The Glycogen Synthase Kinase–3 Inhibitor CHIR99021 Reduces Fatty Infiltration and Muscle Atrophy After Rotator Cuff Tears: An In Vitro Experiment and In Vivo Mouse Model

Background: Rotator cuff tears (RCTs) can cause inflammation, muscle atrophy, and irreversible fatty infiltration, resulting in poor clinical outcomes. Effective therapeutic approaches to inhibit fatty infiltration in rotator cuff muscles remain limited. Purpose: To identify pathways associated with fatty infiltration through RNA sequencing and to evaluate the therapeutic potential of the glycogen synthase kinase–3 (GSK-3) inhibitor CHIR99021 based on enrichment of the Akt/GSK-3 pathway identified by RNA sequencing. Study Design: Controlled laboratory study. Methods: Supraspinatus muscle biopsy specimens from 6 patients with chronic full-thickness RCTs were analyzed by RNA sequencing. Fibro-adipogenic progenitors (FAPs) or C2C12 myoblasts were cultured with different doses of CHIR99021 to assess their effects on adipogenic or myogenic differentiation, respectively. RNA sequencing identified cellular pathways in FAPs treated with or without CHIR99021. A mouse RCT model was established by detaching the supraspinatus tendon, followed by treatment with or without CHIR99021 administered intraperitoneally. Muscle atrophy and fatty infiltration were assessed histologically and through gene expression analysis at 1 and 4 weeks after surgery. Results: RNA sequencing analysis identified a marked upregulation of the Akt/GSK-3 signaling pathway specifically in patients’ samples and FAPs with minimal fat accumulation. CHIR99021 suppressed adipogenic differentiation in FAPs and promoted myogenic differentiation in C2C12 cells. In the mouse RCT model, CHIR99021-treated mice exhibited reduced Oil Red O staining, a larger cross-sectional area, and less muscle weight loss in the supraspinatus muscle compared with the vehicle-treated mice. Gene expression analysis indicated increased myogenesis and reduced fatty infiltration at 1 and 4 weeks after surgery as well as increased expression levels of IL-6 and IL-15 in the CHIR99021 group compared with the control group at 1 week after surgery. Conclusion: The Akt/GSK-3 pathway was enriched in supraspinatus muscle samples and FAPs with low fat accumulation, highlighting its potential as a therapeutic target. The GSK-3 inhibitor CHIR99021 was shown to alleviate fatty infiltration and muscle atrophy after RCTs in vitro and in vivo in a mouse model. Clinical Relevance: The GSK-3 inhibitor CHIR99021 shows potential for treating muscle degeneration after RCTs.