Stem Cell-Associated Proteins and Extracellular Matrix Composition of the Human Atrioventricular Junction

Figures

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  Figure 1. Overview of the atrioventricular junction (AVj). (a) Explanted human heart prior to biopsy collection. (b) Map over a murine heart visualizing the regions corresponding to the biopsies from the human hearts. (c) One representative frozen AVj biopsy, where the black dashed box shows the approximate size of the sample for global quantitative proteomics. (d) The histology of the AVj with the mitral valve and the surrounding left ventricle and atria regions with Haematoxylin–Eosin staining and (e) Picric Sirius staining showing collagen content in red.

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  Figure 2. Overview of the results found in the statistical analyses of the proteomic data. (a) Volcano plot of p-values vs. log2 FC in the AVj region compared with the LV region. Significantly upregulated and downregulated proteins in the AVj are highlighted in red and blue, respectively (p ≤ 0.05, s0 = 0.1). Selected proteins among the most enriched in the AVj region are highlighted and labelled. (b) PCA of the AVj (blue) and the LV (green) based on their proteomic expression profiles. The first and second components segregate the heart areas and account for 72.1 and 6.6% of the variability, respectively. (c) Enrichment analysis of the Gene Ontology biological processes displayed as a dot plot of enriched biological processes derived from the 1133 proteins displaying significantly altered expression patterns (log2 fold change ≥ 0.5, FDR q-value ≤ 0.05). The colour of the dot designates the significance of the change in expression (adjusted p). The size of the dot signifies the number of enriched proteins.

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  Figure 3. Gene expression analysis of AVj and LV was performed using RNA sequencing, followed by paired group comparison of seven individuals. (a) Volcano plot displaying the difference in gene expression between the AVj region and the LV (log2 fold change, FDR q-value < 0.05). (b) Heatmap between the AVj and LV regions showing significantly altered transcripts (log2 fold change, FDR q-value < 0.05). (c) PCA score plot (first three components) of the total number of transcripts detected, showing a separation between the AVj (blue) and left ventricle (green) with regard to gene expression. (d) Pie diagram showing the proportion of transcript categories for the significantly altered transcripts in the AVj compared to the LV.

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  Figure 4. Selected biological processes from the proteomics analysis that were enriched in the AVj. (a) Selected enriched biological processes in the AVj related to stem cell niche biology. The size of the spheres shows the number of proteins involved in the specific biological process. The colour of the spheres shows the FDR q-value. (b–d) Heatmaps over differentially expressed proteins related to stem cell niche biology: fatty acid metabolism, angiogenesis, and hypoxia. The red and blue colour gradients represent the relative increase or decrease in protein expression levels, respectively.

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  Figure 5. Stem cell niche-related signalling pathways from the proteomics analysis that were enriched in the AVj. (a–e) Heatmaps of differentially expressed proteins involved in the TGF-β, Wnt, TNF, FGF, and Notch pathways. The red and blue colour gradients represent the relative increase and decrease in protein levels, respectively. (f–j) Protein–protein interaction network analyses using the STRING database showing the interactions between the differentially expressed proteins involved in the TGF-β, Wnt, TNF, FGF, and Notch pathways. The lines between the nodes are coloured according to the type of interaction. The cyan and purple lines are known interactions from curated databases and experimentally determined interactions, respectively. The green, red, and dark blue lines are predicted interactions based on gene neighbourhood, gene fusion, and gene co-occurrence, respectively. The yellow, black, and light blue lines are other interactions based on textmining, co-expression, and protein homology, respectively.

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  Figure 6. Extracellular matrix (ECM) proteins enriched in the AVj and immunohistological (IHC) verification of proteomic data. (a) Heatmap of differentially expressed ECM proteins. The red, blue, and purple colour gradients represent the relative increase or decrease in protein expression levels, with red corresponding to enrichment in the AVj, blue with reduction, and purple with a further reduction. (b) Immunohistochemistry (IHC) of the human heart tissue sections showing an overview of the AVj region. Periostin (POSTN) is shown in yellow, Natriuretic peptide A (NPPA) in red, cardiac troponin T (cTnT) in green, and cell nuclei stained with DAPI in blue. A subpopulation of POSTN-positive cells that were negative for cTnT is magnified. (c) IHC of cardiomyocytes in the AVj of a human heart. POSTN is shown in yellow, and nuclei stained with DAPI are in blue. (d1–f4) IHC of tissue from three human hearts showing cardiomyocytes stained with POSTN in yellow (d1,e1,f1) and NPPA in red (d2,e2,f2). Both stainings are shown overlayed with each other (d3,e3,f3). A cluster of cells positive for POSTN but negative for NPPA in (d3) is pointed out with an arrow. POSTN and NPPA were overlayed with cTnT,…