Mechanisms of protein balance in skeletal muscle

Abstract

Increased global demand for adequate protein nutrition against a backdrop of climate change and concern for animal agriculture sustainability necessitates new and more efficient approaches to livestock growth and production. Anabolic growth is achieved when rates of new synthesis exceed turnover, producing a positive net protein balance. Conversely, deterioration or atrophy of lean mass is a consequence of a net negative protein balance. During early life and periods of growth, muscle mass is driven by increases in protein synthesis at the level of mRNA translation. Throughout life, muscle mass is further influenced by degradative processes such as autophagy and the ubiquitin proteasome pathway. Multiple signal transduction networks guide and coordinate these processes alongside quality control mechanisms to maintain protein homeostasis (proteostasis). Genetics, hormones and environmental stimuli each influence proteostasis control, altering capacity and/or efficiency of muscle growth. An overview of recent findings and current methods to assess muscle protein balance and proteostasis is presented. Current efforts to identify novel control points have the potential through selective breeding design or development of hormetic strategies to better promote growth and health span during environmental stress.

1. Introduction

Worldwide population growth has increased global demand for adequate protein nutrition [1]. Novel strategies to increase meat production are needed while minimizing the adverse effects on the environment [2]. Genetic approaches to increase production of animal products through selective breeding are successful but also result in economic, environmental and ethical complications [3,4]. Overall, efforts to meet the world’s protein needs against a back drop of environmental stress (i.e., physical, chemical and biological constraints on the productivity of the species [5]) are creating greater pressures on animal agriculture than ever before. For these reasons, a greater understanding of the fundamental control points in determining muscle protein balance is relevant to animal agriculture sustainability.

Over the past two decades, advances in genomics allowed for selective breeding to be more informed and thus targeted. Recent developments in technology have further bolstered if not replaced the genomic age with an age of proteomics and metabolomics. These technologies allow for even more sophisticated questions to be asked, moving the field from monitoring genotype to phenotype [6]. A deeper understanding of the phenotypic mechanisms that regulate muscle mass will in turn provide new insight about how to best address environmental challenges to animal growth and improve overall health of livestock. With the above in mind, the following perspective was crafted to provide a basic overview of recent advances in the study of skeletal muscle protein balance in vivo. This information is aimed to inform the fields of domestic and livestock animal production about ways to better monitor or alter capacity and efficiency of growth, with an emphasis on skeletal muscle.

2. Evolution of methodology in assessing protein balance

A table summarizing past, current and emerging technologies to evaluate skeletal muscle protein synthesis and turnover in animals are found in Table 1. Nitrogen balance has traditionally served as a surrogate marker to assess whole body protein balance and growth [7,8]. Subtracting nitrogen intake from output produces a value which reflects growth (>0), maintenance (=0) or atrophy (<0). While this measure remains the fundamental basis for determining dietary protein requirements in mammals, this technique reflects whole body and not skeletal muscle specifically. MPS capacity can be reflected by the RNA to protein ratio, a classic measurement in skeletal muscle [9–11]. While still in use today, most research settings use instead a variety of techniques that measure the rate of skeletal muscle protein synthesis (MPS) and turnover more directly. These methods use an assortment of metabolic compounds and labeling techniques in combination with highly sensitive and specialized instruments to calculate rates of MPS and/or muscle protein breakdown (MPB) or simply visualize the expression of one or more individual proteins as biomarkers of muscle growth. To estimate global rates of MPS or MPB, the incorporation and/or flux of an injected or infused metabolic tracer (i.e., radioactive or stable amino acid isotope mixed with an unlabeled amino acid or tracee) is measured into and/or out of muscle tissues over a relatively short and defined period of time [12–16]. Analyses of precursor and product tracer/tracee enrichment involve sophisticated methods of chromatography and mass spectrometry and in some cases complex tracer kinetic calculations [17]. MPS measurements over longer periods of time (d or wk) can be accomplished by ingestion of deuterium oxide (heavy water) [18,19]. This method allows for calculation of DNA synthesis in addition to MPS and turnover [20]. Metabolic tracer approaches are very useful in generating tissue averages, changes over short periods of time, assessing muscle fiber-type differences and detecting changes within subcellular organelles (e.g., mitochondrial protein synthesis) [21,22]. Synthesis rates of individual proteins can also be assessed [23,24].

