Mitsunori Miyazaki

Effective fiber hypertrophy in satellite cell-depleted skeletal muscle.

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca2+ sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.

Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals

Growth and maintenance of skeletal muscle mass is critical for long-term health and quality of life. Skeletal muscle is a highly adaptable tissue with well-known sensitivities to environmental cues such as growth factors, cytokines, nutrients, and mechanical loading. All of these factors act at the level of the cell and signal through pathways that lead to changes in phenotype through multiple mechanisms. In this review, we discuss the animal and cell culture models used and the signaling mechanisms identified in understanding regulation of protein synthesis in response to mechanical loading/resistance exercise. Particular emphasis has been placed on 1) alterations in mechanical loading and regulation of protein synthesis in both in vivo animal studies and in vitro cell culture studies and 2) upstream mediators regulating mammalian target of rapamycin signaling and protein synthesis during skeletal muscle hypertrophy.

Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3‐kinase/Akt signalling

Non‐technical summary  Hypertrophy of skeletal muscle in response to resistance exercise is associated with significantly elevated rates of protein synthesis. The protein kinase mTORC1 has been shown to be a key signalling hub through which different anabolic factors (i.e. growth factors, nutrients and mechanical strain) contribute to the regulation of protein synthesis. In this study, we use an in vivo model of muscle hypertrophy to delineate the contribution of different input pathways regulating mTORC1. We found that the insulin/insulin like growth factor 1 pathway is not necessary for early activation of mTORC1 signalling but this probably occurs through activation of the ERK/TSC2 pathway. Knowledge of the key upstream pathways that modulate mTORC1 activity in vivo will provide the necessary foundation for the development of new therapeutic strategies for the maintenance of skeletal muscle mass.

Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis

Muscle growth is associated with an activation of the mTOR signaling pathway and satellite cell regulators. The purpose of this study was to determine whether 17 selected genes associated with mTOR/muscle protein synthesis and the satellite cells/myogenic program are differentially expressed in young and older human skeletal muscle at rest and in response to a potent anabolic stimulus [resistance exercise + essential amino acid ingestion (RE+EAA)]. Twelve male subjects (6 young, 6 old) completed a bout of heavy resistance exercise. Muscle biopsies were obtained before and at 3 and 6 h post RE+EAA. Subjects ingested leucine-enriched essential amino acids at 1 h postexercise. mRNA expression was determined using qRT-PCR. At rest, hVps34 mRNA was elevated in the older subjects (P < 0.05) while there was a tendency for levels of myoD, myogenin, and TSC2 mRNA to be higher than young. The anabolic stimulus (RE+EAA) altered mRNAs associated with mTOR regulation. Notably, REDD2 decreased in both age groups (P < 0.05) but the expression of Rheb mRNA increased only in the young. Finally, cMyc mRNA was elevated (P < 0.05) in both young and old at 6 h post RE+EAA. Furthermore, RE+EAA also increased expression of several mRNAs associated with satellite function in the young (P < 0.05), while expression of these mRNAs did not change in the old. We conclude that several anabolic genes in muscle are more responsive in young men post RE+EAA. Our data provide new insights into the regulation of genes important for transcription and translation in young and old human skeletal muscle post RE+EAA.

REDD2 is enriched in skeletal muscle and inhibits mTOR signaling in response to leucine and stretch

The protein kinase mammalian target of rapamycin (mTOR) is well established as a key regulator of skeletal muscle size. In this study, we determined that the stress responsive gene REDD2 (regulated in development and DNA damage responses 2) is a negative regulator of mTOR signaling and is expressed predominantly in skeletal muscle. Overexpression of REDD2 in muscle cells significantly inhibited basal mTOR signaling and diminished the response of mTOR to leucine addition or mechanical stretch. The inhibitory function of REDD2 on mTOR signaling seems to be mediated downstream or independent of Akt signaling and upstream of Rheb (Ras homolog enriched in brain). Knock down of tuberous sclerosis complex 2 (TSC2) using small interfering (si)RNA potently activated mTOR signaling and was sufficient to rescue REDD2 inhibition of mTOR activity, suggesting that REDD2 functions by modulating TSC2 function. Immunoprecipitation assays demonstrated that REDD2 does not directly interact with either TSC1 or TSC2. However, we found that REDD2 forms a complex with 14-3-3 protein and that increasing expression of REDD2 acts to competitively dissociate TSC2 from 14-3-3 and inhibits mTOR signaling. These findings demonstrate that REDD2 is a skeletal muscle specific inhibitory modulator of mTOR signaling and identify TSC2 and 14-3-3 as key molecular links between REDD2 and mTOR function.

