Down-Regulation of Mitochondrial Metabolism after Tendon Release Primes Lipid Accumulation in Rotator Cuff Muscle
2020; Elsevier BV; Volume: 190; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2020.03.019
ISSN1525-2191
AutoresMartin Flück, Daniel P. Fitze, Severin Ruoss, Paola Valdivieso, Brigitte von Rechenberg, Anna Bratus-Neuenschwander, Lennart Opitz, Junmin Hu, Endre Laczkó, Karl Wieser, Christian Gerber,
Tópico(s)Medical research and treatments
ResumoAtrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of β-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration. Atrophy and fat accumulation are debilitating aspects of muscle diseases and are rarely prevented. Using a vertical approach combining anatomic techniques with omics methodology in a tenotomy-induced sheep model of rotator cuff disease, we tested whether mitochondrial dysfunction is implicated in muscle wasting and perturbed lipid metabolism, speculating that both can be prevented by the stimulation of β-oxidation with l-carnitine. The infraspinatus muscle lost 22% of its volume over the first 6 weeks after tenotomy before the area-percentage of lipid increased from 8% to 18% at week 16. Atrophy was associated with the down-regulation of mitochondrial transcripts and protein and a slow-to-fast shift in muscle composition. Correspondingly, amino acid levels were increased 2 weeks after tendon release, when the levels of high-energy phosphates and glycerophospholipids were lowered. l-Carnitine administration (0.9 g/kg per day) prevented atrophy over the first 2 weeks, and mitigated alterations of glutamate, glycerophospholipids, and carnitine levels in released muscle, but did not prevent the level decrease in high-energy phosphates or protein constituents of mitochondrial respiration, promoting the accumulation of longer lipids with an increasing saturation. We conclude that the early phase of infraspinatus muscle degeneration after tendon release involves the elimination of oxidative characteristics associated with an aberrant accumulation of lipid species but is largely unrelated to the prevention of atrophy with oral l-carnitine administration. Rotator cuff disease is a frequent problem, arising subsequent to the distal rupture of a rotator cuff tendon.1Minagawa H. Yamamoto N. Abe H. Fukuda M. 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We reasoned that the underrated apprehension of the role of mitochondrial metabolism in fatty atrophy reflects the fact that molecular aspects of rotator cuff disease are typically assessed with a narrow focus on late time points of the pathology, and in rodent models, which reflect the active course and degree of rotator cuff degeneration in humans only to a limited degree.13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar,34Rowshan K. Hadley S. Pham K. Caiozzo V. Lee T.Q. Gupta R. Development of fatty atrophy after neurologic and rotator cuff injuries in an animal model of rotator cuff pathology.J Bone Joint Surg Am. 2010; 92: 2270-2278Crossref PubMed Scopus (103) Google Scholar,35Liu X. Laron D. Natsuhara K. Manzano G. Kim H.T. Feeley B.T. 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Effect of tendon release and delayed repair on the structure of the muscles of the rotator cuff: an experimental study in sheep.J Bone Joint Surg Am. 2004; 86-A: 1973-1982Crossref PubMed Scopus (262) Google Scholar identifying that mitochondrial volume density per muscle fiber in sheep infraspinatus muscle is not affected when a massive accumulation of fat is established. The purpose of this study was therefore to investigate the early metabolic processes that precede, and lead to, fatty atrophy of rotator cuff muscle after experimental release of its tendon in sheep,13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar,37Gerber C. Meyer D.C. Schneeberger A.G. Hoppeler H. von Rechenberg B. Effect of tendon release and delayed repair on the structure of the muscles of the rotator cuff: an experimental study in sheep.J Bone Joint Surg Am. 2004; 86-A: 1973-1982Crossref PubMed Scopus (262) Google Scholar with an explorative multilevel approach. In a first hypothesis-generating part, taking the possible main role of mitochondrial processes into consideration, we addressed the extent to which global alterations of mitochondria-related transcript expression, and metabolite and lipid levels, take place in rotator cuff muscle within the early phase of response to tendon release, before irreversible alterations manifest at the macroscopic