ArticleLiterature Review

It's not just about protein turnover: the role of ribosomal biogenesis and satellite cells in the regulation of skeletal muscle hypertrophy

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Abstract

Skeletal muscle has indispensable roles in regulating whole body health (e.g. glycemic control, energy consumption) and, in being central in locomotion, is crucial in maintaining quality-of-life. Therefore, understanding the regulation of muscle mass is of significant importance. Resistance exercise training (RET) combined with supportive nutrition is an effective strategy to achieve muscle hypertrophy by driving chronic elevations in muscle protein synthesis (MPS). The regulation of muscle protein synthesis is a coordinated process, in requiring ribosomes to translate mRNA and sufficient myonuclei density to provide the platform for ribosome and mRNA transcription; as such MPS is determined by both translational efficiency (ribosome activity) and translational capacity (ribosome number). Moreover, as the muscle protein pool expands during hypertrophy, translation capacity (i.e. ribosomes and myonuclei content) could theoretically become rate-limiting such that an inability to expand these pools through ribosomal biogenesis and satellite cell (SC) mediated myonuclear addition could limit growth potential. Simple measures of RNA (ribosome content) and DNA (SC/Myonuclei number) concentrations reveal that these pools do increase with hypertrophy; yet whether these adaptations are a pre-requisite or a limiting factor for hypertrophy is unresolved and highly debated. This is primarily due to methodological limitations and many assumptions being made on static measures or correlative associations. However recent advances within the field using stable isotope tracers shows promise in resolving these questions in muscle adaptation.

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... The adaptive responses to exercise training take many forms, including the increased abundance and/or maximal activity of key proteins with roles in energy provision, remodeling of cellular components such as contractile proteins and the extracellular matrix, and biogenesis of organelles such as ribosomes and mitochondria (Egan and Zierath 2013;Hoppeler et al. 2011;Brook et al. 2019;Hood et al. 2016). The teleological understanding of these coordinated changes is that they occur in order to minimize perturbations to cellular homeostasis, with this better maintenance of cellular homeostasis likely contributing to improved fatigue resistance in response to future bouts of exercise (Booth and Thomason 1991;Holloszy and Coyle 1984). ...
... This suggests the model for exercise-induced signal transduction in skeletal muscle should be updated to include regulators of epigenetic modifications (McGee and Hargreaves 2020; Seaborne and Sharples 2020). Lastly, the regulation of protein translation, the biogenesis and activity of organelles including ribosomes and lysosomes, and the importance of skeletal muscle satellite cells for adaptation are some of the new vistas in the field (Brook et al. 2019). ...
... Additionally, rates of protein synthesis are primarily dependent on translational efficiency and capacity. Both increased translational efficiency (protein synthesis per unit RNA) and elevated translational capacity (total RNA content per unit tissue) as a consequence of ribosome biogenesis have emerged as important regulators of the adaptive response to exercise (Brook et al. 2019). While there has been little focus on these pathways in the context of endurance exercise, a single bout of strength training increases markers of ribosomal DNA transcription (Stec et al. 2016), whereas an increase in RNA content is a rapid (Bickel et al. 2005) and sustained (Brook et al. 2015) response to strength training. ...
Chapter
This chapter reviews the molecular and metabolic responses in human skeletal muscle to exercise training. Acute changes in various stimuli that trigger adaptations largely depend on the type of exercise performed and particularly the intensity and duration of discrete sessions. These stimuli are linked to the activation and/or repression of an array of intracellular signal transduction pathways, pre- and posttranscriptional processes, and the regulation of protein translation. Given the considerable overlap in these underlying molecular processes, the mechanistic basis for how repeated, acute changes are translated into specific training responses remains a topic of much investigation. Endurance training is primarily associated with an enhanced capacity for oxidative energy provision and a shift in substrate utilization, from carbohydrate to lipid, at a given absolute exercise intensity. Strength training mainly results in increased muscle size, force-generating capacity, and enhanced capacity for non-oxidative energy provision. Sprint training also increases the capacity for non-oxidative energy provision, but can elicit a range of responses, including some that resemble endurance or strength training. Training generally enhances fatigue resistance and performance in a manner that is specific, but not exclusive, to the type of exercise performed. These improvements are owed, in part to training-induced changes in both the maximal capacity for, and the specific utilization of, various substrates during exercise.
... After postmitotic maturation, skeletal muscles present two main mechanisms involved with hypertrophy: the activation of protein synthesis conducted by the paracrine signaling and the simultaneous process of myonuclear accretion (23,(85)(86)(87)(88)(89). The paracrine signaling induced by secretion of growth factors can mediate the activation of the Akt/ mammalian target of the rapamycin (mTOR) molecular pathway, culminating in an increase in p70S6K ribosomal activation (90)(91)(92)(93). ...
... Inflammatory cells can also stimulate the hypertrophy mechanism through satellite cell activation, leading to myonuclear accretion and subsequent increases in ribosomal biogenesis, promoting myofiber growth (23,86,92,(94)(95)(96). Indeed, these mechanisms are not independent and are frequently manipulated by supportive anabolic strategies (e.g., better control of exercise overload, supplementation, and pharmacological approaches) to improve muscle mass and training performance (88,89,(97)(98)(99)(100). Furthermore, there is a growing number of studies exploring how inflammatory cells and downstream mediators can contribute to skeletal muscle recovery, mainly after bouts of damaging exercise (e.g., endurance or eccentric exercises) (17,22,87,94,(101)(102)(103)(104). ...
Article
Macrophages are one of the top players when considering immune cells involved with tissue homeostasis. Recently, increasing evidence has demonstrated that these macrophages could also present two major subsets during tissue healing; proliferative macrophages (M1-like), which are responsible for increasing myogenic cell proliferation, and restorative macrophages (M2-like), which are accountable for the end of the mature muscle myogenesis. The participation and characterization of these macrophage subsets is critical during myogenesis, not only to understand the inflammatory role of macrophages during muscle recovery but also to create supportive strategies that can improve mass muscle maintenance. Indeed, most of our knowledge about macrophage subsets comes from skeletal muscle damage protocols, and we still do not know how these subsets can contribute to skeletal muscle adaptation. This narrative review aims to collect and discuss studies demonstrating the involvement of different macrophage subsets during the skeletal muscle damage/regeneration process, showcasing an essential role of these macrophage subsets during muscle adaptation induced by acute and chronic exercise programs.
... In 1973, we introduced the concept that identified the dietary protein-induced regulation of muscle growth acting via stimulation of both the capacity for and efficiency of ribosomal mediated protein synthesis: i.e., total RNA concentration and protein synthesis per unit RNA [251]. Thus, new myonuclei can increase protein synthetic capacity in terms of ribosomal RNA, [58,252] enabling increased global myofibre protein synthesis [60]. Be-cause each myofibre is a multinucleated, post-mitotic single cell, each myonucleus is envisaged as controlling a volume or domain of the fibre, originally defined in operational terms as a DNA-unit with the dimensions of the protein-DNA ratio [253]. ...
... Ribosome biogenesis is a complicated and energetically costly process requiring production of four ribosomal RNAs (rRNAs) and the synthesis of ≈80 ribosomal proteins that make up the mature 80S eukaryotic ribosome. This requires activation of three classes of RNA polymerases (POL I, II, and III), with synthesis of precursor rRNA (i.e., 45S pre-rRNA), by POL I the a major rate-limiting step and its regulation involving multiple signalling pathways [252,275,276] including ERK, AMPK, mTORC1 and P70S6K1. This allows for ribosomal biogenesis to be controlled by hormones, nutrients, including amino acids [277] and contractile activity. ...
Article
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Childhood growth and its sensitivity to dietary protein is reviewed within a Protein-Stat model of growth regulation. The coordination of growth of muscle and stature is a combination of genetic programming, and of two-way mechanical interactions involving the mechanotransduction of muscle growth through stretching by bone length growth, the core Protein-Stat feature, and the strengthening of bone through muscle contraction via the mechanostat. Thus, growth in bone length is the initiating event and this is always observed. Endocrine and cellular mechanisms of growth in stature are reviewed in terms of the growth hormone-insulin like growth factor-1 (GH-IGF-1) and thyroid axes and the sex hormones, which together mediate endochondral ossification in the growth plate and bone lengthening. Cellular mechanisms of muscle growth during development are then reviewed identifying (a) the difficulties posed by the need to maintain its ultrastructure during myofibre hypertrophy within the extracellular matrix and the concept of muscle as concentric “bags” allowing growth to be conceived as bag enlargement and filling, (b) the cellular and molecular mechanisms involved in the mechanotransduction of satellite and mesenchymal stromal cells, to enable both connective tissue remodelling and provision of new myonuclei to aid myofibre hypertrophy and (c) the implications of myofibre hypertrophy for protein turnover within the myonuclear domain. Experimental data from rodent and avian animal models illustrate likely changes in DNA domain size and protein turnover during developmental and stretch-induced muscle growth and between different muscle fibre types. Growth of muscle in male rats during adulthood suggests that “bag enlargement” is achieved mainly through the action of mesenchymal stromal cells. Current understanding of the nutritional regulation of protein deposition in muscle, deriving from experimental studies in animals and human adults, is reviewed, identifying regulation by amino acids, insulin and myofibre volume changes acting to increase both ribosomal capacity and efficiency of muscle protein synthesis via the mechanistic target of rapamycin complex 1 (mTORC1) and the phenomenon of a “bag-full” inhibitory signal has been identified in human skeletal muscle. The final section deals with the nutritional sensitivity of growth of muscle and stature to dietary protein in children. Growth in length/height as a function of dietary protein intake is described in the context of the breastfed child as the normative growth model, and the “Early Protein Hypothesis” linking high protein intakes in infancy to later adiposity. The extensive paediatric studies on serum IGF-1 and child growth are reviewed but their clinical relevance is of limited value for understanding growth regulation; a role in energy metabolism and homeostasis, acting with insulin to mediate adiposity, is probably more important. Information on the influence of dietary protein on muscle mass per se as opposed to lean body mass is limited but suggests that increased protein intake in children is unable to promote muscle growth in excess of that linked to genotypic growth in length/height. One possible exception is milk protein intake, which cohort and cross-cultural studies suggest can increase height and associated muscle growth, although such effects have yet to be demonstrated by randomised controlled trials.
... Ribosomes play a key role in protein translation (11)(12)(13) and recent work in rodents has demonstrated that SCs may supply certain ribosomal proteins to muscle fibers to support adaptation (14). Following an acute bout of resistance exercise, ribosome content increases to support the synthesis of proteins involved in cellular remodeling (15)(16)(17)(18) and muscle contractions (19)(20)(21). ...
