Post-natal muscle growth and protein turnover: a narrative review of current understanding

Abstract

A model explaining the dietary-protein-driven post-natal skeletal muscle growth and protein turnover in the rat is updated, and the mechanisms involved are described, in this narrative review. Dietary protein controls both bone length and muscle growth, which are interrelated through mechanotransduction mechanisms with muscle growth induced both from stretching subsequent to bone length growth and from internal work against gravity. This induces satellite cell activation, myogenesis and remodelling of the extracellular matrix, establishing a growth capacity for myofibre length and cross-sectional area. Protein deposition within this capacity is enabled by adequate dietary protein and other key nutrients. After briefly reviewing the experimental animal origins of the growth model, key concepts and processes important for growth are reviewed. These include the growth in number and size of the myonuclear domain, satellite cell activity during post-natal development and the autocrine/paracrine action of IGF-1. Regulatory and signalling pathways reviewed include developmental mechanotransduction, signalling through the insulin/IGF-1–PI3K–Akt and the Ras–MAPK pathways in the myofibre and during mechanotransduction of satellite cells. Likely pathways activated by maximal-intensity muscle contractions are highlighted and the regulation of the capacity for protein synthesis in terms of ribosome assembly and the translational regulation of 5-TOPmRNA classes by mTORC1 and LARP1 are discussed. Evidence for and potential mechanisms by which volume limitation of muscle growth can occur which would limit protein deposition within the myofibre are reviewed. An understanding of how muscle growth is achieved allows better nutritional management of its growth in health and disease.

Introduction

The regulation of skeletal muscle’s growth in childhood and its maintenance in adult life is of obvious importance given its primary motor function, but is also key to understanding human health and wellbeing throughout the life cycle. Low muscle mass and strength during childhood contributes to several adverse health outcomes⁽¹⁾. Muscle mass and strength in adolescent men is inversely associated with cardiovascular disease (CVD) events and CVD mortality in middle age⁽²⁾, and with risk of all-cause mortality and mortality from CVD and cancer, explaining the obesity paradox of increasing mortality risk associated with low BMI⁽³⁾. One potential mechanism for these relationships is the inverse relationship between muscle mass and insulin resistance⁽⁴⁾ and the development of diabetes.

The phenomenology and potential mechanisms controlling post-natal growth of muscle in children and the interactions between growth in stature and in muscle mass have been recently reviewed⁽⁵⁾, drawing on a large and diverse literature. This included a model linking dietary protein intake to the regulation of post-natal growth suggested some years ago⁽⁶⁾ and identified as a Protein-Stat model of growth, developed from nutritional studies of muscle growth in the rat over its first year of life. At its core is the concept that dietary protein stimulates growth, especially appendicular growth, through its joint action on bone length growth and on muscle weight growth, with their growth interrelated via mechanotransduction mechanisms. The growth of most other organs and tissues, especially the viscera, adipose tissues and skin, was largely a passive consequence of muscle and bone growth influencing energy expenditure and food intake which was regulated at least in part by an aminostatic appetite mechanism.

Several gaps in the knowledge base were indicated, and those addressed here in this narrative review include the physiological and mechanistic basis of mechanotransduction during growth, the relationships between myonuclear domain size and protein turnover, the role of satellite cells specifically in post-natal muscle growth, the role of IGF-1, especially its auto and paracrine action, and detailed mechanisms of the key processes involved. An updated summary model of dietary protein and appendicular muscle–bone interactions is presented in the context of those protein turnover studies which provided the evidence base for the main interactions identified in the model. The signal transduction pathways mediating these processes are reviewed, including insulin/IGF-1 and MAPK pathways, and those acting on and within satellite cells. In addition, studies of the responses to maximal-intensity contractions are included which throw light on the component of mechanotransduction generated through muscle force generation in response to gravitational loading during normal post-natal growth. Also given the importance of the ribosomal capacity for the regulation of muscle protein synthesis (MPS), identified in early studies, a recently discovered mTORC1-mediated regulatory mechanism involved is also included. Finally, the concept that muscle mass is regulated at a fixed capacity due to constraints exerted by the extracellular matrix is examined. The literature search was limited to the main subheadings listed in this paper and was completed up to 21 February 2023 with some updating during the review. Whilst the overall focus is on physiological mechanisms of post-natal growth as observed in the rat, where appropriate, work on muscle growth in mouse and other animal models, on responses to injury and on work-induced hypertrophy in human adult muscle has been referenced, where such work provides potentially important mechanisms relevant to post-natal growth.

Concepts and processes

Proteostasis and turnover during skeletal muscle growth: the evidence base for the growth model

The growth model consistent with the current evidence base is shown in Fig. 1, and some of the experimental basis for the model, that is, early studies of developmental growth and protein turnover within hind limb muscles of the rat over a year⁽⁷⁾ are shown in Fig. 2.

Fig. 1. Dietary protein and appendicular muscle–bone interactions in the rat.

The control of the growth of the major appendicular muscles is directly related to the lengthening of the associated bone which occurs by endochondral ossification in the growth plate^(5,30) . This latter process is regulated through endocrine and nutritional influences, of which dietary protein plays a dominant role, and by a paracrine/autocrine system of bone growth factors^(323,324) within which nutrients including amino acids and zinc have direct roles in signal transduction^(325–329), although specific sites of their action in the growth plate have yet to be described. Bone lengthening stretches muscle, inducing growth in myofibre length through addition of sarcomeres. Force development through muscle contractile activity in the lengthening muscle in response to gravitational loading induces growth in cross sectional area. This mechanotransduction involves mechanosensitive pathways^(330,331) at the cell–matrix interface^(332,333) , which activates satellite cells, fibroblasts and other cell types, enabling increases in the synthesis of collagen^(78,334,335) , proteoglycans^(80,85) , and other components of the ECM essential to its remodelling. This enables an increase in myofibre volume. The activation of satellite cells also induces myogenesis and fusion with the myofibre, adding new myonuclei to myofibers. This sets the capacity for muscle growth in terms of the number of myonuclear domains within the myofibre and this is in part mediated by the autocrine/paracrine action of IGF-1^(81,84,85) . Proteostasis is managed within these myonuclear domains through provision of sufficient protein translational capacity in terms of ribosomal RNA^(291,336) . Maximal translational efficiency is under nutritional control via amino acids from dietary protein, insulin and T3 which optimise myofibre protein synthesis^(26,37–39) . Although dietary protein induces increases in circulating IGF-1 and IGFBP-3 which reflect insulin concentrations, it is not clear if these endocrine changes in IGF-1 influence MPS in addition to any insulin-mediated stimulation. Bone osteocytes, dependent on calcium and vitamin D for their mineralisation, are mechanosensitive to forces exerted by muscle^(337,338) . Thus, muscle and bone growth are intimately connected in a bidirectional relationship in which bone length growth regulates muscle mass and muscle growth regulates bone strength.

Fig. 2. Developmental changes in protein turnover in rat skeletal muscle.

Muscles were the combined gastrocnemius and quadriceps muscle of male littermates of a hooded strain⁽⁷⁾. Growth is shown as the increases in total muscle non-collagen protein, deemed to be that within muscle myofibres. Muscle myofibre protein synthesis, (MPS, %/d) was measured in vivo by the constant intravenous infusion of [¹⁴C]tyrosine, with the rate of protein breakdown (MPB) calculated as the difference between MPS and the growth rate of muscle protein. DNA and RNA were total extractable nucleic acids. The early growth phase up to 60 d, with a high rate of turnover, corresponds to intense myogenesis and myofibre remodelling associated with new fibre formation⁽¹⁶⁾. During the subsequent growth, turnover fell, and after 120 d growth involved mainly enlargement of the myonuclear domain approximated by measurement of the total muscle protein/DNA ratio, with no further change in turnover. However, protein synthesis per unit DNA increased by >50% during this phase of growth mainly through an increase in ribosomal capacity per nucleus (RNA/DNA).

The growth of an appendicular muscle reflects the combined influence of nutrition, especially dietary protein driving both muscle and bone length growth, and mechanotransduction exerted both by stretching, consequent to length growth of the associated bone, and by internal work against gravity.

Growth is identified in the left panel of Fig. 1 under two headings. The first is stem cell activation, extracellular matrix (ECM) remodelling and myonuclear accumulation allowing myofibre expansion, under control of IGF-1. The second, under the heading of proteostasis, involves myofibrillar protein turnover and accretion resulting in myonuclear domain enlargement under the control of insulin. In this section these key features will be elaborated, including a brief review of the experimental work which allowed the model development.

Proteostasis is an umbrella term describing all aspects of the maintenance of an intact proteome within cells, acting through a proteostasis network (PN). Thus, for each protein the PN exerts strict control of the initial production (protein synthesis), folding, conformational maintenance, abundance, subcellular localisation and disposal by breakdown⁽⁸⁾. Various molecular chaperones and co-chaperones are of central importance ensuring correct de novo folding and maintenance of a soluble, non-aggregated state as well as targeting misfolded proteins for degradation or spatial sequestration to protect the rest of the proteome from aberrant interactions. Some of the PN is concerned with environmental stress responses, (thermal, mechanical, pathogen and oxidative stress), which can be triggered and coordinated by proteostasis transcription factors. The accumulation of protein aggregates, such as occurs in the brain with ageing, most likely represents a failure of proteostasis. Protein turnover, the breakdown and resynthesis of cellular proteins, can now be described as the consequence of the action of the ∼2000 components of the PN specifically involved in protein synthesis, chaperone-mediated folding and proteolysis mainly by the lysosomal–autophagic system and ubiquitin–proteasomal systems^(9–11).

The relative importance of protein synthesis and breakdown in muscle growth control was identified in early nutritional studies. Precursor–product methods⁽¹²⁾, as distinct from studies of the decay rates of tracer-labelled proteins^(12–14), proved most useful, allowing accurate measurement of muscle protein synthesis (MPS) in vivo from which the rate of muscle protein breakdown (MPB) can be determined as

$${{\rm{MPB}} = {\rm{MPS }} - {\rm{the \ growth \ rate \ of \ the \ muscle \ protein \ mass}}}$$

Early studies of the developmental changes in muscle protein turnover in the rat obtained in this way are shown in Fig. 2 ⁽⁷⁾. Thus, turnover was intense at weaning at 23 d (MPB >20% d⁻¹), falling to about 5% d⁻¹ by 4 months, which was maintained after this. Furthermore, because intervention studies in young rats which inhibited growth always reduced MPS and often MPB, this identified MPS rather than MPB as the primary site of acute regulation of muscle mass⁽¹⁵⁾. MPB emerged as important for mediating muscle fibre remodelling associated with its rapid growth, both during early development⁽¹⁶⁾ and during stretch-induced hypertrophy in avian muscles^(17–21). In addition, the MPB rate was clearly important in mediating the different rates of turnover in muscle with different fibre types (discussed further below in ‘The myonuclear domain: growth in number and size during development, and relationship with protein turnover’).

