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Reduced Myogenic and Increased Adipogenic Differentiation Capacity of Rotator Cuff Muscle Stem Cells

Schubert, Manuel F., MD, MS1; Noah, Andrew C., MS1; Bedi, Asheesh, MD1; Gumucio, Jonathan P., PhD1; Mendias, Christopher L., PhD, ATC1,2,3

doi: 10.2106/JBJS.18.00509
Scientific Articles
Supplementary Content 1
Supplementary Content 2
Supplementary Content 3
Disclosures

Background: Fat accumulation commonly occurs in chronically torn rotator cuff muscles, and increased fat within the rotator cuff is correlated with poor clinical outcomes. The extent of lipid deposition is particularly pronounced in injured rotator cuff muscles compared with other commonly injured muscles such as the gastrocnemius. Satellite cells, which are a tissue-resident muscle stem-cell population, can differentiate into fat cells. We hypothesized that satellite cells from the rotator cuff have greater intrinsic adipogenic differentiation potential than do gastrocnemius satellite cells, and this difference is due to variations in epigenetic imprinting between the cells.

Methods: Satellite cells from gastrocnemius and rotator cuff muscles of mice were cultured in adipogenic media, and the capacity to differentiate into mature muscle cells and adipogenic cells was assessed (n ≥ 9 plates per muscle group). We also performed DNA methylation analysis of gastrocnemius and rotator cuff satellite cells to determine whether epigenetic differences were present between the 2 groups (n = 5 mice per group).

Results: Compared with the gastrocnemius, satellite cells from the rotator cuff had a 23% reduction in myogenic differentiation and an 87% decrease in the expression of the differentiated muscle cell marker MRF4 (myogenic regulatory factor 4). With respect to adipogenesis, rotator cuff satellite cells had a 4.3-fold increase in adipogenesis, a 12-fold increase in the adipogenic transcription factor PPARγ (peroxisome proliferator-activated receptor gamma), and a 65-fold increase in the adipogenic marker FABP4 (fatty-acid binding protein 4). Epigenetic analysis identified 355 differentially methylated regions of DNA between rotator cuff and gastrocnemius satellite cells, and pathway enrichment analysis suggested that these regions were involved with lipid metabolism and adipogenesis.

Conclusions: Satellite cells from rotator cuff muscles have reduced myogenic and increased adipogenic differentiation potential compared with gastrocnemius muscles. There appears to be a cellular and genetic basis behind the generally poor rates of rotator cuff muscle healing.

Clinical Relevance: The reduced myogenic and increased adipogenic capacity of rotator cuff satellite cells is consistent with the increased fat content and poor muscle healing rates often observed for chronically torn rotator cuff muscles. For patients undergoing rotator cuff repair, transplantation of autologous satellite cells from other muscles less prone to fatty infiltration may improve clinical outcomes.

1Departments of Orthopaedic Surgery (M.F.S., A.C.N., A.B, J.P.G, and C.L.M.) and Molecular and Integrative Physiology (A.C.N, J.P.G., and C.L.M.), University of Michigan Medical School, Ann Arbor, Michigan

2Hospital for Special Surgery, New York, NY

3Departments of Physiology and Biophysics and Orthopaedic Surgery, Weill Cornell Medical College, New York, NY

E-mail address for C.L. Mendias: MendiasC@hss.edu

Investigation performed at the University of Michigan Medical School, Ann Arbor, Michigan

Disclosure: Funding support for this study was provided by the Orthopaedic Research and Education Foundation and NIH grant F31-AR065931. On the Disclosure of Potential Conflicts of Interest forms, which are provided with the online version of the article, one or more of the authors checked “yes” to indicate that the author had a relevant financial relationship in the biomedical arena outside the submitted work and “yes” to indicate that the author had other relationships or activities that could be perceived to influence, or have the potential to influence, what was written in this work (http://links.lww.com/JBJS/F34).

Rotator cuff tears are a common upper-extremity disorder, with >250,000 surgical repairs performed annually in the U.S.1. Achieving positive clinical outcomes following repair can be limited by fatty infiltration or myosteatosis, which is the combined atrophy, fibrosis, and fat accumulation within and around myofibers2-4. The relative amount of fat in torn rotator cuff muscles is often greater than in other injured muscle groups5-7, and many patients develop further myosteatosis even after undergoing a successful rotator cuff repair2. Fat accumulation is also correlated with negative clinical outcomes2,8, and identifying the cellular and molecular mechanisms that induce adipogenesis in the rotator cuff could provide new opportunities for improving muscle healing and recovery.

