Short-term aerobic exercise prevents development of glucocorticoid myopathic features in aged skeletal muscle in a sex-dependent manner.

Older adults are vulnerable to glucocorticoid-induced muscle atrophy and weakness, with sex potentially influencing their susceptibility to those effects. Aerobic exercise can reduce glucocorticoid-induced muscle atrophy in young rodents. However, it is unknown whether aerobic exercise can prevent glucocorticoid myopathy in aged muscle. The objectives of this study were to define the extent to which sex influences the development of glucocorticoid myopathy in aged muscle, and to determine the extent to which aerobic exercise training protects against myopathy development. Twenty-four-month-old female (n = 30) and male (n = 33) mice were randomized to either sedentary or aerobic exercise groups. Within their respective groups, mice were randomized to either daily treatment with dexamethasone (DEX) or saline. Upon completing treatments, the contractile properties of the triceps surae complex were assessed in situ. DEX marginally lowered muscle mass and soluble protein content in both sexes, which was attenuated by aerobic exercise only in females. DEX increased sub-tetanic force and rate of force development only in females, which was not influenced by aerobic exercise. Muscle fatigue was higher in both sexes following DEX, but aerobic exercise prevented fatigue induction only in females. The sex-specific differences to muscle function in response to DEX treatment coincided with sex-specific changes to the content of proteins related to calcium handling, mitochondrial quality control, reactive oxygen species production, and glucocorticoid receptor in muscle. These findings define several important sexually dimorphic changes to aged skeletal muscle physiology in response to glucocorticoid treatment and define the capacity of short-term aerobic exercise to protect against those changes. KEY POINTS: There are sexually dimorphic effects of glucocorticoids on aged skeletal muscle physiology. Glucocorticoid-induced changes to aged muscle contractile properties coincide with sex-specific differences in the content of calcium handling proteins. Aerobic exercise prevents glucocorticoid-induced fatigue only in aged females and coincides with differences in the content of mitochondrial quality control proteins and glucocorticoid receptors.

The mechanosensitive gene arrestin domain containing 2 regulates myotube diameter with direct implications for disuse atrophy with aging.

Arrestin domain containing 2 and 3 (Arrdc2/3) are genes whose mRNA contents are decreased in young skeletal muscle following mechanical overload. Arrdc3 is linked to the regulation of signaling pathways in nonmuscle cells that could influence skeletal muscle size. Despite a similar amino acid sequence, Arrdc2 function remains undefined. The purpose of this study was to further explore the relationship of Arrdc2/Arrdc3 expression with changes in mechanical load in young and aged muscle and define the effect of Arrdc2/3 expression on C2C12 myotube diameter. In young and aged mice, mechanical load was decreased using hindlimb suspension whereas mechanical load was increased by reloading previously unloaded muscle or inducing high-force contractions. Arrdc2 and Arrdc3 mRNAs were overexpressed in C2C12 myotubes using adenoviruses. Myotube diameter was determined 48-h posttransfection, and RNA sequencing was performed on those samples. Arrdc2 and Arrdc3 mRNA content was higher in the unloaded muscle within 1 day of disuse and remained higher up through 10 days. The induction of Arrdc2 mRNA was more pronounced in aged muscle than young muscle in response to unloading. Reloading previously unloaded muscle of young and aged mice restored Arrdc2 and Arrdc3 levels to ambulatory levels. Increasing mechanical load beyond normal ambulatory levels lowered Arrdc2 mRNA, but not Arrdc3 mRNA, in young and aged muscle. Arrdc2 overexpression only was sufficient to lower myotube diameter in C2C12 cells in part by altering the transcriptome favoring muscle atrophy. These data are consistent with Arrdc2 contributing to disuse atrophy, particularly in aged muscle.NEW & NOTEWORTHY We establish Arrdc2 as a novel mechanosensitive gene highly induced in response to mechanical unloading, particularly in aged muscle. Arrdc2 induction in C2C12 myotubes is sufficient to produce thinner myotubes and a transcriptional landscape consistent with muscle atrophy and disuse.

Effects of chronic alcohol intoxication on aerobic exercise-induced adaptations in female mice.

