Isobaric tags for relative and absolute quantitation‑based proteomics reveals potential novel biomarkers for the early diagnosis of acute myocardial infarction within 3 h

Abstract

Acute myocardial infarction (AMI) is one of the most common and life‑threatening cardiovascular diseases. However, the ability to diagnose AMI within 3 h is currently lacking. The present study aimed to identify the differentially expressed proteins of AMI within 3 h and to investigate novel biomarkers using isobaric tags for relative and absolute quantitation (ITRAQ) technology. A total of 30 beagle dogs were used for establishing the MI models successfully by injecting thrombin powder and a polyethylene microsphere suspension. Serum samples were collected prior to (0 h) and following MI (1, 2 and 3 h). ITRAQ‑coupled liquid chromatography‑mass spectrometry (LC‑MS) technology was used to identify the differentially expressed proteins. The bioinformatics analysis selected several key proteins in the initiation of MI. Further analysis was performed using STRING software. Finally, western blot analysis was used to evaluate the results obtained from ITRAQ. In total, 28 proteins were upregulated and 23 were downregulated in the 1 h/0 h group, 28 proteins were upregulated and 26 were downregulated in the 2 h/0 h group, and 24 proteins were upregulated and 19 were downregulated in the 3 h/0 h group. The Gene Ontology (GO) annotation and functional enrichment analysis identified 19 key proteins. Protein‑protein interactions (PPIs) were investigated using the STRING database. GO enrichment analysis revealed that a number of key proteins, including ATP synthase F1 subunit β (ATP5B), cytochrome c oxidase subunit 2 and cytochrome c, were components of the electron transport chain and were involved in energy metabolism. The western blot analysis demonstrated that the expression of ATP5B decreased significantly at all three time points (P<0.01), which was consistent with the ITRAQ results, whereas the expression of fibrinogen γ chain increased at 2 and 3 h (P<0.01) and the expression of integrator complex subunit 4 increased at all three time points (P<0.01), which differed from the ITRAQ results. According to the proteomics of the beagle dog MI model, ATP5B may serve as the potential biomarkers of AMI. Mitochondrial dysfunction and disruption of the electron transport chain may be critical indicators of early MI within 3 h. These finding may provide a novel direction for the diagnosis of AMI.

Figure 1

Entire workflow of proteomics using ITRAQ combined LC-MS technology. Serum protein pre-MI (0 h) was regarded as the control group, the obtained differential expressed proteins were subjected to further bioinformatics analysis. Selected proteins were verified by western blot analysis. MI, myocardial infarction; SCE, strong cation exchange; LC-MS, liquid chromatography-mass spectrometry; GO, Gene Ontology; ITRAQ, isobaric tags for relative and absolute quantitation; DEP, differentially expressed protein.

Figure 2

Coronary angiography images of beagle dogs. (A) Left coronary angiography prior to intervention occlusion surgery (0 h), the red arrows refer to the LAD. (B) Ischemia preconditioning by balloon dilation. (C) Left coronary angiography 2 h post-LAD embolism. (D) Left coronary angiography 3 h post-LAD embolism (E) Left coronary angiography 6 h post-LAD embolism, the second diagonal branch reappeared. (F) Left coronary angiography 3 days post-LAD embolism, the blood-flow of the LAD had returned to normal. LAD, left anterior descending artery.

Figure 3

Venn diagram of all DEPs at the three time points. A Venn diagram of all DEPs at the three time points is presented. There were 51, 54 and 43 DEPs at 1 h/0 h, 2 h/0 h and 3 h/0 h, respectively. In addition, 17 DEPs were common to the 1 h and 2 h time points; eight DEPs were common to the 1 h and 3 h time points; four DEPs were common in the 2 h and 3 h time points; 11 DEPs were common to the 1 h, 2 and 3 h time points. DEPs, differentially expressed proteins.

Figure 4

Heatmap and cluster analysis of 19 DEPs at 1, 2 and 3 h post-MI, vs. pre-MI (0 h). (A) Each line in the heatmap represents a protein with upregulated (red) or downregulated (green) expression at three time points post-MI, vs. pre-MI, whereas white represents no change in expression. A complete list of the 19 selected DEPs is present in Table I. (B) All 19 proteins were divided into four groups according to different expression patterns (patterns 1-4). MI, myocardial infarction; DEPs, differentially expressed proteins.

