Epstein-Barr Virus Lytic Transcripts Correlate with the Degree of Myocardial Inflammation in Heart Failure Patients.

The Epstein-Barr virus (EBV) is frequently found in endomyocardial biopsies (EMBs) from patients with heart failure, but the detection of EBV-specific DNA has not been associated with progressive hemodynamic deterioration. In this paper, we investigate the use of targeted next-generation sequencing (NGS) to detect EBV transcripts and their correlation with myocardial inflammation in EBV-positive patients with heart failure with reduced ejection fraction (HFrEF). Forty-four HFrEF patients with positive EBV DNA detection and varying degrees of myocardial inflammation were selected. EBV-specific transcripts from EMBs were enriched using a custom hybridization capture-based workflow and, subsequently, sequenced by NGS. The short-read sequencing revealed the presence of EBV-specific transcripts in 17 patients, of which 11 had only latent EBV genes and 6 presented with lytic transcription. The immunohistochemical staining for CD3+ T lymphocytes showed a significant increase in the degree of myocardial inflammation in the presence of EBV lytic transcripts, suggesting a possible influence on the clinical course. These results imply the important role of EBV lytic transcripts in the pathogenesis of inflammatory heart disease and emphasize the applicability of targeted NGS in EMB diagnostics as a basis for specific treatment.

MicroRNAs as novel biomarkers and potential therapeutic options for inflammatory cardiomyopathy.

AIMS: Inflammation of the heart is a complex biological and pathophysiological response of the immune system to a variety of injuries leading to tissue damage and heart failure. MicroRNAs (miRNAs) emerge as pivotal players in the development of numerous diseases, suggesting their potential utility as biomarkers for inflammation and as viable candidates for therapeutic interventions. The primary aim of this investigation was to pinpoint and assess particular miRNAs in individuals afflicted by virus-negative inflammatory dilated cardiomyopathy (DCMi). METHODS AND RESULTS: The study involved the analysis of 152 serum samples sourced from patients diagnosed with unexplained heart failure through endomyocardial biopsy. Among these samples, 38 belonged to DCMi patients, 24 to DCM patients, 44 to patients displaying inflammation alongside diverse viral infections, and 46 to patients solely affected by viral infections without concurrent inflammation. Additionally, serum samples from 10 healthy donors were included. The expression levels of 754 distinct miRNAs were evaluated using TaqMan OpenArray. MiR-1, miR-23, miR-142-5p, miR-155, miR-193, and miR-195 exhibited exclusive down-regulation solely in DCMi patients (P < 0.005). These miRNAs enabled effective differentiation between individuals with inflammation unlinked to viruses (DCMi) and all other participant groups (P < 0.005), boasting a specificity surpassing 86%. CONCLUSIONS: The identification of specific miRNAs offers a novel diagnostic perspective for recognizing intramyocardial inflammation within virus-negative DCMi patients. Furthermore, these miRNAs hold promise as potential candidates for tailored therapeutic strategies in the context of virus-negative DCMi.

Advancing Precision Medicine in Myocarditis: Current Status and Future Perspectives in Endomyocardial Biopsy-Based Diagnostics and Therapeutic Approaches.

The diagnosis and specific and causal treatment of myocarditis and inflammatory cardiomyopathy remain a major clinical challenge. Despite the rapid development of new imaging techniques, endomyocardial biopsies remain the gold standard for accurate diagnosis of inflammatory myocardial disease. With the introduction and continued development of immunohistochemical inflammation diagnostics in combination with viral nucleic acid testing, myocarditis diagnostics have improved significantly since their introduction. Together with new technologies such as miRNA and gene expression profiling, quantification of specific immune cell markers, and determination of viral activity, diagnostic accuracy and patient prognosis will continue to improve in the future. In this review, we summarize the current knowledge on the pathogenesis and diagnosis of myocarditis and inflammatory cardiomyopathies and highlight future perspectives for more in-depth and specialized biopsy diagnostics and precision, personalized medicine approaches.

