Unloading-induced Muscle Atrophy Research Articles

Celecoxib attenuates hindlimb unloading-induced muscle atrophy via suppressing inflammation, oxidative stress and ER stress by inhibiting STAT3.

Improving inflammation may serve as useful therapeutic interventions for the hindlimb unloading-induced disuse muscle atrophy. Celecoxib is a selective non-steroidal anti-inflammatory drug. We aimed to determine the role and mechanism of celecoxib in hindlimb unloading-induced disuse muscle atrophy. Celecoxib significantly attenuated the decrease in soleus muscle mass, hindlimb muscle function and the shift from slow- to fast-twitch muscle fibers caused by hindlimb unloading in rats. Importantly, celecoxib inhibited the increased expression of inflammatory factors, macrophage infiltration in damaged soleus muscle. Mechanistically, Celecoxib could significantly reduce oxidative stress and endoplasmic reticulum stress in soleus muscle of unloaded rats. Furthermore, celecoxib inhibited muscle proteolysis by reducing the levels of MAFbx, MuRF1, and autophagy related proteins maybe by inhibiting the activation of pro-inflammatory STAT3 pathway in vivo and in vitro. This study is the first to demonstrate that celecoxib can attenuate disuse muscle atrophy caused by hindlimb unloading via suppressing inflammation, oxidative stress and endoplasmic reticulum stress probably, improving target muscle function and reversing the shift of muscle fiber types by inhibiting STAT3 pathways-mediated inflammatory cascade. This study not only enriches the potential molecular regulatory mechanisms, but also provides new potential therapeutic targets for disuse muscle atrophy.

Mechanical unloading of 3D-engineered muscle leads to muscle atrophy by suppressing protein synthesis

Three-dimensional (3D)-engineered muscle is an useful approach to a more comprehensive understanding of molecular mechanisms underlying unloading-induced muscle atrophy. We investigated the effects of mechanical unloading on molecular muscle protein synthesis (MPS)- and muscle protein breakdown (MPB)-related signaling pathways involved in muscle atrophy in 3D-engineered muscle, and to better understand in vitro model of muscle disuse. The 3D-engineered muscle consisting of C2C12 myoblasts and type-1 collagen gel was allowed to differentiate for 2 wk and divided into three groups: 0 days of stretched-on control (CON), 2 and/or 7 days of stretched-on (ON), in which both ends of the muscle were fixed with artificial tendons, and the stretched-off group (OFF), in which one side of the artificial tendon was detached. Muscle weight (-38.1% to -48.4%), length (-67.0% to -73.5%), twitch contractile force (-70.5% to -75.0%), and myosin heavy chain expression (-32.5% to -50.5%) in the OFF group were significantly decreased on days 2 and 7 compared with the ON group (P < 0.05, respectively), despite that ON group was stable over time. Although determinative molecular signaling could not be identified, the MPS rate reflected by puromysin-labeled protein was significantly decreased following mechanical unloading (P < 0.05, -38.5% to -51.1%). Meanwhile, MPB, particularly the ubiquitin-proteasome pathway, was not impacted. Hence, mechanical unloading of 3D-engineered muscle in vitro leads to muscle atrophy by suppressing MPS, cell differentiation, and cell growth rather than the promotion of MPB.NEW & NOTEWORTHY Three-dimensional (3D)-engineered muscles have recently been shown to closely replicate the in vivo architecture. We found that mechanical unloading of 3D-engineered muscle led to muscle disuse atrophy accompanied by reduced functional properties and contractile protein expression via suppression of muscle protein synthesis. This novel model may improve the in vitro testing of modalities with the potential to reduce mechanical unloading-induced atrophy.