Satellite cells are a heterogenous stem-cell population largely responsible for postnatal skeletal muscle growth, regeneration, and repair, and they are stimulated by trauma to proliferate, differentiate, and fuse into damaged myofibers9,10. Quiescent satellite cells are found between the muscle fibers and basal lamina and express the transcription factor Pax7 (paired box 7)10,11. In addition to myofibers, satellite cells can also differentiate into adipocytes12-14, and in a tenotomy and denervation muscle-injury model, greater fat accumulation and reduced healing were present in rotator cuff muscles compared with gastrocnemius muscles7. Because satellite cells are important in muscle regeneration and can enter the adipogenic lineage, we sought to determine differences in the myogenic and adipogenic differentiation capacity of satellite cells from gastrocnemius and rotator cuff muscles. We tested the hypothesis that, compared with gastrocnemius satellite cells, rotator cuff satellite cells have decreased myogenic and increased adipogenic differentiation capacity. To further explore mechanistic differences, as satellite cell activity can be regulated by epigenetic factors11, we analyzed differential DNA methylation between gastrocnemius and rotator cuff satellite cells.

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Materials and Methods

Animals

This study was approved by the University of Michigan Institutional Animal Care and Use Committee. We crossed 2 lines of genetically modified mice, obtained from The Jackson Laboratory, to generate experimental animals. First, we obtained Pax7 CreERT2 mice, which contain an IRES (internal ribosome entry site)-CreERT2 cassette between the stop codon and 3′ untranslated region of Pax7, resulting in the expression of a tamoxifen-responsive CreERT2 recombinase enzyme when Pax7 is also expressed (strain 017763)15. In the second line of mice, R26R flox-stop-tdTomato , the constitutively expressed Rosa26 (R26R) locus was modified to contain a stop codon cassette flanked by loxP sites upstream of the red fluorescent tdTomato gene (strain 007909)16. In the absence of active Cre recombinase, R26R flox-stop-tdTomato mice do not express tdTomato. However, upon treatment with tamoxifen, which activates the Cre enzyme, a recombination event occurs between loxP sites to remove the stop codons, resulting in the permanent expression of tdTomato, R26R tdTomato . We crossed Pax7 CreERT2 and R26R flox-stop-tdTomato mice to generate Pax7 CreERT2 :R26R flox-stop-tdTomato mice, which were backcrossed for several generations and maintained in the homozygous state. Quiescent satellite cells express Pax715, and upon treatment with tamoxifen, the CreERT2 enzyme complex is activated, causing a recombination event at the R26R locus, resulting in the persistent expression of tdTomato in all cells expressing Pax7, as well as their daughter cells17, which are referred to as Pax7 CreERT2 :R26R tdTomato mice. An overview is presented in Figure 1-A. With the exception of wild-type C57Bl/6 mice that were used to determine baseline flow cytometry fluorescence, all experiments utilized 4-month-old male Pax7 CreERT2 :R26R tdTomato mice.

Fig. 1

Fig. 1

The labeling of satellite cells with tdTomato occurred by treating mice with an intraperitoneal injection of tamoxifen (2 mg) dissolved in corn oil (Sigma) daily for 5 days prior to muscle harvest. On the sixth day, mice were anesthetized with ketamine and xylazine. The supraspinatus and infraspinatus muscles of the rotator cuff and the gastrocnemius muscles were removed and processed for flow cytometry. The plantaris muscle was also removed as a sentinel to verify recombination and labeling of satellite cells. Following muscle removal, mice were killed by cervical dislocation and pneumothorax.

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Muscle Histology

Plantaris muscles were frozen in Tissue-Tek (Sakura) with cold isopentane, and 10-µm sections were incubated with wheat germ agglutinin (WGA) lectin conjugated to Alexa Fluor 488 dye (ThermoFisher Scientific) to identify extracellular matrix. DAPI (4′,6-diamidino-2-phenylindole; Sigma)-labeled nuclei and tdTomato-identified satellite cells. Sections were visualized with a BX51 microscope (Olympus).

