Altered Satellite Cell Responsiveness and Denervation Implicated in Progression of Rotator-Cuff Injury

Abstract

Background: Rotator-cuff injury (RCI) is common and painful; even after surgery, joint stability and function may not recover. Relative contributions to atrophy from disuse, fibrosis, denervation, and satellite-cell responsiveness to activating stimuli are not known.

Methods and findings: Potential contributions of denervation and disrupted satellite cell responses to growth signals were examined in supraspinatus (SS) and control (ipsilateral deltoid) muscles biopsied from participants with RCI (N = 27). Biopsies were prepared for explant culture (to study satellite cell activity), immunostained to localize Pax7, BrdU, and Semaphorin 3A in satellite cells, sectioning to study blood vessel density, and western blotting to measure the fetal (γ) subunit of acetylcholine receptor (γ-AchR). Principal component analysis (PCA) for 35 parameters extracted components identified variables that contributed most to variability in the dataset. γ-AchR was higher in SS than control, indicating denervation. Satellite cells in SS had a low baseline level of activity (Pax7+ cells labelled in S-phase) versus control; only satellite cells in SS showed increased proliferative activity after nitric oxide-donor treatment. Interestingly, satellite cell localization of Semaphorin 3A, a neuro-chemorepellent, was greater in SS (consistent with fiber denervation) than control muscle at baseline. PCAs extracted components including fiber atrophy, satellite cell activity, fibrosis, atrogin-1, smoking status, vascular density, γAchR, and the time between symptoms and surgery. Use of deltoid as a control for SS was supported by PCA findings since "muscle" was not extracted as a variable in the first two principal components. SS muscle in RCI is therefore atrophic, denervated, and fibrotic, and has satellite cells that respond to activating stimuli.

Conclusions: Since SS satellite cells can be activated in culture, a NO-donor drug combined with stretching could promote muscle growth and improve functional outcome after RCI. PCAs suggest indices including satellite cell responsiveness, atrogin-1, atrophy, and innervation may predict surgical outcome.

Fig 1. Changes in the level of the γ-AchR subunit.

A. Graph of the optical density (OD) of bands from western blots probed for the γ-AchR subunit (relative to β-actin) in SS compared to control muscle. The amount of the γ-AchR subunit protein, typically expressed in denervated and fetal muscle, was assessed in protein extracts prepared from homogenized SS and control deltoid muscle (* indicates significant difference, p<<0.001, N = 19 paired samples). B. A representative western blot prepared from protein extracts loaded into lanes for control (C) and Supraspinatus (SS) muscles from different participants, probed to detect the γ-AchR subunit and then re-probed to detect β-actin (as a loading control).

Fig 2. SC activation in response to ISDN in explant cultures.

A and B. Micrographs of immunofluorescence staining for BrdU and Pax7. Images are as labeled: DIC for orientation to the SC; Cy5 channel used to visualize anti-BrdU staining; GFP channel used to visualize anti-Pax7 staining; and a merge of Cy5 and GFP to show any overlap. A. shows staining of a Pax7+/BrdU- cell. B shows a Pax7+/BrdU+ SC where merge of Cy5 and GFP is yellow. C. Graph of the proportion (mean, SEM) of active BrdU+/Pax7+ SCs of the total number of Pax7+ SCs observed in sections of control or supraspinatus (SS) muscles, after culturing for 40 hours in the presence of BrdU, with isosorbide dinitrate (ISDN) or at baseline (no treatment). At baseline, SS had a lower proportion of activated SCs than control muscle. ISDN increased the number of BrdU+ SCs in SS but not control muscle. Asterisk (*) indicates significant difference from untreated SS (p = 0.01, N = 27) from a total of 7661 Pax7+ satellite cells.

Fig 3. Localization of Sema3A protein.

Representative micrographs of Sema3A staining in presumptive SCs, located in the satellite position on fibers (arrow indicates nucleus) in control and Supraspinatus (SS) muscle explant cultures at baseline (without ISDN treatment) or with ISDN (as labeled) [a subset of n = 10 participants from the full dataset shown in Fig 2]. Bar = 20 μm. A. Cells in control muscle ranged from large and activated (left panel) to small and attenuated (middle panel), with low to moderate intensity staining in the cytoplasm. Sema3A staining of an attenuated cell in the satellite position on fibers in ISDN-treated control muscle have dark staining for Sema3A. B. Most SCs in SS muscle at baseline had attenuated moderately-stained cytoplasm (left panel), and were larger with low to moderate intensity staining after ISDN treatment. Right-most panel shows dark Sema3A staining in a Schwann cell close to a NMJ. C. Graph of areal density of Sema3A stain (mean, SEM) in control (C) and supraspinatus (SS) muscle at baseline or after treatment with ISDN. Sema3A staining was higher in SS at baseline than in control muscle at baseline. Asterisk (*) indicates significant difference from control muscle (p = 0.04). D. Graph illustrating the significant correlation of SC areal density after activation by ISDN (y-axis) with that at baseline in the same muscle (control, open diamonds, SS, black diamonds); R2 = 0.4925, p<0.001, N = 10.

Fig 4. Principal component analysis (PCA) biplots.

Biplots show the correlation vectors representing the projection (in 2 dimensions) of loading for each of the variables (for 27 participants) included in the particular PCA. Vectors project onto the 3 axes (dimensions) of the principal components (PCs) 1 (x-axis), 2 (y-axis), and 3 (z-axis, positive is upward, perpendicular to the page). A. PCA-1, on the full dataset (N = 23 variables). B. PCA-2, Supraspinatus variables only (N = 16). C. PCA-3, variables of interest from previous comparisons, including “muscle” as a variable (N = 15 variables). Variables that loaded onto PC3 (as shown in Tables 1–3) are indicated with an asterisk (*) to emphasize that the correlation vector projects upward, perpendicular to PC1 and PC2 axes. Variable labels on the vectors are abbreviated as follows: C (control muscle), SS (Supraspinatus muscle), BMI (body mass index), Sx-to-Surg (weeks from symptom onset to surgery), diam (fiber diameter), SS:C diam (ratio of fiber diameter in SS/C), SC-base (baseline SC activation in explant cultures), SC-ISDN (SC activation in explants cultured with ISDN), dystr (expression of dystrophin protein relative to β-actin), mmp9 (level of matrix metalloproteinase 9 protein relative to β-actin), logRed (log-transformation of average maximum Sirius Red staining), γ:ε (γ:ε ratio of AchR subunits), γ (expression of the γ-subunit of AchR protein relative to β-actin), and BvD (vascular density).