Alcohol Consumption During Muscle Disuse Causes Differential Signaling Responses in a Muscle-Specific Manner in Mice

Abstract

Excessive alcohol consumption promotes clinical myopathy and injury-related immobilization. Because both alcohol and disuse jeopardize muscle health, their combined effects may synergistically accelerate fiber type-dependent muscle wasting. Ten-week-old male C57BL/6J mice were fed a control or 5% alcohol-diet for 3 weeks (NIAAA-model), with or without 1 week of unilateral hindlimb immobilization, generating four sets of limb muscles (n = 9/grp): control (CO), with immobilization (CI), alcohol (AL), with immobilization (AI). Gastrocnemius and soleus muscles were atrophied by CI, AL, and AI, whereas quadriceps atrophy was induced by CI and AI only (all p < 0.05). In soleus, CI, AL, and AL decreased p-mTOR (~40–60%, p < 0.01) and p-p70S6K (~50–87%, p < 0.05), indicating suppressed anabolic signaling. In contrast, in the quadriceps, alcohol increased p-4EBP1 by ~200% (p < 0.01), while p-Akt was elevated by ~180%, only in AI (p < 0.01). Myogenesis signaling was inhibited by alcohol and immobilization. For protein degradation, immobilization increased MAFbx by >50% in both muscles (p < 0.01). Quadriceps exhibited increased p-PERK (+53%) under AI (p < 0.05), whereas several markers of ER stress were reduced by all interventions in soleus (p < 0.05). These findings suggest that alcohol consumption does not exacerbate immobilization-induced atrophy; however, alcohol suppresses anabolic signaling in soleus, suggesting greater susceptibility to myopathy.

1. Introduction

The prevalence of high-risk drinking behaviors, including binge drinking (increasing blood alcohol concentrations (BAC) to ≥0.08%) and heavy alcohol use (≥8 or 15 drinks per week for females and males, respectively) [1], remains substantial and in some populations continues to increase [2]. Notably, more than 55% of adults aged 26 years and older reported consuming alcohol within the past month, and approximately one in four adults engaged in binge drinking [3]. Such excessive alcohol consumption is associated with an annual economic burden exceeding $249 billion in the United States [4]. Beyond its well-recognized systemic and hepatic consequences, chronic alcohol exposure profoundly affects skeletal muscle health. Indeed, skeletal muscle dysfunction (i.e., myopathy) is a common complication in individuals with alcohol use disorder (AUD), with chronic alcoholic myopathy estimated to affect approximately 40–60% of chronic alcohol users [5].

Excessive alcohol consumption contributes to secondary sarcopenia distinctly from aging per se [6,7,8], and promotes clinical myopathy [9,10,11]. Further, alcoholic liver disease leads to more rapid loss of muscle compared to other forms of cirrhosis [12], which is inversely associated with survival, and sarcopenia is linked to worsened outcomes in alcoholic patients [13]. In addition, alcohol consumption is a leading cause of falls [14,15], disease-related complications [16], and other injuries [17]. Such injuries often necessitate limb immobilization or prolonged bed rest, which rapidly and profoundly compromise skeletal muscle, thereby accelerating mobility limitations and disability through the loss of muscle mass and strength [18,19,20,21]. Importantly, the muscle atrophy response to immobilization appears to be augmented by alcohol [22], which also impairs recovery from disuse [22,23] and following myotoxin injury [24]. Despite this limited supportive evidence, the effect of alcohol during disuse has not been confirmed, and the mechanisms underlying the potential deleterious effects of this combination of insults remain unclear.

Therefore, the purpose of this study was to comprehensively investigate the effects of alcohol coupled with hindlimb immobilization on muscle mass and cross-sectional area (CSA) in C57BL/6J male mice. In addition, we sought to elucidate mechanisms underlying these effects by focusing on anabolic and catabolic signaling as well as ER stress in muscles of varying fiber types, thereby identifying specific characteristics and potential therapeutic targets for alcohol-induced muscle myopathy.

2. Results

2.1. NIAAA Model and Hindlimb Immobilization Cause Muscle Atrophy

To identify the independent and combined effects of alcohol intake and muscle disuse on muscle atrophy, the NIAAA (National Institute on Alcohol Abuse and Alcoholism) protocol was combined with unilateral hindlimb immobilization (Figure 1A). Daily body weight measurements showed that mice receiving the 5% ethanol liquid diet gradually lost weight throughout the experimental period, and body weight was significantly reduced beginning on day 11 (Figure 2A). The quantification of final body mass (23.3 ± 0.6 g vs. 27.9 ± 0.6 g) and total kcal of food intake per mouse, averaged per cage (198.6 ± 6.1 kcal vs. 244.8 ± 3.6 kcal), confirmed significantly lower values in alcohol-fed mice compared with controls (p < 0.01) (Figure 2A,B). Average ethanol consumption was ~13.8 g/kg/day per mouse throughout the 14-day period.

Individual muscle weights revealed a marked reduction across alcohol and immobilization interventions. There were main effects of alcohol (p = 0.003) and immobilization (p < 0.001) as well as tendency of an interaction effect (p = 0.057), showing a significant reduction in gastrocnemius muscle mass by alcohol (AL 13%), immobilization (CI 19%), and their combination (AI 23%) compared to CO via post hoc analyses (p < 0.05) (Figure 2C). In the soleus, there was an interaction effect (p = 0.006) and a significant reduction in AL (23%) compared to CO (p < 0.05), and the immobilization decreased muscle mass in both diets (p < 0.01), confirming muscle atrophy (Figure 2D). In the quadriceps, there was a main effect of immobilization (p = 0.002) on muscle weight (CI 19%, p = 0.006 and AI 20%, p = 0.022 vs. CO), whereas there was only a tendency for an interaction (p = 0.092) (Figure 2E).

