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，其渐进的交联会导致肉类韧性增加。本研究计划的长期目标是1)阐明控制食用动物肌肉内胶原合成和交联的机制，以及2)评估改变这些机制对肉类品质的影响。PYR和Ehrlich‘s Chromogen(EC)是两种与肉类韧性有关的肌肉内胶原交联物，其中EC在幼年动物中占主导地位，而PYR在老年动物中占主导地位。对于PYR的优先形成，赖氨酰羟基酶2(LH2)需要比赖氨酰羟基酶1(LH1)更有活性，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。亲环素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最终结构的能力时，CypB的表达增加。CypB的表达受两条细胞保护途径的调控：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)。在某些病理学中，如癌症，组织会经历缺氧并表现出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，这项研究计划将检验以下假设：1)肌肉的快速生长产生内质网(ER)压力和未折叠蛋白反应(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活性增加与肌肉内胶原中EC的增加有关。通过检测与内质网应激和缺氧相关的mRNA和蛋白的表达，将确定快速生长肌肉中有效的细胞保护途径。活性的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的蛋白水平将通过蛋白质印迹分析来确定，并与细胞保护途径的结果以及肌肉内胶原中PYR和EC的浓度有关。肉鸡和肉牛将被用作本计划的动物模型，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商业上可获得的类固醇和β-肾上腺素能激动剂莱克多巴胺来刺激。将分别与限制饲养的肉鸡(自由饲养的80%)和生长缓慢的传统鸡，以及未经处理的牛进行比较。这项研究不仅将有助于控制肉类的质量，而且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。

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.