Figure 1 shows MPS to reflect translational capacity as ribosome content consequent to nucleolar ribosomal biogenesis, and translational control determining translational efficiency. Following initial studies focused on ribosomal activity and polysome profiling^(22,23) , it proved useful to identify changes in MPS in terms of translational capacity (total RNA, of which ≥85% is ribosomal) and ribosomal efficiency (protein synthesis per unit RNA). Thus, in malnourished rats, the reduced MPS mediating growth suppression involved reductions in both translational capacity and efficiency, with the former mediating the major change during longer-term deficiency⁽²⁴⁾. An important newly discovered mechanism involved in the regulation of ribosomal capacity is reviewed below in ‘Regulation of the capacity for protein synthesis’, and the developmental changes in capacity shown in Fig. 2 are discussed below in ‘The myonuclear domain: growth in number and size during development, and relationship with protein turnover’.

Within the growth model, dietary protein is shown to control both muscle and bone growth through the combined effects of amino acids and endocrine responses under behavioural/developmental and nutritional control. Muscle and length growth in the male rat continues throughout its lifespan (Fig. 2), since epiphyseal growth plate fusion does not occur. Their dietary demand for protein is so strong that they can become hyperphagic when given marginally protein-deficient diets^(25,26) , a response which led to the protein leverage theory⁽²⁷⁾. The multiple targets of dietary protein’s influence include endochondral ossification in the bone growth plate mediating bone length growth, and myonuclear domain enlargement through myofibre protein deposition. As shown in Fig. 1, other key nutrients are obligatory for muscle growth, of which zinc is most important^(28–30), in part to enable dietary protein’s anabolic influence.

In early post-natal life, protein-containing food increases circulating insulin and amino acids with a minimal response to a protein-free meal⁽³¹⁾, and each of these responses is important for the stimulation of MPS⁽³²⁾. The role of insulin was established in early studies^(23,32–37) emerging as the main acute, nutritionally sensitive regulator of muscle protein synthesis^(38,39) . The interaction of amino acids with insulin and the extent of their independent influence on MPS in mediating the feeding response initially proved difficult to establish. When they were both controlled independently, it was demonstrated that insulin was permissive for the maximum influence of amino acids, especially leucine^(35,40–42) , with insulin, at low physiological levels, and amino acids acting through independently upstream pathways, (see ‘Insulin/IGF-1 signalling’) to activate an mTORC1-mediated stimulation of MPS. Leucine signals through the Rag GTPase via the cytosolic sensors sestrins^(32,43–45) , probably sestrin1⁽⁴⁶⁾. In human adult muscle, MPS is regulated by amino acids with no discernible influence of insulin^(47–49), at least within the physiological range⁽⁴⁷⁾, although insulin does mediate an inhibition of MPB with no obvious role for amino acids^(50,51) . However Atherton et al. ⁽⁴⁷⁾ have pointed out that insulin is important in mediating postprandial increases in microvascular recruitment and overall blood flow into muscle, increasing amino acid supply and therefore contributing to the postprandial increase in MPS. One suggestion is that insulin’s stimulation of MPS is unique to the immature muscle⁽³²⁾, but there may be a species differences since insulin appears to regulate rat MPS irrespective of age⁽⁵²⁾.

Dietary-protein-mediated changes in thyroid hormones also emerged as important in the regulation of both MPS⁽⁵³⁾, at the level of the ribosomal capacity^(54–56), and MPB⁽⁵⁶⁾, so that reductions in both insulin and thyroid hormones, (specifically circulating free T₃), were shown to mediate protein-deficiency-induced reductions in muscle growth and protein turnover⁽²⁶⁾. Insulin influences T₃ activity by activating type 2 iodothyronine deiodinase to generate T₃ in muscle from T₄ ^(57,58) . The role of thyroid hormones in growth regulation has been briefly reviewed elsewhere⁽⁵⁾ and by Pascual and Aranda⁽⁵⁹⁾.

Catabolic influences on proteostasis in muscle occur in response to energy deficiency and stress mediated in part by increases in corticosterone which exert an increase in MPB^{(38,39,60,61)} . Such changes are observed with severe energy deficiency in zinc-deficient rats^(28,29) , and in response to endotoxaemia⁽⁶²⁾. The role of glucocorticoids in the regulation of muscle mass has been reviewed by Braun and Marks⁽⁶³⁾ which includes a useful summary of the other main growth inhibitory factor, myostatin. Myostatin (GDF8), the best-known ligand of the TGF-β superfamily expressed in muscle, acts through ActRII receptors to suppress early developmental muscle growth, induce muscle atrophy and antagonise the actions of muscle growth promoters^(64–69). Myostatin is therefore described as acting as a molecular brake that prevents excessive muscle hypertrophy: that is, its inhibition allows for muscle growth and its deletion results in massive hypertrophy⁽⁷⁰⁾. It signals via the activin receptors to phosphorylate responsive Smad proteins which form a transcriptional complex with Smad4 to transcribe poorly defined target genes associated with proteolysis and other growth-inhibitory factors^{(66,67,71,72)} . Recently, an interaction between the MAP kinase JNK and myostatin pathway has been described in relation to exercise-induced muscle hypertrophy. Mechanical stress activated JNK, which initiated muscle growth via phosphorylation of the transcription factor SMAD2, which prevented its nuclear translocation and otherwise activation of growth suppression factors⁽⁷³⁾. JNK2 was highly phosphorylated after maximal-intensity contractions⁽⁷⁴⁾. Atwaters and Hughes suggest myostatin is involved in neurotrophic signalling in muscle⁽⁷⁵⁾. Taken together, this literature suggests that, although the TGFβ/myostatin system is unlikely to be involved in the nutritional regulation of muscle growth, it may well play a role in mechanotransduction pathways.

The mechanotransduction shown in Fig. 1 involves bone length growth acting to mediate muscle fibre length through passive stretching and muscle force development acting to increase both muscle fibre cross-sectional area (CSA) and bone mineralisation. The targets in muscle are activation of satellite cells (SCs), remodelling of the extracellular matrix and myogenesis, which increases the capacity for myofibrillar protein accretion. Passive stretch mediates rapid and marked skeletal muscle hypertrophy in avian models in which the wing is weighted, stretching the anterior latissimus dorsi muscle supporting the wing, and was investigated by Laurent in the adult fowl^(20,21,76) , and by others in the adult quail^(17,77) . The treatment induced myogenesis, ECM remodelling as indicated by collagen synthesis⁽⁷⁸⁾, increases in ribosomal capacity and activity and transient increases in MPB.

Protein-mediated bone and linked muscle growth was demonstrated in rat studies of growth regulation of the tibial length and the associated gastrocnemius muscle weight^(79–82), with the observed relationship informative about likely physiological control. In theory, given that post-natal muscle growth must enable it to acquire the necessary strength to support increasing body weight against gravity, the relationship between increase in muscle weight (W) and tibial length (L) is predictable ¹ as W∝L ⁴⁽⁶⁾, and in well-fed rats a similar relationship is observed: that is, W∝L ^3.85(79). This is similar to that observed in boys and girls for quadriceps weight and height (W∝H ⁴)⁽⁸³⁾. Mechanotransduction of muscle growth by gravitational loading in response to increasing body weight is an obvious mechanism explaining this relationship between muscle weight and tibial length growth.

Stretching by tibial length growth was suggested by the time course of growth inhibition in young rats with protein deficiency. Body weight growth stopped more or less immediately, but inhibition of tibial length growth was markedly delayed during which time some muscle growth occurred as the rats got longer and leaner⁽⁷⁹⁾. Taken together, these relationships are consistent with the model shown in Fig. 1: that is, the mechanotransduction of muscle growth derives from both stretching consequent to bone length growth and force development within muscle during its action against the gravitational loading. Subsequent more recent work (discussed below in ‘Mechanotransduction during post-natal growth as indicated by animal models’) has been able to separate and better characterise these two influences.

Finally, remodelling of the extracellular matrix, necessary for increases in muscle cross-sectional area and length during growth, is identified in Fig. 1 as a target of mechanotransduction. Proteoglycan synthesis is active in growing rat muscle and is reduced to very low levels as growth slows and ceases with protein deficiency⁽⁸⁰⁾. Measurements of the time course of changes in insulin, plasma IGF-1, muscle IGF-1, (i.e. IGF-1 extracted from muscle) and rates of myofibrillar and proteoglycan synthesis in muscle during the growth inhibition by protein deficiency^{(80,81,84–86)} pointed to potential mechanisms: the reductions in insulin, (and plasma IGF-1, highly correlated with insulin^(81,87) ), were consistent with insulin mediating the reduced MPS, whereas the delayed reductions in both muscle IGF-1 and proteoglycan synthesis suggested an autocrine/paracrine action of IGF-1 associated with ECM remodelling^{(81,84,85,87)} . As discussed in more detail below, local IGF-1 production is now known to be part of the mechanotransduction of stem cell activation and myogenesis.

The myonuclear domain: growth in number and size during development, and relationship with protein turnover

Given that myofibres form a syncytium containing hundreds or thousands of post-mitotic myonuclei beneath the sarcolemma, with no discernible structural compartmentation between them, the myonuclear domain, (MND), is a necessary concept. It is a volume of cytoplasm within which proteostasis is controlled by a single nucleus. The concept remains valid even with the recent suggestion that in some but not all mouse muscles there may be endoreplication of a very small fraction of myonuclei resulting in some polyploidy⁽⁸⁸⁾. Polyploidy is a strategy which allows a nucleus to manage a larger volume of cytoplasm, and is best understood for the liver⁽⁸⁹⁾.

Our early studies of turnover and growth of the mainly fast type II muscles, (gastrocnemius and quadriceps), shown in Fig. 2, based on total muscle DNA and protein/DNA ratios as measures of MND number and size indicated a complicated two-phase relationship between turnover and growth in MND size during development⁽⁷⁾. The very high turnover at weaning (day 23), and in early post-natal life, most likely related to intense muscle fibre remodelling⁽¹⁶⁾, fell in concert with some growth in apparent MND size. In later life (days 130–330), turnover was low and stable and MND size growth occurred mediated by a >50% increase in the rate of protein synthesis per myonucleus, (per unit DNA), associated with a comparable increase in myonuclear ribosomal capacity indicated by the RNA/DNA ratio. This response enabled the MND size to increase without any further reduction in turnover. Measurements in the adult animals showed that muscles with mainly slow oxidative fibres had faster turnover rates and lower protein/DNA ratios than fast oxidative or glycolytic muscles^(12,90–92) .

