Entacapone alleviates muscle atrophy by modulating oxidative stress, proteolysis, and lipid aggregation in multiple mice models

Abstract

Background: Skeletal muscle atrophy significantly affects quality of life and has socio-economic and health implications. This study evaluates the effects of entacapone (ENT) on skeletal muscle atrophy linked with oxidative stress and proteolysis.

Methods: C2C12 cells were treated with dexamethasone (Dex) to simulate muscle atrophy. Four murine models were employed: diaphragm atrophy from mechanical ventilation, Dex-induced atrophy, lipopolysaccharide (LPS)-induced atrophy, and hyperlipidemia-induced atrophy. Each model utilized entacapone (10 mg/kg), with sample sizes: Control (9), MV (11), MV + ENT (5) for diaphragm atrophy; Control (4), Dex (4), Dex + ENT (5) for Dex model; Control (4), LPS (4), LPS + ENT (5) for LPS model; and similar for hyperlipidemia. Measurements included muscle strength, myofiber cross-sectional area (CSA), proteolysis, oxidative stress markers [uperoxide dismutase 1 (SOD1), uperoxide dismutase 2 (SOD2), 4-hydroxynonenal (4-HNE)], and lipid levels.

Results: Our findings confirm Dex-induced muscle atrophy, evidenced by increased expression of muscle atrophy-associated proteins, including Atrogin-1 and Murf-1, along with decreased diameter of C2C12 myotubes. Atrogin-1 levels rose by 660.6% (p < 0.05) in the Dex group compared to control, while entacapone reduced Atrogin-1 by 84.4% (p < 0.05). Similarly, Murf-1 levels increased by 365% (p < 0.05) in the Dex group and were decreased by 89.5% (p < 0.05) with entacapone. Dexamethasone exposure induces oxidative stress, evidenced by the upregulation of oxidative stress-related proteins Sod1, Sod2, and 4-HNE. Entacapone significantly reduced the levels of these oxidative stress markers, enhancing GSH-PX content by 385.6% (p < 0.05) compared to the Dex-treated group. Additionally, ENT effectively reduced the Dex-induced increase in MDA content by 63.98% (p < 0.05). Furthermore, entacapone effectively prevents the decline in diaphragm muscle strength and myofiber CSA in mice. It also mitigates diaphragm oxidative stress and protein hydrolysis. Additionally, entacapone exhibits the ability to attenuate lipid accumulation in the gastrocnemius muscle of hyperlipidemic mice and alleviate the reduction in muscle fiber CSA.

Conclusion: Our findings suggest that entacapone is a promising therapeutic candidate for muscle atrophy, functioning through the reduction of oxidative stress, proteolysis, and lipid aggregation. Future research should explore the underlying mechanisms and potential clinical applications of entacapone in muscle-wasting conditions.

Keywords: entacapone; lipid aggregation; muscle atrophy; oxidative stress; proteolysis.

FIGURE 1

Entacapone improves Dex-induced muscle atrophy in C2C12 myotubes. (A) Chemical structure of ENT. (B) Cell viability of C2C12 myotubes treated with various concentrations of ENT (50, 100, 150, 200, 250 μM) for 48 h, along with 50 μM Dex during the final 24 h. (C) Viability of C2C12 myotubes treated with different concentrations of ENT (50, 100, 150, 200, 250 μM) for 48 h, along with 150 μM Dex during the last 24 h. Statistical analyses were performed using one-way ANOVA with n = 4 per group. Date was expressed as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 indicate significant differences between the Control and Dex groups. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 indicate significant differences between the ENT and Dex groups. Dex, dexamethasone; Murf-1, muscle ring finger 1; ENT, entacapone.

FIGURE 2

Representative pictures of C2C12 myotubes treated with DMSO or 50 µM Dex, 100 µM ENT. (A) Protein levels of Atrogin-1 and Murf-1 in C2C12 myotubes treated with 50 μM Dex and 100 μM ENT, with GAPDH serving as a loading control. (B) qRT-PCR analysis of Atrogin-1 and Murf-1 in C2C12 myotubes treated with 50 μM Dex and 100 μM ENT. (C) Immunofluorescence staining and phase-contrast microscope images of C2C12 myotubes revealed that Dex induced muscle atrophy, as evidenced by a reduction in myotube diameter, while ENT mitigated Dex-induced muscle atrophy in C2C12 myotubes. Each experiment was conducted with a sample size of n = 4 per group. NC represents the control group, Dex refers to dexamethasone, ENT + Dex indicates entacapone + dexamethasone. The scale bar represents 25 μm.

FIGURE 3

ENT confers protection against Dex-induced muscle atrophy by mitigating oxidative stress in C2C12 myoblasts. The effects of ENT on the activity of GSH-PX (A) and MDA content (B) were evaluated in C2C12 myoblasts treated with 50 µM Dex, with or without ENT for 48 h. (C–F) ENT attenuated the Dex-induced increases in the expression of Murf-1, Atrogin-1, 4-HNE, SOD1, and SOD2 in C2C12 myotubes. Date was expressed as the mean ± SEM. *p < 0.05, **p < 0.01 versus NC group; ^# p < 0.05, ^## p < 0.01 versus Dex group.

FIGURE 4

ENT protects mice against ventilation-induced diaphragm dysfunction and Dex-induced muscle atrophy. (A) Diaphragm force-frequency relationship in VIDD. (B) Protein expression levels of Atrogin-1 and Murf-1. (C) H&E staining of the lungs. (D) Diaphragm force-frequency relationship in Dex-induced muscle atrophy. (E) Changes in mouse body weight. (F) ENT protects against Dex-induced diaphragm and gastrocnemius muscle atrophy. VIDD, ventilator-induced diaphragm dysfunction group; ENT, entacapone; Dex, dexamethasone group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Ctrl; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Dex.

FIGURE 5

Effects of ENT on LPS-induced muscle atrophy. (A–D) The diaphragm’s Murf-1, Atrogin-1, and 4-HNE expression levels were analyzed using Western blotting. The findings demonstrated that the LPS group exhibited an elevation in diaphragmatic oxidative stress and proteolysis levels compared to the Control group. However, ENT treatment was able to mitigate this upregulation of oxidative stress and proteolytic activity. **p < 0.01, ***p < 0.001 vs. control; ^# p < 0.05, ^## p < 0.01 vs. LPS.

FIGURE 6

Effect of ENT on skeletal muscle in APOE^−/− hyperlipidemic mice. (A) Blood lipid concentrations in C57 and APOE^−/− mice. (B) Diaphragm force-frequency relationship in C57 and APOE^−/− mice. (C) Representative immunofluorescence staining images of slow-twitch and fast-twitch fibers in the gastrocnemius. (D) Oil-red O staining of the gastrocnemius was used to evaluate lipid accumulation in C57 and APOE^−/− mice. n = 6–10 per group. TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; TG, triglycerides; LDL-C, low-density lipoprotein cholesterol. Scale bar, 50 μm.