Histological analyses revealed marked alterations in gastrocnemius muscle morphology across interventions. The CSA of gastrocnemius muscle fibers was significantly reduced by alcohol (p = 0.017) and immobilization (p < 0.0001), with a significant interaction effect (p = 0.027) (Figure 2F,G). This interaction indicates that the effect of immobilization in control-fed mice was greater than that in alcohol-fed mice, likely due to the pre-existing reduction in fiber size induced by alcohol. Alcohol feeding alone (AL) resulted in a 15% reduction in fiber CSA compared to CO (p = 0.009), whereas immobilization, regardless of dietary condition, produced a more pronounced reduction in fiber CSA in both CI (24%) and AI (25%) relative to CO (p < 0.0001). Both interventions caused a leftward shift in fiber area distribution, indicating that chronic alcohol intake and immobilization contribute to muscle deterioration that reduced overall muscle mass and fiber size (Figure 2H).

2.2. Muscle-Specific Changes in Anabolic Signaling

We evaluated the effects of alcohol consumption and immobilization on muscle protein synthesis signaling of quadriceps and soleus muscles (Figure 3). The canonical Akt-mTOR pathway is a crucial regulator of anabolic signaling in skeletal muscle, leading to downstream signaling including phosphorylation of protein 70 S6 kinase (p70S6K) and 4E-binding protein 1 (4EBP1) [25]. In the quadriceps, as an initial anabolic signaling regulator, total Akt expression showed a main effect of immobilization (p < 0.001), and Akt was significantly elevated by immobilization in both diet groups (p < 0.05). There were main effects of alcohol and immobilization on phosphorylation of Akt (Ser 473; p-Akt) (alcohol; p = 0.067, immobilization p = 0.002) and main effect of alcohol and interaction on the ratio of p-Akt/Akt (alcohol; p = 0.035, interaction; p = 0.022) due to elevation of protein level in AI compared to CO, CI, and AL (p < 0.05), indicating a synergistic effect of alcohol and immobilization (Figure 3B). Neither alcohol consumption nor immobilization altered the phosphorylation of mTOR (Ser 2448; p-mTOR), total mTOR, or the ratio of mTOR or its downstream target, p70S6K (Figure 3C,D). However, there was a main effect of alcohol on the phosphorylation of 4EBP1 (Thr 37/46;p-4EBP1) (p = 0.003), total 4EBP1 (p = 0.004), and the ratio of 4EBP1 (p = 0.027) with elevation of the phosphorylation of 4EBP1 and total 4EBP1 in alcohol (p < 0.05), resulting in no change in the p-4EBP1/4EBP1 ratio in quadriceps (Figure 3E).

In contrast, patterns of signaling in soleus differed from those observed in quadriceps (Figure 3A,F). In soleus, there was an interaction effect (p = 0.021) on phosphorylation of Akt, a main effect of an increase in alcohol (p = 0.009) on total Akt, and a tendency for a reduction as an effect of immobilization on total Akt (p = 0.084; Figure 3G). AL showed a tendency to reduce phosphorylation of Akt (p = 0.078) and total Akt (p = 0.075), without affecting the ratio (p = 0.693; Figure 3G). However, there were main effects of alcohol and immobilization on phosphorylation of mTOR (alcohol; p = 0.0008, immobilization p = 0.004) and the p-mTOR/mTOR ratio (alcohol; p = 0.012, immobilization; p = 0.006). There was also a main effect of alcohol to reduce total mTOR (p = 0.003). On both diets, immobilization decreased the phosphorylation of mTOR and the p-mTOR/mTOR ratio by ~60% (p < 0.05), whereas AL reduced both by ~42% compared to CO (p < 0.05). Total mTOR was only reduced in AI versus CO (p < 0.05; Figure 3H). Furthermore, main effects of alcohol and immobilization as well as an interaction were observed on phosphorylation of p70S6K (Thr 389; p-p70S6K) (alcohol; p = 0.035, immobilization; p = 0.0002, interaction; p = 0.021) and total p70S6K (alcohol; p = 0.001, immobilization; p < 0.0001, interaction; p < 0.0001), which were reduced by CI, AL, and AI compared to CO (p < 0.05). However, there was only a main effect of immobilization on the p-p70S6K/p70S6K ratio (immobilization; p = 0.004), driven by a trending reduction in CI (p = 0.056) and AI (p = 0.067) compared to CO (Figure 3I). However, the relative protein expressions of phosphorylated and total 4EBP1 were not altered (Figure 3J).

2.3. Myogenesis Signaling Is Inhibited by Alcohol Consumption and Immobilization

Myoblast determination protein 1 (MyoD) and Myogenin are myogenic regulatory factors that play essential roles in satellite cell differentiation and muscle regeneration [26]. In the quadriceps, there were main effects of alcohol (p = 0.035) and immobilization (p = 0.003), but no interaction effect on MyoD (p = 0.680). MyoD tended to be lower in CI vs. CO (p = 0.064), and there was a significant reduction in AI compared to CO (p = 0.003) (Figure 4B). Similarly, there was a main effect of immobilization to lower Myogenin (p = 0.003) and a similar tendency of alcohol (p = 0.052). Compared to CO, CI (p = 0.012) and AI (p = 0.004) were reduced by 40% and 46%, respectively, whereas the 29% reduction in AL did not reach significance (p = 0.099) (Figure 4C).

Similarly, in soleus, there was a main effect of alcohol (p = 0.003) as well as an interaction effect (p = 0.005) on MyoD. MyoD expression was significantly lower (~25–40%) in CI, AL, and AI relative to CO (all p < 0.05) (Figure 4E). Furthermore, there were main effects of alcohol (p = 0.0001), immobilization (p < 0.0001), and an interaction (p = 0.0005) on Myogenin. Compared to CO, Myogenin was significantly suppressed in CI (77%, p < 0.0001), AL (76%, p < 0.0001), and AI (81%, p < 0.0001) (Figure 4F).

