Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in amyotrophic lateral sclerosis.

Arginine methylation is a protein posttranslational modification important for the development of skeletal muscle mass and function. Despite this, our understanding of the regulation of arginine methylation under settings of health and disease remains largely undefined. Here, we investigated the regulation of arginine methylation in skeletal muscles in response to exercise and hypertrophic growth, and in diseases involving metabolic dysfunction and atrophy. We report a limited regulation of arginine methylation under physiological settings that promote muscle health, such as during growth and acute exercise, nor in disease models of insulin resistance. In contrast, we saw a significant remodeling of asymmetric dimethylation in models of atrophy characterized by the loss of innervation, including in muscle biopsies from patients with myotrophic lateral sclerosis (ALS). Mass spectrometry-based quantification of the proteome and asymmetric arginine dimethylome of skeletal muscle from individuals with ALS revealed the largest compendium of protein changes with the identification of 793 regulated proteins, and novel site-specific changes in asymmetric dimethyl arginine (aDMA) of key sarcomeric and cytoskeletal proteins. Finally, we show that in vivo overexpression of PRMT1 and aDMA resulted in increased fatigue resistance and functional recovery in mice. Our study provides evidence for asymmetric dimethylation as a regulator of muscle pathophysiology and presents a valuable proteomics resource and rationale for numerous methylated and nonmethylated proteins, including PRMT1, to be pursued for therapeutic development in ALS.

A critical discussion on the relationship between E3 ubiquitin ligases, protein degradation, and skeletal muscle wasting: it's not that simple.

Ubiquitination is an important post-translational modification (PTM) for protein substrates, whereby ubiquitin is added to proteins through the coordinated activity of activating (E1), ubiquitin-conjugating (E2), and ubiquitin ligase (E3) enzymes. The E3s provide key functions in the recognition of specific protein substrates to be ubiquitinated and aid in determining their proteolytic or nonproteolytic fates, which has led to their study as indicators of altered cellular processes. MuRF1 and MAFbx/Atrogin-1 were two of the first E3 ubiquitin ligases identified as being upregulated in a range of different skeletal muscle atrophy models. Since their discovery, the expression of these E3 ubiquitin ligases has often been studied as a surrogate measure of changes to bulk protein degradation rates. However, emerging evidence has highlighted the dynamic and complex regulation of the ubiquitin proteasome system (UPS) in skeletal muscle and demonstrated that protein ubiquitination is not necessarily equivalent to protein degradation. These observations highlight the potential challenges of quantifying E3 ubiquitin ligases as markers of protein degradation rates or ubiquitin proteasome system (UPS) activation. This perspective examines the usefulness of monitoring E3 ubiquitin ligases for determining specific or bulk protein degradation rates in the settings of skeletal muscle atrophy. Specific questions that remain unanswered within the skeletal muscle atrophy field are also identified, to encourage the pursuit of new research that will be critical in moving forward our understanding of the molecular mechanisms that govern protein function and degradation in muscle.

Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2ß.

Ubiquitination is a posttranslational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2ß, is downregulated in models of muscle growth and that overexpression ASB2ß is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2ß expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2ß-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant diGly-modified peptides. The results show that viral vector-mediated ASB2ß overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2ß knockdown induces mild muscle hypertrophy. ASB2ß-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis, and the cytoskeleton/sarcomere. The overexpression ASB2ß also resulted in marked changes in protein ubiquitination; however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2ß and, therefore, potential ASB2ß targets, Flag-tagged wild-type ASB2ß, and a mutant ASB2ß lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2ß interactome. ASB2ß was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2ß target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction.

Oral digoxin effects on exercise performance, K+ regulation and skeletal muscle Na+ ,K+ -ATPase in healthy humans.