Article
Satellite cells (SC) and ribosomes are key determinants of the skeletal muscle adaptive response. Both are thought to increase acutely after resistance exercise and chronically with resistance training. However, the acute SC and ribosome exercise response with prior aerobic conditioning is unknown. Fourteen young men and women underwent 6 weeks of single-legged aerobic conditioning followed by an acute bout of 300 eccentric contractions. Muscle biopsies were taken from the vastus lateralis of the aerobically conditioned (AC) and the control (CTL) legs before (Pre), 24 (24h) and 48 (48h) hours post-contractions. SC pool expansion (PAX7 ⁺ cells/100 fibres) was greater in type-I (1.3-fold) and mixed-fibres (1.2-fold) in the AC leg compared to the CTL. Pax7 (1.2-fold) and MyoD1 (1.4-fold) mRNA expression was also greater in the AC leg compared to the CTL. AC had greater RNA concentration (1.2-fold) and mRNA expression of Ubf (1.2-fold) and Tif-1a (1.3-fold) compared to CTL. Only the AC leg increased (Pre-48h) c-Myc (3.0-fold), (Pre-24h) 45S pre-rRNA (2.6-fold), 5.8S ITS (2.1-fold) and 28S ITS (2.0-fold) following eccentric contractions. We discovered that aerobic conditioning augmented type-I SC pool expansion and ribosome content following an acute bout of eccentric contractions.
... Considering that muscle hypertrophy may be affected by accumulated periods of increased post-exercise MPS [21], keeping a higher RT frequency would be an effective strategy to contribute to such adaptation [20,22]. Furthermore, TTV can be increased with a higher RT frequency [9], and this increase might be related to ribosomal biogenesis [23], satellite cell activation and proliferation, and myonuclear addition which in turn could influence MPS in long term training conditions [24]. ...
Article
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Several studies comparing resistance training (RT) frequencies may have been affected by the large between-subject variability. This study aimed to compare the changes in lower limbs maximal dynamic strength (1RM) and quadriceps femoris cross-sectional area (CSA) after a RT with different weekly frequencies in strength-trained individuals using a within-subject design. Twenty-four men participated in a 9-week RT program, being randomly divided into two conditions: resistance training with equalized total training volume (RTEV) and with unequalized total training volume (RTUV). The RT protocol used the unilateral leg press 45° exercise and each subject's lower limb executed one of the proposed frequencies (one and three times/week). All conditions effectively increased 1RM and CSA (p<0.001); however, no significant differences were observed in the values of 1RM (p = 0.454) and CSA (p = 0.310) between the RT frequencies in the RTEV and RTUV conditions. Therefore, RT performed three times a week showed similar increases in 1RM and CSA to the program performed once a week, regardless of training volume equalization. Nevertheless, when the higher RT frequency allowed the application of a greater TTV (i.e., RTUV), higher effect size (ES) values (0.51 and 0.63, 1RM and CSA, respectively) were observed for the adaptations.
... The myonuclear domain hypothesis suggests that there is a fixed ratio between the sarcoplasmic volume and the number of myonuclei 4 . Indeed, sufficient myonuclear density facilitates messenger RNA (mRNA) transcription and ribosomemediated protein translation 15 . However, type II muscle fibers have demonstrated the ability to hypertrophy in the absence of myonuclear accretion 16 . ...
Article
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A complex network of biochemical pathways carries out the process of muscle regeneration/growth following resistance exercise. The initial inflammatory response following muscle damage is primarily mediated by the nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), cyclooxygenase enzymes, and prostaglandins. Muscle damage also stimulates the activation, proliferation, differentiation, migration, and fusion of satellite cells onto damaged myofibers, resulting in myofibrillar hypertrophy. The progression of the myogenic lineage is predominantly coordinated by the wingless/integrated family of glycoproteins which engages in crosstalk with NF-κB and the mitogen-activated protein kinase (MAPK)/extracellular signaling-regulated kinase network. The MAPK cascade is essential for mechanotransduction, the process of converting mechanical stimuli into biochemical responses such as accelerated protein synthesis and satellite cell activation. Muscle protein synthesis is primarily governed by the insulin-like growth factor 1/phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathway. Several calcium-dependent pathways are also integrated into the process of myogenesis and influence skeletal muscle plasticity. These dynamic interactions are part of the anabolic priming by resistance exercise effect, which defines resistance exercise as an acute catabolic event that potentiates multiple downstream anabolic pathways. Plateaus in muscle growth are attributed to deteriorating inflammatory signaling with repeated bouts of muscle damage as well as increasing thresholds for continuous adaptations, which ultimately become unreachable beyond a certain point. The physiological ceiling of skeletal muscle mass is also credited to myostatin. However, recent discoveries suggest the role of myostatin is not limited to preventing excessive skeletal muscle hypertrophy.
... Despite contradictory evidence, recent experiments utilising timed in vivo myonuclear labelling suggest that satellite cells play an important role in training-induced hypertrophy [71]. In addition to participation in acute response through myonuclear accretion, MuSCs were suggested to contribute to synthesis of ribosomal proteins and satellite cell-derived myonuclei were shown to have altered methylation status that favours cell-to-cell signalling, possibly "rejuvenating" the myofibers and promoting long-term hypertrophy [71][72][73]. It also appears that MuSC miR-1 expression reflects the epigenetic memory of previous training, as it enhances ongoing growth and likely facilitates re-growth after a period of detraining in mice [74]. ...
... It has been shown that mTORC1 activation by SE upregulates transcription of other components involved in translational machinery itself (e.g. mRNA, ribosomes) (Figueiredo and McCarthy 2019), thus increasing the translational capacity of the skeletal muscle cells over time (Brook et al. 2019;Figueiredo and McCarthy 2019). ...
Chapter
Skeletal muscle is essential in locomotion and plays a role in whole-body metabolism, particularly during exercise. Skeletal muscle is the largest ‘reservoir’ of amino acids, which can be released for fuel and as a precursor for gluconeogenesis. During exercise, whole-body, and more specifically skeletal muscle, protein catabolism is increased, but protein synthesis is suppressed. Metabolism of skeletal muscle proteins can support energy demands during exercise, and persistent exercise (i.e. training) results in skeletal muscle protein remodelling. Exercise is generally classified as being either ‘strength’ or ‘aerobic/endurance’ in nature, and the type of exercise will reflect the phenotypic and metabolic adaptations of the muscle. In this chapter, we describe the impact of various exercise modes on protein metabolism during and following exercise.
... However, we also cannot exclude that existing myonuclei also increase their rates of protein synthesis to elicit the slight hypertrophy. 44,45 Moreover, we did not report change in internalized myonuclei and regenerative fibers, other signs of muscle remodeling, in training patients ( Table 3). The low SC response to the training program may also be explained by the fact that in our study (1) the training intensity was low, (2) the duration of training sessions was short, and (3) the delay between the final training session and the latter biopsy was long (a week). ...
Article
We previously demonstrated that 8 weeks of moderate-intensity endurance training is safe and improves muscle function and characteristics of sickle cell disease (SCD) patients. Here, we investigated skeletal muscle satellite cells (SCs) in SCD patients and their responses to a training program. Fifteen patients followed the training program while 18 control patients maintained a normal lifestyle. Biopsies of the vastus lateralis muscle were performed before and after training. After training, the cross-sectional area and myonuclear content in type I fibers were slightly increased in the training patients compared to non-training patients. The SC pool was unchanged in type I fibers while it was slightly decreased in type II fibers in the training patients compared to non-training patients. No necrotic fibers were detected in patients before or after training. Therefore, the slight myonuclear accretion in type I fibers in trained SCD patients may highlight the contribution of SCs to training-induced slight type I fiber hypertrophy without expansion of the SC pool. The low training intensity and the short duration of training sessions could explain the low SC response to the training program. However, the lack of necrotic fibers suggests that the training program seemed to be safe for patients’ muscle tissue.
... The regulation of protein synthesis is a collaborative process, requiring ribosomes to translate mRNA and enough myonuclei to provide the necessary platform for mRNA transcription. Thus, protein synthesis is determined by both translational efficiency and translation capacity (Brook et al., 2019). Lim et al. (2018) demonstrated that LRT increases the extensor digitorum longus muscle CSA and increases the number of satellite cells. ...
Article
We compared the effects of two ladder-based resistance training (LRT) protocols on the skeletal muscle morphology (biceps brachialis and plantaris) of Wistar rats. Also, we correlated the training parameters with the muscle fiber cross-sectional area (fCSA). After maximum load tests (ML), twenty-nine young adult Wistar rats were divided into: CONTROL (n = 9), LIMITED (n = 10, 6-8 climb [2 × 50%ML, 2 × 75%ML, 2 × 100%ML, and 2 × 100%ML+30 g]) and UNLIMITED (n = 10, ≥4 climbs [50%ML, 75%ML, 90%ML, 100%ML + 30 g until failure) LRT. After eight weeks, the main results were: 1) For biceps brachialis, the type I, IIa, and mean fCSA was statistically larger in the LIMITED than CONTROL. The nuclei/fiber ratio was statistically higher in the LIMITED and UNLIMITED. The correlations found between total load, absolute delta load, and relative load and fCSA were moderate. 2) For plantaris, the type I, IIa, IIx/b, and mean fCSA was statistically larger in the LIMITED than CONTROL. The type IIa, IIx/b, and mean fCSA was statistically larger in the UNLIMITED than CONTROL. The nuclei/fiber ratio was statistically higher in both trained groups than CONTROL. The correlation between the climbing number, total load, and the fCSA was moderate. The correlation between delta absolute load and fCSA was strong. We concluded that rodents submitted to high-intensity, high-volume LRT, but limited climbing volume per session, presented more significant type I, IIa, IIx/b, and mean fCSA, higher nuclei/fiber ratio, and greater maximum carrying capacity. Also, muscle hypertrophy correlated positively with the load progression, training volume, and total load.
... In addition to translational efficiency, translational capacity (i.e. ribosome content) is important in regulating rates of protein synthesis and subsequent muscle adaptation [57]. Previously, we and others have demonstrated diminished ribosomal biogenesis to RET in older muscle that may be fundamental to the blunted muscle hypertrophy observed with age [2,58]. ...
