Next-generation RNA sequencing (RNA-seq) is a method to quantify the expression of all genes in an agnostic fashion 26. In this study, we used RNA-seq to investigate the transcriptomic profiles of SM and WB samples to identify genes and pathways involved in their pathogenesis. We hypothesized that these myopathies have distinct transcriptomic signatures relative to each other and to normal breast samples. We found that, while SM and WB samples had transcriptomic profiles different from normal tissues, they did not differ between each other.
Evaluation of differences between DEGs, as well as PCA and heatmap cluster analyses, showed minimal to no differences between the transcriptomic profiles of SM and WB, while Normal samples clearly clustered separately with a distinct set of differentially regulated genes. This is consistent with the fact that SM and WB share common histological characteristics 10, and suggests that SM and WB share common genetic characteristics in spite of macroscopically distinct phenotypes 4,10.
A large number of DEGs were detected in SM and WB, indicating complex alterations in myocyte homeostasis. This alteration is compatible with the changes observed microscopically, which include myofiber degeneration, acute and chronic inflammation, and repair (mainly in the form of increased fibrous and adipose tissues in the endomysial space) 2,27,28. The following segment of the discussion focuses on the main metabolic pathways enriched by GO and KEGG analyses.
Inflammation and cytokine signaling
In both SM and WB, there was enrichment of GO terms associated with the immune response, as well as of the cytokine–cytokine receptor interaction KEGG pathway, suggesting activation of inflammation and immune processes. This result is in agreement with our previous study, where we showed that both SM and WB (same source as samples in this study) had myositis, predominately characterized by lymphocytic cuffs around venules in the endomysial space 10. Other studies also identified inflammation as a common change in broiler breast myopathies, particularly WB 2,9–11,27.
In both SM and WB, numerous cytokines and their receptors were upregulated, including IL1, IL2, IL5, IL11, IL12, IL13, IL17, IL18, IL20–22. These molecules are generally pro-inflammatory; however, how their net effect influences the phenotype of SM and WB remains to be elucidated. Numerous genes coding for C-C motif chemokines and related receptors were also upregulated in our samples, including CCL4–8. The function of these molecules is to attract inflammatory cells to the site of inflammation, which is consistent observing inflammatory cells in the endomysium in SM and WB. Similarly, Kong and colleagues showed that CCL5 was upregulated in muscle tissue of modern fast-growing broilers, whereas it was downregulated in the a traditional broiler line 29. Additional upregulated genes in the cytokine-cytokine receptor interaction KEGG pathway include transforming growth factor B (TGFB), and platelet derived growth factor receptor (PDGFRA and B). TGF has a pro-fibrotic effect, possibly through exhaustion of satellite cells 30. While PDGFRA and B were upregulated in SM samples only, upregulation of these genes may indicate activation and / or proliferation of fibro-adipogenic precursors, consistent with the accretion of fibrofatty tissue in the endomysium of SM 31.
Three genes associated with inflammation were among the top 20 upregulated genes in both SM and WB: pentraxin 3 (PTX3), macrophage receptor with collagenous structure (MARCO), and P53 apoptosis effector related to PMP-22 (PERP1). PTX3 is a mediator of acute inflammation and innate immunity; in chickens it is upregulated in tissues upon stimulation with lipopolysaccharide and experimental infection with E.coli. 32,33. MARCO is a scavenger receptor expressed on the macrophage surface and mediates opsonin-independent phagocytosis 34; upregulation of MARCO could indicate presence or activation of macrophages in the inflammatory infiltrates of our samples. Lastly, PERP1 is an endoplasmic reticulum-resident protein, which is expressed in B lymphocytes maturing to plasma cells 35. While lymphocytic phlebitis and mononuclear perivascular cuffs have been observed in SM and WB tissues11,28, the majority of inflammatory cells are CD3 + T lymphocytes 2. Future studies are required to identify the contribution of B lymphocytes to the formation of inflammatory infiltrates in both SM and WB tissues.
