Metabolic Processes are Potential Biological Processes Distinguishing Nonischemic Dilated Cardiomyopathy from Ischemic Cardiomyopathy: A Clue from Serum Proteomics

Abstract

Background: Ischemic cardiomyopathy (ICM) and nonischemic dilated cardiomyopathy (DCM) are the two most common causes of heart failure. However, our understanding of the specific proteins and biological processes distinguishing DCM from ICM remains insufficient.

Materials and methods: The proteomics analyses were performed on serum samples from ICM (n=5), DCM (n=5), and control (n=5) groups. Proteomics and bioinformatics analyses, including weighted gene co-expression network analysis (WGCNA) and gene set enrichment analysis (GSEA), were performed to identify the hub circulating proteins and the hub biological processes in ICM and DCM.

Results: The analysis of differentially expressed proteins and WGCNA identified the hub circulating proteins in ICM (GAPDH, CLSTN1, VH3, CP, and ST13) and DCM (one downregulated protein, FGG; 18 upregulated proteins, including HEL-S-276, IGK, ALDOB, HIST1H2BJ, HEL-S-125m, RPLP2, EL52, NCAM1, P4HB, HEL-S-99n, HIST1H4L, HIST2H3PS2, F8, ERP70, SORD, PSMA3, PSMB6, and PSMA6). The mRNA expression of the heart specimens from GDS651 validated that ALDOB, GAPDH, RPLP2, and IGK had good abilities to distinguish DCM from ICM. In addition, GSEA results showed that cell proliferation and differentiation were the hub biological processes related to ICM, while metabolic processes and cell signaling transduction were the hub biological processes associated with DCM.

Conclusion: The present study identified five dysregulated hub circulating proteins among ICM patients and 19 dysregulated hub circulating proteins among DCM patients. Cell proliferation and differentiation were significantly enriched in ICM. Metabolic processes were strongly enhanced in DCM and may be used to distinguish DCM from ICM.

Keywords: dilated cardiomyopathy; gene set enrichment analysis; ischemic cardiomyopathy; proteomics; weighted gene co-expression network analysis.

Graphical abstract

Figure 1

Sample clustering and modules detected in weighted gene co-expression network analysis. (A) The sample clustering plot. The protein expression data from the DCM_5 sample were discarded in the subsequent analysis due to the heterogeneity of the data from other samples. (B) The scale-free network was constructed with the scale-free R²=0.8, where the soft threshold was set at twelve to get the best-fit topology model. (C) According to the hierarchical clustering dendrogram of proteins, nine modules were detected, namely black, blue, brown, green, grey, pink, red, turquoise, and yellow modules. (D) The topological overlap map (TOM) plot of distinct modules detected. The heatmap showed the topological overlap matrix (TOM) among genes in the analysis. Different colors on the x-axis and y-axis represented different modules. The yellow brightness of the middle part represented the strength of connections between modules.

Figure 2

Modules highly associated with ischemic and DCM. Different modules are well distinguished according to the clustering plot of module eigengenes (A) and the module-module correlation plot (B). The module-trait correlation plot (C) showed that the turquoise module was well correlated with ICM. The black module and the green module were strongly associated with DCM. The proteins in the black module were expressed at a high level and the proteins in the green module were at a low level in the DCM group. In addition, the proteins in the turquoise module were significantly down-regulated in the ICM group (D).

Figure 3

Hub proteins enriched in the ICM patients. The hierarchical clustering plot (A) and a volcano plot (B) were constructed to present the dysregulated proteins in the ICM group. The Venn plot (C) showed that five down-regulated proteins were overlapped with the hub proteins in the turquoise module, which was negatively associated with ICM in the module-trait correlation analysis of WGCNA. In addition, the other 70 hub proteins in the turquoise module could be detected only in the DCM group, but not in the ICM group. In addition, the validation results showed that mRNAs of GAPDH, CLSTN1, CP, and ST13 were detected in the heart specimens from GDS651, among which the protein expression change of GAPDH in the serum is consistent with its mRNA expression change in the heart specimens of ICM patients (D). The ROC curve illustrated that GAPDH could distinguish ICM from NF well (E).

Figure 4

Hub proteins enriched in the DCM patients. The hierarchical clustering plot (A) and a volcano plot (B) were constructed to present the dysregulated proteins in the DCM group. The Venn plot (C) showed that 18 up-regulated proteins were overlapped with the hub proteins in the black module, which was positively associated with DCM in the module-trait correlation analysis of WGCNA. FGG was the only protein overlapped with the hub proteins in the green module. In addition, the other 12 hub proteins in the green module could be detected only in the ICM group, but not the DCM group.

Figure 5

ALDOB, GAPDH, RPLP2, and IGK could distinguish DCM from ICM well. The RNA array data of heart specimens of DCM and NF from GDS651 were used to verify the expression of the mRNA corresponding to hub proteins in the heart. The results showed that mRNAs of RPLP2, HIST1H2BJ, HIST1H4L, ALDOB, SORD, IGK, PSMA3, HEL-S-276, NCAM1, HEL-S-99n, F8, P4HB, and HEL-S-125m were detected in the heart specimens, among which the protein expression changes of RPLP2, HIST1H2BJ, HIST1H4L, ALDOB, SORD, and IGK in the serum are consistent with its mRNA expression change in the heart specimens of DCM patients (A). The ROC curve illustrated that RPLP2, HIST1H2BJ, HIST1H4L, ALDOB, SORD, and IGK could distinguish DCM from NF well (B). Furthermore, ALDOB, GAPDH, RPLP2, and IGK could distinguish DCM from ICM well (C).

Figure 6

Cell proliferation and differentiation were the hub biological processes related to ICM. The gene set enrichment analysis (GSEA) results showed that 11 biological processes were positively correlated to ICM (but not correlated to DCM), among which six biological processes were associated with cell proliferation and differentiation (A), three with the structure organization (B). The additional 2 enriched biological processes were the signal transduction by protein phosphorylation and biological adhesion (C).

Figure 7

Metabolic processes were the hub biological processes associated with DCM. A total of thirty-four biological processes were significantly enriched in the DCM patients (but not significantly enriched in the ICM patients) via performing the gene set enrichment analysis (GSEA). The results showed that 11 biological processes were associated with the metabolic processes (A). In addition, there were six biological processes enriched in the cell signaling transduction (B), three in the cell cycle (C), and three in the morphogenesis (D). The other biological processes included the oxidation reduction process, the post-transcriptional regulation of gene expression, the regulation of hemopoiesis, the transmembrane transport, the response to abiotic stimulus, the response to oxygen levels, the immune system development, the epithelium development, the myeloid leukocyte activation, the interspecies interaction between organisms and the cell activation involved in immune response (E).