Skeletal muscle fibrosis and stiffness increase after rotator cuff tendon injury and neuromuscular compromise in a rat model

Abstract

Rotator cuff tears can cause irreversible changes (e.g., fibrosis) to the structure and function of the injured muscle(s). Fibrosis leads to increased muscle stiffness resulting in increased tension at the rotator cuff repair site. This tension influences repairability and healing potential in the clinical setting. However, the micro- and meso-scale structural and molecular sources of these whole-muscle mechanical changes are poorly understood. Here, single muscle fiber and fiber bundle passive mechanical testing was performed on rat supraspinatus and infraspinatus muscles with experimentally induced massive rotator cuff tears (Tenotomy) as well as massive tears with chemical denervation (Tenotomy + BTX) at 8 and 16 weeks post-injury. Titin molecular weight, collagen content, and myosin heavy chain profiles were measured and correlated with mechanical variables. Single fiber stiffness was not different between controls and experimental groups. However, fiber bundle stiffness was significantly increased at 8 weeks in the Tenotomy + BTX group compared to Tenotomy or control groups. Many of the changes were resolved by 16 weeks. Only fiber bundle passive mechanics was weakly correlated with collagen content. These data suggest that tendon injury with concomitant neuromuscular compromise results in extra-cellular matrix production and increases in stiffness of the muscle, potentially complicating subsequent attempts for surgical repair.

Keywords: injury; muscle fibrosis; muscle passive mechanics; rotator cuff; shoulder.

Figure 1

The SS (A) and IS (B) single muscle fibers became less stiff between 8 and 16 weeks, regardless of injury. Whole muscle titin molecular weight was lower in the injured SS (C) and IS (D) muscles compared to controls at 16 weeks. In both muscles, this effect appears to be related to larger titin isoforms in the control group at 16 weeks compared to 8 weeks. N = 6 for each group. * Indicates p < 0.05 for individual comparisons, and ^ indicates p < 0.1.

Figure 2

The SS (A) and IS (B) muscle fiber bundles were more stiff in the Tenotomy + BTX group at both 8 and 16 weeks, although this effect was attenuated by 16 weeks. At 8 weeks, there was also an increase in collagen content in the SS muscle (C). This trend was observed in the IS muscle (D), but it does not achieve significance. By 16 weeks, there were no differences between groups as control and Tenotomy groups had great collagen content at this later time point. N = 6 for each group. *p < 0.05 for individual comparisons, and ^ p < 0.1.

Figure 3

Single fiber stiffness was not related to titin molecular weight at 8 or 16 weeks (A). However, was a positive linear relationship (R² = 0.20, p = 0.0046) between collagen content and muscle fiber bundle tangent modulus at 8 weeks, but not at 16 weeks (B).