Molecular Pathways and Key Genes Associated With Breast Width and Protein Content in White Striping and Wooden Breast Chicken Pectoral Muscle

Growth-related abnormalities affecting modern chickens, known as White Striping (WS) and Wooden Breast (WB), have been deeply investigated in the last decade. Nevertheless, their precise etiology remains unclear. The present study aimed at providing new insights into the molecular mechanisms involved in their onset by identifying clusters of co-expressed genes (i.e., modules) and key loci associated with phenotypes highly related to the occurrence of these muscular disorders. The data obtained by a Weighted Gene Co-expression Network Analysis (WGCNA) were investigated to identify hub genes associated with the parameters breast width (W) and total crude protein content (PC) of Pectoralis major muscles (PM) previously harvested from 12 fast-growing broilers (6 normal vs. 6 affected by WS/WB). W and PC can be considered markers of the high breast yield of modern broilers and the impaired composition of abnormal fillets, respectively. Among the identified modules, the turquoise (r = -0.90, p < 0.0001) and yellow2 (r = 0.91, p < 0.0001) were those most significantly related to PC and W, and therefore respectively named “protein content” and “width” modules. Functional analysis of the width module evidenced genes involved in the ubiquitin-mediated proteolysis and inflammatory response. GTPase activator activity, PI3K-Akt signaling pathway, collagen catabolic process, and blood vessel development have been detected among the most significant functional categories of the protein content module. The most interconnected hub genes detected for the width module encode for proteins implicated in the adaptive responses to oxidative stress (i.e., THRAP3 and PRPF40A), and a member of the inhibitor of apoptosis family (i.e., BIRC2) involved in contrasting apoptotic events related to the endoplasmic reticulum (ER)-stress. The protein content module showed hub genes coding for different types of collagens (such as COL6A3 and COL5A2), along with MMP2 and SPARC, which are implicated inCollagen type IV catabolism and biosynthesis. Taken together, the present findings suggested that an ER stress condition may underly the inflammatory responses and apoptotic events taking place within affected PM muscles. Moreover, these results support the hypothesis of a role of the Collagen type IV in the cascade of events leading to the occurrence of WS/WB and identify novel actors probably involved in their onset

Time course evaluation of collagen type IV in Pectoralis major muscles of broiler chickens selected for different growth-rates

Collagen type IV (COL4) is one of the major components of animals’ and humans’ basement membranes of several tissues, such as skeletal muscles and vascular endothelia. Alterations in COL4 assembly and secretion are associated to muscular disorders in humans and animals among which growth-related abnormalities such as white striping and wooden breast affecting Pectoralis major muscles (PMs) in modern fast-growing (FG) chickens. Considering the high prevalence of these myopathies in FG broilers and that a worsening is observed as the bird slaughter age is increased, the present study was intended to evaluate the distribution and the expression level of COL4 protein and its coding genes in PMs of FG broilers at different stages of muscle development (i.e., 7, 14, 21, 28, 35, and 42 d of age). Medium-growing (MG) chickens have been considered as the control group in consideration of the lower selection pressure on breast muscle growth rate and hypertrophy. Briefly, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. The normalized expression levels of COL4 coding genes showed an overexpression of COL4A2 in FG than MG at d 28, as well as a significant decrease in its expression over their rearing period. Overall, results obtained through the gene expression analysis suggested that selection for the hypertrophic growth of FG broilers may have led to an altered regulation of fibroblast proliferation and COL4 synthesis. Moreover, western blot and IHC analyses suggested an altered secretion and/or degradation of COL4 protein in FG broilers, as evidenced by the fluctuating trend of 2 bands observed in FG over time. In view of the above, the present research supports the evidence about a potential aberrant synthesis and/or degradation of COL4 and corroborates the hypothesis regarding a likely involvement of COL4 in the series of events underlying the growth-related abnormalities in modern FG broilers

The evolution of vimentin and desmin in Pectoralis major muscles of broiler chickens supports their essential role in muscle regeneration