Nox2 Inhibition Regulates Stress Response and Mitigates Skeletal Muscle Fiber Atrophy during Simulated Microgravity

Insufficient stress response and elevated oxidative stress can contribute to skeletal muscle atrophy during mechanical unloading (e.g., spaceflight and bedrest). Perturbations in heat shock proteins (e.g., HSP70), antioxidant enzymes, and sarcolemmal neuronal nitric oxidase synthase (nNOS) have been linked to unloading-induced atrophy. We recently discovered that the sarcolemmal NADPH oxidase-2 complex (Nox2) is elevated during unloading, downstream of angiotensin II receptor 1, and concomitant with atrophy. Here, we hypothesized that peptidyl inhibition of Nox2 would attenuate disruption of HSP70, MnSOD, and sarcolemmal nNOS during unloading, and thus muscle fiber atrophy. F344 rats were divided into control (CON), hindlimb unloaded (HU), and hindlimb unloaded +7.5 mg/kg/day gp91ds-tat (HUG) groups. Unloading-induced elevation of the Nox2 subunit p67phox-positive staining was mitigated by gp91ds-tat. HSP70 protein abundance was significantly lower in HU muscles, but not HUG. MnSOD decreased with unloading; however, MnSOD was not rescued by gp91ds-tat. In contrast, Nox2 inhibition protected against unloading suppression of the antioxidant transcription factor Nrf2. nNOS bioactivity was reduced by HU, an effect abrogated by Nox2 inhibition. Unloading-induced soleus fiber atrophy was significantly attenuated by gp91ds-tat. These data establish a causal role for Nox2 in unloading-induced muscle atrophy, linked to preservation of HSP70, Nrf2, and sarcolemmal nNOS.

Long-term physical inactivity exacerbates hindlimb unloading-induced muscle atrophy in young rat soleus muscle.

This study investigated the effects of long-term physical inactivity in adolescent on subsequent hindlimb unloading-induced muscle atrophy in rat soleus muscle. First, 3-wk-old male Wistar rats were assigned to an age-matched control (n = 6) or a physical inactivity (n = 8) group. Rats in the physical inactivity group were housed in narrow cages with approximately half the usual floor space for 8 wk to limit range of movement. Whole body energy consumption was measured, and the blood, organs, femoral bone, and hindlimb muscles were removed. We found that long-term physical inactivity did not affect the metabolic and physiological characteristics of growing rats. Then, fifty-six 3-wk-old male Wistar rats were assigned randomly into control (n = 28) and physical inactivity (n = 28) groups. After 8 wk, the rats in both groups underwent hindlimb unloading. The soleus muscles were removed before unloading (0 day), and 1, 3, and 7 days after unloading (n = 7 for each). Although the soleus muscle weight was significantly decreased after 7 days of hindlimb unloading in both groups, the decrease was drastic in the inactive group. A significant interaction between inactivity and unloading (P < 0.01) was observed according to the 4-hydroxynonenal-conjugated protein levels and the histone deacetylase 4 (HDAC4) and NF-κB protein levels. HDAC4 and NF-κB p65 protein levels in the physical inactivity group increased significantly 1 day after hindlimb unloading, along with the mRNA levels of their downstream targets myogenin and muscle RING finger protein 1 (MuRF1). Subsequent protein ubiquitination was upregulated by long-term physical inactivity (P < 0.05).NEW & NOTEWORTHY Long-term physical inactivity exacerbates hindlimb unloading-induced disuse muscle atrophy in young rat soleus muscles, possibly mediated by oxidative stress-induced protein ubiquitination via HDAC4- and NF-κB p65-induced MuRF1 mRNA upregulation.

Loss of melusin is a novel, neuronal NO synthase/FoxO3-independent master switch of unloading-induced muscle atrophy.

