The Calmodulin/Striatin Interaction Is Enhanced in Diabetic Hearts and Defines Novel Signaling Clusters Implicated in Cardiac Remodeling

Figures

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  1. CaM pull-down assay and interaction patterns of STRN, PP2A-A, and -C subunits with CaM in diabetic left ventricles at early (8 weeks) and chronic (24 weeks) stages of diabetes. Western blot images and their corresponding histograms showing the interaction between CaM, STRN, and PP2A-A and -C subunits in normal and diabetic LVs at 8 weeks (A) and 24 Weeks (B) post-STZ. Values represent mean ± SEM (n = 7 samples per group for each protein); **p < 0.01.

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  2. Effect of STRN overexpression on its association with CaM in H9c2 cells. (A) Representative Western blot showing STRN levels in CaM pull-down assays and corresponding input (protein lysates) from H9c2 cells transduced with RFP (control) or RFP-tagged STRN (RFP-STRN). GAPDH was used as a loading control for the input samples. (B) Quantification of STRN levels in CaM pull-down assays and input lysates across four independent experiments (p = 0.11, paired t test).

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  3. Proteomic profiling of CaM-interacting proteins in diabetic versus normal left ventricles. (A) Three-dimensional principal component analysis (PCA) plot of proteomics data from normal and diabetic samples. Blue dots represent normal samples (normal 1–5), and red dots represent diabetic samples (diabetic 1–5). Individual samples are labeled to show the distribution of biological replicates within each group. (B) Volcano plot shows differentially interacting CaM-binding proteins between diabetic and normal left ventricles. The results are presented in the upper part of the volcano plots, which represent the significance from the t test (−log10­(p value)) on the y axis and the log2 fold change (FC) on the x axis. (C) Heatmap visualization of the top 10 upregulated and top 10 downregulated CaM-interacting proteins in diabetic versus normal samples. Z-score normalized expression values are shown, with red indicating higher interaction and blue indicating lower interaction with CaM. Samples (normal 1–5 and diabetic 1–5) are presented in columns, and proteins (labeled by gene names) are in rows. Hierarchical clustering highlights distinct expression patterns between both groups.

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  4. Variable Importance in Projection (VIP) analysis of differentially expressed CaM-interacting proteins between diabetic and control LV. (A) Bar plot showing the top 25 proteins ranked by VIP score, highlighting those with the highest discriminatory power between diabetic and control groups. (B) Scatter plot of VIP scores for upregulated (higher interaction with CaM; red) higher interaction with CaM and downregulated (lower interaction with CaM; blue) proteins. Each dot represents an individual protein, with horizontal lines indicating group means (upregulated: 2.49; downregulated: 1.44).

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  5. Functional enrichment analysis of differentially expressed CaM-interacting proteins in diabetic LVs. Enriched GO biological processes (A) and KEGG pathways (B) among proteins with an increased interaction with CaM in diabetic hearts. Top terms include lipid and fatty acid metabolism, acyl-CoA metabolic processes, and the PPAR signaling pathway. Enriched GO biological processes (C) and KEGG pathways (D) among proteins with reduced interaction with CaM. Notable terms include responses to lipids and glucocorticoids, along with enrichment in calcium signaling and amino acid biosynthesis pathways. The bubble size represents gene count, and the color indicates FDR-adjusted significance.

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  6. Comparison and functional profiling of CaM and STRN-IP interacting protein data sets. (A) Venn diagram showing the distribution of protein accessions between STRN IP group (left circle, red) and CaM group (right circle, green). The overlap represents 231 proteins common to both groups. (B) Bar graph representing the top 15 enriched GO terms among proteins overlapping between IP and CaM data sets. GO terms are shown on the y axis, and statistical significance is indicated by −log10­(adjusted p value) on the x axis.

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  7. Comparative analysis of CaM upregulated interactors and STRN IP data sets. (A) Venn diagram comparing CaM upregulated proteins (left circle, red) with IP of STRN in diabetic LVs (right circle, green). The overlap represents three proteins common to both groups. (B) Venn diagram comparing CaM upregulated proteins (left circle, red) with IP of STRN in diabetic and simultaneously normal and diabetic LVs (right circle, green), showing the same three overlapping proteins.