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. 2024 Jul 4;103(10):104047. doi: 10.1016/j.psj.2024.104047

Transcriptomic meta-analysis and exploration of differentially expressed gene functions in wooden breast myopathy of broilers

Xinrui Zhang 1, Tong Xing 1, Liang Zhao 1, Lin Zhang 1, Feng Gao 1,1
PMCID: PMC11332813  PMID: 39068695

Abstract

Wooden breast (WB) myopathy is a common myopathy found in commercial broiler chickens worldwide. Although extensive research on WB has been conducted using transcriptomics, effectively screening and analyzing key target information remains a challenge. In this present study, 5 transcriptomic datasets obtained from the National Center for Biotechnology Information (NCBI) were used. A meta-analysis was conducted to identify meta-differentially expressed genes (meta-DEGs) involved in the response of broilers to WB myopathy. These meta-DEGs were further analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA), supplemented by protein-protein interaction (PPI) network construction to pinpoint hub genes. These analyses help to reveal key genes, pathways, and biological processes associated with WB myopathy. The results showed that 645 up-regulated and 99 down-regulated significant meta-DEGs (|log2FC| ≥0.6, P-Meta < 0.05, and present in at least 4 datasets) were identified. GO analysis showed that multiple fibrosis-related pathways/biological processes, such as cell adhesion, connective tissue development, and collagen-rich extracellular matrix, as well as calcium ion binding were significantly upregulated. PPI analysis identified TGFB3, COL1A1, COL1A2, and COL3A1 as central hub genes involved in the fibrotic processes. KEGG analysis revealed significant upregulation of apoptosis and lysosomal pathways, with an enrichment of Ca2+-related signals and lysosomal cathepsins within the apoptosis pathway. Additionally, GSEA indicated a suppression of the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain (ETC) in WB myopathy, with PPI analysis also identifying specific hub genes associated with these pathways.

In conclusion, our comprehensive analysis of meta-DEGs elucidated key biological processes and pathways implicated in WB myopathy, including fibrosis, apoptosis, altered calcium signaling, and metabolic disruption. The identification of specific hub genes offers avenues for further investigation into the pathogenesis of this condition, potentially guiding targeted therapeutic strategies.

Key words: broiler chicken, wooden breast, transcriptome, meta-analysis

INTRODUCTION

The demand for poultry meat has driven the genetic selection of commercial broiler chickens towards enhanced breast muscle yield and fast growth in recent decades (Papah et al., 2017; Bordignon et al., 2022). However, this selection has led to the occurrence of breast muscle abnormalities, especially wooden breast (WB) myopathy. WB is macroscopically characterized by hardened areas in the pectoralis major (PM) muscle, along with occasional clear viscous fluid and small hemorrhages (Papah et al., 2017). Xing et al. (2020) reported that approximately 61.9% of commercially processed broiler fillets in China exhibited WB myopathy. The occurrence of WB myopathy compromises both the appearance and meat quality, causing significant economic losses in the poultry industry (Petracci et al., 2019; Xing et al., 2020). However, the underlying mechanisms that cause the formation of WB myopathy in broilers are not yet fully understood.

Omics technologies have been widely used to apply pathophysiological mechanisms of WB myopathy. Mutryn et al. (2015) reported that hypoxia, increased intracellular calcium, and oxidative stress are key features of this myopathy through transcriptomic research. Results of RNA-seq analyzed by Zambonelli et al. (2016) revealed that differentially expressed genes were mainly involved in oxidative stress, muscle fiber regeneration, muscle ion homeostasis, lipidosis, glucose metabolism, fibrosis, and other physiological processes. Moreover, Malila et al. (2021) also assessed the transcriptional profiles of normal breast and WB collected from broilers at the ages of 6 and 7 wk. In 6-wk-old broilers, the metabolism of glucose and lipids, along with focal adhesion, apoptosis, tight junction, and gap junction signaling, were identified as the top enriched pathways. For the 7-wk broilers, actin cytoskeleton, protein processing in the endoplasmic reticulum, mitogen-activated protein kinase signaling, and focal adhesion were highlighted as the enriched pathways. Wang et al. (2023a) identified a negative energy balance in WB through the combined analysis of transcriptome and metabolome. Some studies have verified certain pathological processes associated with WB myopathy that were identified through transcriptomic analysis. Pan et al. (2021) reported that the occurrence of WB myopathy significantly impacted muscle structure, which is associated with altered cellular redox status. Xing et al. (2021) reported that the occurrence of WB myopathy was related to the deposition of extracellular matrix (ECM) components and fibrosis. However, due to limited sample sizes, physiological conditions, genetic backgrounds, and experimental conditions (Choi et al., 2003; Zhang et al., 2008), the results of multiple studies may have poor reproducibility. The identified differentially expressed genes and pathways cover a wide range, making it difficult to focus on specific targets (Choi et al., 2003; Zhang et al., 2008). Furthermore, if small changes in gene expression are highly consistent across multiple experiments, they may have significant implications that individual studies could overlook due to focusing on genes with larger differences in expression (Choi et al., 2003; Zhang et al., 2008).