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Satellite Cell Isolation and Flow Cytometry

Satellite cells were isolated from gastrocnemius and rotator cuff muscles, using a method modified from a previous report18. A detailed description is provided in the Appendix. Briefly, muscles were minced and digested to generate a suspension of cells enriched with satellite cells. Cells were then sorted via flow cytometry on the basis of forward scatter area (FSC-A) as a means to measure the cell size and tdTomato fluorescence. TdTomato+ cells were collected and used for in vitro differentiation experiments or DNA sequencing.

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Cell Culture

A detailed description of cell culture is provided in the Appendix. Sorted cells were plated on culture dishes coated with growth factor-reduced Matrigel (Corning). Cells were expanded in growth media containing Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AbAm) (ThermoFisher Scientific) for 2 passages, and on reaching 70% confluence, were switched to adipogenic induction media for a period of 7 days prior to immunocytochemistry and gene-expression analysis14,19.

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Immunocytochemistry

Cells were fixed with 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and blocked in 5% goat serum. Cells were labeled with antibodies against myogenin (F5D, 1:100; Developmental Studies Hybridoma Bank), which is a transcription factor that identifies differentiated muscle cells20,21, or with antibodies against fatty-acid binding protein 4 (FABP4, 1:500; AbCam), which is specifically expressed in adipogenic cells22. Secondary antibodies conjugated to Alexa Fluor 647 dye (ThermoFisher Scientific) detected primary antibodies. DAPI identified nuclei. Three random 10× fields per plate were quantified in an EVOS FL Imaging System (ThermoFisher Scientific). For myogenic differentiation, a cell was considered a differentiated muscle cell if it was a multinuclear myotube or contained a nucleus that was myogenin+, and myogenic cells were calculated as a percentage of total cells. For adipogenic quantification, the number of FABP4+ cells per field was calculated as a percentage of total cells.

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Gene Expression

Gene-expression analysis was performed as previously described23. RNA was isolated from cells using an miRNEasy kit (QIAGEN), reversed transcribed with iScript reagents (Bio-Rad), and amplified in a CFX96 real-time thermal cycler (Bio-Rad). Quantitative polymerase chain reaction (PCR) was performed using iTaq SYBR Green Supermix (Bio-Rad). A list of primers is shown in Appendix Table E-1. Target gene expression was normalized to the stable housekeeping gene β2-microglobulin using the 2−ΔCt method.

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DNA Methylation Analysis

DNA was isolated from freshly sorted tdTomato+ satellite cells, and DNA methylation analysis was performed by the University of Michigan Epigenomics Core using enhanced reduced representation bisulfite sequencing (ERRBS), as previously described24,25. A detailed description is provided in the Appendix. Briefly, genomic DNA was digested with the methylation-insensitive restriction enzyme MspI, followed by end-repair, A-tailing, and ligation of methylated adapters. Bisulfite conversion of methylated sequences was performed prior to PCR amplification and subsequent sequencing. Gene enrichment and pathway analysis was performed with iPathwayGuide (Advaita Bioinformatics)26. A full list of differentially methylated regions is provided in Appendix Table E-2 and differentially methylated cytosines, in Appendix Table E-3.

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Statistical Methods

Data are presented as the mean and standard deviation. Sample sizes were selected on the basis of satellite-cell myogenic differentiation rates21. In immunocytochemistry and gene-expression experiments, differences between gastrocnemius and rotator cuff groups were assessed using a t test (α = 0.05), with Welch correction, in Prism 7.0 (GraphPad). MethylSig R package (The Sartor Lab, University of Michigan)27 was used for differential DNA methylation analysis using a beta-binomial approach, with p values adjusted for multiple testing using a false discovery rate (FDR) adjustment to control for type-I errors. Sites were considered differentially methylated if they had a percent change in methylation of at least 20% and an FDR-adjusted p value of <0.05.