2.4. Selective Activation of Components of the Ubiquitin Proteasome Pathway by Hindlimb Immobilization

Muscle ring finger protein-1 (MuRF1) and muscle atrophy F-box (MAFbx) are E3 ubiquitin ligases that promote ubiquitination and proteasomal degradation of muscle proteins during atrophy [27]. In the quadriceps, no main effect of immobilization (p = 0.648), alcohol (p = 0.649), or an interaction between exposures (p = 0.583) was observed on protein ubiquitination (Figure 5B). Similarly, MuRF1 expression was unchanged across interventions (Figure 5C). In contrast, MAFbx expression was significantly elevated by immobilization (p < 0.05), with a main effect of alcohol to lower MAFbx (p = 0.002), leading to lower MAFbx in AI compared to CI (p = 0.004) (Figure 5D).

In the soleus, total ubiquitin content exhibited a main effect of an increase by immobilization (p = 0.022), although post hoc comparisons did not show individual group differences (Figure 5F). A similar pattern was observed for soleus MuRF1, although the increase caused by immobilization did not reach significance (p = 0.073) (Figure 5G). In contrast, soleus MAFbx expression was markedly increased by immobilization (main effect p < 0.001), driven by 200% and 161% increases in control and alcohol diets, respectively (both p < 0.05), while alcohol tended to lower MAFbx (p = 0.090) (Figure 5H).

2.5. LC3 Is Upregulated by Alcohol in Quadriceps

Microtubule-associated protein 1 light chain 3 (LC3) is a widely used marker of autophagosome formation, where conversion of LC3-I to LC3-II often reflects activation of the autophagy [28]. Excessive skeletal muscle autophagy can contribute to muscle protein breakdown [29]. In the quadriceps, alcohol consumption significantly increased LC3-I expression (main effect p < 0.001), while immobilization alone had no detectable effect (p = 0.546) (Figure 6B). There was also a main effect of alcohol to increase LC3-II (p = 0.014), and a tendency of immobilization (main effect p = 0.089), but no interaction effect on LC-II. However, LC3-II levels were significantly elevated only in AI compared to CO (p = 0.027) (Figure 6C), indicating a combined effect of alcohol and immobilization. On the other hand, in the soleus, there was a tendency for an interaction on LC3-I (p = 0.058), with no other differences. Soleus LC3-II exhibited main effects of both alcohol (p = 0.024) and immobilization (p = 0.011), with no significant differences between specific groups (Figure 6E,F).

2.6. ER-Stress Responses to Alcohol and Immobilization Differ Between Quadriceps and Soleus

One of the functions of the endoplasmic reticulum (ER) is to adjust the synthesis, folding, and maturation of cellular proteins. To alleviate stress and restore ER function, cells initiate a signaling cascade by activating the unfolded protein response (UPR) [30]. ER stress plays a critical role not only in skeletal muscle adaptation to physiological stimuli but also in myofiber formation during embryonic development and regeneration [31,32]. So, we evaluated ER stress in quadriceps and soleus muscles (Figure 7). In the quadriceps, effects were limited to phosphorylation of PERK (Thr 980; p-PERK), ATF4, and CHOP. Specifically, there was a main effect of alcohol (p = 0.045) and a tendency of immobilization (p = 0.056) to increase p-PERK, leading to a 44% increase in AI compared to CO (p = 0.034) (Figure 7B). Quadriceps ATF4 was increased by immobilization (main effect p < 0.001), and there was an interaction (p = 0.043) resulting in an increase of 354% in CI vs. CO (p = 0.026). Likewise, there was a main effect of immolization to increase CHOP (p = 0.026) in quadriceps, but no group differences were apparent. Additional markers of ER stress, total PERK, eIF2α signaling, ATF6, BIP, and XBP1s did not change (all p > 0.1) (Figure 7C).

In contrast, ER stress markers were downregulated in soleus following both alcohol consumption and immobilization (Figure 7E,F). Alcohol and immobilization did not alter the phosphorylation of PERK, total PERK, or the p-PERK/PERK ratio (Figure 7E). Both alcohol and immobilization suppressed the phosphorylation of eIF2α (Ser 51; p-eIF2α), total eIF2α, and the p-eIF2α/eIF2α ratio (all p ≤ 0.001) (Figure 7E). There were also interactions for p-eIF2α, total eIF2α, and the p-eIF2α/eIF2α ratio (all p < 0.05) such that the immobilization effect was more pronounced on the control diet, and CI, AL, and AI were all lower than CO (all p < 0.01). There was a main effect of alcohol (p = 0.0116) to reduce ATF6 but no differences between groups (Figure 7F). However, cleaved ATF6 was inhibited by alcohol and immobilization (both main effects p < 0.001), and there was a tendency for an interaction (p = 0.068). In all groups, cleaved ATF6 was lower than CO, while AI was also reduced 40% vs. AL (p = 0.035). Similarly, both alcohol and immobilization lowered soleus BIP (main effects p < 0.05), and CI, AL, and AI were all lower than CO (all p < 0.05). However, XBP1s protein expression did not differ across groups. Finally, there was a main effect of immobilization to increase CHOP (p = 0.008) and an interaction (p = 0.013), where CHOP was higher in AI compared to AL (p = 0.003), with no other group differences.

