Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Experiment and analysis workflow. Four-day old neonatal mice weighing 3 gram/ pup were infected (per os) with reovirus type-1-lang (T1L). Neonatal mice infected with 1x phosphate buffered saline (PBS) were used as mock controls. Ileum tissue (1 and 4 dpi) and heart tissues (4, 7, 10 dpi) were assayed and used for single-cell RNAseq and spatial transcriptomics. b) UMAP plot of 31,684 single-cell transcriptomes from mock-infected and reovirus-infected hearts at 4, 7, and 10 dpi (one animal per condition), clustered by gene expression and colored by cell type (left). UMAP plots showing cardiac cell type clusters across samples for the heart scRNA-seq data (right). c) 8,243 spatial transcriptomes of cardiac tissue sections from mock-infected and reovirus-infected mice at 4 and 7 dpi (one animal per condition). Hematoxylin and Eosin (H&E) stained image of reovirus-infected myocarditic tissue section used for spatial transcriptomics at 7 dpi (in box). d) UMAP plot of 7,695 single-cell transcriptomes from mock-infected and reovirus-infected ileum at day 1 and 4 dpi, clustered and colored by cell type (left). UMAP plots showing the gaussian kernel density of cells across samples for the ileum scRNA-seq data (right). e) Dot plot showing the percent of cells with non-zero viral transcripts and the mean viral transcript counts (UMIs) in ileal and cardiac cell types. f) RNA FISH labelling of cardiac cell type markers (Tnnt2 for cardiomyocytes, Postn for fibroblasts, and Cdh5 for endothelial cells), and immunofluorescence staining of reovirus antigen on a consecutive section showing infected endothelial cells within the infection foci at 7 dpi. Representative heart images from six biological replicates.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection, Saline, Gene Expression, Staining, Immunofluorescence

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Number of unique genes detected per cell (left), number of unique transcripts per cell (center), and percentage of mitochondrial transcripts (right) in cardiac scRNA-seq datasets from three stages after infection. b) Number of unique genes detected per cell (left), number of unique transcripts per cell (center), and percentage of transcripts from mitochondrial genes (right) in cardiac spatial transcriptomics datasets from two stages after infection. c) UMAP plot of 31,684 single-cell transcriptomes from mock-infected and reovirus-infected hearts at 4, 7, and 10 days post-infection (dpi), clustered by gene expression and colored by cardiac cell type. Dotted lines show the cardiac cell types being grouped as broad endothelial cells and fibroblast cells. d) scRNA-seq UMAP plots showing expression of endothelial markers Cdh5 and Pecam1, smooth muscle cell-specific markers Tagln, Myh11, and Acta2, and pericyte markers Pdgfrb, Kcnj8, and Rgs5 used to define the Cdh5+ Kcnj8+ Pdgfrb+ mesenchymal endothelial cells (Chen Qi et al. Nature Communications 2016). e) Top three differentially expressed genes (two-sided Wilcoxon test, log2 fold-change > 1.0 and p-value < 0.01) for cell types in heart scRNA-seq data. f) Bar plot showing the cell type composition changes in scRNA-seq datasets from reovirus-infected and mock-infected mice hearts.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection, Gene Expression, Expressing

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Number of unique genes detected per cell (left), number of unique transcripts per cell (center), and percentage of mitochondrial transcripts (right) in ileum scRNA-seq datasets (top row) and ileum spatial transcriptomics datasets (bottom row) from two stages after infection. b) Top-three differentially expressed genes (two-sided Wilcoxon test, log2 fold-change > 1.0 and p-value < 0.01) for cell types in ileum scRNA-seq data. c) Bar plot showing the cell type composition changes in scRNA-seq datasets from reovirus-infected and mock-infected mice in ileum tissue. Mock sample bar represents the mean cell type proportions for mock ileum samples at 1 and 4 dpi. d) Spatial transcriptomics map of ileum tissue sections from mock-infected and reovirus-infected mice at 1 and 4 dpi, colored by clusters representing tissue anatomical regions.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Knee plot showing viral UMI counts in the scRNA-seq droplets classified as either empty droplets or with viable/dead cells across ileum (left) and heart (right) scRNA-seq samples. The droplets were labelled using host gene UMI counts detected in scRNA-seq datasets. b) scRNA-seq UMAP plots showing total viral UMI counts per cell before xGen enrichment, after xGen viral transcript enrichment, and after removal of background signal on ileum samples. c) scRNA-seq UMAP plots showing total viral UMI counts per cell before xGen enrichment, after xGen viral transcript enrichment, and after removal of background signal on heart samples. d) Heatmaps showing counts of infected cells of different ileal cell types across two reovirus-infected ileum samples from 1 and 4 dpi. e) Heatmaps showing counts of infected cells of different cardiac cell types across three reovirus-infected heart samples from 4, 7, and 10 dpi.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Single-cell Transcriptomics, Infection