Article
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Background & aims The skeletal muscle anabolic effects of n-3 polyunsaturated fatty acids (n-3 PUFA) appear favoured towards women; a property that could be exploited in older women who typically exhibit poor muscle growth responses to resistance exercise training (RET). Here we sought to generate novel insights into the efficacy and mechanisms of n-3 PUFA alongside short-term RET in older women. Methods We recruited 16 healthy older women (Placebo n=8 (PLA): 67±1y, n-3 PUFA n=8: 64±1y) to a randomised double-blind placebo-controlled trial (n-3 PUFA; 3680mg/day versus PLA) of 6 weeks fully-supervised progressive unilateral RET (i.e. 6×8 reps, 75% 1-RM, 3/wk⁻¹). Strength was assessed by knee extensor 1-RM and isokinetic dynamometry ∼ every 10 d. Thigh fat free mass (TFFM) was measured by DXA at 0/3/6 weeks. Bilateral vastus lateralis (VL) biopsies at 0/2/4/6 weeks with deuterium oxide (D2O) dosing were used to determine MPS responses for 0-2 and 4-6 weeks. Further, fibre cross sectional area (CSA), myonuclei number and satellite cell (SC) number were assessed, alongside muscle anabolic/catabolic signalling via immunoblotting. Results RET increased 1-RM equally in the trained leg of both groups (+23 ± 5 % n-3 PUFA vs. +25 ± 5 % PLA (both P<0.01)) with no significant increase in maximum voluntary contraction (MVC) (+10 ±6 % n-3 PUFA vs. +13 ± 5 % PLA). Only the n-3 PUFA group increased TFFM (3774 ± 158 g to 3961 ± 151 g n-3 PUFA (P<0.05) vs. 3406 ± 201 g to 3561 ± 170 PLA) and type II fibre CSA (3097 ± 339 μm2 to 4329 ± 264 μm2 n-3 PUFA (P<0.05) vs. 2520 ± 316 μm2 to 3467 ± 303 μm2 in PL) with RET. Myonuclei number increased equally in n-3 PUFA and PLA in both type I and type II fibres, with no change in SC number. N-3 PUFA had no added benefit on muscle protein synthesis (MPS), however, during weeks 4-6 of RET, absolute synthesis rates (ASR) displayed a trend to increase with n-3 PUFA only (5.6 ± 0.3 g.d⁻¹ to 7.1 ± 0.5 g.d⁻¹ n-3 PUFA (P=0.09) vs. 5.5 ± 0.5 g.d⁻¹ to 6.5 ± 0.5 g.d⁻¹ PLA). Further, the n-3 PUFA group displayed greater 4EBP1 activation after acute RE at 6 weeks. Conclusion n3-PUFA enhanced RET gains in muscle mass through type II fibre hypertrophy, with data suggesting a role for MPS rather than via SC recruitment. As such, the present study adds to a literature base illustrating the apparent enhancement of muscle hypertrophy with RET in older women fed adjuvant n3-PUFA.
... Skeletal muscle mass is indispensable due to its central roles in energy metabolism, amino acid (AA) storage, and locomotion [18]. This important tissue is in a continuous state of turnover, with old/ damaged proteins being broken down (muscle protein breakdown; MPB) and their constituent AAs providing substrates for the synthesis of new proteins in their place (muscle protein synthesis; MPS) [19][20][21]. ...
Article
Purpose of review: The practice of time-restricted feeding (TRF) has received fervent interest in recent years as a strategy to mitigate obesity and metabolic disease. We sought to review the implications of TRF for skeletal muscle health and function in aging. Recent findings: TRF has high adherence and can promote body weight loss in older populations. Body weight reductions favor fat mass in the young, however, there is also the potential for undesirable losses in lean mass. There is currently no evidence to support TRF for skeletal muscle function and metabolism in older persons, and only tentative findings in the young. With a narrow eating window of 6–8 h and a prolonged fasting period to minimize daily insulin exposure, TRF may contradict recommended dietary practices for optimizing skeletal muscle anabolism in older people. Summary: TRF might represent a promising intervention to address obesity and its associated metabolic diseases, however, at present there is insufficient evidence for optimizing skeletal muscle mass or health in older individuals. Further research is needed to: (1) ascertain the impact of TRF on body composition, skeletal muscle anabolism, and autophagy in aging, and; (2) delineate the potentially myoprotective roles of dietary protein and exercise within the framework of TRF in older persons.
... The fact that RT-induced muscle hypertrophy varies substantially between individuals highlights a strong intrinsic (i.e., resident within the muscle itself) component to hypertrophy (74,75). Clearly, part of the innate responses to RT comes from changes in MPS; however, changes in ribosomal content and satellite cell number and activation also contribute to hypertrophy (76). Thus, it is unsurprising that changes in MPS, measured acutely or in the medium-term, do not capture all aspects of hypertrophy. ...
Article
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Hypertrophy can be operationally defined as an increase in the axial cross-sectional area of a muscle fiber or whole muscle, and is due to increases in the size of pre-existing muscle fibers. Hypertrophy is a desired outcome in many sports. For some athletes, muscular bulk and, conceivably, the accompanying increase in strength/power, are desirable attributes for optimal performance. Moreover, bodybuilders and other physique athletes are judged in part on their muscular size, with placings predicated on the overall magnitude of lean mass. In some cases, even relatively small improvements in hypertrophy might be the difference between winning and losing in competition for these athletes. This position stand of leading experts in the field synthesizes the current body of research to provide guidelines for maximizing skeletal muscle hypertrophy in an athletic population. The recommendations represent a consensus of a consortium of experts in the field, based on the best available current evidence. Specific sections of the paper are devoted to elucidating the constructs of hypertrophy, reconciliation of acute vs long-term evidence, and the relationship between strength and hypertrophy to provide context to our recommendations.
... What is abundantly clear from our previous work (184), as well as others (103,178), is that transient postexercise rises in systemic concentrations of various anabolic hormones, such as testosterone, growth hormone, and insulin-like growth factor 1 (IGF-1) are unrelated to skeletal muscle anabolism. Also, skeletal muscle contains a pool of resident muscle stem cells-satellite cells-which possess mitotic potential (36). Using a cluster analysis, Petrella and colleagues (209) demonstrated that "higher-responders" to RT-induced myofiber hypertrophy had an increased number of satellite cells at baseline, and also expanded myonuclei number in response to RT, compared with "lower-responders" to RT. Stimulation of MPS can occur due to increased translational efficiency, with more messenger ribonucleic acid (mRNA) translated per ribosomal unit, or to increased translational capacity, which ensues by adding more ribosomes to translate existing mRNA (44,125). ...
Article
Skeletal muscle is the organ of locomotion, its optimal function is critical for athletic performance, and is also important for health due to its contribution to resting metabolic rate and as a site for glucose uptake and storage. Numerous endogenous and exogenous factors influence muscle mass. Much of what is currently known regarding muscle protein turnover is owed to the development and use of stable isotope tracers. Skeletal muscle mass is determined by the meal- and contraction-induced alterations of muscle protein synthesis and muscle protein breakdown. Increased loading as resistance training is the most potent nonpharmacological strategy by which skeletal muscle mass can be increased. Conversely, aging (sarcopenia) and muscle disuse lead to the development of anabolic resistance and contribute to the loss of skeletal muscle mass. Nascent omics-based technologies have significantly improved our understanding surrounding the regulation of skeletal muscle mass at the gene, transcript, and protein levels. Despite significant advances surrounding the mechanistic intricacies that underpin changes in skeletal muscle mass, these processes are complex, and more work is certainly needed. In this article, we provide an overview of the importance of skeletal muscle, describe the influence that resistance training, aging, and disuse exert on muscle protein turnover and the molecular regulatory processes that contribute to changes in muscle protein abundance. © 2021 American Physiological Society. Compr Physiol 11:2249-2278, 2021.
... Transcriptional control of genes required for the initiation of mRNA translation and nuclear export of ribosomal subunits are also modulated by c-Myc [133]. c-Myc is considered as an essential driver of ribosome biogenesis as it regulates the transcription of UBF, TIF-1A, TIF-1B, Pol I, Pol II, and Pol III [133][134][135][136][137][138]. Muscle loading, contractile activity, as well as nutrients availability, hormones, and other growth stimuli may impact ribosome biogenesis through the regulation of the aforementioned pathways [124,139,140]. ...
Article
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The regulation of skeletal muscle mass and organelle homeostasis is dependent on the capacity of cells to produce proteins and to recycle cytosolic portions. In this investigation, the mechanisms involved in skeletal muscle mass regulation—especially those associated with proteosynthesis and with the production of new organelles—are presented. Thus, the critical roles of mammalian/mechanistic target of rapamycin complex 1 (mTORC1) pathway and its regulators are reviewed. In addition, the importance of ribosome biogenesis, satellite cells involvement, myonuclear accretion, and some major epigenetic modifications related to protein synthesis are discussed. Furthermore, several studies conducted on the topic of exercise training have recognized the central role of both endurance and resistance exercise to reorganize sarcomeric proteins and to improve the capacity of cells to build efficient organelles. The molecular mechanisms underlying these adaptations to exercise training are presented throughout this review and practical recommendations for exercise prescription are provided. A better understanding of the aforementioned cellular pathways is essential for both healthy and sick people to avoid inefficient prescriptions and to improve muscle function with emergent strategies (e.g., hypoxic training). Finally, current limitations in the literature and further perspectives, notably on epigenetic mechanisms, are provided to encourage additional investigations on this topic.
... Therefore, we conducted a parallel-group study to investigate the impact of dietary protein source (plantbased vs. mixed dietary proteins) on RT-induced changes in muscle mass and strength in healthy young habitual vegans and omnivores under conditions of optimal protein intake (1.6 g kg −1 day −1 ) [21]. Considering that the acute intermittent elevations in MPS in response to, and with persistent practice of, resistance exercise in combination with sufficient protein feeding are considered the major drivers of muscle protein accretion and skeletal muscle hypertrophy [22], and given the acute differences in anabolic responses between plant and animal-based proteins, we hypothesized that an exclusive consumption of plant-based dietary protein would be less effective in supporting RT-induced muscle adaptations than an omnivorous diet. ...
Article
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Background Acute protein turnover studies suggest lower anabolic response after ingestion of plant vs. animal proteins. However, the effects of an exclusively plant-based protein diet on resistance training-induced adaptations are under investigation. Objective To investigate the effects of dietary protein source [exclusively plant-based vs. mixed diet] on changes in muscle mass and strength in healthy young men undertaking resistance training. Methods Nineteen young men who were habitual vegans (VEG 26 ± 5 years; 72.7 ± 7.1 kg, 22.9 ± 2.3 kg/m²) and nineteen young men who were omnivores (OMN 26 ± 4 years; 73.3 ± 7.8 kg, 23.6 ± 2.3 kg/m²) undertook a 12-week, twice weekly, supervised resistance training program. Habitual protein intake was assessed at baseline and adjusted to 1.6 g kg⁻¹ day⁻¹ via supplemental protein (soy for VEG or whey for OMN). Dietary intake was monitored every four weeks during the intervention. Leg lean mass, whole muscle, and muscle fiber cross-sectional area (CSA), as well as leg-press 1RM were assessed before (PRE) and after the intervention (POST). Results Both groups showed significant (all p < 0.05) PRE-to-POST increases in leg lean mass (VEG: 1.2 ± 1.0 kg; OMN: 1.2 ± 0.8 kg), rectus femoris CSA (VEG: 1.0 ± 0.6 cm²; OMN: 0.9 ± 0.5 cm²), vastus lateralis CSA (VEG: 2.2 ± 1.1 cm²; OMN: 2.8 ± 1.0 cm²), vastus lateralis muscle fiber type I (VEG: 741 ± 323 µm²; OMN: 677 ± 617 µm²) and type II CSA (VEG: 921 ± 458 µm²; OMN: 844 ± 638 µm²), and leg-press 1RM (VEG: 97 ± 38 kg; OMN: 117 ± 35 kg), with no between-group differences for any of the variables (all p > 0.05). Conclusion A high-protein (~ 1.6 g kg⁻¹ day⁻¹), exclusively plant-based diet (plant-based whole foods + soy protein isolate supplementation) is not different than a protein-matched mixed diet (mixed whole foods + whey protein supplementation) in supporting muscle strength and mass accrual, suggesting that protein source does not affect resistance training-induced adaptations in untrained young men consuming adequate amounts of protein. Clinical Trial Registration NCT03907059. April 8, 2019. Retrospectively registered.
