Xuhui Liu

Background: Volumetric muscle loss (VML) often results in chronic muscle weakness, limb dysfunction, and even permanent disability. Current research primarily focuses on cell and extracellular matrix transplantation, but there is a lack of studies on the physiologically supportive intervention to enhance endogenous muscle recovery after VML. Recent studies have shown that β3-adrenergic receptor (β3AR) agonists can increase intramuscular UCP-1 expression and the proportion of PDGFRα-GFP/UCP-1 double-positive cells, a process associated with muscle recovery. This study aims to elucidate the role of the β3AR agonist Amibegron in promoting intramuscular brown/beige-like adipogenic activity to improve muscle atrophy, fibrosis and limb function after VML injury in mice. Methods: Three-month-old PDGFRα-GFP reporter mice and C57BL/6J mice were used in this study. A VML injury model was created in the tibialis anterior (TA) muscle using a biopsy punch. To induce fibro/adipogenic progenitors (FAPs) to differentiate into beige-like adipocytes, the β3AR agonist Amibegron was administered via intraperitoneal injection. Limb function was assessed by gait analysis at 2 and 6 weeks post-VML, and muscle histomorphological examination and RT-qPCR for muscle gene expression analysis were performed at the corresponding time points. Results: Compared with the vehicle control group, histology and gait analysis showed that Amibegron administration for 2 and 6 weeks increased intramuscular brown/beige adipogenic activity and modestly improved VML-injured muscle mass, fibrosis, and limb function. Moreover, at 6 weeks of Amibegron administration, limb function showed a significant positive association with uncoupling protein-1 (UCP-1) expression. Conclusions: β3AR agonist Amibegron attenuates VML-induced muscle atrophy and fibrosis and improves limb motor function. These improvements are associated with increased brown/beige activity, but the causal role of FAP-derived BAT is not yet established. Given systemic administration, benefits likely involve both local and systemic β3AR effects. This study provides a potential therapeutic strategy for VML.

Background: Secondary muscle degeneration after a rotator cuff tear (RCT) critically affects clinical outcomes. Vascular compromise after a tendon injury creates a complex microenvironment that may be associated with the degeneration of rotator cuff muscle. The role of hypoxia-inducible factor–1α (HIF-1α), a master regulator of cellular stress responses to hypoxia, in modulating muscle abnormalities after an RCT remains undefined. Purpose: To define the role of HIF-1α in stem cell differentiation and muscle degeneration after an RCT in a murine model. Study Design: Controlled laboratory study. Methods: A supraspinatus tendon transection and suprascapular nerve transection (TTDN) model was established in C57BL/6J, platelet-derived growth factor receptor α (PDGFRα)–green fluorescent protein (GFP) reporter, and inducible cell-specific HIF-1α knockout mice. Vascularity and HIF-1α colocalization with fibroadipogenic progenitor (FAP) cells and satellite cells were analyzed. Fibrosis, fatty infiltration, and myofiber cross-sectional area were assessed. In vitro, HIF-1α was modulated in isolated FAP cells via CRISPR-Cas9 or prolyl hydroxylase domain inhibitors to evaluate FAP cell differentiation. Results: TTDN induced significant capillary density reduction (CD31 ⁺ ) at 1, 2, and 6 weeks after an injury. Global HIF-1α expression decreased after TTDN compared to the sham side (1 week: 0.78 ± 0.22 vs 1.40 ± 0.42, respectively [ P = .019]; 2 weeks: 0.74 ± 0.51 vs 1.70 ± 0.48, respectively [ P = .015]). The percentage of PDGFRα ⁺ FAP cells increased at 6 weeks after TTDN compared to the sham side (15.69% ± 1.90% vs 12.76% ± 0.78%, respectively; P = .013). The percentage of HIF-1α ⁺ FAP cells relative to total PDGFRα ⁺ cells significantly decreased in the late stage (6 weeks) of an RCT compared to the sham side (2.78% ± 0.90% vs 7.38% ± 2.29%, respectively; P = .003). Knocking out HIF-1α in FAP cells in vivo resulted in increased fibrosis (Cre ⁺ : 4.43% ± 2.16% vs Cre ^– : 1.72% ± 0.39%; P = .047), decreased fatty infiltration (Cre ⁺ : 0.62% ± 0.42% vs Cre ^– : 1.55% ± 0.45%; P = .016), and reduced cross-sectional area (Cre ⁺ : 664.71 ± 354.45 vs Cre ^– : 1195.81 ± 338.66; P = .041). Neither satellite cell–specific nor myocyte-specific HIF-1α deletion resulted in significant phenotypic changes. The downregulation of HIF-1α led to a decrease in uncoupling protein 1 expression and an increase in α–smooth muscle actin expression in FAP cells. Conclusion: Although vascularity was reduced after TTDN, pronounced global tissue hypoxia was not directly evidenced. Decreased global HIF-1α expression may reflect denervation-induced reductions in metabolic demand. HIF-1α emerges as a key player in FAP cell differentiation within the injury microenvironment, promoting brown adipose tissue differentiation and inhibiting fibrogenesis. Clinical Relevance: Targeting HIF-1α in FAP cells offers a novel therapeutic strategy to mitigate secondary muscle atrophy and fibrosis after an RCT.