These interpretations were made on the assumption that total muscle DNA could serve as a measure of muscle myonuclei and hence MND number. In fact, in adult skeletal muscle at least ten mononucleated cell types are present in addition to the myonuclei in the multinucleated myofibre⁽⁹³⁾. This means that our observations about changes in MND number and size based on total muscle DNA could have been artefacts of changes in numbers of other cell types in muscle. Recently, the changes in the cellular sources of nuclei in muscle during development have been quantified⁽⁹⁴⁾. Myonuclei account for 59% or less, up to weaning, rising to over 80% in the adult (Fig. 3). This means that the early, post-weaning increase in total DNA which we took to indicate intense myogenesis is, if anything, an underestimate of the actual accumulation rate of new myonuclei, given the increasing proportion of myonuclei among the other cell types. Nevertheless, calculation of changes in MND size during the intense fibre remodelling occurring in this early growth phase⁽¹⁶⁾ cannot be made from the whole muscle protein/DNA ratio with any certainty. However, changes in MND size and number during the subsequent growth phase after turnover had stabilised were likely to be a reasonably accurate representation of the reality.

Fig. 3. Distribution of cell types in the tibialis anterior hind limb muscle across the life span of the mouse.

Cell types were identified by their pattern of gene expression measured by single-nucleus RNA sequencing. Myonuclear number varied from 55% in early life to 73–84% in adult and aged animals, with satellite cells ranging from 3–5% in early life to 1–2% in adult and aged muscle. FAPS, fibro-adipogenic progenitor cells are a muscle interstitial mesenchymal cell population, which support satellite cell differentiation during muscle growth. Modified from Petrany et al. 2020⁽⁹⁴⁾ under a Creative Commons Attribution BY-NC-ND License.

As for the inverse MND size and turnover relationship with fibre type, the higher protein/DNA ratios of fast compared with slow muscle were consistent with early histological studies of fibre cross-sectional area and myonuclear number in different muscle types. In fast compared with slow muscle fibres, half the myonuclear number per fibre was observed in fibres of the same breadth, resulting in a 75% greater CSA per nucleus⁽⁹⁵⁾. Furthermore, more recent discussions of the determination of MND size based on isolated fibre studies indicate that the fast and slow fibre type–MND size relationship has become a more widely known feature, including the inverse relationship between MND size and turnover rate⁽⁹⁶⁾. Direct measurement of MND size calculated from three-dimensional reconstructions in single muscle fibre segments⁽⁹⁷⁾ confirmed that actual MND volumes do correspond to our measured protein/DNA ratios. The largest MND size was observed in muscle fibres expressing fast myosin heavy chain (MyHC) isoforms. For the rat at 6 months, the soleus muscle, with 92% type 1 MyHC isoforms, had a MND size which was 60% of the weighted mean value of the gastrocnemius with type IIX (62.5%) and IIXB (37.5%) MyHC isoforms. For comparison, the relative protein/DNA ratios of these muscles indicated that MND size in the soleus was 54% that of the gastrocnemius muscles⁽¹²⁾.

Most recent discussions of the myonuclear domain have focused on whether and/or how MND size is limited^{(96,98–100)} , and the linkage of MND size and number to the role of SCs during induced muscle hypertrophy rather than post-natal development. Murach et al. ⁽⁹⁸⁾ note the evidence that type 2 fibres can undergo hypertrophy without myonuclei acquisition as evidence of myonuclear domain flexibility. The evidence base in these discussions comes from the synergist ablation model of hypertrophy of the plantaris muscle in which, normally, DNA accumulation occurs from SC activation. However, hypertrophy still occurs in the absence of SCs in transgenic mice⁽¹⁰¹⁾, through increased myonuclear transcriptional activity resulting in an increase in MND size. This is consistent with our observations⁽⁷⁾ shown in Fig. 2 of an apparent increase in MND size and myonuclear transcriptional and translational activity during the adult growth phase in the male rat as a feature of normal growth and development of muscles with predominantly type 2 fibres.

As for the differences in turnover rate in muscles characterised by fast or slow fibre types identified above which determine MND size, the assembly and turnover of the contractile apparatus remains largely mysterious apart from a much better understanding of the proteolytic systems which are likely to be involved⁽⁸⁾. We demonstrated a heterogeneity of turnover of individual myofibrillar proteins⁽¹⁰²⁾, and suggested a turnover mechanism in which exchange of protein subunits within the sarcomere occurred prior to the interaction of subunits in peripheral myofilaments with the proteolytic system⁽¹⁰³⁾. We speculated that subunit exchange could be mediated by the cytoplasmic flow during each contraction cycle, and this is supported by recent observations. Contracting sarcomeres do change volume with flow into and out of the myofilament lattice, enabling delivery of ATP and other metabolites, that is, ‘advective flow’⁽¹⁰⁴⁾. We also speculated that the less regular structures of type 1 slow muscle could aid subunit exchange and interaction with the proteolytic systems. Recent work on muscle structure and force transmission has demonstrated that within myofibres there is a continuous myofibrillar matrix linked together by frequently branching sarcomeres. Furthermore, the frequency of myofibril branching and splitting in mouse muscles is three to four times higher in slow fibres (soleus) than fast fibres (gastrocnemius) and higher in early compared with late post-natal development⁽¹⁰⁵⁾. These structural differences and the need for sustained contractions in type 1 slow fibre muscles compared with type 2 fast muscles with a consequent more sustained cytoplasmic flow could explain the higher turnover rates of tonic slow muscles.

Satellite cell activity during post-natal development

Although the myonuclear accumulation from SCs was verified by the radiographic–histochemical determination of SC-to-myonuclei conversion in growing rat soleus and extensor digitorum longus (EDL) muscles⁽¹⁰⁶⁾, the issue of whether SC addition is/is not obligatory for work-induced skeletal muscle hypertrophy was debated in a series of point–counterpoint articles^(107–109). Moreover, the issue was reviewed again more recently^(110–112). Whilst it appears clear that any work-induced hypertrophy, (e.g. progressive weighted wheel running), occurring in the absence of SCs results in functionally impaired muscle⁽¹¹³⁾, there is a consensus that post-natal muscle growth requires the activation, proliferation and fusion of SCs as the source of new myonuclei which enable an increased number of MNDs. Bachman and Chakkalakal have recently reviewed the role of SCs in growth of mouse muscle from birth to puberty onset to young adulthood⁽¹¹⁴⁾. They argue that the mouse is an appropriate model for human muscle growth given that, in terms of myofibre size and myonuclear number, human myofibres scale similarly to that of mice. In fact, whilst active accretion of new myonuclei from SCs from birth to weaning has been widely accepted following early observations from Moss and Leblond⁽¹¹⁵⁾, the developmental stage when myonuclear accretion finally ceases and SC quiescence is established has been controversial. For example, in our rat muscle data, total muscle DNA present at weaning had increased by 2.5 fold at day 46 and by 4.1 fold at 330 d (Fig. 2)⁽⁷⁾. However, White et al. reported that, in mice, myonuclear addition ceased after weaning at day P21⁽¹¹⁶⁾. Nevertheless, Bachman and Chakkalakal argue that evidence from a variety of approaches, including lineage tracing, (where actual transfer of SC DNA into myonuclear DNA is traced), indicates that relatively robust SC activity and progression to myonuclei occurs up to puberty or even into young adulthood. They also document much higher accumulation of new myonuclei in myofibres of slow oxidative soleus muscle compared with faster glycolytic EDL muscle, as previously also observed by Schultz⁽¹⁰⁶⁾. This is consistent with our assumptions from total DNA measurements that the MND size is smaller in the slow than in fast muscles⁽⁹⁰⁾.

As discussed in more detail elsewhere⁽⁵⁾, after the activation of SCs, their transformation into myogenic cells and eventual myonuclei is mediated by a series of transcription factors, of which the best known⁽¹¹⁷⁾ are the MyoD family of four myogenic regulatory factors. Myf5 and MyoD are early markers for myogenic commitment to the myogenic programme, myogenin is a direct target of MyoD, and both myogenin and Mrf4 control expression of the terminal differentiation genes in differentiated myocytes. These fuse with the myofibre and express muscle genes such as myosin heavy chain (MHC). A detailed account of these developmental changes in SCs isolated from mouse hind limb muscles at different ages between birth and adulthood at 49–56 d as revealed by flow cytometry⁽¹¹⁸⁾ is shown in Fig. 4. During this time the composition of the myogenic population changed from a mixture of myogenic cells in various stages to an essentially single population of quiescent cells with two peak periods of terminal differentiation at the early post-natal and prepubertal phases of muscle growth.

Fig. 4. Schematic representation of the mechano-transduction of skeletal muscle myogenic cell dynamics during muscle post-natal growth.

Post-natal myogenesis is mediated by forces imposed on and generated by muscle, acting on SCs in their niche. Signalling pathways known to be activated include p38 mitogen activated protein kinase (MAPK)^(258,262) , IGF1/Pi3K/AKT⁽³³⁹⁾, Wnt⁽³⁴⁰⁾, Ca²⁺/calmodulin-dependent protein kinase (CaMK)⁽³⁴¹⁾, TNFα⁽³⁴²⁾ and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)⁽¹²¹⁾. The myogenic programme of SC activation and myogenesis is mediated by a group of specific transcription factors, the myogenic regulatory factors. The sequence shown was identified in mouse muscle⁽¹¹⁸⁾ on the basis of the expression markers shown. Myogenesis commences with the activation of SCs expressing PAX7 in their niche, which then express MYOD as progenitors and cease to express PAX7, allowing progression towards differentiation as precursors (PAX⁻MYOD⁺). These differentiate expressing MYOG and start to express muscle genes such as myosin heavy chain while fusing with the myofibre to provide a new myonucleus. At each of these stages these cells can enter the cell cycle and replicate, recognised by expression of Ki-67 protein. Also the progenitor cells can return to quiescence as PAX7⁺MYOD⁻ cells. Two successive phases of expansion and differentiation were identified: an early post-natal phase and a post-weaning/prepubertal phase, when precursor production, differentiation and myofibre fusion is most rapid. Activated SCs mediate remodelling of the basal lamina, secreting both metalloproteinases and laminin proteins as well as communicating with fibrogenic cells to regulate an appropriate amount of collagen production. In adulthood, activation and differentiation ceases with SCs converted to quiescence, a transition mediated in part by sex hormones through Notch signalling. Notch also regulates production of collagen V by the SCs to stabilise the niche. SCs secrete vascular endothelial growth factor mediating angiogenesis which also helps to maintain quiescence.