... To examine these null findings further, we analysed muscle RNA data post-hoc. As~80% of the total cellular RNA is ribosomal (rRNA), a rapid increase in RNA is indicative of rRNA synthesis [28], which is associated with increases in translational efficiency, capacity and myoPS response to anabolic stimuli [10,[29][30][31]. Indeed, a cross-study analysis revealed that an untrained age-matched cohort had significantly lower RNA content, myoFSR and muscle protein content compared to our resistance-trained cohort [10]. ...
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... Animal studies suggest that although muscle growth can occur in satellite cell ablated conditions (2 weeks overload) (McCarthy et al., 2011), satellite cells may be necessary to support more extensive muscle fiber hypertrophy (8 weeks overload) (Fry et al., 2014). Work in humans showing the upregulation of MRFs following resistance exercise (McKay et al., 2008;Nederveen et al., 2019), and positive correlations between muscle growth and increased satellite cell content during prolonged resistance exercise training (Petrella et al., 2006;Verdijk et al., 2010;Bellamy et al., 2014), has contributed to the idea that satellite cells play a role in human muscle fiber hypertrophy (Snijders et al., 2015;Brook et al., 2019). The MuIS (+) phenotype and age-related impairments in satellite cell function may therefore limit hypertrophy of skeletal muscle after an atrophy-inducing event. ...
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Resistance exercise training (RET) is widely used to increase muscle mass in athletes and also aged/cachectic populations. However, the time course and metabolic and molecular control of hypertrophy remain poorly defined. Using newly developed deuterium oxide (D2O)-tracer techniques, we investigated the relationship between long-term muscle protein synthesis (MPS) and hypertrophic responses to RET. A total of 10 men (2361 yr) undertook 6 wk of unilateral (1-legged) RET [6 x 8 repetitions, 75% 1 repetition maximum (1-RM) 3/wk], rendering 1 leg untrained (UT) and the contralateral, trained (T). After baseline bilateral vastus lateralis (VL) muscle biopsies, subjects consumed 150 ml D2O (70 atom percentage; thereafter 50 ml/wk) with regular body water monitoring in saliva via high-temperature conversion elemental analyzer:isotope ratio mass spectrometer. Further bilateral VL muscle biopsies were taken at 3 and 6 wk to temporally quantify MPS via gas chromatography: pyrolysis: isotope ratio mass spectrometer. Expectedly, only the T leg exhibited marked increases in function [i.e., 1-RM/maximal voluntary contraction (60 degrees)] and VL thickness (peaking at 3 wk). Critically, whereas MPS remained unchanged in the UT leg (e.g., similar to 1.35 +/- 0.08%/d), the T leg exhibited increased MPS at 0-3wk(1.6 +/- 0.01%/d), but not at3-6wk(1.29 +/- 0.11%/d); this was reflected by dampened acute mechanistic target of rapamycin complex 1 signaling responses to RET, beyond 3 wk. Therefore, hypertrophic remodeling is most active during the early stages of RET, reflecting longer-term MPS. Moreover, D2O heralds promise for coupling MPS and muscle mass and providing insight into the control of hypertrophy and efficacy of anabolic interventions.
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Skeletal muscles comprise a substantial portion of whole body mass, and are integral for locomotion and metabolic health. Increasing age is associated with declines in both muscle mass and function (e.g. strength-related performance, power) with declines in muscle function quantitatively outweighing those in muscle volume. The mechanisms behind these declines are multi-faceted involving both intrinsic age-related metabolic dysregulation and environmental influences such as nutritional and physical activity. Ageing is associated with a degree of "anabolic resistance" to these key environmental inputs, which likely accelerates the intrinsic processes driving ageing. On this basis, strategies to sensitize and/or promote anabolic responses to nutrition and physical activity are likely to be imperative in alleviating the progression and trajectory of sarcopenia. Both resistance and aerobic type exercises are likely to confer functional and health benefits in older age, and a clutch of research suggests that enhancement of anabolic responsiveness to exercise and/or nutrition may be achieved by optimizing modifications of muscle-loading paradigms (workload, volume, blood flow restriction) or nutritional support (e.g. EAA/ leucine) patterns. Nonetheless, more work is needed in which a more holistic view in ageing studies is taken into account. This should include improved characterization of older study recruits i.e. physical activity/nutritional behaviours, to limit confounding variables influencing whether findings are attributable to age, or other environmental influences. Nonetheless, on balance, ageing is associated with declines in muscle mass and function and a partially related decline in aerobic capacity. There is also good evidence that metabolic flexibility is impaired in older age. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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A key determinant of geriatric frailty is sarcopenia, the age-associated loss of skeletal muscle mass and strength. Although the etiology of sarcopenia is unknown, the correlation during aging between the loss of activity of satellite cells, which are endogenous muscle stem cells, and impaired muscle regenerative capacity has led to the hypothesis that the loss of satellite cell activity is also a cause of sarcopenia. We tested this hypothesis in male sedentary mice by experimentally depleting satellite cells in young adult animals to a degree sufficient to impair regeneration throughout the rest of their lives. A detailed analysis of multiple muscles harvested at various time points during aging in different cohorts of these mice showed that the muscles were of normal size, despite low regenerative capacity, but did have increased fibrosis. These results suggest that lifelong reduction of satellite cells neither accelerated nor exacerbated sarcopenia and that satellite cells did not contribute to the maintenance of muscle size or fiber type composition during aging, but that their loss may contribute to age-related muscle fibrosis.
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We investigated architectural, functional, and molecular responses of human skeletal muscle to concentric (CON) or eccentric (ECC) resistance training (RT). Twelve young males performed 10 weeks of concentric (CON) or eccentric (ECC) resistance training (RT) (n = 6 CON, 6 ECC). An additional 14 males were recruited to evaluate acute muscle fascicle behaviour and molecular signalling in biopsies collected from vastus lateralis (VL) after 30 min of single bouts of CON or ECC exercise. VL volume was measured by magnetic resonance imaging. Muscle architecture (fascicle length, Lf; pennation angle, PA) was evaluated by ultrasonography. Muscle remodelling signals to CON or ECC loading (MAPK/AKT-mammalian target of rapamycin (mTOR) signalling) and inflammatory pathway (TNFα/Murf-1-MAFbx) were evaluated by immunoblotting. Despite the ~1.2 fold greater load of the ECC group, similar increases in muscle volume (+8% CON and +6% ECC) and in maximal voluntary isometric contraction (+9% CON and +11% ECC) were found after RT. However, increases in Lf were greater after ECC than CON (+12 vs. +5%) while increases in PA were greater in CON than ECC (+30 vs. +5%). Distinct architectural adaptations were associated with preferential growth in the distal regions of VL for ECC (+ECC +8% vs. +CON +2) and mid-belly for CON (ECC +7 vs. CON +11%). While MAPK activation (p38MAPK, ERK1/2, p90RSK) was specific to ECC, neither mode affected AKT-mTOR or inflammatory signalling 30 min after exercise. Muscle growth with CON and ECC RT occurs with different morphological adaptations reflecting distinct fibre fascicle behaviour and molecular responses. This article is protected by copyright. All rights reserved.
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Quantification of muscle protein synthesis (MPS) remains a cornerstone to understanding the control of muscle mass. Traditional (13)C-amino-acid tracer methodologies necessitate sustained bed-rest and intravenous cannulation(s), restricting studies to ~12h, and thus cannot holistically inform on diurnal MPS. This limits insight into the regulation of habitual muscle metabolism in health, ageing and disease while querying the utility of tracer-techniques to predict the long-term efficacy of anabolic/anti-catabolic interventions. We tested the efficacy of the D2O tracer for quantifying MPS over a period not feasible with (13)C tracers and too short to quantify changes in mass. Eight men (22±3.5y) undertook one-legged resistance-exercise over 8-d (4×8-10 repetitions: 80%-1RM every second-day, to yield 'non-exercised' vs. 'exercise' leg-comparisons) with Vastus Lateralis biopsies taken bi-laterally: 0, 2, 4 and 8-days. After day-0 biopsies, participants consumed a D2O bolus (150ml; 70-Atom%); saliva was collected daily. Fractional synthetic rates (FSR) of myofibrillar (MyoPS), sarcoplasmic (SPS) and collagen (CPS) protein-fractions were measured by GC-Pyrolysis-IRMS and TC/EA-IRMS. Body-water initially enriched at 0.16-0.24 APE, decayed at ~0.009%.d-1. In the non-exercised-leg, MyoPS was: 1.45±0.10%.d-1, 1.47±0.06%.d(-1), 1.35±0.07%.d-1 at 0-2, 0-4 and 0-8d respectively (~0.05-0.06%.h-1). MyoPS was greater in the exercised-leg (0-2d 1.97±0.13%.d(-1), 0-4d 1.96±0.15%.d-1; P<0.01, 0-8d 1.79±0.12%.d(-1); P<0.05). CPS was slower than MyoPS, but followed a similar pattern, with the exercised-leg tending to yield greater FSR's (0-2d; 1.14±0.13%.d-1 vs. 1.45±0.15%.d(-1), 0-4d; 1.13±0.07%.d(-1) vs. 1.47±0.18%.d(-1), 0-8d; 1.03±0.09%.d(-1) vs. 1.40±0.11%.d(-1)). SPS remained unchanged. Therefore, D2O has unrivaled utility to quantify day-to-day MPS in humans and inform on short-term changes in anabolism, and presumably, catabolism alike.
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Our aim in the current study was to determine the necessity of satellite cells for long-term muscle growth and maintenance. We utilized a transgenic Pax7-DTA mouse model, allowing for the conditional depletion of > 90% of satellite cells with tamoxifen treatment. Synergist ablation surgery, where removal of synergist muscles places functional overload on the plantaris, was used to stimulate robust hypertrophy. Following 8 wk of overload, satellite cell-depleted muscle demonstrated an accumulation of extracellular matrix (ECM) and fibroblast expansion that resulted in reduced specific force of the plantaris. Although the early growth response was normal, an attenuation of hypertrophy measured by both muscle wet weight and fiber cross-sectional area occurred in satellite cell-depleted muscle. Isolated primary myogenic progenitor cells (MPCs) negatively regulated fibroblast ECM mRNA expression in vitro, suggesting a novel role for activated satellite cells/MPCs in muscle adaptation. These results provide evidence that satellite cells regulate the muscle environment during growth.-Fry, C. S., Lee, J. D., Jackson, J. R., Kirby, T. J., Stasko, S. A., Liu, H., Dupont-Versteegden, E. E., McCarthy, J. J., Peterson, C. A. Regulation of the muscle fiber microenvironment by activated satellite cells during hypertrophy.