Table 1.

Common and emerging technologies to assess muscle protein balance in domestic animals and livestock.

Method	Applications	Strengths	Limitations	Refs
Nitrogen Balance	Whole body protein balance and growth	Simple, sensitive, noninvasive	Cannot assess muscle-specific effects; difficult to measure losses precisely	[7,8]
RNA/protein ratio	Estimates protein synthesis capacity	Simple, inexpensive	Crude	[9–11]
Amino acid isotope tracer kinetics	Whole body protein synthesis and breakdown, Tissue and fiber-type-specific protein synthesis and breakdown, individual protein synthesis and breakdown	Sensitive, can assess change over short time frames	May not capture proteins with longer half-lives; cannot assess free-living environments	[12–17,21,22,24,29]
Deuterium oxide/heavy water enrichment	Protein synthesis, protein breakdown and DNA synthesis in whole body, skeletal muscle, specific fiber-types and individual proteins	Can assess free living conditions over extended periods of time	Isotope expense, sophisticated equipment and calculations required	[18–20,23]
Two-dimensional electrophoresis	Differential expression of specific tissue proteins using mass spectrometry	Can visualize modifications affecting protein activity in addition to changes in expression	Protein identification is often targeted versus global	[10]
SUnSET - non-isotopic immunodetection of puromycin	Skeletal muscle protein synthesis (whole tissue and fiber-type)	Simple, inexpensive snapshot of nascent MPS	Qualitative, puromycin dose may inhibit protein synthesis in other tissues; cannot assess breakdown	[25,26]
PUNCH-P - puromycin-associated nascent chain proteomics	Genome-wide identification and quantification of protein synthesis in tissues	Ex vivo, relatively simple, less expensive than Ribo-Seq	Qualitative, sophisticated equipment required; cannot assess breakdown	[27,28]
Ribosomal profiling	Tissue-specific global quantification of mRNA translation	Ex vivo, global snapshot of both translated and untranslated mRNAs	Conducting gene expression across polysome fractions is time consuming and expensive; cannot assess breakdown	[30,35]
Ribo-Seq	Global quantification of the average ribosome density on mRNA	Ex vivo, comprehensive evaluation of nascent mRNA translation	Expensive; technology not yet optimized for use in animal tissues; unable to reveal the proportion of untranslated mRNA relative to polysome bound mRNA; cannot assess breakdown	[31–34]

Applications that rely on antibody-based detection methods such as immunoblotting, immunofluorescence, and flow cytometry are commonly used to visualize protein expression and provide a qualitative measure of the proteome. The tagging of newly synthesized proteins with puromycin is a more recent method being used to estimate new protein synthesis in muscle [25,26]. Another approach uses biotinylated puromycin to label newly synthesized proteins in cell-free conditions, followed by proteomic analysis to generate a snapshot of the translatome [27,28]. These protein tagging approaches to assess the proteome are faster and easier than two-dimensional gel electrophoresis methods to assess the proteome [10,29].

The use of whole genome sequencing is now emerging as a powerful approach to assess the translatome [30]. Genome-wide analyses of mRNA expression by microarray or RNA sequencing is now being applied to monitor in vivo mRNA translation. One of these approaches is called ribosomal profiling or Ribo-Seq. This method conducts deep sequencing of ribosome-protected RNA fragments to determine which mRNAs are being actively translated at a specific point in time [31–34]. A related approach to examine gene-specific translation involves whole genome sequencing of mRNA bound to varying numbers of polysomes separated via density gradient centrifugation [35]. The technology and instrumentation behind many of these high-throughput systems-based methods are advancing at a pace that precludes independent validation against other more traditional measures of growth. Furthermore, the value of next-generation sequencing data requires bioinformatics pipelines that are not only fast, accurate and easy to use but also take into account animal to animal variability and complex experimental designs. As such, the usefulness of these findings as a phenotypic measure or accurate reflection of animal physiology is uncertain. Nevertheless, there is great potential for these emerging technologies to improve assessment of protein balance and meat quality in livestock as the cost of conducting genome-wide sequencing becomes more affordable and bioinformatics pipelines become more widely accessible and applicable to animal physiology [33,36,37].

