Upregulation of transforming growth factor-β signaling in a rat model of rotator cuff tears

Abstract

Background: Muscle atrophy, fatty infiltration, and fibrosis of the muscle have been described as important factors governing outcome after rotator cuff injury and repair. Muscle fibrosis is also thought to have a role in determining muscle compliance at the time of surgery. The transforming growth factor-β (TGF-β) pathways are highly conserved pathways that exert a potent level of control over muscle gene expression and are critical regulators of fibrosis in multiple organ systems. It has been shown that TGF-β can regulate important pathways of muscle atrophy, including the Akt/mammalian target of rapamycin pathway. The purpose of this study was to evaluate the expression of TGF-β and its downstream effectors of fibrosis after a massive rotator cuff tear (RCT) in a previously established rat model.

Methods: To simulate a massive RCT, infraspinatus and supraspinatus tenotomy and suprascapular nerve transection were performed on Sprague-Dawley rats with use of a validated model. Two and 6 weeks after surgery, supraspinatus muscles were harvested to study alterations in TGF-β signaling by Western blotting, quantitative polymerase chain reaction, and histologic analysis.

Results: There was a significant increase in fibrosis in the rotator cuff muscle after RCT in our animal model. There was a concomitant increase in TGF-β gene and protein expression at both 2 and 6 weeks after RCT. Evaluation of the TGF-β signaling pathway revealed an increase in SMAD2 activation but not in SMAD3. There was an increase in profibrotic markers collagen I, collagen III, and α-smooth muscle actin.

Conclusions: TGF-β signaling is significantly upregulated in rat supraspinatus muscles after RCTs.

Keywords: Massive rotator cuff tear; fibrosis; transforming growth factor-β.

Figure 1

Typical Masson trichrome staining of supraspinatus muscles from sham and surgical sides at 2 and 6 weeks after surgery. At 2 weeks after surgery, a large amount of fibroblast-like cells infiltrated into the space between myofibers in the muscle after RCT (lower left panel). At 6 weeks after surgery, a significant amount of collagen was deposited between myofibers in the muscle after RCT (lower right panel).

Figure 2

Real-time RT-PCR showed a significant increase in TGF-β1 and TGF-β2 but not in TGF-β3 expression in the supraspinatus muscle at both 2 and 6 weeks after RCT (compared with the sham side) (N = 6; *P < .05).

Figure 3

Western blot showed a significant increase in TGF-β protein expression in the supraspinatus muscle at both 2 and 6 weeks after RCT (compared with the sham side) (N = 6; *P < .05).

Figure 4

Western blot showed a significant increase in p-SMAD2 but not in p-SMAD3 protein expression in the supraspinatus muscle at both 2 and 6 weeks after RCT (compared with the sham side) (N = 6; *P < .05).

Figure 5

Real-time RT-PCR showed a significant increase in collagen I, collagen III, and α-SMA gene expression in the supraspinatus muscle at both 2 and 6 weeks after RCT (compared with the sham side) (N = 6; *P < .05).