioned between inflammatory/non-inflammatory subjects, irrespective of the algorithm. AUC of CMR and EMB versus hs-TroponinT Conclusions Poor agreement between CMR and EMB-based diagnostic algorithms suggests non-overlapping definitions of myocardial inflammatory involvement. Excellent agreement between CMR algorithm and hs-TropT reiterates its high sensitivity for inflammatory myocardial injury. Acknowledgement/Funding 1. National Institute for Health Research (NIHR) Biomedical Research Centre 2. German Centre for Cardiovascular Research (DZHK)

Myocarditis is often associated with parvovirus B19 (B19V) persistence, which can induce vascular damage. Based on the antiviral and anti‐inflammatory properties of telbivudine, we aimed to evaluate its efficacy to protect B19V‐infected endothelial cells in vitro and to treat chronic lymphocytic myocarditis patients with B19V transcriptional activity.

Rationale: Remodeling and fibrosis of the right ventricle (RV) may cause RV dysfunction and poor survival in patients with pulmonary hypertension. Objectives: To investigate the consequences of RV fibrosis modulation and the accompanying cellular changes on RV function. Methods: Expression of fibrotic markers was assessed in the RV of patients with pulmonary hypertension, the murine pulmonary artery banding, and rat monocrotaline and Sugen5416/hypoxia models. Invasive hemodynamic and echocardiographic assessment was performed on galectin-3 knockout or inhibitor-treated mice. Measurements and Main Results: Established fibrosis was characterized by marked expression of galectin-3 and an enhanced number of proliferating RV fibroblasts. Galectin-3 genetic and pharmacologic inhibition or antifibrotic treatment with pirfenidone significantly diminished RV fibrosis progression in the pulmonary artery banding model, without improving RV functional parameters. RV fibrotic regions were populated with mesenchymal cells coexpressing vimentin and PDGFRα (platelet-derived growth factor receptor-α), but generally lacked αSMA (α-smooth muscle actin) positivity. Serum levels of galectin-3 were increased in patients with idiopathic pulmonary arterial hypertension but did not correlate with cardiac function. No changes of galectin-3 expression were observed in the lungs. Conclusions: We identified extrapulmonary galectin-3 as an important mediator that drives RV fibrosis in pulmonary hypertension through the expansion of PDGFRα/vimentin-expressing cardiac fibroblasts. However, interventions effectively targeting fibrosis lack significant beneficial effects on RV function.

Aims:Foxo3 is a transcription factor involved in cell metabolism, survival, and inflammatory disease. However, mechanistic insight in Foxo3 effects is still limited. Here, we investigated the role of Foxo3 on natural killer (NK) cell responses and its effects in viral myocarditis.Methods and results:Effects of Foxo3 on viral load and immune responses were investigated in a model of coxsackie virus B3 myocarditis in wild-type (WT) and Foxo3 deficient mice. Reduced immune cell infiltration, viral titres, and pro-inflammatory cytokines in cardiac tissue were observed in Foxo3-/- mice 7 days post-infection (p.i.). Viral titres were also attenuated in hearts of Foxo3-/- mice at Day 3 while interferon-γ (IFNγ) and NKp46 expression were up-regulated suggesting early viral control by enhanced NK cell activity. CD69 expression of NK cells, frequencies of CD11b+CD27+ effector NK cells and cytotoxicity of Foxo3-/- mice was enhanced compared to WT littermates. Moreover, microRNA-155 expression, essential in NK cell activation, was elevated in Foxo3-/- NK cells while its inhibition led to diminished IFNγ production. Healthy humans carrying the longevity-associated FOXO3 single nucleotide polymorphism (SNP) rs12212067 exhibited reduced IFNγ and cytotoxic degranulation of NK cells. Viral inflammatory cardiomyopathy (viral CMI) patients with this SNP showed a poorer outcome due to less efficient virus control.Conclusion:Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.

Enteroviral cardiomyopathy is a life-threatening disease, and detection of enterovirus (EV) RNA in the initial endomyocardial biopsy is associated with adverse prognosis and increased mortality. Some patients with EV infection may spontaneously eliminate the virus and recover, whereas those with virus persistence deteriorate and progress to heart failure. Interferon-beta (IFN-β) therapy eliminates the virus, resulting in increased survival of treated patients. CCR5 is expressed on antigen-presenting cells (both macrophages and dendritic cells) and immune effector cells (T-lymphocytes with memory/effector phenotype and natural killer cells). Its 32-bp deletion (CCR5del32) is the most frequent human coding sequence mutation. This study addresses the correlation of CCR5 polymorphism to the clinical course of EV infection and the necessity for IFN-β treatment.