BackgroundUnloading/disuse induces skeletal muscle atrophy in bedridden patients and aged people, who cannot prevent it by means of exercise. Because interventions against known atrophy initiators, such as oxidative stress and neuronal NO synthase (nNOS) redistribution, are only partially effective, we investigated the involvement of melusin, a muscle‐specific integrin‐associated protein and a recognized regulator of protein kinases and mechanotransduction in cardiomyocytes.MethodsMuscle atrophy was induced in the rat soleus by tail suspension and in the human vastus lateralis by bed rest. Melusin expression was investigated at the protein and transcript level and after treatment of tail‐suspended rats with atrophy initiator inhibitors. Myofiber size, sarcolemmal nNOS activity, FoxO3 myonuclear localization, and myofiber carbonylation of the unloaded rat soleus were studied after in vivo melusin replacement by cDNA electroporation, and muscle force, myofiber size, and atrogene expression after adeno‐associated virus infection. In vivo interference of exogenous melusin with dominant‐negative kinases and other atrophy attenuators (Grp94 cDNA; 7‐nitroindazole) on size of unloaded rat myofibers was also explored.ResultsUnloading/disuse reduced muscle melusin protein levels to about 50%, already after 6 h in the tail‐suspended rat (P < 0.001), and to about 35% after 8 day bed rest in humans (P < 0.05). In the unloaded rat, melusin loss occurred despite of the maintenance of β1D integrin levels and was not abolished by treatments inhibiting mitochondrial oxidative stress, or nNOS activity and redistribution. Expression of exogenous melusin by cDNA transfection attenuated atrophy of 7 day unloaded rat myofibers (−31%), compared with controls (−48%, P = 0.001), without hampering the decrease in sarcolemmal nNOS activity and the increase in myonuclear FoxO3 and carbonylated myofibers. Infection with melusin‐expressing adeno‐associated virus ameliorated contractile properties of 7 day unloaded muscles (P ≤ 0.05) and relieved myofiber atrophy (−33%) by reducing Atrogin‐1 and MurF‐1 transcripts (P ≤ 0.002), despite of a two‐fold increase in FoxO3 protein levels (P = 0.03). Atrophy attenuation by exogenous melusin did not result from rescue of Akt, ERK, or focal adhesion kinase activity, because it persisted after co‐transfection with dominant‐negative kinase forms (P < 0.01). Conversely, melusin cDNA transfection, combined with 7‐nitroindazole treatment or with cDNA transfection of the nNOS‐interacting chaperone Grp94, abolished 7 day unloaded myofiber atrophy.ConclusionsDisuse/unloading‐induced loss of melusin is an early event in muscle atrophy which occurs independently from mitochondrial oxidative stress, nNOS redistribution, and FoxO3 activation. Only preservation of melusin levels and sarcolemmal nNOS localization fully prevented muscle mass loss, demonstrating that both of them act as independent, but complementary, master switches of muscle disuse atrophy.

PO-264 Target Mitochondria Metabolism and Energy Expenditure in Muscle Atrophy, Sarcopenia and Cachexia

Objective Muscle atrophy is a common and clinically important outcome of many diseases and hospitalization bed rest. Mechanical unloading of skeletal muscles may result in a rapid loss of muscle mass and mitochondria dysfunction. And the recovery from disuse muscle atrophy is usually complete in young healthy adults but is delayed and often incomplete in older patients. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. To this end, we employed multiple approaches to address the role of mitochondrial function and metabolism in muscle atrophy and sarcopenia. And we are also engaged to develop novel mitochondria-targeted antioxidant peptides to mitigate this mitochondria-related functional impairment by ROS and improve the recovery from muscle atrophy and cachexia.&#x0D; Methods Tail Suspension Hind Limb Unloading for adult mice (6-month-old) and aged mice (22-24month old). Cardiometabolic Phenotyping：Measurement of fat and lean mass (g) is accomplished using a LF90II TD-NMR (Bruker, Madison, WI). Locomotor activity measurements are obtained using a Promethion Mouse Multiplexed Metabolic System(Sable Systems International, Las Vegas, NV).Mitochondrial Respiration :Respirometry assays were conducted using an Oxygraph-2k (Oroboros Instruments, Innsbruck, Austria), Mitochondria H2O2 emission was measured with Amplex Red reagent which reacts with H2O2 to produce the stable fluorescent compound resorufin. Resorufin fluorescence was monitored using a Fluorescence. A continuous, spectrophotometric assay was utilized to measure mitochondrial calcium retention capacity (CRC) within soleus fiber bundles. RNA Sequencing and Informatics, KEGG Pathway and Upstream Regulator Analysis, Real-Time Quantitative-PCR. Multidimensional mass spectrometry-based shotgun lipidomics was employed to measure and characterize the lipid patterns inmouse soleus muscle. Metabolomics Acylcarnitines A panel of acylcarnitines was quantitated by LC/ MS/MS (Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer). Amino Acids. Quantitation of amino acids was achieved using LC/MS/MS (Agilent 1290 HPLC/6490 triple quadrupole mass spectrometer).&#x0D; Results Old mice have impaired early recovery of soleus mass following unloading induced muscle atrophy. And mitochondrial function and metabolism does not Improve during early recovery in aged Mice. Divergent metabolomic response was found between adult and aged Mice during unloading and recovery. However, transcriptomic response to unloading and reloading is similar in adult and aged Mice.&#x0D; Conclusions Here, we report that aged mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass after disuse muscle atrophy. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid β-oxidation and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. Our findings also showed cellular metabolic changes during muscle atrophy and sarcopenia may induce higher production of oxygen radicals that play a significant role in the progression of age-related sarcopenia. These findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse. We are engaged to develop novel mitochondria-targeted antioxidant peptides to mitigate this mitochondria-related functional impairment by ROS and improve the recovery from muscle atrophy and cachexia. Co-administration of other targeted compounds was also promising to improve recovery from muscle atrophy and cachexia.&#x0D;

