Inhibition of calpain delays early muscle atrophy after rotator cuff tendon release in sheep

Abstract

Chronic rotator cuff (RC) tears are characterized by retraction, fat accumulation, and atrophy of the affected muscle. These features pose an intractable problem for surgical repair and subsequent recovery, and their prevention may be easier than reversal. Using an established ovine model, we tested the hypothesis that inhibition of the protease calpain mitigates m. infraspinatus atrophy by preservation of the myofibers’ structural anchors in the sarcolemma (the costameres). Already 2 weeks of distal tendon release led to a reduction in muscle volume (−11.6 ± 9.1 cm3, P = 0.038) and a 8.3% slow‐to‐fast shift of the fiber area (P = 0.046), which were both entirely abolished by chronic local administration of the calpain inhibitor calpeptin alone, and in combination with sildenafil. Calpain inhibition blunted the retraction of the muscle‐tendon unit by 0.8–1.0 cm (P = 0.020) compared with the control group, and prevented cleavage of the costameric protein talin. Calpain 1 and 2 protein levels increased in the medicated groups after 4 weeks, counteracting the efficacy of calpeptin. Hence atrophic changes emerged after 4 weeks despite ongoing treatment. These findings suggest that the early muscular adaptations in the specific case of RC tear in the ovine model are indistinguishable from the atrophy and slow‐to‐fast fiber transformation observed with conventional unloading and can be prevented for 2 weeks. Concluding, calpain is a potential target to extend the temporal window for reconstruction of the ruptured RC tendon before recovery turns impossible.

Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle

“…5e7 Here we explored the course of alterations in the cellular composition of rotator cuff muscle in sheep during the first 16 weeks after tendon release, and assessed molecular composition at selected time points, in order to identify metabolic processes that are associated with the degradation of muscle fibers and lipid accumulation. 13,15,38 Our multilevel characterization exposed that atrophy and lipid accumulation in sheep infraspinatus muscle after tendon release took place in two different phases. Atrophy was essentially confined to the first 6 weeks after tendon release and was related to a slow-to-fast fiber shift of muscle composition that became manifest as early as 2 weeks after tendon release.…”

“…Aspects of the anatomic data (ie, the muscle volume and histologic examination) that serve as the biological background for this study have been reported previously in other form for the T2e6 and T16 groups. 13,38 Computed tomography and/or magnetic resonance imaging were performed immediately after tendon release and at the end of the experiment, in the upper body of anesthetized sheep, to estimate the volume and compositional alterations in the released infraspinatus muscle and its contralateral control. Additionally, in group T16, magnetic resonance imaging was performed at 6 weeks after tendon release.…”

“…The abundance of selected proteins for gene ontologies, the transcript levels of which were affected by 2 weeks after tendon release, was analyzed by separation of 10 mg of total muscle protein in homogenate by 12% SDS-PAGE, immunoblot analysis with validated antibodies, and enhanced chemiluminescence-based detection, as described. 13,38 Specifically, this process concerned the detection of protein constituents of mitochondrial respiratory chain complexes I to V [I, NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9); II, succinate dehydrogenase complex flavoprotein subunit A (SDHA); III, ubiquinol-cytochrome c reductase core protein 1 (UQCRC1); IV, cytochrome c oxidase subunit 4I1 (COX4I1); V, ATP synthase F1 subunit a (ATP5A1)] through the use of the anti-OxPhos Complex Kit (InvitroGen, Carlsbad, CA), or detection of slow-type myosin heavy chain, fast-type myosin heavy chain with the primary antibodies [catalog number MAB1628 (Merck & Cie, Altdorf, Switzerland); My-32 (Sigma-Aldrich, Buchs, Switzerland)], followed by incubation with horseradish peroxidaseecoupled secondary antimouse IgG (Fab Specific-Peroxidase; Sigma-Aldrich, St. Louis, MO). Equal protein loading and blotting were verified based on Ponceau S staining before the immunodetection.…”

“…13 The staining of slow-and fast-type myosin heavy chain was performed as described. 38 In brief, the thawed sections were fixed in 4% paraformaldehyde, quenched with 0.3% H 2 O 2 , and incubated after serial washes with phosphate-buffered saline in blocking solution of 5% normal goat serum in phosphate-buffered saline. Subsequently, the sections were incubated with 1:100 dilutions of mouse antibody against slow-type myosin heavy chain (catalog number MAB1628; Millipore Corp., Temecula, CA) and rabbit antibody against fast-type myosin heavy chain (catalog number ab91506; Abcam, Cambridge, UK), followed by washes in phosphate-buffered saline and incubation with a 1:200 dilution of immunofluorescence-labeled secondary antibodies (anti-mouse Alexa Fluor 488, catalog number A11017; and anti-rabbit Alexa Fluor 555, catalog number A21428; Thermo Fisher Scientific).…”

“…Both radiologic data sets were evaluated using an open-source DICOM viewer (OsiriX version 5.6, 32-bit; Pixmeo SARL, Bernex, Switzerland) from transverse sections of the shoulders perpendicular to the glenoid cavity essentially as established. 18,38 Magnetic resonance images were evaluated for the determination of fat fraction for the mean over the different muscle regions. Computed tomography scans were inspected for the retraction of infraspinatus muscle based on the distance of the bone chip respective to its original site of insertion, and segmented to reveal the density of muscle tissue from the mean over the different muscle regions.…”