sirolimus and Muscular-Atrophy

Atrogin-1 and Muscle-specific RING finger protein 1 (MuRF1) are highly expressed in multiple conditions of skeletal muscle atrophy. The phosphoinositide 3-kinase (PI3K)/Akt/forkhead box (FoxO) signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mechanistic target of rapamycin complex 1 (mTORC1), which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 with a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 μM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased by PF-4708671 treatment for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by the rapamycin-sensitive mTOR-S6K1-dependent signaling pathway.

Topics: Humans; Mechanistic Target of Rapamycin Complex 1; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; SKP Cullin F-Box Protein Ligases; TOR Serine-Threonine Kinases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Ubiquitins

Prolonged inactivity of skeletal muscles due to limb immobilization, bedrest, and exposure to microgravity results in a significant muscle atrophy. Inactivity-induced muscle atrophy is caused by a downregulation of protein synthesis (PS) and increased proteolysis. Mechanistic target of rapamycin complex 1 (mTORC1) is considered to be one of the main regulators of translational capacity (quantity of ribosomes), a key determinant of PS. Using a specific mTORC1 inhibitor (rapamycin) we aimed to determine if mTORC1 activity would influence ribosome biogenesis in rat soleus muscle at both early and later stages of mechanical unloading. Wistar rats were subjected to 1- and 7-day hindlimb suspension (HS) with and without rapamycin injections (1.5 mg/kg) and compared to weight-bearing control animals. The key markers of ribosome biogenesis were assessed by RT-PCR or agarose gel electrophoresis. The rate of PS was measured by SUnSET method. Both 1-day and 7-day HS resulted in a significant downregulation of ribosome biogenesis markers (c-Myc, 47S pre-rRNA, 18S + 28S rRNAs) and the rate of PS. Rapamycin administration during 1-day HS fully prevented a decrease in 47S pre-rRNA expression and amount of 18S + 28S rRNAs (without affecting c-Myc mRNA expression) and partially attenuated a decline in PS. Rapamycin treatment during 7-day HS significantly decreased p70S6K phosphorylation but failed to rescue a reduction in both the markers of ribosome biogenesis and the rate of PS. All together, our results suggest that mTORC1 inhibition at the initial (1 day), but not later (7 days) stage of HS can be beneficial for the maintenance of translational capacity (ribosome biogenesis) and the rate of PS in rat soleus muscle.

Topics: Animals; Hindlimb Suspension; Mechanistic Target of Rapamycin Complex 1; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases, 70-kDa; Ribosomes; RNA Precursors; RNA, Messenger; Sirolimus

The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading.

Topics: Animals; Eukaryotic Initiation Factor-4F; Female; Immobilization; Immunohistochemistry; Isometric Contraction; Mice; Muscle Contraction; Muscle, Skeletal; Muscular Atrophy; Protein Biosynthesis; Ribosomes; Signal Transduction; Sirolimus; Stress, Mechanical; TOR Serine-Threonine Kinases

The purpose of our study was to compare two acquired muscle atrophies and the use of myostatin inhibition for their treatment. Myostatin naturally inhibits skeletal muscle growth by binding to ActRIIB, a receptor on the cell surface of myofibers. Because blocking myostatin in an adult wild-type mouse induces profound muscle hypertrophy, we applied a soluble ActRIIB receptor to models of disuse (limb immobilization) and denervation (sciatic nerve resection) atrophy. We found that treatment of immobilized mice with ActRIIB prevented the loss of muscle mass observed in placebo-treated mice. Our results suggest that this protection from disuse atrophy is regulated by serum and glucocorticoid-induced kinase (SGK) rather than by Akt. Denervation atrophy, however, was not protected by ActRIIB treatment, yet resulted in an upregulation of the pro-growth factors Akt, SGK and components of the mTOR pathway. We then treated the denervated mice with the mTOR inhibitor rapamycin and found that, despite a reduction in mTOR activation, there is no alteration of the atrophy phenotype. Additionally, rapamycin prevented the denervation-induced upregulation of the mTORC2 substrates Akt and SGK. Thus, our studies show that denervation atrophy is not only independent from Akt, SGK and mTOR activation but also has a different underlying pathophysiological mechanism than disuse atrophy.

Topics: Activin Receptors, Type II; Animals; Autophagy; Biomarkers; Enzyme Activation; Male; Mice; Muscle Denervation; Muscular Atrophy; Myostatin; Phenotype; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta; Up-Regulation

