Atrophy, inducible satellite cell activation, and possible denervation of supraspinatus muscle in injured human rotator-cuff muscleAm J Physiol Cell Physiol


Deanna Gigliotti, Jeff R. S. Leiter, Bryce Macek, Michael J. Davidson, Peter B. MacDonald, Judy E. Anderson
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CALL FOR PAPERS Stem Cell Biology

Atrophy, inducible satellite cell activation, and possible denervation of supraspinatus muscle in injured human rotator-cuff muscle

Deanna Gigliotti,1 Jeff R. S. Leiter,2 Bryce Macek,3 Michael J. Davidson,4 Peter B. MacDonald,5 and Judy E. Anderson1 1Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada; 3College of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; 4Department of Radiology, University of Manitoba, Winnipeg, Manitoba,

Canada; 5Section of Orthopedics, Department of Surgery, University of Manitoba, Winnipeg, Manitoba, Canada; and 2Pan

Am Clinic, Winnipeg, Manitoba, Canada

Submitted 20 May 2015; accepted in final form 29 June 2015

Gigliotti D, Leiter JR, Macek B, Davidson MJ, MacDonald PB,

Anderson JE. Atrophy, inducible satellite cell activation, and possible denervation of supraspinatus muscle in injured human rotator-cuff muscle. Am J Physiol Cell Physiol 309: C383–C391, 2015. First published July 1, 2015; doi:10.1152/ajpcell.00143.2015.—The high frequency of poor outcome and chronic pain after surgical repair of shoulder rotator-cuff injury (RCI) prompted this study to explore the potential to amplify muscle regeneration using nitric oxide (NO)based treatment. After preoperative magnetic resonance imaging (MRI), biopsies of supraspinatus and ipsilateral deltoid (as a control) were collected during reparative surgery for RCI. Muscle fiber diameter, the pattern of neuromuscular junctions observed with alphabungarotoxin staining, and the :ε subunit ratio of acetylcholine receptors in Western blots were examined in tandem with experiments to determine the in vitro responsiveness of muscle satellite cells to activation (indicated by uptake of bromodeoxyuridine, BrdU) by the

NO-donor drug, isosorbide dinitrate (ISDN). Consistent with MRI findings of supraspinatus atrophy (reduced occupation ratio and tangent sign), fiber diameter was lower in supraspinatus than in deltoid.

ISDN induced a significant increase over baseline (up to 1.8-fold), in the proportion of BrdU (activated) Pax7 satellite cells in supraspinatus, but not in deltoid, after 40 h in culture. The novel application of denervation indices revealed a trend for supraspinatus muscle to have a higher :ε subunit ratio than deltoid (P  0.13); this ratio inversely with both occupancy ratio (P  0.05) and the proportion of clusters at neuromuscular junctions (P  0.05). Results implicate possible supraspinatus denervation in RCI and suggest NO-donor treatment has potential to promote growth in atrophic supraspinatus muscle after RCI and improve functional outcome.

Pax7; nitric oxide; acetylcholine receptor; supraspinatus; deltoid “ROTATOR CUFF” MUSCLES (supraspinatus, infraspinatus, teres minor, and subscapularis) function to rotate and abduct the arm and stabilize the shoulder joint. Rotator-cuff injury (RCI) from damage to cuff muscles or their tendons may be either chronic and age-related (4, 5) or an acute result of sports injury (23, 29). RCI most commonly affects the supraspinatus, and secondary pathology, including muscle atrophy and fibro-fatty infiltration, results in pain, weakness, loss of function, and biomechanical shoulder-joint instability (3, 12, 15). Most tears require reparative surgery; however, recurrence of tears following surgery is common, with failure rates ranging from 30 to 94% (12).

Regulation of muscle satellite cells (SCs), the stem cells for muscle regeneration and growth, is implicated in the response to and rehabilitation from RCI, since there are reportedly fewer

SCs in RCI muscle (12). The SC population and its responsiveness to an activating stimulus needed for regeneration could potentially be targeted for new treatments to improve recovery, since surgical repair of a torn tendon may not restore joint stability and function.

SC are mitotically quiescent and identifiable by their satellite position between the plasma membrane and myofiber basal lamina (27), and by the proteins they express, including Pax7 (4, 6, 27). Transit of a SC from the quiescent G0 state into the

G1 (activated) stage of the cell cycle is regulated by hepatocyte growth factor (5, 35) and nitric oxide (NO) (2, 3). Declining

SC function can lead to myopathy and accompanies conditions such as muscular dystrophy and age-related atrophy. However, while strategies such as treatment with the NO-donor drug, isosorbide dinitrate (ISDN), have been explored as possible treatment for age-related atrophy and muscular dystrophy (19, 26, 28), the potential of NO-based treatment for RCI has not been explored (3, 26). This study aimed to investigate whether

NO treatment in culture would increase SC activation in pathological supraspinatus compared with uninjured deltoid muscle (as an internal control).

Acetylcholine receptor (AChR) distribution on the muscle fiber membrane and subunit composition reveal the innervation status of muscle, histologically and in protein assays, respectively. During development, before fibers are innervated,

AChRs are arranged in a linear pattern on fibers. With innervation, AChRs cluster (45) at neuromuscular junctions (NMJs).

Five subunits form each receptor, and the identity of one subunit changes during development: specifically, the gamma () subunit, which shows fetal characteristics, transitions to a more efficient adult epsilon (ε) subunit after innervation. After muscle is denervated, both histological and subunit character change: AChR distribution becomes more linear on the sarcolemma, and the AChR subunit pattern reverts to the fetal,  isoform (45). These functional transitions make AChR distribution and composition useful indicators of innervation status.

This study aimed to investigate myogenic and neurogenic aspects of RCI, by examining differences in fiber morphometry

Address for reprint requests and other correspondence: J. E. Anderson,

Univ. of Manitoba, 50 Sifton Rd., Winnipeg, MB, Canada R3T 2N2 (e-mail: