Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons

Abstract

Injury to the energy-storing superficial digital flexor tendon is common in equine athletes and is age-related. Tenocytes in the superficial digital flexor tendon of adult horses appear to have limited ability to respond adaptively to exercise or prevent the accumulation of strain-induced microdamage. It has been suggested that conditioning exercise should be introduced during the growth period, when tenocytes may be more responsive to increased quantities or intensities of mechanical strain. Tenocytes are linked into networks by gap junctions that allow coordination of synthetic activity and facilitate strain-induced collagen synthesis. We hypothesised that there are reductions in cellular expression of the gap junction proteins connexin (Cx) 43 and 32 during maturation and ageing of the superficial digital flexor tendon that do not occur in the non-injury-prone common digital extensor tendon. Cryosections from the superficial digital flexor tendon and common digital extensor tendon of 5 fetuses, 5 foals (1-6 months), 5 young adults (2-7 years) and 5 old horses (18-33 years) were immunofluorescently labelled and quantitative confocal laser microscopy was performed. Expression of Cx43 and Cx32 protein per tenocyte was significantly higher in the fetal group compared with all other age groups in both tendons. The density of tenocytes was found to be highest in immature tissue. Higher levels of cellularity and connexin protein expression in immature tendons are likely to relate to requirements for tissue remodelling and growth. However, if further studies demonstrate that this correlates with greater gap junctional communication efficiency and synthetic responsiveness to mechanical strain in immature compared with adult tendons, it could support the concept of early introduction of controlled exercise as a means of increasing resistance to later injury.

Fig. 1

Tenocyte density (expressed as number per mm²) in the SDFT and CDET from each age group. Asterisk indicates a significant difference compared to the CDET. Error bars are indicated as ±SEM. SDFT, superficial digital flexor tendon; CDET, common digital extensor tendon.

Fig. 2

Western blots showing bands for Cx43 (43 kDa, black arrow, plus a band usually identified at 47kDa indicating different states of phosphorylation, blue arrow) and (b) Cx32 (27 kDa, black arrow). Cx32, connexin 32; Cx43, connexin 43.

Fig. 3

Longitudinal cryosections from the SDFT and CDET immunolabelled with Cx32 (green plaques) from a fetus, foal and an old horse. Tenocyte nuclei counterstained with propidium iodide (red). Bar = 80 µm.

Fig. 4

Longitudinal cryosections from the SDFT and CDET immunolabelled with Cx43 (green plaques) from a fetus, foal and an old horse. Tenocyte nuclei counterstained with propidium iodide (red). Bar = 80 µm.

Fig. 5

High magnification of longitudinal cryosections from a fetus. SDFT and CDET immunolabelled with Cx32 and Cx43. Tenocyte nuclei counterstained with propidium iodide. Bar = 15 µm.

Fig. 6

(a) Total area of Cx32 and Cx43 plaques per tenocyte (expressed per µm²) in the SDFT and CDET from each age group. Asterisk indicates a significant difference compared with all other age groups. The mean area of Cx plaques per tenocyte was significantly higher in the fetal group than in any other group for both Cx43 and Cx32, but did not differ between the SDFT and CDET. Error bars are indicated as ±SEM. (b) Cx32 and Cx43 plaque number per tenocyte (expressed per mm²) in the SDFT and CDET from each age group. Double asterisk indicates a significant difference compared with all other age groups. The number of Cx plaques was significantly higher in the fetal group than in any other group for both Cx43 and Cx32, but did not differ between tendons.