3. The proteostasis network in skeletal muscle

Skeletal muscle mass is regulated by dynamic control of the processes of protein synthesis and degradation in combination with systems in place to manage folding, trafficking and clearance. In this regard, protein balance does not function as a binary operation akin to a balance scale (i.e., synthesis or degradation) but instead is a summation of multiple processes that operate according to highly interconnected signaling networks. These signaling networks coordinate quality control mechanisms to maintain protein homeostasis, or proteostasis [38]. Proteostasis control is complex and dynamic, manifesting phenotypic outcomes over months or years. In this regard, visualizing proteostasis as gears to a clock mechanism is an effective analogy [39]. Each step or process in the making and degrading of a protein impacts the function of the entire mechanism. External pressure on the network can alter the pace of the mechanism (i.e., aging) or result in its collapse (i.e., cellular damage or disease). The proteostasis network is a subject of intense study in humans, aimed toward discovering new approaches to delay aging and prevent disease [40]. Proteostasis control concepts are relevant throughout lifespan of domestic animals, even in rapid growth states, particularly under conditions of environmental stress (e.g., changes in temperature, humidity, nutrition), where growth among breeds or species can differ [41]. A greater understanding of how proteostasis is regulated in the different muscle fiber types in each species will help shape future genetic, hormonal and environmental approaches to maximize muscle growth and animal production. It will also help understand differences in meat quantity between breeds, provide novel targets that have efficient growth potential through selective breeding design or perhaps assist in the development of novel in vitro strategies to produce meat [2,42].

4. Muscle protein synthesis is controlled at the level of mRNA translation

Studies in rodents and pigs show that postnatal skeletal muscle growth occurs via increases in MPS that are controlled at the initiation step of mRNA translation [43,44]. Two events in translation initiation are identified as contributing factors in the regulation of muscle mass: 1) the creation of a ternary initiation complex, which consists of methionyl tRNA, eukaryotic initiation factor 2 (eIF2) and GTP; and 2) formation of the eIF4 ribosomal complex. Both of these cellular events are regulated by phosphorylation of specific eIFs which can be monitored using immunoblotting techniques and then used as biomarkers of MPS in response to environmental factors. Among various environmental factors, nutritional factors are of primary importance. Feeding increases MPS, via enhanced formation of the eIF4 complex [45,46] whereas fasting and low protein reduces MPS in concert with reduced eIF4 formation [47]. Stimulation of MPS by feeding is developmentally regulated, with the capacity of the translational machinery reducing with age [43]. Furthermore, recent evidence points to bolus feeding as more effective in stimulating MPS than continuous feeding in neonates [48]. Catabolic stimuli such as infection, inflammation, disuse and aging blunt or block feeding-induced stimulations in MPS at the level of translation initiation and correspond with reductions in the formation of the ternary initiation complex and/or the eIF4 complex [49–51].

Among nutritional strategies examined in recent years, dietary amino acid supply and composition have been a focus [52]. Some of this work began with the seminal observations that the branched chain amino acids and especially leucine stimulated MPS independent of insulin or energy intake [53,54]. More recent efforts have further identified a potentially important role for the leucine metabolite, beta-hydroxy-beta-methylbutyrate [55–57]. Other related research efforts have studied the impact of dose and timing of amino acid intake on maximizing MPS over time, particularly in relation to physical activity and aging [58–61]. The logic behind these studies is clear: maximize MPS with each meal and over time muscle mass will increase. Many examples are to be found in the literature showing impressive increases in MPS in response to feeding protein or leucine more frequently and/or in greater amounts [58,62]. Nonetheless, long term studies examining the impact of protein or amino acid supplementation on lean mass gains are inconsistent or equivocal [63,64] with significant differences between breeds and among phases of growth evident [65,66]. Thus, it is difficult to assign a single conclusion about the effect of supplementing dietary protein or specific amino acids on skeletal muscle growth; sometimes a reduction rather than an increase in dietary protein or amino acids improves quality of finished product [67]. The use of dietary protein and amino acids must therefore be utilized according to the biological goal in mind alongside an appreciation for the underlying genetic and hormonal background.

