Mitochondrial Transplantation Preserves Regenerative Capacity of Denervated Skeletal Muscle by Disrupting a Ca 2+ Overload‐Mitophagy‐Ferroptosis Circuit
Haiqi Ding, Jiagu Huang, Jiayao Zhang, Zhizhen He, Jun Luo, Zhao Lin, Linhai Yang, Yibin Su, Mingjuan Zhang, Fuli Wen, Yuan Lin, Cheng Chang, Hanhao Dai, Xuehui Ge, Jie XuABSTRACT
Background
Denervation‐induced muscle atrophy progresses during the interval between nerve injury and reinnervation, and depletion of muscle satellite cells (MuSCs) contributes to impaired functional recovery. Although mitochondrial dysfunction and impaired quality control are implicated in muscle wasting, whether excessive mitochondrial turnover drives MuSC failure after denervation remains unclear. This study aimed to identify the pathogenic mechanisms underlying MuSC depletion and determine whether mitochondrial transplantation therapy (MTT) preserves regenerative capacity.
Methods
A sciatic nerve transection model was established in mice to induce denervation‐associated muscle atrophy. Mitochondria isolated from differentiated MuSCs were transplanted into denervated gastrocnemius muscles beginning 6 h after injury and repeated every 3 days for 6 weeks. RNA sequencing, calcium imaging, mitochondrial ultrastructural analysis, mito‐mKeima‐based mitophagic flux assessment, flow cytometry and molecular analyses were performed to investigate disease mechanisms and therapeutic effects. In vitro calcium overload models combined with Mdivi‐1 treatment and Pink1 knockdown were used to define the regulatory pathway linking calcium stress, mitophagy and ferroptosis.
Results
Denervated muscle exhibited early calcium dyshomeostasis (Fluo‐4 AM fluorescence increased 6.7‐fold vs. Sham, p < 0.0001) and mitochondrial dysfunction within 3 days, followed by iron accumulation (nonheme iron increased 1.5‐fold, p = 0.0046) and ferroptotic changes by 2 weeks. Mechanistically, sustained calcium overload activated the Ampk‐Tsc2‐mTorc1 pathway in MuSCs, inducing Pink1‐dependent excessive mitophagic flux. Mito‐mKeima flow cytometry confirmed increased mitophagic flux after denervation, with approximately tenfold elevation at Day 3 and persistent activation at 2 weeks. Excessive mitophagy promoted Fth1 degradation (reduced by 47.5%, p = 0.0181) and increased Tfr1 expression (1.9‐fold, p = 0.0035), thereby enhancing ferroptotic susceptibility. Inhibition of mitochondrial fission or Pink1 knockdown reduced iron accumulation (Fe 2+ fluorescence reduced by 58.3% and 69.2%, respectively, both p < 0.01) and protected MuSCs from ferroptosis. MTT restored mitochondrial homeostasis by reducing calcium overload (73.7% decrease vs. A23187, p < 0.0001), increasing ATP production (36.9%, p < 0.01), suppressing excessive mitophagic flux, normalizing iron metabolism (39.9% reduction, p = 0.0015) and preserving MuSCs (30.0% increase vs. DN_PBS, p = 0.0196). Consequently, MTT attenuated denervation‐induced muscle fibre atrophy and improved functional recovery following delayed nerve repair.
Conclusions
This study identifies an early pathogenic cascade linking calcium overload, excessive mitophagic flux and ferroptotic susceptibility as a critical driver of MuSC dysfunction during denervation‐induced muscle wasting. MTT preserves regenerative capacity by interrupting this degenerative circuit and restoring mitochondrial quality control, providing a potential therapeutic strategy for denervation‐associated muscle atrophy and other muscle wasting disorders.