An interferon gamma response signature links myocardial aging and immunosenescence

Figures

• 

  Figure 1. Dissecting aging kinetics using single-cell transcriptomics from cardiac cells and T cells in heart-draining mediastinal lymph node simultaneously. Panel A: experimental outline for the sc-seq pipeline. Panel B: UMAP plot representation of the sorted 7243 mediastinal lymph node effector/memory T cells from 2-, 6-, 12-, to18-month-old male and female C57BL/6J mice shown on a UMAP dimensionality reduction plot. Panel C: bar graph representation for the lymph node T cell subsets identified based on the Seurat clustering among the different age groups. Age groups were identified based on the hashtag antibody signal, which was demultiplexed prior to Seurat clustering. Panel D: UMAP plot representation of the sorted 12 613 non-cardiomyocyte cells from 2- (left) to 18- (right) month-old male and female hearts. Panel E: bar graph representation for the cardiac cell subset distribution among the 2- and 18-month-old hearts. Panel F: dot plot of the compiled scores for the average expression levels of gene sets ascribing a gene set of senescence and effector functions within mediastinal lymph node CD8-cytotoxic, CD4-EM, and Tregs among the different age groups. Size of the dot represents…

• 

  Figure 2. A clonally expanded, effector and IFN-γ producing profile of T cells in mediastinal lymph node T cells from aged mice. Panel A: T cell receptor sequencing analysis overlaid on UMAP plots of the T cell subsets from the different age groups. Cells are colour-coded according to how many times the T cell receptor is repeated i.e. clonal size. The identity of the T cell receptor clones is based on the variable chain usage (VDJ gene segments) and the CDR3 sequences. Panel B: dot plot of the complied score for gene sets ascribed to Th1/17 effector, cytotoxicity functions, glycolysis activity, cellular senescence-related genes, and exhaustion classified based on the definition of the clonally expanding class. Panel C: feature plot for the expression score of IFN-γ production gene set in each cell projected onto dimension reduction ‘UMAP’ plots to identify the signature expression in the different age groups in lymph node T cells. Panel D: dot plot of the averaged expression of Tbx21, Eomes, Cxcr3, and Itgb1 within CD4-EM, Tregs, and CD8 cytotoxic T cells along the aging axis. Panel E: feature plot showing the overlapping expression score of IFN-γ production gene set (red) and clonal…

• 

  Figure 3. An IFN-γ signature builds up in aged hearts. Panel A: (left) feature plot of Cd3e indicating T cells within the heart sc-seq dataset. (Right) Feature plot for the expression score of interferon-γ production gene set within the cardiac T cells in 2- and 18-month-old hearts after subsetting and re-clustering. Panel B: frequency of CD29+ CD4+ and CD8+ T cells from heart tissue of 2- and 18-month-old female C57BL/6J mice. The bar graphs indicate the group mean values ± SEM per group (n = 6), and the distribution of each individual value. Statistical test: unpaired t-test. Panel C: (left) gene set enrichment analysis of inflammatory response, IFN- γ response, and IFN- γ production genes in an age range of 3–27 months from cardiac tissue of C57BL/6J mice from the Tabula Muris Senis bulk RNA-seq data. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated in the graphs. (Right) heatmap of the normalized score of IFN-γ response gene set in cardiac tissue of the different ages. Blue indicates down-regulation and rosa indicates up-regulation of the signature. All replicates from the Tabula Muris Senis per age group were used for the analysis. An FDR value <0.25 was…

• 

  Figure 4. Dynamics of interferon-γ response and metabolic gene signatures in aged cardiomyocytes. Panel A: dot plot of the complied score for OxPhos, FAO, glycolysis, and interferon-γ response gene sets in young and old cardiomyocytes from the Tabula Muris Senis data.25 Panel B: dot plot of the complied score for OxPhos, FAO, glycolysis, and interferon-γ response gene sets in young and old endothelial cells, leucocytes, and fibroblasts from in-house generated sc-seq data. Panel C: feature plot showing the overlapping expression score of IFN-γ response gene set (red) and the expression score of down-regulation of OxPhos gene set (green) per cell base in young, and old cardiomyocytes. Cells with a high overlapping score are indicated with a yellow colour. Panel D: gene set enrichment analysis (GSEA) of Oxphos, FAO, glycolysis gene sets in an age range of 3–27 months from cardiac tissue of C57BL/6J mice from the Tabula Muris Senis bulk RNA-seq data. Normalized enrichment score (NES) and false discovery rate (FDR) are indicated in the graphs. All replicates from the Tabula Muris Senis per age group were used for the analysis. An FDR value <0.25 was considered significant. Each age group…

• 

  Figure 5. Effect of IFN-γ stimulation on iPSC derived cardiomyocytes. Panel A: schematic representation of the experiment outline. Panel B: visualization of sarcomeric (α-actinin/green) and mitochondrial (Mitospy/red) structure of iPSC-CM with treatment of IFN-γ. Scale bars = 50 µm Panel C: quantification of mitochondrial network structures based on the Mitospy-staining. Statistical analysis was performed with Mann–Whitney. Every dot represents one analysed picture. Panels D and E: basal respiration (OCR) and H+ production (ECAR) of iPSC-derived cardiomyocytes after 1 (D) or 4 (E) weeks of IFN-γ stimulation or control cultures. Each point indicates group mean value ± SEM per group (n = 3 differentiations of five measurements each). Statistical test: unpaired t-test.