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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.


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).
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pone.0162494.g001: 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).

Mentions: The ε subunit in AchRs reverts to the γ subunits upon denervation. Here, the expression of the two AchR subunit proteins was quantified by western blot. Expression of the γ-AchR subunit (standardized to β-actin) was significantly higher in SS than in the control muscle (p<<0.001, N = 19), implicating some impact of SS denervation at the time of the biopsy (Fig 1). The γ:ε ratio of AchR subunits, which can be used as an indicator of innervation status in a muscle [22], also tended to be higher than control (p = 0.09, N = 20).


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

License
Show All Figures
getmorefigures.php?uid=PMC5036792&req=5

pone.0162494.g001: 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).
Mentions: The ε subunit in AchRs reverts to the γ subunits upon denervation. Here, the expression of the two AchR subunit proteins was quantified by western blot. Expression of the γ-AchR subunit (standardized to β-actin) was significantly higher in SS than in the control muscle (p<<0.001, N = 19), implicating some impact of SS denervation at the time of the biopsy (Fig 1). The γ:ε ratio of AchR subunits, which can be used as an indicator of innervation status in a muscle [22], also tended to be higher than control (p = 0.09, N = 20).

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.