Fatty infiltration and fibrosis drive poor outcomes after chronic muscle injury. Here, we characterized fibroadipogenic progenitor cell (FAP) subpopulations from healthy and injured human rotator cuff muscle by single-cell RNA sequencing, full-spectrum flow cytometry, and functional assays and found distinct subpopulations, including a preadipogenic population that expresses delta-like noncanonical notch ligand 1 (DLK1 ⁺ ) and a prefibrogenic population that expresses decay-accelerating factor (CD55 ⁺ ). We used in vitro and flow cytometry experiments to show that human FAP lineages displayed unique surface marker expression across adipogenic and fibrogenic differentiation. In chronic human rotator cuff injury, expression of DLK1 RNA and DLK1 protein was decreased compared with that in healthy muscle. Overexpression of DLK1 in primary human FAPs suppressed differentiation into adipocytes in vitro, whereas DLK1 knockdown increased adipogenesis. In an immunodeficient murine model of glycerol-induced acute muscle injury, xenotransplantation of DLK1-overexpressing human FAPs into the injured murine muscle resulted in reduced fatty infiltration after 14 days by histological assessment, supporting a functional role for DLK1 in restraining adipogenesis. Together, we defined molecularly distinct, clinically relevant human FAP subpopulations, established DLK1 as a regulator of adipogenic potential in vivo, and provided a framework to identify pathogenic FAP states that may be used in the pursuit to prevent maladaptive muscle remodeling.

Aims: Fibroadipogenic progenitors (FAPs) are a group of resident muscle stem cells capable of differentiating into fibroblasts and adipocytes, contributing to intramuscular fibrotic and fatty degeneration after injury. However, FAPs are also thought to play a crucial role in muscle regeneration by facilitating satellite cell myogenesis. Despite this dual role, the precise functions of FAPs in muscle degeneration and regeneration remain unclear. This study aimed to utilize a FAP depletion mouse model to define the role of FAPs in two distinct, clinically relevant injury models of rotator cuff tears (RCTs) and tibialis anterior (TA) volumetric muscle loss (VML). Methods: Six PDGFRα-Cre ^ERT /DTA mice were applied for fluorescence-activated cell sorting (FACS) evaluation of FAP depletion efficiency with tamoxifen induction. Then, two groups of ten PDGFRα-Cre ^ERT /DTA mice and five DTA mice underwent either unilateral supraspinatus and infraspinatus tendons and suprascapular nerve transection (RCT model) or the creation of a 4 mm diameter defect in the unilateral TA (VML model). To induce FAP depletion, tamoxifen or corn oil (control) was administered daily for two weeks before surgery. Gait analysis was conducted at six weeks post-surgery to evaluate shoulder or hindlimb function. Supraspinatus or TA muscles were harvested to assess muscle atrophy and for histological analysis. Results: FACS analysis confirmed a 50% reduction in FAPs following tamoxifen administration in PDGFRα-Cre ^ERT /DTA mice. In the RCT model, FAP depletion significantly attenuated muscle atrophy and improved fibrosis, fatty infiltration (FI), and global shoulder function. Conversely, in the VML model, FAP depletion significantly decreased muscle fibrosis but had no effect on FI and functional outcomes. Conclusion: Our results suggest that FAPs play distinct roles in muscle regeneration depending on the specific clinical context. These models can be used to further understand the different injury mechanisms and muscle-specific properties influencing FAP roles in musculoskeletal pathology. Cite this article: Bone Joint Res 2026;15(8):927–936.