Skeletal muscle mass is regulated by activity, metabolism, and the availability of nutrients. During muscle atrophy, MNK2 expression increases. We found that MNK2 (mitogen-activated protein kinase-interacting kinase 2), but not MNK1, inhibited proteins involved in promoting protein synthesis, including eukaryotic translation initiation factor 4G (eIF4G) and mammalian target of rapamycin (mTOR). Phosphorylation at serine 1108 (Ser¹¹⁰⁸) of eIF4G, which is associated with enhanced protein translation, is promoted by insulin-like growth factor 1 and inhibited by rapamycin or starvation, suggesting that phosphorylation of this residue is regulated by mTOR. In cultured myotubes, small interfering RNA (siRNA) knockdown of MNK2 increased eIF4G Ser¹¹⁰⁸ phosphorylation and overcame rapamycin's inhibitory effect on this phosphorylation event. Phosphorylation of Ser¹¹⁰⁸ in eIF4G, in gastrocnemius muscle, was increased in mice lacking MNK2, but not those lacking MNK1, and this increased phosphorylation was maintained in MNK2-null animals under atrophy conditions and upon starvation. Conversely, overexpression of MNK2 decreased eIF4G Ser¹¹⁰⁸ phosphorylation. An siRNA screen revealed that serine-arginine-rich protein kinases linked increased MNK2 activity to decreased eIF4G phosphorylation. In addition, we found that MNK2 interacted with mTOR and inhibited phosphorylation of the mTOR target, the ribosomal kinase p70S6K (70-kD ribosomal protein S6 kinase), through a mechanism independent of the kinase activity of MNK2. These data indicate that MNK2 plays a unique role, not shared by its closest paralog MNK1, in limiting protein translation through its negative effect on eIF4G Ser¹¹⁰⁸ phosphorylation and p70S6K activation.

Topics: Animals; Arginine; Blotting, Western; Cell Line; Dexamethasone; Eukaryotic Initiation Factor-4G; Insulin-Like Growth Factor I; Mice; Mice, Knockout; Muscle, Skeletal; Muscular Atrophy; Myoblasts; Phosphorylation; Protein Binding; Protein Serine-Threonine Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA Interference; Serine; Signal Transduction; Sirolimus; Starvation; TOR Serine-Threonine Kinases

Sarcopenia is the loss of muscle mass and strength which occurs with aging. Whether the molecular basis of sarcopenia differs with muscle type and across sex is not well understood. Here we examine how aging affects the regulation of protein kinase B (Akt), the mammalian target of rapamycin (mTOR), AMP activated kinase (AMPK), p70 ribosomal S6 kinase (p70s6k), S6 ribosomal protein (rps6) and calcineurin (CaN) in the slow soleus and fast extensor digitorum longus (EDL) muscles of 6- (adult), 30- (aged), and 36-month (very aged) male and 6- (adult), 26- (aged), and 30-month (very aged) female Fischer 344xBrown Norway (F344BN) rats. In male animals, soleus and EDL muscle to body weight ratios decreased steadily with age while in the females, losses remained unchanged after 26 months. These age-related changes in the degree of muscle atrophy across sex were associated with differences in the regulation of Akt, mTOR, and p70s6k in the slow-twitch soleus and the regulation of AMPK, 4EBP1, p70s6k and rpS6 in the fast-twitch EDL. Irrespective of muscle type, aging in both the genders was associated with increased calcineurin expression. Taken together, these data suggest that indices of protein synthesis and muscle adaptation are regulated differently with aging in different muscle types and sex.

Topics: Aging; Animals; Calcineurin; Female; Male; Mammals; Muscle, Skeletal; Muscular Atrophy; Muscular Diseases; Proto-Oncogene Proteins c-akt; Rats; Rats, Inbred BN; Rats, Inbred F344; Ribosomal Protein S6 Kinases, 70-kDa; Sarcopenia; Signal Transduction; Sirolimus

Although it is well established that chronic hypoxia leads to an inexorable loss of skeletal muscle mass in healthy subjects, the underlying molecular mechanisms involved in this process are currently unknown. Skeletal muscle atrophy is also an important systemic consequence of chronic obstructive pulmonary disease (COPD), but the role of hypoxemia in this regulation is still debated. Our general aim was to determine the molecular mechanisms involved in the regulation of skeletal muscle mass after exposure to chronic hypoxia and to test the biological relevance of our findings into the clinical context of COPD. Expression of positive and negative regulators of skeletal muscle mass were explored 1) in the soleus muscle of rats exposed to severe hypoxia (6,300 m) for 3 wk and 2) in vastus lateralis muscle of nonhypoxemic and hypoxemic COPD patients. In rodents, we observed a marked inhibition of the mammalian target of rapamycin (mTOR) pathway together with a strong increase in regulated in development and DNA damage response 1 (REDD1) expression and in its association with 14-3-3, a mechanism known to downregulate the mTOR pathway. Importantly, REDD1 overexpression in vivo was sufficient to cause skeletal muscle fiber atrophy in normoxia. Finally, the comparative analysis of skeletal muscle in hypoxemic vs. nonhypoxemic COPD patients confirms that hypoxia causes an inhibition of the mTOR signaling pathway. We thus identify REDD1 as a negative regulator of skeletal muscle mass during chronic hypoxia. Translation of this fundamental knowledge into the clinical investigation of COPD shows the interest to develop therapeutic strategies aimed at inhibiting REDD1.