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Nutrient timing is a popular nutritional strategy involves the consumption of combinations of nutrients--primarily protein and carbohydrate--in and around an exercise session. Some have claimed that this approach can produce dramatic improvements in body composition. It has even been postulated that the timing of nutritional consumption may be more important than the absolute daily intake of nutrients. The post-exercise period is widely considered the most critical part of nutrient timing. Theoretically, consuming the proper ratio of nutrients during this time not only initiates the rebuilding of damaged muscle tissue and restoration of energy reserves, but it does so in a supercompensated fashion that enhances both body composition and exercise performance. Several researchers have made reference to an anabolic "window of opportunity" whereby a limited time exists after training to optimize training-related muscular adaptations. However, the importance - and even the existence - of a post-exercise 'window' can vary according to a number of factors. Not only is nutrient timing research open to question in terms of applicability, but recent evidence has directly challenged the classical view of the relevance of post-exercise nutritional intake with respect to anabolism. Therefore, the purpose of this paper will be twofold: 1) to review the existing literature on the effects of nutrient timing with respect to post-exercise muscular adaptations, and; 2) to draw relevant conclusions that allow practical, evidence-based nutritional recommendations to be made for maximizing the anabolic response to exercise.
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Resident muscle stem cells, known as satellite cells, are thought to be the main mediators of skeletal muscle plasticity. Satellite cells are activated, replicate, and fuse into existing muscle fibers in response to both muscle injury and mechanical load. It is generally well-accepted that satellite cells participate in postnatal growth, hypertrophy, and muscle regeneration following injury; however, their role in muscle regrowth following an atrophic stimulus remains equivocal. The current study employed a genetic mouse model (Pax7-DTA) that allowed for the effective depletion of >90% of satellite cells in adult muscle upon the administration of tamoxifen. Vehicle and tamoxifen-treated young adult female mice were either hindlimb suspended for 14 days to induce muscle atrophy or hindlimb suspended for 14 days followed by 14 days of reloading to allow regrowth, or they remained ambulatory for the duration of the experimental protocol. Additionally, 5-bromo-2'-deoxyuridine (BrdU) was added to the drinking water to track cell proliferation. Soleus muscle atrophy, as measured by whole muscle wet weight, fiber cross-sectional area, and single-fiber width, occurred in response to suspension and did not differ between satellite cell-depleted and control muscles. Furthermore, the depletion of satellite cells did not attenuate muscle mass or force recovery during the 14-day reloading period, suggesting that satellite cells are not required for muscle regrowth. Myonuclear number was not altered during either the suspension or the reloading period in soleus muscle fibers from vehicle-treated or satellite cell-depleted animals. Thus, myonuclear domain size was reduced following suspension due to decreased cytoplasmic volume and was completely restored following reloading, independent of the presence of satellite cells. These results provide convincing evidence that satellite cells are not required for muscle regrowth following atrophy and that, instead, the myonuclear domain size changes as myofibers adapt.
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We explored the relationships between resistance exercise volume/intensity and muscle myofibrillar protein synthetic (MPS) responses in young and older men. In a crossover design, four groups of six young (24±6 years) and older (70±5 years) men performed two volumes of resistance exercise: either 40% one repetition maximum (1RM) (3 × 14, then 6 × 14 repetitions) or 75% 1RM (3 × 8, then 6 × 8 repetitions), such that at the same volume, work was identical between intensities. Muscle biopsies were taken 0, 1, 2, and 4hours after exercise to measure MPS via myofibrillar bound [1,2-13C2]leucine and indices of mammalian target of rapamycin signaling by immunoblotting. In younger men, doubling exercise volume produced limited added effects, whereas in older men, it resulted in greater MPS and p70S6 kinase (p70S6KThr389) phosphorylation at both intensities, that is, MPS area under the curve: 75% (1× volume: 0.07±0.01 vs 2× volume: 0.14% ± 0.02% protein synthesized/4hours (p < .001). Doubling exercise volume is a valid strategy to maximize postexercise MPS in ageing.
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We have reported that the acute postexercise increases in muscle protein synthesis rates, with differing nutritional support, are predictive of longer-term training-induced muscle hypertrophy. Here, we aimed to test whether the same was true with acute exercise-mediated changes in muscle protein synthesis. Eighteen men (21 ± 1 yr, 22.6 ± 2.1 kg/m(2); means ± SE) had their legs randomly assigned to two of three training conditions that differed in contraction intensity [% of maximal strength (1 repetition maximum)] or contraction volume (1 or 3 sets of repetitions): 30%-3, 80%-1, and 80%-3. Subjects trained each leg with their assigned regime for a period of 10 wk, 3 times/wk. We made pre- and posttraining measures of strength, muscle volume by magnetic resonance (MR) scans, as well as pre- and posttraining biopsies of the vastus lateralis, and a single postexercise (1 h) biopsy following the first bout of exercise, to measure signaling proteins. Training-induced increases in MR-measured muscle volume were significant (P < 0.01), with no difference between groups: 30%-3 = 6.8 ± 1.8%, 80%-1 = 3.2 ± 0.8%, and 80%-3= 7.2 ± 1.9%, P = 0.18. Isotonic maximal strength gains were not different between 80%-1 and 80%-3, but were greater than 30%-3 (P = 0.04), whereas training-induced isometric strength gains were significant but not different between conditions (P = 0.92). Biopsies taken 1 h following the initial resistance exercise bout showed increased phosphorylation (P < 0.05) of p70S6K only in the 80%-1 and 80%-3 conditions. There was no correlation between phosphorylation of any signaling protein and hypertrophy. In accordance with our previous acute measurements of muscle protein synthetic rates a lower load lifted to failure resulted in similar hypertrophy as a heavy load lifted to failure.
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The purpose of this study was to investigate associations between acute exercise-induced hormone responses and adaptations to high intensity resistance training in a large cohort (n = 56) of young men. Acute post-exercise serum growth hormone (GH), free testosterone (fT), insulin-like growth factor (IGF-1) and cortisol responses were determined following an acute intense leg resistance exercise routine at the midpoint of a 12-week resistance exercise training study. Acute hormonal responses were correlated with gains in lean body mass (LBM), muscle fibre cross-sectional area (CSA) and leg press strength. There were no significant correlations between the exercise-induced elevations (area under the curve—AUC) of GH, fT and IGF-1 and gains in LBM or leg press strength. Significant correlations were found for cortisol, usually assumed to be a hormone indicative of catabolic drive, AUC with change in LBM (r = 0.29, P < 0.05) and type II fibre CSA (r = 0.35, P < 0.01) as well as GH AUC and gain in fibre area (type I: r = 0.36, P = 0.006; type II: r = 0.28, P = 0.04, but not lean mass). No correlations with strength were observed. We report that the acute exercise-induced systemic hormonal responses of cortisol and GH are weakly correlated with resistance training-induced changes in fibre CSA and LBM (cortisol only), but not with changes in strength. Electronic supplementary material The online version of this article (doi:10.1007/s00421-011-2246-z) contains supplementary material, which is available to authorized users.
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Chronic mechanical loading (CML) of skeletal muscle induces compensatory growth and the drug rapamycin has been reported to block this effect. Since rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), many have concluded that mTOR plays a key role in CML-induced growth regulatory events. However, rapamycin can exert mTOR-independent actions and systemic administration of rapamycin will inhibit mTOR signalling in all cells throughout the body. Thus, it is not clear if the growth inhibitory effects of rapamycin are actually due to the inhibition of mTOR signalling, and more specifically, the inhibition of mTOR signalling in skeletal muscle cells. To address this issue, transgenic mice with muscle specific expression of various rapamycin-resistant mutants of mTOR were employed. These mice enabled us to demonstrate that mTOR, within skeletal muscle cells, is the rapamycin-sensitive element that confers CML-induced hypertrophy, and mTOR kinase activity is necessary for this event. Surprisingly, CML also induced hyperplasia, but this occurred through a rapamycin-insensitive mechanism. Furthermore, CML was found to induce an increase in FoxO1 expression and PKB phosphorylation through a mechanism that was at least partially regulated by an mTOR kinase-dependent mechanism. Finally, CML stimulated ribosomal RNA accumulation and rapamycin partially inhibited this effect; however, the effect of rapamycin was exerted through a mechanism that was independent of mTOR in skeletal muscle cells. Overall, these results demonstrate that CML activates several growth regulatory events, but only a few (e.g. hypertrophy) are fully dependent on mTOR signalling within the skeletal muscle cells.
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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, Ca(2+) 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.
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Skeletal muscle tissue provides mechanical force for locomotion of all vertebrate animals. It is prone to damage from acute physical trauma and physiological stress. To cope with this, it possesses a tremendous capacity for rapid and effective repair that is widely held to be accomplished by the satellite cells lying between the muscle fiber plasmalemma and the basement membrane. Cell transplantation and lineage-tracing studies have demonstrated that Pax7-expressing (Pax7(+)) satellite cells can repair damaged muscle tissue repeatedly after several bouts of acute injury. These findings provided evidence that Pax7(+) cells are muscle stem cells. However, stem cells from a variety of other origins are also reported to contribute to myofibers upon engraftment into muscles, questioning whether satellite cells are the only stem cell source for muscle regeneration. Here, we have engineered genetic ablation of Pax7(+) cells to test whether there is any significant contribution to muscle regeneration after acute injury from cells other than this source. We find that such elimination of Pax7(+) cells completely blocks regenerative myogenesis either following injury to the tibialis anterior (TA) muscle or after transplantation of extensor digitorum longus (EDL) muscles into nude mice. As Pax7 is specifically expressed in satellite cells, we conclude that they are essential for acute injury-induced muscle regeneration. It remains to be established whether there is any significant role for stem cells of other origins. The implications of our results for muscle stem cell-based therapy are discussed.