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

OBJECTIVE: Inflammatory 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 inflammation.The effects of the gain of function FOXO3A single-nucleotide polymorphism (SNP) rs12212067 on inflammation and outcome were studied in a cohort of patients with inflammatory dilated cardiomyopathy (DCMi) in relation to cardiac viral presence. METHODS: Distribution of the SNP was determined in virus-positive and virus-negative DCMi patients and in control subjects without myocardial pathology. Baseline and outcome data were compared in 221 virus-negative patients with detection of cardiac inflammation and reduced LVEF according to their carrier status of the SNP. RESULTS: Distribution of SNP rs12212067 did not differ between virus-positive (n=22, 19.3%), virus-negative (n=45, 20.4 %) and control patients (n=18, 23.4 %), indicating the absence of susceptibility for viral infection or inflammation per se (p=0.199). Patients in the virus-negative DCMi group were characterised by reduced LVEF 35.5% (95% CI) 33.5 to 37.4) and increased LVEDD (LV end-diastolic diameter) 59.8 mm (95% CI 58.5 to 61.2). Within the group, SNP and non-SNP carriers had similarly impaired LVEF 39.2% (95% CI 34.3% to 44.0%) vs 34.5% (95% CI 32.4 to 36.5), p=0.083, and increased LVEDD 58.9 mm (95% CI 56.3 to 61.5) vs 60.1 mm (95% CI 58.6 to 61.6), p=0.702, respectively. The number of inflammatory infiltrates was not different in both SNP groups at baseline. Outcome after 6 months showed a significant improvement in LVEF and clinical symptoms in SNP rs12212067 carriers 50.9% (95% CI 45.4 to 56.3) versus non-SNP carriers 41.7% (95% CI 39.2 to 44.2), p≤0.01. The improvement in clinical symptoms and LVEF was associated with a significant reduction in cardiac inflammation (ΔCD45RO+ p≤0.05; ΔMac-1+ p≤0.05; ΔLFA-1+ p≤0.01; ΔCD54+ p≤0.01) in the SNP cohort versus non-SNP cohort, respectively. Subgroup analyses identified ΔMac-1+, ΔLFA-1+, ΔCD3+ and Δperforin+ as predictors for improvement in cardiac function in SNP-positive patients. CONCLUSION: FOXO3A might act as modulator of the cardiac immune response, diminishing cardiac inflammation and injury in pathogen-negative DCMi.

Intramyocardial Inflammation after COVID-19 Vaccination: An Endomyocardial Biopsy-Proven Case Series.

Myocarditis in response to COVID-19 vaccination has been reported since early 2021. In particular, young male individuals have been identified to exhibit an increased risk of myocardial inflammation following the administration of mRNA-based vaccines. Even though the first epidemiological analyses and numerous case reports investigated potential relationships, endomyocardial biopsy (EMB)-proven cases are limited. Here, we present a comprehensive histopathological analysis of EMBs from 15 patients with reduced ejection fraction (LVEF = 30 (14-39)%) and the clinical suspicion of myocarditis following vaccination with Comirnaty® (Pfizer-BioNTech) (n = 11), Vaxzevria® (AstraZenica) (n = 2) and Janssen® (Johnson & Johnson) (n = 2). Immunohistochemical EMB analyses reveal myocardial inflammation in 14 of 15 patients, with the histopathological diagnosis of active myocarditis according the Dallas criteria (n = 2), severe giant cell myocarditis (n = 2) and inflammatory cardiomyopathy (n = 10). Importantly, infectious causes have been excluded in all patients. The SARS-CoV-2 spike protein has been detected sparsely on cardiomyocytes of nine patients, and differential analysis of inflammatory markers such as CD4+ and CD8+ T cells suggests that the inflammatory response triggered by the vaccine may be of autoimmunological origin. Although a definitive causal relationship between COVID-19 vaccination and the occurrence of myocardial inflammation cannot be demonstrated in this study, data suggest a temporal connection. The expression of SARS-CoV-2 spike protein within the heart and the dominance of CD4+ lymphocytic infiltrates indicate an autoimmunological response to the vaccination.

Interferon-ß Suppresses Transcriptionally Active Parvovirus B19 Infection in Viral Cardiomyopathy: A Subgroup Analysis of the BICC-Trial.

