Proteomics of stress-induced cardiomyopathy: insights from differential expression, protein interaction networks, and functional pathway enrichment in an isoproterenol-induced TTC mouse model

Figures

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  Figure 1. Echocardiographic assessment of ISO-induced TTC-like cardiomyopathy in female mice. (A) Heart rate (beats per minute, bpm) measured at baseline (before ISO injection) and 1, 3, and 7 days post-injection. (B) Respiratory rate (breaths per minute, bpm) measured at the same time points. (C) Representative echocardiographic images demonstrating apical ballooning in ISO-treated mice compared to the control group. (D) Left ventricular ejection fraction (LVEF, %), (E) left ventricular end-diastolic volume (LVEDV, µL), and (F) left ventricular end-systolic volume (LVESV, µL) measured at the same time points. Data are presented as mean ± SD from 5 animals per group at each time point. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (ns, not significant; *p < 0.05, **p < 0.01, compared to baseline or control group).

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  Figure 2. Histopathological changes in myocardial tissues from ISO-induced TTC-like cardiomyopathy. (A) Hematoxylin and eosin (H & E) staining of myocardial tissue at baseline (control), and 1, 3, and 7 days after ISO injection. Representative images show progressive myocardial cell thickening, dissolution, and structural disorganization. Scale bar = 50 µm. (B) Quantitative scoring of myocardial damage based on injury area, defined as: 0 = no damage, 1 = <25%, 2 = 25–50%, 3 = 50–75%, and 4 = >75% damage. Data represent mean ± SD from five animals per group at each time point. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test (ns, not significant; *p < 0.05, **p < 0.01 compared to baseline or control group).

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  Figure 3. Proteomic profiling of myocardial tissues from ISO-induced and control groups. (A) Boxplot of protein expression levels in myocardial tissues from control and ISO-treated mice. Data represent log-transformed normalized protein intensities from five biological replicates per group. (B) Principal component analysis (PCA) of protein expression data showing distinct clustering of control and ISO-treated samples, indicating significant differences in proteomic profiles.

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  Figure 4. Identification and validation of differentially expressed proteins (DEPs) between ISO-treated and control groups. (A) Total number of DEPs identified (81 proteins) with 39 upregulated (red) and 42 downregulated (blue) proteins (|log2FC| ≥ 1.5, p < 0.05). (B) Volcano plot displaying the significance (−log10 p-value) and fold change (log2FC) of all detected proteins. Upregulated proteins are shown in red, downregulated proteins in blue, and non-significant proteins in gray.

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  Figure 5. Gene Ontology (GO) enrichment analysis of differentially expressed proteins (DEPs). (A) GO analysis of upregulated DEPs reveals significant enrichment in pathways related to collagen fibril organization, cholesterol biosynthesis, and elastic fiber assembly. (B) Downregulated DEPs are significantly enriched in pathways including positive regulation of vascular smooth muscle differentiation, complement activation (classical pathway), and response to hypoxia. Data are derived from five biological replicates per group, with p < 0.05 considered significant.

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  Figure 6. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEPs. (A) KEGG analysis of upregulated DEPs shows significant enrichment in pathways such as ferroptosis, glutathione metabolism, and biosynthesis of unsaturated fatty acids. (B) Downregulated DEPs are enriched in pathways including primary immunodeficiency, complement and coagulation cascades, and cholesterol metabolism. Pathways were considered significantly enriched based on p < 0.05, with data derived from five biological replicates per group.

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  Figure 7. Protein-protein interaction (PPI) network analysis of DEPs. Protein-protein interaction network was constructed using the STRING database, visualized in Cytoscape. Hub proteins were identified based on degree centrality, including Ntrk2, Fdft1, Serpine1, and Cyp1a1. The network highlights key interactions among DEPs, with nodes representing proteins and edges indicating functional associations. Confidence score threshold = 0.7.

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  Figure 8. Gene Set Enrichment Analysis (GSEA) of ISO-treated vs. control groups. GSEA results reveal significantly enriched upregulated pathways in ISO-treated mice, including oxidative phosphorylation, ferroptosis, and terpenoid backbone biosynthesis (normalized enrichment score [NES] > 1.91, p < 0.01). Downregulated pathways include systemic lupus erythematosus, Rap1 signaling, and chemokine signaling (NES < −2.00, p < 0.01). Data represent normalized enrichment scores based on five biological replicates per group.

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  Figure 9. Dual validation of DEPs using qPCR and Western Blot. (A–J) qPCR validation of the top 5 upregulated and top 5 downregulated DEPs, showing consistent expression trends between qPCR and proteomics data. Data represent mean ± SD from 3 biological replicates per group. Statistical significance was determined using Student ’ s t-test (**p < 0.01 compared to control). (K, L) Western Blot validation of key hub proteins (Ntrk2, Fdft1, Serpine1, and Cyp1a1) identified through PPI network analysis. Representative Western Blot images and quantification of protein expression normalized to beta-actin (loading control) are shown. Data represent mean ± SD from three biological replicates per group. Statistical significance was determined using Student’s t-test (**p < 0.01 compared to control).