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We applied K-means cluster analysis to test the hypothesis that muscle-specific factors known to modulate protein synthesis and satellite cell activity would be differentially expressed during progressive resistance training (PRT, 16 wk) in 66 human subjects experiencing extreme, modest, and failed myofiber hypertrophy. Muscle mRNA expression of IGF-I isoform Ea (IGF-IEa), mechanogrowth factor (MGF, IGF-IEc), myogenin, and MyoD were assessed in muscle biopsies collected at baseline (T1) and 24 h after the first (T2) and last (T3) loading bouts from previously untrained subjects clustered as extreme responders (Xtr, n=17), modest responders (Mod, n=32), and nonresponders (Non, n=17) based on mean myofiber hypertrophy. Myofiber growth averaged 2,475 microm2 in Xtr, 1,111 microm2 in Mod, and -16 microm2 in Non. Main training effects revealed increases in all transcripts (46-83%, P<0.005). For the entire cohort, IGF-IEa, MGF, and myogenin mRNAs were upregulated by T2 (P<0.05), while MyoD did not increase significantly until T3 (P<0.001). Within clusters, MGF and myogenin upregulation was robust in Xtr (126% and 65%) and Mod (73% and 41%) vs. no changes in Non. While significant in all clusters by T3, IGF-IEa increased most in Xtr (105%) and least in Non (44%). Although MyoD expression increased overall, no changes within clusters were detected. We reveal for the first time that MGF and myogenin transcripts are differentially expressed in subjects experiencing varying degrees of PRT-mediated myofiber hypertrophy. The data strongly suggest the load-mediated induction of these genes may initiate important actions necessary to promote myofiber growth during PRT, while the role of MyoD is less clear.
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Consuming protein following exercise has been shown to stimulate protein synthesis acutely in skeletal muscle and has been recommended to prevent sarcopenia. It is not known, however, whether acute stimulation persists long term or includes muscle cell division. We asked here whether consuming protein following exercise during aerobic training increases long-term protein and DNA synthesis rates in skeletal muscle of adult humans. Sixteen previously untrained participants (50 ± 8 yr) consumed either a carbohydrate or carbohydrate and protein drink following each session during 6 wk of treadmill training. A younger untrained group provided a nonexercising comparison. Participants were administered heavy water (²H₂O; deuterium oxide) continuously for 6 wk to isotopically label newly synthesized skeletal muscle proteins and DNA. Muscle biopsies were performed after 6 wk of training. Contrary to acute studies, consuming protein after exercise did not increase skeletal muscle protein synthesis rates. In contrast, muscle protein synthesis, DNA, and phospholipid synthesis were significantly higher in the older exercise groups than the younger sedentary group. The higher DNA replication rate could not be attributed to mitochondrial DNA and may be due to satellite cell activation. We conclude that postexercise protein supplementation does not increase rates of mixed protein synthesis over 6 wk and that aerobic exercise may stimulate long-term cell division (DNA synthesis) in skeletal muscle of humans. Measurements of long-term synthesis rates provide important insights into aging and exercise adaptations.
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Skeletal muscle hypertrophy is typically considered to be a slow process. However, this is partly because the time course for hypertrophy has not been thoroughly examined. The purpose of this study was to use weekly testing to determine a precise time course of skeletal muscle hypertrophy during a resistance training program. Twenty-five healthy, sedentary men performed 8 weeks of high-intensity resistance training. Whole muscle cross-sectional area (CSA) of the dominant thigh was assessed using a peripheral quantitative computed tomography scanner during each week of training (W1-W8). Isometric maximum voluntary contractions (MVC) were also measured each week. After only two training sessions (W1), the mean thigh muscle CSA increased by 5.0 cm(2) (3.46%; p < 0.05) from the pre-testing (P1) and continued to increase with each testing session. It is possible that muscular edema may have influenced the early CSA results. To adjust for this possibility, with edema assumedly at its highest at W1, the next significant increase from W1 was at W3. W4 was the first significant increase of MVC over P1. Therefore, significant skeletal muscle hypertrophy likely occurred around weeks 3-4. Overall, from the pre-testing to W8, there was an increase of 13.9 cm(2) (9.60%). These findings suggested that training-induced skeletal muscle hypertrophy may occur early in a training program.
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The mammalian target of rapamycin complex 1 (mTORC1) functions as a central integrator of a wide range of signals that modulate protein metabolism and cell growth. However, the contributions of individual pathways regulating mTORC1 activity in skeletal muscle are poorly defined. The purpose of this study was to determine the regulatory mechanisms that contribute to mTORC1 activation during mechanical overload-induced skeletal muscle hypertrophy. Consistent with previous studies, mechanical overload induced progressive hypertrophy of the plantaris muscle which was associated with significant increases in total RNA content and protein metabolism. mTORC1 was activated after a single day of overload as indicated by a significant increase in S6K1 phosphorylation at T389 and T421/S424. In contrast, Akt activity, as assessed by Akt phosphorylation status (T308 and S473), phosphorylation of direct downstream targets (glycogen synthase kinase 3 β, proline-rich Akt substrate 40 kDa and tuberous sclerosis 2 (TSC2)) and a kinase assay, was not significantly increased until 2–3 days of overload. Inhibition of phosphoinositide 3-kinase (PI3K) activity by wortmannin was sufficient to block insulin-dependent signalling but did not prevent the early activation of mTORC1 in response to overload. We identified that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)-dependent pathway was activated at day 1 after overload. In addition, a target of MEK/ERK signalling, phosphorylation of TSC2 at S664, was also increased at this early time point. These observations demonstrate that in vivo, mTORC1 activation at the early phase of mechanical overload in skeletal muscle occurs independently of PI3K/Akt signalling and provide evidence that the MEK/ERK pathway may contribute to mTORC1 activation through phosphorylation of TSC2.
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We previously showed that human muscle protein synthesis (MPS) increased during infusion of amino acids (AAs) and peaked at ≈120 min before returning to baseline rates, despite elevated plasma AA concentrations. We tested whether a protein meal elicited a similar response and whether signaling responses that regulate messenger RNA translation matched MPS changes. Eight postabsorptive healthy men (≈21 y of age) were studied during 8.5 h of primed continuous infusion of [1,2-¹³C₂]leucine with intermittent quadriceps biopsies for determination of MPS and anabolic signaling. After 2.5 h, subjects consumed 48 g whey protein. At 45-90 min after oral protein bolus, mean (± SEM) myofibrillar protein synthesis increased from 0.03 ± 0.003% to 0.10 ± 0.01%/h; thereafter, myofibrillar protein synthesis returned to baseline rates even though plasma essential AA (EAA) concentrations remained elevated (+130% at 120 min, +80% at 180 min). The activity of protein kinase B (PKB) and phosphorylation of eukaryotic initiation factor 4G preceded the rise of MPS and increases in phosphorylation of ribosomal protein kinase S6 (S6K1), and 4E-binding protein 1 (4EBP1) was superimposable with MPS responses until 90 min. However, although MPS decreased thereafter, all signals, with the exception of PKB activity (which mirrored insulin responses), remained elevated, which echoed the slowly declining plasma EAA profile. The phosphorylation of eukaryotic initiation factor 2α increased only at 180 min. Thus, discordance existed between MPS and the mammalian target of rapamycin complex 1 (mTORC1) and signaling (ie, S6K1 and 4EBP1 phosphorylation). We confirm our previous findings that MPS responses to AAs are transient, even with oral protein bolus. However, changes in MPS only reflect elevated mTORC1 signaling during the upswing in MPS.
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Adult skeletal muscle fibers are among the few cell types that are truly multinucleated. Recently, evidence has accumulated supporting a role for the modulation of myonuclear number during muscle remodeling in response to injury, adaptation, and disease. These studies have demonstrated that muscle hypertrophy is associated with, and is dependent on, the addition of newly formed myonuclei via the fusion of myogenic cells to the adult myofiber, whereas muscle atrophy and disease appear to be associated with the loss of myonuclei, possibly through apoptotic‐like mechanisms. Moreover, these studies also have demonstrated that myonuclear domain size, i.e., the amount of cytoplasm per myonucleus, is unchanged following the acute phase of hypertrophy but is reduced following atrophy. Together these data demonstrate that modulation of myonuclear number or myonuclear domain size (or both) is a mechanism contributing to the remodeling of adult skeletal muscle in response to alterations in the level of normal neuromuscular activity. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 1350–1360, 1999
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Current methods to quantify RNA dynamics are beset with limitations. Here, we developed a novel stable isotope (D2O)-methodology to quantify RNA synthesis (i.e. ribosomal biogenesis) in cells, animal models, and humans. Firstly, proliferating C2C12 cells were incubated in D2O-enriched media and myotubes (±) 50ng.ml(-1) IGF-1. Secondly, rat quadriceps [untrained n=9; 7-wks interval-"like" training n=13] were collected after ~3-wks D2O (70-Atom%) administration, with body-water enrichment (BWE) monitored via blood sampling. Finally, 10 (23±1y) men consumed 150ml D2O followed by 50ml/wk and undertook 6-wks resistance-exercise (RE; 6×8 repetitions, 75%-1RM 3/wk) with BWE monitored by saliva sampling and muscle biopsies (for determination of RNA synthesis) 0-3-6-wks. Ribose mole percent excess (r-MPE) from purine nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cells r-MPE exhibited a rise-to-plateau while IGF-1 increased myotube RNA from 76±3ng/ul to 123±3ng/ul and r-MPE by 0.39±0.1% (both P<0.01). After 3-wks, rat quadriceps r-MPE had increased to 0.25±0.01% (P<0.01) and was greater with running-exercise (0.36±0.02%; P<0.01)). Human muscle r-MPE increased to 0.06±0.01% and 0.13±0.02% at 3/6-wks respectively equating to synthesis rates of ~0.8%/d, increasing with RE to 1.7±0.3%/d (P<0.01) and 1.2±0.1%/d (P<0.05) at 3/6-wks, respectively. Therefore, we have developed and physiologically validated a novel technique to explore ribosomal biogenesis in a multi-modal fashion.
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Purpose of review: Methods that inform on dynamic metabolism that can be applied to clinical populations to understand disease progression and responses to therapeutic interventions are of great importance. This review perspective will highlight recent advances, development, and applications of the multivalent stable isotope tracer deuterium oxide (D2O) to the study of substrate metabolism with particular reference to protein, lipids, and nucleic acids, and how these methods can be readily applied within clinical and pharmaceutical research. Recent findings: Advances in the application of D2O techniques now permit the simultaneous dynamic measurement of a range of substrates (i.e. protein, lipid, and nucleic acids, along with the potential for OMICs methodologies) with minimal invasiveness further creating opportunities for long-term 'free living' measures that can be used in clinical settings. These techniques have recently been applied to ageing populations and further in cancer patients revealing altered muscle protein metabolism. Additionally, the efficacy of numerous drugs in improving lipoprotein profiles and controlling cellular proliferation in leukaemia have been revealed. Summary: D2O provides opportunities to create a more holistic picture of in-vivo metabolic phenotypes, providing a unique platform for development in clinical applications, and the emerging field of personalized medicine.
