Role of timp2 in Ctenopharyngodon idellus muscle collagen deposition and the regulatory mechanism by Cu2

Collagen serves as one of determinants of fish muscle quality. Timp2 is a key enzyme in Collagen metabolism. Its role in grass carp (Ctenopharyngodon idellus) and related nutritional regulation mechanism are unknown. Given the important role of copper as a micronutrient in Collagen biosynthesis, it was hypothesized that Cu²⁺ up-regulates timp2 expression through the TGFβ/Smads signaling pathway, thus modulating grass carp Collagen content. In the present study, this hypothesis validation was performed through parallel experiments using the TGF-β/Smads inhibitor SB431542 and timp2-specific siRNA (sitimp2) in cultured fibroblasts. Results showed that both sitimp2 and SB431542 treatment decreased the Collagen content (to 90.2 % ± 0.52 % and 61.8 % ± 1.59 % of control, respectively), down-regulated timp2 expression (by 53.8 % ± 1.30 % and 11.3 % ± 1.91 % relative to control) and up-regulated mmp2 expression (by 33.1 % ± 9.70 % and 10.9 % ± 2.51 % relative to control). Additionally, SB431542 inhibited the TGFβ/Smads signaling pathway (P < 0.05). Coversely, Cu²⁺ supplementation significantly increased the Collagen content, up-regulated timp2 expression and enhanced TGFβ/Smads signaling pathway activity, while simultaneously suppressing mmp2 expression (P < 0.05). Futhermore, after sitimp2 or SB431542 treatment, 10 μM Cu²⁺ reversed the changes in the relevant indicators induced by both sitimp2 and SB431542 treatment (P < 0.05). These results confirmed the hypothesis that Cu²⁺ enhanced Collagen deposition through TGFβ/Smads-mediated up-regulation of timp2. The present study provided novel mechanistic insights into copper-dependent Collagen regulation and suggested that dietary copper supplementation could be a practical nutritional strategy to improve muscle texture and product quality in farmed grass carp.

Fibroblast; Grass carp; Muscle quality; TGFβ/Smads signaling pathway.