3. Discussion

Chronic alcohol consumption is a well-established contributor to skeletal muscle dysfunction, which can cause muscle myopathy [29,33,34,35,36]. Alcohol misuse dramatically increases the risk of injury, often requiring immobilization. In such scenarios, muscle disuse induces a rapid and pronounced activation of catabolic signaling, causing loss of muscle mass [37,38,39,40]. However, the extent to which alcohol modifies immobilization-induced muscle atrophy in a muscle-dependent manner, particularly whether alcohol exacerbates disuse-induced muscle loss, and the underlying mechanisms responsible, has remained unclear. The present study demonstrates that, independently, both alcohol exposure and hindlimb immobilization induced muscle atrophy with magnitudes varying across different muscles, but that their combination did not exacerbate reductions in muscle mass and myofiber cross-sectional area. However, alcohol coupled with immobilization was associated with distinct alterations in anabolic and catabolic signaling pathways, including markers of ER stress in different skeletal muscles. Collectively, these findings indicate that alcohol does not simply amplify disuse-induced muscle atrophy globally but instead modulates susceptibility in a muscle-specific manner, providing mechanistic insight into alcohol-induced myopathy and identifying potential targets for therapeutic intervention.

The results of this study revealed that alcohol and immobilization each elicited similar atrophy of the gastrocnemius (~23%) and soleus (~40%) muscles, but immobilization induced a more pronounced quadriceps atrophy (~20%) than alcohol (~11%). The skeletal muscle atrophy response to hindlimb immobilization is very well established, and our findings are consistent with a large body of literature demonstrating ~20–40% reductions in muscle size and CSA following 7–14 days of disuse [41,42,43]. The effects of alcohol, on the other hand, are much more conflicting, inducing atrophy in some studies [29,44,45,46] but not others [22,23,47,48]. This discrepancy appears to be mediated, at least in part, by method of administration, dose, duration, and model, where high doses appear to acutely induce atrophy [49], whereas dietary approaches may be less impactful [50,51]. Studies using the Lieber–DeCarli liquid diet, as used in the current study, often do not elicit muscle atrophy, even when consumed for extended periods of time, which elicits other alcohol-related pathologies [23].

In a similar study to ours, in rats subjected to 3 days of unilateral hindlimb immobilization along with daily ethanol gavage (50 mM/kg; ~2.3 g/kg/day), alcohol alone did not induce gastrocnemius atrophy; however, it augmented muscle loss from ~12% with immobilization alone to ~23% when combined [22]. In contrast, ~8 weeks of the Lieber–DeCarli 5% ethanol diet in female mice did not result in atrophy of the quadriceps or overtly alter recovery of muscle mass following 7 days of hindlimb immobilization [23], but did not overtly alter recovery of muscle mass [23]. Notably, here, alcohol alone produced a modest but significant reduction in CSA (15%), whereas immobilization produced a larger reduction (25%) regardless of diet. Our results indicate that short-term alcohol exposure and immobilization independently promote skeletal muscle atrophy, with mechanical unloading serving as the primary driver of rapid muscle loss, showing that the combined interventions did not produce a synergistic effect. In the context of the current literature, it appears that the interaction between alcohol consumption and immobilization on skeletal muscle atrophy is not uniform and may vary according to muscle type and experimental paradigm.

The rather large discrepancy in atrophy across muscles was intriguing and necessitated a deeper investigation. Skeletal muscle is composed of a mixture of fast-twitch (type II) and slow-twitch (type I) fibers, each with distinct metabolic and contractile properties [52] and sensitivity to specific atrophy-inducing stimuli. For example, slow-twitch type I fibers are more vulnerable to inactivity, whereas type II fibers are more susceptible to alcohol-induced myopathy [53,54,55]. Given this, coupled with a body of literature suggesting that alcohol and immobilization may disrupt proteostasis through distinct yet overlapping mechanisms, we evaluated relevant intracellular anabolic and catabolic signaling pathways in quadriceps (predominantly type II) and soleus (~40–50% type I) muscles.

Akt-mTOR signaling is known as a central regulator of protein synthesis in skeletal muscle, and suppression of this pathway leads to a reduction in muscle protein synthesis [25]. Acutely, alcohol consumption (~24 h) suppresses Akt–mTOR signaling [56,57,58]. Similarly, our previous study using the Drinking in the Dark (DID) model showed that voluntary binge-patterned ethanol consumption for 14 days caused a significant suppression of mTOR phosphorylation, indicating impaired anabolic signaling [59]. Consistent with the present findings in soleus, a previous study using alcohol administration for 4 weeks via the Lieber–DeCarli liquid diet in female mice reported dephosphorylation of mTORC1 and its downstream target p70S6K [45], as did 20 weeks in the gastrocnemius of female rats [60]. Furthermore, 26 weeks of a 40% ethanol-agar diet impaired protein synthesis in rat gastrocnemius muscle by disrupting the 4EBP1-eIF4G pathway [44]. Similarly, hindlimb unloading consistently suppresses phosphorylation of Akt, mTOR, and p70S6K by approximately 50–60% [61], in agreement with our data, indicating a robust inhibitory effect of mechanical unloading on translational signaling [37,38]. Together, the results of the present study demonstrate that both alcohol consumption and immobilization potently suppress anabolic signaling in the soleus muscle.

In contrast to these canonical responses in soleus, our study revealed a muscle-specific divergence in signaling patterns. In the quadriceps, both alcohol and immobilization unexpectedly increased Akt–mTOR signaling, with the p-Akt/Akt ratio elevated only in the combined alcohol and immobilization (AI) group, suggesting synergistic activation of proximal Akt signaling. However, this activation did not translate into downstream mTOR or p70S6K activation, indicating a partial uncoupling between proximal Akt signaling and translational control. This observation aligns with previous findings showing increased anabolic signaling in the gastrocnemius following five days of unilateral immobilization [40]. Notably, the soleus muscle exhibited heightened vulnerability to alcohol-induced suppression of Akt–mTOR signaling, displaying pronounced reductions in both phosphorylation and total protein abundance of key anabolic regulators. Given its highly oxidative and chronically load-bearing nature, the soleus appears to undergo a more substantial suppression of translational signaling, which may be further exacerbated by alcohol. Collectively, these data suggest that alcohol and immobilization converge in a muscle-specific manner to establish a robust anti-anabolic state, particularly in the soleus.

