Exercise-induced mitochondrial protection in skeletal muscle of ovariectomized mice: A myogenic E2 synthesis-independent mechanism

Abstract

Background: Skeletal muscle, a 17β-estradiol (E₂)-sensitive tissue, is prone to accelerated aging due to postmenopausal E₂ deficiency and subsequent mitochondrial dysfunction. While exogenous E₂ treatment has been shown to protect against mitochondrial damage in ovariectomized rodents, the impact of exercise-induced local E₂ production in skeletal muscle on mitochondrial function remains to be determined. This study investigated exercise-mediated mitochondrial protection in ovariectomized mice and the contribution of myogenic E₂.

Methods: Female C57BL/6J mice (8-week-old) were divided into Sham, OVX, and OVX + ET groups (N = 12). OVX mice underwent bilateral ovariectomy, with the OVX + ET group performing 8 weeks of treadmill exercise starting 10 weeks post-surgery. Functional tests (grip strength, fatigue resistance) and gastrocnemius analyses (morphology, mitochondrial function, E2/antioxidant levels, and protein expression) were conducted. Parallel experiments in muscle-specific aromatase knockout (MS-ARO-CKO) mice included E2 supplementation via subdermal pellets.

Results: 18 weeks after ovariectomy (OVX), C57BL/6J mice exhibited significant reductions in grip strength (∼30 %), rotarod performance (∼57 %), and grid hanging performance (∼92 %). Concomitantly, OVX led to marked decreases in mitochondrial respiration (p < 0.05) and antioxidant capacity (p < 0.05) in the gastrocnemius muscle, accompanied by alterations in mitochondrial quality control and antioxidant signaling proteins (p < 0.05). Exercise intervention effectively attenuated these OVX-induced deficits, accompanied by a 66 % increase in E₂ levels and upregulation of aromatase (ARO) activity and expression (p < 0.05). In MS-ARO-CKO mice model, exercise failed to improve the impaired antioxidant capacity induced by OVX. However, exercise, similar to estrogen supplementation, restored mitochondrial function and related protein expression abnormalities induced by OVX (p < 0.05).

Conclusions: Our findings demonstrate that the protective effects of exercise on skeletal muscle mitochondria involve multiple mechanisms, independent myogenic E₂ Synthesis, providing novel insights for improving skeletal muscle health in postmenopausal women.

Keywords: Exercise training; Mitochondrial function; Muscle weakness; Myogenic 17β-estradiol.

Fig. 1

Characterizations of the effects of exercise training on ovariectomized mice. A,B,C, Changes in grip strength, inverted grid fatigability, and Rota-rod endurance of mice. D, Representative images of HE staining, MGT staining, and NADH-TR staining. Scale bar: 50 μm. E, Frequency of cross-sectional area (CSA) distribution for GAS muscles. F, Average CSA of GAS muscles. G, %NADH-TR positive fibers was quantified by calculating the percentage of weak, intermediate, or strong staining intensity of the whole muscle. H, NADH-TR activity was quantified by calculating the average of total pixel intensity. n = 3∼6 mice per group. ∗P < 0.05, ∗∗P < 0.01.

Fig. 2

Exercise training restores respiratory capacity, mitochondrial morphology, biogenesis, and dynamics in skeletal muscle of ovariectomized mice. A, Oroboros O2k representative tracings of red gastrocnemius (RG). B, Mitochondrial-specific O2 flux in permeabilized fibers with Krebs cycle substrates and inhibitors. C, ATP content in GAS muscle. D, Mitochondrial DNA content evaluated by the ratio of a mitochondrial encoded gene (mt-Cytb) and a nuclear-encoded gene (Cycs). E, Representative electron micrograph of the intermyofibrillar area of the GAS muscle. Scale bar = 1 μm. F, Representative images of Western blot and semiquantitative analyses of the bands of OXPHOS proteins in GAS muscle. G, Representative images of Western blot and semiquantitative analyses of the bands of mitochondrial biogenesis and fusion/fission proteins in GAS muscle. n = 3∼6 mice per group. ∗P < 0.05, ∗∗P < 0.01.