Meta-analysis approaches are increasingly used as an effective method to address these limitations (Tseng et al., 2012). By combining and analyzing data from multiple independent studies, meta-analysis can provide more reliable results, identify common patterns, and reveal associations that may not be apparent in individual studies (Tihagam and Bhatnagar, 2023). For example, Bordignon et al. (2022) identified high growth (daily weight gain and slaughter weight) could be a factor increasing the risk of occurrence of WB myopathy by meta-analysis. The data used in meta-analysis are not the original data from each study, but the existing statistical results of different studies, such as effect sizes and P-value (Tihagam and Bhatnagar, 2023). In transcriptomic studies applying meta-analysis, it is essential to first collect publicly available data from relevant studies. For instance, a gene expression matrix is obtained from public databases such as the gene expression omnibus (GEO) of the National Center for Biotechnology Information (NCBI) (Chen et al., 2021; Tihagam and Bhatnagar, 2023). One application of meta-analysis in transcriptomics is the identification of disease biomarkers. Chen et al. (2021) utilized meta-analysis to integrate transcriptome datasets to identify tuberculosis biomarkers in patients positive for human immunodeficiency virus. Additionally, meta-analysis can be used to understand the molecular mechanisms of complex diseases. By analyzing common patterns of gene expression across different studies, researchers can identify the signaling pathways and molecular processes that play a key role in the occurrence and development of diseases (Sass et al., 2017; Tihagam and Bhatnagar, 2023). Sass et al. (2017) conducted a meta-analysis on gene expression profiles from different tissues, revealing consistent injury response features and the transcriptional induction of inflammatory genes in both the initial response and during the healing phases. Therefore, in the context of transcriptomics, meta-analysis of the transcriptome can help us understand the gene expression patterns and regulatory mechanisms related to specific diseases or biological processes (Sass et al., 2017; Tihagam and Bhatnagar, 2023). However, there are no reports of transcriptomic meta-analysis studies for WB myopathy currently.

In the present study, a meta-analysis was conducted using 5 transcriptomic studies obtained from NCBI to identify meta-differentially expressed genes (meta-DEGs) involved in the response of broilers to WB myopathy. Analyses including the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), gene set enrichment analysis (GSEA), and protein-protein interaction (PPI) network construction were performed on the meta-DEGs. Additionally, real-time quantitative PCR was used to validate the differential expression of the meta-DEGs. Conducting transcriptomic meta-analysis and enrichment analysis helps to reveal key genes, pathways, and biological processes, and provides regulatory targets for mitigating the formation of WB myopathy.

MATERIAL AND METHODS

Collection of Microarray Data

The transcriptome datasets utilized in this study were all obtained from the GEO public database at NCBI, using the search term "Wooden/Woody breast." The criteria for including datasets in the meta-analysis were as follows: (1) the dataset must be genome-wide mRNA-expression chip/sequencing data supported by the literature; (2) all samples within each dataset must have consistent experimental conditions and origins; (3) the dataset must include normal breast and WB; (4) each dataset must contain at least 3 samples per group, sourced from the PM muscle of male broilers. Ultimately, 5 datasets met these criteria for inclusion in the meta-analysis: GSE127806, GSE144000, GSE107362_6 wk, GSE107362_7 wk, and GSE79276. Detailed information about these transcriptome datasets was found in Supplementary Table S1.

Meta-Analysis of Differential Gene Expression

Gene expression matrices were obtained using the GEO query package, and differential expression analysis was subsequently performed using the DESeq2 package, yielding P-values and fold change (FC). A meta-analysis of DEGs was conducted following the methodology described by De Toma et al. (2021). This analysis employed Fisher's method within the metaseqR package to combine the P-values of the same gene from multiple studies, thereby generating a consolidated P-Meta value. Significant meta-DEGs were identified based on the combined P-Meta<0.05, in conjunction with meeting the absolute value of log2FC ≥ 0.6 (|log2 FC| ≥ 0.6), and the gene was present in at least 4 datasets (n ≥ 4). These meta-DEGs were subsequently utilized for GO and KEGG enrichment analysis.