Chronic alcohol intoxication decreases muscle strength/function and causes mitochondrial dysfunction. Aerobic exercise training improves mitochondrial oxidative capacity and increases muscle mass and strength. Presently, the impact of chronic alcohol on aerobic exercise-induced adaptations was investigated. Female C57BL/6Hsd mice were randomly assigned to one of four groups: control sedentary (CON SED; n = 26), alcohol sedentary (ETOH SED; n = 27), control exercise (CON EX; n = 28), and alcohol exercise (ETOH EX; n = 25). Exercise mice had running wheel access for 2 h a day, 7 days a week. All mice were fed either control or an alcohol-containing liquid diet. Grip strength testing and EchoMRI were performed before and after the interventions. After 6 wk, hindlimb muscles were collected for molecular analyses. A subset of mice performed a treadmill run to fatigue (RTF), then abstained from alcohol for 2 wk and repeated the RTF. Alcohol decreased lean mass and forelimb grip strength compared with control-fed mice. Alcohol blunted the exercise-induced increase in muscle mass (plantaris and soleus), type IIa fiber percentage in the plantaris, and run time to fatigue. Mitochondrial markers (Citrate synthase activity and Complex I-IV, COXIV and Cytochrome C protein expression) were increased with exercise regardless of ETOH in the gastrocnemius but not tibialis anterior muscle. Two weeks of alcohol abstinence improved RTF time in ETOH EX but not in ETOH SED. These data suggest that alcohol impairs some exercise-induced adaptations in skeletal muscle, but not all were negatively affected, indicating that exercise may be a beneficial behavior even while consuming alcohol.NEW & NOTEWORTHY Alcohol consumption during an aerobic exercise training period prevented training-induced increases in run to fatigue time and grip strength. Cessation of alcohol allowed for recovery of endurance performance within 2 wk. The worsened exercise performance after alcohol was unrelated to impairments in markers of mitochondrial health. Therefore, some adaptations to exercise training are impaired with alcohol use (endurance performance, muscle growth, and strength), while others remain mostly unaffected (mitochondrial health).

SIRT1 induction in the skeletal muscle of male mice partially attenuates changes to whole-body metabolism in response to androgen deprivation.

In males, androgens regulate whole body metabolism. The components in androgen target organs contributing to whole-body metabolic function remain ill defined. Sirtuin1 (SIRT1) protein levels are lower in the limb muscle of male mice subjected to androgen deprivation. Because SIRT1 can influence whole-body metabolism, the purpose was to assess whether muscle specific SIRT1 induction attenuated changes to whole-body metabolism in response to androgen deprivation. Physically mature male mice containing an inducible muscle specific SIRT1 transgene (SIRT1) were subjected to a sham or castration surgery and compared to sham and castrated male mice where the SIRT1 transgene was not induced (WT). The respiratory exchange ratio (RER), energy expenditure, and carbohydrate and fat oxidation rates were determined using metabolic cages. Castration lowered RER in WT mice and the lower RER coincided with lower energy expenditure, lower carbohydrate oxidation rates, and higher fat oxidation rates. SIRT1 induction attenuated the castration-induced changes to RER and fat oxidation rates. Changes to energy expenditure and glucose oxidation rates were not affected by SIRT1. Decreases in muscle SIRT1 protein in males may partially contribute to the dysregulation of whole-body metabolism in response to androgen deprivation.

SIRT1 induction in the skeletal muscle of male mice partially preserves limb muscle mass but not contractile force in response to androgen deprivation.

In males, the factors that decrease limb muscle mass and strength in response to androgen deprivation are largely unknown. Sirtuin1 (SIRT1) protein levels are lower in the limb muscle of male mice subjected to androgen deprivation. The present study aimed to assess whether SIRT1 induction preserved limb muscle mass and force production in response to androgen deprivation. Physically mature male mice containing an inducible muscle-specific SIRT1 transgene were subjected to a sham or castration surgery and compared to sham and castrated male mice where the SIRT1 transgene was not induced. SIRT1 induction partially preserved whole-body lean mass, tibialis anterior (TA) mass and triceps surae muscle mass in response to castration. Further analysis of the TA muscle showed that muscle-specific SIRT1 induction partially preserved limb muscle soluble protein content and fibre cross-sectional area. Unilateral AAV9-mediated SIRT1 induction in the TA muscle showed that SIRT1 partially preserved mass by acting directly in the muscle. Despite those positive outcomes to limb muscle morphology, muscle-specific SIRT1 induction did not preserve the force generating capacity of the TA or triceps surae muscles. Interestingly, SIRT1 induction in females did not alter limb muscle mass or limb muscle strength even though females have naturally low androgen levels. SIRT1 also did not alter the androgen-mediated increase in limb muscle mass or strength in females. In all, these data suggest that decreases in SIRT1 protein in the limb muscle of males may partially contribute to the loss of limb muscle mass in response to androgen deprivation. KEY POINTS: SIRT1 induction in skeletal muscle of male mice subjected to androgen deprivation partially preserved limb muscle mass and fibre cross-sectional area. SIRT1 induction in skeletal muscle of male mice subjected to androgen deprivation did not prevent preserve limb muscle force generating capacity. SIRT1 induction in skeletal muscle of females did not alter baseline limb muscle mass, nor did it affect the androgen-mediated increase in limb muscle mass.