Modified from Gattazo et al. 2020⁽¹¹⁸⁾ under the CC BY-NC-ND license with additions as described in the text.

The signalling pathways involved and the molecular mechanisms at the level of transcriptional and epigenetic regulation of the transitions shown in Fig. 4 have been recently reviewed by Segalés et al. ^(119,120) and by Rugowska, Starosta and Konieczny⁽¹²¹⁾, and are discussed further below in ‘Satellite cell signalling’.

After emerging in embryogenesis, a key property of SCs like all true stem cells is their ability to survive long term in their ‘niche’ between the myofibre sarcolemma and the basal lamina, in a cell-cycle-arrested specific state of low cellular activity called quiescence^(122–126). This allows for any subsequent growth and regeneration late in life through activation, replication and differentiation into a fully competent myonucleus which can direct myofibre reconstruction. Notch signalling is particularly important for the maintenance of quiescence⁽¹²⁶⁾. Notch signalling promotes cell–cell communication through the interaction between transmembrane ligands on one cell and the Notch protein receptor on the other. Unlike activated myogenic cells, quiescent SCs express high levels of the Notch receptors and intracellular mediators of Notch nuclear activation of quiescence genes^(127,128) . Endocytosis of the Notch ligand allows the intracellular domain of Notch receptors to translocate into the nucleus and activate Notch target genes which maintain quiescence. This sequence is initiated by E3 ubiquitin ligases, one of which, Mib1, has been shown to be induced by the sex hormones within the myofibre face of the SC niche⁽¹²⁹⁾. This activates Notch signalling in cycling juvenile satellite cells, causing them to be converted into adult quiescent satellite cells. Notch also regulates the expression of collagen genes. Autocrine production of collagen V by SCs is used as a feed-forward mechanism to stabilise the niche and reinforce the quiescent state⁽¹³⁰⁾. Finally, Notch signalling in SCs is involved in angiogenesis within the niche. Quiescent SCs secrete the vascular endothelial growth factor (VEGF) to attract endothelial cells and bind the endothelial-cell-derived Dll4 Notch ligand to maintain quiescence⁽¹²²⁾. Some of these mechanisms around notch signalling are shown in Fig. 4.

Another important issue related to MND expansion is the need for associated ECM remodelling to allow the increased CSA of muscle fibres (i.e. ‘bag’ enlargement as discussed below in ‘Evidence for limitation of muscle growth through a ‘bag full’ signal’). The maintenance of this complex connective tissue structure (see Purslow, 2020⁽¹³¹⁾) is essential to enable force transmission from contracting sarcomeres within muscle fibres to the adjacent tendon and bone^(132,133) . Increased collagen synthesis was a very early event in our weighted wing muscle studies⁽⁷⁸⁾, and SCs play an important role in regulating this process. Early after activation they secrete both metalloproteinases (MMPs), which can degrade the ECM, and also substrates for remodelling such as the laminin glycoproteins⁽¹³⁴⁾. Activated SCs also interact with interstitial fibrogenic cells, the source of most of the ECM proteins, which require regulation to prevent excessive collagen synthesis and fibrosis. Fry and colleagues⁽¹³⁵⁾ showed that this involves release of exosomes by activated SCs into the extracellular niche containing specific microRNA molecules which regulate ribosomal binding protein 1 (RBP1). This is a master regulator of collagen biosynthesis, preventing excessive collagen production by fibrogenic cells. This group subsequently showed SC-derived exosomes can also transfer microRNAs to the myofibre which regulates MMP synthesis⁽¹³⁶⁾. The pivotal role of extracellular matrix remodelling during muscle hypertrophy has recently been reviewed with the suggestion that ECM remodelling is a rate-limiting step in load-induced hypertrophy⁽¹³⁷⁾, and this may also be true for post-natal growth.

Taken together, the evidence base for a central role for SC-derived myogenesis and ECM remodelling in post-natal muscle growth is now overwhelming.

Role of IGF-1

It is commonly accepted (e.g.⁽¹³⁸⁾) that the primary action of IGF1 in mammals is to promote structural growth, with the primary action of insulin limited to regulation of postprandial nutrient utilisation: and in the present context, nutrient utilisation includes protein deposition within the myofibre. While there is no dispute about insulin’s role^{(42,44,139–143)} , equivalent detail is, however, lacking for the role of IGF-1, especially in relation to whether any role in proteostasis can be identified which is distinct from that of insulin. Furthermore, whilst the GH–IGF-1 axis has become much better understood in relation to bone growth⁽¹⁴⁴⁾, and despite the considerable evidence from gene knockout studies, of both IGF-1 and its receptor, of its importance for post-natal growth^{(138,145–148)} , details of the action of IGF-1 in post-natal muscle growth have been difficult to untangle and controversial for several reasons. Firstly, the biology of the two hormones, their receptors and the hormone receptor interactions is extremely complex^(149,150) . Secondly, the IGF-1 literature is controversial with some issues not entirely resolved. Thirdly, although IGF-1 and insulin are generally identified as ‘mitogenic’ and ‘metabolic’, respectively, they each signal through common pathways and it was argued in 2012⁽¹⁵¹⁾ that no evidence had yet emerged for signalling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, with only limited evidence for differential activation of signalling mechanisms that are common to both receptors. What this means is that the differences between IGF-1 and insulin must relate to the ligand–receptor interactions and the cellular context in which they occur, and this will be explored here.

The IGF/insulin system consists of three primary ligands (IGF-1, IGF-2 and insulin), six ligand-binding proteins, (IGFBP1–6), and five receptors, IGF1R, IGF2R, IR-A and IR-B, and a hybrid IGF-1/insulin receptor (HybR)⁽¹⁵²⁾. Furthermore, unlike insulin, which comprises mainly a single species acting in an endocrine role to mediate postprandial responses to food intake, the IGF-1 ligand exists in multiple isoforms^(149,153) and can exert both endocrine and autocrine/paracrine actions, with the latter assumed to be particularly important for muscle growth. The isoforms of IGF-1 result from alternate splicing during transcription of the six exons of the IGF-1 gene, which gives rise to a pre-pro-IGF-1 transcript comprising the 70-amino-acid IGF-1 peptide, preceded by one of two signal peptides, which direct its secretion, and followed by one of three different C-terminal E-peptide extensions^(154,155) . After cleavage of the signal peptide, the pro-IGF-1, either IGF-1A, IGF-1B or IGF-1C, can be cleaved to give the IGF-1 and E-peptide, or secreted as the pro IGF-1 without cleavage⁽¹⁵⁶⁾. All three isoforms are expressed in human muscle (A>>B>C)⁽¹⁵⁷⁾, with the A and B isoforms predominant in rodent skeletal muscle, although the A isoform is by far the most abundant^(156,158) . To increase the complexity, N-glycosylation in the E-peptide of the predominant IGF-1 isoform (IGF-1A) can occur. Glycosylation reduces the potency of activation of the IGF-1 receptor but protects the ligand from degradation by the proteasome⁽¹⁵⁹⁾. This may enable it to be stored in the ECM until needed.

As for the relative biological significance of the different isoforms of IGF-1, there is a contentious literature relating to whether the B (rodent)/C (human) isoform is a specific factor involved in the mechanical stimulation of muscle growth identified as mechano-growth factor (MGF)⁽¹⁶⁰⁾. MGF was also used to describe the biological activity of a peptide encoded within the C-terminal E-peptide reported to have an independent mitogenic biological activity⁽¹⁶¹⁾. However, the independent biological activity of this peptide was questioned⁽¹⁶²⁾, and an editorial entitled ‘The Fall of Mechanogrowth Factor?’ published in 2014 called for experiments that critically and objectively test the MGF hypothesis⁽¹⁶³⁾. It was subsequently showed that viral expression in mouse muscle of the A and B IGF-1 isoforms in which the IGF-1 component was inactivated demonstrated that the B peptide did induce muscle hypertrophy that was IGF-1 receptor dependent but the increased mass was associated with a loss of muscle strength⁽¹⁶⁴⁾. This raised questions about its physiological relevance. More recently, overexpression of the proIGF-1A in mice induced a pronounced hypertrophic phenotype in mice at 6 months, which was not observed with proIGF-1B overexpression⁽¹⁶⁵⁾. In these mice, the EDL muscle exhibited a shift to larger fibres and was capable of production of >30% higher tetanic force. Also, most of the IGF-1 in the IGF-1A transgenic muscle was the mature peptide, whereas overexpression of the IGF-1B pro peptide resulted in mainly unprocessed or partially processed isoforms in muscle. Barton’s group investigated the relative roles of the IGF-1 isoforms⁽¹⁵³⁾ and concluded that, although it appears beneficial for muscle to accumulate glycosylated pro-IGF-1Ea within a local muscle reservoir, glycosylation of IGF-1A reduces its activity. They speculated that, although multiple strategies exist to eliminate the glycosylated form, the expression of a non-glycosylated form as IGF-1B would be simpler when a non-glycosylated form was needed, for example after mechanical stimulation of muscle. Overall, the physiological importance of the IGF-1 isoforms remains by no means clear, and in any case it is most likely that E-peptides are removed in the extracellular milieu, leaving the mature and active IGF-1 to interact with the receptor⁽¹⁴⁹⁾.

Knowledge of the regulation of the autocrine/paracrine expression of the IGF-1 gene in muscle is limited. While expression can be mediated in response to growth hormone which signals through the JAK/STAT pathway⁽¹⁶⁶⁾, it is not exclusively dependent on GH⁽¹⁴⁷⁾. It is likely that myogenic regulatory factors recognise translation initiation sites on the IGF-1 gene. It has been shown in C2C12 myocytes that an IGF-1 promotor contains a response element to an nuclear factor of activated T cells (NFAT) transcription factor which is responsive to calcineurin signalling⁽¹⁶⁷⁾. As discussed elsewhere⁽⁵⁾, changes in free Ca²⁺ in response to stretch-induced Ca²⁺ influx into satellite cells is an important mechanism for regulating gene expression. Calcium binds to calmodulin and then activates calcineurin, the calcium- and calmodulin-dependent serine/threonine protein phosphatase which dephosphorylates NFAT. Also in mouse myoblasts GH induced the nuclear localisation of NFATc2 and IL-4 expression: IL-4 is a cytokine essential for myoblast recruitment⁽¹⁶⁸⁾. There is very little information about any nutritional regulation of muscle IGF-1 expression, but in non-proliferating rat L6 muscle cells, increased amino acid levels increased expression of IGF-1 mRNA⁽¹⁶⁹⁾.

