Formation of intramuscular collagen cross-links in cattle

牛肌内胶原交联的形成

• 批准号: RGPIN-2021-02714
• 负责人: Bruce, Heather
• 金额: $ 2.91万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=742173
• 关键词: Formation; intramuscular; collagen; cross; links

Collagen is the main protein in connective tissue, and its progressive crosslinking leads to increased meat toughness as animals age. The long-term objectives of this research program are to 1) elucidate the mechanisms controlling the synthesis and crosslinking of intramuscular collagen in food animals, and 2) evaluate the impact of altering these mechanisms on meat quality. Pyridinoline (PYR) and Ehrlich's Chromogen (EC) are two intramuscular collagen crosslinks associated with toughness of meat, with EC predominating in young animals and PYR in old animals. For the preferentially formation of PYR, lysyl hydroxylase 2 (LH2) would need to be more active than lysyl hydroxylase 1 (LH1) because LH2 hydroxylates lysine in the telopeptides of the collagen molecule, a necessary step in the formation of PYR. The activities of LH1 and LH2 are respectively enhanced and inhibited by cyclophilin B (CypB). CypB expression increases when cells experience low oxygen (hypoxia) or when intracellular protein synthesis exceeds cellular capacity to fold proteins into their final structure. CypB expression is controlled through two cytoprotective pathways: the first pathway is through hypoxia-inducible factor 1 (HIF-1) while the second is through the unfolded protein response (UPR). In certain pathologies like cancer, tissues experience hypoxia and exhibit UPR, but whether this occurs in muscle during normal rapid growth is unknown. This research program, therefore, will test the hypotheses that 1) rapid growth of muscle produces endoplasmic reticulum (ER) stress and an unfolded protein response (UPR), 2) rapid growth increases the expression of CypB, 3) increased CypB expression is associated with increased LH1 activity, and 4) increased LH1 activity is associated with an increase in EC in intramuscular collagen. The operative cytoprotective pathways in rapidly growing muscle will be determined by measuring expression of mRNA and proteins associated with ER stress and hypoxia. Active UPR pathways will be determined by measuring mRNA expression and protein levels of ER stress proteins and protein levels of LH1 and LH2 will be determined using Western blot analysis and related to cytoprotective pathway results and concentrations of PYR and EC in intramuscular collagen. Broiler chickens and beef cattle will be used as the animal models in this program as rapid growth in broiler chickens is ensured by provision of unrestricted fed access, while rapid growth in beef cattle will be provoked using commercially-available steroids and the beta-adrenergic agonist ractopamine. Respective comparisons will be made to broiler chickens that are restricted-fed (80% of ad libitum) and slow-growing heritage chickens, and to non-treated cattle. This research will not only benefit control of meat quality but will impact understanding of mechanisms associated with rapid muscle growth and repair in human injury or disease.

胶原蛋白是结缔组织中的主要蛋白质，随着动物年龄的增长，其逐渐交联会导致肉的韧性增加。该研究计划的长期目标是 1) 阐明控制食用动物肌内胶原蛋白合成和交联的机制，2) 评估改变这些机制对肉质的影响。吡啶啉 (PYR) 和艾利希色原 (EC) 是两种与肉的韧性相关的肌内胶原蛋白交联，其中 EC 在年轻动物中占主导地位，而 PYR 在老年动物中占主导地位。为了优先形成 PYR，赖氨酰羟化酶 2 (LH2) 需要比赖氨酰羟化酶 1 (LH1) 更具活性，因为 LH2 羟基化胶原分子端肽中的赖氨酸，这是形成 PYR 的必要步骤。 LH1 和 LH2 的活性分别被亲环蛋白 B (CypB) 增强和抑制。当细胞经历低氧（缺氧）或细胞内蛋白质合成超过细胞将蛋白质折叠成最终结构的能力时，CypB 表达就会增加。 CypB 表达通过两条细胞保护途径控制：第一条途径是通过缺氧诱导因子 1 (HIF-1)，第二条途径是通过未折叠蛋白反应 (UPR)。在癌症等某些疾病中，组织会经历缺氧并表现出 UPR，但在正常快速生长过程中肌肉中是否会发生这种情况尚不清楚。因此，本研究计划将测试以下假设：1) 肌肉的快速生长会产生内质网 (ER) 应激和未折叠蛋白反应 (UPR)，2) 快速生长会增加 CypB 的表达，3) CypB 表达的增加与 LH1 活性的增加有关，4) LH1 活性的增加与肌内胶原蛋白中 EC 的增加有关。快速生长肌肉中有效的细胞保护途径将通过测量与 ER 应激和缺氧相关的 mRNA 和蛋白质的表达来确定。活性 UPR 途径将通过测量 ER 应激蛋白的 mRNA 表达和蛋白水平来确定，LH1 和 LH2 的蛋白水平将使用蛋白质印迹分析来确定，并与细胞保护途径结果以及肌内胶原蛋白中 PYR 和 EC 的浓度相关。肉鸡和肉牛将被用作该计划的动物模型，因为肉鸡的快速生长是通过提供不受限制的饲喂途径来确保的，而肉牛的快速生长将使用市售类固醇和β-肾上腺素激动剂莱克多巴胺来激发。将分别与限制饲喂的肉鸡（80％随意采食）和生长缓慢的传统鸡以及未经处理的牛进行比较。这项研究不仅有利于肉类质量的控制，还将影响对人类损伤或疾病中肌肉快速生长和修复相关机制的理解。