Extracellular space and collagen
The GO terms collagen-containing extracellular space (GO:0005615) and extracellular region (GO:0005576), in the Cellular Component ontology, were enriched in both SM and WB samples. Different subunits of collagen I–III, V–VI, VIII–IX, and XII–XV were variably upregulated in SM and WB, although the magnitude of increased expression was modest and never greater than eight-fold. Indeed, our previous microscopic analysis showed increased amounts of connective tissue in the endomysium in both myopathies10. Together, these findings are consistent with a body of evidence implicating the quality and quantity of collagen as a contributor to the textural changes observed in breast myopathies 36–38. For instance, it was suggested that the increased firmness of WB may be caused by higher concentrations of collagen (hydroxyproline) 39, higher levels of collagen cross-linking 40 or more tightly packed spatial arrangement of collagen fibers 37, when compared to normal samples. While Baldi and colleagues reported that collagen content was not increased in SM samples 9, Sanden and colleagues observed that both SM and WB had an increased amount of collagen fibers compared to normal breast muscle 32. This same study also showed that WB had thicker collagen fibers compared to SM, suggesting that collagen fibers may be more mature in WB compared to SM 41.
Supporting the results of the GO enrichment analysis, the cell adhesion molecules KEGG pathway was also enriched in both SM and WB groups. As part of this pathway, the cluster of differentiation 80 (CD80) and CD86 genes were upregulated in both SM and WB. These molecules have a co-stimulatory function for numerous immune cells, mainly lymphocytes, and are upregulated in inflammation and autoimmune diseases 42,43. Other upregulated genes in this enriched pathway included those of the integrin family (integrin subunit beta 2 and 7 [ITGB2 and 7]), and Ig superfamily (cell adhesion molecule 1 and 3 [CADM1, 3]; neuronal cell adhesion molecule [NRCAM], vascular cell adhesion molecule [VCAM]). Similarly, Marchesi and colleagues reported that cell adhesion molecule 1 (CADM1) was upregulated in white striping (WS), another phenotype of breast myopathy 44, while Papah and colleagues showed that ITGB2 was upregulated in WB 20. It has been suggested that cell adhesion pathways might be implicated in myofiber hypertrophy by promoting muscle cell development 45.
It remains unclear if upregulation of cell adhesion molecules is directly implicated in the pathogenesis of broiler breast myopathies, or may simply be an indicator of muscle hypertrophy, which in turn predisposes to development of such conditions 10. Alternatively, upregulation of these genes may be contextual with the inflammation observed in SM and WB myopathies, as cell adhesion molecules are associated with diapedesis of white blood cells at the site of inflammation 46.
Other genes associated with the extracellular space, which were among the top 20 upregulated in both SM and WB, include the C-type lectin domain family 3 (CLEC-3A), metalloproteinase 10 (MMP10), and thrombospondin-2 (THBS2). The C-type lectins encompass a class of proteins found in the extracellular space, which carry the C-type carbohydrate recognition domains that bind proteins, lipids, or carbohydrates 47. The CLEC3A enables carbohydrate binding activity and is active in the extracellular space, and it has been shown to be involved in ossification and development of the nucleus pulposus 48,49.
Metalloproteinases play diverse roles, including cleaving extracellular matrix components, participating in myoblast fusion and migration 50,51, and degradation of fibrous tissue 52. The MMP10 is upregulated by thrombin in endothelial cells during inflammation 53, and it has an important role in regeneration of striated muscle, as shown in gene knock-down experiments in mice 54. Upregulation in myopathic samples could be caused by inflammation, and / or regeneration in response to myofiber degeneration and loss 2,11,28.
THBS2 is a pro-fibrotic and anti-angiogenic matricellular protein 55, and its upregulation may be associated with the increased amounts of collagen in endomysial space of SM and WB. Sihvo and colleagues suggested that fibrosis of WB might result from insufficient delivery of oxygen and nutrients to skeletal muscle 2; upregulation of THBS2 in turn may inhibit angiogenesis, thereby leading to fibrosis in WB. Similarly, Brothers and colleagues also reported that THBS2 was upregulated in male broilers with WB 56.
Tissue morphogenesis and proliferation
The neuroactive ligand-receptor interaction KEGG pathway was enriched in both SM and WB. This finding is similar to that of Marchesi and colleagues who reported that the pathway was enriched in broilers affected with WB 44. This pathway includes dopamine and serotonin receptors and is associated with signaling molecules and interaction 57. How this pathway may affect muscle metabolism is unclear.
The cellular communication network factor 3 (CCN3), melan-A (MLANA-A), and myosin heavy chain 15 (MYH15) were among the 20 most upregulated genes in both SM and WB. The CCN3 gene encodes secreted matrix-associated proteins that impair myocyte differentiation in the dermomyotome during fetal development 58, and its overexpression results in aberrant muscle repair and fibrosis 30. Upregulation in our samples suggests altered myofiber differentiation in both SM and WB.