Across both muscles in this study, alcohol and immobilization suppressed markers of myogenic programming. In soleus, specifically, interaction effects indicated that alcohol and immobilization exert both independent and interactive suppressive effects on regenerative signaling in a muscle-specific manner. Consistent with these in vivo findings, exposure to 100 mM ethanol significantly reduced MyoD mRNA expression during the early stages (days 1–3) of C2C12 myoblast differentiation [62]. Likewise, MyoD mRNA expression was significantly decreased in alcohol-fed 3-month-old female rats following 3 days of recovery after immobilization using the Lieber–DeCarli liquid diet model [23], supporting the concept that alcohol impairs early myogenic activation. Previous studies examining the effects of immobilization alone on myogenic regulators have yielded inconsistent results, with some reports showing increased MyoD and Myogenin expression in the gastrocnemius muscle [63,64], whereas others demonstrate reduced Pax7 expression in the soleus without changes in MyoD or Myogenin mRNA following 7 days of hindlimb immobilization in mice [65]. Despite these discrepancies, our findings suggest that alcohol exposure in the context of disuse not only promotes muscle atrophy but also disrupts the normal temporal regulation of myogenic signaling. Notably, increased MyoD expression observed after 14 days of recovery following immobilization in alcohol-fed animals [23] may reflect delayed or dysregulated regeneration rather than a true enhancement of myogenic capacity.

As a major regulator of protein turnover, overactivation of the ubiquitin–proteasome pathway contributes to skeletal muscle atrophy [27]. Interestingly, MuRF1 protein expression remained largely unchanged in both the quadriceps and soleus muscles, whereas MAFbx expression was consistently elevated following immobilization. In contrast, increased protein ubiquitination was observed exclusively in the soleus. Immobilization has been shown to robustly increase MuRF1/MAFbx mRNA levels, accompanied by corresponding elevations in protein abundance [40,64,66]. Collectively, these findings are consistent with the results of the present study, demonstrating that accelerated muscle protein breakdown plays a dominant role in immobilization-induced muscle wasting. This expression profile suggests that, in the current model, immobilization activates a selective component of the ubiquitin–proteasome system, characterized by enhanced MAFbx expression in both muscles and a broad increase in ubiquitinated proteins, at least in soleus.

Unlike the effects of immobilization, the effects of alcohol on the ubiquitin–proteosome pathway are inconsistent. For example, voluntary binge-patterned alcohol consumption of ~8 g/kg/day for 5 days activates muscle atrophy-related MuRF1 and MAFbx mRNA signaling [51]. Similarly, rats fed the Lieber–DeCarli liquid ethanol diet for 12 or 20 weeks exhibited increased MuRF1 and MAFbx mRNA expression [46,60]; however, protein expression was not reported. In direct disagreement, various models of alcohol consumption over relatively short-term (~14 days) or long-term (~14 weeks) did not alter MuRF1 and MAFbx protein expression [59] or mRNA [67], respectively. The lack of a detectable alcohol effect on these measures indicates that alcohol-driven atrophy in this timeframe may not primarily operate through MuRF1- or MAFbx-dependent proteasomal activation.

Accumulating evidence suggests that alcohol exposure is associated with activation of autophagy-related pathways in skeletal muscle [68]. In vitro studies have shown that ethanol treatment increases LC3-II expression along with other indicators of autophagy activation in C2C12 myotubes [33,34,45], and chronic alcohol consumption has been linked to muscle atrophy in the absence of consistent activation of ubiquitin–proteasome-related genes [29]. Our data are in partial agreement, as alcohol tended to increase LC3-II in quadriceps, although additional markers of autophagy were not assessed to support activation or flux. Interestingly, knockdown of Atg7, a key component of the autophagy pathway, attenuated ethanol-induced atrophy of C2C12 myotubes [29], supporting its role in this process. Surprisingly, in the present study, unlike in the quadriceps, LC3-II was reduced by alcohol in soleus, suggesting differential regulation of autophagy by alcohol that may not contribute to atrophy in all muscles, which may be fiber type or metabolism-dependent.

Autophagy signaling appears to be stimulated by disuse as well. In humans subjected to short-term immobilization, reductions in myofiber cross-sectional area have been reported alongside increased LC3-II expression without parallel changes in MuRF1 or MAFbx protein levels [69]. In the present study, immobilization increased LC3-II abundance, with a trending main effect of immobilization in quadriceps, whereas the changes in the soleus muscle were more robust. Given prior reports that autophagosome abundance is greater in glycolytic type II fibers than in oxidative type I fibers [70], these findings suggest that LC3-II accumulation may be regulated in a muscle fiber-dependent manner. This concept may have relevance for muscle degradation as well, as type II fibers are more vulnerable to alcohol [53,71]. However, because only LC3 was assessed, autophagic flux cannot be assessed based on the present data, and we are unable to distinguish between increased autophagosome formation and impaired autophagosome clearance. Thus, the observed changes in LC3-II should be interpreted as altered autophagy-associated signaling rather than definitive changes in autophagic flux. Collectively, these findings, in the context of the current literature, suggest a potential role for autophagy-related pathways in alcohol- and disuse-associated muscle remodeling, while highlighting the need for future studies incorporating additional markers of autophagic flux.

