Beneficial Effects of Resveratrol in Mouse Gastrocnemius: A Hint to Muscle Phenotype and Proteolysis

Abstract

We hypothesized that the phenolic compound resveratrol mitigates muscle protein degradation and loss and improves muscle fiber cross-sectional area (CSA) in gastrocnemius of mice exposed to unloading (7dI). In gastrocnemius of mice (female C57BL/6J, 10 weeks) exposed to a seven-day period of hindlimb immobilization with/without resveratrol treatment, markers of muscle proteolysis (tyrosine release, systemic troponin-I), atrophy signaling pathways, and muscle phenotypic features and function were analyzed. In gastrocnemius of unloaded mice treated with resveratrol, body and muscle weight and function were attenuated, whereas muscle proteolysis (tyrosine release), proteolytic and apoptotic markers, atrophy signaling pathways, and myofiber CSA significantly improved. Resveratrol treatment of mice exposed to a seven-day period of unloading prevented body and muscle weight and limb strength loss, while an improvement in muscle proteolysis, proteolytic markers, atrophy signaling pathways, apoptosis, and muscle fiber CSA was observed in the gastrocnemius muscle. These findings may have potential therapeutic implications in the management of disuse muscle atrophy in clinical settings.

Keywords: apoptosis; atrophy signaling pathways; chronic conditions; disuse muscle atrophy; limb muscles; muscle fiber cross-sectional areas; proteolysis; resveratrol.

Figure 1

Graphical timeline representation of all the groups and the treatments administered to the mice in the study. Definition of abbreviations: PBS, phosphate-buffered saline; DMSO, dimethyl sulfoxide; mL, milliliter; mg, milligram; kg, kilogram; h, hour; I, immobilization.

Figure 2

Representative examples of the gastrocnemius muscle in animals of the all study groups of mice. Myofibers are stained in green in the top and middle panels, type I in the top panel and type II in the middle-up panel, and negative controls with no staining (middle-down panels). Hybrid fibers (white arrows) are seen in both up panels. Myofibers of positively stained nuclei (brown color, black arrowheads) and negatively stained nuclei (no staining, black arrows) for the TUNEL assay are seen in the bottom panel. Definition of abbreviations: MyHC, myosin heavy chain; TUNEL, terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate nick-end labeling; NI, non-immobilized; I, immobilization.

Figure 3

(A) Mean values and standard deviations of the variable tyrosine release (nmol/mg/2 h) of the gastrocnemius muscle of the different study groups of mice. (B) Mean values and standard deviations of the variable plasma troponin-I (ng/mL) of the gastrocnemius muscle of the different study groups of mice. Statistical significance is represented as follows: * p < 0.05 between 7dI animals and the non-immobilized mice; $ p < 0.05 the group of resveratrol-treated mice compared with the 7dI group. Definition of abbreviations: NI, non-immobilized; I, immobilization; nmol, nanomol; mg, milligram; h, hour; ng, nanogram; mL, mililiter.

Figure 4

(A) Representative immunoblots of atrogin-1, MuRF-1, 20 S proteasome alpha subunit C8, total ubiquitinated proteins, and GAPDH proteins in the gastrocnemius muscle of all study groups of mice. The analyzed bands are indicated with specific arrows. (B) Mean values and standard deviations of gene expression of Atrogin-1 in the gastrocnemius muscle of the different study groups of mice. (C) Mean values and standard deviations of atrogin-1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (D) Mean values and standard deviations of gene expression of MuRF-1 in the gastrocnemius muscle of the different study groups of mice. (E) Mean values and standard deviations of MuRF-1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (F) Mean values and standard deviations of C8-20S protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (G) Mean values and standard deviations of total ubiquitinated proteins content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). Statistical significance is represented as follows: * p < 0.05 between 7dI animals and the non-immobilized mice, $ p < 0.05 the group of resveratrol-treated mice compared with the 7dI group, *** p < 0.001 between 7dI animals and the non-immobilized mice, $$$ p < 0.001 the group of resveratrol-treated mice compared with the 7dI group. Definition of abbreviations: MuRF-1, muscle RING-finger protein-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; kDa, kilodalton; NI, non-immobilized; I, immobilization; mRNA, messenger ribonucleic acid; a.u., arbitrary units; OD, optical densities.

Figure 5

(A) Representative immunoblots of FoxO1, acetylated FoxO1, FoxO3, acetylated FoxO3, and GAPDH proteins in the gastrocnemius muscle of all study groups of mice. The analyzed bands are indicated with specific arrows. (B) Mean values and standard deviations of gene expression of FoxO1 in the gastrocnemius muscle of the different study groups of mice. No statistical differences were observed between groups. (C) Mean values and standard deviations of FoxO1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). No statistical differences were observed between groups. (D) Mean values and standard deviations of acetylated FoxO1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (E) Mean values and standard deviations of gene expression of FoxO3 in the gastrocnemius muscle of the different study groups of mice. (F) Mean values and standard deviations of FoxO3 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (G) Mean values and standard deviations of acetylated FoxO3 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). No statistical differences were observed between groups. Statistical significance is represented as follows: * p < 0.05 between 7dI animals and the non-immobilized mice; $ p < 0.05 the group of resveratrol-treated mice compared with the 7dI group. Definition of abbreviations: FoxO1, transcription factor fork-head box O1; FoxO3, transcription factor fork-head box O3; ac-, acetylated; p-, phosphorylated; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; kDa, kilodalton; NI, non-immobilized; I, immobilization; mRNA, messenger ribonucleic acid; a.u., arbitrary units; OD, optical densities.

Figure 5

(A) Representative immunoblots of FoxO1, acetylated FoxO1, FoxO3, acetylated FoxO3, and GAPDH proteins in the gastrocnemius muscle of all study groups of mice. The analyzed bands are indicated with specific arrows. (B) Mean values and standard deviations of gene expression of FoxO1 in the gastrocnemius muscle of the different study groups of mice. No statistical differences were observed between groups. (C) Mean values and standard deviations of FoxO1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). No statistical differences were observed between groups. (D) Mean values and standard deviations of acetylated FoxO1 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (E) Mean values and standard deviations of gene expression of FoxO3 in the gastrocnemius muscle of the different study groups of mice. (F) Mean values and standard deviations of FoxO3 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). (G) Mean values and standard deviations of acetylated FoxO3 protein content in the gastrocnemius muscle of the different study groups of mice, as measured by optical densities in arbitrary units (OD, a.u.). No statistical differences were observed between groups. Statistical significance is represented as follows: * p < 0.05 between 7dI animals and the non-immobilized mice; $ p < 0.05 the group of resveratrol-treated mice compared with the 7dI group. Definition of abbreviations: FoxO1, transcription factor fork-head box O1; FoxO3, transcription factor fork-head box O3; ac-, acetylated; p-, phosphorylated; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MW, molecular weight; kDa, kilodalton; NI, non-immobilized; I, immobilization; mRNA, messenger ribonucleic acid; a.u., arbitrary units; OD, optical densities.