Effect of Eukarion-134 on Akt-mTOR signalling in the rat soleus during 7days of mechanical unloading.

What is the central question of this study? Translocation of nNOSμ initiates catabolic signalling via FoxO3a and skeletal muscle atrophy during mechanical unloading. Recent evidence suggests that unloading-induced muscle atrophy and FoxO3a activation are redox sensitive. Will a mimetic of superoxide dismutase and catalase (i.e. Eukarion-134) also mitigate suppression of the Akt-mTOR pathway? What is the main finding and its importance? Eukarion-134 rescued Akt-mTOR signalling and sarcolemmal nNOSμ, which were linked to protection against the unloading phenotype, muscle fibre atrophy and partial fibre-type shift from slow to fast twitch. The loss of nNOSμ from the sarcolemma appears crucial to Akt phosphorylation and is redox sensitive, although the mechanisms remain unresolved. Mechanical unloading stimulates rapid changes in skeletal muscle morphology, characterized by atrophy of muscle fibre cross-sectional area and a partial fibre-type shift from slow to fast twitch. Recent studies revealed that oxidative stress contributes to activation of forkhead box O3a (FoxO3a), proteolytic signalling and unloading-induced muscle atrophy via translocation of the μ-splice variant of neuronal nitric oxide synthase (nNOSμ) and activation of FoxO3a. There is limited understanding of the role of reactive oxygen species in the Akt-mammalian target of rapamycin (mTOR) pathway signalling during unloading. We hypothesized that Eukarion-134 (EUK-134), a mimetic of the antioxidant enzymes superoxide dismutase and catalase, would protect Akt-mTOR signalling in the unloaded rat soleus. Male Fischer 344 rats were separated into the following three study groups: ambulatory control (n=11); 7days of hindlimb unloading+ saline injections (HU, n=11); or 7days of HU + EUK-134; (HU+EUK-134, n=9). EUK-134 mitigated unloading-induced dephosphorylation of Akt, as well as FoxO3a, in the soleus. Phosphorylation of mTOR in the EUK-treated HU rats was not different from that in control animals. However, EUK-134 did not significantly rescue p70S6K phosphorylation. EUK-134 attenuated translocation of nNOSμ from the membrane to the cytosol, reduced nitration of tyrosine residues and suppressed upregulation of caveolin-3 and dysferlin. EUK-134 ameliorated HU-induced remodelling, atrophy of muscle fibres and the 12% increase in type II myosin heavy chain-positive fibres. Attenuation of the unloaded muscle phenotype was associated with decreased reactive oxygen species, as assessed by ethidium-positive nuclei. We conclude that oxidative stress affects Akt-mTOR signalling in unloaded skeletal muscle. Direct linkage of abrogation of nNOSμ translocation with Akt-mTOR signalling during unloading is the subject of future investigation.

The Role of IGF-1 Signaling in Skeletal Muscle Atrophy.