Human parvovirus B19 (B19V) is the predominant virus currently detected in endomyocardial biopsies (EMBs). Recent findings indicate that, specifically, transcriptionally active B19V with detectable viral RNA is of prognostic relevance in inflammatory viral cardiomyopathy. We aimed to evaluate B19V replicative status (viral RNA) and beneficial effects in a sub-collective of the prospective randomized placebo-controlled phase II multi-center BICC-Trial (Betaferon In Chronic Viral Cardiomyopathy) after interferon beta-1b (IFN-ß) treatment. EMBs of n = 64 patients with B19V mono-infected tissue were retrospectively analyzed. Viral RNA could be detected in n = 18/64 (28.1%) of B19V DNA positive samples (mean age 51.7 years, 12 male), of whom n = 13 had been treated with IFN-ß. Five patients had received placebo. PCR analysis confirmed in follow-up that EMBs significantly reduced viral RNA loads in n = 11/13 (84.6%) of IFN-ß treated patients (p = 0.001), independently from the IFN-ß dose, in contrast to the placebo group, where viral RNA load was not affected or even increased. Consequently, a significant improvement of left ventricular ejection fraction (LVEF) after treatment with IFN-ß was observed (LVEF mean baseline 51.6 ± 14.1% vs. follow-up 61.0 ± 17.5%, p = 0.03). In contrast, in the placebo group, worsening of LVEF was evaluated in n = 4/5 (80.0%) of patients. We could show for the first-time the beneficial effects from treatment with IFN-ß, suppressing B19V viral RNA and improving the hemodynamic course. Our results need further verification in a larger prospective randomized controlled trial.

Cardiac SARS-CoV-2 infection is associated with pro-inflammatory transcriptomic alterations within the heart.

AIMS: Cardiac involvement in COVID-19 is associated with adverse outcome. However, it is unclear whether cell-specific consequences are associated with cardiac SARS-CoV-2 infection. Therefore, we investigated heart tissue utilizing in situ hybridization, immunohistochemistry, and RNA-sequencing in consecutive autopsy cases to quantify virus load and characterize cardiac involvement in COVID-19. METHODS AND RESULTS: In this study, 95 SARS-CoV-2-positive autopsy cases were included. A relevant SARS-CoV-2 virus load in the cardiac tissue was detected in 41/95 deceased (43%). Massive analysis of cDNA ends (MACE)-RNA-sequencing was performed to identify molecular pathomechanisms caused by the infection of the heart. A signature matrix was generated based on the single-cell dataset 'Heart Cell Atlas' and used for digital cytometry on the MACE-RNA-sequencing data. Thus, immune cell fractions were estimated and revealed no difference in immune cell numbers in cases with and without cardiac infection. This result was confirmed by quantitative immunohistological diagnosis. MACE-RNA-sequencing revealed 19 differentially expressed genes (DEGs) with a q-value <0.05 (e.g. up: IFI44L, IFT3, TRIM25; down: NPPB, MB, MYPN). The upregulated DEGs were linked to interferon pathways and originate predominantly from endothelial cells. In contrast, the downregulated DEGs originate predominately from cardiomyocytes. Immunofluorescent staining showed viral protein in cells positive for the endothelial marker ICAM1 but rarely in cardiomyocytes. The Gene Ontology (GO) term analysis revealed that downregulated GO terms were linked to cardiomyocyte structure, whereas upregulated GO terms were linked to anti-virus immune response. CONCLUSION: This study reveals that cardiac infection induced transcriptomic alterations mainly linked to immune response and destruction of cardiomyocytes. While endothelial cells are primarily targeted by the virus, we suggest cardiomyocyte destruction by paracrine effects. Increased pro-inflammatory gene expression was detected in SARS-CoV-2-infected cardiac tissue but no increased SARS-CoV-2 associated immune cell infiltration was observed.

Transcriptional Active Parvovirus B19 Infection Predicts Adverse Long-Term Outcome in Patients with Non-Ischemic Cardiomyopathy.