The duration of glucocorticoid treatment alters the anabolic response to high-force muscle contractions.

Glucocorticoids induce a myopathy that includes loss of muscle mass and strength. Resistance exercise may reverse the muscle loss because it induces an anabolic response characterized by increases in muscle protein synthesis and potentially suppressing protein breakdown. Whether resistance exercise induces an anabolic response in glucocorticoid myopathic muscle is unknown, which is a problem because long-term glucocorticoid exposure alters the expression of genes that may prevent an anabolic response by limiting activation of pathways such as the mechanistic target of rapamycin in complex 1 (mTORC1). The purpose of this study was to assess whether high-force contractions initiate an anabolic response in glucocorticoid myopathic muscle. The anabolic response was analyzed by treating female mice with dexamethasone (DEX) for 7 days or 15 days. After treatment, the left tibialis anterior muscle of all mice was contracted via electrical stimulation of the sciatic nerve. Muscles were harvested 4 h after contractions. Rates of muscle protein synthesis were estimated using the SUnSET method. After 7 days of treatment, high-force contractions increased protein synthesis and mTORC1 signaling in both groups. After 15 days of treatment, high-force contractions activated mTORC1 signaling equally in both groups, but protein synthesis was only increased in control mice. The failure to increase protein synthesis may be because baseline synthetic rates were elevated in DEX-treated mice. The LC3 II/I ratio marker of autophagy was decreased by contractions regardless of treatment duration. These data show duration of glucocorticoid treatment alters the anabolic response to high-force contractions.NEW & NOTEWORTHY Glucocorticoid myopathy is the most common, toxic, noninflammatory myopathy. Our work shows that high-force contractions increase protein synthesis in skeletal muscle following short-term glucocorticoid treatment. However, longer duration glucocorticoid treatment results in anabolic resistance to high-force contractions despite activation of the mechanistic target of rapamycin in complex 1 (mTORC1) signaling pathway. This work defines potential limits for high-force contractions to activate the processes that would restore lost muscle mass in glucocorticoid myopathic patients.

REDD1 deletion and treadmill running increase liver hepcidin and gluconeogenic enzymes in male mice.

The iron-regulatory hormone hepcidin is transcriptionally up-regulated by gluconeogenic signals. Recent evidence suggeststhat increases in circulating hepcidin may decrease dietary iron absorption following prolonged exercise, however evidence is limited on whether gluconeogenic signals contribute to post-exercise increases in hepcidin. Mice with genetic knockout of regulated in development and DNA response-1 (REDD1) display greater glycogen depletion following exercise, possibly indicating greater gluconeogenesis. The objective of the present study was to determine liver hepcidin, markers of gluconeogenesis and iron metabolism in REDD1 knockout and wild-type mice following prolonged exercise. Twelve-week-old male REDD1 knockout and wild-type mice were randomised to rest or 60 min treadmill running with 1, 3 or 6 h recovery (n = 5-8/genotype/group). Liver gene expression of hepcidin (Hamp) and gluconeogenic enzymes (Ppargc1a, Creb3l3, Pck1, Pygl) were determined by qRT-PCR. Effects of genotype, exercise and their interaction were assessed by two-way ANOVAs with Tukey's post-hoc tests, and Pearson correlations were used to assess the relationships between Hamp and study outcomes. Liver Hamp increased 1- and 4-fold at 3 and 6 h post-exercise, compared to rest (P-adjusted < 0⋅009 for all), and was 50% greater in REDD1 knockout compared to wild-type mice (P = 0⋅0015). Liver Ppargc1a, Creb3l3 and Pck1 increased with treadmill running (P < 0⋅0001 for all), and liver Ppargc1a, Pck1 and Pygl were greater with REDD1 deletion (P < 0⋅02 for all). Liver Hamp was positively correlated with liver Creb3l3 (R = 0⋅62, P < 0⋅0001) and Pck1 (R = 0⋅44, P = 0⋅0014). In conclusion, REDD1 deletion and prolonged treadmill running increased liver Hamp and gluconeogenic regulators of Hamp, suggesting gluconeogenic signalling of hepcidin with prolonged exercise.