Vimentin (VIM) and desmin (DES) are muscle-specific proteins having crucial roles in maintaining the lateral organization and alignment of the sarcomeric structure during myofibrils’ regeneration. The present experiment was designed to ascertain the evolution of VIM and DES in Pectoralis major muscles (PM) of fast-growing (FG) and medium-growing (MG) meat-type chickens both at the protein and gene levels. MG broilers were considered as a control group whereas the evolution of VIM and DES over the growth period was evaluated in FG by collecting samples at different developmental stages (7, 14, 21, 28, 35, and 42 days). After performing a preliminary classification of the samples based on their histological features, 5 PM/sampling time/genotype were selected for western blot, immunohistochemistry (IHC), and gene expression analyses. Overall, the findings obtained at the protein level mirrored those related to their encoding genes, although a potential time lag required to observe the consequences of gene expression was evident. The two- and 3-fold higher level of the VIM-based heterodimer observed in FG at d 21 and d 28 in comparison with MG of the same age might be ascribed to the beginning and progressive development of the regenerative processes. This hypothesis is supported by IHC highlighting the presence of fibers to coexpressing VIM and DES. In addition, gene expression analyses suggested that, unlike VIM common sequence, VIM long isoform may not be directly implicated in muscle regeneration. As for DES content, the fluctuating trends observed for both the native protein and its heterodimer in FG might be ascribed to its importance for maintaining the structural organization of the regenerating fibers. Furthermore, the higher expression level of the DES gene in FG in comparison with MG further supported its potential application as a marker of muscle fibers’ regeneration. In conclusion, the findings of the present research seem to support the existence of a relationship between the occurrence of muscle regeneration and the growth rate of meat-type chickens and corroborate the potential use of VIM and DES as molecular markers of these cellular processes

Differential expression patterns of genes associated with metabolisms, muscle growth and repair in Pectoralis major muscles of fast- and medium-growing chickens

The aim of this study was to investigate the expression of genes related to muscle growth, hypoxia and oxidative stress responses, a multi-substrate serine/threonine-protein kinase (AMPK) and AMPK-related kinases, carbohydrate metabolism, satellite cells activities and fibro- adipogenic progenitors (FAPs) in fast-growing (FG) (n = 30) and medium-growing (MG) chickens (n = 30). Pectoralis major muscles were collected at 7d, 14d, 21d, 28d, 35d and 42d of age. According to their macroscopic features, the samples from FG up to 21d of age were classified as unaffected, while all samples collected at an older age exhibited macroscopic features ascribable to white striping and/or wooden breast abnormalities. In contrast, MG samples did not show any feature associated to muscle disorders. The absolute transcript abundance of 33 target genes was examined by droplet digital polymerase chain reaction. The results showed differential gene expression profiles between FG and MG chickens at different ages. While most genes remained unchanged in MG chickens, the expression patterns of several genes in FG were significantly affected by age. Genes encoding alpha 1, alpha 2, beta 2 and gamma 3 isoforms of AMPK, as well as AMPK-related kinases, were identified as differentially expressed between the two strains. The results support the hypothesis of oxidative stress-induced muscle damage with metabolic alterations in FG chickens. An increased expression of ANXA2, DES, LITAF, MMP14, MYF5 and TGFB1 was observed in FG strain. The results suggest the occurrence of dysregulation of FAP proliferation and differentiation occurring during muscle repair. FAPs could play an important role in defining the proliferation of connective tissue (fibrosis) and deposition of intermuscular adipose tissue which represents distinctive traits of muscle abnormalities. Overall, these findings demonstrate that dysregulated molecular processes associated with myopathic lesions in chickens are strongly influenced by growth rate, and, to some extent, by age

Weighted gene co-expression network analysis identifies molecular pathways and hub genes involved in broiler White Striping and Wooden Breast myopathies