Muscle fibrosis is a key pathological feature of Duchenne muscular dystrophy (DMD) and is closely associated with disease progression. Fibroadipogenic progenitors (FAPs) are major contributors to fibrosis, yet the precise mechanisms remain unclear. To investigate FAP dynamics and lineage specification, we generated dual-reporter mice (PRURD2) by crossing D2.B10-Dmdmdx/J (D2-mdx) mice with FAP and brown/beige adipose tissue (BAT) reporter lines. Corresponding control mice (PRURDBA) were established on the DBA/2J background. At 12 months, heart, diaphragm, and tibialis anterior (TA) muscles were collected for histological analysis. FAPs were isolated via FACS and subjected to single-cell RNA sequencing. PRURD2 mice exhibited increased fibrosis across all muscles compared to controls ( p < 0.01) and a significant rise in PDGFRα-GFP + FAPs ( p < 0.05). UMAP clustering identified 11 distinct FAP subpopulations, with the fibrosis-associated CD55 cluster enriched in PRURD2 mice. Pseudotime analysis showed lineage progression from progenitor clusters toward the fibrogenic CD55 cluster. CellChat analysis indicated increased interactions in PRURD2 mice involving fibrosis-related pathways like COLLAGEN, TGF-β, WNT, NOTCH, and ANGPTL. Additionally, fibrosis-related signaling pathways such as THY1, TWEAK, EPHA, EPHB, and SEMA6 showed increased interactions among FAP clusters in PRURD2 mice. Differential gene expression analysis revealed top upregulated genes including Cxcl13, Cxcl3, Ly6d, Klk1, Fgf23, Serpinb2, Mmp13, Ccl17 , Postn , and Adam12 . PRURD2 mice develop severe fibrosis in skeletal and cardiac muscle, driven by FAP-induced signaling pathways and genes. This model is valuable for understanding muscle fibrosis in DMD and developing anti-fibrotic therapies.

Background: Rotator cuff (RC) tears are among the most common causes of shoulder dysfunction in sports medicine. Muscle atrophy and degeneration are important risk factors for RC tendon retearing and suboptimal recovery of shoulder function after tendon repair. Although blood flow restriction (BFR) can stimulate muscle regeneration after lower extremity trauma and anterior cruciate ligament reconstruction, the mechanisms that underlie BFR remain unknown, and its application to RC tears has not yet been explored. Hypothesis: The authors hypothesized that BFR induces transfer of mitochondria from intramuscular fibro-adipogenic progenitors (FAPs) to myocytes, enhances muscle regeneration, and improves shoulder function after RC injury. Study Design: Controlled laboratory study. Methods: To assess mitochondrial transfer after BFR, the authors used Prrx1-Cre/MitoTag reporter mice, in which FAP mitochondria are labeled. Mice underwent unilateral forelimb BFR, and supraspinatus (SS) muscles were collected at baseline and days 1, 2, 3, 5, and 7 for histology. To model massive RC tears, mice received unilateral SS and infraspinatus tendon transection with denervation (TT+DN) and then were randomized to a BFR (every 3 days) or control group. At 2 or 6 weeks after surgery, SS muscles were analyzed for mitochondrial transfer, fiber size, and fiber-type distribution. Additionally, forelimb gait and weightbearing were captured using the Blackbox system. Results: BFR was associated with increased FAP-mediated mitochondrial transfer in healthy SS muscle as early as 1 day after BFR treatment and lasted for up to 3 days after BFR. The authors observed an enhanced effect of BFR-induced FAP mitochondrial transfer in SS muscle after RC injury, compared with the control, at both 2 and 6 weeks after TT+DN. BFR-treated mice had significantly reduced muscle atrophy, fatty infiltration, and fibrosis after RC injury. They also observed a significant improvement in forepaw weightbearing ratio and ipsilateral forepaw stride length at 6 weeks after injury in BFR-treated mice compared with controls. Conclusion: BFR significantly improves muscle quality and shoulder function after RC injury. These effects occur alongside increased mitochondrial transfer from FAPs to myocytes. Clinical Relevance: Understanding the mechanism of BFR by which BFR enhances muscle regeneration could pave the way for its use as a novel rehabilitation strategy to improve recovery in patients with RC injuries and other muscle-related conditions.