Insulin-like growth factor 1 (IGF-1) is a key anabolic growth factor stimulating phosphatidylinositol 3-kinase (PI3K)/Akt signaling which is well known for regulating muscle hypertrophy. However, the role of IGF-1 in muscle atrophy is less clear. This review provides an overview of the mechanisms via which IGF-1 signaling is implicated in several conditions of muscle atrophy and via which mechanisms protein turnover is altered. IGF-1/PI3K/Akt signaling stimulates the rate of protein synthesis via p70S6Kinase and p90 ribosomal S6 kinase and negatively regulates protein degradation, predominantly by its inhibiting effect on proteasomal and lysosomal protein degradation. Caspase-dependentprotein degradation is also attenuated by IGF/PI3K/Akt signaling, whereas evidence for an effect on calpain-dependentprotein degradation is inconclusive. IGF-1/PI3K/Akt signaling reduces during denervation-, unloading-, and joint immobilization-induced muscle atrophy, whereas IGF-1/PI3K/Akt signaling seems unaltered during aging-associated muscle atrophy. During denervation and aging, IGF-1 overexpression or injection counteracts denervation- and aging-associated muscle atrophy, despite enhanced anabolic resistance with regard to IGF-1 signaling with aging. It remains unclear whether pharmacological stimulation of IGF-1/PI3K/Akt signaling attenuates immobilization- or unloading-induced muscle atrophy. Exploration of the possibilities to interfere with IGF-1/PI3K/Akt signaling reveals that microRNAs targeting IGF-1 signaling components are promising targets to counterbalance muscle atrophy. Overall, the findings summarized in this review show that in disuse conditions, but not with aging, IGF-1/PI3K/Akt signaling is attenuated and that in some conditions stimulation of this pathway may alleviate skeletal muscle atrophy.

The effect of Bu Zhong Yi Qi decoction on simulated weightlessness-induced muscle atrophy and its mechanisms

Microgravity has been previously demonstrated to induce skeletal muscle atrophy, loss of muscle force and disorders in myogenesis and metabolism. Current pharmacological strategies exhibit poor efficacy. Bu Zhong Yi Qi decoction (BZ) is a well-known traditional Chinese medicine decoction used for myasthenia gravis. In the present study, its effect on unloading induced muscle atrophy was investigated. The mousetail suspension model was used to simulate weightlessness induced muscle atrophy. The results indicated that BZ could significantly protect muscles from simulated weightlessness-induced atrophy. To elucidate the underlying mechanisms, drugCIPHER-CS methods were introduced to predict its potential targets, significantly enriched pathways and biological processes. The results demonstrated that the calcium signaling pathway, citrate cycle, biosynthetic and lipid metabolic process are affected by BZ. Among the targets, nuclear receptor corepressor 1 (NCoR1) is one of the most important proteins involved in myogenesis and metabolism. The results indicated that BZ significantly downregulated NCoR 1 expression, and further induced muscle differentiation and metabolism by regulating NCoR1-associated gene expression in vivo and in vitro. In summary, the present study indicated that may be effective in combating weightlessness-induced muscle atrophy. Combined with bioinformatics, the underlying mechanism for this decoction was investigated, which provided an improved understanding of this decoction.

Paradoxical effect of IKKβ inhibition on the expression of E3 ubiquitin ligases and unloading-induced skeletal muscle atrophy.

We tested whether NF‐κB pathway is indispensable for the increase in expression of E3‐ligases and unloading‐induced muscle atrophy using IKKβ inhibitor IMD‐0354. Three groups of rats were used: nontreated control (C), 3 days of unloading/hindlimb suspension with (HS+IMD) or without (HS) IMD‐0354. Levels of IκBα were higher in HS+IMD (1.16‐fold) and lower in HS (0.82‐fold) when compared with C group. IMD‐0354 treatment during unloading: had no effect on loss of muscle mass; increased mRNA levels of MuRF1 and MAFbx; increased levels of pFoxO3; and had no effect on levels of Bcl‐3, p105, and p50 proteins. Our study for the first time showed that inhibiting IKK β in vivo during 3‐day unloading failed to diminish expression of ubiquitin ligases and prevent muscle atrophy.

Trichostatin A, a histone deacetylase inhibitor, modulates unloaded-induced skeletal muscle atrophy.

