A20 (TNFAIP3) alleviates viral myocarditis through ADAR1/miR-1a-3p-dependent regulation

Objective To investigate the effect of A20 and how A20 is regulated in viral myocarditis (VMC). Methods BABL/C mice, primary neonatal rat cardiomyocytes and H9c2 cells were infected with Coxsackie virus B3 (CVB3) to establish animal and cellular models of VMC. H&E staining revealed the pathologic condition of myocardium. ELISA measured the serum levels of creatine kinase, creatine kinase isoenzyme and cardiac troponin I. The effects of A20, miR-1a-3p and ADAR1 were investigated using gain and loss of function approaches. ELISA measured the levels of IL-6, IL-18 and TNF-α in serum or cell culture supernatant. TUNEL staining and flow cytometry assessed the apoptosis of myocardium and cardiomyocytes, respectively. RNA-binding protein immunoprecipitation and dual-luciferase reporter assays verified the binding between A20 and miR-1a-3p. Co-immunoprecipitation assay verified the binding between ADAR1 and Dicer. Results A20 was underexpressed and miR-1a-3p was overexpressed in the myocardium of VMC mice as well as in CVB3-infected cardiomyocytes. Overexpression of A20 suppressed cardiomyocyte inflammation and apoptosis in vivo and in vitro. miR-1a-3p promoted CVB3-induced inflammation and apoptosis in cardiomyocytes by binding to A20. The expression of miR-1a-3p was regulated by ADAR1. ADAR1 promoted the slicing of miR-1a-3p precursor by binding to Dicer. Conclusion A20, regulated by ADAR1/miR-1a-3p, suppresses inflammation and cardiomyocyte apoptosis in VMC.

Dapagliflozin Alleviates Myocardial Inflammation by Regulating the Macrophage Polarization and Stat3-related Pathways in Coxsackie Virus B3-induced Acute Viral Myocarditis

Viral myocarditis (VMC), which is most prevalently caused by Coxsackievirus B3 (CVB3) infection, is a serious clinical condition characterized by cardiac inflammation. Dapagliflozin, a kind of sodium glucose co-transporters 2(SGLT-2) inhibitor, exhibited protective effects on plenty of inflammatory diseases, while its effect on viral myocarditis has not been studied. Recently we found the protective effect of dapagliflozin on VMC. After CVB3 infection, dapagliflozin were given orally to Balb/c male mice for 8 days and then the severity of myocarditis was assessed. Our results indicated that dapagliflozin significantly alleviated the severity of viral myocarditis, elevated the survival rate, and ameliorated cardiac function. Besides, dapagliflozin can decrease the level of proinflammatory cytokines included IL-1β, IL-6, TNF-α. Furthermore, dapagliflozin can inhibit macrophages differentiate to classically activated macrophages (M1) in cardiac tissue and activate the Stat3 signal pathway which is reported to promote polarization of the alternatively activated macrophage (M2). In conclusion, our study demonstrates that dapagliflozin alleviates myocardial inflammation by regulating the macrophage polarization and Stat3-related pathways in coxsackie virus B3-induced acute viral myocarditis.

NMR-Based Metabolomic Analysis of Sera in Mouse Models of CVB3-Induced Viral Myocarditis and Dilated Cardiomyopathy

Viral myocarditis (VMC) is an inflammatory heart condition which can induce dilated cardiomyopathy (DCM). However, molecular mechanisms underlying the progression of VMC into DCM remain exclusive. Here, we established mouse models of VMC and DCM by infecting male BALB/c mice with Coxsackievirus B3 (CVB3), and performed NMR-based metabonomic analyses of mouse sera. The mouse models covered three pathological stages including: acute VMC (aVMC), chronic VMC (cVMC) and DCM. We recorded 1D 1H-NMR spectra on serum samples and conducted multivariate statistical analysis on the NMR data. We found that metabolic profiles of these three pathological stages were distinct from their normal controls (CON), and identified significant metabolites primarily responsible for the metabolic distinctions. We identified significantly disturbed metabolic pathways in the aVMC, cVMC and DCM stages relative to CON, including: taurine and hypotaurine metabolism; pyruvate metabolism; glycine, serine and threonine metabolism; glycerolipid metabolism. Additionally, we identified potential biomarkers for discriminating a VMC, cVMC and DCM from CON including: taurine, valine and acetate for aVMC; glycerol, valine and leucine for cVMC; citrate, glycine and isoleucine for DCM. This work lays the basis for mechanistically understanding the progression from acute VMC to DCM, and is beneficial to exploitation of potential biomarkers for prognosis and diagnosis of heart diseases.