Enrichment Analysis of GO, KEGG, GSEA, and the Construction of PPI Networks

The gene annotation file was retrieved from the Ensembl Genome Browser database (http://www.ensembl.org/index.html). The R package ClusterProfiler 3.4.4 was utilized to perform enrichment analysis of genes using GO and KEGG. The Q-value was obtained by adjusting P-value using the Benjamini-Hochberg method, with a threshold of Q < 0.05 set to determine the significance of pathways in the GO and KEGG analyses. Additionally, meta-DEGs that appeared in at least 4 datasets were analyzed using the GSEA tool available on the Sangerbox online platform (http://vip.sangerbox.com/), which included calculations of the normalized enrichment score (NES) and the false discovery rate (FDR). In GSEA analysis, gene sets with |NES|≥2 and FDR<0.01 were considered for significantly enriched. The STRING database (https://string-db.org/) and Cytoscape software were used to construct and visualize PPI networks. Within the PPI network, the term “degree” indicates the number of connections a particular protein has with others. A higher degree value reflects the centrality and importance of that protein in biological processes. Accordingly, the top 10 genes ranked by degree were selected as hub genes for visualization.

Experimental Broiler Chickens Selection and Sample Collection

Experimental procedures and bird management were sanctioned by the Institutional Animal Care and Use Committee of Nanjing Agricultural University. The protocol number is SYXK 2021-0014. In this study, broiler chickens (Arbor Acres male) were raised in 3-tiered battery cages (100 × 60 × 40 cm per cage) and received commercial diets. The room was maintained at a temperature ranging from 33°C for the first 7 d and then was reduced by 3°C per week to a final temperature of around 26°C until the end of the experiment. The relative humidity was maintained at around 70%. Birds were exposed to light for 23 h/d and dark for 1 h/d throughout the whole experimental period. Water and feed were provided ad libitum to broiler chickens. According to the method of Pan et al. (2021) and the WB myopathy scoring system proposed by Papah et al. (2017), a total of 80 broilers aged 42 d were examined for WB myopathy, involving visual observations for posture, and wing contact, as well as bilateral manual palpation for hardness of the PM muscle in a cranio-caudal direction. 12 suspected WB-unaffected (score 1; easily lift their wings and no detectable hardness of the breast area) and 12 WB-affected live birds (scores 4–5; unable to lift their wings sufficiently, >75% of the breast muscle belly markedly firm on palpation exhibiting widespread/diffuse cover-age, with the score of 5 being given to birds with exceptionally firm breast muscles) were selected, and then electrically stunned (alternating current, 400 Hz, 50 V, 5 s each). The birds were immediately slaughtered via exsanguination. Broiler chickens were necropsied according to the standard of the WB myopathy scoring system reported by Papah et al. (2017). 8 WB-unaffected fillets (score 0; showing no detectable increase in firmness of the breast area) and 8 WB fillets (scores 4–5; >75% of muscle belly palpably firmer than normal in widespread multifocal to diffuse pattern, with the score of 5 being given to specimens which were exceptionally firm on palpation) were eventually selected through palpation of the PM muscle by 3 trained personnel. Samples were collected from the superficial layer of the left PM muscles (cranial part) and stored at −80°C.

RNA Purification and Real-Time Quantitative PCR Analysis

Total mRNA from PM muscles was isolated using RNAiso Plus reagent (Takara Biotechnology Co., Ltd., Dalian, China), and then reversed transcribed into cDNA with a commercial kit from the same company. Real-time quantitative polymerase chain reaction (qPCR) was performed using SYBR Premix Ex Taq (Vazyme Biotechnology Co., Ltd., Nanjing, China) on an ABI PRISM 7500 system (Applied Biosystems, Foster City, CA, USA). Table S3 shows the primer sequences used for qPCR. Relative mRNA expression was calculated using the 2−∆∆Ct method with Tubulin as the internal reference gene.

Statistical Analysis

SPSS Statistics (SPSS, Inc. Chicago, IL) was used for data analysis and Student's t-tests were used to compare the differences between the groups of CON (normal breast) and WB (n = 8). Data were reported as mean ± standard error, and significance was indicated at P ≤ 0.05.

RESULTS

General Description of Datasets and Meta-Analysis of Differentially Expressed Genes

Gene expression responses to WB myopathy of broiler chickens were investigated by meta-analysis. Meta-analysis was conducted on total of 30 normal breast samples and 37 WB samples from 5 publicly accessible microarray datasets to identify meta-DEGs. Refer to Chen et al. (2021) for the organization of the transcriptome dataset information, and detailed information can be found in Table S1. GSE127806 was run using the Agilent-037360 gallus_exp_microarray_SLagarrigue_8×60k_V1. GSE144000 implemented on Illumina HiSeq 2500. GSE107362 was processed using the Agilent-084792 SurePrint G3 Custom GE 8×60K Gallus gallus, while GSE79276 was run using The Affymetrix Chicken Gene 1.1 ST Array. A total of 744 significant metaDEGs (|log2FC| ≥ 0.6, P-Meta <0.05, and present in at least 4 datasets) were identified, including 645 up-regulated and 99 down-regulated meta-DEGs (Figure 1).