Skeletal muscle atrophy is commonly associated with immobilization, ageing, and catabolic diseases such as diabetes and cancer cachexia. Epigenetic regulation of gene expression resulting from chromatin remodeling through histone acetylation has been implicated in muscle disuse. The present work was designed to test the hypothesis that treatment with trichostatin A (TSA), a histone deacetylase inhibitor, would partly counteract unloading-induced muscle atrophy. Soleus muscle atrophy (-38%) induced by 14 days of rat hindlimb suspension was reduced to only 25% under TSA treatment. TSA partly prevented the loss of type I and IIa fiber size and reversed the transitions of slow-twitch to fast-twitch fibers in soleus muscle. Unloading or TSA treatment did not affect myostatin gene expression and follistatin protein. Soleus protein carbonyl content remained unchanged, whereas the decrease in glutathione vs. glutathione disulfide ratio and the increase in catalase activity (biomarkers of oxidative stress) observed after unloading were abolished by TSA treatment. The autophagy-lysosome pathway (Bnip3 and microtubule-associated protein 1 light chain 3 proteins, Atg5, Gabarapl1, Ulk1, and cathepsin B and L mRNA) was not activated by unloading or TSA treatment. However, TSA suppressed the rise in muscle-specific RING finger protein 1 (MuRF1) caused by unloading without affecting the forkhead box (Foxo3) transcription factor. Prevention of muscle atrophy by TSA might be due to the regulation of the skeletal muscle atrophy-related MuRF1 gene. Our findings suggest that TSA may provide a novel avenue to treat unloaded-induced muscle atrophy.

Electrical stimulation using sine waveform prevents unloading-induced muscle atrophy in the deep calf muscles of rat

The aim of this study was to compare the effects of electrical stimulation by using rectangular and sine waveforms in the prevention of deep muscle atrophy in rat calf muscles. Rats were randomly divided into the following groups: control, hindlimb unloading (HU), and HU plus electrical stimulation (ES). The animals in the ES group were electrically stimulated using rectangular waveform (RS) on the left calves and sine waveform (SS) on the right calves, twice a day, for 2 weeks during unloading. HU for 2 weeks resulted in a loss of the muscle mass, a decrease in the cross-sectional area of the muscle fibers, and overexpression of ubiquitinated proteins in the gastrocnemius and soleus muscles. In contrast, electrical stimulation with RS attenuated the HU-induced reduction in the cross-sectional area of muscle fibers and the increase of ubiquitinated proteins in the gastrocnemius muscle. However, electrical stimulation with RS failed to prevent muscle atrophy in the deep portion of the gastrocnemius and the soleus muscles. Nevertheless, electrical stimulation with SS attenuated the HU-induced muscle atrophy and the up-regulation of ubiquitinated proteins in both gastrocnemius and soleus muscles. This indicates that SS was more effective in the prevention of deep muscle atrophy than RS. Since the skin muscle layers act like the plates of a capacitor, separated by the subcutaneous adipose layer, the SS can pass through this capacitor more easily than the RS. Hence, SS can prevent the progressive loss of muscle fibers in the deep portion of the calf muscles.

Longitudinal characterization of functional, morphologic, and biochemical adaptations in mouse skeletal muscle with hindlimb suspension

Hindlimb unloading-induced muscle atrophy is often assessed after a homeostatic state is established, thus overlooking the early adaptations that are critical to developing this pattern of atrophy. Muscle function and physiology were characterized at 0, 1, 3, 7, and 14 days of hindlimb suspension (HS). Reductions in muscle mass were maximal by Day 14 of HS. Functional strength and isolated muscle strength were reduced. MyHC-I and -IIa expressing fibers were reduced in size by Day 7 in the soleus and by Day 14 in the gastrocnemius (MyHC-I fibers only). Atrogin-1 and MuRF1 expression was increased by Day 1 in both the calf and tibialis anterior while IGF-1 expression was significantly reduced on Day 3. Phosphorylation of Akt was reduced on Day 14. Insight into these early changes in response to HS improves understanding of the molecular and functional changes that lead to muscle atrophy.

Inhibition of Xanthine Oxidase by Allopurinol Prevents Skeletal Muscle Atrophy: Role of p38 MAPKinase and E3 Ubiquitin Ligases