Impact of prior alcohol use on the subsequent development of cancer cachexia in male and female mice.

BACKGROUND: Alcohol is a carcinogen and its intake prior to developing cancer and throughout its duration exacerbates cancer cachexia in rodent models. However, the effects on cancer cachexia of stopping alcohol prior to tumor establishment are unknown. METHODS: Male and female mice consumed either a nonalcohol control liquid diet (CON) or a 20% ethanol (kcal/day) liquid diet (EtOH) for 6 weeks. All mice then consumed a control diet and mice in the cancer groups were inoculated with C26 colon cancer cells. Gastrocnemius muscles were collected and analyzed after ~2 weeks. RESULTS: Skeletal muscle weight and male epididymal and female perigonadal fat mass were reduced more by the combination of cancer and prior EtOH than either exposure alone in both males and females. In males, protein synthesis was reduced by 30% following alcohol exposure, while no reductions were observed in female mice. AMPK Thr172 phosphorylation was increased in both male and female EtOH-Cancer groups, while Akt Thr308 phosphorylation was reduced only among males in EtOH-Cancer mice. Substrates in the mTORC1 pathway were reduced by cancer in both males and females, but prior alcohol intake only reduced phosphorylation of 4E-BP1 Ser65 and rpS6 Ser240/244 to a greater extent in male, but not female, mice. Autophagic and proteasomal signaling were largely unaffected by prior alcohol intake in cancer mice, despite a greater increase in Murf1 mRNA in both sexes. CONCLUSIONS: Prior alcohol consumption accelerates or worsens the onset of certain aspects of cancer cachexia in a sex-dependent manner, with males being more sensitive to these exposures, even with abstinence from alcohol prior to tumor initiation.

Mealtime alcohol consumption suppresses skeletal muscle mTORC1 signaling in female mice.

OBJECTIVE: To determine whether alcohol consumed within the meal influences the feeding induced increase in mTORC1 signaling. METHODS: Alcohol provided in the liquid diet was consumed by alcohol naïve, fasted, C57BL/6Hsd female mice and gastrocnemius was collected 1hr after the refeeding. Subsequent experiments determined the extent to which changes in mTORC1 signaling persisted across the day. RESULTS: Compared with control mice, protein synthesis, mTORC1 (Ser2448), 4EBP1 (Ser65), S6K1 (Thr389), rpS6 (Ser240/244), Akt (Thr308), and ULK1 (Ser757) were lower in EtOH. Similar suppressive patterns were observed in the hours following consumption of alcohol containing food throughout the dark cycle. Higher peak blood alcohol concentrations induced by intraperitoneal injection of alcohol extended the time and magnitude of mTORC1 pathway suppression. CONCLUSION: Alcohol administered as part of the meal results in lower skeletal muscle mTORC1 signaling while subsequent models show that alcohol may influence this pathway across the day.

Chronic Alcohol Consumption Disrupts the Skeletal Muscle Circadian Clock in Female Mice.

The intrinsic skeletal muscle core clock has emerged as a key feature of metabolic control and influences several aspects of muscle physiology. Acute alcohol intoxication disrupts the core molecular clock, but whether chronic consumption, like that leading to alcoholic myopathy, is also a zeitgeber for skeletal muscle remains unknown. The purpose of this work was to determine whether chronic alcohol consumption dysregulates the skeletal muscle core molecular clock and clock-controlled genes (CCGs). C57BL/6Hsd female mice (14 weeks old) were fed a control (CON) or alcohol (EtOH) containing liquid diet for 6 weeks. Gastrocnemius muscles and serum were collected from CON and EtOH mice every 4-h for 24-h. Chronic alcohol consumption disrupted genes of the core clock including suppressing the rhythmic peak of expression of Bmal1, Per1, Per2, and Cry2. Genes involved in the regulation of Bmal1 also exhibited lower rhythmic peaks including Reverb α and Myod1. The CCGs, Dbp, Lpl, Hk2, and Hadh were also suppressed by alcohol. The nuclear expression patterns of MYOD1, DBP, and REVERBα were shifted by alcohol, while no change in BMAL1 was detected. Overall, these data indicate that alcohol disrupted the skeletal muscle core clock but whether these changes in the core clock are causative or a consequence of alcoholic myopathy requires future mechanistic confirmation.

