Chicken Health: Preventing wooden breast by identifying and editing regulators of severe chicken myopathy

Summary

With the rapid growth and intensive breeding of chickens, a severe myopathy known as wooden breast (WB) has emerged during the last decade. This myopathy is a myodegenerative disease in chickens, characterized by hardened muscle fibers, extensive extracellular matrix (ECM) remodeling, skeletal muscle fibrosis, tissue degeneration, and disrupted metabolic processes. This PhD thesis aims to investigate the molecular mechanisms driving the progression of WB, with a special focus on ECM remodeling and the roles of syndecans, a family of transmembrane proteoglycans. Syndecan shedding, which involves the proteolytic release of their extracellular domains, is considered fundamental in regulating fibrotic processes, influencing cellular signaling pathways and ECM deposition. Syndecan shedding is also involved in muscle development, regeneration, and homeostasis. Using a multi-omics approach with immunostaining, histology, gene and protein expression analyses, transcriptomics, and proteomics, the research provides a comprehensive exploration of ECM proteins, metabolic processes, myogenesis, syndecan expression and their shedding in the pathophysiology of WB, both in vivo and in vitro. The involvement of syndecans in WB myopathy is a novel area of study. This thesis highlights their various roles in cell-specific activities. In Paper I, we investigated the histological and molecular characteristics of WB myopathy in chickens, revealing extensive ECM remodeling in muscle tissue. This included increased expression of collagens, small leucine-rich proteins, glycosaminoglycan biosynthesis, ECM-receptor interactions, and cell adhesion, along with a disrupted collagen structure. Additionally, the study characterized syndecan expression and their shedding in WB myopathy. The investigation revealed elevated gene expression of syndecan-1 (SDC1) and syndecan-4 (SDC4) in WB myopathy, with all syndecan family members exhibiting increased shedding in WB-affected chickens. This was accompanied by enhanced matrix metalloproteinase (MMP) activity and alterations in key signaling pathways, including MAPK, Akt, and Wnt. These findings suggest that syndecans contribute significantly to ECM remodeling and fibrosis in WB myopathy, highlighting them as a potential part of further research interest. Paper II in this thesis focused on the role of muscle dysfunction and the potential role of syndecan-4 in the development of WB myopathy, using both an in vivo and in vitro approach. In this study, significant metabolic alterations linked to glycolysis, oxidative phosphorylation and metabolic pathways were identified in WB. Affected chickens showed varied muscle fiber sizes, more PAX7-expressing muscle stem cells, but no change in the proliferation marker PCNA, suggesting ongoing regeneration, as well as a defect in proliferation. Muscle satellite cells (MuSCs) derived from WB-affected chickens showed impaired proliferation, retained differentiation potential, and had altered gene expression of myogenic markers. Furthermore, we observed increased shedding of syndecan-4 in WB-affected MuSCs. The overexpression of SDC4 in non-affected MuSCs resulted in decreased proliferation and lower levels of myogenesis markers. In this paper, we found that the use of blocking peptides derived from syndecan-4 ectodomain partially enhanced the proliferation of MuSCs and reduced syndecan-4 shedding, indicating the pivotal role of syndecan-4 in MuSCs with an impact on WB. Paper III focused on the hallmark of WB – fibrosis and the role of syndecan-4. An in vitro approach using embryonic chicken fibroblasts revealed that overexpression of SDC4 reduced the expression of fibrotic markers like collagen I and III, while inducing shedding of syndecan-4 and increasing gene expression of pro-inflammatory cytokines such as transforming growth factor-beta 1 (TGF-β1, TGFB1 gene) and interleukin-1 beta (IL-1β, IL1B gene). Additionally, the study explored the use of blocking peptides derived from the syndecan-4 ectodomain and found that they significantly decreased syndecan-4 shedding, as evidenced by the 20 kDa cytoplasmic remaining fragment on the Western blot, without affecting fibrotic markers. TGF-β1 stimulation on the other hand increased this syndecan-4 shedding and triggered production of fibrotic markers, identifying interactions between syndecan-4 and TGF-β1. Through these findings, this thesis extends the understanding of WB myopathy in broiler chickens and contributes to the development of strategies to improve poultry health while addressing broader challenges in research on skeletal myopathies and fibrotic diseases. Although various aspects of the investigations require further exploration, we suggest that this work will lay the foundation for new hypotheses and research directions regarding WB myopathy and the role of syndecans.