Integrative Proteomic Analysis Reveals the Cytoskeleton Regulation and Mitophagy Difference Between Ischemic Cardiomyopathy and Dilated Cardiomyopathy

Figures

• 

  Fig. 1. Morphology transfection of ICM and DCM.A, clinical cardiac markers in normal control, ICM, and DCM serum. B, representative H&E staining images of human heart samples (the scale bar represents 100 μm) and corresponding quantification. C, representative Masson images of human heart samples for fibrosis and corresponding quantification of collagen area ratio. Statistical significance was calculated using a two-tailed Student’s t test (mean ± SD.). ns, no significance, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy.

• 

  Fig. 2. Proteomics profiling and phosphoproteomic landscape of human heart samples.A, overview workflows of the experimental design and the number of samples used for proteomics and phosphoproteomics analyses. B, number of proteins, phosphoproteins, and phosphosites identified in NC, ICM, and DCM. C, proportion of subcellular localizations in proteomics and phosphoproteomics data. D and E, principal component analysis (PCA) of proteomics and phosphoproteomics data in heart samples. F, proteins driving the segregation between NC, ICM, and DCM. G, expression of cardiac markers in heart tissues among NC, ICM, and DCM. Statistical significance was calculated using a two-tailed Student’s t test (mean ± SD). ns, no significance, ∗p < 0.05, ∗∗p < 0.01. DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy; NC, normal control.

• 

  Fig. 3. Proteomics Features of ICM and DCM.A, the contribution proportion of each organelle to cellular protein mass among NC, ICM, and DCM. B, the summation profiling protein intensities in extracellular matrix and mitochondria. C, volcano plots depicting the differential expression of proteins between ICM and DCM. D, the schematic picture of upregulated and downregulated proteins in ICM and DCM. E, GSEA of ICM and DCM revealed distinct pathways (p value from log rank test). F, the heat map generated by unsupervised hierarchical clustering of z-scored protein intensities highlights the differential expression proteins, with significantly upregulation and downregulation. Statistical significance was calculated using a two-tailed Student’s t test. ns, no significance, ∗p < 0.05. DCM, dilated cardiomyopathy; GSEA, gene set enrichment analysis; ICM, ischemic cardiomyopathy; NC, normal control.

• 

  Fig. 4. Phosphoproteomic analysis of ICM and DCM.A, the heat map generated by unsupervised hierarchical clustering of differentially expressed phosphoproteins reveals significantly enriched pathways. B, phospho-regulatory network in ICM and DCM. The center cycle linking the upregulated (blue diamonds) and downregulated (blue diamonds) kinases and phosphoproteins (ellipses) enriched in signaling pathways. Bold lines indicate higher correlations (r > 0.7 or r < −0.7). C, fold changes of proteins and phosphoproteins in ICM. Red dots defined as activated phosphoproteins in ICM, which calculated as the fold changes of phosphoproteins are greater than changes of their corresponding protein intensities. D, comparison of the differently regulated phosphosites involved in cytoskeleton pathways among the three groups and KSEA analysis of those phosphosites (p value from log-rank test). Statistical significance was calculated using a two-tailed Student’s t test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy; KSEA, kinase-substrate enrichment analysis.

• 

  Fig. 5. Cytoskeleton remodeling and mitochondrial dysfunction in mouse and cell models.A, representative image of echocardiography in mouse model. B, representative images of WGA staining showing cardiomyocyte atrophy. C, electron microscopy of myocardial tissue within sarcomeres, gap junctions, and mitochondria after MI and DOX injury. D, representative images of cTnT and CD31 staining showing cardiomyocytes and vessels, respectively. E, different expression of kinases and different regulated phosphosites in NC, ICM, and DCM. F, expression of PRKACA and phosphorylation of Ser339, GSK3β, and phosphorylation of Ser9 in animal models detected by Western blotting analysis. G, expression of ROCK1, vimentin1, and phosphorylation of Ser72 in animal models detected by Western blotting analysis. H, cell viabilities in different concentration gradient in tBHP and DOX were detected by CCK8 assay in three different cell lines. I, representative images of filamentous actin detected by phalloidin-Rhodamine (red) in cardiomyocytes and CMECs. J, mitochondria morphology was detected by mitotracker (red). Statistical significance was calculated using a two-tailed Student’s t test. ns, no significance, ∗p…

