Abstract
Introduction
Exercise increases tendon collagen synthesis and cross-link formation. Exercise also increases the expression of TGF-β1. TGF-β1 may contribute to the upregulation of tendon collagen synthesis during exercise, but this relationship has not been established in vivo. The purpose of this study was to evaluate the effects of TGF-β1 receptor inhibition on tendon collagen.
Materials and Methods
Male Wistar rats were divided into sedentary (SED, n = 9) or exercised (RUN, n=15) groups. Exercised animals completed four days of treadmill exercise (60 minutes/days). The peritendinous space of one Achilles tendon was injected with LY-364947 (ALK5 inhibitor; INHIB) while the opposite leg was injected with a vehicle (SHAM). Injections were given daily after each exercise bout. ERK and Smad 2/3 phosphorylation was evaluated by Western blotting. Collagen I and III gene expression were evaluated via qRT-PCR. Tendon hydroxyproline and hydroxylyslpyridinoline (HP) cross-linking were assayed via HPLC. A longitudinal section of tendon was stained with H&E for evaluation of cell numbers and fibril organization.
Results
Phosphorylation of ERK increased by 2.5-fold in both legs given LY-364947 (p<0.05) but was not influenced by exercise (p>0.05). Smad 3 phosphorylation was reduced (p<0.05) in tendons treated with LY-364947. Neither type I nor type III collagen gene expression was affected by TGF-β1 receptor inhibition or exercise (p>0.05). Collagen content was not altered by either exercise or LY-364947 (p>0.05). HP cross-linking was 3-fold lower in the RUN-INHIB when compared to the RUN-SHAM tendon (p<0.05). No effect of inhibitor on HP was noted in the sedentary animals. Cell density was greater (p<0.05) in the Achilles tendon of exercised animals (SED: 7.5 cell/100 μm2, RUN: 10.3 cell/100 μm2) but was not influenced by TGF-β1 receptor inhibition (p>0.05). Fiber structure scores were 45% lower (p<0.07) in SED animals treated with inhibitor but normal in RUN animals given inhibitor.
Discussion
The changes in ERK and Smad phosphorylation suggest that LY-364947 was effective at altering TGF-β1 signaling. Our data suggest that neither acute exercise nor TGF-β1 receptor inhibition altered collagen gene expression. In contrast, TGF-β1 appears to be important for regulating Achilles tendon cross-link formation during exercise training and inhibition of TGF-β1 impacts fiber structure.
