Identifying molecular and functional similarities and differences between human primary cardiac valve interstitial cells and ventricular fibroblasts

FIGURE 1. RNA-sequencing of primary CFBs. (A) Principal component analysis (PCA) plot of human primary LVCFB and VIC samples showing distinct clusters by cell type. (B) Heatmap visualizing the z-score distribution across each row (gene) in VIC and LVCFB samples with log-2-fold change and average expression displayed on right bars. (C) Volcano plot displaying selected differentially regulated genes including genes with the highest fold change, including NTRK2 and EBF2, and SUSD2 across log-2-fold change on the x-axis and -log10(P-adj) on the y-axis with identified potential genes that distinguish VICs and LVCFBs labelled. (D) Selected pathways of interest from Gene Ontology (GO) enrichment analysis with the Bonferroni corrected p-values of the upstream regulator genes obtained from QuatenaryProd show common developmental pathways being differentially regulated between primary CFBs. Cells in this experiment were passage 1. Full list of GO analysis results can be found in Supplementary Material S2.

FIGURE 2. CFB contraction and stress activation. (A) CFB contractility was assayed through collagen gel contraction using passage 3 LVCFBs and VICs. Example brightfield image of collagen gel 24 h post-seeding. Gel area was analyzed using ImageJ. (B) Quantification of collagen gel contraction; gel areas with samples were normalized to an averaged area of control gels lacking cells at 24 h. Dots represent 3 wells and color represents donor. Statistics are a two-way ANOVA with Tukey’s post hoc test where * is p < 0.05 and ** is p < 0.01. (C) Primary CFB stress activation potential measured by treatment of passage 4 cells with control primary CFB maintenance medium, DMEM/F12 medium, or DMEM/F12 medium supplemented with 10 ng/mL TGFβ1. Example images of cells stained for phalloidin (red) and Hoechst (blue) that were categorized as containing stress fibers (yes) or not containing stress fibers (no). Scale bar is 100 µm. (D) Quantification of fraction of cells containing stress fibers. Dots represent the percentage of cells for each sample with 20–750 cells per condition and color represents the donor. Statistics are a four-way ANOVA on donor, cell source, medium, and cell source cross medium.…

FIGURE 3. Alizarin red mineralization staining of primary CFBs. (A) Brightfield images of primary CFBs (passage 4) stained with Alizarin Red after 4 weeks of treatment with maintenance or osteogenic medium. Images shown are representative of three well replicates. Scale bar is 250 μm. (B) Quantification of Alizarin red staining by measuring absorbance at 560 nm. Dots represent three well replicates and donors are represented by color. Statistics are a two-way ANOVA on media controlling for donor where * is p < 0.05. (C) Percentage change in Alizarin Red staining comparing osteogenic to maintenance medium where dots represent three well replicates and donors are represented by color. Statistics are a two-way ANOVA on cell source controlling for donor where ** is p < 0.01.

FIGURE 4. Calcification of primary CFBs. Primary CFBs (passage 4) were treated with maintenance or osteogenic medium for 4 weeks. (A) Von Kossa and Nuclear Fast Red Staining of primary CFBs. Calcium in mass deposits are black, calcium in dispersed deposits are gray, nuclei are red, and cytoplasm is light pink. Images are representative of three well replicates and scale bar is 100 μm. (B) Relative ALP activity in primary CFBs is quantified by measuring absorbance at 405 nm. Dots represent well replicates and color represents donor. Statistics are a two-way ANOVA for medium controlling for donor where * is p < 0.05 and ** is p < 0.01. (C) Percentage change in relative ALP activity between maintenance to osteogenic medium. Dots represent well replicates and color represents donor. Statistics are a two-way ANOVA for cell source controlling for donor where * is p < 0.05.

FIGURE 5. Calcification of primary CFBs post-activation. (A) Schematic showing experimental setup. Primary CFB samples (passages 5 and 6) were either pre-treated with activation medium or maintained in CFB maintenance medium prior to mineralization activation to mimic dystrophic and metastatic mineralization. (B) Quantification of αSMA immunocytochemistry (ICC) staining of cells after 3 days in activation medium. Graph is image αSMA mean intensity minus background normalized by number of nuclei. Dots represent well replicates and color represents donor. (C) Example ICC images used for quantification in B. Magenta is anti-αSMA antibody and blue is Hoechst stain. Scale bar is 100 μm. (D) Left: Relative ALP activity for primary CFBs upon activation. Dots represent well replicates and color represents donor. Right: Percentage change in relative ALP activity of metastatic and dystrophic compared to control. Dots represent well replicates and color represents donor. Statistics are a two-way ANOVA for cell source controlling for donor where * is p < 0.05, ** is p < 0.01, and ***is p < 0.005.

FIGURE 6. Mass Spectrometry for ECM and Secreted Factors. (A) The relative abundance ratio of VIC/LVCFB ECM and secreted factors as detected by mass spectrometry was used to determine the percentage of proteins detected with decreased abundance (VIC/LVCFB Abundance ratio <0.5), increased abundance (>2.0) or those that remained unchanged (0.5–2.0). Cells used in this experiment were of passages 4 and 5. (B) Hierarchical Clustering using the one minus Pearson’s correlation of n = 3 replicates from three different donors determines that the ECM and secreted factors of VICs and LVCFB cluster distinctly. (C) ECM and secreted factors of interest that were determined to be statistically significantly (p < 0.05) up or downregulated in VICs in comparison to LVCFB (n = 9 grouped across the three different donors).