We investigated whether digoxin lowered muscle Na+ ,K+ -ATPase (NKA), impaired muscle performance and exacerbated exercise K+ disturbances. Ten healthy adults ingested digoxin (0.25 mg; DIG) or placebo (CON) for 14 days and performed quadriceps strength and fatiguability, finger flexion (FF, 105%peak-workrate , 3 × 1 min, fourth bout to fatigue) and leg cycling (LC, 10 min at 33% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ and 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , 90% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ to fatigue) trials using a double-blind, crossover, randomised, counter-balanced design. Arterial (a) and antecubital venous (v) blood was sampled (FF, LC) and muscle biopsied (LC, rest, 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , fatigue, 3 h after exercise). In DIG, in resting muscle, [3 H]-ouabain binding site content (OB-Fab ) was unchanged; however, bound-digoxin removal with Digibind revealed total ouabain binding (OB+Fab ) increased (8.2%, P = 0.047), indicating 7.6% NKA-digoxin occupancy. Quadriceps muscle strength declined in DIG (-4.3%, P = 0.010) but fatiguability was unchanged. During LC, in DIG (main effects), time to fatigue and [K+ ]a were unchanged, whilst [K+ ]v was lower (P = 0.042) and [K+ ]a-v greater (P = 0.004) than in CON; with exercise (main effects), muscle OB-Fab was increased at 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ (per wet-weight, P = 0.005; per protein P = 0.001) and at fatigue (per protein, P = 0.003), whilst [K+ ]a , [K+ ]v and [K+ ]a-v were each increased at fatigue (P = 0.001). During FF, in DIG (main effects), time to fatigue, [K+ ]a , [K+ ]v and [K+ ]a-v were unchanged; with exercise (main effects), plasma [K+ ]a , [K+ ]v , [K+ ]a-v and muscle K+ efflux were all increased at fatigue (P = 0.001). Thus, muscle strength declined, but functional muscle NKA content was preserved during DIG, despite elevated plasma digoxin and muscle NKA-digoxin occupancy, with K+ disturbances and fatiguability unchanged. KEY POINTS: The Na+ ,K+ -ATPase (NKA) is vital in regulating skeletal muscle extracellular potassium concentration ([K+ ]), excitability and plasma [K+ ] and thereby also in modulating fatigue during intense contractions. NKA is inhibited by digoxin, which in cardiac patients lowers muscle functional NKA content ([3 H]-ouabain binding) and exacerbates K+ disturbances during exercise. In healthy adults, we found that digoxin at clinical levels surprisingly did not reduce functional muscle NKA content, whilst digoxin removal by Digibind antibody revealed an ∼8% increased muscle total NKA content. Accordingly, digoxin did not exacerbate arterial plasma [K+ ] disturbances or worsen fatigue during intense exercise, although quadriceps muscle strength was reduced. Thus, digoxin treatment in healthy participants elevated serum digoxin, but muscle functional NKA content was preserved, whilst K+ disturbances and fatigue with intense exercise were unchanged. This resilience to digoxin NKA inhibition is consistent with the importance of NKA in preserving K+ regulation and muscle function.

The regulation of polyamine pathway proteins in models of skeletal muscle hypertrophy and atrophy: a potential role for mTORC1.

Polyamines have been shown to be absolutely required for protein synthesis and cell growth. The serine/threonine kinase, the mechanistic target of rapamycin complex 1 (mTORC1), also plays a fundamental role in the regulation of protein turnover and cell size, including in skeletal muscle, where mTORC1 is sufficient to increase protein synthesis and muscle fiber size, and is necessary for mechanical overload-induced muscle hypertrophy. Recent evidence suggests that mTORC1 may regulate the polyamine metabolic pathway, however, there is currently no evidence in skeletal muscle. This study examined changes in polyamine pathway proteins during muscle hypertrophy induced by mechanical overload (7 days), with and without the mTORC1 inhibitor, rapamycin, and during muscle atrophy induced by food deprivation (48 h) and denervation (7 days) in mice. Mechanical overload induced an increase in mTORC1 signaling, protein synthesis and muscle mass, and these were associated with rapamycin-sensitive increases in adenosylmethione decarboxylase 1 (Amd1), spermidine synthase (SpdSyn), and c-Myc. Food deprivation decreased mTORC1 signaling, protein synthesis, and muscle mass, accompanied by a decrease in spermidine/spermine acetyltransferase 1 (Sat1). Denervation, resulted increased mTORC1 signaling and protein synthesis, and decreased muscle mass, which was associated with an increase in SpdSyn, spermine synthase (SpmSyn), and c-Myc. Combined, these data show that polyamine pathway enzymes are differentially regulated in models of altered mechanical and metabolic stress, and that Amd1 and SpdSyn are, in part, regulated in a mTORC1-dependent manner. Furthermore, these data suggest that polyamines may play a role in the adaptive response to stressors in skeletal muscle.

The role of raptor in the mechanical load-induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy.