Long-term administration of resveratrol and MitoQ stimulates cavernosum antioxidant gene expression in a mouse castration model of erectile dysfunction.

AIMS: Erectile dysfunction is a common complication within many pathological conditions associated with low testosterone. Testosterone deficiency increases oxidative stress in the penile tissue that contributes to endothelial dysfunction and subsequent erectile dysfunction. Current therapies do not ameliorate oxidative stress so targeting oxidative stress may improve erectile dysfunction. Resveratrol and MitoQ are two prospective drugs that have antioxidant-like properties and may be useful to improve erectile dysfunction induced by androgen deprivation. MATERIALS AND METHODS: We castrated 12-week-old male C57BL/6 mice and performed an eight-week intervention with oral delivery of resveratrol or MitoQ at low and high doses. We assessed vascular reactivity of the corpus cavernosum and internal pudendal arteries (IPA) through dose-dependent responses to vasodilatory, vasocontractile, and neurogenic stimuli in a myograph system. We performed qRT-PCR to measure expression changes of 18 antioxidant genes in the corpus cavernosum. KEY FINDINGS: Castration significantly impaired erectile function via impaired endothelial-dependent and-independent relaxation, and increased constriction of the corpus cavernosum, and induced severe endothelial dysfunction of the IPA. Castration decreased expression of 8 of the antioxidant genes investigated. Resveratrol and MitoQ were ineffective in reversing the effects of androgen deprivation on vascular reactivity, however high-dose resveratrol treatment upregulated several key antioxidant genes, including Cat, Sod1, Gstm1, and Prdx3. SIGNIFICANCE: Our findings suggest that oral resveratrol and MitoQ treatment may provide protection to the corpus cavernosum under androgen deprived conditions by stimulating endogenous antioxidant systems. However, they may need to be paired with vasoactive drugs to reverse erectile dysfunction under androgen deprived conditions.

Acute binge alcohol alters whole body metabolism and the time-dependent expression of skeletal muscle-specific metabolic markers for multiple days in mice.

Alcohol is a myotoxin that disrupts skeletal muscle function and metabolism, but specific metabolic alternations following a binge and the time course of recovery remain undefined. The purpose of this work was to determine the metabolic response to binge alcohol, the role of corticosterone in this response, and whether nutrient availability mediates the response. Female mice received saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle. Whole body metabolism was assessed for 5 days. In a separate cohort, gastrocnemius muscles and liver were collected every 4 h for 48 h following intoxication. Metyrapone was administered before alcohol and gastrocnemius was collected 4 h later. Lastly, alcohol-treated mice were compared with fed or fasted controls. Alcohol disrupted whole body metabolism for multiple days. Alcohol altered the expression of genes and proteins in the gastrocnemius related to the promotion of fat oxidation (Pparα, Pparδ/ß, AMPK, and Cd36) and protein breakdown (Murf1, Klf15, Bcat2). Changes to select metabolic genes in the liver did not parallel those in skeletal muscle. An alcohol-induced increase in circulating corticosterone was responsible for the initial change in protein breakdown factors but not the induction of FoxO1, Cebpß, Pparα, and FoxO3. Alcohol led to a similar, but distinct metabolic response when compared with fasting animals. Overall, these data show that an acute alcohol binge rapidly disrupts macronutrient metabolism including sustained disruption to the metabolic gene signature of skeletal muscle in a manner similar to fasting at some time points.NEW & NOTEWORTHY Herein, we demonstrate that acute alcohol intoxication immediately alters whole body metabolism coinciding with rapid changes in the skeletal muscle macronutrient gene signature for at least 48 h postbinge and that this response diverges from hepatic effects and those of a fasted animal.

The influence of nutrients on mechanical overload-induced changes to skeletal muscle mRNA content.