In recent years, the poultry industry has experienced an increased incidence of myopathies affecting breasts of fast-growing broilers, such as White Striping (WS) and Wooden Breast (WB) defects. To explore the molecular mechanisms and genes involved in WS and WB onset, we decided to perform a Weighted Gene Co-expression Network Analysis (WGCNA) using the gene expression profile and meat quality parameters of Pectoralis major muscles analysed in our previous study. Among the 212 modules identified by WGCNA, the red, darkred, midnightblue and paleturquoise4 modules were chosen for subsequent analysis. Functional analysis evidenced pathways involved in extracellular matrix (ECM) organization, collagen metabolism, cellular signaling and unfolded protein response. The hub gene analysis showed several genes coding for ECM components as the most interconnected nodes in the gene network (e.g. COL4A1, COL4A2, LAMA2, LAMA4, FBLN5 and FBN1). In this regard, this study suggests that alterations in ECM composition could somehow activate the cascade of biological reactions that result in the growth-related myopathies onset, and the involvement of Collagen IV alterations in activating the endoplasmic reticulum (ER) stress response may be hypothesized. Therefore, our findings provide further and innovative knowledge concerning the molecular mechanisms related to the breast abnormalities occurrence in modern broilers

Distribution and Expression of Vimentin and Desmin in Broiler Pectoralis major Affected by the Growth-Related Muscular Abnormalities

Desmin (DES) and Vimentin (VIM) exert an essential role in maintaining muscle cytoarchitecture and since are considered reliable markers for muscle regeneration, their expression has been extensively investigated in dystrophic muscles. Thus, exhibiting features similar to those of human dystrophic muscles, the present study aimed at assessing the distribution of VIM and DES proteins and the expression of the corresponding genes in Pectoralis major muscles affected by white striping (WS), wooden breast (WB), and spaghetti meat (SM) abnormalities as well as in those having macroscopically normal appearance (NORM). For this purpose, 20 Pectoralis major muscles (5/group) were collected from the same flock of fast-growing broilers to perform immunohistochemistry, immunoblotting and gene expression. Immunohistochemical analyses showed an increased number of fibers immunoreactive to both VIM and DES in WS and WB, while only a few immunoreactive fibers were observed in NORM. Concerning the protein level, if compared with NORM, a 55% increase in VIM content was found in WB affected cases (P < 0.05) thus suggesting the development of intense regenerative processes in an early-stage within these muscles. The significantly higher amount of DES (+53%) found in WS might be attributed to a progression of the regenerative processes that require its synthesis to preserve the structural organization of the developing fibers. On the other hand, significantly lower VIM and DES contents were found in SM. About gene expression, VIM mRNA levels gradually increased from the NORM to the SM group, with significantly higher gene expressions in WB and SM samples compared to the NORM group (P = 0.009 for WB vs. NORM and P = 0.004 for SM vs. NORM). Similarly, the expression of DES gene showed an increase from the NORM to WB group (P = 0.05). Overall, the findings of the present study suggest that intense regenerative processes take place in both WB and WS muscles although a different progression of regeneration might be hypothesized. On the other hand, the lack of correspondence between VIM gene expression and its protein product observed in SM suggests that VIM may also exert a role in the development of the SM phenotype

Using Transesophageal Echocardiography in Liver Transplantation with Veno-Venous Bypass Is a Tool with Many Applications: A Case Series from an Italian Transplant Center

Background: Hemodynamic instability (HDI) is common during liver transplantation (LT); veno-venous bypass (VVB) is a tool used in selected cases to ensure hemodynamic stability and for surgical needs. Transesophageal echocardiography (TEE) allows the transplant team to identify the causes of HDI and to guide therapies. We present a case series of four patients showing the valuable role of TEE during LT in VVB. Methods: We report four explicative cases of TEE use in LT with VVB performed at IRCCS Azienda Ospedaliero–Universitaria di Bologna. Four transplants were performed between 2016 and 2022. Results: Many authors have highlighted the diagnostic value of TEE during LT in the case of HDI. However, its specific role during LT with VVB is poorly described. This paper illustrates multiple potential uses of TEE in LT with VVB: TEE as a guide for catheterization and optimal cannula positioning, TEE as a tool for intraoperative Patent Foramen Ovale management, TEE as help for anticoagulation therapy and finally, TEE as support when evaluating bypass efficiency and correcting hypovolemia. Conclusion: TEE is a useful instrument during LT with VVB. However, further studies are needed to assess the suitable applications of TEE during LT in patients with HDI requiring VVB. TEE should be part of the anesthetist’s cultural background