It is well known that an increase in mechanical loading can induce skeletal muscle hypertrophy, and a long standing model in the field indicates that mechanical loads induce hypertrophy via a mechanism that requires signaling through the mechanistic target of rapamycin complex 1 (mTORC1). Specifically, it has been widely proposed that mechanical loads activate signaling through mTORC1 and that this, in turn, promotes an increase in the rate of protein synthesis and the subsequent hypertrophic response. However, this model is based on a number of important assumptions that have not been rigorously tested. In this study, we created skeletal muscle specific and inducible raptor knockout mice to eliminate signaling by mTORC1, and with these mice we were able to directly demonstrate that mechanical stimuli can activate signaling by mTORC1, and that mTORC1 is necessary for mechanical load-induced hypertrophy. Surprisingly, however, we also obtained multiple lines of evidence that indicate that mTORC1 is not required for a mechanical load-induced increase in the rate of protein synthesis. This observation highlights an important shortcoming in our understanding of how mechanical loads induce hypertrophy and illustrates that additional mTORC1-independent mechanisms play a critical role in this process.-You, J.-S., McNally, R. M., Jacobs, B. L., Privett, R. E., Gundermann, D. M., Lin, K.-H., Steinert, N. D., Goodman, C. A., Hornberger, T. A. The role of raptor in the mechanical load-induced regulation of mTOR signaling, protein synthesis, and skeletal muscle hypertrophy.

Resistance Exercise-Induced Hypertrophy: A Potential Role for Rapamycin-Insensitive mTOR.

The mechanistic target of rapamycin (mTOR) exerts both rapamycin-sensitive and rapamycin-insensitive signaling events, and the rapamycin-sensitive components of mTOR signaling have been widely implicated in the pathway through which resistance exercise induces skeletal muscle hypertrophy. This review explores the hypothesis that rapamycin-insensitive components of mTOR signaling also contribute to this highly important process.

The Hippo Signaling Pathway in the Regulation of Skeletal Muscle Mass and Function.

The Hippo signaling pathway regulates the activity of the proteins Yes-associated protein (Yap) and transcriptional co-activator with PDZ-binding motif (Taz) to control tissue growth in many different cell types. Previously, we demonstrated that Yap is a critical regulator of skeletal muscle mass. We hypothesize that alterations in Yap and Taz activity modulate the anabolic adaptations of skeletal muscle to resistance exercise.

The role of mTORC1 in regulating protein synthesis and skeletal muscle mass in response to various mechanical stimuli.

Skeletal muscle plays a fundamental role in mobility, disease prevention, and quality of life. Skeletal muscle mass is, in part, determined by the rates of protein synthesis, and mechanical loading is a major regulator of protein synthesis and skeletal muscle mass. The mammalian/mechanistic target of rapamycin (mTOR), found in the multi-protein complex, mTORC1, is proposed to play an essential role in the regulation of protein synthesis and skeletal muscle mass. The purpose of this review is to examine the function of mTORC1 in relation to protein synthesis and cell growth, the current evidence from rodent and human studies for the activation of mTORC1 signaling by different types of mechanical stimuli, whether mTORC1 signaling is necessary for changes in protein synthesis and skeletal muscle mass that occur in response to different types of mechanical stimuli, and the proposed molecular signaling mechanisms that may be responsible for the mechanical activation of mTORC1 signaling.

PGC-1α overexpression by in vivo transfection attenuates mitochondrial deterioration of skeletal muscle caused by immobilization.

Prolonged immobilization (IM) causes skeletal muscle atrophy characterized by mitochondrial deterioration and proteolysis. Muscle remobilization (RM) increases reactive oxygen species generation, proinflammatory cytokine expression, and oxidative stress, preventing muscle from quick recovery. Thus, we hypothesized that overexpression of peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) via in vivo transfection would promote mitochondrial biogenesis and antioxidant defense, thus ameliorating the aforementioned deteriorations in a mouse model with 14-d IM followed by 5-d RM. PGC-1α transfection in tibialis anterior muscle resulted in a 7.2- and 4-fold increase in PGC-1α content in cytosol and nucleus, respectively. Mitochondrial biogenic (cytochrome c, mitochondrial transcription factor A), morphologic (mitochondrial density, mDNA/nDNA ratio), and functional (cytochrome c oxidase activity, ATP synthesis rate) markers, as well as fiber cross-sectional area, significantly increased in IM-RM muscle by PGC-1α overexpression. These effects were accompanied by an 18% decrease in H2O2, 30% decrease in nuclear factor-κB-DNA binding, and 25% reduction of IL-1ß and-6 production in IM-RM muscle. There was a 34% increase in superoxide dismutase-2 activity, along with a 3.5-fold increase in NAD-dependent deacetylase sirtuin-3 expression caused by enhanced PGC-1α-estrogen-related receptor α binding. Our findings highlighted the importance of PGC-1α in protecting muscle from metabolic and redox disturbances caused by IM.

The effect of taurine and ß-alanine supplementation on taurine transporter protein and fatigue resistance in skeletal muscle from mdx mice.