Topics: Animals; Atrophy; Down-Regulation; Humans; Hypoxia; Male; Mammals; Muscle, Skeletal; Muscular Atrophy; Proto-Oncogene Proteins c-akt; Pulmonary Disease, Chronic Obstructive; Rats; Rats, Wistar; Signal Transduction; Sirolimus

This study was conducted to examine whether stretch-related mechanical loading on skeletal muscle can suppress denervation-induced muscle atrophy, and if so, to depict the underlying molecular mechanism. Denervated rat soleus muscle was repetitively stretched (every 5 s for 15 min/day) for 2 weeks. Histochemical analysis showed that the cross-sectional area of denervated soleus muscle fibers with repetitive stretching was significantly larger than that of control denervated muscle (P<0.05). We then examined the involvement of the Akt/mammalian target of the rapamycin (mTOR) cascade in the suppressive effects of repetitive stretching on muscle atrophy. Repetitive stretching significantly increased the Akt, p70S6K, and 4E-BP1 phosphorylation in denervated soleus muscle compared to controls (P<0.05). Furthermore, repetitive stretching-induced suppression of muscle atrophy was fully inhibited by rapamycin, a potent inhibitor of mTOR. These results indicate that denervation-induced muscle atrophy is significantly suppressed by stretch-related mechanical loading of the muscle through upregulation of the Akt/mTOR signal pathway.

Topics: Animals; Carrier Proteins; Immunosuppressive Agents; Intracellular Signaling Peptides and Proteins; Male; Muscle Denervation; Muscle Stretching Exercises; Muscle, Skeletal; Muscular Atrophy; Phosphoproteins; Phosphorylation; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Stress, Mechanical; TOR Serine-Threonine Kinases; Up-Regulation; Weight-Bearing

Clenbuterol and other beta2-adrenergic agonists are effective at inducing muscle growth and attenuating muscle atrophy through unknown mechanisms. This study tested the hypothesis that clenbuterol-induced growth and muscle sparing is mediated through the activation of Akt and mammalian target of rapamycin (mTOR) signaling pathways. Clenbuterol was administered to normal weight-bearing adult rats to examine the growth-inducing effects and to adult rats undergoing muscle atrophy as the result of hindlimb suspension or denervation to examine the muscle-sparing effects. The pharmacological inhibitor rapamycin was administered in combination with clenbuterol in vivo to determine whether activation of mTOR was involved in mediating the effects of clenbuterol. Clenbuterol administration increased the phosphorylation status of PKB/Akt, S6 kinase 1/p70(s6k), and eukaryotic initiation factor 4E binding protein 1/PHAS-1. Clenbuterol treatment induced growth by 27-41% in normal rats and attenuated muscle loss during hindlimb suspension by 10-20%. Rapamycin treatment resulted in a 37-97% suppression of clenbuterol-induced growth and a 100% reduction of the muscle-sparing effect. In contrast, rapamycin was unable to block the muscle-sparing effects of clenbuterol after denervation. Clenbuterol was also shown to suppress the expression of the MuRF1 and MAFbx transcripts in muscles from normal, denervated, and hindlimb-suspended rats. These results demonstrate that the effects of clenbuterol are mediated, in part, through the activation of Akt and mTOR signaling pathways.

Topics: Adrenergic beta-Agonists; Animals; Clenbuterol; Drug Interactions; Female; Hindlimb Suspension; Immunosuppressive Agents; Muscle Proteins; Muscle, Skeletal; Muscular Atrophy; Protein Kinases; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; SKP Cullin F-Box Protein Ligases; TOR Serine-Threonine Kinases; Tripartite Motif Proteins; Ubiquitin-Protein Ligases

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR.

Topics: Animals; Autophagy; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Elongation Factor 2 Kinase; Eukaryotic Initiation Factors; Hepatocytes; Insulin; Leucine; Liver; Male; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Muscle Development; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Biosynthesis; Protein Kinases; Ribosomal Protein S6; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitin-Protein Ligases

Nerve activity controls fiber size and fiber type in skeletal muscle, but the underlying molecular mechanisms remain largely unknown. We have previously shown that Ras-mitogen-activated protein kinase and calcineurin control fiber type but not fiber size in regenerating rat skeletal muscle. Here we report that constitutively active protein kinase B (PKB), also known as Akt, increases fiber size and prevents denervation atrophy in regenerating and adult rat muscles but does not affect fiber type profile. The coexistence of hypertrophic muscle fibers overexpressing activated PKB with normal-size untransfected fibers within the same muscle points to a cell-autonomous control of muscle growth by PKB. The physiological role of this pathway is confirmed by the finding that PKB kinase activity and phosphorylation status are significantly increased in innervated compared with denervated regenerating muscles in parallel with muscle growth. Muscle fiber hypertrophy induced by activated PKB and by a Ras double mutant (RasV12C40) that activates selectively the phosphoinositide 3-kinase-PKB pathway is completely blocked by rapamycin, showing that the mammalian target of rapamycin kinase is the major downstream effector of this pathway in the control of muscle fiber size. On the other hand, nerve activity-dependent growth of regenerating muscle is only partially inhibited by dominant negative PKB and rapamycin, suggesting that other nerve-dependent signaling pathways are involved in muscle growth. The present results support the notion that fiber size and fiber type are regulated by nerve activity through different mechanisms.

Topics: Animals; Electric Stimulation; Hypertrophy; Male; Muscle Denervation; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Regeneration; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases