Formation of intramuscular collagen cross-links in cattle

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，胶原蛋白的逐渐交联会增加肉的韧性。本研究计划的长期目标是：1)阐明控制食用动物肌内胶原合成和交联的机制；2)评估改变这些机制对肉质的影响。吡啶啉（PYR）和埃利希氏色素原（EC）是与肉质韧性相关的两种肌内胶原交联，其中EC在幼龄动物中占优势，PYR在老年动物中占优势。为了优先形成PYR，赖基羟化酶2 （LH2）需要比赖基羟化酶1 （LH1）更活跃，因为LH2羟基化胶原分子端肽中的赖氨酸，这是PYR形成的必要步骤。亲环蛋白B （cyclophilin B, CypB）分别增强和抑制LH1和LH2的活性。当细胞经历低氧（缺氧）或当细胞内蛋白质合成超过细胞将蛋白质折叠成其最终结构的能力时，CypB表达增加。CypB的表达通过两种细胞保护途径控制：第一种途径是通过缺氧诱导因子1 (HIF-1)，第二种途径是通过未折叠蛋白反应（UPR）。在某些疾病中，如癌症，组织经历缺氧并表现出UPR，但这种情况是否发生在正常快速生长的肌肉中尚不清楚。因此，本研究计划将验证以下假设：1)肌肉的快速生长产生内质网（ER）应激和未折叠蛋白反应（UPR）； 2)快速生长增加CypB的表达；3)CypB表达增加与LH1活性增加有关；4)LH1活性增加与肌内胶原中EC增加有关。通过测量与内质网应激和缺氧相关的mRNA和蛋白质的表达，可以确定快速生长肌肉的手术细胞保护途径。通过测量内质网应激蛋白的mRNA表达和蛋白水平来确定活跃的UPR通路，使用Western blot分析来确定LH1和LH2的蛋白水平，并与细胞保护通路结果和肌内胶原蛋白中PYR和EC的浓度相关。肉鸡和肉牛将作为本项目的动物模型，因为肉鸡的快速生长通过提供无限制的饲料来保证，而肉牛的快速生长将使用市售的类固醇和β -肾上腺素能激动剂莱克多巴胺来刺激。将分别与限制饲养（80%自由采食）的肉鸡和生长缓慢的传统鸡以及未处理的牛进行比较。这项研究不仅有利于肉类品质的控制，而且将影响对人类损伤或疾病中肌肉快速生长和修复机制的理解。

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.