Nestin Regulates Autophagy-Dependent Ferroptosis Mediated Skeletal Muscle Atrophy by Ubiquitinating MAP 1LC3B

IF 9.4 1区医学 Q1 GERIATRICS & GERONTOLOGY

Journal of Cachexia Sarcopenia and Muscle Pub Date : 2025-04-04 DOI:10.1002/jcsm.13779

Shunshun Han, Xiyu Zhao, Chunlin Yu, Can Cui, Yao Zhang, Qing Zhu, Mohan Qiu, Chaowu Yang, Huadong Yin

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Abstract

Background

Programmed cell death plays a critical role in skeletal muscle atrophy. Ferroptosis, an iron-dependent form of programmed cell death driven by lipid peroxidation, has been implicated in various diseases, but its role in skeletal muscle atrophy remains unclear.

Methods

Ferroptosis in skeletal muscle atrophy was investigated using two models: dexamethasone (Dex)-induced atrophy (n = 6 independent cell cultures per group) and simulated microgravity (n = 6 mice per group). Conditional Nestin knockout (KO) mice were generated using CRISPR/Cas9 (n = 6–8 mice per group), with wild-type (WT) controls (n = 6–8). Phenotypic analyses included histopathology (HE staining), functional assessments (muscle strength, weight analysis, treadmill), and dystrophy evaluation (dystrophin staining). Molecular analyses involved flow cytometry, ELISA, transmission electron microscopy, PI staining, and IP/MS to delineate Nestin-regulated ferroptosis pathways in skeletal muscle atrophy.

Results

Ferroptosis was significantly activated in both atrophy models, with a 2.5-fold increase in lipid peroxidation (p < 0.01), a 2-fold accumulation of Fe²⁺ (p < 0.01) and a 50% reduction in Nestin expression (p < 0.001). Nestin KO mice exhibited exacerbated muscle atrophy, showing a 40% decrease in muscle weight (p < 0.01) and a 30% reduction in muscle strength (p < 0.05) compared to WT mice. Nestin overexpression mitigated Dex-induced ferroptosis, reducing lipid peroxidation by 40%, decreasing Fe²⁺ accumulation by 50% (p < 0.01), and improving muscle function by 30% (p < 0.05). Mechanistically, Nestin interacted with MAP 1LC3B (LC3B) to catalyse LC3B polyubiquitination at lysine-51, reducing LC3B availability for autophagy and inhibiting autophagy flux by 60% (p < 0.01), leading to a 50% reduction in ferroptosis (p < 0.001).

Conclusions

Our study identifies Nestin as a critical regulator of ferroptosis-autophagy crosstalk in skeletal muscle atrophy. Targeting Nestin-LC3B ubiquitination may offer novel therapeutic strategies for preventing muscle wasting in diseases such as cachexia and sarcopenia.

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巢蛋白通过泛素化MAP 1LC3B调控自噬依赖性铁下垂介导的骨骼肌萎缩

细胞程序性死亡在骨骼肌萎缩中起关键作用。铁凋亡是一种由脂质过氧化引起的铁依赖性程序性细胞死亡形式，与多种疾病有关，但其在骨骼肌萎缩中的作用尚不清楚。方法采用地塞米松（Dex）诱导的骨骼肌萎缩模型（每组n = 6个独立细胞培养）和模拟微重力（每组n = 6只小鼠）两种模型研究骨骼肌萎缩中的铁下垂。使用CRISPR/Cas9技术生成条件巢蛋白敲除（KO）小鼠（每组n = 6-8只），野生型（WT）对照组（n = 6-8只）。表型分析包括组织病理学（HE染色）、功能评估（肌力、体重分析、跑步机）和营养不良评估（肌营养不良蛋白染色）。分子分析包括流式细胞术、ELISA、透射电镜、PI染色和IP/MS来描绘巢蛋白调节的骨骼肌萎缩中的铁下垂途径。结果在两种萎缩模型中，铁下垂均被显著激活，脂质过氧化增加2.5倍（p < 0.01）， Fe2+积累2倍（p < 0.01）， Nestin表达减少50% （p < 0.001）。与WT小鼠相比，Nestin KO小鼠肌肉萎缩加剧，肌肉重量减少40% (p < 0.01)，肌肉力量减少30% （p < 0.05）。Nestin过表达减轻了dex诱导的铁下垂，减少了40%的脂质过氧化，减少了50%的Fe2+积累（p < 0.01），改善了30%的肌肉功能（p < 0.05）。在机制上，Nestin与MAP 1LC3B （LC3B）相互作用，在赖氨酸-51位点催化LC3B多泛素化，降低LC3B自噬的可用性，抑制自噬通量60% (p < 0.01)，导致铁死亡减少50% （p < 0.001）。结论本研究确定Nestin是骨骼肌萎缩中铁凋亡-自噬串扰的关键调节因子。针对巢蛋白lc3b泛素化可能为预防恶病质和肌肉减少症等疾病的肌肉萎缩提供新的治疗策略。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Cachexia Sarcopenia and Muscle MEDICINE, GENERAL & INTERNAL-

CiteScore

13.30

自引率

12.40%

发文量

234

审稿时长

16 weeks

期刊介绍： The Journal of Cachexia, Sarcopenia and Muscle is a peer-reviewed international journal dedicated to publishing materials related to cachexia and sarcopenia, as well as body composition and its physiological and pathophysiological changes across the lifespan and in response to various illnesses from all fields of life sciences. The journal aims to provide a reliable resource for professionals interested in related research or involved in the clinical care of affected patients, such as those suffering from AIDS, cancer, chronic heart failure, chronic lung disease, liver cirrhosis, chronic kidney failure, rheumatoid arthritis, or sepsis.