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.
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.
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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