Age-Associated Disruption of Molecular Clock Expression in Skeletal Muscle of the Spontaneously Hypertensive Rat

It is well known that spontaneously hypertensive rats (SHR) develop muscle pathologies with hypertension and heart failure, though the mechanism remains poorly understood. Woon et al. (2007) linked the circadian clock gene Bmal1 to hypertension and metabolic dysfunction in the SHR. Building on these findings, we compared the expression pattern of several core-clock genes in the gastrocnemius muscle of aged SHR (80 weeks; overt heart failure) compared to aged-matched control WKY strain. Heart failure was associated with marked effects on the expression of Bmal1, Clock and Rora in addition to several non-circadian genes important in regulating skeletal muscle phenotype including Mck, Ttn and Mef2c. We next performed circadian time-course collections at a young age (8 weeks; pre-hypertensive) and adult age (22 weeks; hypertensive) to determine if clock gene expression was disrupted in gastrocnemius, heart and liver tissues prior to or after the rats became hypertensive. We found that hypertensive/hypertrophic SHR showed a dampening of peak Bmal1 and Rev-erb expression in the liver, and the clock-controlled gene Pgc1α in the gastrocnemius. In addition, the core-clock gene Clock and the muscle-specific, clock-controlled gene Myod1, no longer maintained a circadian pattern of expression in gastrocnemius from the hypertensive SHR. These findings provide a framework to suggest a mechanism whereby chronic heart failure leads to skeletal muscle pathologies; prolonged dysregulation of the molecular clock in skeletal muscle results in altered Clock, Pgc1α and Myod1 expression which in turn leads to the mis-regulation of target genes important for mechanical and metabolic function of skeletal muscle.

Insulin like growth factor-1-induced phosphorylation and altered distribution of tuberous sclerosis complex (TSC)1/TSC2 in C2C12 myotubes

Insulin like growth factor‐1 (IGF‐1) is established as an anabolic factor that can induce skeletal muscle growth by activating the phosphoinositide 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Although this signaling pathway has been the subject of much study, the molecular mechanisms linking IGF‐1 binding to mTOR activation remain poorly defined in muscle. The present study aimed to test the hypothesis that IGF‐1 activation of mTOR in C2C12 myotubes requires a phosphorylation‐dependent, altered distribution of the tuberous sclerosis complex (TSC)1/TSC2 complex from the membrane to the cytosol. We found that IGF‐1 treatment does not affect complex formation between TSC1 and TSC2, but rather IGF‐1 induces an altered distribution of the TSC1/TSC2 complex in C2C12 myotubes. In response to IGF‐1 treatment, there was a relative redistribution of the TSC1/TSC2 complex, composed of TSC1 and phosphorylated TSC2, from the membrane to the cytosol. IGF‐1‐stimulated TSC1/TSC2 phosphorylation and redistribution were completely prevented by the phosphoinositide 3‐kinase inhibitor wortmannin, but were not with the downstream mTOR inhibitor, rapamycin. When a nonphosphorylatable form of TSC2 (S939A) was overexpressed, phosphorylation‐dependent binding of the scaffold protein 14‐3‐3 to TSC2 was diminished and no redistribution of the TSC1/TSC2 complex was observed after IGF‐1 stimulation. These results indicate that TSC2 phosphorylation in response to IGF‐1 treatment is necessary for the altered distribution of the TSC1/TSC2 complex to the cytosol. We suggest that this translocation is likely critical for mTOR activation by dissociating the interaction between the GTPase activating protein activity of the TSC1/TSC2 complex and its downstream target, Ras homolog enriched in brain.

IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes

Insulin like growth factor‐1 (IGF‐1) is established as an anabolic factor that can induce skeletal muscle growth by activating the phosphoinositide 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Although this signaling pathway has been the subject of much study, the molecular mechanisms linking IGF‐1 binding to mTOR activation remain poorly defined in muscle. The present study aimed to test the hypothesis that IGF‐1 activation of mTOR in C2C12 myotubes requires a phosphorylation‐dependent, altered distribution of the tuberous sclerosis complex (TSC)1/TSC2 complex from the membrane to the cytosol. We found that IGF‐1 treatment does not affect complex formation between TSC1 and TSC2, but rather IGF‐1 induces an altered distribution of the TSC1/TSC2 complex in C2C12 myotubes. In response to IGF‐1 treatment, there was a relative redistribution of the TSC1/TSC2 complex, composed of TSC1 and phosphorylated TSC2, from the membrane to the cytosol. IGF‐1‐stimulated TSC1/TSC2 phosphorylation and redistribution were completely prevented by the phosphoinositide 3‐kinase inhibitor wortmannin, but were not with the downstream mTOR inhibitor, rapamycin. When a nonphosphorylatable form of TSC2 (S939A) was overexpressed, phosphorylation‐dependent binding of the scaffold protein 14‐3‐3 to TSC2 was diminished and no redistribution of the TSC1/TSC2 complex was observed after IGF‐1 stimulation. These results indicate that TSC2 phosphorylation in response to IGF‐1 treatment is necessary for the altered distribution of the TSC1/TSC2 complex to the cytosol. We suggest that this translocation is likely critical for mTOR activation by dissociating the interaction between the GTPase activating protein activity of the TSC1/TSC2 complex and its downstream target, Ras homolog enriched in brain.

