Chen Mao, Huang Xiaoyu, Li Bingshu, Xiao Ya, Chen Liying, Zhu Fangyi, Hong Shasha, Tang Jianming, Li Suting, Min Jie, Jin Wenyi, Zhang Yubiao, Yang Lian, Li Yang, Zhang Shufei, Hong Li
Department of Gynecology and Obstetrics, Renmin Hospital of Wuhan University, Wuhan, Hubei, China.
Hubei Provincial Clinical Medical Research Center for Pelvic Floor Disease, Wuhan, Hubei, China.
J Cachexia Sarcopenia Muscle. 2025 Feb;16(1):e13725. doi: 10.1002/jcsm.13725.
Few researches have investigated the molecular mechanism responsible for the age-related loss of the pelvic floor muscle (PFM) mass and functionality-a pivotal contributor to pelvic organ prolapse and diminished physical well-being. ADP ribosylation factor GTPase activating protein 3 (ArfGAP3) is a member of ArfGAPs, which regulates the vesicular trafficking pathway and intracellular proteins transporting. However, its effects on skeletal muscle ageing remain largely unknown.
Mouse models of natural ageing and D-gal (D-galactose)-induced ageing were subject to analyse the structure, function and pathological alterations of the PFM and the expression of ArfGAP3. Stable ArfGAP3 knockdown and overexpression C2C12 cell lines were established to investigate the anti-senescence effects of ArfGAP3 and the underlying mechanisms in ageing process, complemented by Rab5a genetic intervention and mRFP-GFP-LC3 adenoviral particles transfection. In vivo experiments entailed ArfGAP3 overexpression in mice alongside autophagy inhibitor treatment, with assessments encompassing tissue mass, bladder leak point pressure (BLPP), submicroscopic structure, antioxidative stress system and muscle regeneration.
Aged (24-month-old) mice exhibited significant physiological alterations in PFMs, including decreased muscle mass, diminished cross-sectional area (CSA), deteriorated supporting function (as evidenced by reduced BLPP), impaired autophagy and increased levels of oxidative stress (p < 0.001). Utilizing ageing C2C12 model, we observed a dose-dependent relationship between D-gal induction and cellular senescence, impaired differentiation and mitochondrial damage. Remarkably, the expression levels of ArfGAP3 were markedly downregulated in both in vitro and in vivo ageing models. Knockdown of ArfGAP3 exacerbated impaired differentiation potential and induced aberrant mitochondrial morphology and functional dysfunction in ageing C2C12 myoblasts, whereas ArfGAP3 overexpression largely mitigated these effects. Mechanistically, our findings revealed an interplay between ArfGAP3 and Rab5a, indicating their coordinated regulation. ArfGAP3-mediated activation of Rab5a-associated autophagy and IRS1-AKT-mTOR signalling pathways during cellular senescence and myogenesis was identified, leading to enhanced autophagic flux and improved resistance to oxidative stress. In vivo, ArfGAP3 overexpression ameliorated D-gal-induced loss of muscle mass and function, while promoting antioxidant responses and muscle regeneration in mice. However, these protective effects of ArfGAP3 overexpression were extinguished by autophagy inhibition.
Our study uncovers the significant role of ArfGAP3 in enhancing differentiation capacity and mitochondrial function through mediating Rab5a expression to activate IRS1-AKT-mTOR signalling pathways and promote autophagy during the ageing process. These findings underscore the potential of ArfGAP3 as a promising therapeutic target for ameliorating the decline in skeletal muscle function associated with ageing.
很少有研究探讨导致盆底肌（PFM）质量和功能随年龄增长而丧失的分子机制，而盆底肌质量和功能的丧失是盆腔器官脱垂和身体健康下降的关键因素。ADP核糖基化因子GTP酶激活蛋白3（ArfGAP3）是ArfGAPs家族的成员，它调节囊泡运输途径和细胞内蛋白质运输。然而，其对骨骼肌衰老的影响在很大程度上仍不清楚。
对自然衰老和D-半乳糖（D-gal）诱导衰老的小鼠模型进行分析，以研究盆底肌的结构、功能和病理改变以及ArfGAP3的表达。建立稳定的ArfGAP3基因敲低和过表达的C2C12细胞系，以研究ArfGAP3的抗衰老作用及其在衰老过程中的潜在机制，并通过Rab5a基因干预和mRFP-GFP-LC3腺病毒颗粒转染进行补充。体内实验包括在小鼠中过表达ArfGAP3并进行自噬抑制剂处理，评估内容包括组织质量、膀胱漏点压力（BLPP）、亚微观结构、抗氧化应激系统和肌肉再生。
老年（24月龄）小鼠的盆底肌出现明显的生理改变，包括肌肉质量下降、横截面积（CSA）减小、支撑功能恶化（如BLPP降低所示）、自噬受损和氧化应激水平升高（p<0.001）。利用衰老的C2C12模型，我们观察到D-半乳糖诱导与细胞衰老、分化受损和线粒体损伤之间存在剂量依赖性关系。值得注意的是，在体外和体内衰老模型中，ArfGAP3的表达水平均显著下调。敲低ArfGAP3会加剧衰老的C2C12成肌细胞分化潜能受损，并导致线粒体形态异常和功能障碍，而ArfGAP3过表达则在很大程度上减轻了这些影响。从机制上讲，我们的研究结果揭示了ArfGAP3与Rab5a之间的相互作用，表明它们的协同调节。在细胞衰老和肌生成过程中，ArfGAP3介导的Rab5a相关自噬和IRS1-AKT-mTOR信号通路的激活被确定，从而导致自噬通量增加和对氧化应激的抵抗力提高。在体内，ArfGAP3过表达改善了D-半乳糖诱导的肌肉质量和功能丧失，同时促进了小鼠的抗氧化反应和肌肉再生。然而，自噬抑制消除了ArfGAP3过表达的这些保护作用。
我们的研究揭示了ArfGAP3在衰老过程中通过介导Rab5a表达来激活IRS1-AKT-mTOR信号通路并促进自噬，从而增强分化能力和线粒体功能方面的重要作用。这些发现强调了ArfGAP3作为改善与衰老相关的骨骼肌功能下降的有前景的治疗靶点的潜力。