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Heatmap showing the expression of the 25 most upregulated genes in the reovirus-infected heart as compared to mock at 4 dpi. b) Infection response score for cardiac cell types in scRNA-seq data across mock-infected and reovirus-infected hearts at three distinct stages. The infection response score represents the gene module score for a panel of 230 genes that are significantly upregulated (two-sided Wilcoxon test, log fold-change > 1.0 and p-value < 0.01) in the reovirus-infected heart as compared to the mock-infected heart at 4 dpi (n = 23,812 total cells were examined over six independent experiment conditions; one biologically independent sample was used for each experiment). Boxes in the boxplots indicates 25th and 75th percentile, the band in the box indicated the median and whiskers extend to 1.5 × Interquartile Range (IQR) of the hinge. Outliers (beyond 1.5 × IQR) are plotted individually. c) Infection response score (defined above) across spots in spatial transcriptomics data. d) Infection response score calculated for five common cell types across 10 tissues from the tabula-muris mouse atlas data (n = 16,651 total cells were examined over 10 independent tissues derived from seven biologically independent animals). e) Infection response score for ileal cell types in scRNA-seq data across mock-infected and reovirus-infected ileum at two distinct stages. The infection response score represents the gene module score for a panel of 438 genes significantly upregulated (two-sided Wilcoxon test, log fold-change > 1.0 and p-value < 0.01) in the reovirus-infected ileum at 1 dpi as compared to the mock-infected ileum (n = 5,101 total cells were examined over four independent experiment conditions; one biologically independent sample was used for each experiment). d,e) Boxes in the boxplots indicates 25th and 75th percentile, the band in the box indicated the median and whiskers extend to 1.5 × Interquartile Range (IQR) of the hinge. Outliers (beyond 1.5 × IQR) are plotted individually. f) Infection response score for spatial transcriptomics data from mock-infected and reovirus-infected ileum at two distinct stages.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Expressing, Infection, Derivative Assay

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) UMAP plot of 9,786 single-cell endothelial cell transcriptomes from mock-infected and reovirus-infected hearts at 4, 7, and 10 dpi colored by endothelial cell (EC) subtype clusters (phenotypes) (top) and condition (bottom). b) Heatmap showing top-five differentially expressed genes (two-sided Wilcoxon test, log fold-change > 1.0 and p-value < 0.01) for endothelial cell subtypes. c) UMAP plot showing the expression of genes upregulated in Cxcl9-high endothelial cells. d) Spatial transcriptomic maps of cardiac tissue from reovirus infected hearts at 7 dpi showing gene module scores calculated for four GO terms enriched in Cxcl9-high endothelial cells. e) UMAP plot of 2,205 single-cell T cell (TC) transcriptomes from mock-infected and reovirus-infected hearts at 4, 7, and 10 dpi colored by T cell subtype clusters (top) and condition (bottom). f) Heatmap showing top-five differentially expressed genes (two-sided Wilcoxon test, log fold-change > 1.0 and p-value < 0.01) for T cell subtypes. g) UMAP plot showing the expression of genes upregulated in cytotoxic T cells from myocarditic heart at 7 dpi. h) Spatial transcriptomics maps of cardiac tissue from reovirus infected hearts at 7 dpi showing gene module scores calculated for four GO terms enriched in cytotoxic T cells. i,j) RNA FISH staining for i) endothelial marker Cdh5, and chemokine Cxcl9 j) T cell marker Trbc2 and lytic molecule Prf1 on consecutive sections from myocarditic hearts at 7 dpi. Representative images from 14 biological replicates (n=7 males and n=7 females). k,l) Immunofluorescence staining for k) cleaved Caspase1 protein-subunit (Casp1 p20 subunit) l) cleaved Gasdermin D protein (GSDMD N terminus fragment) on myocarditic hearts at 7 dpi. Representative images from 14 reovirus-infected biological replicates (n=7 males and n=7 females). Immunofluorescence signal from reovirus-infected hearts was compared to mock-infected hearts using two-sided Wilcoxon statistical test. Boxes in the boxplots indicates 25th and 75th percentile, the band in the box indicated the median and whiskers extend to 1.5 × Interquartile Range (IQR) of the hinge. Outliers (beyond 1.5 × IQR) are plotted individually. p-value annotation legend: ns: p <= 1.00e+00, *: 1.00e−02 < p <= 5.00e−02, **: 1.00e−03 < p <= 1.00e−02, ***: 1.00e−04 < p <= 1.00e−03, ****: p <= 1.00e−04.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection, Expressing, Staining, Marker, Immunofluorescence