5. Protein degradation pathways regulate muscle mass

Skeletal muscle cell mass is influenced by protein turnover/degradation in all phases of life. Enhanced degradation not only reduces muscle mass but also alters muscle fiber type composition [68]. Fasting, glucocorticoids, sepsis and aging shifts fast (Type II) fiber types to slow (Type I) while inactivity and denervation causes a slow to fast fiber type shift [68]. How the different proteolytic systems regulate the capacity and efficiency of growth in the different muscle fiber types, particularly during environmental stress, are important research questions without clearly defined answers. The difficulty in answering these questions lies in the complexity of proteolysis. The stability or half-life of any single protein is modulated by the activity of assorted, overlapping degradative systems in the body. The main proteolytic processes influencing skeletal muscle mass are the 1) autophagy lysosomal system, 2) ubiquitin proteasome pathway, 3) calcium-dependent calpains and 4) cysteine protease caspase enzyme cascade (for recent review see [69]). The relative contribution of these processes in determining muscle mass fluctuates according to genetics, life stage, hormones and environmental stimuli. Furthermore, these catabolic processes interact through associated quality-control signaling networks and gene expression events that modulate one another [70]. As such, a deeper understanding of the regulatory processes guiding activation of each of these modes of proteolysis is a subject of intense investigation.

The two most influential cellular processes in muscle protein turnover and especially during muscle wasting are the autophagy lysosomal system and ubiquitin proteasome pathway [71]. The autophagy lysosomal system is well-recognized for its important role in conducting bulk degradation and recycling of cellular material and organelles. Recent descriptions of the different types or forms of autophagy relevant to muscle metabolism and mass are published [69,72–77] and include microautophagy, macroautophagy, and chaperone mediated autophagy. Microautophagy describes direct engulfment of small portion of cellular material (e.g., glycogen) into the lysosome; its contribution to normal skeletal muscle metabolism is not well-defined. On the other hand, macroautophagy is recognized as a major regulator of muscle mass and activation of macroautophagy corresponds with muscle atrophy. During macroautophagy, the process of “self-eating” occurs through highly-regulated expansion of a double-membraned vesicle that encapsulates cytoplasmic material. Concomitant maturation and closure of the membrane produces an autophagic vacuole which subsequently fuses with a lysosome. While the cellular material degraded can be nonselective, targeted forms of macroautophagy do exist and are increasingly becoming recognized as important for maintenance of muscle homeostasis. The selectivity of client substrate is also specified during chaperone-mediated autophagy (CMA). During CMA, soluble cytosolic proteins containing a targeting motif (KFERQ) are shuttled to the lysosomal surface without the need for vesicular transfer. A role for CMA in normal muscle maintenance is identified in flies, rodents and humans [72] and certain degenerative muscle disease states indicate involvement of CMA [78].

Targeted mutations in components of the autophagic machinery in the skeletal muscle of mice have revealed that macroautophagy is essential for muscle remodeling and quality-control. In particular, selective forms of macroautophagy such as mitophagy have important roles in mitochondrial function and oxidative stress defense during aging [79]. Macroautophagy is regulated by nutritional state, with fasting stimulating and feeding inhibiting biomarkers of autophagosome formation [77]. In neonatal pigs, both insulin and amino acids play a role in inhibiting macroautophagy whereas CMA is not altered [80]. It should be noted that functional assessment of autophagy is complex and some controversy surrounding valid biomarkers of autophagosome maturation exists. The reader is directed to a formal position statement describing these considerations [81].