Figure 4

Heatmap and cluster analysis of 19 DEPs at 1, 2 and 3 h post-MI, vs. pre-MI (0 h). (A) Each line in the heatmap represents a protein with upregulated (red) or downregulated (green) expression at three time points post-MI, vs. pre-MI, whereas white represents no change in expression. A complete list of the 19 selected DEPs is present in Table I. (B) All 19 proteins were divided into four groups according to different expression patterns (patterns 1-4). MI, myocardial infarction; DEPs, differentially expressed proteins.

Figure 5

PPI regulatory network of DEPs. All DEPs were combined together to form a complex interaction network using STRING software. The network contained 19 nodes and 15 edges. Average node degree: 1.58; local clustering coefficient: 0.558; expected number of edges: one. The PPI enrichment P-value was 4.11e-13. HBD represents LOC476825, ENSCAFG00000032615 represents LOC1008-55540. PPI, protein-protein interaction; DEPs, differentially expressed proteins.

Figure 6

GO enrichment analysis of 19 selected differentially expressed proteins. Data enrichment analysis was performed using the STRING database. It includes the biological process, molecular function, and cellular component. When FDR<0.05, the GO term was considered significant. All significant GO terms and respective -LOG₁₀ (FDR) values are presented. GO, Gene Ontology; FDR, false discovery rate.

Figure 7

KEGG pathway enrichment analysis of 19 selected DEPs. The KEGG pathway analysis was performed using the STRING database. A pathway was considered significant at a FDR of <0.05. All significant pathways are presented. KEGG, Kyoto Encyclopedia of Genes and Genomes; FDR, false discovery rate.

Figure 8

Validation of the expression of TFRC, INTS4, FGG and ATP5B by western blot analysis. The serum levels of TFRC, INTS4, ATP5B and FGG were determined by western blot analysis prior to MI model establishment (0 h) and following MI model establishment at 1, 2 and 3 h (mean ± standard error of the mean, n=10). P-values of multiple groups was calculated using one-way analysis of variance, and pairwise comparison within was performed using the Least Significant Difference test. (A) Representative bands of different proteins at different time points. (B) Relative expression of TFRC at the three time points, 1 h/0 h: 0.95±0.12 (P>0.05); 2 h/0 h: 1.03±0.10 (P>0.05), 3 h/0 h: 1.05±0.10 (P>0.05). (C) Relative expression of ATP5B at the three time points, 1 h/0 h: 0.58±0.06 (P<0.01); 2 h/0 h: 0.56±0.09 (P<0.01), 3 h/0 h: 0.35±0.08 (P<0.01). (D) Relative expression of FGG at the three time points, 1 h/0 h: 1.07±0.21 (P>0.05); 2 h/0 h: 1.72±0.21 (P<0.01), 3 h/0 h: 1.51±0.18 (P<0.01). (E) Relative expression of INTS4 at the three time points, 1 h/0 h: 2.12±0.41 (P<0.01); 2 h/0 h: 1.99±0.35 (P<0.01), 3 h/0 h: 2.20±0.25 (P<0.01). ^**P<0.01, vs. 0 h/0 h group; ^#P<0.05 and ^##P<0.01, vs. 1 h/0 h group; ^&P<0.05, vs. 2 h/0 h group. MI, myocardial infarction; ATP5B, ATP synthase F1 subunit β; FGG, fibrinogen γ chain; INTS4, integrator complex subunit 4; TFRC, transferrin receptor 1.

Figure 9

ECG of beagle dogs pre-MI (0 h) and post-MI (3 h). Adapted from Ref (62). (A) ECG of beagle dogs prior to surgery. There was no notable abnormality in the ECG of normal beagles. (B) ECG of beagle dogs at 3 h post-surgery. The ST segment of beagle dogs between V2 and V6 elevated. ECG, electrocardiogram; MI, myocardial infarction.