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BIONÆR-Bionæringsprogram

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

Alternative title: Kylling Helse: Reduksjon av wooden breast ved å identifisere og editere regulatorer av kyllingmyopati

Awarded: NOK 12.1 mill.

Project Number:

323939

Application Type:

Project Period:

2021 - 2025

Funding received from:

Partner countries:

The main goal of Chicken-Health is to elucidate the role of relevant extracellular matrix (ECM) components, with a particular focus on the proteoglycan family syndecans in wooden breast (WB) severe chicken myopathy, and if they can serve as therapeutic targets. In addition, understanding syndecan molecular mechanisms in WB will help to establish reliable and appropriate strategies, not just to understand but to be able to treat the disease. The PhD student allocated in the project is now at a research stay in Stony Brook University. Several presentations at international ECM conferences (MBE2022, Nordic PG-meeting) and national (NBS-meeting) and seminar have been performed. A kick-off meeting, yearly-project meeting and WP-meetings between consortium partners has been performed. Scientific paper with the PhD student as first author is accepted for publication in Frontiers in Physiology. A master student has been allocated in the project. WP1: A chicken model with feeding-induced WB at 36-day growth has been successfully performed. Skeletal muscle, heart, blood, and primary cells were collected. NIR-spectroscopy and palpation of skeletal muscle samples were used for scoring the level of wooden breast. Furthermore, HE histology together with WGA and Picro-Sirius Red was used to confirm groups based on the level of fibrosis. NIR-spectroscopy and grouping by histology showed similar pattern regarding WB-detection, but with some discrepancy for some of the individuals. Comprehensive extracellular matrix remodeling was observed between normal and WB-affected samples. RNA- sequencing and bioinformatic cluster analysis pointed out syndecans to be located in clusters defining WB-affected samples. A distinct expression pattern of the syndecan family members has been verified at mRNA by real-time PCR, and increased shedding and upregulation of specific signaling pathways by western. Proteomic analysis identified upregulation of metabolic enzymes in WB-affected animals. A reduced regeneration properties in WB-affected animals has been identified by immunohistology, and changes in ECM-complexes at protein and mRNA level. Parts of results from WP1 are included in the accepted publication in Frontiers in Physiology. WP2: A syndecan-1,2,3 and-4 chicken specific ab tool-box has been developed by antibody production and specificity testing by peptide blocking, laying the foundation for in depth molecular analysis and investigation of this family group as potential regulators of WB-myopathy. Screening of commercial antibodies with specific staining for chicken has also been performed and still ongoing, laying the foundation for a toolbox for skeletal muscle fibrosis in chicken. We have further developed a toolbox of blocking-peptides for further in depth studies of syndecan regulatory mechanisms. WP3: Primary cells were isolated for further in vitro investigation of molecular mechanisms at the cellular level in the project period. Growth media and cell culture conditions have been optimized. Further in vitro culturing of normal and affected MuSCs revealed large differences in growth and mRNA expression pattern. The planned phosfoproteomic-proteomic analysis has been changed to RNA-sequencing because of impaired growth of MuSCs from affected individuals. Preparation of manuscript from our in vivo and in vitro data on skeletal muscle regeneration are ongoing. The establishment of a new cell system for further in detalied study of syndecan regulatory mechanisms in relation to fibrosis is ongoing. Growth conditions for fibroblast cells has been established. Transfection studies are ongoing. WP4: Investigating the profile of promising biomarkers of wooden breast in blood samples is ongoing, and a reduced level of specific sugar-structures revealed in serum samples from affected animals. At present, using ELISA method for detection of syndecans was unsuccessful. The use of FTIR and NMR to investigate potential biomarkers of wooden breast in serum is ongoing. This is in collaboration with another internal project. Shedding of syndecan-4 in vivo has been detected by chicken specific antibody produced in the project,and revealed shedding fragments of 15 kDa and 20 kDa of syndecan-4. Increased MMP mRNA expression and activity in WB-affected samples support MMPs as potential sheddases. Crisper/Cas9-gene editing of syndecan-4 in fibroblasts in vitro is now under planning. WP5: Combining NIR-spectroscopic analysis, transcriptomics and histology shows an overlapping grouping, with a clear separation between normal and affected individuals. Further multistatestic analysis at individual level is initiated to increase knowledge of syndecans potential regulatory role in WB and fibrosis.

Intensive breeding and genetic manipulation have led to severe myopathic defects named Wooden breast (WB). Chicken suffering from these illnesses usually must be withdrawn from the food chain due to insufficient food quality and less nutritional value. The underlying molecular mechanisms of WB is still only partially understood. Morphological characteristics of WB demonstrate severe skeletal muscle fibrosis, necrosis and multifocal degeneration of muscle tissue. Fibrosis in general is characterized by extensive extracellular matrix (ECM) remodeling and is caused by a dysfunctional muscle satellite cell activity in combination with an imbalanced regulation of fibroblast/myofibroblast activity. A family of ECM receptors called syndecans are known to be involved in human skeletal muscle myopathies, they regulate fibrosis in cardiac muscle and have crucial functions in normal MuSC and fibroblast activity. These molecules have a wide spectrum of biological functions and are master regulators of tissue homeostasis, and gene expression of this family is up- regulated in WB chicken. However, no studies of syndecans and their possible role in WB exist. And more importantly, how can these complex molecules be modified to avoid developing the disease, and at the same time retaining functions important for key cellular processes? The main goal is to elucidate the role of syndecans and relevant ECM components in severe chicken myopathy and if they can serve as therapeutic targets. Understanding syndecan molecular mechanisms in WB will help establish reliable and relevant strategies, not just to understand but to be able to treat the disease. In addition, this project has a potential for a broader societal impact with benefits to the food industry, public sector and civil society in a longer term. Reducing WB will secure healthy chickens without suffering, will provide healthy meat for humans and will contribute with a solution to the increasing protein demand of the future.

Funding scheme:

BIONÆR-Bionæringsprogram