Figure 1.

Figure 1

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Volcano plot of meta-DEGs in WB myopathy. Note: Gray dots represent genes that do not meet the criteria for |log2FC|≥0.6 (up or down) and significant P-Meta < 0.05; Red dots represent genes that meet both the log2FC ≥ 0.6 (up) and P-Meta < 0.05 criteria; Blue dots represent genes that meet both the log2FC≤ −0.6 (down) and P-Meta value < 0.05 criteria; The horizontal dashed line is located at a value equivalent to the P-Meta value (0.05); Vertical lines are located at log2FC= −0.6 and log2FC = 0.6.

GO Annotation Analysis of Meta-DEGs

GO annotation analysis is divided into biological process (BP), cellular component (CC), and molecular function (MF). In this present study, gene functional category analysis was performed to investigate the functions of the significantly upregulated meta-DEGs (Figure 2A). Regarding the results of the BP analysis, the meta-DEGs were primarily annotated with terms related to cell adhesion and the ECM, including connective tissue development and ECM organization. Meanwhile, the meta-DEGs categorized under CC were primarily located in the collagen trimer, collagen-containing ECM, and extracellular region. The MF analysis indicated that calcium ion binding predominated. Subsequently, a PPI analysis integrating factors identified in the GO analysis related to fibrosis pathways was constructed. Then the top 10 hub genes were displayed based on degree, including transforming growth factor-Β3 (TGFB3), collagen type I alpha 1 chain (COL1A1), collaen type I alpha 2 chain (COL1A2), collagen type III alpha 1 (COL3A1), among others (Figure 2B). These genes typically encode proteins that are associated with the regulation of collagen synthesis and the ECM.

Figure 2.

Figure 2

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GO enrichment analysis of upregulated meta-DEGs in WB myopathy. Note: (A) GO terms enrichment analysis, (B) The PPI network shows the interaction of the hub genes involved in fibrosis. BP, biological processes; CC, cellular compartment; MF, molecular function. WB, wooden breast.

KEGG Pathway Analysis of Meta-DEGs

As shown in Figure 3A, KEGG analysis revealed that 7 upregulated pathways were enriched (Q<0.05), including apoptosis, lysosomes, and pathways related to cell adhesion and interactions between cells and the ECM. Moreover, the metabolism pathways of starch and sucrose, nucleotide and pentose phosphate, as well as glycolysis/gluconeogenesis showed significant downregulation (Q<0.05; Figure 3B). Figure 3C showed gene-concept network (cnetplot) of overlapping KEGG pathways between apoptosis and lysosomes. The inositol 1,4,5-trisphosphate receptor, type 2 gene (ITPR2), which is primarily associated with Ca2+ signaling, was enriched in the apoptosis pathway. The genes of cathepsin (CTS) B, CTSC, CTSK, and CTSS were all simultaneously enriched in both the apoptosis and lysosome pathways.

Figure 3.

Figure 3

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The KEGG pathways of meta-DEGs in WB myopathy. Note: (A) The KEGG pathways enrichment analysis of up meta-DEGs, (B) The KEGG pathways enrichment analysis of down meta-DEGs, (C) Gene-concept network (cnetplot) of overlapping KEGG pathways between apoptosis and lysosomes. WB, wooden breast.

GSEA of meta-DEGs

GSEA was employed to pinpoint enriched biological pathways and functional categories within the meta-DEGs in this present study. Table S2 presented the gene sets that were significantly downregulated in the GSEA. This was determined based on |NES| ≥ 2 and FDR < 0.01. Notably, pathways and functional categories associated with the tricarboxylic acid (TCA) cycle and mitochondrial electron transport chain (ETC) were found to be significantly enriched. Figure 4A displayed the TOP 4 gene sets related to the TCA cycle in GSEA. Then a PPI network was constructed to integrate factors from the significant gene sets related to the TCA cycle in GSEA (Figure 4B). The top 10 hub genes selected based on degree were pyruvate dehydrogenase E1 component subunit beta (PDHB), pyruvate dehydrogenase complex component X (PDHX), succinate-CoA ligase, ADP-forming, beta subunit (SUCLA2), succinate-CoA ligase GDP/ADP-forming subunit alpha (SUCLG1), SUCLG2, and so on.

