Evaluation of SS-31 as a Potential Therapeutic Target in the Treatment of Tendinopathy
Authors: Szeto Hazel , Zhang Ying , Nakagawa Yusuke , Carballo Camila , Green Samuel , Deng Xianghua , Rodeo Scott , Zhang Xueying 
Published: July 2020

Objectives: The purpose of this study is to investigate the therapeutic potential of SS-31, a target to improve mitochondria function for the treatment of tendinopathy using human tenocytes. Methods: After IRB approval (IRB# 2018-0490), samples of non-injured hamstring tendon were obtained from 9 patients at the time of surgery (Fig 1 and Table 1). Tenocytes Isolation (Fig 2): Tendons were carefully dissected to remove adjacent connective tissue, minced into small pieces and digested in collagenase1 and dispase for 1h at 37oC. The cells were then plated on culture dishes with α-MEM+10% FBS supplemented with β-ME. Tenocytes were used at passages 3-5 and assigned to 4 groups: 1. Control group: tenocytes only; 2. Healthy group with treatment: tenocytes with 1mM SS-31 treatment for 72 hours; 3. Degenerative group: cells were treated with 10 ng/ml TNF-α for 72 hours; 4. Degenerative group with treatment: cells were treated with 10 ng/ml TNF-α for 72 hours and 1mM SS-31 was added for 72 hours. Reactive Oxygen Species (ROS): The production of ROS by tenocytes were measured using a DCFDA Cellular ROS Detection Assay kit (Abcam) following the manufacturer’s protocol. qRT-PCR: cDNA was generated from 200ng of RNA from cells and qRT-PCR was performed to analyze 4 mitochondrial genes: FXN, SNRPB, ATP5F1, and oPA1; and 2 tendon genes: Col III, TNMD. Transmission Electron Microscopy (TEM): Cells were fixed, prepared for sagittal sections and stained. Micrographs were taken using Philips CM-12 transmission electron microscope. Statistical analyses: T-test and One-way ANOVA analysis were used to compare groups with significance level defined as p ≤ 0.05. Results: There was a significantly higher level of ROS in degenerative tenocytes compared to healthy tenocytes (Fig 3). The expression of ROS decreased in the degenerative group after 72 hours of treatment with SS-31. The expression of FXN (iron transport and respiration) and ATP5F1A (ATP synthase) increased significantly in degenerative tenocytes but the expression levels of all 4 genes were close to the healthy tenocyte after SS-31 treatment (Fig 4), suggesting that mitochondrial activity was reduced in degenerative tenocytes but activated by treatment. Col3 and TNMD, tendon-specific gene markers, were higher in degenerative tenocytes and decreased after treatment, suggesting an elevated extracellular matrix remodeling process in tendons under degenerative conditions (Fig 5). TEM images (Fig 6) revealed that the number of mitochondria per cell and the ratio of area/perimeter significantly decreased in the degenerative group, suggesting mitochondrial fission and degradation. The disorganized cristae were observed in degenerative tenocytes compare to the healthy tenocytes, which may be caused by different ATP synthesis needs. The number of cristae per cell are under evaluation. TEM images for the treatment groups are under analysis. Conclusion: Degenerative tenocytes had higher levels of ROS that was decreased with SS-31 treatment. High ROS levels can lead to activation of apoptosis/autophagy pathways capable of inducing cell death. Expression levels in four mitochondrial genes were upregulated in degenerative tenocytes but normalized by SS-31 treatment. In summary, our data demonstrate that mitochondrial dysfunction is associated with tendinopathy and could be a possible mechanism leading to tendinopathy. SS-31, as a mitochondria protective agent, may be a potential therapeutic agent in the treatment of tendinopathy. [Figure: see text]