The other important proteolytic system in skeletal muscle is the ubiquitin proteasome pathway [71]. The ubiquitin proteasome pathway accomplishes selective protein degradation via ATP hydrolysis and the tagging of client protein with a polyubiquitin chain. In a series of three cooperative catalytic reactions mediated by the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes, four or more ubiquitin monomers are covalently attached to proteins selected for destruction by the 26S proteasome. Because E3 ligases catalyze the final and rate-limiting step of the ubiquitination cascade, research efforts have been directed toward identifying the determinants of substrate selection. However, less than half of the known E3 ligases harbor enzymatic activity by themselves; most require interaction with the proper E2 in order to correctly target substrates for degradation [82]. These E2–E3 relationships in developing or atrophying skeletal muscle are for the most part unknown.

Many catabolic conditions correspond with an increase the expression or activity of a number of E3 ligases, some which exist broadly in different cell types and others with expression exclusive to skeletal muscle [83–85]. Two muscle-specific E3 ubiquitin ligases, named Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx)/Atrogin-1 are well-studied in their expression levels during various states of muscle catabolism and atrophy [84]. Other E3 ligases such as Nedd4-1, Trim32 and TRAF6 play critical roles in different models of atrophy and different stages of muscle development [71]. A full list of E3 ligases relevant to the control of muscle mass has yet to be generated, as does the identity of the E2s that pair with them. A detailed understanding of this level of control will reveal novel means to improve environmental resilience.

6. Signaling pathways coordinate muscle protein balance

Intracellular signaling events are initiated by a variety of chemical signals that reflect nutrition and hormonal status (e.g., insulin/IGF-I), energy state and activity (e.g., AMP kinase, phosphatidic acid), and other mediators of environmental stress (e.g., glucocorticoids, cytokines) (Figure 1). A key point of integration in muscle growth and development is the protein kinase B/Akt kinase [86]. The insulin/IGF-I- Akt pathway increases MPS via inhibiting glycogen synthase kinase 3β (an inhibitor of eIF2 ternary complex formation) and activating mechanistic target of rapamycin complex 1 (mTORC1) signaling [87]. Akt also reduces MPB via phosphorylation of the Forkhead box class O (FOXO) transcription factors [70,88].

Figure 1.

Signaling pathways coordinate muscle protein balance. Anabolic and catabolic stimuli are integrated through the PKB/Akt-mTORC1 signaling to regulate mechanisms that control muscle protein synthesis and breakdown.

Assembly of mTORC1, a major hub for sensing nutrients, redox and energy state, consists of the core proteins mTOR, Raptor and mLST8, and can include associated ancillary proteins such as Rag A–D, Deptor, PRAS40 and Rheb-GTP [89]. A second complex called mTORC2 is required for Akt signaling to FOXO but the role of mTORC2 in muscle growth is unclear [87] [90]. The activity of mTORC1 is controlled by its localization and assembly at or near the lysosome [91]. Increased mTORC1 lysosomal assembly and signaling corresponds with increased MPS at the translation initiation step and inhibition of autophagy whereas reduced mTORC1 assembly and signaling lowers MPS and increases autophagy. The ubiquitin proteasome pathway is not directly inhibited by mTORC1 but rather is regulated by PI3K/Akt signaling upstream of mTORC1 [86]. In general, anabolic stimuli (e.g., growth hormone, insulin/IGF-I, amino acids, testosterone, β-agonist) activates the mTORC1 signaling pathway whereas catabolic stimuli (e.g., inflammatory cytokines, glucocorticoids, myostatin, fasting, low protein) represses mTORC1 signaling [73,92,93]. Nutrients, especially the branched chain amino acids, are potent activators of mTORC1 in muscle independent of insulin/IGF-I-Akt [52,54]. Likewise growth factors can stimulate mTORC1 signaling in skeletal muscle independent of amino acid nutrition [94]. A variety of plant steroid compounds called phytoecdysteroids are found to increase protein synthesis and activate Akt signaling similarly to IGF-I in cultured myocytes [95,96]. Feeding these and other phytoecdysteroids produce an anti-obesity effect in mice [97]. Nonetheless, recent feeding trials are unable to identify an acute impact of 20-hydroxyecdysone on Akt or mTORC1 signaling in skeletal muscle [98] suggesting that phytoecdysteroids may require other factors for activity and/or regulate longer-term transcriptional changes in MPB versus signaling mechanisms that regulate MPS.