As for the cellular source of IGF-1 within muscle, it appears to derive from the muscle fibres as well as from satellite cells and other cells within the extracellular matrix including fibro-adipogenic progenitor cells⁽¹⁷⁰⁾, endothelial cells of the microvasculature⁽¹⁷¹⁾, and the nervous system⁽¹⁵³⁾. However, regardless of its source, IGF-1 in the extracellular matrix of muscle occurs as a complex with an IGF-1 binding protein so that expression of IGF-1 activity requires binding protein cleavage by protease action allowing the IGF-1 to bind to its receptor.

Of the six IGF-1 binding proteins, all of which bind IGFs with an affinity that is one order of magnitude higher than the binding of IGFs to their receptors,⁽¹⁴⁹⁾, IGFBP-3 is the most abundant in the circulation where, together with an acid-labile subunit glycoprotein, it forms a stable ternary complex with IGF-1⁽¹⁷²⁾. Thus, any endocrine action of circulating IGF-1 requires cleavage of the ternary complex prior to IGF-1 receptor action. This means that, in contrast to insulin, serum IGF-1 is less influenced by the immediate fed state. However, the hepatic, GH-mediated IGF-1 expression pathway, the source of most circulating IGF-1, is regulated by insulin, enabling hepatic IGF-1 secretion in the fed state^(173,174) . This explains the mainly parallel changes in serum insulin and IGF-1 in response to feeding and protein deficiency which we observed in our early studies^(81,84,85) . Release of the circulating IGF-1 from the ternary complex is achieved by the action of a highly specific metalloproteinase, binding tightly to glycosaminoglycans present on the surface of cells⁽¹⁷⁵⁾. By cleaving the IGFBP, the protease can function within tissues as a growth-promoting enzyme, releasing bioactive IGF in close proximity to the IGF receptor⁽¹⁷²⁾.

Within muscle the biology of the IGFBPs is by no means understood, but IGFBP 3, 4, 5 and 6 are expressed, and deletion of IGFBP 3, 4 and 5 reduces post-natal muscle growth⁽¹⁵³⁾. Thus, these IGFBPs are required to coordinate IGF-1 bioavailability within muscle, both maintaining stored or recently secreted IGF ligands away from receptors and making them available when required through cleavage by proteinases within muscle.

As for IGF-1 signalling, it can be assumed that the ‘anabolic’ signalling of IGF-1 and metabolic signalling of insulin are mediated through their respective highly homologous tyrosine kinase receptors, although, as indicated above, once signalling has been initiated it may well be that similar target processes can be activated⁽¹⁵¹⁾. Of the five receptors available to IGF-1 within muscle, IGF-1R, IGF-2R, IR-A and IR-B, and a hybrid IGF-1/insulin receptor, HybR^(152,176) , the latter hybrid receptors present the major form of IGF-1 receptors, with which IGF-1 has high affinity and insulin has a lower affinity^(177,178) . IGF-1 has low affinity for the two insulin receptors, IR-A and IR-B. These result from alternative splicing of the primary IR transcript, with IR-B, containing an extra twelve amino acid residues, most likely acting as the functionally important insulin-specific receptor in the main insulin target tissues in post-natal life. Although the IGF-1R and IR are very similar in the kinase domains and respond similarly to ligand bindings in terms of autophosphorylation of tyrosine residues, the IGF-1R C-terminal tail differs with only 44% of sequence identity with IR and may signal differently from the IR⁽¹⁷⁹⁾. Thus, the tyrosine kinase activity of the IGF-1R is normally inhibited by GSK-3β-mediated serine phosphorylation in the IGF-1R C terminus, but IGF-1 binding reverses this, allowing kinase activity.

In mouse muscle, the relative amounts of the insulin and IGF-1 receptors change from a 4:1 excess of IGF-1 receptor in myoblasts (day −2) and myotubes (day +7) to a 4:1 excess of the insulin receptor at 8 weeks⁽¹⁸⁰⁾. Various knock-out studies suggest that IR and IGF-1R have overlapping anabolic and metabolic roles in skeletal muscle^{(153,181–183)} , and at high concentrations, insulin and IGF-1 can bind and initiate signalling with the opposite receptor. In each case, intracellular signalling occurs via similar cascades, beginning with tyrosine phosphorylation of insulin receptor substrates (IRSs), which leads to activation of the phosphatidylinositol 3-kinase PI3K/Akt pathway, as well as other downstream signals (see ‘Insulin/IGF-1 signalling’ and Fig. 5 for more discussion). Mice with single-receptor deletions show only a modest fall in muscle mass for the IR^−/−, no change with the IGF-1R^−/− but a marked fall with both deletions^(180,184) . Similarly, Spangenburg et al. ⁽¹⁸⁵⁾ reported that, in a transgenic mouse model expressing a dominant negative IGF-1 receptor in skeletal muscle and with the insulin receptor also dysfunctional, muscle growth was impaired. However, in these animals, muscle hypertrophy and Akt–mTOR signalling induced by functional overload was not impaired, suggesting that growth in response to functional overload must involve mechanisms other than the insulin/IGF-1 receptor.

Fig. 5. Cross-talk between insulin/IGF and Ras/Raf/ERK Map Kinase signalling cascade.

The insulin receptor subunits are illustrated at the top in red and blue. Ligand binding triggers tyrosine phosphorylation of the insulin receptor substrate (IRS) or SH3-containing protein (SHC). The IRS protein binds and activates phosphoinositide 3-kinase (PI3K), which generates phosphatidylinositol (3,4,5)-trisphosphate (PIP3) that recruits phosphoinositide-dependent kinase-1 (PDK1), SIN1 (a MAPK-associated protein 1) and AKT to the plasma membrane. AKT is activated upon phosphorylation by PDK1 and by the SIN1–mTORC2 complex. AKT acts on a number of downstream pathways, as illustrated, of which phosphorylation of the Tuberous Sclerosis 1 & 2 Protein (TSC1/TSC2) complex activates mTORC1 via RHEB GTP. mTORC1 mediates phosphorylation of S6K and SREBP1, which promote protein and lipid synthesis, respectively. AKT-mediated phosphorylation of the FOXO transcriptional factors causes their sequestration in the cytoplasm, which inhibits their influence upon transcriptional activity. Insulin and or IGF-1 activation of MAPK signalling by the small GTPase ras, commences by targeting the GTP exchange factor SOS associated with the small adapter protein Grb2 via either phosphorylation of IRS or by the signalling adapter protein SHC. In each case this promotes GDP/GTP exchange on p21ras, which activates the ras/raf/MEK/ERK1/2 cascade. Mitogens such as fibroblast growth factors (FGFs) and mechanical signals also activate Map Kinase signalling at this point. Recent studies suggest effective insulin receptor activity requires its linkage via plakoglobin, a scaffold protein at the sarcolemma, to the dystrophin glycoprotein complex which anchors the myofibre cytoskeleton to the extracellular matrix⁽³⁴³⁾. It has yet to be shown whether mechanical activation of insulin signalling involves this linkage. Activated ERK can stimulate transcriptional activity by inducing expression of the proto-oncogene C-MYC which activates the ribosomal RNA Polymerase complex (Pol I regulon)⁽²⁸⁷⁾ (see ‘Regulation of the capacity for protein synthesis’). ERK also mediates direct phosphorylation of ELK1 (ETS domain-containing protein) and by indirect phosphorylation of cFOS through MAPKAPK1 (MAPK-activated protein kinase-1). Transcriptional targets of ERK also include mTORC1 and consequent protein synthesis via the TSC1/TSC2 complex⁽¹⁴²⁾. Insulin stimulates protein synthesis by altering the intrinsic activity or binding properties of key translation initiation and elongation factors (eIFs and eEFs, respectively) as well as critical ribosomal proteins (see also Fig. 7). mTORC1-mediated phosphorylation of 4E-BP1 and S6K plays an important role in stimulating translation initiation and elongation^(142,304) . Stimulatory phosphorylation sites (tyrosine pY, threonine pT, serine pS and both serine and tyrosine pSY) are highlighted in green, and inhibitory sites are highlighted in red. Copied and modified from White and Kahn⁽¹³⁹⁾ under the terms of the Creative Commons Attribution License BY-NC-ND.

As for any endocrine action of IGF-1 on MPS on muscle, no influence which is distinct from insulin, long identified as the primary regulator of MPS in rodent muscle^{(33,37,44,141)} , has been unequivocally established. Experimental studies involving the infusion of recombinant IGF-1 with in vivo measurement of MPS have either failed⁽¹⁸⁶⁾ or succeeded to show an influence⁽¹⁸⁷⁾, although in the latter case it was not shown whether the action of IGF-1 was achieved via the insulin receptor.

The cross-receptor action of insulin and IGF-1 and the role of IGF-1BPs in regulating IGF-1 signal transduction at its receptor in vivo makes for difficulties in interpreting in vitro studies of IGF-1 action on muscle cells. For example, a widely cited cell culture study with C2C12 myotubes showing hypertrophy in response to added IGF-1⁽¹⁸⁸⁾ could well have achieved the effect of IGF-1 on proteostasis through its signalling via the insulin receptor. This would have been shown if the response of these cells to insulin had also been examined. As already referred to above, rat L6 muscle cells respond to increased amino acid concentrations by increasing expression of IGF-1 mRNA⁽¹⁶⁹⁾, and this is associated with an increased rate of MPS. However, the investigators showed that the increased IGF-1 was not responsible for the increased MPS because neither inhibition of IGF-1 signalling by a variety of inhibitors nor knockdown of the IGF-1 gene altered stimulation of protein synthesis by amino acids. In fact, the increased IGF-1 expression was shown to be related to myogenesis within the L6 muscle cells. Thus, these studies imply that the signalling induced by the amino acids not only activates MPS but also induces expression of IGF-1, which mediates satellite cell proliferation and myogenesis.

