The Influence of Stress and Binge-Patterned Alcohol Drinking on Mouse Skeletal Muscle Protein Synthesis and Degradation Pathways

Abstract

Adverse experiences (e.g., acute stress) and alcohol misuse can both impair skeletal muscle homeostasis, resulting in reduced protein synthesis and greater protein breakdown. Exposure to acute stress is a significant risk factor for engaging in alcohol misuse. However, little is known about how these factors together might further affect skeletal muscle health. To that end, this study investigated the effects of acute stress exposure followed by a period of binge-patterned alcohol drinking on signaling factors along mouse skeletal muscle protein synthesis (MPS) and degradation (MPD) pathways. Young adult male C57BL/6J mice participated in the Drinking in the Dark paradigm, where they received 2-4 h of access to 20% ethanol (alcohol group) or water (control group) for four days to establish baseline drinking levels. Three days later, half of the mice in each group were either exposed to a single episode of uncontrollable tail shocks (acute stress) or remained undisturbed in their home cages (no stress). Three days after stress exposure, mice received 4 h of access to 20% ethanol (alcohol) to model binge-patterned alcohol drinking or water for ten consecutive days. Immediately following the final episode of alcohol access, mouse gastrocnemius muscle was extracted to measure changes in relative protein levels along the Akt-mTOR MPS, as well as the ubiquitin-proteasome pathway (UPP) and autophagy MPD pathways via Western blotting. A single exposure to acute stress impaired Akt singling and reduced rates of MPS, independent of alcohol access. This observation was concurrent with a potent increase in heat shock protein seventy expression in the muscle of stressed mice. Alcohol drinking did not exacerbate stress-induced alterations in the MPS and MPD signaling pathways. Instead, changes in the MPS and MPD signaling factors due to alcohol access were primarily observed in non-stressed mice. Taken together, these data suggest that exposure to a stressor of sufficient intensity may cause prolonged disruptions to signaling factors that impact skeletal muscle health and function beyond what could be further induced by periods of alcohol misuse.

Keywords: autophagy; binge-patterned drinking; drinking in the dark; muscle protein degradation; muscle protein synthesis.

Figure 1

The experimental design. Male mice participated in “Drinking in the Dark” (DID), where they received access to 20% alcohol or water (controls) two hours daily over three days and four hours on the fourth day. Seventy-two hours later, mice were exposed to a single episode of acute stress or left undisturbed in home cages (no stress). Seventy-two hours later, mice participated in daily bouts of DID, where they received 4 h of access to 20% alcohol or water for 10 consecutive days.

Figure 2

Changes in relative protein expression of the Akt-mTOR signaling pathway following acute stress and alcohol access. Representative blots (A) and quantification (B–E) are presented for several key proteins in the Akt-mTOR pathway. Data are presented as mean ± SEM (n = 9/group). Two-way ANOVA was used to compare both the main effects and an interaction between stress and alcohol (listed below the x-axis). Tukey’s post hoc analysis was used for multiple comparisons, and statistical significance was set at p < 0.05. * Denotes significantly decreased compared to no-stress/water group (p < 0.05); ^† Denotes a non-statistically significant trend compared to no-stress/water group (p < 0.10).

Figure 3

Changes in the ubiquitin-proteasome pathway and myostatin following acute stress and alcohol access. Both representative blots (A) and relative protein expression (B–E) are presented. Data are presented as mean ± SEM (n = 9/group). A two-way ANOVA was used to compare the individual effects of stress exposure and binge-patterned drinking, as well as to compare and interaction between factors (reported next to each figure).

Figure 4

The effects of stress exposure and binge-patterned alcohol drinking on autophagy signaling and HSP70. Representative blots (A) and relative protein expression (B–G) are presented. Data are presented as mean ± SEM (n = 9/group). To compare the effects of acute stress, alcohol consumption, and an interaction between factors, a two-way ANOVA was used. Statistical significance was set at p < 0.05 (reported next to each figure).

Figure 5

Puromycin incorporation to peptide chains following acute stress and alcohol access. Representative blots (A) and quantification of puromycin levels (B) are presented. Data are presented as mean ± SEM (n = 9/group). Two-way ANOVA assessed the significant main effects and interactions between alcohol and stress (reported next to the figure).