Summary

Blood flow restriction (BFR) induces promyogenic mitochondrial transfer and improves motor function in chronic rotator cuff injury murine model.

Abstract

Disclosure: All authors have no disclosure.

Introduction

While blood flow restriction (BFR) has been shown to stimulate muscle regeneration and pain relief after injury, its mechanism of action remains unknown. Since it has not been investigated in rotator cuff (RC) injury, our goal was to explore BFR’s ability to promote muscle regeneration and improve mobility after RC tears in a mouse model. We hypothesized that BFR induces mitochondrial transfer from FAPs to myocytes, thus enhancing muscle regeneration and kinematic function after RC injury.

Methods

To administer BFR, an orthodontic rubber band 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 from both the experimental and sham/uninjured mice. To assess the impact of BFR on injured RC muscles, unilateral SS and IS tendon transection and denervation (TT+DN) were performed on Prrx1-Cre/MitoTag mice to induce RC tears. Mice were then randomized to receive ipsilateral arm BFR every three days or no treatment as a control. Mice were sacrificed at 2, 4, or 6 weeks after surgery, and SS muscles were analyzed for MitoTag signaling and myofiber size. Kinematic function was assessed using BlackBox and DeepLabCut at an acute and chronic timepoint of 2 and 6 weeks post-op, respectively.

Results

BFR was shown to significantly increase FAP mitochondria transfer in SS compared to non-BFR controls at 2 weeks (Percentage of GFP+ myofibers/total number of myofibers; BFR 12.8% vs control 3.3%, p<.0001) and 6 weeks (BFR 25.3% vs control 6.6%, p<.0001) after TT+DN injury. Kinematically, at 2 weeks post-op, right forepaw stride length was the only significantly different parameter, with BFR-treated mice interestingly exhibiting worse stride length compared to control mice (1.37 ± 0.22 cm vs 1.82 ± 0.11 cm, p < 0.05); however, at 6 weeks, stride length was significantly improved in BFR-treated mice, (2.16 ± 0.40 cm vs 1.73 ± 0.34 cm, p < 0.05). Additionally at 6 weeks, right forepaw stepping correlation was significantly lower in BFR-treated mice, (-0.30 ± 0.02 vs -0.26 ± 0.03, p < 0.05); signifying a more synchronous gait. Lastly, forepaw weightbearing ratio (injured/uninjured) was significantly greater in BFR-treated mice compared to controls at 6 weeks post-injury (0.99 ± 0.14 vs 0.55 ± 0.15, p<0.001); with a ratio of 1 denoting equal weightbearing between forepaws.

Discussion

Our data demonstrates that BFR induces mitochondrial transfer from FAPs to myocytes after RC injury, suggesting that FAP mitochondria transfer is a crucial underlying mechanism of BFR-induced muscle regeneration and reduction of atrophy after injury. In addition, this study shows that BFR-treated mice exhibited improved mobility, as measured by stride length, stepping correlation, and weightbearing ratio; notably, these kinetic improvements were appreciated only at our chronic injury timepoint of 6 weeks. Future studies will aim to further elucidate BFR-mediated promyogenic pathways, along with identifying additional motion kinematics pertaining to both acute and chronic RC-injury pain.