Parvovirus B19 (B19V) is the predominant cardiotropic virus currently found in endomyocardial biopsies (EMBs). However, direct evidence showing a causal relationship between B19V and progression of inflammatory cardiomyopathy are still missing. The aim of this study was to analyze the impact of transcriptionally active cardiotropic B19V infection determined by viral RNA expression upon long-term outcomes in a large cohort of adult patients with non-ischemic cardiomyopathy in a retrospective analysis from a prospective observational cohort. In total, the analyzed study group comprised 871 consecutive B19V-positive patients (mean age 50.0 ± 15.0 years) with non-ischemic cardiomyopathy who underwent EMB. B19V-positivity was ascertained by routine diagnosis of viral genomes in EMBs. Molecular analysis of EMB revealed positive B19V transcriptional activity in n = 165 patients (18.9%). Primary endpoint was all-cause mortality in the overall cohort. The patients were followed up to 60 months. On the Cox regression analysis, B19V transcriptional activity was predictive of a worse prognosis compared to those without actively replicating B19V (p = 0.01). Moreover, multivariable analysis revealed transcriptional active B19V combined with inflammation [hazard ratio 4.013, 95% confidence interval 1.515-10.629 (p = 0.005)] as the strongest predictor of impaired survival even after adjustment for age and baseline LVEF (p = 0.005) and independently of viral load. The study demonstrates for the first time the pathogenic clinical importance of B19V with transcriptional activity in a large cohort of patients. Transcriptionally active B19V infection is an unfavourable prognostic trigger of adverse outcome. Our findings are of high clinical relevance, indicating that advanced diagnostic differentiation of B19V positive patients is of high prognostic importance.

Viral Myocarditis-From Pathophysiology to Treatment.

The diagnosis of acute and chronic myocarditis remains a challenge for clinicians. Characterization of this disease has been hampered by its diverse etiologies and heterogeneous clinical presentations. Most cases of myocarditis are caused by infectious agents. Despite successful research in the last few years, the pathophysiology of viral myocarditis and its sequelae leading to severe heart failure with a poor prognosis is not fully understood and represents a significant public health issue globally. Most likely, at a certain point, besides viral persistence, several etiological types merge into a common pathogenic autoimmune process leading to chronic inflammation and tissue remodeling, ultimately resulting in the clinical phenotype of dilated cardiomyopathy. Understanding the underlying molecular mechanisms is necessary to assess the prognosis of patients and is fundamental to appropriate specific and personalized therapeutic strategies. To reach this clinical prerequisite, there is the need for advanced diagnostic tools, including an endomyocardial biopsy and guidelines to optimize the management of this disease. The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has currently led to the worst pandemic in a century and has awakened a special sensitivity throughout the world to viral infections. This work aims to summarize the pathophysiology of viral myocarditis, advanced diagnostic methods and the current state of treatment options.

Human myocardial mitochondrial oxidative capacity is impaired in mild acute heart transplant rejection.