Overuse‐induced muscle disorders (OIMD) frequently occur in athletes due to excessive and improper use under high physical demand, often leading to muscle pain and weakness. Limited studies have shown intramuscular fibrosis in OIMD, with fibro‐adipogenic progenitors (FAPs), also known as mesenchymal stromal cells (MSCs), playing a crucial role in this fibrosis. This study aimed to develop a rat OIMD model using neuromuscular electrical stimulation‐induced isometric exercise (NMES‐ISO) and to investigate mechanisms by which excessive exercise without adequate rest leads to OIMD. We hypothesize that daily NMES‐ISO would cause muscle weakness and fibrogenesis mediated by FAP activation triggered by muscle fiber damage. Male Wistar rats received daily NMES‐ISO loading on the plantar flexor muscles at a lengthened position. Two weeks of NMES‐ISO led to decreased muscle torque without muscle mass reduction. Masson‐Trichrome stain revealed collagen‐rich fibrogenic lesions in the gastrocnemius muscles, while Evans blue stain detected no muscle fiber damage during the first 5 days. MRI showed increased T2*wi signals correlating with fibrogenic areas. Excessive NMES‐ISO stimulated FAP proliferation. This study established a rat model of OIMD using NMES‐ISO, characterized by muscle weakness and intramuscular fibrogenesis. Contrary to our hypothesis, FAP activation occurred without overt muscle injury, suggesting excessive mechanical loading may directly trigger FAP proliferation and fibrogenesis. It was cautioned that even relatively safe isometric contraction training could lead to FAP activation and intramuscular fibrosis without proper methods and recovery periods.

For efficient regeneration, muscle stem cells (MuSCs) transition out of quiescence through a series of progressively more activated states. During MuSC aging, transition through the earliest steps is the slowest and delayed, with the molecular regulators that govern this transition not well characterized. By analyzing the dynamic changes of MuSCs at the molecular (scRNA-Seq and Cell Painting) and phenotypic (heteromotility) level at single cell resolution we found that the Integrated Stress Response (ISR) Pathway is a critical regulator of MuSC transition states. Aged MuSCs have increased baseline ISR activity in quiescence that does not increase during activation to levels observed in adult MuSCs. Rapid and transient pharmacological ISR activation in vitro was sufficient to increase aged MuSC activation rate and migratory behavior as well as alter the transcriptional states toward a younger phenotype. ISR activation also improved aged MuSC potency and aged mouse muscle regeneration in vivo . Therefore, pharmacological activation of the ISR has therapeutic potential to improve MuSC function and skeletal muscle repair during aging.

Amibegron, a β3‐adrenergic receptor (B3AR) agonist, has recently been shown to provide therapeutic effects for chronic rotator cuff (RC) tears by inducing the expression of uncoupling protein 1 (UCP‐1), a marker of brown fat, in fibroadipogenic progenitors (FAPs). However, it remains to be seen if these beneficial effects hold true with age and in older, more clinically relevant populations. This study seeks to understand the impacts of aging on the efficacy of amibegron to treat chronic RC tears. Young (4‐month‐old) and aged (33‐month‐old) C57BL/6 mice underwent a RC injury procedure with delayed repair (DR). Mice were equally randomized to receive amibegron or dimethyl sulfoxide (DMSO) treatments after repair. Functional ability was measured at baseline and 6‐weeks after DR. Wet muscle weight and histology of injured and contralateral supraspinatus were also analyzed 6‐weeks post‐DR. For in vitro histology and real‐time quantitative PCR experiments, FAPs were isolated from young and aged mice via fluorescence‐activated cell sorting. Young and aged FAPs were treated with amibegron or DMSO either immediately after seeding (early exposure) or 8‐days after seeding (late exposure). In vitro results showed that amibegron‐mediated FAP UCP‐1 expression decreases with age. In vivo data demonstrated that aged mice have a decreased responsiveness to amibegron and decreased propensity for intramuscular FAP UCP‐1 expression. Further, delayed amibegron treatment with RC repair did not lead to improvements in muscle atrophy and functional outcomes. Our findings demonstrate that age and the timing of interventions play a critical role in FAP‐targeted therapeutics for chronic injuries.