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Results

To efficiently isolate satellite cells from gastrocnemius and rotator cuff muscles, and to verify that adipogenic markers were observed in myogenic lineage cells, we used a genetic approach to label the satellite cells of Pax7 CreERT2 :R26R tdTomato mice with tdTomato (Fig. 1-B). Across several runs, approximately 12% of cells isolated from both muscle groups were tdTomato+ (Fig. 1-C). We then evaluated the potential of satellite cells from the gastrocnemius and rotator cuff muscles to differentiate into myogenic and adipogenic cells (n ≥ 9 plates analyzed per group). There was a 23% reduction in the number of differentiated rotator cuff muscle cells per field compared with gastrocnemius cells, and a 4.3-fold increase in the number of adipogenic rotator cuff cells per field (Figs. 2-A through 2-D). Next, we measured the expression of myogenesis and adipogenesis-related genes (n ≥ 6 plates analyzed per group). While we did not see a difference between the rotator cuff and gastrocnemius groups in the early myogenic marker desmin or the muscle-fusion gene myomaker, we did see an 87% reduction in the expression of the late muscle differentiation marker MRF4 (myogenic regulatory factor 4) in rotator cuff cells (Fig. 3-A). For genes related to adipogenesis, we observed a 12-fold increase in the adipogenic transcription factor PPARγ (peroxisome proliferator-activated receptor gamma) and a 65-fold increase in FABP4 in rotator cuff cells, although no differences were observed between the rotator cuff and gastrocnemius muscle groups with respect to the adipocyte signaling molecule adiponectin or adipogenic transcription factor C/EBPα (CCAAT-enhancer-binding protein-alpha) (Fig. 3-B).

Fig. 2

Fig. 2

Fig. 3

Fig. 3

Because there was an increased capacity for adipogenic differentiation in rotator cuff cells, we sought to determine whether epigenetic differences existed between sorted gastrocnemius and rotator cuff satellite cells (Figs. 4-A and 4-B; n = 5 mice per group). We identified 180 hypomethylated regions and 175 hypermethylated regions in satellite cells from the rotator cuff compared with cells from the gastrocnemius (Fig. 4-C). The top 50 hypermethylated regions and hypomethylated regions are shown in Tables I and II. Finally, to identify biological processes and biochemical pathways that might be impacted by the difference in DNA methylation, we performed gene ontology analysis. For biological processes, the top 15 pathways identified were related to embryonic development and limb morphogenesis (Fig. 4-D). With regard to molecular function, the top pathways were related to transcription-factor activity and lipid metabolism, which is consistent with the in vitro findings related to adipogenesis.

Fig. 4

Fig. 4

TABLE I - Top 50 Differentially Hypermethylated Regions of DNA in Satellite Cells from the Rotator Cuff Compared with the Gastrocnemius
ID Symbol Gene Name Methylation Difference (%) Distance (bp) P Value
NM_001113412 Fggy FGGY carbohydrate kinase domain containing 99.03 177,851 0.035
NR_046076 Gm4251 Predicted gene 4251 82.28 33,232 0.038
NM_025642 Mis18a MIS18 kinetochore protein A 70.18 45,772 0.004
NM_025642 Mis18a MIS18 kinetochore protein A 67.24 45,822 0.021
NM_029441 Cdyl2 Chromodomain protein, Y chromosome-like 2 60.33 183,549 0.007
NM_013813 Epb41l3 Erythrocyte membrane protein band 4.1 like 3 56.46 196,484 0.029
NM_198610 Igsf21 Immunoglobulin superfamily, member 21 55.18 0 0.001
NM_001286033 Stx2 Syntaxin 2 55.06 280,368 <0.001
NM_001286033 Stx2 Syntaxin 2 54.42 280,443 0.008
NM_015803 Atp8a2 ATPase, aminophospholipid transporter-like, class I, type 8A, member 2 54.25 48,132 0.040
NM_001033228 Itga1 Integrin alpha 1 53.62 245,091 0.040
NM_015803 Atp8a2 ATPase, aminophospholipid transporter-like, class I, type 8A, member 2 52.84 48,082 0.018
NM_001033228 Itga1 Integrin alpha 1 52.23 245,041 0.001
NM_029441 Cdyl2 Chromodomain protein, Y chromosome-like 2 51.93 183,449 0.005
NR_024085 BC006965 cDNA sequence BC006965 51.80 1,001,517 0.003
NM_029441 Cdyl2 Chromodomain protein, Y chromosome-like 2 50.96 183,424 0.038
NR_030709 Gm16386 Predicted gene 16386 50.32 41,229 0.009
NR_015496 1700031M16Rik RIKEN cDNA 1700031M16 gene 48.16 9,442 0.021
NM_152895 Kdm5b Lysine (K)-specific demethylase 5B 47.71 0 0.004
NM_152895 Kdm5b Lysine (K)-specific demethylase 5B 46.90 0 0.012
NM_198610 Igsf21 Immunoglobulin superfamily, member 21 46.79 0 0.004
NM_009723 Atp2b2 ATPase, Ca++ transporting, plasma membrane 2 46.16 0 0.015
NR_015496 1700031M16Rik RIKEN cDNA 1700031M16 gene 46.08 9,267 0.002
NR_015496 1700031M16Rik RIKEN cDNA 1700031M16 gene 46.07 9,292 0.015
NM_010462 Hoxc10 Homeobox C10 45.99 717 <0.001
NM_010462 Hoxc10 Homeobox C10 45.53 692 <0.001
NR_131145 Gm29683 Predicted gene 29683 43.80 351,254 <0.001
NM_177544 Ang4 Angiogenin, ribonuclease A family, member 4 43.46 20,976 <0.001
NM_180662 Trappc9 Trafficking protein particle complex 9 43.22 0 0.029
NR_102286 4933432K03Rik RIKEN cDNA 4933432K03 gene 43.10 37,697 0.007
NM_009834 Noct Nocturnin 42.86 19,402 0.005
NM_172462 Zfp11 Zinc finger protein 11 42.51 187,078 0.015
NR_131145 Gm29683 Predicted gene 29683 42.34 351,279 0.011
NR_102286 4933432K03Rik RIKEN cDNA 4933432K03 gene 42.18 37,647 0.009
NM_177544 Ang4 Angiogenin, ribonuclease A family, member 4 42.14 20,901 0.001
NR_045702 AW549542 Expressed sequence AW549542 42.10 21,482 0.049
NM_009834 Noct Nocturnin 42.05 19,377 0.026
NM_001310738 Siglech Sialic acid binding Ig-like lectin H 41.27 26,345 0.001
NR_035497 Mir1970 MicroRNA 1970 41.20 0 0.016
NM_180662 Trappc9 Trafficking protein particle complex 9 40.80 0 <0.001
NM_001029872 Itgad Integrin, alpha D 40.64 22,757 0.001
NM_009834 Noct Nocturnin 40.48 19,327 0.042
NM_001042617 Cadps Ca2+-dependent secretion activator 40.33 0 0.003
NM_009309 T Brachyury, T-box transcription factor T 40.30 0 0.008
NM_027188 Smyd3 SET and MYND domain containing 3 39.79 0 0.010
NM_009309 T Brachyury, T-box transcription factor T 39.59 0 0.033
NM_015764 Greb1 Gene regulated by estrogen in breast cancer protein 39.20 0 0.005
NR_038085 Six3os1 SIX homeobox 3, opposite strand 1 38.87 66,806 0.013
NR_045342 4933411E08Rik RIKEN cDNA 4933411E08 gene 38.21 0 0.020
NM_013723 Podxl Podocalyxin-like 38.01 246,814 0.003