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Stable isotope tracers have been invaluable assets in physiological research for over 80 years. The application of substrate-specific stable isotope tracers has permitted exquisite insight into amino acid, fatty-acid and carbohydrate metabolic regulation (i.e. incorporation, flux, oxidation, and in a tissue-specific and whole-body fashion) in health, disease and in response to acute and chronic exercise. Yet, despite many breakthroughs, there are limitations to "substrate specific" stable isotope tracers, which limit physiological insight e.g. the need for I.V infusions and restriction to short term studies (hours) in controlled laboratory settings. In recent years significant interest has developed in alternative stable isotope tracer techniques that overcome these limitations; in particular deuterium oxide (D2 O or heavy water). The unique properties of this tracer mean that through oral administration, the turnover and flux through a number of different substrates (muscle proteins, lipids, glucose, DNA [satellite cells]) can be monitored simultaneously and flexibly (hours/weeks/months) without the need for restrictive experimental control. This makes it uniquely suited for the study of 'real world' human exercise physiology (amongst many other applications). Moreover, using D2 O permits evaluation of turnover of plasma/muscle proteins (e.g. dynamic proteomics) in addition to metabolomics (e.g. fluxomics) to seek molecular underpinnings e.g. of exercise adaptation. Herein, we provide insight into the role of stable isotope tracers, from substrate-specific to novel D2 O approaches, in facilitating our understanding of metabolism. Further novel potential applications of stable isotope tracers are also discussed in the context of integrating with the snowballing field of OMIC technologies. This article is protected by copyright. All rights reserved.
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Resistance exercise training (RT) is the most effective method for increasing skeletal muscle mass in older adults, however, the amount of RT-induced muscle growth is highly variable between individuals. Recent evidence from our lab and others suggests ribosome biogenesis may be an important factor regulating RT-induced hypertrophy, and we hypothesized that the extent of hypertrophy is at least partly regulated by the amount of RT-induced ribosome biogenesis. To examine this, forty-two older adults underwent 4 wk of RT aimed at inducing hypertrophy of the knee extensors (e.g. 2 sets of squat, leg press, and knee extension, 10-12RM, 3d/wk), and vastus lateralis muscle biopsies were performed pre- and post-RT. Post hoc K-means cluster analysis revealed distinct differences in Type II myofiber hypertrophy among subjects. The percent change in Type II myofiber size in non-responders (Non; n=17) was -7%, moderate responders (Mod; n=19) +22%, and extreme responders (Xtr; n=6) +83%. Total muscle RNA increased only in Mod (+9%, p<0.08) and Xtr (+26%, p<0.01), and only Xtr increased rRNA content (+40%, p<0.05) and myonuclei/type II fiber (+32%, p<0.01). Additionally, Mod and Xtr had a greater increase in c-Myc protein levels when compared to Non (e.g. ≈+350% and ≈+250% vs. +≈50%, respectively; p<0.05). In vitro studies showed that growth factor-induced human myotube hypertrophy is abolished when rRNA synthesis is knocked down using the Pol I-specific inhibitor, CX-5461. Overall, these data implicate ribosome biogenesis as a key process regulating the extent of RT-induced myofiber hypertrophy in older adults.
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Key points: Ribosome biogenesis is the primary determinant of translational capacity, but its regulation in skeletal muscle following acute resistance exercise is poorly understood. Resistance exercise increases muscle protein synthesis acutely, and muscle mass with training, but the role of translational capacity in these processes is unclear. Here, we show that acute resistance exercise activated pathways controlling translational activity and capacity through both rapamycin-sensitive and -insensitive mechanisms. Transcription factor c-Myc and its downstream targets, which are known to regulate ribosome biogenesis in other cell types, were upregulated after resistance exercise in a rapamycin-independent manner and may play a role in determining translational capacity in skeletal muscle. Local inhibition of myostatin was also not affected by rapamycin and may contribute to the rapamycin-independent effects of resistance exercise. Abstract: This study aimed to determine (1) the effect of acute resistance exercise on mechanisms of ribosome biogenesis, and (2) the impact of mammalian target of rapamycin on ribosome biogenesis, and muscle protein synthesis (MPS) and degradation. Female F344BN rats underwent unilateral electrical stimulation of the sciatic nerve to mimic resistance exercise in the tibialis anterior (TA) muscle. TA muscles were collected at intervals over the 36 h of exercise recovery (REx); separate groups of animals were administered rapamycin pre-exercise (REx+Rapamycin). Resistance exercise led to a prolonged (6-36 h) elevation (30-50%) of MPS that was fully blocked by rapamycin at 6 h but only partially at 18 h. REx also altered pathways that regulate protein homeostasis and mRNA translation in a manner that was both rapamycin-sensitive (proteasome activity; phosphorylation of S6K1 and rpS6) and rapamycin-insensitive (phosphorylation of eEF2, ERK1/2 and UBF; gene expression of the myostatin target Mighty as well as c-Myc and its targets involved in ribosome biogenesis). The role of c-Myc was tested in vitro using the inhibitor 10058-F4, which, over time, decreased basal RNA and MPS in a dose-dependent manner (correlation of RNA and MPS, r(2) = 0.98), even though it had no effect on the acute stimulation of protein synthesis. In conclusion, acute resistance exercise stimulated rapamycin-sensitive and -insensitive mechanisms that regulate translation activity and capacity.
Article
Constituting ∼40% of body mass, skeletal muscle has essential locomotory and metabolic functions. As such, an insight into the control of muscle mass is of great importance for maintaining health and quality-of-life into older age, under conditions of cachectic disease and with rehabilitation. In healthy weight-bearing individuals, muscle mass is maintained by the equilibrium between muscle protein synthesis (MPS) and muscle protein breakdown; when this balance tips in favour of MPS hypertrophy occurs. Despite considerable research into pharmacological/nutraceutical interventions, resistance exercise training (RE-T) remains the most potent stimulator of MPS and hypertrophy (in the majority of individuals). However, the mechanism(s) and time course of hypertrophic responses to RE-T remain poorly understood. We would suggest that available data are very much in favour of the notion that the majority of hypertrophy occurs in the early phases of RE-T (though still controversial to some) and that, for the most part, continued gains are hard to come by. Whilst the mechanisms of muscle hypertrophy represent the culmination of mechanical, auto/paracrine and endocrine events, the measurement of MPS remains a cornerstone for understanding the control of hypertrophy - mainly because it is the underlying driving force behind skeletal muscle hypertrophy. Development of sophisticated isotopic techniques (i.e. deuterium oxide) that lend to longer term insight into the control of hypertrophy by sustained RE-T will be paramount in providing insights into the metabolic and temporal regulation of hypertrophy. Such technologies will have broad application in muscle mass intervention for both athletes and for mitigating disease/age-related cachexia and sarcopenia, alike.
Article
The hypertrophic response to resistance training is generally attenuated with aging; yet the mechanisms regulating this phenomenon are largely unknown. Several studies to date have shown blunted translational efficiency following acute resistance exercise in older adults; however, the effects on translational capacity (i.e. ribosome biogenesis) have not yet been examined. Thus, the purpose of this study was to examine changes in markers of ribosome biogenesis following an acute bout of resistance loading (RL; 9 sets x 10 repetitions of knee extensions) in younger (Y; n = 14; 39.2 ± 4.1y) and older (O; n = 12; 75.7 ± 5.7y) adults. Vastus lateralis biopsies were taken pre- and 24h post-RL, and muscle samples were analyzed for total RNA content, 45S pre-rRNA expression, ribosomal protein content, and levels of signaling proteins that regulate ribosome biogenesis. Prior to RL, O had higher total RNA content (+28%; p < 0.05), a trend toward higher 45S pre-rRNA expression (+59%; p = 0.08), and greater protein content of several ribosomal components (≈ +50-80%; p < 0.05) than Y. However, 24h post-RL, only Y increased 45S pre-rRNA expression (+34%; p< 0.01), possibly driven by higher basal p-Rb (Ser780) (+61%; p = 0.10), and a robust TIF-1a response (+75%; p < 0.05). RL tended to increase protein components of the 40S ribosomal subunit in Y only (≈ +20-25%; p ≤ 0.12). Overall, the data suggest blunted ribosome biogenesis in response to RL in O, which may be a potential mechanism driving the age-related attenuation of resistance training-induced hypertrophy. Copyright © 2015, Journal of Applied Physiology.
Article
The effects of denervation were investigated in mice with transgenes containing promoter elements from the muscle acetylcholine receptor epsilon- and alpha-subunit genes. The promoter sequences were coupled to a nuclear localization signal-beta-galactosidase fusion gene (nlacZ) as a reporter. While many postsynaptic specializations form in the embryo, expression of the epsilon subunit is induced during the first two postnatal weeks. When muscles were denervated at birth, before the onset of epsilon expression, epsilon nlacZ still appeared at the former synaptic sites on schedule. This result suggests that the nerve leaves a localized "trace" in the muscle that can continue to regulate transcription. An additional finding was that epsilon nlacZ expression was much stronger in denervated than in intact muscles. This suggests that the epsilon promoter is similar to the other subunits in containing elements that are activated on cessation of neural activity. However, even after denervation, epsilon nlacZ expression was always confined to the synaptic region whereas alpha nlacZ expression increased in nuclei along the entire length of the fiber. This suggests that while the epsilon gene is similar in its activity dependence to other subunit genes, it is unique in that local nerve-derived signals are essential for its expression. Consequently, inactivity enhances epsilon expression only in synaptic nuclei where such signals are present, but enhances expression throughout the muscle fiber. Truncations and an internal deletion of the epsilon promoter indicate that cis-elements essential for the response to synaptic signals are contained within 280 bp of the transcription start site. In contrast to these results in young animals, denervation in older animals leads to an unexpected reduction in nlacZ activity. However, mRNA measurements indicated that transgene expression was increased in these animals. This discordance between nlacZ mRNA and enzyme activity, demonstrates a previously unknown limitation of nlacZ as a reporter gene in transgenic animals.
Article
Maintaining proteostasis is thought to be a key factor in slowed aging. In several growth-restricted models of long-life, we have shown evidence of increased proteostatic mechanisms, suggesting that proteostasis may be a shared characteristic of slowed aging. The Snell dwarf mouse is generated through the mutation of the Pit-1 locus causing reductions in multiple hormonal growth factors and mTORC1 signaling. Snell dwarfs are one of the longest lived rodent models of slowed aging. We hypothesized that proteostatic mechanisms would be increased in Snell compared to control (Con) as in other models of slowed aging. Using D2 O, we simultaneously assessed protein synthesis in multiple subcellular fractions along with DNA synthesis in skeletal muscle, heart, and liver over 2 weeks in both sexes. We also assessed mTORC1-substrate phosphorylation. Skeletal muscle protein synthesis was decreased in all protein fractions of Snell compared to Con, varied by fraction in heart, and was not different between groups in liver. DNA synthesis was lower in Snell skeletal muscle and heart but not in liver when compared to Con. The new protein to new DNA synthesis ratio was increased threefold in Snell skeletal muscle and heart compared to Con. Snell mTORC1-substrate phosphorylation was decreased only in heart and liver. No effect of sex was seen in this study. Together with our previous investigations in long-lived models, we provide evidence further supporting proteostasis as a shared characteristic of slowed aging and show that increased proteostatic mechanisms may not necessarily require a decrease in mTORC1. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
Article
The ribosome is a supramolecular ribonucleoprotein complex that functions at the heart of the translation machinery to convert mRNA into protein. Ribosome biogenesis is the primary determinant of translational capacity of the cell and accordingly has an essential role in the control of cell growth in eukaryotes. Cumulative evidence supports the hypothesis that ribosome biogenesis has an important role in the regulation of skeletal muscle mass. The purpose of this review is to, first, summarize the main mechanisms known to regulate ribosome biogenesis and, second, put forth the hypothesis that ribosome biogenesis is a central mechanism used by skeletal muscle to regulate protein synthesis and control skeletal muscle mass in response to anabolic and catabolic stimuli. The mTORC1 and Wnt/β-catenin/c-myc signaling pathways are discussed as the major pathways that work in concert with each of the three RNA polymerases (RNA Pol I, II and III) in regulating ribosome biogenesis. Consistent with our hypothesis, activation of these two pathways has been shown to be associated with ribosome biogenesis during skeletal muscle hypertrophy. Although further study is required, the finding that ribosome biogenesis is altered under catabolic states, in particular during disuse atrophy, suggests that its activation represents a novel therapeutic target to reduce or prevent muscle atrophy. Lastly, the emerging field of ribosome specialization is discussed and its potential role in the regulation of gene expression during periods of skeletal muscle plasticity.Contract grant sponsor : National Institues of Health Contract grant number : AR061939 J. Cell. Physiol. © 2014 Wiley Periodicals, Inc.