This study investigated the effect of taurine and ß-alanine supplementation on muscle function and muscle taurine transporter (TauT) protein expression in mdx mice. Wild-type (WT) and mdx mice (5 months) were supplemented with taurine or ß-alanine for 4 weeks, after which in vitro contractile properties, fatigue resistance and force recovery, and the expression of the TauT protein and proteins involved in excitation-contraction (E-C) coupling were examined in fast-twitch muscle. There was no difference in basal TauT protein expression or basal taurine content between mdx than WT muscle. Supplementation with taurine and ß-alanine increased and reduced taurine content, respectively, in muscle from WT and mdx mice but had no effect of TauT protein. Taurine supplementation reduced body and muscle mass, and enhanced fatigue resistance and force recovery in mdx muscle. ß-Alanine supplementation enhanced fatigue resistance in WT and mdx muscle. There was no difference in the basal expression of key E-C coupling proteins [ryanodine receptor 1 (RyR1), dihydropyridine receptor (DHPR), sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1) or calsequestrin 1 (CSQ1)] between WT and mdx mice, and the expression of these proteins was not altered by taurine or ß-alanine supplementation. These findings suggest that TauT protein expression is relatively insensitive to changes in muscle taurine content in WT and mdx mice, and that taurine and ß-alanine supplementation may be viable therapeutic strategies to improve fatigue resistance of dystrophic skeletal muscle.

The role of diacylglycerol kinase ζ and phosphatidic acid in the mechanical activation of mammalian target of rapamycin (mTOR) signaling and skeletal muscle hypertrophy.

The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

Prioritization of skeletal muscle growth for emergence from hibernation.

Mammalian hibernators provide an extreme example of naturally occurring challenges to muscle homeostasis. The annual hibernation cycle is characterized by shifts between summer euthermy with tissue anabolism and accumulation of body fat reserves, and winter heterothermy with fasting and tissue catabolism. The circannual patterns of skeletal muscle remodelling must accommodate extended inactivity during winter torpor, the motor requirements of transient winter active periods, and sustained activity following spring emergence. Muscle volume in thirteen-lined ground squirrels (Ictidomys tridecemlineatus) calculated from MRI upper hindlimb images (n=6 squirrels, n=10 serial scans) declined from hibernation onset, reaching a nadir in early February. Paradoxically, mean muscle volume rose sharply after February despite ongoing hibernation, and continued total body mass decline until April. Correspondingly, the ratio of muscle volume to body mass was steady during winter atrophy (October-February) but increased (+70%) from February to May, which significantly outpaced changes in liver or kidney examined by the same method. Generally stable myocyte cross-sectional area and density indicated that muscle remodelling is well regulated in this hibernator, despite vastly altered seasonal fuel and activity levels. Body composition analysis by echo MRI showed lean tissue preservation throughout hibernation amid declining fat mass by the end of winter. Muscle protein synthesis was 66% depressed in early but not late winter compared with a summer fasted baseline, while no significant changes were observed in the heart, liver or intestine, providing evidence that could support a transition in skeletal muscle regulation between early and late winter, prior to spring emergence and re-feeding.

The mechanical activation of mTOR signaling: an emerging role for late endosome/lysosomal targeting.

It is well recognized that mechanical signals play a critical role in the regulation of skeletal muscle mass, and the maintenance of muscle mass is essential for mobility, disease prevention and quality of life. Furthermore, over the last 15 years it has become established that signaling through a protein kinase called the mammalian (or mechanistic) target of rapamycin (mTOR) is essential for mechanically-induced changes in protein synthesis and muscle mass, however, the mechanism(s) via which mechanical stimuli regulate mTOR signaling have not been defined. Nonetheless, advancements are being made, and an emerging body of evidence suggests that the late endosome/lysosomal (LEL) system might play a key role in this process. Therefore, the purpose of this review is to summarize this body of evidence. Specifically, we will first explain why the Ras homologue enriched in brain (Rheb) and phosphatidic acid (PA) are considered to be direct activators of mTOR signaling. We will then describe the process of endocytosis and its involvement in the formation of LEL structures, as well as the evidence which indicates that mTOR and its direct activators (Rheb and PA) are all enriched at the LEL. Finally, we will summarize the evidence that has implicated the LEL in the regulation of mTOR by various growth regulatory inputs such as amino acids, growth factors and mechanical stimuli.

Growth restriction in the rat alters expression of metabolic genes during postnatal cardiac development in a sex-specific manner.

