Impaired contractile function of the supraspinatus in the acute period following a rotator cuff tear

Abstract

Background: Rotator cuff (RTC) tears are a common clinical problem resulting in adverse changes to the muscle, but there is limited information comparing histopathology to contractile function. This study assessed supraspinatus force and susceptibility to injury in the rat model of RTC tear, and compared these functional changes to histopathology of the muscle.

Methods: Unilateral RTC tears were induced in male rats via tenotomy of the supraspinatus and infraspinatus. Maximal tetanic force and susceptibility to injury of the supraspinatus muscle were measured in vivo at day 2 and day 15 after tenotomy. Supraspinatus muscles were weighed and harvested for histologic analysis of the neuromuscular junction (NMJ), intramuscular lipid, and collagen.

Results: Tenotomy resulted in eventual atrophy and weakness. Despite no loss in muscle mass at day 2 there was a 30% reduction in contractile force, and a decrease in NMJ continuity and size. Reduced force persisted at day 15, a time point when muscle atrophy was evident but NMJ morphology was restored. At day 15, torn muscles had decreased collagen-packing density and were also more susceptible to contraction-induced injury.

Conclusion: Muscle size and histopathology are not direct indicators of overall RTC contractile health. Changes in NMJ morphology and collagen organization were associated with changes in contractile function and thus may play a role in response to injury. Although our findings are limited to the acute phase after a RTC tear, the most salient finding is that RTC tenotomy results in increased susceptibility to injury of the supraspinatus.

Keywords: Collagen organization; Contractility; Eccentric injury; Muscle force; Neuromuscular junction; Rat.

Fig. 1

Supraspinatus tendon in a model of a RTC tear. a Top panel . Normal anatomy of the rat RTC (used as control) is shown, with the supraspinatus muscle (SS) and infraspinatus muscle (IS), including attachment of their tendons to the greater tubercle of the humerus (yellow circle). RTC tear was surgically induced by tenotomizing the supraspinatus and infraspinatus tendons. Bottom panel . After 2 days (2D), the RTC tear results in retraction of the tendons. Fifteen days after RTC tear (15D), the space between the muscle tendon and insertion site is filled by a fibrous-connective tissue (arrow) that reattaches the supraspinatus to the humeral head (inset). b As expected, supraspinatus muscle mass was slightly altered after tenotomy and significantly reduced by day 15. c Western blot analysis was used to detect ubiquitinated proteins in total protein extracts of supraspinatus muscles. Equal amounts of protein were loaded and confirmed with Ponceau and probed with anti-ubiquitin antibody. Total protein ubiquitination was upregulated at 2 and 15 days after tenotomy in supraspinatus muscle compared to control (CTRL). All data are presented as mean ± SD, p < 0.05. *, indicates statistical significance compared to control

Fig. 2

Maximal isometric force is lower in tenotomized supraspinatus at 2D and 15D. a Apparatus to measure muscle in vivo contractility and susceptibility to injury in the supraspinatus muscle. The insertion of the supraspinatus was released and the tendon tied to a load cell. The suprascapular nerve was stimulated via subcutaneous needle electrodes to activate the supraspinatus maximally. A series of maximal twitches was used to determine optimal length (L_o) and the force-frequency relationship was determined to obtain maximal isometric force. b When compared to control, the mean of maximal isometric force per group was 30% lower at 2D and 20% lower at 15D. Maximal force was not different between the tenotomized groups. All data are presented as mean ± SD, p < 0.05. *, indicates statistical significance compared to control

Fig. 3

NMJ morphology is altered in tenotomized supraspinatus at 2D, but recovers at 15D. Neuromuscular junctions (NMJs) of at least three supraspinatus muscles per group were fluorescently stained with an acetylcholine receptor binding neurotoxin (α-Bungarotoxin, BTX, green) and imaged using confocal microscopy. Z-stacked images were analyzed and quantified using ImageJ software. Skeletonized images are shown in the white panel for each NMJ to further illustrate continuity and branching of NMJs. At 2D, NMJs were smaller and morphology was altered, as evidenced by decreased continuity of NMJ branches. No significant differences were seen in NMJ morphology compared to control at 15D. Scale bar represents 10 μm. All data are presented as mean ± SD, p < 0.05. *, indicates statistical significance compared to control, and ŧ indicates statistical significance compared to 2D

Fig. 4

Lipid content is not altered in tenotomized supraspinatus at 2D or 15D. a Axial sections of in vivo magnetic resonance imaging (MRI) of a rat 15 days after unilateral RTC tear. When fat suppression is turned off, the white signal represents fat (note the obvious white signal from subcutaneous fat, red arrow). Fat was not detected in any axial sections in the torn supraspinatus (outlined by white dotted line). A longer (> 2 h), more detailed MRI scan of supraspinatus muscles ex vivo corroborated this finding. b Neutral lipids were stained in cross-sections from the mid-belly of the supraspinatus using Bodipy-493/503 and quantified (c) using ImageJ. No differences in intramyocellular lipid at 2D and 15D were found compared to controls. Scale bar represents 50 μm. All data are presented as mean ± SD, p < 0.05

Fig. 5

Collagen organization is altered in tenotomized supraspinatus at 15D. a Representative images from the mid-belly of muscle cross-sections stained with Picosirius red viewed under brightfield microscopy. Collagen content was visualized using Picosirius red staining, and quantified using a threshold on ImageJ to calculate the percentage of pixels stained per area. No differences in total collagen content are evident between groups. b Collagen organization was assessed using Picosirius red-stained sections viewed under polarized light. Red represents more densely packed collagen that is perpendicular to muscle fibers, and green represents loosely packed collagen that is parallel to the fibers. When analyzed as a proportion to total colored pixels, supraspinatus muscle at 15D has a lower proportion of red pixels and greater proportion of yellow and green pixels compared to control, indicating altered collagen organization (decreased collagen density, crosslinking, and thickness) at 15D after tear. No differences were evident at 2D compared to control. Scale bar represents 50 μm. All data are presented as mean ± SD, p < 0.05. *, indicates statistical significance compared to control

Fig. 6

Tenotomized supraspinatus becomes more susceptible to injury at 15D but not at 2D. The same apparatus used to collect isometric force (Fig. 2) was also used to induce injury. The suprascapular nerve is used to stimulate the supraspinatus maximally while movement of the lever arm resulted in forced linear lengthening of the muscle (15% L_o). a Representative screen shot showing force from a single eccentric contraction (closed arrow) superimposed onto a maximal isometric contraction (open arrow; y-axis in volts, which is converted to units of force based on calibration). b Representative rep-by-rep isometric force loss for one animal in each group throughout eccentric contraction protocol. c The mean loss of force for each group after the injury protocol. Despite an identical injury protocol, there is a greater drop in isometric force after injury at 15D (51.8 ± 2.5%) compared to control. All data are presented as mean ± SD, p < 0.05. *, indicates statistical significance compared to control