level. Subsequently the extent to which nutraceutical treatment with l-carnitine would halt early signs of muscle degeneration was assessed. Animal experiments were performed according to Swiss law on animal welfare (TSch455) and with approval of the Veterinary Office of the Canton (Zurich, Switzerland; protocols ZH219/2014 and ZH72/2013). Female Swiss Alpine sheep (Ovis aries) were subjected to release of the infraspinatus tendon for 2, 6, and 16 weeks, for: (1) characterization of the course of infraspinatus muscle degeneration based on measurements of molecular parameters (emphasis on mitochondrial metabolism) and cellular parameters on collected bioptic material and radiologic imaging; and (2) study of the effects of the oral administration of l-carnitine (Supplemental Figure S1). Six sheep per group were studied at 2 weeks (T2 group; means ± SEM age and weight, 16.6 ± 0.0 months and 59.7 ± 2.5 kg), 2 to 6 weeks (T2–6 group; means ± SEM age and weight, 26.7 ± 0.6 months and 63.3 ± 1.4 kg), and 16 weeks (T16; means ± SEM age and weight, 23.2 ± 1.2 months and 45.3 ± 4.8 kg) after tendon release. l-Carnitine was administered starting 1 week prior to tendon release in another group of sheep (T2-C group; 29.6 ± 0.3 months and 59.9 ± 2.4 kg; n = 3). 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 T2–6 and T16 groups.13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar,38Ruoss S. Kindt P. Oberholzer L. Rohner M. Jungck L. Abdel-Aziz S. Fitze D. Rosskopf A. Klein K. von Rechenberg B. Gerber C. Wieser K. Flück M. Inhibition of calpain delays early muscle atrophy after rotator cuff tendon release in sheep.Physiol Rep. 2018; 6: e13833Crossref PubMed Scopus (7) Google Scholar 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. Biopsy samples (approximately 50 mg) were collected intraoperatively with 5-mm Bergstrom needles (Dixon instruments, Wickford, UK) from the lateral aspect within the belly of the operated infraspinatus muscle immediately before tendon release (pre), and from the released muscle and its contralateral control at the end of the experiment; samples were frozen in melting isopentane. Sites being selected for repeated biopsy sampling were separated by at least 2 cm from the previous collection site. This distance was ensured by the insertion of a suture in the fascia above the site selected for the previous biopsy sampling. The sampled tissue was stored at −80°C until subjected to histologic, transcriptomic, and protein analysis. Additional tissue pieces were extracted with a scalpel (approximately 500 mg) from the lateral aspect within the belly of the released and contralateral infraspinatus muscle that were excised and frozen at the end of the experiment. These samples were used for the metabolomic/lipidomic measurements. The right shoulder of each sheep was subjected to release of the infraspinatus tendon by osteotomy of the greater tuberosity, as described.13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar Animals were housed in standard housing, monitored for health, and medicated to control eventual pain and discomfort. l-Carnitine (Carnipure tartrate; Lonza, Basel, Switzerland) was dissolved in tap water and administered with the help of a syringe at a daily dose of 0.9 g/kg. The dose applied corresponded to the dose of the related compound, acetyl-l-carnitine, effective in reducing the degeneration of slow oxidative muscle fibers with 2 weeks of disuse.27Cassano P. Fluck M. Giovanna Sciancalepore A. Pesce V. Calvani M. Hoppeler H. Cantatore P. Gadaleta M.N. Muscle unloading potentiates the effects of acetyl-L carnitine on the slow oxidative muscle phenotype.Biofactors. 2010; 36: 70-77PubMed Google Scholar During the days after tendon release, animals were confined to a quiet area with limited space for gait activities. At the end of the experiment, euthanasia was performed. Gene expression was analyzed by RNA sequencing and expressed as a concentration per biopsy volume essentially as described.13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar In brief, total RNA was isolated from 15 mm3 of cryosections for three biological replicas for the pre and post time points from the T2 and T16 groups using the RNeasy Kit (Qiagen, Basel, Switzerland) and verified for quantity and quality using the Qubit 1.0 RNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA) and Agilent RNA 6000 Nano Kit with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. RNA libraries were prepared from 1 μg of total RNA using the Illumina TruSeq