This study investigated the impact of uteroplacental insufficiency and growth restriction on the expression of genes related to mitochondrial biogenesis, glucose transport, and antioxidant defenses in cardiac tissue at embryonic day 20 (E20) and postnatal days 1, 7, and 35 in male and female Wistar rats (8-10 per group). Bilateral uterine vessel ligation to induce growth restriction (Restricted) or sham surgery was performed at pregnancy day 18. In male and female Controls, expression of most cardiac genes decreased during postnatal life, including genes involved in mitochondrial biogenesis regulation such as PGC-1α, NRF-2, and mtTFA and the glucose transporter GLUT-1 (P < 0.05). However, the pattern of gene expression during cardiac development differed in male and female Restricted rats compared with their respective Controls. These effects of restriction were observed at postnatal day 1, with female Restricted rats having delayed reductions in PGC-1α and GLUT-1, whereas males had exacerbated reductions in PGC-1α and mtTFA (P < 0.05). By day 35, cardiac gene expression in Restricted hearts was similar to Controls, except for expression of the antioxidant enzyme MnSOD, which was significantly lower in both sexes. In summary, during postnatal life male and female Control rats have similar patterns of expression for genes involved in mitochondrial biogenesis and glucose transport. However, following uteroplacental insufficiency these gene expression patterns diverge in males and females during early postnatal life, with MnSOD gene expression reduced in later postnatal life.

Eccentric contractions increase the phosphorylation of tuberous sclerosis complex-2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome.

The goal of this study was to determine whether the mechanical activation of mechanistic target of rapamycin (mTOR) signalling is associated with changes in phosphorylation of tuberous sclerosis complex-2 (TSC2) and targeting of mTOR and TSC2 to the lysosome. As a source of mechanical stimulation, mouse skeletal muscles were subjected to eccentric contractions (ECs). The results demonstrated that ECs induced hyper-phosphorylation of TSC2 and at least part of this increase occurred on residue(s) that fall within RxRxxS/T consensus motif(s). Furthermore, in control muscles, we found that both mTOR and TSC2 are highly enriched at the lysosome. Intriguingly, ECs enhanced the lysosomal association of mTOR and almost completely abolished the lysosomal association of TSC2. Based on these results, we developed a new model that could potentially explain how mechanical stimuli activate mTOR signalling. Furthermore, this is the first study to reveal that the activation of mTOR is associated with the translocation of TSC2 away from the lysosome. Since a large number of signalling pathways rely on TSC2 to control mTOR signalling, our results have potentially revealed a fundamental mechanism via which not only mechanical, but also various other types of stimuli, control mTOR signalling.

Altering the rest interval during high-intensity interval training does not affect muscle or performance adaptations.

It has been hypothesized that exercise-induced changes in metabolites and ions are crucial in the adaptation of contracting muscle. We tested this hypothesis by comparing adaptations to two different interval-training protocols (differing only in the rest duration between intervals), which provoked different perturbations in muscle metabolites and acid-base status. Prior to and immediately after training, 12 women performed the following tests: (1) a graded exercise test to determine peak oxygen uptake (V(O2)); (2) a high-intensity exercise bout (followed 60 s later by a repeated-sprint-ability test; and (3) a repeat of the high-intensity exercise bout alone with muscle biopsies pre-exercise, immediately postexercise and after 60 s of recovery. Subjects performed 5 weeks (3 days per week) of training, with either a short (1 min; HIT-1) or a long rest period (3 min; HIT-3) between intervals; training intensity and volume were matched. Muscle [H(+)] (155 ± 15 versus 125 ± 8 nmol l(-1); P < 0.05) and muscle lactate content (84.2 ± 7.9 versus 46.9 ± 3.1 mmol (g wet weight)(-1)) were both higher after HIT-1, while muscle phosphocreatine (PCr) content (52.8 ± 8.3 versus 63.4 ± 9.8 mmol (g wet weight)(-1)) was lower. There were no significant differences between the two groups regarding the increases in , repeated-sprint performance or muscle Na(+),K(+)-ATPase content. Following training, both groups had a significant decrease in postexercise muscle [H(+)] and lactate content, but not postexercise ATP or PCr. Postexercise PCr resynthesis increased following both training methods. In conclusion, intense interval training results in marked improvements in muscle Na(+),K(+)-ATPase content, PCr resynthesis and . However, manipulation of the rest period during intense interval training did not affect these changes.

Measuring protein synthesis with SUnSET: a valid alternative to traditional techniques?

Protein synthesis rates commonly are measured using isotopic tracers to quantify the incorporation of a labeled amino acid into muscle proteins. Here we provide evidence supporting our hypothesis that the nonisotopic SUnSET technique is a valid and accurate method for the measurement of in vivo changes in protein synthesis at the whole-muscle and single-muscle fiber levels.