Poster 20: Muscle ERRγ Overexpression Mitigates Muscle Fibrosis and Fatty Infiltration After Rotator Cuff Injury

Abstract

Objectives:

Rotator cuff (RC) pathology is a common, age-related degenerative musculoskeletal disorder that occurs in more than 30% of individuals over age 60. RC tears are associated with pathology that extends beyond tendon damage to include degenerative changes in the RC muscles, including atrophy, fatty infiltration, and fibrosis. Preserving muscle health after RC tears is important for both maintaining function after conservative treatment and improving outcomes with surgical repair. Our laboratory and others have shown that the promotion of angiogenesis through the delivery of angiogenic factors can significantly improve muscle healing after injury. Our team has identified that Estrogen-Related Receptor gamma (ERRγ) is a crucial regulator of paracrine angiogenesis in skeletal muscle. Selective over-expression of ERRγ in skeletal muscle (Err-gamma transgenic mice, TG) activates a robust paracrine angiogenic gene program involving myofibrillar induction and secretion of a battery of angiogenic factors resulting in muscle vascularization. The aim of this study was to determine if ERRγ-driven muscle angiogenesis can reduce muscle fibrosis and fatty infiltration after RC injury by comparing TG mice to age-matched wild-type (WT) littermate control mice using a mouse model of massive rotator cuff tears.

Methods:

Animals: 12-week-old male and female TG (provided by Dr. Narkar) and WT mice were used for this study. This study was approved by IACUC. RC injury: Mice in different groups received supraspinatus and infraspinatus tendon transection by following the published protocol. The contralateral shoulder was served as a control. The mice were euthanized six weeks after injury. Histological analysis and immune staining were used to evaluate muscle fibrosis and fatty infiltration after RC injury. H&E, Sirius red and oil red O staining were performed on 10µm cryosections from supraspinatus and infraspinatus muscle and Mesenchymal Stem Cells (MSCs) according to the manufacturer’s instructions. The Sirius red and oil red O + area was measured using Nikon Eclipse software. Immunohistochemical staining:  Muscle sections were fixed with 4% paraformaldehyde. PDGFRα, perilipin and desmin staining were performed as previously described (6). The number of PDGFRα + cells and the perilipin + area was counted and measured using ImageJ software. Cell isolation and adipogenic differentiation assay: Mesenchymal stem cells (MSCs) from TG and WT mice were isolated using a modified preplate technique and adipogenic differentiation assay was performed by following the protocols from Lonza. Statistical analysis: All results are presented as mean ± standard deviation (SD). Means from RC injured and non-injured WT and TG mice were compared using One-way and two-way ANOVA with a significance value of p < 0.05.

Results:

The results from H&E and perilipin immune staining revealed a significant reduction in fatty infiltration in supraspinatus muscle of TG mice compared to the supraspinatus muscle of WT mice after RC injury (Fig. 1A, B, C). To evaluate the muscle fibrosis, we performed sirius red staining on the supraspinatus muscles of TG and WT mice. The result demonstrated significantly less muscle fibrosis in TG muscle compared to WT muscle after RC injury (Fig. 2A, C). It has been reported that (PDGFRα)-positive mesenchymal stem cells are induced after RC injury and are responsible for fatty infiltration in the muscle. Based on PDGFRα staining we found that the number of PDGFRα+ cells was significantly higher in the WT supraspinatus muscle compared to TG supraspinatus muscle after ACL injury (Fig. 2B, D). In addition, we isolated muscle MSCs from the supraspinatus and infraspinatus muscles of TG and WT mice 2 weeks after RC injury and performed adipogenic differentiation assay and oil red O staining. The result indicated that the muscle MSCs isolated from WT mice significantly increased their adipogenic potential after RC injury (Fig. 3A, B). Conversely, the muscle MSCs isolated from TG mice significantly decreased their adipogenic potential after RC injury (Fig. 3A, B). Interestingly, we found that adipogenic potential of TG MSCs was significantly higher than WT MSCs without RC injury (Fig. 3A, B), indicating that Err-gamma may play a role for adipogenisis in the muscle.

Conclusions:

Degenerative muscle changes occur even in asymptomatic RC tears and may eventually contribute to pain and functional limitations. Exercise therapy is often prescribed over surgical repair for initial treatment of rotator cuff tears and can be effective for relieving symptoms and restoring function. However, it is challenging for older individuals and may be less effective. The TG mice generated in our laboratory is a unique exercise mimetic model because it recapitulates exercise effects such as angiogenesis, improved muscle contractility, and exercise tolerance by simply activating a single nuclear receptor, ERRγ, in the skeletal muscle. In this study, we observed that the TG mice with muscle specific ERRγ activation have the capacity to reduce muscle fibrosis and fatty infiltration after RC injury. Additionally, the MSCs isolated from TG muscle decreased their adipogenic potential after RC injury compared to non-injured muscle. It has been reported that ERRγ positively regulates the adipocyte differentiation with modulating the expression of various adipogenesis-related genes, this can explain why adipogenic potential of TG MSCs was significantly higher than WT MSCs without RC injury. Future development of pharmaceuticals targeting ERRγ could provide a safe and effective therapy for improving outcomes after RC injury or potentially other musculoskeletal disorders.

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