TABLE II - Top 50 Differentially Hypomethylated Regions of DNA in Satellite Cells from the Rotator Cuff Compared with the Gastrocnemius
ID Symbol Gene Name Methylation Difference (%) Distance (bp) P Value
NR_047528 Hotair HOX transcript antisense RNA −65.29 5,258 0.005
NR_047528 Hotair HOX transcript antisense RNA −64.92 5,283 0.001
NR_047528 Hotair HOX transcript antisense RNA −64.29 5,308 0.001
NM_010465 Hoxc6 Homeobox C6 −61.24 12,733 0.003
NM_010465 Hoxc6 Homeobox C6 −61.14 12,808 <0.001
NR_027899 Hoxd3os1 Homeobox D3, opposite strand 1 −61.05 13,110 <0.001
NM_010465 Hoxc6 Homeobox C6 −61.03 12,833 <0.001
NM_010465 Hoxc6 Homeobox C6 −60.90 12,858 <0.001
NR_027899 Hoxd3os1 Homeobox D3, opposite strand 1 −60.74 13,135 <0.001
NM_010465 Hoxc6 Homeobox C6 −60.72 12,883 0.002
NR_027899 Hoxd3os1 Homeobox D3, opposite strand 1 −59.88 13,060 0.005
NM_010466 Hoxc8 Homeobox C8 −58.96 0 0.031
NR_027899 Hoxd3os1 Homeobox D3, opposite strand 1 −58.94 13,185 <0.001
NR_037977 Gm53 Predicted gene 53 −57.17 0 <0.001
NM_010452 Hoxa3 Homeobox A3 −56.94 0 0.015
NM_010466 Hoxc8 Homeobox C8 −56.17 0 0.005
NR_027899 Hoxd3os1 Homeobox D3, opposite strand 1 −55.79 13,235 0.002
NM_172839 Ccnj Cyclin J −54.26 0 0.015
NM_010451 Hoxa2 Homeobox A2 −51.90 0 0.033
NM_010466 Hoxc8 Homeobox C8 −51.73 0 0.003
NM_010451 Hoxa2 Homeobox A2 −51.06 0 0.040
NM_008274 Hoxd12 Homeobox D12 −50.78 0 0.009
NM_010465 Hoxc6 Homeobox C6 −50.04 0 0.044
NR_131758 Hoxb5os Homeobox B5 and homeobox B6, opposite strand −50.01 25,927 0.023
NM_010465 Hoxc6 Homeobox C6 −48.18 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −47.36 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −47.06 0 0.003
NM_008274 Hoxd12 Homeobox D12 −47.05 0 <0.001
NR_037977 Gm53 Predicted gene 53 −47.00 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −46.63 0 0.002
NM_010465 Hoxc6 Homeobox C6 −46.52 0 0.011
NM_008274 Hoxd12 Homeobox D12 −46.48 0 0.005
NM_008274 Hoxd12 Homeobox D12 −46.38 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −46.36 0 0.008
NR_037977 Gm53 Predicted gene 53 −46.34 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −46.08 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −46.03 0 0.012
NR_037977 Gm53 Predicted gene 53 −45.94 0 <0.001
NM_008274 Hoxd12 Homeobox D12 −45.78 0 <0.001
NR_037977 Gm53 Predicted gene 53 −45.72 0 <0.001
NR_037977 Gm53 Predicted gene 53 −45.65 0 0.001
NR_037977 Gm53 Predicted gene 53 −45.64 0 <0.001
NM_026080 Mrps24 Mitochondrial ribosomal protein S24 −45.29 11,448 0.031
NM_008274 Hoxd12 Homeobox D12 −45.27 0 0.008
NR_037977 Gm53 Predicted gene 53 −44.81 0 <0.001
NM_175730 Hoxc5 Homeobox C5 −44.42 0 0.011
NM_008274 Hoxd12 Homeobox D12 −43.48 0 0.003
NR_037977 Gm53 Predicted gene 53 −43.23 0 0.004
NM_007967 Evx2 Even-skipped homeobox 2 −43.02 15,125 0.015
NM_175730 Hoxc5 Homeobox C5 −42.62 0 0.001