Gene expression trend changes in breast cancer populations over two decades: insights from The Cancer Genome Atlas database

Background Breast cancer has remained the most common malignancy in women over the past two decades. As lifestyle and living environments have changed, alterations to the disease spectrum have inevitably occurred in this time. As molecular profiling has become a routine diagnostic and objective indicator of breast cancer etiology, we analyzed changes in gene expression in breast cancer populations over two decades using The Cancer Genome Atlas (TCGA). Methods We performed Heatmap and Venn diagram analyses to identify constantly up- and down-regulated genes in this cohort. We used Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to visualize associated functional pathways. Results We determined that three oncogenes, PD-L2, ETV5, and MTOR and 113 long intergenic non-coding RNAs (lincRNAs) were constantly up-regulated, whereas two oncogenes, BCR and GTF2I, one tumor suppression gene (TSG) MEN1, and 30 lincRNAs were constantly down-regulated. Up-regulated genes were enriched in “focal adhesion” and “PI3K-Akt signaling” pathways, et al, and down-regulated genes were significantly enriched in “metabolic pathways” and “viral myocarditis”. Eight up-regulated genes exhibited doubled or higher expression, and the expression of three down-regulated genes was halved or lowered and correlated with long-term survival. Conclusions In this study, we determined that genes and molecular pathways are constantly changing, importantly, some altered genes were associated with prognostics and are potential therapeutic targets, suggesting molecular typing technologies must keep pace with this dynamic situation.

3D virtual histopathology of cardiac tissue from Covid-19 patients based on phase-contrast X-ray tomography

We have used phase-contrast X-ray tomography to characterize the three-dimensional (3d) structure of cardiac tissue from patients who succumbed to Covid-19. By extending conventional histopathological examination by a third dimension, the delicate pathological changes of the vascular system of severe Covid-19 progressions can be analyzed, fully quantified and compared to other types of viral myocarditis and controls. To this end, cardiac samples with a cross section of 3:5mm were scanned at a laboratory setup as well as at a parallel beam setup at a synchrotron radiation facility. The vascular network was segmented by a deep learning architecture suitable for 3d datasets (V-net), trained by sparse manual annotations. Pathological alterations of vessels, concerning the variation of diameters and the amount of small holes, were observed, indicative of elevated occurrence of intussusceptive angiogenesis, also confirmed by high resolution cone beam X-ray tomography and scanning electron microscopy. Furthermore, we implemented a fully automated analysis of the tissue structure in form of shape measures based on the structure tensor. The corresponding distributions show that the histopathology of Covid-19 differs from both influenza and typical coxsackie virus myocarditis.

Mechanism of Blood–Heart-Barrier Leakage: Implications for COVID-19 Induced Cardiovascular Injury

