Abstract
Objectives:
Muscle atrophy and fatty degeneration negatively impact clinical outcomes after rotator cuff (RC) injury and repair. Blood flow restriction (BFR) is a therapeutic approach involving the temporary restriction of blood flow that has been used for stimulating muscle regeneration and pain relief after lower extremity trauma and ACL reconstruction. However, the underlying mechanisms of BFR remain unknown, and it has not been applied to RC injury. Fibroadipogenic progenitors (FAPs) are resident skeletal muscle stem cells that have been shown to have the capacity to donate mitochondria to myogenic cells for reducing muscle degeneration and improving shoulder function after RC tears. The purpose of this study was to investigate the ability and mechanism of BFR to promote muscle regeneration after RC tears in a mouse model and consequently translate to improved functional outcomes. We hypothesized that BFR induces horizontal mitochondrial transfer from FAPs to myocytes, thus enhancing muscle regeneration and kinematic function after RC injury.
Methods:
Due to its anatomical location, directly applying BFR to the RC muscle is technically challenging. Instead, we applied BFR to the ipsilateral arm adjacent to the shoulder, presenting a more feasible and translational approach. To define the role of BFR on healthy rotator cuff muscles, an orthodontic rubber band (4.0 oz) was applied to the right arm of healthy Prrx1-Cre/MitoTag FAP-mitochondria reporter mice for 10 minutes, then removed for 10 minutes, for 3 cycles. Mice were then sacrificed at 1, 2, 3, 5, and 7 days post-BFR. Ipsilateral supraspinatus (SS) muscles were harvested for histologic analysis. To assess the impact of BFR on injured RC muscles, unilateral supraspinatus and infraspinatus tendon transection (TT) and denervation (DN) were performed on Prrx1-Cre/MitoTag mice to simulate a massive RC tear as previously described (Liu et al, 2012, JBJS). Mice were then randomized into BFR or the control groups. Mice in the BFR group received ipsilateral arm BFR every three days, while mice in the control group were left untreated Mice were sacrificed at 2 or 6 weeks after surgery, and SS muscles were analyzed for mitochondria transfer and myofiber size analysis. All images were analyzed using ImageJ. Kinematic function was also recorded using BlackBox, a novel AI-based animal gait analysis system, at baseline, 2, 4, and 6 weeks after RC injury in BFR and control groups. Forepaw stride length and weightbearing ratio were then analyzed using DeepLabCut, an open-source software for training deep neural networks. Unpaired t-tests were used for statistical analysis between experimental groups. Two-way analysis of variance with Tukey post hoc honestly significant difference test was used in the case of multiple comparisons. Significance was considered when p<0.05. All data is presented in the form of mean ± SD.
Results:
Ipsilateral arm BFR showed a significant effect in inducing mitochondrial transfer from FAPs to myocytes in SS muscle (Figure 1A-G). This effect lasted for up to 3 days post-BFR, with approximately 10.7% of myofibers still containing FAP-transferred mitochondria (Figure 1D,G). At both 2 and 6 weeks after TT+DN injury, BFR significantly increased FAP mitochondria transfer in SS compared to non-BFR controls (Figure 2A-F). No significant difference in myofiber size was found between BFR and control at 6 weeks after TT+DN injury (Figure 2C,D,G). These data suggest that BFR increases myofiber size in both healthy and injured muscle. However, this effect diminishes in the context of chronic tendon and nerve injury. Functionally, while BFR treatment decreased right forepaw stride length at 2 weeks after RC injury, by 6 weeks, it significantly improved right forepaw stride length compared to the control (2.2 ± 0.4 cm vs 1.7 ± 0.3 cm, p<0.05; Figure 3A). Forepaw weightbearing ratio (injured/non-injured forepaw) was significantly greater in BFR-treated mice compared to non-treated mice at 6 weeks post-TT+DN (1.0 ± 0.1 vs 0.6 ± 0.2, p<0.001; Figure 3B-D). Because reduced weightbearing pressure ratio is sensitive marker of pain, this result suggests that BFR reduces shoulder pain after rotator cuff tears.
Conclusions:
In this study, we evaluated the role of ipsilateral arm BFR in improving rotator cuff muscle atrophy and shoulder function following cuff tendon tear in our mouse model. Our data demonstrates that BFR induces horizontal mitochondrial transfer from FAPs to myocytes and increases both average myofiber size in healthy SS muscle and injured muscle after RC tears. As horizontal mitochondrial transfer from MSCs has been implicated in restoring tissue respiration and promoting regeneration, these results highlight that FAP mitochondria transfer is a crucial underlying mechanism of BFR-induced muscle regeneration after injury. Additionally, we showed that BFR-treated mice had both improved long-term functional outcomes and reduction in pain metrics after RC injury. The novel findings from this study suggest that BFR improves shoulder function and reduces pain after rotator cuff injury. This effect may be attributed to BFR-induced horizontal mitochondria transfer from FAPs. These data reveal an exciting rehabilitation method that has the potential to be translated into clinical care for rotator cuff tear patients.