Stranded total RNA Sample Preparation Kit (Illumina, San Diego, CA); quantity and quality of the libraries were checked using quantitative PCR (Roche, Basel, Switzerland) with Illumina adapter-specific primers and Agilent 2200 TapeStation, respectively. Diluted indexed libraries (10 nmol/L) were pooled and used for cluster generation according to the manufacturer's recommendations using the Illumina TruSeq SR Cluster Kit v3-cBot-HS reagents and sequenced on Illumina HiSeq 2500 [single-read approach (1 × 100 bp) with 20 to 30 million reads per sample]. Raw reads were cleaned by removing adapter sequences and the first three and last four bases. The resulting reads were aligned, that is, annotated, respective to the current build of the sheep genome (Oar version 3.1.75; Ensembl, https://useast.ensembl.org, last accessed September 9, 2019) using STAR aligner software version 2.3.0e.39Dobin A. Davis C.A. Schlesinger F. Drenkow J. Zaleski C. Jha S. Batut P. Chaisson M. Gingeras T.R. STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013; 29: 15-21Crossref PubMed Scopus (19483) Google Scholar RNA signals were normalized to the total RNA signal intensity per analyzed sample, and multiplied with the amount of RNA being analyzed per muscle biopsy (ie, concentration), to reveal relative concentrations per species. Data sets were deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo; accession number GSE144394). 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 μg of total muscle protein in homogenate by 12% SDS-PAGE, immunoblot analysis with validated antibodies, and enhanced chemiluminescence-based detection, as described.13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar,38Ruoss S. Kindt P. Oberholzer L. Rohner M. Jungck L. Abdel-Aziz S. Fitze D. Rosskopf A. Klein K. von Rechenberg B. Gerber C. Wieser K. Flück M. Inhibition of calpain delays early muscle atrophy after rotator cuff tendon release in sheep.Physiol Rep. 2018; 6: e13833Crossref PubMed Scopus (7) Google Scholar 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 α (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 peroxidase–coupled secondary anti-mouse IgG (Fab Specific-Peroxidase; Sigma-Aldrich, St. Louis, MO). Equal protein loading and blotting were verified based on Ponceau S staining before the immunodetection. Experimental samples from the same sheep, and an internal standard consisting of the pre sample of one sheep, were loaded on the same SDS-PAGE. Protein signals were background-corrected, and related to the signal of the internal standard to reveal relative protein levels per total protein. Combined protein levels were calculated as the means of the relative values of the assessed proteins in each sample. Nonpolar and polar compounds were extracted from 10 mm3 of biopsy material from infraspinatus muscle in cold methanol/methyl tert-butyl ether/H2O 360:1200:348 vol/vol/vol using a full glass Potter-type homogenizer and subjected to ultrahigh-performance liquid chromatography–tandem mass spectrometry essentially as described.13Fluck M. Ruoss S. Mohl C.B. Valdivieso P. Benn M.C. von Rechenberg B. Laczko E. Hu J. Wieser K. Meyer D.C. Gerber C. Genomic and lipidomic actions of nandrolone on detached rotator cuff muscle in sheep.J Steroid Biochem Mol Biol. 2017; 165: 382-395Crossref PubMed Scopus (12) Google Scholar The nonpolar phase and the polar phase, containing the majority of lipids and metabolites, respectively, were stored at −30°C until being dried down and reconstituted for separation via ultra performance liquid chromatography. Lipids were separated with a C18 reversed-phase column (HSS T3; 1.7 mm external diameter, 0.2 × 150 mm) by a NanoAquity ultra performance liquid chromatography (Waters, Baden-Dättwil, Switzerland), with the application of a gradient of 5 μmol/L ammonium acetate in acetonitrile/water 40:60 vol/vol (A) and 5 μmol/L ammonium acetate in acetonitrile/isopropanol 10:90 vol/vol. The eluate was injected at 6-second intervals into a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Reinach, Switzerland) by a nano-ESI source that was operated in positive-polarization mode. Metabolites were separated on the NanoAquity UPLC system (Waters) equipped with a BEH-Amide capillary column (200 μm × 150 mm, 1.7-μm particle size; Waters), applying a gradient of 0.5 μmol/L ammonium acetate in acetonitrile (A) and 0.5 μmol/L ammonium acetate in water (B) from 90% to 50% A and injected into a Q Exactive H
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