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Volcano plot showing differentially expressed genes (two-sided Wilcoxon Rank-Sum test, -log2 fold change > 2.0 and p-value < 10-4) upregulated in Cxcl9-high inflamed endothelial cells from heart at 7 dpi. Dotted lines show the thresholds for significantly enriched genes (red). b) Top Gene Ontology (GO) terms of interest enriched for genes upregulated in Cxcl9-high endothelial cells in myocarditic heart at 7 dpi. c) Volcano plot showing differentially expressed genes (two-sided Wilcoxon Rank-Sum test, -log2 fold change > 2.0 and p-value < 10-4) upregulated in cytotoxic T cells at 7 and 10 dpi. Dotted lines show thresholds for significantly enriched genes (red). d) Top GO terms of interest enriched for genes upregulated in cytotoxic T cells in myocarditic hearts at 7 and 10 dpi. e-h) Spatial transcriptomics maps of cardiac tissue sections from reovirus-infected mice pups at 7 dpi showing: e) The expression of genes enriched in Cxcl9-high inflamed endothelial cells. f) Gene module scores for four GO terms of interest enriched in Cxcl9-high inflamed endothelial cells. g) The expression of genes enriched in cytotoxic T cells from myocarditic heart. h) Gene module scores calculated for four GO terms of interest enriched in cytotoxic T cells from myocarditic heart.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection, Expressing

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Spatial transcriptomics map of cardiac tissue section from reovirus-infected mice at 7 dpi colored by spot clusters representing transcriptionally distinct tissue regions. b) Spatial transcriptomics maps of cardiac tissue sections from reovirus-infected mice at 7 dpi showing the expression of differentially expressed genes of interest in the myocarditic and the border zone. c) Changes in average predicted cell-type proportions across the infected ventricle, for cell types enriched in the myocarditic region and the border zone. d) UMAP plot of 502 single-cell cardiomyocyte cell transcriptomes from mock-infected and reovirus-infected hearts at 4, 7, and 10 dpi colored by myocyte cell subtype (phenotypes) (left) and condition (right). e) Heatmap showing top-five differentially expressed genes (two-sided Wilcoxon test, log fold-change > 1.0 and p-value < 0.01) for cardiomyocyte cell subtypes. f) Venn Diagram showing myocyte-specific genes upregulated in the border zone around the myocarditic regions (left). UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters. Zoom-in shows the spatial arrangement of Slide-seq clusters within a myocarditic region. h) Spatial transcriptomic maps showing Slide-seq expression of four cardiomyocyte specific genes enriched in inflamed cardiomyocytes as compared to uninflamed myocytes. i) RNA FISH staining for cardiomyocyte marker Tnnt2, and border-zone cardiomyocyte markers such as Clu and Nppa on tissue sections from myocarditic hearts and mock controls at 7 dpi. Representative images from 14 reovirus-infected biological replicates (n=7 males and n=7 females) and 6 mock-infected biological replicates (n=3 males and n=3 females).

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection, Expressing, Staining, Marker

Journal: Nature cardiovascular research

Article Title: Spatiotemporal transcriptomics reveals pathogenesis of viral myocarditis

doi: 10.1038/s44161-022-00138-1

Figure Lengend Snippet: a) Number of unique UMIs detected per cell (left), number of unique genes detected per cell (center), and percentage of mitochondrial transcripts (right) in slide-seq datasets from reovirus infected heart at 7 dpi. b) Heatmap showing top-five differentially expressed genes (two-sided Wilcoxon test, log2 fold-change > 1.0 and p-value < 0.01) for slide-seq spatial transcriptomics clusters. c) UMAP plot of > 40,000 slide-seq spatial transcriptomes from reovirus-infected heart at 7 dpi, clustered by gene expression and colored by putative cardiac cell types based on differential gene expression and marker analysis. d) Slide-seq spatial transcriptomics maps showing three slide-seq clusters at a time. e) Heatmap of permutation test scores for neighborhood enrichment of slide-seq clusters. Enrichment scores reflect enrichment of spatial proximity of slide-seq clusters. f) Slide-seq spatial transcriptomics maps showing the expression of border-zone cardiomyocyte specific genes. g) UMAP plot showing the scRNAseq expression of cardiomyocyte-specific genes which are upregulated in border-zone myocytes in the slide-seq data.

Article Snippet: UMAP plot showing the expression of myocyte-specific genes which are upregulated in the border zone of myocarditic regions (right). g) High-resolution Slide-seq spatial transcriptomics map of cardiac ventricular tissue from reovirus infected mice at 7 dpi colored by Slide-seq bead clusters.

Techniques: Infection, Gene Expression, Marker, Expressing