AIMS: Acute cellular rejection (ACR) following heart transplantation (HTX) is associated with long-term graft loss and increased mortality. Disturbed mitochondrial bioenergetics have been identified as pathophysiological drivers in heart failure, but their role in ACR remains unclear. We aimed to prove functional disturbances of myocardial bioenergetics in human heart transplant recipients with mild ACR by assessing myocardial mitochondrial respiration using high-resolution respirometry, digital image analysis of myocardial inflammatory cell infiltration, and clinical assessment of HTX patients. We hypothesized that (i) mild ACR is associated with impaired myocardial mitochondrial respiration and (ii) myocardial inflammation, systemic oxidative stress, and myocardial oedema relate to impaired mitochondrial respiration and myocardial dysfunction. METHODS AND RESULTS: We classified 35 HTX recipients undergoing endomyocardial biopsy according International Society for Heart and Lung Transplantation criteria to have no (0R) or mild (1R) ACR. Additionally, we quantified immune cell infiltration by immunohistochemistry and digital image analysis. We analysed mitochondrial substrate utilization in myocardial fibres by high-resolution respirometry and performed cardiovascular magnetic resonance (CMR). ACR (1R) was diagnosed in 12 patients (34%), while the remaining 23 patients revealed no signs of ACR (0R). Underlying cardiomyopathies (dilated cardiomyopathy 50% vs. 65%; P = 0.77), comorbidities (type 2 diabetes mellitus: 50% vs. 35%, P = 0.57; chronic kidney disease stage 5: 8% vs. 9%, P > 0.99; arterial hypertension: 59% vs. 30%, P = 0.35), medications (tacrolimus: 100% vs. 91%, P = 0.54; mycophenolate mofetil: 92% vs. 91%, P > 0.99; prednisolone: 92% vs. 96%, P > 0.99) and time post-transplantation (21.5 ± 26.0 months vs. 29.4 ± 26.4 months, P = 0.40) were similar between groups. Mitochondrial respiration was reduced by 40% in ACR (1R) compared with ACR (0R) (77.8 ± 23.0 vs. 128.0 ± 33.0; P < 0.0001). Quantitative assessment of myocardial CD3+ -lymphocyte infiltration identified ACR (1R) with a cut-off of >14 CD3+ -lymphocytes/mm2 (100% sensitivity, 82% specificity; P < 0.0001). Myocardial CD3+ infiltration (r = -0.41, P < 0.05), systemic oxidative stress (thiobarbituric acid reactive substances; r = -0.42, P < 0.01) and myocardial oedema depicted by global CMR derived T2 time (r = -0.62, P < 0.01) correlated with lower oxidative capacity and overt cardiac dysfunction (global longitudinal strain; r = -0.63, P < 0.01). CONCLUSIONS: Mild ACR with inflammatory cell infiltration associates with impaired mitochondrial bioenergetics in cardiomyocytes. Our findings may help to identify novel checkpoints in cardiac immune metabolism as potential therapeutic targets in post-transplant care.

Nucleoside Analogue Reverse Transcriptase Inhibitors Improve Clinical Outcome in Transcriptional Active Human Parvovirus B19-Positive Patients.

Human parvovirus B19 (B19V) is the predominant cardiotropic virus associated with dilated inflammatory cardiomyopathy (DCMi). Transcriptionally active cardiotropic B19V infection is clinically relevant and triggers adverse long-term mortality. During the study; we evaluated whether antiviral treatment with the nucleoside analogue telbivudine (LTD) is effective in suppressing transcriptional active B19V in endomyocardial biopsies (EMBs) of B19V positive patients and improving clinical outcomes. Seventeen B19V-positive patients (13 male; mean age 45.7 ± 13.9 years; mean left ventricular ejection fraction (LVEF) 37.7 ± 13.5%) with positive B19V DNA and transcriptional activity (B19V mRNA) in EMBs were treated with 600 mg/d LTD over a period of six months. Patients underwent EMBs before and after termination of the LTD treatment. B19V RNA copy numbers remained unchanged in 3/17 patients (non-responder) and declined or disappeared completely in the remaining 14/17 patients (responder) (p ≤ 0.0001). Notably; LVEF improvement was more significant in patients who reduced or lost B19V RNA (responder; p = 0.02) in contrast to non-responders (p = 0.7). In parallel; responder patients displayed statistically significant improvement in quality of life (QoL) questionnaires (p = 0.03) and dyspnea on exertion (p = 0.0006), reflecting an improvement in New York Heart Association (NYHA) Classification (p = 0.001). Our findings demonstrated for the first time that suppression of B19V transcriptional activity by LTD treatment improved hemodynamic and clinical outcome significantly. Thus; the present study substantiates the clinical relevance of detecting B19V transcriptional activity of the myocardium.

Plasminogen activator inhibitor-1 reduces cardiac fibrosis and promotes M2 macrophage polarization in inflammatory cardiomyopathy.