Alterations in muscle play an important role in common diseases and conditions. Reactive oxygen species (ROS) are generated during hindlimb unloading due, at least in part, to the activation of xanthine oxidase (XO). The major aim of this study was to determine the mechanism by which XO activation causes unloading-induced muscle atrophy in rats, and its possible prevention by allopurinol, a well-known inhibitor of this enzyme. For this purpose we studied one of the main redox sensitive signalling cascades involved in skeletal muscle atrophy i.e. p38 MAPKinase, and the expression of two well known muscle specific E3 ubiquitin ligases involved in proteolysis, the Muscle atrophy F-Box (MAFbx; also known as atrogin-1) and Muscle RING (Really Interesting New Gene) Finger-1 (MuRF-1). We found that hindlimb unloading induced a significant increase in XO activity and in the protein expression of the antioxidant enzymes CuZnSOD and Catalase in skeletal muscle. The most relevant new fact reported in this paper is that inhibition of XO with allopurinol, a drug widely used in clinical practice, prevents soleus muscle atrophy by ∼20% after hindlimb unloading. This was associated with the inhibition of the p38 MAPK-MAFbx pathway. Our data suggest that XO was involved in the loss of muscle mass via the activation of the p38MAPK-MAFbx pathway in unloaded muscle atrophy. Thus, allopurinol may have clinical benefits to combat skeletal muscle atrophy in bedridden, astronauts, sarcopenic, and cachexic patients.

Electrical Stimulation Influences Satellite Cell Proliferation and Apoptosis in Unloading-Induced Muscle Atrophy in Mice

Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.

Therapeutic potential of ghrelin treatment for unloading-induced muscle atrophy in mice

Ghrelin is a growth hormone (GH) secretagogue secreted mainly from the stomach that functions in controlling muscle volume and energy homeostasis. We here studied the effects of ghrelin on unloading-induced muscle atrophy using a mouse model of hindlimb suspension (HS). Ghrelin administration during 2-week HS alleviated reductions of muscle mass in the fast-twitch fiber-rich plantaris muscle and the slow-twitch fiber-rich soleus muscle of the hindlimb. Ghrelin administration during a 5-day recovery period following 2-week HS enhanced food intake and facilitated recovery from atrophy in both muscles. Ghrelin administration normalized hypercorticosteronemia in these studies. Ghrelin’s anti-muscle atrophy effect was found even under pair-feeding condition, but not in mice given des-acyl ghrelin. Insulin-like growth factor (IGF)-1 mRNA expression was significantly reduced in the atrophied plantaris muscle compared with control muscles. A single ghrelin administration to HS mice acutely increased plasma GH and also amplified phosphorylation of signal transducer and activator of transcription (STAT) 5 and increased IGF-1 mRNA expression in the plantaris muscle, but not in the soleus muscle. This study demonstrated that ghrelin stimulated the GH-STAT5-IGF-1 axis in the locally atrophied plantaris muscle, and its administration alleviated muscle atrophy and facilitated recovery from muscle atrophy. Ghrelin’s effects represent a novel therapeutic paradigm for the treatment of unloading-induced muscle atrophy induced by factors such as bed rest, injury, and joint immobilization.

Ubiquitin Ligase Cbl-b Is a Negative Regulator for Insulin-Like Growth Factor 1 Signaling during Muscle Atrophy Caused by Unloading

Skeletal muscle atrophy caused by unloading is characterized by both decreased responsiveness to myogenic growth factors (e.g., insulin-like growth factor 1 [IGF-1] and insulin) and increased proteolysis. Here, we show that unloading stress resulted in skeletal muscle atrophy through the induction and activation of the ubiquitin ligase Cbl-b. Upon induction, Cbl-b interacted with and degraded the IGF-1 signaling intermediate IRS-1. In turn, the loss of IRS-1 activated the FOXO3-dependent induction of atrogin-1/MAFbx, a dominant mediator of proteolysis in atrophic muscle. Cbl-b-deficient mice were resistant to unloading-induced atrophy and the loss of muscle function. Furthermore, a pentapeptide mimetic of tyrosine(608)-phosphorylated IRS-1 inhibited Cbl-b-mediated IRS-1 ubiquitination and strongly decreased the Cbl-b-mediated induction of atrogin-1/MAFbx. Our results indicate that the Cbl-b-dependent destruction of IRS-1 is a critical dual mediator of both increased protein degradation and reduced protein synthesis observed in unloading-induced muscle atrophy. The inhibition of Cbl-b-mediated ubiquitination may be a new therapeutic strategy for unloading-mediated muscle atrophy.

