Defining Endogenous Mitochondrial Transfer in Muscle After Rotator Cuff Injury

Abstract

Background: Rotator cuff muscle degeneration leads to poor clinical outcomes for patients with rotator cuff tears. Fibroadipogenic progenitors (FAPs) are resident muscle stem cells with the ability to differentiate into fibroblasts as well as white and beige adipose tissue. Induction of the beige adipose phenotype in FAPs has been shown to improve muscle quality after rotator cuff tears, but the mechanisms of how FAPs exert their beneficial effects have not been fully elucidated. Purpose: To study the horizontal transfer of mitochondria from FAPs to myogenic cells and examine the effects of β-agonism on this novel process. Study Design: Controlled laboratory study. Methods: In mice that had undergone a massive rotator cuff tear, single-cell RNA sequencing was performed on isolated FAPs for genes associated with mitochondrial biogenesis and transfer. Murine FAPs were isolated by fluorescence-activated cell sorting and treated with a β-agonist versus control. FAPs were stained with mitochondrial dyes and cocultured with recipient C2C12 myoblasts, and the rate of transfer was measured after 24 hours by flow cytometry. PdgfraCreERT/MitoTag mice were generated to study the effects of a rotator cuff injury on mitochondrial transfer. PdgfraCreERT/tdTomato mice were likewise generated to perform lineage tracing of PDGFRA+ cells in this injury model. Both populations of transgenic mice underwent tendon transection and denervation surgery, and MitoTag-labeled mitochondria from Pdgfra+ FAPs were visualized by fluorescent microscopy, spinning disk confocal microscopy, and 2-photon microscopy; overall mitochondrial quantity was compared between mice treated with β-agonists and dimethyl sulfoxide. Results: Single-cell RNA sequencing in mice that underwent rotator cuff tear demonstrated an association between transcriptional markers of adipogenic differentiation and genes associated with mitochondrial biogenesis. In vitro cocultures of murine FAPs with C2C12 cells revealed that treatment of cells with a β-agonist increased mitochondrial transfer compared to control conditions (17.8% ± 9.9% to 99.6% ± 0.13% P