The control of gene expression through key transcription factors plays a major role in regulating muscle mass. Many of these proteins such as the FOXO family promotes muscle atrophy through increased expression of both E3 ubiquitin ligases as well as inducing autophagosome membrane components [70]. These discoveries are revealing how the ubiquitin proteasome and autophagy lysosome pathways often work together versus separate from each other. Furthermore, hormones and other growth factors can alter the activities and function of both mTORC1 and transcription factors [73,83]. For example, insulin/IGF-I treatment promotes increases Akt signaling, which promotes mTORC1 complex assembly and MPS but also inhibits MPB via reduced proteolytic gene expression under control of the FOXO transcription factors [86,87]. Myostatin treatment blocks Akt, which reduces MPS via mTORC1 complex assembly and increases MPB by activating the FOXO transcription factors [99] as well as other transcriptional regulators such as SMAD family members [100].

An emerging pathway of muscle mass control involves regulation of the Activating Transcription Factor 4 (ATF4) transcription factor [101]. Early studies in non-myofibrillar tissues characterized ATF4 as part of the integrated stress response, a signaling pathway that controls adaptation to nutrient and oxidative stress by altering gene-specific translation and the transcriptome [102,103]. Knockout and transgenic studies in rodents find that ATF4 is necessary and sufficient for muscle atrophy during fasting and aging [101,104]. Subsequently, unbiased mRNA expression signatures of muscle atrophy in combination with connectivity maps identified two natural compounds, ursolic acid and tomatidine, as small molecule inhibitors of ATF4 activity which prevent age-related loss of muscle mass [105–107]. Because these compounds are naturally occurring, the potential for their use as dietary additives is feasible. This innovative approach highlights the potential of mRNA expression signatures to discover novel regulators of protein muscle mass for use in domestic animal production [108].

7. Proteostasis meets hormesis

A loss of proteostasis control can speed up the aging process whereas enhancement of the proteostasis network extends lifespan and promotes disease resistance [40]. Current evidence suggests that dietary restriction or reductions in insulin/IGF-I signaling improves proteostasis in association with increased life span [20,109–111]. However, these life span benefits can associate with reduced fertility and/or increased development time [112,113]. The balance between adverse (reduced fecundity and growth) versus beneficial (increased resistance to environmental stress and disease) outcomes can be seen as a function of preconditioning or hormesis in which physiological outcomes are influenced by exposure level or dose [114]. Hormetic responses by definition are multi-phasic, and in certain dose ranges, both adverse and beneficial outcomes can coexist or overlap [115]. Benefits of hormesis can also be ‘off-target’, so that exposure to one type of stress can impart protection from another, different types of stress. These concepts are relevant to the study of quality versus quantity of muscle mass, not only with respect to an animal’s age and sex, but also with respect to future progeny. For example, previous work in cows suggested that during nutrient restriction, muscle protein degradation pathways were activated in pregnant cows but not their fetuses [116]. However, a recent study in rats shows that protein restriction during pregnancy limits muscle growth capacity of male progeny later in life [117] and a another study in pigs shows that feeding a high protein diet during gestation permanently alters the offspring’s genome [118]. These data highlight how adaptive responses to protein nutrition can cross generations in their influence. In addition to nutrition, climate conditions are a major influence on the development, performance and health of livestock [119] yet much remains unknown regarding the regulation of protein balance under these conditions. In an age of increased environmental pressures, these factors are important to consider in the search for novel therapeutic and prophylactic strategies to optimize if not maximize muscle growth and meat production.

Highlights.

• Global demand for protein challenges animal agriculture sustainability.

• A variety of sophisticated approaches to studying the translatome in muscle are revealing novel targets.

• The proteostasis network in skeletal muscle imparts quality control and cytoprotection.

• Signaling pathways in skeletal muscle coordinate protein synthesis and degradation to regulate protein mass.