The ER stress response can be either adaptive or maladaptive depending on the magnitude and duration of proteostatic stress [72]. Transient activation of the PERK–eIF2α axis suppresses global protein synthesis to facilitate restoration of ER homeostasis, whereas unresolved ER stress promotes sustained CHOP activation and pro-apoptotic signaling [73,74,75]. Importantly, CHOP exerts context-dependent effects: while early CHOP activation may facilitate recovery by permitting translational reinitiation, untimely resumption of protein synthesis in the setting of unresolved ER stress can impair cell viability, thereby necessitating subsequent CHOP suppression for full restoration of ER homeostasis [73,76]. Thus, elevated CHOP expression in the absence of coordinated activation of UPR signaling is often indicative of maladaptive ER stress. In skeletal muscle, mechanical unloading increased ATF4 mRNA in parallel with muscle atrophy [77], with ER stress markers such as CHOP and BIP preferentially induced in glycolytic rather than oxidative muscles [62]. Consistent with these observations, the present study revealed marked muscle-specific regulation of ER stress signaling. In the quadriceps muscle, alcohol exposure combined with immobilization increased PERK phosphorylation, and immobilization alone elevated ATF4, which is downstream in this same signaling arm, indicating engagement of an adaptive PERK-mediated UPR. In contrast, the soleus exhibited coordinated attenuation of UPR output, as alcohol and immobilization reduced eIF2α phosphorylation, ATF6 activation, and BIP expression despite increased CHOP levels. While intriguing, it cannot be concluded that the soleus is undergoing a maladaptive ER response to these stressors.

The possibility exists that the soleus has, in fact, adapted to the exposures of alcohol and immobilization, thereby suppressing the need to activate ER stress signaling pathways. Nonetheless, given that transient eIF2α phosphorylation and ATF6 signaling are required for normal myogenic differentiation [78,79], and that PERK–eIF2α signaling preserves myogenic competence in satellite cells [80], attenuation of ER stress signaling likely destabilizes the cellular environment required for effective regeneration. This is further supported by both pharmacological and genetic inhibition of ER stress, which attenuates anabolic signaling through the Akt/mTOR pathway and results in muscle atrophy [81,82,83]. This raises the possibility that the apparent disruption in ER stress signaling seen here in soleus may contribute to reduced anabolic signaling and myogenic capacity in oxidative muscle during combined alcohol exposure and disuse, although this has not been empirically examined. Alternatively, excessive ER stress, which may be present in quadriceps here, promotes muscle atrophy [84]. Our data suggest a potential disconnect between ER stress and regeneration, since MyoD and Myogenin were similarly reduced in soleus and quadriceps, although in quadriceps, the PERK arm of ER stress is elevated, and other signaling is unchanged.

Several considerations should be noted when interpreting the present findings. In this study, individual food intake and blood alcohol concentration (BAC) were not directly assessed; however, previous studies indicate that mice consuming a 5% alcohol-containing diet typically achieve consistent BAC levels of approximately 180 mg/dL [85]. Alcohol exposure resulted in reductions in caloric intake and body mass [46,85], consistent with the NIAAA model, and a pair-feeding control group was not included. These limitations may influence the signaling changes of interest and impede our ability to fully distinguish the direct effects of alcohol from those secondary to negative energy balance. Although AST and ALT levels were not analyzed, the increased liver weight-to-body weight ratio (p = 0.0002; Figure S1) observed in the alcohol-fed group is nevertheless consistent with expected alcoholic liver injury in the NIAAA alcohol-feeding model [86].

Prior studies report substantial differences in fiber-type distribution between the quadriceps and soleus muscles of mice, with the quadriceps predominantly composed of type IIx and IIb fibers and the soleus enriched in type I and IIa fibers [87,88,89]. However, myofiber composition was not specifically examined here, limiting claims regarding fiber-type related-difference. Nevertheless, previous evidence suggests differential vulnerability across fiber types, with slow-twitch type I fibers being more sensitive to inactivity, whereas fast-twitch type II fibers appear more susceptible to alcohol-induced myopathy [53,54,55]. Finally, although we found decreased muscle mass, CSA, and a change in anabolic signaling, protein synthesis and muscle force production were not directly measured, which limit interpretation of the functional consequences of the observed signaling changes.

4. Materials and Methods

4.1. Animal Housing

Eight-week-old C57BL/6J male mice, purchased from Jackson Labs (Bar Harbor, ME, USA), were housed three mice per cage. Mice were acclimated to a reversed 12:12 light–dark cycle in standard laboratory cages for two weeks. During the acclimatization, mice received a normal chow diet and ad libitum access to water. Throughout the experiment, mice were housed 3 per cage. Body weight and food intake were measured daily. All procedures were conducted in accordance with institutional and national guidelines for the care and use of laboratory animals and approved by the University of Massachusetts Lowell Institutional Animal Care and Use Committee (IACUC-24-08; approved on 6 August 2024).

4.2. NIAAA Model

At the beginning of the study, mice were randomly assigned to either a control or an alcohol liquid diet, based on body weight (n = 9 mice per group). We adopted the NIAAA method of binge alcohol consumption [85]. During the experiment, mice had free access to a Liebe Decarli ’82 liquid diet containing control (Cat No. F1259SP, Bio-Serv™, Prospect, CT, USA) or isocalorically substituted ethanol (Cat No. F1258SP, Bio-Serv™, Prospect, CT, USA) via standard feeding tube (Cat No. 9010, Bio-Serv™, Prospect, CT, USA) (Figure 1B). At the onset of the experiment, mice were acclimated to the control liquid diet for 2 days. Mice assigned to the control group received the control liquid diet for 3 weeks. In contrast, mice in the alcohol feeding group were given a liquid diet with 1.4% alcohol for 2 days, 3.1% alcohol for 2 days, 4.2% alcohol for 1 day, and 5% alcohol for 2 weeks, following the NIAAA model [85,90]. All liquid diets were replaced with fresh ones daily at 5:00 PM. On day 18, ethanol-fed and control-fed mice received a single dose of alcohol (5 g/kg⁻¹ body weight) or isocaloric maltose dextrin via oral gavage in the early morning [85]. To quantify food intake, liquid diet volume was measured daily when placing fresh diet and divided by the number of mice in the cage. All researchers were aware of group assignments throughout the study.

