Spermidine coupled with exercise rescues skeletal muscle atrophy from D-gal-induced aging rats through enhanced autophagy and reduced apoptosis via AMPK-FOXO3a signal pathway

Abstract

The quality control of skeletal muscle is a continuous requirement throughout the lifetime, although its functions and quality present as a declining trend during aging process. Dysfunctional or deficient autophagy and excessive apoptosis may contribute to the atrophy of senescent skeletal muscle. Spermidine, as a natural polyamine, can be involved in important cellular functions for lifespan extension and stress resistance in several model organisms through activating autophagy. Similarly, cellular autophagic responses to exercise have also been extensively investigated. In the present study, in order to confirm the mitigation or amelioration of skeletal muscle atrophy in aging rats through spermidine coupled with exercise intervention and explore corresponding mechanisms, the rat model with aging-related atrophy of skeletal muscle was established by intraperitoneal injection of D-galactose (D-gal) (200 mg/kg∙d), and model rats were subjected to the intervention with spermidine (5 mg/kg∙d)) or swimming (60 min/d, 5 d/wk) or combination for 42 days. Spermidine coupled with exercise could attenuate D-gal-induced aging-related atrophy of skeletal muscle through induced autophagy and reduced apoptosis with characteristics of more autophagosomes, activated mitophagy, enhanced mitochondrial quality, alleviated cell shrinkage, and less swollen mitochondria under transmission scanning microscopic observation. Meanwhile, spermidine coupled with exercise could induce autophagy through activating AMPK-FOXO3a signal pathway with characterization of increased Beclin1 and LC3-II/LC3-I ratio, up-regulated anti-apoptotic Bcl-2, down-regulated pro-apoptotic Bax and caspase-3, as well as activated AMPK and FOXO3a. Therefore, spermidine combined with exercise can execute the prevention or treatment of D-gal-induced aging-related skeletal muscle atrophy through enhanced autophagy and reduced apoptosis mediated by AMPK-FOXO3a signal pathway.

Keywords: AMPK-FOXO3a signal pathway; D-galactose; Gerotarget; autophagy; exercise; spermindine.

Figure 1. Spermidine coupled with exercise training reduced D-gal-induced damage of skeletal muscle, and suppressed or rescued the decline of sectional area of skeletal muscle fibers

Cross sectional area was measured and calculated from 150 skeletal muscle fibers in H&E stained sections using imageJ software. All rats were divided into five groups (n = 7) including rats treated with saline (Con), rats treated with D-gal (D), rats treated with D-gal coupled with spermidine (DS), rats treated with D-gal coupled with exercise training (DE), and rats treated with D-gal and spermidine as well as exercise training (DES) (×200).

Figure 2. Effects of spermidine and/or exercise on the damage of gastrocnemius muscle in D-gal-induced aging rats through the evaluation of SA-β-gal staining (×40)

The positive cells with SA-β-gal staining (blue) due to D-gal-induced senescence exhibited a decrease in spermidine and exercise groups when compared with D-gal-treated group.

Figure 3. Transmission electron microscopic images showed damaged or swollen or fusion mitochondria, and disordered fiber arrangement or dysfunctional fiber structure in D-gal-induced aging gastrocnemius muscle (×20000)

Spermidine and/or exercise improved the disordered fiber arrangement and reduced the population of damaged or fusion mitochondria of D-gal-induced aging skeletal muscle fibers. White arrows represent the region of mitochondria, and black arrows point to the autophagosomes in gastrocnemius muscle.

Figure 4. Effects of spermidine and exercise on SOD activity and MDA content of aging model rats (n = 5)

*P < 0.05, ***P < 0.001 when compared with the normal control group. ^#P < 0.05 when compared with the D-gal group.

Figure 5. Spermidine and exercise resulted in the activation of autophagy in skeletal muscle and the recovered autophagy during the D-gal-induced atrophy process of skeletal muscle due to up-regulated Beclin-1 and down-regulated p62 as well as increased LC3-II/LC3-I ratio

GAPDH was used as the loading control. *P < 0.05, **P < 0.01 when compared with the control group; ^#P < 0.05 when compared with the D-gal group.

Figure 6. Apoptosis of skeletal muscle cells in different groups was evaluated by TUNEL assay, and the images were recorded by an Olympus fluorescence microscope, captured with a CCD monochrome camera Sensystem1401E at the magnification of ×800

***P < 0.001 when compared with the control group; ^###P < 0.001 when compared with the D-gal group.

Figure 7. Spermidine combined with exercise could reduce apoptosis in D-gal-induced atrophy of gastrocnemius muscle

A. Immunofluorescence assay was used to localize the expression of cleaved caspase-3 (green). Nuclei were marked with DAPI (blue), magnification (×800). B. Expression of apoptosis-associated proteins and quantitative analysis for Western blotting. GAPDH was used as the loading control. Data were expressed as mean ± standard error (M ± SE). **P < 0.01, ***P < 0.001 when compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 when compared with the D-gal group.

Figure 8. Spermidine combined with exercise enhanced AMPK-FOXO3a signal pathway in D-gal-induced aging gastrocnemius muscle

A. Protein expression of p-AMPK (Thr72)/AMPKα (D5A2) and FOXO3a. B. Quantitative analysis for Western blotting. GAPDH was used as the loading control. Data were expressed as mean ± standard error (M ± SE). *P < 0.05, ***P < 0.001 when compared with the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 when compared with the D-gal group.

Figure 9. The synergistic effect of spermidine and exercise on the attenuation of aging-related skeletal muscle atrophy through regulating autophagy and apoptosis

via AMPK-FOXO3a signal pathway.