Acupuncture treatment preserves soleus muscle mass and improves mitochondrial function in a rat model of disuse atrophy

Abstract

Background: Muscle atrophy leads to debilitating loss of physical capacity, particularly when alternative treatments are needed. Acupuncture is proposed as a potential therapy for disuse atrophy, but its effects on muscle biology remain unclear. This study evaluated the effects of acupuncture on soleus muscle mass and mitochondrial function in a rat model of immobilization-induced atrophy.

Methods: Female Sprague Dawley rats were assigned to three groups: Control (CON), casting-induced immobilization (CT), and CT with acupuncture (CT-A) (n = 8). Immobilization of the left hindlimb lasted for 14 days, and acupuncture was performed at specific acupoints (stomach-36, gallbladder-34) three times per week for 15 min. Mitochondrial function was assessed in saponin-permeabilized fibers, and signaling molecules regulating muscle mass were analyzed by Western blot.

Results: CT-A attenuated soleus muscle atrophy compared to CT. Under fatty acid substrate conditions, CT reduced complex I and II-supported oxidative phosphorylation (OXPHOS) compared to CON, while CT-A decreased respiratory leak and enhanced OXPHOS coupling relative to CT. Without fatty acids, CT-A decreased both respiratory leak and complex I and II-supported OXPHOS compared to CON, but differences between CT and CT-A were not significant. AMPKα activity (p-AMPKα/AMPKα) was significantly elevated in the CT group compared to the CON group, but returned to CON levels in the CT-A group. However, there were no changes in proteins associated with muscle atrophy or autophagy markers among the groups.

Conclusion: Acupuncture mitigates immobilization-induced muscle atrophy and preserves mitochondrial function, suggesting its potential as a therapeutic approach for muscle disuse conditions.

Keywords: Acupuncture; Immobilization; Mitochondrial respiration; Muscle atrophy.

Fig. 1

The effects of acupuncture on body and muscle weight. (A) Final BW; (B) Soleus muscle; (C) Sol wt./BW. Values are expressed as mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001. n = 8/group. Each filled dot, square, or triangle represents an individual animal. CON: control; CT: cast; CT-A: acupuncture treatment during the 14-day immobilization period; BW: body weight.

Fig. 2

Mitochondrial respiratory function. (A-D) OXPHOS capabilities without fatty acids: (A) Leak respiration observed with pyruvate and malate (PM_L); (B) Pyruvate and malate (PM)-stimulated OXPHOS respiration following ADP addition (PMp); (C) Coupling efficiency under pyruvate and malate; (D) Peak OXPHOS rates across Complex I and II (CI+CIIp). (E-I) OXPHOS in the presence of fatty acid substrates; (E) Fatty acid-induced leak respiration (FAO_L); (F) OXPHOS activation by ADP addition (FAOp); (G) Coupling efficiency under fatty acid conditions. (H) OXPHOS performance with PalM support (FAOp CIp); (I) Maximum OXPHOS respiration facilitated by Complex I and II (FAOp CI+CIIp). Values are expressed as mean ± SEM. n = 6–7/group. Each filled dot, square, or triangle represents an individual animal. CON: control; CT: cast; CT-A: acupuncture treatment during the 14-day immobilization period; CI: complex I; CII: complex II; FAO: fatty acid oxidation; JO₂: oxygen flux.

Fig. 3

Proteins associated with OXPHOS capacity in tissue lysates. (A) Citrate synthase (CS) activity normalized to mg of protein. (B) Cytochrome c oxidase (COX) activity normalized to mg of protein. (C) Quantification of ETC proteins with representative Western blot images. (D) Quantification of UCP3 protein normalized to vinculin as a loading control, with representative Western blot images. Values are presented as mean ± SEM, derived from 4–7 animals per group, with 4 technical replicates per animal. Ponceau staining served as the loading control for OXPHOS protein content. Each filled dot, square, or triangle represents an individual animal. *P < 0.05, **P < 0.01. CON: control; CT: cast; CT-A: acupuncture treatment during the 14-day immobilization period. CS: citrate synthase; CI-NDUFB8: NADH dehydrogenase 1, Complex I; CII-SDHB: succinate dehydrogenase iron-sulfur subunit B, Complex II; CIII-UQCRC2: ubiquinol-cytochrome c reductase core protein 2, Complex III; CIV-MTCO1: mitochondrially encoded cytochrome c oxidase 1, Complex IV; CV-ATP5F1A: ATP synthase F1 subunit alpha, Complex V.

Fig. 4

Proteins involved in the regulation of muscle mass. (A) mTOR activity, (B) AKT activity, and (C) AMPKα activity. (D) Beclin-1 expression, (E) the LC3B II/I ratio, (F) Atrogin-1 levels; The fourth lane from the left (CON group) was excluded from analysis due to sample degradation and is not included in the quantification, and (G) MuRF-1 levels. Protein activity is expressed as the phosphorylated protein to total protein ratio (A-C). For panels (D-G), total protein levels were normalized to vinculin. Representative Western blot images are provided below each quantified graph. Ponceau staining was used to verify transfer efficiency and equal protein loading. Values are presented as mean ± SEM, derived from 5–8 animals per group, with 4 technical replicates per animal. Each filled dot, square, or triangle represents an individual animal. * P < 0.05, ** P < 0.01. CON: control; CT: cast; CT-A: acupuncture treatment during the 14-day immobilization period.