Figure 4.

Figure 4

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GSEA reveals the inhibition of tricarboxylic acid cycle in WB myopathy. Note: (A) Ranked by NES score and FDR, the top 4 downregulated enriched gene sets related to tricarboxylic acid (TCA) cycle were screened, (B) The PPI network shows the interaction of the hub genes involved in TCA cycle. NES, normalized enrichment score, FDR, false discovery rate. WB, wooden breast.

Figure 5A displayed the top 4 gene sets related to the mitochondrial ETC, including respirasome, mitochondrial electron transport NADH to ubiquinone, nicotinamide adenine dinucleotide dehydrogenase complex, and ATP synthesis coupled electron transport. Then a PPI network was also constructed to integrate factors from the significant gene sets related to mitochondrial ETC in GSEA analysis (Figure 5B). The top 10 hub genes according to degree were displayed, including NADH: ubiquinone oxidoreductase subunit A (NDUFA) 9, NDUFA10, NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), NADH dehydrogenase ubiquinone Fe-S Protein (NDUFS) 1, NDUFS3, ubiquinol-cytochrome c reductase core protein 1 (UQCRC1), and others.

Figure 5.

Figure 5

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GSEA reveals the inhibition of mitochondrial electron transport chain in WB myopathy. Note: (A) Ranked by NES score and FDR, the top 4 downregulated enriched gene sets related to mitochondrial electron transport chain (ETC) were screened; (B) The PPI network shows the interaction of the hub genes involved in mitochondrial ETC. NES, normalized enrichment score, FDR = false discovery rate. WB, wooden breast.

Fluorescent quantitative PCR validation

As shown in Figure 6A-D, the relative expression of genes related to fibrosis (TGFβ3, COL1A1, COL1A2, and COL3A1) and apoptosis (BAK1, BID, ITPR2, CTSB, CTSK and CTSS) was considerably higher in the WB group than in the CON group. Moreover, in contrast to CON, the relative expression of genes related to the TCA cycle (PDHB, PDHX, SUCLA2, and SUCLG1) and mitochondrial ETC (NDUFA9, NDUFA10, NDUFV2, NDUFS1, NDUFS3, and UQCRC1) in WB was significantly decreased.

Figure 6.

Figure 6

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Relative mRNA expression of genes related to fibrosis, apoptosis, tricarboxylic acid cycle and mitochondrial electron transport chain of normal breast and wooden breast. Note: (A) Relative mRNA expressions of transforming growth factor B3 (TGFB3), collagen type I alpha 1 chain (COL1A1), collaen type I alpha 2 chain (COL1A2) and collagen type III alpha 1 (COL3A1). (B) Relative mRNA expressions of Bcl-2 antagonist/killer1 (Bak1), BH3 interacting domain death agonist (Bid), Inositol 1,4,5-trisphosphate receptor type 2 (ITPR2), cathepsin (CTS) B, CTSK, and CTSS. (C) Relative mRNA expressions of pyruvate dehydrogenase E1 component subunit beta (PDHB), pyruvate dehydrogenase complex component X (PDHX), succinate-CoA ligase, ADP-forming, beta subunit (SUCLA2), succinate-CoA ligase GDP/ADP-forming subunit alpha (SUCLG1). (D) Relative mRNA expressions of NADH: ubiquinone oxidoreductase subunit A (NDUFA) 9, NDUFA10, NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2), NADH dehydrogenase ubiquinone Fe-S protein (NDUFS) 1, NDUFS3, and ubiquinol-cytochrome c reductase core protein 1 (UQCRC1). Data were expressed as the mean ± SE (n = 8), **P < 0.01, 0.01≤*P < 0.05. CON, normal breast; WB, wooden breast.

