Formation of intramuscular collagen cross-links in cattle

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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.