MLANA is located in the endoplasmic reticulum membrane and is involved in the biogenesis of melanosomes, which synthesize and store melanin pigments 59,60. MLANA was upregulated in the breast muscles of black-boned chickens compared to the breast muscles of commercial broiler lines 61; the authors of that study suggested that melanogenesis in breast muscle may result in improved flavour by production of aromatic compounds. Since our samples originated from white breast muscles, upregulation of MLANA was an unexpected finding.
The MYH15 gene encodes for a protein involved in the contraction and regeneration of the avian skeletal muscle 62. Praud and colleagues reported that MYH15 could be a potential biomarker for WB, as demonstrated by the increased expression of MYH15 in regenerating fibers within the WB-affected area 63. This evidence suggests a potential role for MYH15 in the pathogenesis of WB.
The taste 2 receptor (TAS2R) consist of a family of G-protein-coupled receptors primary implicated in the taste of bitter compounds 64, as well as extra-gustatory effects, including thyroid function, smooth muscle relaxation, and adipogenesis 65–67. While chickens appear to have a much smaller repertoire of TAS2R genes compared to mammals, with only three bitter receptors identified, their distribution in extra-sensory organs suggests additional functions, as seen in their mammalian counterparts 70. The effect of TAS2R downregulation in development of breast myopathies remains unclear.
Two genes involved in tissue differentiation were downregulated in both SM and WB samples: Rho BTB containing 3 (RHOBTB3) and amphiphysin (AMPH), which are part of the Rho GTPase signaling pathway. The RHOBTB3 is a subgroup of the Rho GTPase family, which is thought to contain key regulatory molecules that link surface receptors to the organization of the actin cytoskeleton 71. Specifically, the RHOBTB3 encodes for a protein involved with the retrograde transport of proteins from the endosomes to the Golgi complex, as well as cell cycle regulation 72. A similar study found that another member of the Rho GTPase family, the rho-associated coiled-coil containing protein kinase 2 (ROCK2) 73, was also downregulated in WS 44. The authors suggested that this might have affected cell adhesion, and the assembly and disassembly of actin stress fibers 44,74.
Amphiphysin (AMPH) encodes for a protein associated with endocytosis 75,76 and membrane integrity of myocytes during muscle maturation and regeneration (including fusion of myocytes) 77,78. Downregulation of AMPH may indicate decreased regenerative ability of affected muscle, leading to repair by scarring (fibrosis).
Hypoxia and oxidative stress
Three genes associated with oxidative balance were among the 20 most highly downregulated genes in both SM and WB samples: ChaC glutathione-specific gamma-glutamylcyclotransferase 1 (CHAC1), retinol-binding protein type 1 (RBP1), and mitochondrial carrier protein family (solute carrier family 25, SLC25). The CHAC1 genes encode a protein related to unfolded protein response, and the regulation of glutathione levels and oxidative balance 79. Brothers and colleagues 56 found upregulation of CHAC1 in the WB of 3-week-old male broilers, and suggested that CHAC1 may degrade glutathione in the P. major, making the tissue more susceptible to oxidative stress, ultimately contributing to the development of myopathies. The reasons for CHAC1 downregulation in both SM and WB in our cohort are uncertain, however, differences with previous studies may reflect different stages of myopathy progression.
The product of the RBP1 gene protects retinoids from non-specific oxidation, and delivers them to specific enzymes to facilitate biosynthesis of retinoic acid 80. RBP1 is downregulated in humans with breast cancer, as low glucose concentration and hypoxia in the tumor environment reduce its expression 81. Downregulation of RBP1 in our samples may reflect local hypoxia, a condition that has been associated with WB in multiple studies 8,82,83.
The SLC25 gene family encodes for a set of proteins implicated with exchange of molecules between the mitochondrial matrix and the cytoplasm, as well as mitochondrial viability 84. Downregulation of this gene could negatively affect mitochondrial function and integrity, which could ultimately lead to myocyte degeneration. Ultrastructural studies of WB showed mitochondrial damage, including cristolysis and matrix dissolution28. Similar to our study, Marchesi and colleagues reported the downregulation of SLC25A4 44 in WS.
Calcium signaling and phagosome
Calcium signaling and phagosome KEGG pathways were enriched in WB only. Enrichment of the calcium signaling pathway has also been shown previously in WS 44. Increase of cytoplasmic calcium levels could have an apoptogenic function, promoting cell death and myofiber loss, as also suggested by others 47. The phagosome pathway has been shown to be enriched in denervation-induced skeletal muscle atrophy 85, and its upregulation in our samples could be associated with myofiber loss. Enrichment of this pathway could be also a sequel of inflammation, partly characterized by influx of cells with marked phagocytic functions75.