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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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.
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pone.0162494.g003: 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.

Mentions: The neural chemorepellent, Sema3A, secreted by activated SCs, is implicated in muscle re-innervation [23–25, 45]. Observations of staining for Sema3A in muscles cultured with or without ISDN treatment (Fig 3A and 3B) showed that the area of cytoplasmic staining of cells in the satellite position (interpreted as SCs) varied in sections, with generally larger cells stained in control muscle compared to the more attenuated cells stained in SS muscle at baseline. This observation is consistent with the finding that SC in SS muscle at baseline had a lower level of activation than in control muscle at baseline (Fig 2). In muscles treated with the SC-activating NO-donor, ISDN, there was a subjective increase in the size of these cells in SS but not control muscle. Staining varied from low to high, in both muscles; the stained area of cytoplasm varied from a narrow attenuated region in thin cells to larger areas of cytoplasm in the cells that were apparently more activated as they contained larger nuclei, as previously reported [15]. Sema3A staining appeared quite dense in the cytoplasm of Schwann cells observed in proximity to neuromuscular junctions (Fig 3B, right image); Schwann cells are another source of this secreted protein [46].


Altered Satellite Cell Responsiveness and Denervation Implicated in Progression of Rotator-Cuff Injury
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.
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Related In: Results  -  Collection

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pone.0162494.g003: 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.
Mentions: The neural chemorepellent, Sema3A, secreted by activated SCs, is implicated in muscle re-innervation [23–25, 45]. Observations of staining for Sema3A in muscles cultured with or without ISDN treatment (Fig 3A and 3B) showed that the area of cytoplasmic staining of cells in the satellite position (interpreted as SCs) varied in sections, with generally larger cells stained in control muscle compared to the more attenuated cells stained in SS muscle at baseline. This observation is consistent with the finding that SC in SS muscle at baseline had a lower level of activation than in control muscle at baseline (Fig 2). In muscles treated with the SC-activating NO-donor, ISDN, there was a subjective increase in the size of these cells in SS but not control muscle. Staining varied from low to high, in both muscles; the stained area of cytoplasm varied from a narrow attenuated region in thin cells to larger areas of cytoplasm in the cells that were apparently more activated as they contained larger nuclei, as previously reported [15]. Sema3A staining appeared quite dense in the cytoplasm of Schwann cells observed in proximity to neuromuscular junctions (Fig 3B, right image); Schwann cells are another source of this secreted protein [46].

View Article: PubMed Central - PubMed

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 (&gamma;) subunit of acetylcholine receptor (&gamma;-AchR). Principal component analysis (PCA) for 35 parameters extracted components identified variables that contributed most to variability in the dataset. &gamma;-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, &gamma;AchR, and the time between symptoms and surgery. Use of deltoid as a control for SS was supported by PCA findings since &ldquo;muscle&rdquo; 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.

No MeSH data available.