The importance of IGF-1 for exercise-induced muscle growth was the subject of a vigorous debate. This involved a proposal by Stewart and Pell⁽¹⁸⁹⁾ that IGF-1 was the main driver of muscle growth, which was disputed^(190,191) . With hindsight it is difficult to understand much of the debate which in part was semantic in terms of the meaning of muscle growth (work-induced hypertrophy as distinct from post-natal growth) and in part was related to the relevance of MGF as an important mediator of IGF-1 action work induced hypertrophy⁽¹⁹²⁾. In fact, at the time an autocrine/paracrine role for IGF-1 in the regulation of SC activity following intense damaging muscle contractions in adult human muscle was known⁽¹⁹³⁾, and subsequently IGF-1 was shown to co-localise with muscle satellite cells following acute exercise in human adults⁽¹⁹⁴⁾. Furthermore, there was no disagreement about the importance of IGF-1 for embryonic growth, and physiological myofibre hypertrophy during post-natal development (e.g.⁽¹⁹⁵⁾) through its paracrine/autocrine role in the stimulation and proliferation of satellite cells. Early studies showed muscle IGF-1 to be regulated by muscle stretching^(196–199), and by responses to injury when monocytes and macrophages in muscle secrete IGF-1 probably through a GH-independent mechanism⁽²⁰⁰⁾.

As indicated in ‘Proteostasis and turnover during skeletal muscle growth: the evidence base for the growth model’, our early studies of the responses of bone and muscle growth to graded protein deficiency pointed to the inhibition of MPS reflecting reductions in insulin, and the inhibition of muscle connective tissue synthesis reflecting reductions in muscle IGF-1. Although these were only associative studies, the findings are consistent with the separate regulation during post-natal growth of IGF-1 within muscle and circulating IGF-1, and with muscle IGF-1 being a driver of ECM remodelling in response to mechanotransduction associated with bone length growth^{(79–81,84–86)} .

Since our early studies, the case for an autocrine/paracrine role of IGF-1 in mediating muscle growth has become very strong, yet major gaps remain. The importance of both insulin and IGF-1 for post-natal muscle growth has been clearly shown by the knock-out models which have also shown the separate roles for insulin, in mediating growth in terms of protein deposition and IGF-1 in terms of myogenesis as shown in Fig. 1. To some extent there has been more confusion over the role of insulin than that of IGF-1 because of failure by some reviewers to recognise that insulin’s influence on ‘growth’, that is, protein deposition within the myofibre, is not different in kind from its ‘metabolic’ action in relation to glucose disposal and lipogenesis, (e.g.⁽¹⁸¹⁾). As for the ligand–receptor interactions which regulate IGF-1 action and make it distinct from insulin, the means for regulation clearly exists in terms of both post-translational processing of the IGF-1 transcript isoforms, and interactions with the various IGF-1BPs known to be active in muscle. However, detail is lacking as to how this works during normal growth. Thus, although we can be reasonably certain that satellite cell activation and myogenesis is a target, we have no detailed map describing target processes for IGF-1 action in muscle. Although there is no unequivocal evidence for any role for IGF-1 in myofibre growth in length and CSA other than through myogenesis, it would be surprising if it played no direct role in proteostasis at the level of MPS or MPB. Furthermore, assuming IGF-1 does play an important role in the physiological mechanotransduction of post-natal muscle growth mediated by bone length growth-induced stretching and gravitational loading (as discussed further below), and seems to be involved in activating satellite cells in human muscle following intense damaging muscle contractions⁽¹⁹³⁾ and acute exercise⁽¹⁹⁴⁾, how does this differ from the growth induced by surgically induced functional overload⁽²⁰¹⁾? Growth in the latter case can occur without a functioning IGF-I or insulin receptor⁽¹⁸⁵⁾. This is a puzzle to be resolved.

Regulatory mechanisms and signalling pathways involved in muscle growth

When post-natal muscle growth is considered from a proteostasis perspective, it can be expected to involve the increase in myonuclear capacity for protein synthesis through SC-mediated myogenesis as shown in Fig. 4, and the maximisation of myonuclear transcriptional and ribosomal translational activity as identified⁽⁷⁾. The developmental and nutritionally activated hormones, other mediators such as metabolites⁽²⁰²⁾, including amino acids^(203,204) , cytokines and the mechanical signals⁽²⁰⁵⁾ which provoke muscle growth and hypertrophy, exert their influences on both SCs and on the myofibre through either receptors or focal adhesion complexes of one sort or another⁽²⁰⁶⁾. Each of these connects to signal transduction pathways which activate or inhibit target processes – transcription, translation or proteolysis, which together regulate proteostasis.

Of these pathways driving growth, the multiprotein complex, mTOR complex 1 (mTORC1), plays a central role as a conduit between growth signals and the metabolic processes underlying growth^{(204,205,207)} . It is now known that its cellular localisation may be fundamentally important in the regulation of mTOR activity in response to physiological stimuli. Thus, after mitogen or amino acid stimulation, mTOR translocates to the lysosome where it associates with GTP-bound RHEB (ras-homologue enriched in brain) to achieve full activation, and this translocation is reversed with the lack of anabolic signals^(208,209) . This spatial regulation of mTOR is particularly important in skeletal muscle with mTOR co-localising with the lysosome in basal conditions and then translocating to the cell periphery, in close proximity to capillaries, in response to feeding and resistance exercise⁽²¹⁰⁾. One current suggestion is that mTOR trafficking is important for translational control, allowing close proximity with focal adhesion complexes involved with mechanotransduction, with the microvasculature and associated amino acid transporters, and with the ribosomal translational apparatus which is also close to the sarcolemma⁽²¹¹⁾.

However, in the case of mechanical stimuli, Goodman has argued that, depending on its duration and intensity, rapamycin-insensitive mTORC1-dependent or even mTORC1-independent mechanisms may play a role⁽²¹²⁾. Also, as discussed below, Hornberger’s group have identified some apparent disconnects between mTORC1-dependent hypertrophy and MPS⁽²¹³⁾, and shown that some of the signals from maximal-intensity contractions which mediate muscle hypertrophy involve rapamycin-insensitive pathways⁽⁷⁴⁾. Whilst the roles of insulin/IGF-1^{(180,184,214,215)} , AKT/PKB signalling^(216,217) and the MAPK pathway^{(120,218,219)} have become much better understood, the initiation of mechanotransduction has yet to be described in detail, although plausible focal adhesion-related mechanisms can be suggested⁽⁵⁾.

Mechanotransduction during post-natal growth as indicated by animal models

Any mechanism linking muscle growth to bone length growth must mediate increases in both length and cross-sectional area of individual myofibres within the ECM which supports them. As recently reviewed⁽⁵⁾, the evidence base now includes substantial information about the mechanotransduction of muscle stem cells in their niche and the role of other stromal cells within the ECM and the muscle myofibre. However, this evidence base is heterogeneous in its origins, in terms of both the experimental models from which it derives, and the type of muscle growth which is the specific focus of investigators. Also, the application of knowledge gained from some models of hypertrophy, such as the avian weighted wing model or synergist ablation has been questioned: both models are often described as supra-physiological in terms of the extent of the muscle overload imposed (see Goh et al., 2019⁽²²⁰⁾).

Despite the plethora of recent reviews on skeletal muscle hypertrophy^{(111,221–227)} , there has been only limited reference to post-natal growth (e.g.^(114,228) ). Attwaters and Hughes reviewed cellular and molecular pathways controlling muscle size in response to exercise and argued that muscle stretch and force development need to be considered separately as drivers of muscle hypertrophy⁽⁷⁵⁾. They argue that, although muscle stretch is a specific mechanical stimulus for increases in muscle mass as bones elongate during development, this only involves adding nuclei and sarcomeres at the ends of fibres. This cellular mode of growth is distinct from the increase in fibre CSA at a fixed muscle length observed after increased force development during training. Certainly during post-natal muscle growth of limb muscles, any bone length-growth mediated muscle length increase must be matched by sufficient cross sectional growth to enable the increased force development necessary to support the increasing body weight. In fact there is a four- to five-fold increase in human muscle fibre diameters from birth to adulthood⁽²²⁹⁾. This means that post-natal muscle growth could be considered to reflect both externally applied, bone length-increase stretching and internally generated forces by the increasing work demands made on the muscle by the increasing body weight. Animal models demonstrate each of these separate influences.

Hind limb unloading (hind limbs suspended at the tail) is a model for gravitational unloading as occurs, for example, during spaceflight⁽²³⁰⁾. Very little muscle contraction and force development can occur. Indeed, if started early, this leads to a lack of development, or in older animals, a reduction in the CSA of limb muscles^(231,232) . With suspension of newborn rat pups (day 4) for 3 months continuously, both femur and tibial length growth occurred as usual (although their mineralisation is markedly depressed⁽²³³⁾), and length growth of the hindlimb soleus muscle was unchanged⁽²³⁰⁾. However muscle weight and fibre CSA was reduced by ∼70% and myonuclear number by 60% at 3 months⁽²³⁰⁾. After removal of the tail suspension with a return to normal ambulation, fibre CSA and myonuclear number increases towards control size. What this model shows is that both bone length growth-induced muscle stretch as well as gravitational loading and tension development are needed for post-natal growth and recruitment of satellite cells.

The importance and consequences of force development during a mechanical load has been demonstrated in many different studies (e.g. see Zhu et al., 2021⁽²³⁴⁾). One rat muscle training model⁽²³⁵⁾ involved electrical stimulation of the dorsiflexor muscles in vivo under deep anaesthesia, with force output induced in a regulated way with either high-load pure isometric contractions or low-load concentric contractions. The trained muscles exhibited up to 15% additional growth and increased myonuclei compared with untrained muscles, demonstrating load-dependent muscle hypertrophy. Other models include non-invasive high-intensity treadmill protocols which induced progressive myonuclear accretion and muscle hypertrophy⁽²²⁰⁾. Progressive resistance exercise can be induced with mice trained to pull an increasingly weighted cart with their tail⁽²³⁴⁾. The latter protocol induced a 6–23% increase in mass of muscle from both fore and hind limbs, associated with an overall 50% increase in grip strength, increases in myofibre CSA and myonuclear number, and decreases in interstitial nuclei in several muscles analysed. There is little dispute that, in human adults, resistance training sufficient to induce measurable increases in strength and muscle size (CSA) can activate and increase SCs and myonuclei^(236,237) . For example, an 8-week resistance training regime in young men and women increased muscle strength and type II fibre hypertrophy, and increased SC and myonuclear accretion in proportion to the hypertrophy⁽²³⁸⁾.