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Discussion

Satellite cells are activated after a rotator cuff tear in mice, and their biological activity is thought to play an important role in pathological changes in rotator cuff disease28. Meyer and colleagues found that cells from patients with partial-thickness tears had reduced proliferative capacity in vitro, but no difference in fusion, when compared with cells from untorn rotator cuff muscles and full-thickness tears29. In an vivo study, Lundgreen et al. reported reduced satellite cell density and fewer proliferating cells in full-thickness rotator cuff tears compared with partial tears30. While these studies identified differential activity of satellite cells within rotator cuff and shoulder girdle muscles in various states of injury, in the current study, we compared the activity of satellite cells from normal rotator cuff and gastrocnemius muscles, as the gastrocnemius is another commonly injured muscle that generally has better outcomes than the rotator cuff when recovering from a tendon tear2,31,32. We observed a reduced myogenic capacity of rotator cuff satellite cells, along with a decreased expression of the differentiated myogenic transcription factor MRF420. No differences in the muscle cell-fusion gene myomaker33 were observed, indicating a similar capacity of myogenic cells from the gastrocnemius and rotator cuff to fuse into myotubes. These results were also in agreement with a previous study of rats, which demonstrated greater fatty infiltration and reduced healing of the rotator cuff compared with the gastrocnemius7.

There are 3 types of differentiated adipogenic cells, including white, brown, and beige adipocytes34. White adipose cells store fat and are the classical adipocyte cell type35,36. Within muscle tissue, the primary progenitor cell for white adipocytes is thought to be fibro/adipogenic progenitor (FAP) cells, which are a distinct lineage from satellite cells35,36, although myogenic cells can also differentiate into this lineage12,13, with debate ongoing34. Brown and beige adipocytes are related in function but arise from distinct populations of cells, with brown adipocytes having a myogenic origin and beige cells originating from a lineage that is likely similar to white adipocytes34,37. It can be difficult to morphologically discern the 3 cell types in culture; however, a common feature among all adipogenic cells is the expression of FABP422,38. In our findings, all FABP4+ cells were also Pax7-tdTomato+, indicating that these adipocytes originated from a myogenic progenitor population. Further, we observed a 4-fold increase in adipogenic cells from rotator cuff muscles, and a 12-fold increase in the common adipogenic master regulator PPARγ, providing additional support for the finding of greater adipogenic differentiation capacity of rotator cuff satellite cells.