DISCUSSION

WB is a common myopathy in global commercial broiler farming, affecting both meat quality and nutritional value, and causing significant economic losses to the poultry industry (Kuttappan et al., 2016; Petracci et al., 2019). Transcriptomics has been employed to study the complex pathogenesis of WB, but screening and in-depth analysis of key genes and physiological pathways remain challenging. Applying meta-analysis in transcriptomics helps identify critical signaling pathways and molecular processes, enhancing our understanding of the molecular mechanisms of complex diseases. In this present study, meta-analysis was conducted on a total of 30 normal breast samples and 37 WB samples from 5 publicly accessible microarray datasets. A total of 744 significant meta-DEGs (|log2 FC| ≥ 0.6, P-Meta<0.05, and n ≥ 4) were identified, including 645 up-regulated and 99 down-regulated meta-DEGs. GO enrichment analysis was performed on the significantly increased meta-DEGs, which included BP, CC, and MF. Functional analysis (BP and CC) revealed their involvement in a series of pathway/biological processes related to fibrosis, including cell adhesion, connective tissue development and ECM organization, collagen trimer, and collagen-containing ECM. The ECM, primarily composed of collagen, provides structural support and facilitates cell adhesion (Bhattacharyya et al., 2012; Kular et al., 2014). Fibrosis is a pathological condition that occurs during tissue repair processes, characterized by excessive accumulation of collagen proteins in the ECM, leading to tissue abnormal hardening and causing changes of tissue structure and function (Bhattacharyya et al., 2012; Kular et al., 2014). The palpation hardness of WB-affected tissues is linked to an increase in highly cross-linked collagen fibrils (Papah et al., 2017; Xing et al., 2021). Next, a PPI analysis was conducted on genes related to fibrosis pathways identified in GO, which revealed hub genes that mediated the occurrence of fibrosis in WB myopathy, including TGFB3, COL1A1, COL1A2, COL3A1, and others. Activated mesenchymal fibroblasts produce ECM proteins in response to fibrogenic cytokines, with TGFB3 identified as the most potent driver of fibrotic disorder pathogenesis (Bhattacharyya et al., 2016; Xing et al., 2021). Herein, our results showed that the relative mRNA expression of TGFB3 was elevated in WB-affected PM muscle. Guo et al. (2021) reported that TGFB3 regulated the initiation of liver fibrosis progression as dynamic network biomarker. Moreover, COL1A1, COL1A2, and COL3A1 are genes that belong to the collagen protein family. Notably, the proteins encoded by COL1A1 and COL1A2 are key components of collagen type I (Alcaide-Ruggiero et al., 2021). COL1A1 and COL1A2 were identified as central genes in obesity-induced cardiac fibrosis using single-cell transcriptomics (Pan et al., 2022). The protein encoded by COL3A1 is a component of collagen III. Type I and III collagen, key components of reticular fibers in various parenchymal tissues including the liver, muscles, and spleen, are essential for preserving their structural integrity and functionality (Alcaide-Ruggiero et al., 2021; Kuivaniemi and Tromp, 2019). In this present study, the relative gene expressions of COL1A1, COL1A2, and COL3A1 were up-regulated in WB. Overall, these results indicated that WB myopathy may induce severe fibrosis in the PM muscle of broiler chickens by increasing the expression of TGFB3 and collagen-related factors thereby enhancing collagen cross-linking. Furthermore, MF analysis indicated that calcium ion binding played a predominant role. Specifically, PLA2G4A (P-Meta=2.06E-06, log2FC=0.9754) was enriched in the calcium ion binding pathway. PLA2G4A encodes cytosolic phospholipase A2 (PLA2), which could be related with the increase in cytoplasmic Ca2+ levels in WB myopathy (González et al., 2017; Zhang et al., 2023). Activated PLA2 degrades phosphatidylcholine, the major component of cell membranes, leading to muscle membrane damage (González et al., 2017).