4.3. Unilateral Hindlimb Immobilization

All mice received unilateral hindlimb immobilization. Each mouse was anesthetized using isoflurane, and the left hindlimb was immobilized using the following procedure on a heat pad. First, a free adhesive band was wrapped to minimize skin damage, and then a thermo-plastic wrap (Cat No. 4030, Orfit Industries, Wijnegem, Belgium) was heated to maintain softness/plasticity and wrapped to immobilize the left hindlimb, while maintaining the left ankle in a plantar-flexed position. Nontoxic polyvinyl siloxane (Cat No. VPS-RF4, 3D Dental, Cleveland, OH, USA) was used for adhesion between the two wraps (Figure 1C). The procedure of immobilization was completed within 5 min. The immobilization status was checked daily along with body weight measurements. Mice were monitored to ensure the ability to move freely about the cage, exhibit no difficulties feeding, and show no irritation of the immobilized limb. Replacement of the thermo-plastic wrap was performed as needed due to loosening (n = 2). The combination of diet and unilateral immobilization resulted in 4 groups of limb muscles: a control liquid diet without immobilized leg (CO, n = 9), a control liquid diet with an immobilized leg (CI, n = 9), an alcohol liquid diet without an immobilized leg (AL, n = 9), and an alcohol liquid diet with an immobilized leg (AI, n = 9). After 7 days of immobilization, mice were brought to a surgical level of anesthesia using pentobarbital (60 mg/kg), and then quadriceps, gastrocnemius, soleus muscle, and liver tissue were harvested, weighed, and flash frozen in liquid nitrogen and stored at −80 °C for subsequent assays, with the exception of gastrocnemius, which was fixed for histology. Euthanasia was confirmed by cardiac removal [91].

4.4. Tissue Fixation and Dehydration/Infiltration

Gastrocnemius muscles were placed in 10% buffered formalin for fixation. After 24 h, muscles were transferred into 70% ethanol for storage until analysis. The samples were dehydrated by incubation in gradually increased ethanol (EtOH) concentrations, consisting of two times for 20 min in 70%, 95%, and 100% EtOH and two times for 30 min in 100% xylene. After dehydration, tissues underwent paraffin infiltration and incubation steps of 1 h and 12 h. The paraffin-infiltrated muscle samples were then embedded using a paraffin embedding console (Tissue-Tek TEC™ 6, Sakura Finetek, Torrance, CA, USA), sectioned at a thickness of 10 μm using a microtome (HistoCore Multicut, Nussloch, Germany), and placed on glass slides.

4.5. Hematoxylin and Eosin Staining

Sectioned gastrocnemius tissues were stained with hematoxylin and eosin (H&E) to assess muscle fiber size. Slides were first deparaffinized in xylene (two changes, 2 min each) and then rehydrated through a graded ethanol series (100%, 95%, and 70% EtOH, 2 min each). The sections were stained in hematoxylin for 2 min and rinsed in water for 1 min. Nuclear staining was differentiated with 1% HCl in 70% EtOH, followed by another 1 min water rinse. Slides were then stained in eosin for 1 min, dehydrated through 70%, 95%, and 100% EtOH, cleared in xylene for 2 min, and coverslipped.

4.6. Muscle Fiber Cross-Sectional Area

Fiber size was measured by acquiring images from standardized cross-sectional areas (CSA) at ×40 magnification using Moticonnect software v1.5.3 (Motic, Hong Kong, China) with a light microscope (Cat No. M3802CT-4, Motic, Hong Kong, China). Each section was analyzed in ImageJ v2.1.0 to determine the area of individual muscle fibers, with an average of ~400 fibers used for each sample. The measured values were averaged for each sample, and CSA was distributed by experimental group.

4.7. Western Blot

Quadriceps and soleus muscles were powdered in liquid nitrogen and homogenized with RIPA buffer (Cat No. 20-188, Millipore, Burlington, MA, USA) containing protease (Cat No. 78439, Thermo Fisher Scientific, Waltham, MA, USA) and phosphatase (Cat No. P0044, Sigma-Aldrich, St. Louis, MO, USA) inhibitors (20:1 per mg muscle tissue). Protein concentrations were then quantified using a Pierce™ BCA protein assay kit (Cat no. 23227, Thermo Fisher Scientific, Waltham, MA, USA), as described in the manufacturer’s protocol. Equal amounts of proteins (10–15 µg) were denatured at 95 °C for 5 min and loaded on a 4–15% gradient Stain-Free Criterion TGX free gel, separated by electrophoresis (Cat No. 5678084, Bio-Rad, Hercules, CA, USA) using a Criterion™ Vertical Electrophoresis Cell (Cat No. 1656001, Bio-Rad, Hercules, CA, USA), and transferred onto polyvinylidene difluoride (PVDF) membranes (Cat No. IPFL00010, Millipore Sigma, Burlington, MA, USA) using a Trans-Blot^® Turbo™ Transfer system (Cat No. 1704150, Bio-Rad, Hercules, CA, USA). Total protein was assessed by imaging according to Bio-Rad’s Stain-Free protocol to allow for normalizing proteins of interest to protein loading [92,93]. After measuring total protein, the membranes were blocked in 5% non-fat dry milk with Tris-buffered saline (pH 7.5) with 0.1% Tween-20 (TBST) for 1 h at room temperature, followed by incubation for 24–48 h in primary antibody (1:1000 dilution) at 4 °C.