Mechanical overload and nutrients influence skeletal muscle phenotype, with the combination sometimes having a synergistic effect. Muscle phenotypes influenced by these stimuli are mediated in part by changes to the muscle mRNA signature. However, the mechanical overload-sensitive gene programs that are influenced by nutrients remain unclear. The purpose of this study was to identify mechanical overload-sensitive gene programs that are influenced by nutrients and identify potential transcription factors that may differentiate the change in mRNA in response to mechanical overload versus nutrients. Nutrient-deprived 12-wk-old male mice were randomized to remain fasted or allowed access to food. All mice underwent a single bout of unilateral high force contractions of the tibialis anterior (TA). Four hours postcontractions TA muscles were extracted and the content of 12 contraction-sensitive mRNAs was analyzed. The mRNA content of genes associated with transcription, PI3K-Akt signaling pathway, Z-disc, intracellular signal transduction, cell cycle, and amino acid transport was altered by contractions without the influence of nutrient consumption. Conversely, the mRNA content of genes associated with transcription, cell cycle, FoxO signaling pathway, and amino acid transport was altered by contractions with nutrition consumption influencing the change. We identified the signal transducer and activator of transcription 3 (STAT3) and activator protein 1 (AP-1) as transcription factors common among mRNAs that were primarily altered by mechanical overload regardless of feeding. Overall, these data provide a deeper molecular basis for the specific muscle phenotypes exclusive to mechanical overload versus those regulated by the addition of nutrients.

Aerobic exercise-mediated changes in the expression of glucocorticoid responsive genes in skeletal muscle differ across the day.

Glucocorticoids are released in response to acute aerobic exercise. The objective was to define changes in the expression of glucocorticoid target genes in skeletal muscle in response to acute aerobic exercise at different times of day. We identified glucocorticoid target genes altered in skeletal muscle by acute exercise by comparing data sets from rodents subjected to acute aerobic exercise in the light or dark cycles to data sets from C2C12 myotubes treated with glucocorticoids. The role of glucocorticoid receptor signaling and REDD1 protein in mediating gene expression was assessed in exercised mice. Changes to expression of glucocorticoid genes were greater when exercise occurred in the dark cycle. REDD1 was required for the induction of genes induced at both times of day. In all, the time of day at which aerobic exercise is conducted dictates changes to the expression of glucocorticoid target genes in skeletal muscle with REDD1 contributing to those changes.

REDD1 deletion attenuates cancer cachexia in mice.

Cancer cachexia is a wasting disorder associated with advanced cancer that contributes to mortality. Cachexia is characterized by involuntary loss of body weight and muscle weakness that affects physical function. Regulated in DNA damage and development 1 (REDD1) is a stress-response protein that is transcriptionally upregulated in muscle during wasting conditions and inhibits mechanistic target of rapamycin complex 1 (mTORC1). C2C12 myotubes treated with Lewis lung carcinoma (LLC)-conditioned media increased REDD1 mRNA expression and decreased myotube diameter. To investigate the role of REDD1 in cancer cachexia, we inoculated 12-wk-old male wild-type or global REDD1 knockout (REDD1 KO) mice with LLC cells and euthanized 28 days later. Wild-type mice had increased skeletal muscle REDD1 expression, and REDD1 deletion prevented loss of body weight and lean tissue mass but not fat mass. We found that REDD1 deletion attenuated loss of individual muscle weights and loss of myofiber cross-sectional area. We measured markers of the Akt/mTORC1 pathway and found that, unlike wild-type mice, phosphorylation of both Akt and 4E-BP1 was maintained in the muscle of REDD1 KO mice after LLC inoculation, suggesting that loss of REDD1 is beneficial in maintaining mTORC1 activity in mice with cancer cachexia. We measured Foxo3a phosphorylation as a marker of the ubiquitin proteasome pathway and autophagy and found that REDD1 deletion prevented dephosphorylation of Foxo3a in muscles from cachectic mice. Our data provide evidence that REDD1 plays an important role in cancer cachexia through the regulation of both protein synthesis and protein degradation pathways.NEW & NOTEWORTHY Cancer cachexia is a debilitating and lethal consequence of many advanced cancers. REDD1, a negative regulator of mTORC1 activity, is an emerging target in cachexia. Our data show that skeletal muscle REDD1 expression is increased in LLC-induced cancer cachexia. Mice lacking REDD1 have attenuated skeletal muscle atrophy that is likely due to maintaining both protein synthesis and inhibiting protein degradation.

