Cathepsin K as a key regulator of myocardial fibrosis in dilated cardiomyopathy and a promising therapeutic target

Figures

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  Figure 1. Overview of the workflow and GEO data. A, flow chart of the present study. B, the box plots of gene expression in human non-failing (NF) and failing (DCM) heart tissues. The x-axis is the sample and the y-axis is the gene expression level. The black line in the middle is the median of the expressed values. C, principal component analysis plot of the gene expression profiles in GSE165303. It shows the separate clustering of the two groups, with a few samples overlapping by two principal components of the variance. D, Volcano plot of DEGs between NF and DCM heart tissues. The red plots represent upregulated genes, the grey plots represent nonsignificant genes, and the blue plots represent downregulated genes.

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  Figure 2. Gene co-expression modules associated with DCM. A, network topology for different soft-thresholding powers. The scale-free topology can be attained at the soft-thresholding power of 6. B, Cluster dendrogram and gene modules identified by WGCNA. Each color represents one specific co-expression module, and the branches above represent genes. C, correlation heat map of gene modules and traits, labeled with correlation coefficient and p-value. D, biological processes enriched genes within each module.

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  Figure 3. The PPI network of these interested modules. Visualization of the interested modules' hub genes. The circles represent the hub genes in the modules, and the lines show the interaction between the hub genes. The different colors represent the different modules' genes. The size of the nodes denotes the total number of predicted interactions initiated from each node. PPI, protein-protein interaction.

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  Figure 4. Identification of CTSK as a driver gene to cardiac fibrosis. A, heatmap of DEGs between the non-failing and dilated cardiomyopathy samples. Red rectangles represent high expression, and blue rectangles represent low expression. B, Venn diagram illustrating shared genes in different groups. C and D, RNA-seq for 11 shared genes in GSE165303 and GSE29819.The black arrow indicates genes that are consistently expressed in both datasets (GSE165303 and GSE29819). E, cluster analysis for single-cell RNA sequencing data from all cell types. F, heatmap of CTSK expression in each cardiomyocyte cluster projected on UMAP graph. Color scale represents the expression of CTSK. G, Masson's staining of left ventricular specimens from patients with dilated cardiomyopathy (DCM) and normal left ventricular samples (NF). H, the quantification of fibrosis. The statistical analysis employed the Mann-Whitney test. I, immunofluorescence analysis of CTSK and fibroblast marker COL1A1 in human paraffin-embedded tissue sections. Representative images show co-localization of CTSK (red) and COL1A1 (green) in human tissues (arrows indicate positive cells). I, fluorescence intensity was quantified using ImageJ.…

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  Figure 5. Effects of CTSK knockdown and PDGF-BB treatment on HCFs mRNA and protein expression, proliferation markers, and PDGFR signaling pathway components. A, real-time quantitative PCR was used to quantify the levels of CTSK mRNA in HCFs 48 h after knockdown with specific siRNA, with GAPDH mRNA level as internal control. Data are mean values ± SD for independent experiments (n = 3). The unpaired t test (two-tailed) was used to calculate the presented p values. B, Western blot detection of CTSK in HCFs transfected with CTSK siRNA (si-CTSK). C, RT-qPCR was used to quantify the levels of Ki67 in HCFs 48 h after knockdown with specific siRNA, with GAPDH mRNA level as internal control. Data are mean values ± SD for independent experiments (n = 3). The unpaired t test (two-tailed) was used to calculate the presented p values. D, RT-qPCR method was used to determine the levels of CTSK and Ki67 in HCFs treated with different concentrations of PDGF-BB (platelet-derived growth factor subunit B homodimer) for 24 h. Plots are showing duplicate RT-qPCR assays using GAPDH as an internal control. E, Western blot detection of CTSK, ERK1/2, and p-ERK1/2 in HCFs incubated with 20 ng/ml PDGF-BB at…

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  Figure 6. Impact of CTSK knockdown on HCF proliferation, and PDGFR signaling pathway activation in response to PDGF-BB stimulation. A, proliferation assay of HCF cells. Cells were transfected with siRNA on Day 1, and cell viability was assessed every 24 h using the CCK8 method until Day 6. Each data point represents the mean of three independent experiments (n = 3). Error bars indicate standard deviations. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparisons test. The p-values comparing the Ctrl siRNA group and the si-CTSK group within the PDGF-BB group are indicated. p-values that are not indicated denote no statistical significance. B, RT-qPCR was used to quantify the levels of α-SMA mRNA in HCFs 48 h post-knockdown with specific siRNA. with GAPDH mRNA level as internal control. Data are mean values ± SD for independent experiments (n = 3). The unpaired t test (two-tailed) was used to calculate the presented p values. C, Western blot detection of CTSK, α-SMA, and PDGFR downstream signaling proteins (p-ERK1/2) in HCFs with CTSK knockdown and treated with 20 ng/ml PDGF-BB for 48 h. D, protein levels quantified from three independent samples by…

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  Figure 7. TGF-β1 induces cardiac fibroblast-to-myofibroblast transdifferentiation independent of CTSK. A, the mRNA levels of α-SMA, Ki67, and CTSK in HCFs treated with different concentrations of TGF-β1 for 24 h were measured by RT-qPCR. Plots are showing duplicate RT-qPCR assays using GAPDH as an internal control. B, the mRNA levels of α-SMA, Ki67, and CTSK in HCFs treated with 10 ng/ml TGF-β1 for different durations were measured by RT-qPCR. Plots are showing duplicate RT-qPCR assays using GAPDH as an internal control. C, the protein expression levels of CTSK, COL1A1, p-SMAD2, and α-SMA in HCFs treated with CTSK knockdown and 10 ng/ml TGF-β1 for 48 h were detected by Western blotting. D, protein levels quantified from three independent samples by Western blot assays as represented in C. Protein bands were normalized to that of GAPDH. Levels of p-SMAD2 were shown as ratios to its total protein level. p values were calculated by ordinary 1-way ANOVA with Tukey multiple comparisons test. E, the mRNA levels of CTSK, Ki67, and α-SMA in HCFs treated with CTSK knockdown and 10 ng/ml TGF-β1 for 48 h were measured by RT-qPCR, with GAPDH mRNA level as internal control. p values were calculated by…