• 

  Fig. 6. Differences incytoskeleton associated proteins following myocardial infraction and DOX-induced injury.A and B, expression of PRKACA and phosphorylation of Ser339, GSK3β, and phosphorylation of Ser9 in tBHP treatment detected by Western blotting analysis. C and D, expression of PRKACA and phosphorylation of Ser339, GSK3β, and phosphorylation of Ser9 in tBHP and BLU0588 treatment detected by Western blotting analysis. E, representative images of f-actin detected by phalloidin-Rhodamine (red) in tBHP and CHIR99021 treatment in cardiomyocytes. F, representative images of VE-cadherin (red) and β-catenin (green) detected by immunofluorescence in tBHP- and CHIR99021-treated CMECs. G, statistical analysis of f-actin in tBHP and CHIR99021 treatment in cardiomyocytes. H, statistical analysis of VE-cadherin and β-catenin in tBHP and CHIR99021 treatment in CMECs. I, statistical analysis of f-actin in site-mutant cardiomyocytes. J, statistical analysis of VE-cadherin and β-catenin in site-mutant CMECs. K, representative images of f-actin detected by phalloidin-Rhodamine (red) in site-mutant cardiomyocytes. L, representative images of VE-cadherin (red) and β-catenin (green) detected by…

• 

  Fig. 7. Differences in mitophagy protein changes following myocardial infraction and DOX-induced injury.A and B, Western blot analysis of revealing the expression levels of ROCK1, vimentin, and phosphorylation of Ser72 under tBHP and DOX treatment. C and D, Western blot analysis of revealing the expression levels of ROCK1, vimentin, and phosphorylation of Ser72 under DOX and ROCK-IN-1 treatment. E, representative fluorescent images of mito-Keima-488/555 transfection in DOX and ROCK-IN-1–treated cardiomyocytes. F, representative fluorescent images mito-Keima-488/555 transfection in DOX and ROCK-IN-1–treated CMECs. G, statistical analysis of mito-keima-488/555 transfection in DOX and ROCK-IN-1–treated cardiomyocytes. Mitophagy levels were assessed by the ratio of red/green. H, statistical analysis of mito-Keima-488/555 transfection in DOX and ROCK-IN-1–treated CMECs. Mitophagy levels were assessed by the ratio of red/green. I, statistical analysis of mito-Keima-488/555 transfection in DOX and ROCK-IN-1–treated cardiomyocytes. Mitophagy levels were assessed by the ratio of red/green. J, statistical analysis of mito-Keima-488/555 transfection in DOX and ROCK-IN-1–treated CMECs. Mitophagy…

• 

  Supplemental Figure S1. Proteomics profiling and phosphoproteomic landscape of human heart samples.A–C, density plot of the protein intensities distribution. A unimodal distribution (dip test) was observed as all of samples passed proteomic quality control. D, intensity-based ranks (log2 transformed) of the proteomics profile of NC, ICM and DCM. The highest- and lowest-abundance proteins are shown in the box. E, the numbers of phosphoproteins and phosphosites in NC, ICM and DCM. F, comparison of the collagen protein expression among samples from NC, ICM and DCM. Statistical significance was calculated using a two-tailed Student’s t-test. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

• 

  Supplemental Figure S2. Proteomics Features of ICM and DCM.A–C, volcano plots of different expressed proteins in NC, ICM and DCM. D, heatmap of different expressed protein (left) and the cluster of significantly enriched pathways in ICM and DCM (right).

• 

  Supplemental Figure S3. Phosphoproteomics Features of ICM and DCM.A and B, volcano plots of different expressed phosphoproteins and phosphosites in NC, ICM and DCM. C, fold-changes of proteins and phosphoproteins in DCM. Red dots defined as activated phosphoproteins in DCM, which calculated as the fold changes of phosphoproteins are greater than changes of their corresponding protein intensities. D, KSEA of kinase activities in NC, ICM and DCM. E, GSEA of differently altered phosphoproteins enriched pathways in NC, ICM and DCM. F, different regulated phosphosites involved in mitophagy and their intensities in the three groups, and KSEA of those phosphosites. G, heatmap of different expressed phosphoprotein (left) and the cluster of significantly enriched pathways in ICM and DCM (right). Statistical significance was calculated using a two-tailed Student’s t-test. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

• 

  Supplemental Figure S4. Differences in mitophagy protein changes after myocardial infraction and DOX injury.A, representative fluorescent images of VIM (green) and Tomm20 (red). B, representative fluorescent images of LC3-GFP-RFP transfection with ROCK-IN-1 treatment. Autolysosomes were identified by an LC3 signal with single-color fluorescence (red). C, representative fluorescent images of LC3-GFP-RFP transfection in site-mutate transfection. Autolysosomes were identified by an LC3 signal with single-color fluorescence (red). Statistical significance was calculated using a two-tailed Student’s t-test. ns, no significance, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.