The activation of apoptosis factor in hindlimb unloading-induced muscle atrophy under normal and low-temperature environmental conditions

In order to identify the apoptosis-induced factors and apoptosis pathway in hindlimb unloading muscle atrophy, the reciprocal relationships between caspase-3 activation and factors related to mitochondria, other organelle pathways, oxidative stress and nitric oxide were investigated. Male Wistar rats were divided into four groups, two groups of hindlimb-unloaded rats were maintained under normal (25 degrees C) and low-temperature (10 degrees C) environmental conditions for a 3-week experimental period, plus two corresponding control groups. Active caspase-3-containing myofibers were observed in the hindlimb-unloaded rats in normal and low-temperature environments, but not in the control rats. In these caspase-3-containing fibers, DNA fragmentation, dystrophin breakdown, increased immunolabeling of mu-calpain, decreased cytochrome c, cathepsin-D effusion from the lysosomes and increased lipid peroxidation were observed, while no changes in active caspase-12, eNOS or nNOS immunolabeling were seen. Furthermore, although caspase-3 activation was observed in type-I fibers, caspase-12 labeling was observed in fibers of the hybrid type. These results show that the apoptosis observed in hindlimb unloading-induced muscle atrophy is caused by activation of the caspase cascade via the lysosome pathway. Moreover, the results suggest that caspase-12 does not activate caspase-3 due to differences in the cell differentiation or the apoptosis-inducing stimulation.

Combined isometric, concentric, and eccentric resistance exercise prevents unloading-induced muscle atrophy in rats

Previously, we reported that an isometric resistance training program that was effective in stimulating muscle hypertrophy in ambulatory rats could not completely prevent muscle atrophy during unloading (Haddad F, Adams GR, Bodell PW, Baldwin KM. J Appl Physiol 100: 433-441, 2006). These results indicated that preventing muscle atrophy does not appear to be simply a function of providing an anabolic stimulus. The present study was undertaken to determine if resistance training, with increased volume (3-s contractions) and incorporating both static and dynamic components, would be effective in preventing unloading-induced muscle atrophy. Rats were exposed to 5 days of muscle unloading via tail suspension. During that time one leg received electrically stimulated resistance exercise (RE) that included an isometric, concentric, and eccentric phase. The results of this study indicate that this combined-mode RE provided an anabolic stimulus sufficient to maintain the mass and myofibril content of the trained but not the contralateral medial gastrocnemius (MG) muscle. Relative to the contralateral MG, the RE stimulus increased the amount of total RNA (indicative of translational capacity) as well as the mRNA for several anabolic/myogenic markers such as insulin-like growth factor-I, myogenin, myoferlin, and procollagen III-alpha-1 and decreased that of myostatin, a negative regulator of muscle size. The combined-mode RE protocol also increased the activity of anabolic signaling intermediates such as p70S6 kinase. These results indicate that a combination of static- and dynamic-mode RE of sufficient volume provides an effective stimulus to stimulate anabolic/myogenic mechanisms to counter the initial stages of unloading-induced muscle atrophy.

NO production results in suspension-induced muscle atrophy through dislocation of neuronal NOS

Forkhead box O (Foxo) transcription factors induce muscle atrophy by upregulating the muscle-specific E3 ubiquitin ligases MuRF-1 and atrogin-1/MAFbx, but other than Akt, the upstream regulators of Foxos during muscle atrophy are largely unknown. To examine the involvement of the dystrophin glycoprotein complex (DGC) in regulation of Foxo activities and muscle atrophy, we analyzed the expression of DGC members during tail suspension, a model of unloading-induced muscle atrophy. Among several DGC members, only neuronal NOS (nNOS) quickly dislocated from the sarcolemma to the cytoplasm during tail suspension. Electron paramagnetic resonance spectrometry revealed production of NO in atrophying muscle. nNOS-null mice showed much milder muscle atrophy after tail suspension than did wild-type mice. Importantly, nuclear accumulation of dephosphorylated Foxo3a was not evident in nNOS-null muscle, and neither MuRF-1 nor atrogin-1/MAFbx were upregulated during tail suspension. Furthermore, an nNOS-specific inhibitor, 7-nitroindazole, significantly prevented suspension-induced muscle atrophy. The NF-kappaB pathway was activated in both wild-type and nNOS-null muscle during tail suspension. We also show that nNOS was involved in the mechanism of denervation-induced atrophy. We conclude that nNOS/NO mediates muscle atrophy via regulation of Foxo transcription factors and is a new therapeutic target for disuse-induced muscle atrophy.