Binge alcohol disrupts skeletal muscle core molecular clock independent of glucocorticoids.

Circadian rhythms are central to optimal physiological function, as disruption contributes to the development of several chronic diseases. Alcohol (EtOH) intoxication disrupts circadian rhythms within liver, brain, and intestines, but it is unknown whether alcohol also disrupts components of the core clock in skeletal muscle. Female C57BL/6Hsd mice were randomized to receive either saline (control) or alcohol (EtOH) (5 g/kg) via intraperitoneal injection at the start of the dark cycle [Zeitgeber time (ZT12)], and gastrocnemius was collected every 4 h from control and EtOH-treated mice for the next 48 h following isoflurane anesthetization. In addition, metyrapone was administered before alcohol intoxication in separate mice to determine whether the alcohol-induced increase in serum corticosterone contributed to circadian gene regulation. Finally, synchronized C2C12 myotubes were treated with alcohol (100 mM) to assess the influence of centrally or peripherally mediated effects of alcohol on the muscle clock. Alcohol significantly disrupted mRNA expression of Bmal1, Per1/2, and Cry1/2 in addition to perturbing the circadian pattern of clock-controlled genes, Myod1, Dbp, Tef, and Bhlhe40 (P < 0.05), in muscle. Alcohol increased serum corticosterone levels and glucocorticoid target gene, Redd1, in muscle. Metyrapone prevented the EtOH-mediated increase in serum corticosterone but did not normalize the EtOH-induced change in Per1, Cry1 and Cry2, and Myod1 mRNA expression. Core clock gene expression (Bmal, Per1/2, and Cry1/2) was not changed following 4, 8, or 12 h of alcohol treatment on synchronized C2C12 myotubes. Therefore, binge alcohol disrupted genes of the core molecular clock independently of elevated serum corticosterone or direct effects of EtOH on the muscle.NEW & NOTEWORTHY Alcohol is a myotoxin that impairs skeletal muscle metabolism and function following either chronic consumption or acute binge drinking; however, mechanisms underlying alcohol-related myotoxicity have not been fully elucidated. Herein, we demonstrate that alcohol acutely interrupts oscillation of skeletal muscle core clock genes, and this is neither a direct effect of ethanol on the skeletal muscle, nor an effect of elevated serum corticosterone, a major clock regulator.

Effects of alcohol on skeletal muscle contractile performance in male and female mice.

BACKGROUND: Acute and chronic alcohol use can cause skeletal muscle myopathy in concert with impairments in skeletal muscle strength, function and fatigue resistance. However, the fundamental contractile deficits induced in the presence of alcohol versus those observed in the recovery period following the clearance of alcohol have not yet been characterized nor is it known whether sex influences these outcomes. METHODS: Male and female mice received an intraperitoneal injection of either saline (Control) or ethanol (EtOH; 5g/kg body weight). Muscle force, fatigue, fatigue recovery and twitch characteristics of the posterior crural muscle complex were measured in situ 1 hour and 24 hours post alcohol. RESULTS: In the presence of alcohol (1-hour post treatment) absolute and normalized force generated at 80-150 Hertz was decreased in male and female mice with concurrent reductions in the rate of force development and increases in ½ relaxation time. When expressed as a percentage of maximum force, both males and females also displayed an alcohol-induced leftward shift in the force frequency curve indicative of a type I contractile phenotype. Alcohol enhanced fatigue in both males and females but had no effect on force recovery. Following clearance of alcohol (24-hour post treatment), contractile function was completely restored in females while alcohol treated males experienced sustained reductions in absolute force and had enhanced fatigue compared with male controls. CONCLUSIONS: In the presence of alcohol, both males and females exhibited significant declines in muscle force production and enhanced fatigue; however, following complete clearance of the alcohol, females recovered all functional parameters, while males did not.

Systemic delivery of a mitochondria targeted antioxidant partially preserves limb muscle mass and grip strength in response to androgen deprivation.

