ArfGAP3 Protects Mitochondrial Function and Promotes Autophagy Through Rab5a-Mediated Signals in Ageing Skeletal Muscle

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: ArfGAP3; Rab5a; ageing; autophagy; pelvic floor muscle.

FIGURE 1

ArfGAP3 was downregulated with skeletal muscle ageing. (A) Weights of four types of muscles (PFM: pelvic floor muscle, GA: gastrocnemius, TA: tibialis anterior and SOL: soleus) in mice from four different groups (n = 15). (B) The average bladder leak point pressure (BLPP) values from the mice in the four groups (n = 15). (C) H&E staining of PFM myofibres (magnification 200×; scale bar = 50 μm) alongside measurement of the mean cross‐sectional area (CSA) of PFM fibres via ImageJ software (upper panels) is presented. The analysis included seven mice, with CSA measurement of more than 50 muscle fibres per mouse as evaluated through HE staining (n = 7). The lower panel shows IHC images of ArfGAP3 in PFM of mice (magnification 200×; scale bar = 50 μm) and the quantitative assessment of ArfGAP3 expression intensity using ImageJ software. (D) Western blot analysis and quantification of ArfGAP3, P16 and P53 protein levels in mouse PFM. (E) qRT‐PCR analysis of the expression of Arfgap1, Arfgap2 and Arfgap3. β‐actin was used as the loading control. (F) Representative images of the ultrastructure showing muscle bundles, autophagosomes (pink arrows) and abnormal mitochondrial organelles (white arrows) of PFM from the four groups (magnification = × 12.0k). Quantification of the mean number of autophagosomes per area. All data were presented as mean ± SD and analysed using one‐way ANOVA. *p < 0.05/**p < 0.01/***p < 0.001 versus 3 months group; ^& p < 0.05/^&& p < 0.01 /^&&& p < 0.001 versus 8 months group; ^# p < 0.05/^## p < 0.01/^### p < 0.05 versus 12 months group.

FIGURE 2

ArfGAP3 was downregulated in aged skeletal muscle and senescent C2C12 myoblasts induced by D‐gal. (A) Wet weights of PFM muscle samples (n = 15). (B) Average bladder leak point pressure (BLPP) values of mice (n = 15). (C) Western blots and quantification of ArfGAP3, P16 and P53 protein levels in mouse PFM. (D) SA‐β‐gal staining and quantification for C2C12 myoblasts treated with different concentrations of D‐gal on day 0 (D0) (top panels) and day 5 (D5) (bottom panels) after differentiation induction, and light microscopic images of unstained C2C12 myotubes. (E) Western blot analysis and quantification of ArfGAP3, P16 and P53 proteins in C2C12 myoblasts after D‐gal treatment for 48 h (D0) and protein levels of MyHC, MyoG and MyoD after differentiation for 5 days (D5). (F) Fluorescence staining and quantification for the measure of cellular ROS using DCFH in C2C12 myoblasts (scale bar = 100 μm). Data were expressed as the mean ± SD and statistically analysed via one‐ or two‐way ANOVA. **p < 0.05/**p < 0.01/***p < 0.001 versus Con group or 0 g/L group; ^&& p < 0.01/^&&& p < 0.001 versus 2 months group.