The primary antibodies were directed against antibodies p-mTOR (S2448, #2971, 1:1000), mTOR (#2983, 1:1000), p-Akt (Ser473, #9271, 1:1000), Akt (#9272, 1:1000), p-p70S6K (T389, #9234, 1:1000), p70S6K (#2708, 1:1000), p-4EBP1 (Thr37/46, #2855, 1:1000), 4EBP1 (#9644, 1:1000), ubiquitin (#8395, 1:1000), p-PERK (Thr980, #3179, 1:1000), PERK (#5683, 1:1000), ATF6 (#65880S, 1:1000), BIP (#3177, 1:1000), ATF4 (#11815, 1:1000), XBP1s (#40435, 1:1000), p-eIF2α (#3398, 1:1000), eIF2α (#9722, 1:1000), and CHOP (#2895, 1:1000) were purchased from Cell Signaling Technology (Danvers, MA, USA). MuRF1 (sc-398608, 1:1000), MAFbx (sc-166806, 1:1000), and LC3-I/II (sc-376404, 1:1000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). After 24 h (total protein) or 48 h (phosphorylated protein) incubation with primary antibodies, membranes were washed three times for 5 min with TBST and incubated in 5% non-fat dry milk TBST with horseradish peroxidase-conjugated secondary antibodies against mouse (#7076) or rabbit (#7074; Cell Signaling Technology, Danvers, MA, USA) at a 1:2000 (phosphorylated protein)–1:5000 (total protein) dilution and then washed three times for 5 min with TBST. The membranes were incubated in chemiluminescence solution (Cat No. 170-5061 or 170-5062, Bio-Rad, Hercules, CA, USA), and the immunoreactive bands were captured using the Chemi Doc™ MP imaging system (Cat No. 12003154, Bio-Rad, Hercules, CA, USA), and quantified using Image Lab V6.1 (Bio-Rad, Hercules, CA, USA). For Western blot analyses, some membranes were stripped (Cat No. BP98, Boston BioProducts Inc., Milford, MA, USA) for 10 min after initial detection and then washed three times for 5 min with TBST. Stripped membranes were reprobed following the same procedure to detect subsequent proteins. Phosphorylated proteins were always detected prior to stripping. Signal intensity of proteins of interest in each lane was normalized to the total protein quantification in the same lane, and phosphorylated proteins were normalized to their respective total protein, as indicated. Full Western blot membranes are shown in Figures S2–S21.

4.8. Sample Size

Sample size was determined based on the detection of an alcohol × immobilization interaction on skeletal muscle atrophy. Based on a previous study employing a 2 × 2 alcohol and hindlimb immobilization design, an effect size of the interaction was determined, whereby the atrophic response to immobilization was markedly exacerbated by alcohol exposure [22]. To avoid overestimation of effect size and to provide a conservative estimate suitable for the present mouse model, the study was powered to detect approximately 50% of the interaction magnitude reported in the previous study (f ≈ 0.8). Power analysis was performed using G*Power (version 3.1; ANOVA: fixed effects, special, main effects and interactions; α = 0.05; power = 0.80; numerator df = 1; 4 groups), which indicated that a minimum of 6–8 animals per group was required to detect a statistically significant alcohol × immobilization interaction. To account for biological variability and potential experimental attrition, the calculated sample size was increased by 10%, resulting in a final group size of nine animals per group. This sample size also provides sufficient power to detect the main effect of immobilization, which is known to induce substantial muscle atrophy in rodent disuse models.

4.9. Statistics

Statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software Inc., San Diego, CA, USA). Data are presented as mean ± standard error of the mean (SEM). An independent t-test was used for total caloric intake and liver weight. Two-way repeated-measures analysis of variance (RM-ANOVA) using Sidak’s multiple comparisons test was used to assess body weight throughout the intervention. Two-way ANOVA using Tukey’s post hoc test was used for diet (i.e., alcohol vs. control) and immobilization (i.e., immobilization vs. non-immobilization) as factors for muscle mass, CSA, anabolic (Akt, mTOR, p70S6K, 4EBP1, MyoD, and Myogenin), catabolic (ubiquitin, MuRF1, MAFbx, and LC3-I/II), and ER-stress signaling (PERK, eIF2α, ATF6, BIP, XBP1s, ATF4, and CHOP) protein expression. Potential outliers were identified using the robust regression and outlier removal (ROUT) method with a false discovery rate (Q) of 1%. Outliers identified as statistically significant were excluded from the analysis, and corresponding sample sizes are indicated in each figure legend line. All remaining data were analyzed using the indicated statistical analyses. Differences were determined statistically significant at p < 0.05 for all analyses, and statistically non-significant tendencies were reported at p < 0.1.

5. Conclusions

In conclusion, the NIAAA model of alcohol consumption and hindlimb immobilization independently induced skeletal muscle atrophy but interacted in a muscle-specific manner. At the mechanistic level, both stressors converge on shared downstream pathways, including anabolic and catabolic signaling, and ER stress responses that collectively regulate muscle atrophy programs. However, the relative contribution and engagement of these pathways differ between conditions, underscoring the importance of context-specific therapeutic strategies. Notably, alcohol exposure and immobilization synergistically suppressed anabolic signaling and strongly inhibited myogenic regulators while broadly downregulating multiple ER stress components in the soleus, suggesting the possibility of an anti-anabolic and anti-regenerative state in oxidative muscle. These findings indicate that although both conditions ultimately result in muscle wasting, the initiating molecular drivers and temporal progression of atrophy differ substantially. Collectively, our data demonstrate that alcohol exposure and hindlimb immobilization elicit distinct yet converging pathological responses in skeletal muscle, supporting the concept that these stressors impair muscle homeostasis through both independent and overlapping mechanisms that vary depending on muscle group. Importantly, these results highlight the need to consider alcohol use as a modifying factor in disuse-induced muscle atrophy, particularly in immobilized populations.