Taken together, these various animal models show that mechanotransduction of post-natal growth most likely involves not only passive stretching induced by bone length growth but also the separate influence of force generation during muscle contraction in response to gravity-induced load bearing with increasing body weight. This should allow muscle size to increase in response to overall mechanical demands made upon it. One aspect of the signalling in response to force generation is described below in ‘Phosphoproteomics of maximal-intensity contractions’ and Fig. 6.

Fig. 6. Activation of satellite cells and myogenesis by maximal-intensity muscle contractions.

Phosphoproteomic data from mouse muscle, subject to electrically imposed maximal intensity contractions, identified the three giant spring-like proteins titin, obscurin and the striated preferentially expressed protein kinase (SPEG), and the transcription intermediary factor TRIM 28, as highly phosphorylated. Titin links the Z disk to each end of the sarcomere, obscurin is another giant sarcomeric signalling protein and SPEG interacts with proteins of the sarcoplasmic reticulum and the Z-band protein, desmin. It is highly likely that these proteins initiate force transmission from the sarcomere out of the myofibre activating satellite cells in their niche. The pathway is likely to involve the actin cytoskeleton, integrin-based focal adhesions, the dystrophin-associated glycoprotein complex and transmembrane calcium-dependent cadherin adhesion proteins (see Millward, 2021⁽⁵⁾). In satellite cells, p38 MAPK signalling to MSK1 phosphorylates TRIM28, (also known as KAP1) a transcription intermediary factor which mediates MYOD activation, enabling proliferation, differentiation, and myogenesis. Scheme based on data published by Potts et al., 2017⁽²⁴⁵⁾, Steinert et al., 2021⁽⁷⁴⁾, Lin et al., 2022⁽²⁷⁸⁾, and Singh et al., 2015⁽²⁷⁷⁾.

Insulin/IGF-1 signalling

It is likely that insulin and IGF-1 post-receptor signalling is similar⁽¹⁵¹⁾. Recent increases in our understanding of signal transduction mechanisms driving muscle growth have come from technologies such as transgenics probing specific pathways such as AKT signalling during mouse muscle growth⁽²³⁹⁾, and phosphoproteomics^(240–242) which can reveal both targets and kinases involved in phosphorylations of proteins during signal transduction⁽²⁴³⁾. The latter technology can reveal personalised phosphoproteomic profiles regulated by exercise obtained from single muscle biopsies⁽²⁴²⁾; during the differentiation of proliferating myoblasts into multinuclear myotubes enabling muscle growth⁽²⁴⁴⁾; and following repeated maximal intensity contractions which result in muscle hypertrophy^(74,214,245) (see ‘Phosphoproteomics of maximal-intensity contractions’). The combination of phosphoproteomics with cellular fractionation into different cellular compartments (spatial proteomics) allows spatio-temporal signalling to be investigated such as the stress-induced cellular relocation of ribosomal proteins in response to the in vivo electrical stimulation of muscle contraction⁽²⁴⁶⁾. Key results from both transgenics and phosphoproteomics will be highlighted here.

Insulin and IGF-1 receptors are both very similar tyrosine kinases so that ligand binding induces autophosphorylation of the receptor leading to the recruitment and phosphorylation of a cascade of receptor substrates which activate specific pathways^{(140,215,247)} . Those important for proteostasis and growth include both the PI3K–Akt pathway^(216,217) and the Ras–MAPK pathway⁽²¹⁹⁾. Proud has specifically addressed the regulation of protein synthesis by these pathways⁽¹⁴²⁾. One scheme showing current understanding of these transduction pathways is shown in Fig. 5 ⁽¹³⁹⁾. A phosphoproteomic analysis of insulin signalling through this pathway in human myoblasts obtained from cultured human SC cells obtained from adult human skeletal muscle by biopsy indicates that this pathway is expressed in satellite cells⁽²⁴⁸⁾.

As shown in Fig. 5, insulin/IGF-1 binding with its receptor enables activation of AKT through phosphorylation by the phosphoinositide-dependent protein kinase 1 (PDK1). The importance of insulin–P13K–AKT signalling for muscle growth is illustrated by the marked skeletal muscle hypertrophy induced by the muscle-specific over-expression of an activated form of AKT1⁽²⁴⁹⁾. Activated AKT signalling promotes growth and proliferation and regulates proteostasis via a number of downstream substrates, of which tuberous sclerosis complex, (TSC1/2) controls mTORC1 and protein synthesis, and the forkhead box O (FOXO) transcription factors mainly control proteolysis. Phosphorylation of TSC1/2 allows activation of mTORC1 by relieving the TSC-mediated inhibition of Ras homologue enriched in brain (RHEB) which activates mTORC1. This regulates protein synthesis through phosphorylation of its two best-established substrates, S6 kinase 1 and eukaryotic translation initiation factor 4e (eIF4e)-binding protein (4E-BP1). Phosphorylation of FOXO by AKT inhibits their ability to induce proteasomal protein degradation through the muscle atrogenes, Murf-1 and Atrogin-1 (E3 ligases which ubiquitinate target proteins resulting in their proteolysis). As discussed below in ‘Regulation of the capacity for protein synthesis’ and Fig. 7, the role of mTORC1 in regulating mRNA translation is best understood in relation to the 5′TOP group of mRNAs.

Fig. 7. Model for LARP1-mediated regulation of 5′TOP mRNA stabilisation.

As shown, LARP1 is a multimeric protein of which the carboxy-terminal DM15 domain and the mid-domain La-module play important roles in the stabilisation of 5′TOP mRNAs and inhibition of their translation. Such stabilisation is important to enable rapid production of ribosomal proteins and biogenesis factors enabling ribosome assembly when transcription of rDNA is activated during a growth stimulation which activates mTORC1. According to this model when mTORC1 is inactive and the eIF-4E initiation factor is bound to 4E-BPs, DM15 engages with the m⁷Gppp cap and 5′TOP motif, acting like a ‘pendular hook’. This blocks the 7-methyl-GTP (m7GTP) cap initiation site, blocking initiation of translation. At the same time the La-module binds with polyA-binding protein (PABP) at the polyA tail of the mRNA thereby stabilising it⁽³⁴⁴⁾. Activated mTORC1 phosphorylates multiple serine and threonine residues on 4E-BPs releasing eIF4E (Fig. 5) and also on DM15 enabling its release from the m7Gppp cap. This allows for the assembly of the eIF4F initiation complex involving the association of eIF4E with eIF4G which together with eIF4A and eIF3 recruit the 40S subunit of the ribosome for translation initiation. The translation initiation complex is then recruited stimulating mRNA translation, including the unwinding of mRNA secondary structure via the eIF4A helicase, and engagement with the 40S ribosome subunit after phosphorylation of its S6 protein component. Phosphorylation of the La-module does not interfere with its binding with PABP at the polyA tail. Proximity of the mRNA ends in the closed-loop structure is considered to facilitate the re-initiation during translation, that is, ribosomes are more easily engaged in the next round of initiation after termination. Copied and modified from Jia et al. (2021)⁽²⁹⁷⁾ under the terms of the Creative Commons Attribution License BY-NC-ND.

AKT exists in three isoforms in mammals AKT 1–3 with AKT1 (<10%) and AKT2 (∼90%) expressed in skeletal muscle. On the basis that whole-body knockout of both AKT isoforms results in organismal growth retardation and early post-natal lethality, Jaiswal and colleagues utilised both congenital and tamoxifen-inducible mouse models of skeletal muscle-specific AKT deficiency to investigate insulin signalling through the AKT pathway⁽²³⁹⁾. If AKT stimulates protein synthesis via activating mTORC1 and inhibits proteolysis via inhibiting FOXO it might be predicted that deletion of AKT would inhibit, in the fed state, protein synthesis and stimulate proteolysis. In fact, the induced deletion of both AKT1 and AKT2 isoforms reduced muscle size (by 40%), fibre CSA and exercise performance, impaired contractile properties of individual muscles and reduced fed-state protein synthesis. However, it had no effect on fed-state proteolysis, atrogene expression or the expression of key autophagy markers, all of which were increased in the fasted state. This indicated that skeletal muscle AKT deletion reduces muscle mass and fibre size through a reduction in protein synthesis but independent of multiple protein degradation pathways. Restoration of mTORC1 signalling downstream of AKT by deletion of both AKT and TSC1, or removal of any FOXO-mediated inhibitory influence by deletion of AKT and FOXO only partially restored muscle growth. However coordinated inhibition of FOXO1 and activation of mTORC1 pathways by the induced deletion of AKT1/2, TSC1 and FOXO1 not only rescued muscle mass and restored protein synthesis and body performance but also increased both body weight and muscle mass. They then applied a casting-induced immobilisation model of muscle wasting to stimulate a reduction in muscle mass, the inhibition of protein synthesis and a fibre-type shift. This was shown to reflect a decrease in insulin-stimulated phosphorylation status of AKT: that is, induced AKT deletion partially rescued the immobilisation-induced atrophy and inhibition of protein synthesis, as did induced deletion of FOXO1 and TSC⁽²³⁹⁾. Whilst these authors did not investigate the role of GSK3 as a downstream mediator of AKT signalling, they argued that as far as insulin–AKT signalling as a mediator of normal growth and development, their finding that deletion of FOXO1 and TSC1 were sufficient to restore protein synthesis, muscle oxidative capacity and muscle function in the absence of AKT in vivo, suggested that GSK3 was not a necessary factor for AKT-mediated muscle growth. This is in line with other studies of the role of GSK3⁽²⁵⁰⁾.

These findings suggest that the insulin/IGF-1 signalling network in rodent muscle responsible for muscle proteostasis appeared to be more complex than a simple AKT–mTORC1-dependent anabolic versus AKT–FOXO-dependent catabolic mechanism. This is consistent with our early findings that MPS rather than MPB is the main target for muscle growth regulation⁽¹⁵⁾. Furthermore other work suggests that the interactions between FOXO, mTOR and Akt include FOXO having direct inhibitory effects on mTORC1 and protein synthesis⁽²⁵¹⁾. In cultured fibroblast cells, activated FOXO induces the expression of sestrin 3, which activates AMPK to phosphorylate TSC and inhibit mTORC1⁽²⁵²⁾. Sestrins are multifunctional inhibitors of mTORC1⁽²⁵³⁾ also acting as amino acid sensors, which is important because muscle growth requires both insulin and amino acids^(35,44,141) . Leucine specifically activates mTORC1 by binding with sestrin. This prevents the inhibition by sestrin of mTORC1 by promoting its dissociation from GATOR2^(207,254) . Relative sestrin concentrations vary between tissues, but in rat skeletal muscle sestrin 1 acts as the main leucine sensor, via the GATOR2 complex⁽⁴⁶⁾. If FOXO1 is involved in regulating sestrin 1 in muscle, which at low leucine levels would inhibit mTORC1 via GATOR2, it may be that if FOXO is not fully inhibited by AKT, it could inhibit mTORC1 when leucine levels are not high enough to bind sestrin1 and relieve its inhibitory influence on mTORC1. Hence, FOXO may act as a rheostat in terms of its modulatory influence between insulin–AKT–mTORC1 and amino-acid signalling as suggested by Hay⁽²⁵¹⁾.