Plasminogen activator inhibitor-1 (PAI-1) has a cardioprotective function in mice by repressing cardiac fibrosis through TGF-ß and plasminogen-mediated pathways. In addition it is known to be involved in the recruitment and polarization of monocytes/macrophages towards a M2 phenotype in cancer. Here, we investigated the expression of PAI-1 in human dilated cardiomyopathy (DCM) and inflammatory dilated cardiomyopathy (DCMi) and its effect on cardiac fibrosis and macrophage polarization. We retrospectively analyzed endomyocardial biopsies (EMBs) of patients with DCM or DCMi for PAI-1 expression by immunohistochemistry. Furthermore, EMBs were evaluated for the content of fibrotic tissue, number of activated myofibroblasts, TGF-ß expression, as well as for M1 and M2 macrophages. Patients with high-grade DCMi (DCMi-high, CD3+ lymphocytes > 30 cells/mm2) had significantly increased PAI-1 levels compared to DCM and low-grade DCMi patients (DCMi-low, CD3+ lymphocytes = 14-30 cells/mm2) (15.5 ± 0.4% vs. 1.0 ± 0.1% and 4.0 ± 0.1%, p ≤ 0.001). Elevated PAI-1 expression in DCMi-high subjects was associated with a diminished degree of cardiac fibrosis, decreased levels of TGF-ß and reduced number of myofibroblasts. In addition, DCMi-high patients revealed an increased proportion of non-classical M2 macrophages towards classical M1 macrophages, indicating M2 macrophage-favoring properties of PAI-1 in inflammatory cardiomyopathies. Our findings give evidence that elevated expression of cardiac PAI-1 in subjects with high-grade DCMi suppresses fibrosis by inhibiting TGF-ß and myofibroblast activation. Moreover, our data indicate that PAI-1 is involved in the polarization of M2 macrophages in the heart. Thus, PAI-1 could serve as a potential prognostic biomarker and as a possible therapeutic target in inflammatory cardiomyopathies.

MicroRNA profiling as a novel diagnostic tool for identification of patients with inflammatory and/or virally induced cardiomyopathies.

AIMS: MicroRNAs (miRNAs) might be used as prospective biomarkers for the identification of unexplained heart failure caused by a viral and/or inflammatory process. The aim of this study was to identify and to evaluate prognostic miRNAs in serum of patients with inflammatory heart diseases diagnosed by endomyocardial biopsies. METHODS AND RESULTS: After TaqMan® OpenArray® screening of 754 unique circulating miRNAs in serum of biopsy-proven patients [184 patients with inflammatory and/or virally induced myocardial diseases (DCMi), 25 patients with dilated cardiomyopathy (DCM), and 25 healthy donors], we identified seven miRNAs of interest (P < 0.05). These data have been verified by single qRT-PCR assays in other biopsy-proven patients (159 patients with viral and/or inflammatory myocardial diseases, 46 patients with DCM, and 60 healthy donors). The expression of let-7f, miR-197, miR-223, miR-93, and miR-379 allowed us to differentiate between patients with a virus and/or inflammation and healthy donors (P < 0.05) with the specificity over 93%. Based on the expression of miR-21 and miR-30a-5p, we could sort out patients with DCM from all other study groups (P < 0.05) with the specificity over 95%. CONCLUSIONS: This miRNA profile provides for the first time a new non-invasive diagnostic perspective to identify patients with intramyocardial inflammation and/or viral persistence only from single serum sample, independently of prescribed therapy and time of symptoms onset. It allows the early finding of those patients relevant for myocardial biopsy for exact diagnosis and further proscription of causal aetiology-driven specific treatment.

Proof of SARS-CoV-2 genomes in endomyocardial biopsy with latency after acute infection.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has reached pandemic levels. Cardiovascular complications in COVID-19 have been reported frequently, however evidence for a causal relationship has not been established. This report describes the detection of SARS-CoV-2 viral genomes in a patient with symptoms of heart failure, in whom endomyocardial biopsy was investigated following a latency period of 4 weeks after the onset of pulmonary symptoms. The viral infection was accompanied by myocardial inflammation indicating an infection of the heart muscle.

Detection of parvovirus mRNAs as markers for viral activity in endomyocardial biopsy-based diagnosis of patients with unexplained heart failure.

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.

Tissue Engineering of Cartilage Using a Random Positioning Machine.