Numerous changes in DNA methylation were observed between rotator cuff and gastrocnemius satellite cells, and bioinformatics analysis identified several biochemical pathways involving adipogenesis and lipid metabolism that were predicted to be different between gastrocnemius and rotator cuff muscles. Many of the genes that were differentially methylated in rotator cuff samples were members of the HOX family of genes. The HOX genes encode transcription factors that were originally identified by their role in instructing the positional identity of progenitor cells along the anterior-to-posterior body axis39. HOX genes also play important roles in myogenesis40, and some of the differences in HOX methylation may be related to the more proximal location of rotator cuff muscles compared with the gastrocnemius in the limb, in particular with HOX9, HOX10, HOX11, and HOX13, which display a proximal-to-distal gradient of restricted expression in the developing limb39. However, some of the HOX genes are also important in brown and beige adipogenesis, in particular HOXC4 and HOXC841. In satellite cells from rotator cuff muscles, there were 2 hypomethylated regions for HOXC4, and 6 for HOXC8. HOXA3 has also been reported to be important in white adipogenesis42, and 8 hypomethylated regions for HOXA3 were found in rotator cuff satellite cells. As hypomethylation of a gene is associated with an increased expression of that gene, the combined results of this study indicate that satellite cells from the rotator cuff are more likely to become adipogenic cells, and this may be explained by differential methylation of adipogenic genes.

There were several limitations to this work. Humans frequently develop more profound and severe atrophy and fat accumulation than found in mouse models of rotator cuff disease4,43,44. We only evaluated changes in adult male mice, which allows for examination of DNA methylation on both the X and Y chromosomes, although the observed results are likely applicable to both sexes. Differentiation experiments were performed in vitro, but it is possible that these findings do not entirely translate to the in vivo setting. We did not identify the specific type of adipocytes in our studies, but white and beige fat cells are known to be present in rotator cuff muscles45, and there are brown fat depots located close to the rotator cuff14,46. We also did not evaluate changes in chromatin packaging and histone methylation, which are also epigenetic regulatory mechanisms. ERRBS analysis also focuses gene promoter regions25, but methylation of other regions of DNA could also play important roles in regulating the activity of satellite cells. Despite these limitations, the current work provides important insight into our understanding of the cellular development of rotator cuff disease.

The rotator cuff is a clinically unique muscle group with regard to pathophysiology, surgical treatment, and rehabilitation7,47. In the current study, we sought to determine if satellite cells from gastrocnemius and rotator cuff muscles differ in their biological activity and epigenetic imprinting. Supporting our hypothesis, we found reduced myogenic and increased adipogenic differentiation of satellite cells from rotator cuff muscles, and differences in DNA methylation patterns that correspond to observed phenotypic differences between the 2 muscle groups, which helps to identify a cellular and genetic basis of the generally poor rates of rotator cuff muscle healing. As satellite cells are activated after injury to repair damaged muscle fibers9, and animal models have demonstrated that the repair of chronically torn rotator cuffs causes extensive injury throughout the muscle48, it is possible that increased differentiation of myogenic cells into the adipogenic lineage contributes to the continued accumulation of fat that is observed in many patients after rotator cuff repair2. Further, transplantation of satellite cells from a healthy muscle to heal diseased muscles within the same patient has shown some promise in early clinical trials49 and has been proposed as a therapy for patients with chronic rotator cuff tears50. Our results provide additional support for the potential use of autologous satellite cell transplantation to improve the treatment of patients with chronic rotator cuff disease.

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Appendix

Details of satellite cell isolation and flow cytometry, cell culture, and DNA methylation analysis and a table listing the primer sequences used for quantitative PCR; as well as tables presenting a full list of the differentially methylated regions and the differentially methylated cytosines are available with the online version of this article as a data supplement at jbjs.org (http://links.lww.com/JBJS/F35, http://links.lww.com/JBJS/F36, http://links.lww.com/JBJS/F37).

Note: The authors thank Claudia Lalancette, PhD, and Karthik Padmanabhan, PhD, for assistance with DNA methylation analysis, and Richard McEachin, PhD, for assistance with bioinformatics.

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References

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