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BACKGROUND

Satellite cells (SCs) are critical to skeletal muscle regeneration. Inactivation of SCs is linked to skeletal muscle loss. Transferrin receptor 1 (Tfr1) is associated with muscular dysfunction as muscle-specific deletion of Tfr1 results in growth retardation, metabolic disorder, and lethality, shedding light on the importance of Tfr1 in muscle physiology. However, its physiological function regarding skeletal muscle ageing and regeneration remains unexplored.

METHODS

RNA sequencing is applied to skeletal muscles of different ages to identify Tfr1 associated to skeletal muscle ageing. Mice with conditional SC ablation of Tfr1 were generated. Between Tfr1 and Tfr1 (n = 6-8 mice per group), cardiotoxin was intramuscularly injected, and transverse abdominal muscle was dissected, weighted, and cryosectioned, followed by immunostaining, haematoxylin and eosin staining, and Masson staining. These phenotypical analyses were followed with functional analysis such as flow cytometry, tread mill, Prussian blue staining, and transmission electron microscopy to identify pathological pathways that contribute to regeneration defects.

RESULTS

By comparing gene expression between young (2 weeks old, n = 3) and aged (80 weeks old, n = 3) mice among four types of muscles, we identified that Tfr1 expression is declined in muscles of aged mice (~~80% reduction, P < 0.005), so as to its protein level in SCs of aged mice. From in vivo and ex vivo experiments, Tfr1 deletion in SCs results in an irreversible depletion of SCs (~~60% reduction, P < 0.005) and cell-autonomous defect in SC proliferation and differentiation, leading to skeletal muscle regeneration impairment, followed by labile iron accumulation, lipogenesis, and decreased Gpx4 and Nrf2 protein levels leading to reactive oxygen species scavenger defects. These abnormal phenomena including iron accumulation, activation of unsaturated fatty acid biosynthesis, and lipid peroxidation are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis further exacerbates SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor, could not rescue ferroptosis. However, intramuscular administration of lentivirus-expressing Tfr1 could partially reduce labile iron accumulation, decrease lipogenesis, and promote skeletal muscle regeneration. Most importantly, declined Tfr1 but increased Slc39a14 protein level on cellular membrane contributes to labile iron accumulation in skeletal muscle of aged rodents (~80 weeks old), leading to activation of ferroptosis in aged skeletal muscle. This is inhibited by ferrostatin-1 to improve running time (P = 0.0257) and distance (P = 0.0248).

CONCLUSIONS

Satellite cell-specific deletion of Tfr1 impairs skeletal muscle regeneration with activation of ferroptosis. This phenomenon is recapitulated in skeletal muscle of aged rodents and human sarcopenia. Our study provides mechanistic information for developing novel therapeutic strategies against muscular ageing and diseases.

背景

卫星细胞（SCs）对骨骼肌再生至关重要。SCs 的失活与骨骼肌丢失有关。转铁蛋白受体 1（Tfr1）与肌肉功能障碍有关，因为肌肉特异性敲除 Tfr1 会导致生长迟缓、代谢紊乱和致死，这表明 Tfr1 在肌肉生理学中很重要。然而，它在骨骼肌衰老和再生方面的生理功能仍未被探索。

方法

应用 RNA 测序鉴定与骨骼肌衰老相关的 Tfr1。生成了条件性 SC 缺失 Tfr1 的小鼠。在 Tfr1 缺失和 Tfr1 缺失（每组 n=6-8 只小鼠）的小鼠中，肌肉内注射心脏毒素，并分离、称重和冷冻切片，然后进行免疫染色、苏木精和伊红染色和 Masson 染色。对这些表型分析进行功能分析，如流式细胞术、跑步机、普鲁士蓝染色和透射电子显微镜，以鉴定导致再生缺陷的病理途径。

结果

通过比较四种肌肉中年轻（2 周龄，n=3）和年老（80 周龄，n=3）小鼠之间的基因表达，我们发现 Tfr1 在年老小鼠的肌肉中表达下降（~~80%，P<0.005），因此在年老小鼠的 SC 中 Tfr1 蛋白水平也下降。从体内和体外实验来看，SC 中的 Tfr1 缺失导致 SC 的不可逆耗竭（~~60%，P<0.005）和细胞自主的 SC 增殖和分化缺陷，导致骨骼肌再生受损，随后出现不稳定铁积累、脂肪生成和谷胱甘肽过氧化物酶 4（Gpx4）和核因子红细胞 2 相关因子 2（Nrf2）蛋白水平降低，导致活性氧清除缺陷。这些异常现象包括铁积累、不饱和脂肪酸生物合成的激活和脂质过氧化，与骨骼肌中铁死亡的发生有关。铁死亡进一步加剧了 SC 增殖和骨骼肌再生。铁死亡抑制剂 ferrostatin-1 不能挽救铁死亡。然而，肌肉内注射表达 Tfr1 的慢病毒可以部分减少不稳定铁的积累，减少脂肪生成，并促进骨骼肌再生。最重要的是，细胞膜上 Tfr1 蛋白水平的降低和 Slc39a14 蛋白水平的升高导致衰老啮齿动物（~80 周龄）骨骼肌中不稳定铁的积累，从而激活衰老骨骼肌中的铁死亡。这可以通过 ferrostatin-1 抑制来改善跑步时间（P=0.0257）和距离（P=0.0248）。