High-intensity eccentric training ameliorates muscle wasting in colon 26 tumor-bearing mice

Eccentric (ECC) contractions are used to maintain skeletal muscle mass and strength in healthy subjects and patients. Here we investigated the effects of ECC training induced by electrical stimulation (ES) on muscle wasting in colon 26 (C-26) tumor-bearing mice. Mice were divided into four groups: control (CNT), CNT + ECC, C-26, and C-26 + ECC. Cancer cachexia was induced by a subcutaneous injection of C-26 cells and developed for four weeks. In experiment 1, muscle protein synthesis rate and mammalian target of rapamycin complex (mTORC) 1 signaling were investigated six hours after one bout of ECC-ES (2 s contraction given every 6 s, 20°/s, 4 sets of 5 contractions). In experiment 2, ECC-ES training, a total of 14 sessions, was performed every other day starting one day after C-26 injection. Compared to the CNT mice, the gastrocnemius muscle weight was significantly decreased in the tumor-bearing mice. This change was accompanied by a reduction in protein synthesis rate and a marked increase in the expression levels of genes including regulated in development and DNA damage responses (REDD) 1, forkhead box protein O1 (FoxO1), muscle-specific E3 ubiquitin ligases atrogin-1, and muscle ring finger 1 (MuRF-1) mRNA. ECC-ES increased the protein synthesis rate and the phosphorylation levels of p70S6K (Thr389) and rpS6 (Ser240/244), markers for mTORC1 signaling, and reversed an upregulation of MuRF-1 mRNA in muscles from C-26 mice. Our findings suggest that ECC-ES training reduces skeletal muscle atrophy in C-26 tumor-bearing mice through activation of mTORC1 signaling and the inhibition of ubiquitin-proteasome pathway. Thus, ECC-ES training might be used to effectively ameliorate muscle wasting in patients with cancer cachexia.

Insulin like growth factor-1-induced phosphorylation and altered distribution of tuberous sclerosis complex (TSC)1/TSC2 in C2C12 myotubes: TSC1/TSC2 distribution with IGF-1 stimulation

Insulin like growth factor‐1 (IGF‐1) is established as an anabolic factor that can induce skeletal muscle growth by activating the phosphoinositide 3‐kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Although this signaling pathway has been the subject of much study, the molecular mechanisms linking IGF‐1 binding to mTOR activation remain poorly defined in muscle. The present study aimed to test the hypothesis that IGF‐1 activation of mTOR in C2C12 myotubes requires a phosphorylation‐dependent, altered distribution of the tuberous sclerosis complex (TSC)1/TSC2 complex from the membrane to the cytosol. We found that IGF‐1 treatment does not affect complex formation between TSC1 and TSC2, but rather IGF‐1 induces an altered distribution of the TSC1/TSC2 complex in C2C12 myotubes. In response to IGF‐1 treatment, there was a relative redistribution of the TSC1/TSC2 complex, composed of TSC1 and phosphorylated TSC2, from the membrane to the cytosol. IGF‐1‐stimulated TSC1/TSC2 phosphorylation and redistribution were completely prevented by the phosphoinositide 3‐kinase inhibitor wortmannin, but were not with the downstream mTOR inhibitor, rapamycin. When a nonphosphorylatable form of TSC2 (S939A) was overexpressed, phosphorylation‐dependent binding of the scaffold protein 14‐3‐3 to TSC2 was diminished and no redistribution of the TSC1/TSC2 complex was observed after IGF‐1 stimulation. These results indicate that TSC2 phosphorylation in response to IGF‐1 treatment is necessary for the altered distribution of the TSC1/TSC2 complex to the cytosol. We suggest that this translocation is likely critical for mTOR activation by dissociating the interaction between the GTPase activating protein activity of the TSC1/TSC2 complex and its downstream target, Ras homolog enriched in brain.