Targeting SIRT3 by a ROS-responsive hydrogel promotes skeletal muscle regeneration and functional restoration

The functional regeneration of skeletal muscle following traumatic injury-induced volumetric muscle loss (VML) remains a significant challenge in orthopedic clinics. Myoblast mitochondrial dysfunction at the VML defect site hinders the formation of mature myotubes by disrupting energy metabolism and redox homeostasis. Here we demonstrate that activation of mitochondrial Sirtuin 3 (SIRT3) by honokiol (HKL) enhances skeletal muscle regeneration in a mouse model of cardiotoxin-induced acute injury and promotes functional restoration in a rat VML model. HKL treatment dose-dependently increased mitochondrial respiratory chain activity, activated mitochondrial antioxidant defense mechanisms, and enhanced the myogenic differentiation of myoblast cells via the SIRT3-mediated pathway. Conversely, silencing Sirt3 abrogated the protective effects of HKL on mitochondrial function and redox homeostasis. To evaluate the therapeutic potential for VML injuries, we developed a reactive oxygen species (ROS)-responsive hydrogel based on hyaluronic acid methacrylate (HAMA) by incorporating phenylboronic acid (PBA) with HKL (HAMA-PBA@HKL) through the formation of boronic ester bonds. When applied to rat tibialis anterior defects, the HAMA-PBA@HKL hydrogel significantly enhanced myofiber formation, improved vascularization, restored neuromuscular junction innervation, and recovered muscle contractile performance, while effectively inhibiting fibrotic tissue formation. Single-cell RNA sequencing analysis of the newly regenerated muscle demonstrated that the implantation of the HAMA-PBA@HKL hydrogel significantly increased the proportions of myonuclei cells, satellite cells, endothelial cells, and Schwann cells, while reducing the presence of macrophages, monocytes, and T lymphocytes. Transcriptomic profiling analysis confirmed that treatment with the HAMA-PBA@HKL hydrogel up-regulated genes associated with myoblast differentiation, oxidative phosphorylation, and mitochondrial fusion, while down-regulating gene expression related to immune response, fibroblast proliferation, and collagen deposition. Collectively, these findings highlight the translational potential of the HAMA-PBA@HKL hydrogel that targets mitochondrial SIRT3 in promoting functional skeletal muscle regeneration following severe traumatic injuries.