Although blood–heart-barrier (BHB) leakage is the hallmark of congestive (cardio-pulmonary) heart failure (CHF), the primary cause of death in elderly, and during viral myocarditis resulting from the novel coronavirus variants such as the severe acute respiratory syndrome novel corona virus 2 (SARS-CoV-2) known as COVID-19, the mechanism is unclear. The goal of this project is to determine the mechanism of the BHB in CHF. Endocardial endothelium (EE) is the BHB against leakage of blood from endocardium to the interstitium; however, this BHB is broken during CHF. Previous studies from our laboratory, and others have shown a robust activation of matrix metalloproteinase-9 (MMP-9) during CHF. MMP-9 degrades the connexins leading to EE dysfunction. We demonstrated juxtacrine coupling of EE with myocyte and mitochondria (Mito) but how it works still remains at large. To test whether activation of MMP-9 causes EE barrier dysfunction, we hypothesized that if that were the case then treatment with hydroxychloroquine (HCQ) could, in fact, inhibit MMP-9, and thus preserve the EE barrier/juxtacrine signaling, and synchronous endothelial-myocyte coupling. To determine this, CHF was created by aorta-vena cava fistula (AVF) employing the mouse as a model system. The sham, and AVF mice were treated with HCQ. Cardiac hypertrophy, tissue remodeling-induced mitochondrial-myocyte, and endothelial-myocyte contractions were measured. Microvascular leakage was measured using FITC-albumin conjugate. The cardiac function was measured by echocardiography (Echo). Results suggest that MMP-9 activation, endocardial endothelial leakage, endothelial-myocyte (E-M) uncoupling, dyssynchronous mitochondrial fusion-fission (Mfn2/Drp1 ratio), and mito-myocyte uncoupling in the AVF heart failure were found to be rampant; however, treatment with HCQ successfully mitigated some of the deleterious cardiac alterations during CHF. The findings have direct relevance to the gamut of cardiac manifestations, and the resultant phenotypes arising from the ongoing complications of COVID-19 in human subjects.

Identification of Immunogenic Epitopes That Permit the Detection of Antigen-Specific T Cell Reponses to Coxsackievirus B3

Background and Hypothesis:Coxsackievirus B3 (CVB3) is a non-enveloped RNA virus from the Picornaviridae family and is a primary cause of viral myocarditis in the United States. Approximately 5% of all symptomatic CVB3 infections are fatal. Therefore, there is a need to identify the mechanism(s) that regulate a protective immune response to CVB3. However, viral epitopes that stimulate T cell responses to CVB3 remain poorly characterized. To this end, we used a mouse model of CVB3 infection to identify the viral immunogenic CD8 T cell epitopes. We hypothesized that isolated antigen-experienced CD8 T cells from infected mice would be stimulated in the presence of predicted viral epitopes, confirming CVB3-specific T cells. Experimental Design: To identify novel CD8 T cell epitopes, predicted 9-mer MHC binding peptides from the CVB3-Nancy polyprotein were identified using the Immune Epitope Database (IEDB) analysis resource consensus tool. The top ten predicted peptides were synthesized for our assays. Splenocytes from CVB3-infected male and female IFNAR -/- mice were stimulated with each peptide in the presence of brefeldin A for 6 hours at 37˚C. Following stimulation, cells were surfaced stained with antibodies specific for antigen-experienced CD8 T cells. Next, we performed intracellular staining for IFN-gamma. Cells were analyzed using flow cytometry. Candidate epitopes were identified as having results ≥2 standard deviations over the control. Results: Thus far, our analysis has revealed responses to three novel CD8 T cell epitopes within the peptide library, including the viral epitopes within VP1 protein and the RNA-dependent RNA polymerase. Conclusion and Potential ImpactOverall, these data provide an advancement in CVB3 immunology. Further, these data generate new tools like MHC-tetramers to track endogenous T cell responses to CVB3 infection.

Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

A significant fraction of sudden death in children and young adults is due to myocarditis, an inflammatory disease of the heart, most often caused by viral infection. Here we used integrated single-cell and spatial transcriptomics to create a high-resolution, spatially resolved map of reovirus-induced myocarditis in neonatal murine hearts. We assayed hearts collected at three timepoints after reovirus infection and studied the temporal, spatial, and cellular heterogeneity of host-virus interactions. We further assayed the intestine, the primary site of reovirus infection to establish a full chronology of molecular events that ultimately lead to myocarditis. We implemented targeted enrichment of viral transcripts to establish the cellular targets of the virus in the intestine and the heart. Our data give insight into the cell-type specificity of innate immune responses, and into the transcriptional states of inflamed cardiac cells that recruit circulating immune cells, including cytotoxic T cells which induce pyroptosis in the myocarditic tissue. Analyses of spatially restricted gene expression in myocarditic regions and the border zone around those regions identified immune-mediated cell-type specific injury and stress responses. Overall, we observe a dynamic and complex network of cellular phenotypes and cell-cell interactions associated with viral myocarditis.