Moreover, Ca2+-related signals were also found to be associated with apoptosis in WB myopathy by KEGG analysis. Apoptosis is a self-destruct mechanism initiated by cells in response to damage, which helps to remove damaged parts with minimal harm, thereby maintaining overall health and balance (Rudzińska et al., 2019; Ziegler et al., 2021). In particular, the pro-apoptotic factors BCL2 antagonist/killer 1 (BAK1) and BH3 interacting domain death agonist (BID), along with the Ca2+ channel ITPR2, were observed in the apoptosis pathway. ITPR2 is a member of the inositol trisphosphate receptor (IP3R) family, which is located on the sarcoplasmic reticulum (SR). The transfer of Ca2+ from the SR to the mitochondria through ITPR is a key trigger of the apoptosis signal, which triggers the release of cytochrome c (Cytc) through the BAK pore, ultimately resulting in apoptosis (Ziegler et al., 2021). The mice with an ITPR2 knockout could alleviate neuronal apoptosis after focal ischemic stroke by disrupting the ITPR2-mediated Ca2+ signaling pathways in astrocytes (Li et al., 2015). Additionally, the lysosome pathway was also found to be enriched in the KEGG analysis, playing an important role in cell apoptosis. The cnetplot highlighted the enrichment of CTS in the lysosome and apoptosis pathways, including CTSB, CTSK, and CTSS. CTS can target BID and degrade prosurvival BCL-2 homologs, thereby promoting the pro-apoptotic activity of BAK and ultimately leading to apoptosis (Rudzińska et al., 2019). Wang et al. (2023b) found that CTSB could promote acute kidney injury induced by sepsis through activation of apoptosis. Moreover, silencing CTSS in C2C12 cells prevented cell apoptosis (Wan et al., 2023). Liu et al. (2023b) found that infection with an infectious hematopoietic necrosis virus significantly activated apoptosis, as evidenced by the significant upregulation of gene expression levels of CTSB, CTSK, and Cytc. The current study showed that the relative mRNA expressions of BAK1, BID, ITPR2, CTSB, CTSK, and CTSS were significantly up-regulated in the WB, suggesting that Ca2+ signaling and lysosomes may mediate apoptosis by upregulating BAK1 and BID. Furthermore, KEGG analysis also indicated inhibition of the glycolysis/gluconeogenesis pathway in this present study. Glycolysis breaks down glucose into pyruvic acid, which are then utilized by the TCA cycle, while simultaneously generating a small amount of ATP. In this present study, glucose-6-phosphate isomerase (GPI) and phosphoglucose mutase 1 (PGM1) were found to be enriched in this pathway. GPI catalyzes the conversion of glucose-6-phosphate (G6P) to fructose-6-phosphate (Han et al., 2021). A decrease in GPI activity can impair this conversion process, potentially leading to reduced efficiency in glucose metabolism and consequently affecting cellular energy production (Han et al., 2021). PGM1 is involved in glycogen metabolism and the glycolysis pathway, catalyzing the interconversion of glucose-1-phosphate (G1P) and G6P (Liu et al., 2023a). A decrease in PGM1 reduces glycogen reserves, potentially affecting cell energy balance (Liu et al., 2023a). Herein, the downregulation of GPI (P-Meta = 9.75E-06, log2FC = -0.6124) and PGM1 (P-Meta = 1.40E-04, log2FC = -0.6199) may negatively impact glycolysis and glycogen metabolism, thereby affecting energy production (Liu et al., 2023a).

In addition to glycolysis, the TCA cycle is another crucial energy-producing process within cells. In the present study, GSEA revealed significant downregulation of multiple gene sets related to the TCA cycle. The TCA cycle primarily occurs in the mitochondrial matrix, converting acetyl-CoA into carbon dioxide and generating energy molecules such as ATP, flavin adenine dinucleotide (FADH2), and NADH (Martínez-Reyes and Chande, 2020). Inhibition of the TCA cycle may lead to reduced cellular energy production, potentially affecting the normal function of muscle tissue (Martínez-Reyes and Chande, 2020). Wang et al. (2023a) reported that there was a negative energy balance in WB through an integrative analysis of transcriptomic and metabolomic, which may be related to the pathogenesis of WB myopathy and the pathological changes in muscle tissue. Through PPI of genes associated with the TCA cycle pathways in GSEA, hub genes affecting the TCA cycle in WB were identified, notably PDHB, PDHX, SUCLA2, SUCLG1 and SUCLG2. Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the TCA cycle (Danileviciute et al., 2022). The proteins encoded by PDHB and PDHX are subunits of the pyruvate dehydrogenase complex, which is involved in the conversion of pyruvate to acetyl-CoA, thus facilitating the entry of glycolysis products into the TCA cycle (Lazzarino et al., 2019). In this present study, we discovered that the relative mRNA expressions of PDHB and PDHX were significantly downregulated in WB, potentially resulting in a decreased rate of pyruvate entry into the TCA cycle (Danileviciute et al., 2022; Lazzarino et al., 2019). This finding shed light on the link between abnormalities in the TCA cycle and glycolysis in WB myopathy. Additionally, the proteins encoded by SUCLA2, SUCLG1, and SUCLG2 are involved in a critical step of the TCA cycle, namely the conversion of succinyl-CoA to succinate (Lancaster and Graham, 2023). SUCLG1, a shared subunit of succinyl CoA synthetase, catalyzes the reversible conversion of succinyl-CoA and ADP or GDP into succinate and ATP (SUCLA2) or GTP (SUCLG2) through substrate-level phosphorylation in the TCA cycle (Chinopoulos et al., 2019). Herein, the relative mRNA expressions of SUCLA2 and SUCLG1 in WB were significantly decreased. Jia et al. (2023) found that the arrest of the TCA cycle was closely related to the decrease of SUCLA2 and PDHB. Overall, abnormalities in the TCA cycle associated with WB myopathy may impact cellular energy production and metabolic stability.