Muscle mass is important for health. Decreased testicular androgen production (hypogonadism) contributes to the loss of muscle mass, with loss of limb muscle being particularly debilitating. Androgen replacement is the only pharmacological treatment, which may not be feasible for everyone. Prior work showed that markers of reactive oxygen species and markers of mitochondrial degradation pathways were higher in the limb muscle following castration. Therefore, we tested whether an antioxidant preserved limb muscle mass in male mice subjected to a castration surgery. Subsets of castrated mice were treated with resveratrol (a general antioxidant) or MitoQ (a mitochondria targeted antioxidant). Relative to the non-castrated control mice, lean mass, limb muscle mass, and grip strength were partially preserved only in castrated mice treated with MitoQ. Independent of treatment, markers of mitochondrial degradation pathways remained elevated in all castrated mice. Therefore, a mitochondrial targeted antioxidant may partially preserve limb muscle mass in response to hypogonadism.

A clinically relevant decrease in contractile force differentially regulates control of glucocorticoid receptor translocation in mouse skeletal muscle.

Muscle atrophy decreases physical function and overall health. Increased glucocorticoid production and/or use of prescription glucocorticoids can significantly induce muscle atrophy by activating the glucocorticoid receptor, thereby transcribing genes that shift protein balance in favor of net protein degradation. Although mechanical overload can blunt glucocorticoid-induced atrophy in young muscle, those affected by glucocorticoids generally have impaired force generation. It is unknown whether contractile force alters the ability of resistance exercise to mitigate glucocorticoid receptor translocation and induce a desirable shift in protein balance when glucocorticoids are elevated. In the present study, mice were subjected to a single bout of unilateral, electrically induced muscle contractions by stimulating the sciatic nerve at 100 Hz or 50 Hz frequencies to elicit high or moderate force contractions of the tibialis anterior, respectively. Dexamethasone was used to activate the glucocorticoid receptor. Dexamethasone increased glucocorticoid signaling, including nuclear translocation of the receptor, but this was mitigated only by high force contractions. The ability of high force contractions to mitigate glucocorticoid receptor translocation coincided with a contraction-mediated increase in muscle protein synthesis, which did not occur in the dexamethasone-treated mice subjected to moderate force contractions. Though moderate force contractions failed to increase protein synthesis following dexamethasone treatment, both high and moderate force contractions blunted the glucocorticoid-mediated increase in LC3 II:I marker of autophagy. Thus, these data show that force generation is important for the ability of resistance exercise to mitigate glucocorticoid receptor translocation and promote a desirable shift in protein balance when glucocorticoids are elevated.NEW & NOTEWORTHY Glucocorticoids induce significant skeletal muscle atrophy by activating the glucocorticoid receptor. Our work shows that muscle contractile force dictates glucocorticoid receptor nuclear translocation. We also show that blunting nuclear translocation by high force contractions coincides with the ability of muscle to mount an anabolic response characterized by increased muscle protein synthesis. This work further defines the therapeutic parameters of skeletal muscle contractions to blunt glucocorticoid-induced atrophy.

Androgen depletion alters the diurnal patterns to signals that regulate autophagy in the limb skeletal muscle.

Hypogonadism contributes to limb skeletal muscle atrophy by increasing rates of muscle protein breakdown. Androgen depletion increases markers of the autophagy protein breakdown pathway in the limb muscle that persist throughout the diurnal cycle. However, the regulatory signals underpinning the increase in autophagy markers remain ill-defined. The purpose of this study was to characterize changes to autophagy regulatory signals in the limb skeletal muscle following androgen depletion. Male mice were subjected to a castration surgery or a sham surgery as a control. Seven weeks post-surgery, a subset of mice from each group was sacrificed every 4 hr over a 24 hr period. Protein and mRNA from the Tibialis Anterior (TA) were subjected to Western blot and RT-PCR. Consistent with an overall increase in autophagy, the phosphorylation pattern of Uncoordinated Like Kinase 1 (ULK1) (Ser555) was elevated throughout the diurnal cycle in the TA of castrated mice. Factors that induce the progression of autophagy were also increased in the TA following androgen depletion including an increase in the phosphorylation of c-Jun N-terminal Kinase (JNK) (Thr183/Tyr185) and an increase in the ratio of BCL-2 Associated X (BAX) to B-cell lymphoma 2 (BCL-2). Moreover, we observed an increase in the protein expression pattern of p53 and the mRNA of the p53 target genes Cyclin-Dependent Kinase Inhibitor 1A (p21) and Growth Arrest and DNA Damage Alpha (Gadd45a), which are known to increase autophagy and induce muscle atrophy. These data characterize novel changes to autophagy regulatory signals in the limb skeletal muscle following androgen deprivation.