iTRAQ-based proteomic analysis reveals key proteins affecting cardiac function in broilers that died of sudden death syndrome

Abstract

Sudden death syndrome (SDS), which is a cardiac-related condition commonly observed in chickens selected for rapid growth, causes significant economic losses to the global poultry industry. Its pathogenesis in broilers is poorly understood, and little is known about the proteome of the heart tissue of SDS broilers. A quantitative proteomic approach using isobaric tags for relative and absolute quantification labeling of peptides was used to characterize the protein expression profiles in the left ventricle of SDS broilers. These proteins were further analyzed by bioinformatics, and two proteins were validated by western blot analysis. We identified 186 differentially expressed proteins (DEPs), of which 72 were upregulated, and 114 were downregulated in the SDS group. Functional annotation suggested that 7 DEPs were related to cardiac muscle contraction, and another 7 DEPs were related to cardiac energy metabolism. Protein interaction network predictions indicated that differences in cardiac muscle contraction between SDS and healthy groups were regulated by troponin T, tropomyosin alpha-1 chain, fast myosin heavy chain HCIII, myosin-1B, coronin, and myoglobin, whereas differences in cardiac energy metabolism and biosynthesis of amino acids were regulated by gamma-enolase, phosphoglycerate mutase, NADH-ubiquinone oxidoreductase chain 2, serine/threonine-protein kinase, myoglobin, and alpha-amylase. Our expression profiles provide useful information and new insights into key proteins to elucidate SDS for further studies.

Keywords: broiler chicken; gene ontology analysis; iTRAQ analysis; sudden death syndrome.

Figure 1

Histological and ultrastructural examination of the left ventricle. (A and B) Representative HE-stained longitudinal sections of the heart tissue of healthy broilers and sudden death syndrome (SDS) broilers. Blue arrow () indicates the inflammatory cells. Yellow arrows () indicate the myocardial cells showed obvious granular degeneration. (C and D) Electron microscopy pictures showing the differences in myocardial fibers and mitochondria between healthy broilers and SDS broilers (×10,000). Red arrows () indicate the myocardial fibers of SDS broilers were broken, edema and irregular arrangement. Black arrows (→) indicate the damaged mitochondria.

Figure 2

Protein identification and analysis. (A) Basic information of protein identification. (B) Distribution of the identified proteins among the different molecular weight classes (in kDa). (C) Coverage of proteins by the identified peptides. (D) Distribution of proteins containing different number of identified peptides.

Figure 3

Volcano plot of differentially expressed proteins (DEPs) obtained from the labelled samples. The x -axis represents log2 expression fold-change (FC) in sudden death syndrome (SDS) vs. normal samples and the y -axis represents false discovery rate (FDR) (−log10). Up-regulated genes (FDR <0.01 and FC >1.2) are shown in red while down-regulated genes are shown in green (FDR <0.01 and FC <0.833).

Figure 4

Gene ontology analysis results of differentially expressed proteins in sudden death syndrome (SDS). Results of significantly enriched biological process classification (A), cell component (B) and molecular function enrichment (C) are illustrated. (D) Percentages of proteins involved in each of the biological process, cellular component and molecular function terms.

Figure 5

Class of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways participated by the differentially expressed proteins (DEPs).

Figure 6

Distribution of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways arranged in ascending order according to the P -value.

Figure 7

Protein–protein associations analyzed by the String software. The interaction network showing important proteins involved in the regulation of cardiac muscle contraction and glycolysis or biosynthesis of amino acids, respectively.

Figure 8

Validation of differentially expressed proteins (DEPs) using western blot. (A) Western blot result illustration. Group comparison based on gray value western blot result of myoglobin (B) and PGAM1 (C). β -actin was used as an internal standard. **: P < 0.01; *:P < 0.05.