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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.


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.
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pone.0162494.g002: 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.

Mentions: The initial phase-1 study of SC responsiveness to activation by ISDN in culture found that SS had a lower baseline level of active proliferation (BrdU+/Pax7+) SCs than control muscle (p = 0.03, N = 13). In that study, ISDN increased the proportion of BrdU+ SCs only in SS, and the level of activity after ISDN increased up to the baseline level found in SCs of control muscle. The current, independent-repeat experiment on phase-2 samples gave identical results (p = 0.03, N = 14) from data on a total of 5016 Pax7+ satellite cells photographed and counted from the four groups of muscles (Control baseline: N = 117; Control ISDN: N = 1171; SS baseline: N = 1371; SS ISDN: N = 1295). Fig 2 shows results from the full dataset; the level of BrdU+/Pax7+ SCs in baseline SS cultures was significantly lower than the level in all other groups (p = 0.016, N = 27). The data represented in Fig 2 include counts of the following number of Pax7+ satellite cells (N = 27 participants): Control baseline: N = 1915; Control ISDN: N = 1862; SS baseline: N = 1946; and SS ISDN: N = 1938.


Altered Satellite Cell Responsiveness and Denervation Implicated in Progression of Rotator-Cuff Injury
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.
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pone.0162494.g002: 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.
Mentions: The initial phase-1 study of SC responsiveness to activation by ISDN in culture found that SS had a lower baseline level of active proliferation (BrdU+/Pax7+) SCs than control muscle (p = 0.03, N = 13). In that study, ISDN increased the proportion of BrdU+ SCs only in SS, and the level of activity after ISDN increased up to the baseline level found in SCs of control muscle. The current, independent-repeat experiment on phase-2 samples gave identical results (p = 0.03, N = 14) from data on a total of 5016 Pax7+ satellite cells photographed and counted from the four groups of muscles (Control baseline: N = 117; Control ISDN: N = 1171; SS baseline: N = 1371; SS ISDN: N = 1295). Fig 2 shows results from the full dataset; the level of BrdU+/Pax7+ SCs in baseline SS cultures was significantly lower than the level in all other groups (p = 0.016, N = 27). The data represented in Fig 2 include counts of the following number of Pax7+ satellite cells (N = 27 participants): Control baseline: N = 1915; Control ISDN: N = 1862; SS baseline: N = 1946; and SS ISDN: N = 1938.

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.