Article
The intake of whey, compared with casein and soy protein intakes, stimulates a greater acute response of muscle protein synthesis (MPS) to protein ingestion in rested and exercised muscle. We characterized the dose-response relation of postabsorptive rates of myofibrillar MPS to increasing amounts of whey protein at rest and after exercise in resistance-trained, young men. Volunteers (n = 48) consumed a standardized, high-protein (0.54 g/kg body mass) breakfast. Three hours later, a bout of unilateral exercise (8 × 10 leg presses and leg extensions; 80% one-repetition maximum) was performed. Volunteers ingested 0, 10, 20, or 40 g whey protein isolate immediately (∼10 min) after exercise. Postabsorptive rates of myofibrillar MPS and whole-body rates of phenylalanine oxidation and urea production were measured over a 4-h postdrink period by continuous tracer infusion of labeled [(13)C6] phenylalanine and [(15)N2] urea. Myofibrillar MPS (±SD) increased (P < 0.05) above 0 g whey protein (0.041 ± 0.015%/h) by 49% and 56% with the ingestion of 20 and 40 g whey protein, respectively, whereas no additional stimulation was observed with 10 g whey protein (P > 0.05). Rates of phenylalanine oxidation and urea production increased with the ingestion of 40 g whey protein. A 20-g dose of whey protein is sufficient for the maximal stimulation of postabsorptive rates of myofibrillar MPS in rested and exercised muscle of ∼80-kg resistance-trained, young men. A dose of whey protein >20 g stimulates amino acid oxidation and ureagenesis. This trial was registered at http://www.isrctn.org/ as ISRCTN92528122.
Article
Physical activity and molecular ageing presumably interact to precipitate musculoskeletal decline in humans with age. Herein, we have delineated molecular networks for these two major components of sarcopenic risk using multiple independent clinical cohorts. We generated genome-wide transcript profiles from individuals (n = 44) who then undertook 20 weeks of supervised resistance-exercise training (RET). Expectedly, our subjects exhibited a marked range of hypertrophic responses (3% to +28%), and when applying Ingenuity Pathway Analysis (IPA) up-stream analysis to ∼580 genes that co-varied with gain in lean mass, we identified rapamycin (mTOR) signaling associating with growth (P = 1.4×10(-30)). Paradoxically, those displaying most hypertrophy exhibited an inhibited mTOR activation signature, including the striking down-regulation of 70 rRNAs. Differential analysis found networks mimicking developmental processes (activated all-trans-retinoic acid (ATRA, Z-score = 4.5; P = 6×10(-13)) and inhibited aryl-hydrocarbon receptor signaling (AhR, Z-score = -2.3; P = 3×10(-7))) with RET. Intriguingly, as ATRA and AhR gene-sets were also a feature of endurance exercise training (EET), they appear to represent "generic" physical activity responsive gene-networks. For age, we found that differential gene-expression methods do not produce consistent molecular differences between young versus old individuals. Instead, utilizing two independent cohorts (n = 45 and n = 52), with a continuum of subject ages (18-78 y), the first reproducible set of age-related transcripts in human muscle was identified. This analysis identified ∼500 genes highly enriched in post-transcriptional processes (P = 1×10(-6)) and with negligible links to the aforementioned generic exercise regulated gene-sets and some overlap with ribosomal genes. The RNA signatures from multiple compounds all targeting serotonin, DNA topoisomerase antagonism, and RXR activation were significantly related to the muscle age-related genes. Finally, a number of specific chromosomal loci, including 1q12 and 13q21, contributed by more than chance to the age-related gene list (P = 0.01-0.005), implying possible epigenetic events. We conclude that human muscle age-related molecular processes appear distinct from the processes regulated by those of physical activity.
Article
In response to muscle injury, satellite cells activate the p38α/β MAPK pathway to exit quiescence, then proliferate, repair skeletal muscle, and self-renew, replenishing the quiescent satellite cell pool. Although satellite cells are capable of asymmetric division, the mechanisms regulating satellite cell self-renewal are not understood. We found that satellite cells, once activated, enter the cell cycle and a subset undergoes asymmetric division, renewing the satellite cell pool. Asymmetric localization of the Par complex activates p38α/β MAPK in only one daughter cell, inducing MyoD, which permits cell cycle entry and generates a proliferating myoblast. The absence of p38α/β MAPK signaling in the other daughter cell prevents MyoD induction, renewing the quiescent satellite cell. Thus, satellite cells employ a mechanism to generate distinct daughter cells, coupling the Par complex and p38α/β MAPK signaling to link the response to muscle injury with satellite cell self-renewal.
Article
Adult skeletal muscle fibers are among the few cell types that are truly multinucleated. Recently, evidence has accumulated supporting a role for the modulation of myonuclear number during muscle remodeling in response to injury, adaptation, and disease. These studies have demonstrated that muscle hypertrophy is associated with, and is dependent on, the addition of newly formed myonuclei via the fusion of myogenic cells to the adult myofiber, whereas muscle atrophy and disease appear to be associated with the loss of myonuclei, possibly through apoptotic-like mechanisms. Moreover, these studies also have demonstrated that myonuclear domain size, i.e., the amount of cytoplasm per myonucleus, is unchanged following the acute phase of hypertrophy but is reduced following atrophy. Together these data demonstrate that modulation of myonuclear number or myonuclear domain size (or both) is a mechanism contributing to the remodeling of adult skeletal muscle in response to alterations in the level of normal neuromuscular activity. © 1999 John Wiley & Sons, Inc. Muscle Nerve 22: 1350–1360, 1999
Article
Muscle protein synthesis (MPS) is the driving force behind adaptive responses to exercise and represents a widely adopted proxy for gauging chronic efficacy of acute interventions, (i.e. exercise/nutrition). Recent findings in this arena have been progressive. Nutrient-driven increases in MPS are of finite duration (∼1.5 h), switching off thereafter despite sustained amino acid availability and intramuscular anabolic signalling. Intriguingly, this 'muscle-full set-point' is delayed by resistance exercise (RE) (i.e. the feeding × exercise combination is 'more anabolic' than nutrition alone) even 24 h beyond a single exercise bout, casting doubt on the importance of nutrient timing vs. sufficiency per se. Studies manipulating exercise intensity/workload have shown that increases in MPS are negligible with RE at 20-40% but maximal at 70-90% of one-repetition maximum when workload is matched (according to load × repetition number). However, low-intensity exercise performed to failure equalises this response. Analysing distinct subcellular fractions (e.g. myofibrillar, sarcoplasmic, mitochondrial) may provide a readout of chronic exercise efficacy in addition to effect size in MPS per se, i.e. while 'mixed' MPS increases similarly with endurance and RE, increases in myofibrillar MPS are specific to RE, prophetic of adaptation (i.e. hypertrophy). Finally, the molecular regulation of MPS by exercise and its regulation via 'anabolic' hormones (e.g. IGF-1) has been questioned, leading to discovery of alternative mechanosensing-signalling to MPS.
Article
The main goal of the present study was to investigate the regulation of ribosomal DNA (rDNA) gene transcription at the onset of skeletal muscle hypertrophy. Mice were subjected to functional overload of the plantaris by bilateral removal of the synergist muscles. Mechanical loading resulted in muscle hypertrophy with an increase in rRNA content. rDNA transcription, as determined by 45S pre-rRNA abundance, paralleled the increase in rRNA content and was consistent with the onset of the hypertrophic response. Increased transcription and protein expression of c-Myc and its downstream polymerase I (Pol I) regulon (POL1RB, TIF-1A, PAF53, TTF1, TAF1C) was also consistent with the increase in rRNA. Similarly, factors involved in rDNA transcription, such as the upstream binding factor and the Williams syndrome transcription factor, were induced by mechanical loading in a corresponding temporal fashion. Chromatin immunoprecipitation revealed that these factors, together with Pol I, were enriched at the rDNA promoter. This, in addition to an increase in histone H3 lysine 9 acetylation, demonstrates that mechanical loading regulates rRNA synthesis by inducing a gene expression program consisting of a Pol I regulon, together with accessory factors involved in transcription and chromatin remodeling at the rDNA promoter. Altogether, these data indicate that transcriptional and epigenetic mechanisms take place in the regulation of ribosome production at the onset of muscle hypertrophy.
Article
Insulin-like growth factor I (IGF-I) peptide levels have been shown to increase in overloaded skeletal muscles (G. R. Adams and F. Haddad. J. Appl. Physiol. 81: 2509-2516, 1996). In that study, the increase in IGF-I was found to precede measurable increases in muscle protein and was correlated with an increase in muscle DNA content. The present study was undertaken to test the hypothesis that direct IGF-I infusion would result in an increase in muscle DNA as well as in various measurements of muscle size. Either 0.9% saline or nonsystemic doses of IGF-I were infused directly into a non-weight-bearing muscle of rats, the tibialis anterior (TA), via a fenestrated catheter attached to a subcutaneous miniosmotic pump. Saline infusion had no effect on the mass, protein content, or DNA content of TA muscles. Local IGF-I infusion had no effect on body or heart weight. The absolute weight of the infused TA muscles was approximately 9% greater (P < 0.05) than that of the contralateral TA muscles. IGF-I infusion resulted in significant increases in the total protein and DNA content of TA muscles (P < 0.05). As a result of these coordinated changes, the DNA-to-protein ratio of the hypertrophied TA was similar to that of the contralateral muscles. These results suggest that IGF-I may be acting to directly stimulate processes such as protein synthesis and satellite cell proliferation, which result in skeletal muscle hypertrophy.