FIGURE 3

Downregulation of ArfGAP3 in C2C12 myoblasts impaired differentiation, proliferation capacity and mitochondrial function. (A) SA‐β‐gal staining and corresponding quantification in C2C12 myoblasts; scale bar = 100 μm. (B) Representative images from EdU staining and quantification of the relative percentages of EdU‐positive myoblasts in EdU assays after transfection with lentiviral vector with or without 20 g/L D‐gal treatment; scale bar = 50 μm. (C) Representative confocal microscopy images of MitoTracker (red) staining in C2C12 myoblasts. Scale bar = 20 μm; (D) Western blot analysis and quantification of ArfGAP3, P16 and P53 protein levels. (E) Microscopic visualization of myotube formation (upper panels) and representative immunofluorescence images of C2C12 myotubes with MyhC stain (bottom panels). Quantifications were for myotube diameter and the percentage of MyhC‐positive nuclei (differentiation index). Scale bar = 20 μm. (F) Western blot analysis and respective quantification for MyhC, MyoG and MyoD after differentiation for 5 days. All data were presented as mean ± SD. All analyses were conducted using two‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Overexpression of ArfGAP3 improved the differentiation capacity by rescuing mitochondrial function in D‐gal–treated C2C12 myoblasts. (A) SA‐β‐gal staining and quantification in C2C12 myoblasts; scale bar = 100 μm. (B) Representative images of EdU staining and quantification of the relative percentages of EdU‐positive myoblasts after transfection with lentiviral vector with or without 20 g/L D‐gal treatment; scale bar = 100 μm. (C) Representative confocal microscopy images of MitoTracker (red) staining in C2C12 myoblasts. Scale bar = 20 μm; (D) Western blot analysis and quantification of ArfGAP3, P16 and P53 protein levels. (E) Microscopic visualization of myotube formation (upper panels) and representative immunofluorescence images of C2C12 myotubes with MyhC stain (bottom panels). Quantifications were for myotube diameter and the percentage of MyhC‐positive nuclei (differentiation index). Scale bar = 20 μm. (F) Western blot analysis and respective quantification for MyhC, MyoG and MyoD after differentiation for 5 days. All data were presented as mean ± SD. All analyses were conducted using two‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Rab5a played a critical role in the effect of ArfGAP3. (A) Comparison of relative mRNA expressions of Rab1, Rab5, Rab7, Rab11 and Rab34 in PFM of 3‐, 8‐, 12‐ and 24‐month‐old mice. (B) Correlation graphs between the mRNA and protein expression of ArfGAP3 and Rab5a in PFM of 3‐, 8‐, 12‐ and 24‐month‐old mice and in C2C12 myoblasts treated with D‐gal. (C) Images of immunofluorescence staining to present spatial expression patterns of ArfGAP3 (red) and Rab5a (green) in C2C12 myoblasts after transfection. Scale bar = 10 μm. The colocalization scatter plot (bottom panels) represents fluorescence intensity profiles extracted for quantification. (D) Coimmunoprecipitation (CoIP) experiment using an antibody against ArfGAP3 to detect the interaction between ArfGAP3 and Rab5a in C2C12 myoblasts with or without 20 g/L D‐gal treatment, and the CoIP products were subjected to immunoblotting assay of ArfGAP3 and Rab5a. (E) Western blot analysis and quantification of the temporal expression of MyHC, ArfGAP3, Rab5a, P16, P53 and LC3 in C2C12 myoblasts during myotube differentiation from 0 (D0) to 14 days (D14). (F) Western blot analysis and quantification of LC3 II/LC3I ratio in C2C12 myoblasts after ArfGAP3 overexpression with or without 20 g/L D‐gal treatment. All data were presented as mean ± SD. All analyses were done using one‐ or two‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Rab5a‐mediated autophagy was activated in the regulatory effect of ArfGAP3. (A) SA‐β‐gal staining and quantification in C2C12 myoblasts with Rab5a overexpression in the presence of 20 g/L D‐gal; scale bar = 100 μm. (B) Western blot analysis and quantification of ArfGAP3, Rab5a, P16 and P53 proteins. (C) Contents of MDA in C2C12 cells and activities of antioxidant enzymes including CAT, GST and SOD. (D) ROS production evaluated by DCFH‐DA and ImageJ used for quantitative analysis. (E) Immunofluorescent staining for MyHC in myotubes after differentiation for 5 days and quantification for myotube diameter and the percentage of MyhC‐positive nuclei (differentiation index). Scale bar = 20 μm. (F) Western blot analysis and respective quantification for MyhC, MyoG and MyoD after differentiation for 5 days. Data were presented as mean ± SD. Statistical analyses were conducted using one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Rab5a overexpression rescued autophagy and activated signalling IRS1‐AKT‐mTOR pathway. C2C12 transfected with sh‐ArfGAP3 or/and Rab5a‐overexpression plasmid, followed by treatment with or without 20 g/L D‐gal. (A) Western blot analysis of LC3 and associated quantification. (B) Representative phase contrast, fluorescence photomicrographs and the quantification of GFP‐RFPLC3 puncta in C2C12 myoblasts were shown. Yellow puncta denote autophagic vesicle structures. The scale bars represent 20 μm. (C) Protein levels of Rab5a‐activated IRS1‐AKT‐mTOR signalling by western blotting analysis. All data were presented as mean ± SD. All analyses were done using one‐way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8

ArfGAP3 overexpression improved muscle mass and promoted the antioxidant response and autophagy in ageing mice. (A) Schematic diagram of the mouse experiment and the mRNA expression levels of ArfGAP3 were validated using qRT‐PCR. (B) The average bladder leak point pressure (BLPP) values from the mice. (C) H&E staining of PFM muscle fibres (magnification 200×, scale bar = 50 μm) and the CSA of muscle fibres measured by ImageJ software (upper panels). IHC images and quantification for ArfGAP3 in mouse PFM muscle (magnification 200×; scale bar = 50 μm; bottom panels). (D) Western blotting showing the protein levels of ArfGAP3, Rab5a, myogenic markers (MyHC, MyoD and MyoG), ageing‐related markers (P16 and P53) and autophagy‐related proteins (LC3). β‐Actin was used as the loading controls to quantify the relative protein levels. (E) Representative mitochondria were observed via transmission electron micrographs (magnification 12 000×) and quantification of the mean number of autophagosomes per area. Scale bar = 1.0 μm. (F) Representative images for Evans blue dye (EBD) assay, HE staining and immunofluorescence staining of PFM after 3 days of CTX injection in different groups were shown for the muscle histology. Scale bar = 50 μm. Quantification of EBD fluorescence intensity (red), CSA of centralized nuclear muscle fibres (μm²) and immunofluorescence intensities of ArfGAP3 and Pax 7 were showed on the right. The data are expressed as the mean ± SD and were analysed using one‐way ANOVA, ns, no significance, *p < 0.05, **p < 0.01 and ***p < 0.001; n = 5 mice per group.