Moreover, the ETC located on the mitochondrial inner membrane generates ATP through oxidative phosphorylation (OXPHOS), serving as the core of cellular energy supply (Tang et al., 2014). In this present study, GSEA also revealed significant downregulation of gene sets associated with the mitochondrial ETC, indicating a potential inhibition of mitochondrial ETC function. The ETC transfers electrons from the energy-rich molecules NADH and FADH2 to oxygen through a series of redox reactions, thereby generating ATP, which provides the essential energy for cellular activities (Tang et al., 2014). Complex I and Complex III are crucial in the ETC, creating a proton gradient by transferring protons from the mitochondrial matrix to the intermembrane space, which facilitates ATP synthesis (Tang et al., 2014). Furthermore, inhibition of the ETC promotes the production of ROS and affects the mitochondrial redox state (Nolfi-Donegan et al., 2020; Tang et al., 2014). Our previous study indicated mitochondrial dysfunction in WB, characterized by decreased activities of respiratory chain complexes I and III (Zhang et al., 2023). Shakeri et al. (2023) and Shakeri et al. (2024) also identified mitochondrial dysfunction in WB-affected PM muscle, which may affect ATP synthesis (Shakeri et al., 2023) as well as increase ROS and oxidative stress in the tissue (Shakeri et al., 2024). Through PPI analysis of genes identified in the ETC-related gene sets by GSEA, we also identified hub genes that influence the ETC in WB. These included subunits of mitochondrial ETC complex I, such as NDUFA9, NDUFA10, NDUFV2, NDUFS1, and NDUFS3, as well as UQCRC1, which encodes a subunit of mitochondrial ETC complex III. NDUFA9 and NDUFA10 are crucial for the assembly or stability of Complex I (Singh et al., 2023). Baertling et al. (2018) reported that a point mutation in NDUFA9 may lead to defects in the assembly process of mitochondrial Complex I. In addition, NDUFS1 and NDUFS3 are involved in forming the core structure of Complex I, and their inhibition may lead to decreased activity and assembly defects of Complex I (He et al., 2013; Qi et al., 2022). Qi et al. (2022) found that overexpression of NDUFS1 alleviated myocardial infarction (MI)/hypoxia-induced ROS production and apoptosis. Additionally, NADH: ubiquinone oxidoreductase subunit B7 (NDUFB7) is also a subunit of mitochondrial Complex I. Shakeri et al. (2023) reported that a reduction in NDUFB7 may be a potential factor affecting ATP production and normal mitochondrial function in WB myopathy. Moreover, UQCRC1 is an essential component of mitochondrial Complex III, directly involved in the transfer of electrons from coenzyme Q to Cytc (Kriaucionis et al., 2006). Under conditions of impaired mitochondrial function, the expression level of UQCRC1 decreased (Shibanuma et al., 2011). Shan et al. (2019) found that knocking out 1 allele of the UQCRC1 gene in mice led to decreased formation, activity, and ATP content of Complex III in the brain under baseline conditions. Hung et al. (2021) also found that the loss of UQCRC1 led to an increase in Cytc in the cytoplasm, triggering a caspase cascade and promoting apoptosis. Herein, the relative mRNA expressions of NDUFA9, NDUFA10, NDUFV2, NDUFS1, NDUFS3, and UQCRC1 were decreased in WB, suggesting the function of the mitochondrial respiratory ETC was inhibited. Overall, these findings indicated that the mitochondrial ETC in WB was inhibited, which may impair mitochondrial function, leading to decreased energy production and elevated ROS levels (Nolfi-Donegan et al., 2020; Tang et al., 2014), as well as the occurrence of cell apoptosis (Hung et al., 2021; Qi et al., 2022), thereby triggering the development or exacerbation of WB myopathy.

CONCLUSION

In summary, meta-analysis proved to be an effective method for integrating findings from multiple transcriptome studies. Enrichment analysis of meta-DEGs has revealed key pathological processes in WB myopathy, including fibrosis, apoptosis, and alterations in Ca2+-related signaling. Notably, it identified the suppression of the TCA cycle and mitochondrial ETC as pivotal factors in the occurrence and development of WB myopathy. Additionally, hub genes identified through PPI analysis play significant roles in mediating fibrosis, influencing the TCA cycle, and affecting mitochondrial ETC functionality. These findings enhance our understanding of WB myopathy and contribute to the development of strategies for the prevention of WB myopathy.

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ACKNOWLEDGMENTS

This study was supported by the National Natural Science Foundation of China (32072780 and 32272900), and the Earmarked Fund for Jiangsu Agricultural Industry Technology System (JATS[2023]418).

Data availability statement: The datasets presented in this study can be found in online repositories.

Availability of data and materials: All data generated during this study are available from the corresponding authors on reasonable request.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104047.

Appendix. Supplementary materials

mmc1.docx (27.9KB, docx)

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