Articular cartilage is a skeletal tissue of avascular nature and limited self-repair capacity. Cartilage-degenerative diseases, such as osteoarthritis (OA), are difficult to treat and often necessitate joint replacement surgery. Cartilage is a tough but flexible material and relatively easy to damage. It is, therefore, of high interest to develop methods allowing chondrocytes to recolonize, to rebuild the cartilage and to restore joint functionality. Here we studied the in vitro production of cartilage-like tissue using human articular chondrocytes exposed to the Random Positioning Machine (RPM), a device to simulate certain aspects of microgravity on Earth. To screen early adoption reactions of chondrocytes exposed to the RPM, we performed quantitative real-time PCR analyses after 24 h on chondrocytes cultured in DMEM/F-12. A significant up-regulation in the gene expression of IL6, RUNX2, RUNX3, SPP1, SOX6, SOX9, and MMP13 was detected, while the levels of IL8, ACAN, PRG4, ITGB1, TGFB1, COL1A1, COL2A1, COL10A1, SOD3, SOX5, MMP1, and MMP2 mRNAs remained unchanged. The STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) analysis demonstrated among others the importance of these differentially regulated genes for cartilage formation. Chondrocytes grown in DMEM/F-12 medium produced three-dimensional (3D) spheroids after five days without the addition of scaffolds. On day 28, the produced tissue constructs reached up to 2 mm in diameter. Using specific chondrocyte growth medium, similar results were achieved within 14 days. Spheroids from both types of culture media showed the typical cartilage morphology with aggrecan positivity. Intermediate filaments form clusters under RPM conditions as detected by vimentin staining after 7 d and 14 d. Larger meshes appear in the network in 28-day samples. Furthermore, they were able to form a confluent chondrocyte monolayer after being transferred back into cell culture flasks in 1 g conditions showing their suitability for transplantation into joints. Our results demonstrate that the cultivation medium has a direct influence on the velocity of tissue formation and tissue composition. The spheroids show properties that make them interesting candidates for cellular cartilage regeneration approaches in trauma and OA therapy.

Evaluation of Myocardial Gene Expression Profiling for Superior Diagnosis of Idiopathic Giant-Cell Myocarditis and Clinical Feasibility in a Large Cohort of Patients with Acute Cardiac Decompensation.

AIMS: The diagnostic approach to idiopathic giant-cell myocarditis (IGCM) is based on identifying various patterns of inflammatory cell infiltration and multinucleated giant cells (GCs) in histologic sections taken from endomyocardial biopsies (EMBs). The sampling error for detecting focally located GCs by histopathology is high, however. The aim of this study was to demonstrate the feasibility of gene profiling as a new diagnostic method in clinical practice, namely in a large cohort of patients suffering from acute cardiac decompensation. Methods and Results: In this retrospective multicenter study, EMBs taken from n = 427 patients with clinically acute cardiac decompensation and suspected acute myocarditis were screened (mean age: 47.03 ± 15.69 years). In each patient, the EMBs were analyzed on the basis of histology, immunohistology, molecular virology, and gene-expression profiling. Out of the total of n = 427 patient samples examined, GCs could be detected in 26 cases (6.1%) by histology. An established myocardial gene profile consisting of 27 genes was revealed; this was narrowed down to a specified profile of five genes (CPT1, CCL20, CCR5, CCR6, TLR8) which serve to identify histologically proven IGCM with high specificity in 25 of the 26 patients (96.2%). Once this newly established profiling approach was applied to the remaining patient samples, an additional n = 31 patients (7.3%) could be identified as having IGCM without any histologic proof of myocardial GCs. In a subgroup analysis, patients diagnosed with IGCM using this gene profiling respond in a similar fashion to immunosuppressive therapy as patients diagnosed with IGCM by conventional histology alone. Conclusions: Myocardial gene-expression profiling is a promising new method in clinical practice, one which can predict IGCM even in the absence of any direct histologic proof of GCs in EMB sections. Gene profiling is of great clinical relevance in terms of a) overcoming the sampling error associated with purely histologic examinations and b) monitoring the effectiveness of therapy.

Association of Cardiac Infection With SARS-CoV-2 in Confirmed COVID-19 Autopsy Cases.