Fig. 3

Exercise training rescues antioxidant capacity in skeletal muscle of ovariectomized mice. A, Representative images of Western blot and semiquantitative analyses of the bands of antioxidant signaling pathway proteins in GAS muscle. B, C, D, E, SOD, CAT and GPx activities and MDA content in mice GAS muscle. n = 6. ∗P < 0.05, ∗∗P < 0.01.

Fig. 4

Alterations in aromatase activity, E₂ levels, and ER expression in GAS muscle. A, Representative image of aromatase immunofluorescence assay. B, Semiquantitative analyses of the fluorescence intensity of aromatase protein in GAS muscle. C, Representative images of Western blot and semiquantitative analyses of the bands of aromatase protein in GAS muscle. D, Aromatase activity analysis. E, Representative image of estradiol immunofluorescence assay. F, Semiquantitative analyses of the fluorescence intensity of estradiol in GAS muscle. G, Representative images of Western blot and semiquantitative analyses of the bands of ERα and ERβ proteins in GAS muscle. H, Quantitative analysis of the mRNA expression of ERα and ERβ in GAS muscle. n = 6 mice per group. ∗P < 0.05, ∗∗P < 0.01.

Fig. 5

Construction of muscle-specific ARO knockout mice. A, A chart described an organization-specific knockout strategy. B, C, PCR detected ARO gene and Cre gene expression in mouse tail genomic DNA. D, Western blot showed the expression of ARO in the heart, kidney, brain, and liver from ARO^fl/fl and ARO;Ckm-Cre female mice. E, Western blot revealed the expression of ARO protein in the GAS, TA, SOL, and EDL muscles from ARO^fl/fl and ARO;Ckm-Cre female mice. F, Immunofluorescence confirmation of aromatase expression (red) in GAS, TA, Sol, and EDL muscle. Scale bar: 50 μm. G, Relative intensity of aromatase in GAS, TA, SOL, and EDL muscle was quantified following confocal analysis above. H, Immunofluorescence confirmation of E₂ level (green) in GAS, TA, SOL, and EDL muscle. Scale bar: 50 μm. I, Relative quantification of E₂ levels in GAS, TA, SOL, and EDL muscle. n = 6 mice per group. ∗P < 0.05, ∗∗P < 0.01 vs. Flox group.

Fig. 6

Exercise training or E₂ supplementation restores respiratory capacity, mitochondrial morphology, biogenesis, and dynamics in skeletal muscle of ovariectomized mice with ARO gene knockout. A, Oroboros O2k representative tracings of red GAS (RG). B, Mitochondrial-specific O2 flux in permeabilized fibers with Krebs cycle substrates and inhibitors. C, ATP content in GAS muscle. D, Mitochondrial DNA content evaluated by the ratio of a mitochondrial encoded gene (mt-Cytb) and a nuclear-encoded gene (Cycs). E, Representative electron micrograph of the intermyofibrillar area of the GAS muscle. Scale bar = 1 μm. F, Representative images of Western blot and semiquantitative analyses of the bands of OXPHOS proteins in GAS muscle. G, Representative images of Western blot and semiquantitative analyses of the bands of mitochondrial biogenesis and fusion/fission proteins in GAS muscle. n = 3∼6 mice per group. ∗P < 0.05, ∗∗P < 0.01.

Fig. 7

E₂ supplementation, but not exercise training, restored antioxidant systems in OVX mice with ARO knockout. A, Representative images of Western blot and semiquantitative analyses of the bands of antioxidant signaling pathway proteins in GAS muscle. B, C, D, E, SOD, CAT and GPx activities and MDA content in mice GAS muscle. n = 6. ∗P < 0.05, ∗∗P < 0.01.

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