Importance: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be documented in various tissues, but the frequency of cardiac involvement as well as possible consequences are unknown. Objective: To evaluate the presence of SARS-CoV-2 in the myocardial tissue from autopsy cases and to document a possible cardiac response to that infection. Design, Setting, and Participants: This cohort study used data from consecutive autopsy cases from Germany between April 8 and April 18, 2020. All patients had tested positive for SARS-CoV-2 in pharyngeal swab tests. Exposures: Patients who died of coronavirus disease 2019. Main Outcomes and Measures: Incidence of SARS-CoV-2 positivity in cardiac tissue as well as CD3+, CD45+, and CD68+ cells in the myocardium and gene expression of tumor necrosis growth factor α, interferon γ, chemokine ligand 5, as well as interleukin-6, -8, and -18. Results: Cardiac tissue from 39 consecutive autopsy cases were included. The median (interquartile range) age of patients was 85 (78-89) years, and 23 (59.0%) were women. SARS-CoV-2 could be documented in 24 of 39 patients (61.5%). Viral load above 1000 copies per µg RNA could be documented in 16 of 39 patients (41.0%). A cytokine response panel consisting of 6 proinflammatory genes was increased in those 16 patients compared with 15 patients without any SARS-CoV-2 in the heart. Comparison of 15 patients without cardiac infection with 16 patients with more than 1000 copies revealed no inflammatory cell infiltrates or differences in leukocyte numbers per high power field. Conclusions and Relevance: In this analysis of autopsy cases, viral presence within the myocardium could be documented. While a response to this infection could be reported in cases with higher virus load vs no virus infection, this was not associated with an influx of inflammatory cells. Future investigations should focus on evaluating the long-term consequences of this cardiac involvement.

Detection of viral SARS-CoV-2 genomes and histopathological changes in endomyocardial biopsies.

AIMS: Since December 2019, the novel coronavirus SARS-CoV-2 has spread rapidly throughout China and keeps the world in suspense. Cardiovascular complications with myocarditis and embolism due to COVID-19 have been reported. SARS-CoV-2 genome detection in the heart muscle has not been demonstrated so far, and the underlying pathophysiological mechanisms remain to be investigated. METHODS AND RESULTS: Endomyocardial biopsies (EMBs) of 104 patients (mean age: 57.90 ± 16.37 years; left ventricular ejection fraction: 33.7 ± 14.6%, sex: n = 79 male/25 female) with suspected myocarditis or unexplained heart failure were analysed. EMB analysis included histology, immunohistochemistry, and detection of SARS-CoV-2 genomes by real-time reverse transcription polymerase chain reaction in the IKDT Berlin, Germany. Among 104 EMBs investigated, five were confirmed with SARS-CoV-2 infected by reverse real-time transcriptase polymerase chain reaction. We describe patients of different history of symptoms and time duration. Additionally, we investigated histopathological changes in myocardial tissue showing that the inflammatory process in EMBs seemed to permeate vascular wall leading to small arterial obliteration and damage. CONCLUSIONS: This is the first report that established the evidence of SARS-CoV-2 genomes detection in EMBs. In these patients, myocardial injury ischaemia may play a role, which could explain the ubiquitous troponin increases. EMB-based identification of the cause of myocardial injury may contribute to explain the different evolution of complicated SARS-CoV-2-infection and to design future specific and personalized treatment strategies.

Detection of SARS-CoV-2 in Human Retinal Biopsies of Deceased COVID-19 Patients.

PURPOSE: To report the presence of viral ribonucleic acid (RNA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human retina in deceased patients with confirmed novel coronavirus disease 2019 (COVID-19). PATIENTS AND METHODS: Fourteen eyes of 14 deceased patients with confirmed COVID-19 disease were enucleated during autopsy. A sample of human retina was secured and fixed in RNAlater™. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to detect three different viral RNA sequences (RdRp-gene, E-gene and Orf1 gene) of SARS-CoV-2. RESULTS: In three out of 14 eyes SARS-CoV-2 viral RNA was detected in the retina of deceased COVID-19 patients. As analysis for three different sequences (RdRp-gene, E-gene and Orf1 gene) revealed positive results in RT-PCR, the existence of SARS-CoV-2 viral RNA in human retina is proven according to the standards of the World-Health-Organization. CONCLUSION: Viral RNA of SARS-CoV-2 is detectable in the retina of COVID-19 patients.