Finally, the insulin–AKT–mTORC1 signalling described here as revealed by the transgenic work involved the pathways presumed to occur within the myofibre. However, the insulin stimulation of MYOD1/myogenin-expressing myoblasts derived from satellite cells isolated from biopsies of adult human muscle⁽²⁴⁸⁾ may well illustrate the pathway activated in SCs in vivo by IGF-1. This means that insulin/IGF-1–AKT–mTORC1 signalling can be considered as a key driver of protein synthesis and other events during both myogenesis and myofibre proteostasis.

MAPK signalling

Mitogen-activated protein kinases (MAPKs) comprise a complex, multi-branched signalling system of protein Ser/Thr kinases that convert extracellular stimuli including mechanical, mitogens and insulin/IGF-1 (as shown in Fig. 5) into a wide range of cellular responses^{(218,255–257)} . In the present context, MAPK-mediated cell proliferation, differentiation and migration are particularly relevant to SC activation and myogenesis^(120,258) . Signalling commences through three sequentially acting kinases: a MAPKK kinase (MAPKKK, or MAP3K), which acts on a MAPK kinase (MAPKK, MAP2K or MEK) to activate the MAPK. Fourteen mammalian MAPKs have been characterised, but the most extensively studied are the extracellular signal-related kinases, ERK(1/2), Jun amino-terminal kinases JNK(1/2/3) and p38 isoforms, p38(α–δ). Mechanical stimuli can activate MAPK signalling, which is not only required for the maintenance of myofibre size but also plays a role in the induction of skeletal muscle hypertrophy^(259–261). In the present context, MAPK signalling is important in both the regulation of protein synthesis and in SC activation and myogenesis as discussed above.

Both ERK, which can be activated by insulin/IGF-1 (Fig. 5), and p38 MAPKs, which are turned on by other distinct stimuli such as cytokines (not shown in Fig. 5), participate in the regulation of protein synthesis (see Proud⁽¹⁴²⁾). ERK activates transcription and translation by at least two pathways as shown in Fig. 5. It phosphorylates TSC1/2, activating mTORC1 and translation. It can also activate both transcription and translation initiation independently from mTORC1 via MAPK-activated protein kinases (MAPKAPK1) and MNK1 & 2 (Mitogen-Activated Protein Kinase–Interacting Kinase 1/2) which activates one or more of the eIF4 initiation factors. p38 MAPKα/β can also activate MNK1 & 2 and translation initiation.

As for the regulation of myogenesis, p38α MAPK has been shown to be a global regulator of skeletal muscle differentiation⁽²⁶²⁾. This was indicated by chromatin-wide and transcriptome profiling integration in satellite cells derived from mice with muscle-specific deletion of p38α, and in the C2C12 murine myoblast cell line cultured in the absence or presence of the p38α/β inhibitor. p38α was shown to bind to a large set of active promoters during the transition of myoblasts from proliferation to differentiation stages.

Satellite cell signalling

The initial activation of SCs during post-natal growth at the molecular level remains incompletely understood, although Martino et al. has recently reviewed cellular mechanotransduction in general terms⁽²⁶³⁾. Much of the muscle-focused literature deals with responses to muscle damage⁽²⁶⁴⁾. However direct cell-to-cell interactions with their microenvironment offer potential mechanisms by which they can respond to mechanical force, so that the progression shown in Fig. 4 is assumed to be mainly mediated by mechano-transduction. SCs are anchored to the sarcolemma of the myofiber by cadherins, and on their apical side to the basal lamina of the endomysium via glycocalyx, integrins, syndecans, dystroglycans and sarcoglycans^(265,266) . Mechanical overload by exercise or stretching could directly exert both shear and tensile stress and activate SCs in their niche⁽²⁶⁷⁾. The latter group have recently employed an ex vivo muscle myofibre bundle experimental model to show that stretching by pulsating fluid shear stress caused compression as well as considerable tensile and shear deformation of SCs in their native niche⁽²⁶⁸⁾. Isolated SCs responded to pulsating fluid shear stress with increased nitric oxide production, known to release hepatocyte growth factor from the ECM and to mediate SC activation, proliferation and fusion⁽²⁶⁹⁾. There was enhanced gene expression of c-Fos and Cdk4, known to promote cell proliferation. Also there was upregulation of IL-6 production, reported to be essential for muscle regeneration and SC proliferation⁽²⁷⁰⁾, and for ERK 1/2 and p38 MAPK signalling.

The fibroblast growth factor (FGF) family have been implicated in skeletal muscle regeneration and satellite cell activity⁽²⁷¹⁾, with mRNAs of FGF1, FGF2, FGF4 and FGF6 detected in satellite cells which also express high levels of two of the FGF receptors (1 and 4). FGF2 and FGF6 particularly have been shown to regulate satellite cell function in vivo. FGF2 is present in the extracellular matrix and basal lamina of skeletal muscles, and is produced by fibroblasts, myofibres and SCs. FGF6 can be detected in both embryonic and adult skeletal muscle tissues, and isolated myofibres⁽²⁷²⁾. In adult mice, FGF6 is secreted by fast-twitch fibres, and its expression is increased after skeletal muscle injury.

Receptor-mediated FGF signalling involves activation of four major intracellular signalling pathways, RAS–MAPK, PI3K–AKT, phosphoinositide-specific phospholipase C (PLCγ) and signal transducer and activator of transcription (STAT)^(273,274) . The complexity of FGF signalling in satellite cells reflects the capability of FGFs to control multiple cell functions that include self-renewal, expansion and terminal differentiation.

One direct downstream target of FGF signalling in satellite cells is MYOD. Upon satellite cell activation, MYOD mRNA is stabilised, permitting commitment of the satellite cell to a myoblast fate and promoting cell cycle entry. In the absence of pro-proliferative signals, MYOD drives and is necessary for satellite cell differentiation. FGF2 can also activate MYOD in SCs by increasing calcium influx through the transient receptor potential canonical (TRPC1) calcium channel⁽²⁷⁵⁾. This is accompanied by nuclear translocation of the NFAT transcription factors NFATc2 and NFATc3, which induces MYOD expression. Thus, depending on the context, FGF signalling can promote satellite cell activation and expansion as well as promote cell cycle exit to generate either a self-renewed quiescent stem cell or a terminally differentiated myonucleus. Specific mechanisms by which these mitogens are made available to SCs in their niche have not been reported.

Phosphoproteomics of maximal-intensity contractions

The growth model shown in Fig. 1 includes mechanotransduction deriving from both stretching from bone length growth and work-induced force generation against gravity as discussed in ‘Mechanotransduction during post-natal growth as indicated by animal models’. New evidence has emerged about how force development within muscle can signal to SCs. This involves work with a maximal-intensity contractions (MICs) mouse muscle model by Hornberger’s’ group. They report phosphoproteomic studies, identifying specific MICs-regulated phosphorylation sites⁽²⁴⁵⁾ including those which were rapamycin sensitive⁽⁷⁴⁾. They also were able to predict which kinases were involved and which were phosphorylated themselves by the MICS.

The pathways identified⁽²⁴⁵⁾ included the regulation of mTOR, MAPK signalling, insulin signalling and ubiquitin-mediated protein degradation. These were mirrored by strong signatures for alterations in kinase activity, ubiquitin–protein transferase activity and the regulation of transcription, translation and proteolysis. The most frequent kinases identified were MAPK1 (ERK2), MAPK3 (ERK1) and cyclic AMP kinase (cAMK2α). Also identified were protein kinase C (PKCα/β), GSK3β, p38γ&δ (MAPK12/13), protein kinase A (PKAα,γ) and PKCδ. The largest group of phosphorylated kinases were members of the cAMP family.

Likely mechanotransduction signalling centres were highly phosphorylated sites on the actin cytoskeleton and especially within the contractile apparatus at the level of the Z-disc. They argued that this is consistent with the role of the Z-disc as facilitator of both longitudinal force transmission between sarcomeres and lateral transmissions via costameres. Furthermore they identified the giant protein titin and two of the cAMP mechanically sensitive kinases, obscurin and SPEG (striated muscle-specific serine/threonine protein kinase) as mediators of Z-disc phosphorylations. These two kinases are structurally similar to titin, which is known to act like a molecular spring, so all three proteins have the potential to act as mechanically sensitive kinases and mediate force transmission from the Z-disk to beyond the plasmalemma. The interaction between obscurin and titin in the sarcomere is illustrated by Mukund and Subramaniam⁽²⁷⁶⁾.

In a subsequent study involving pre-treatment with rapamycin, the inhibitor of a subset of the mTOR signalling events, Hornberger’s group showed that only a minority of the MIC-activated phosphorylation sites were mediated through a rapamycin-sensitive (RSmTOR) mechanism⁽⁷⁴⁾. The phosphorylation patterns suggested the activation by MICS of various isoforms of the MEK, ERK, JNK (MAPK), p38 and RSK (ribosomal s6 kinase), kinases upstream or parallel to RSmTOR. Kinases such as the 70-kDa ribosomal protein S6 kinase (p70s6K) are activated downstream of RSmTOR.

Importantly, they showed that one of the most robust MIC-regulated rapamycin-insensitive phosphorylation sites was a residue on Tripartite Motif-Containing 28 (TRIM28). TRIM28 proved to be particularly important because skeletal muscle-specific TRIM28 KO mice exhibited an attenuated hypertrophic response to myotenectomy. In addition, overexpression of phosphorylated (S473) TRIM28 induced hypertrophy. This supports the concept that MICS-induced TRIM28 phosphorylation is a key mediator of signalling events that promote mechanical load-induced hypertrophy. The importance of TRIM28 had been shown in previous work⁽²⁷⁷⁾. This protein, also known as KAP1 ((Krüppel-like associated box)-associated protein 1) is part of a dynamic transcriptional regulatory system acting with transcription factors MYOD and Mef2 in a signal-dependent fashion to modulate the efficient initiation of the myogenic gene expression program in SCs. This was under the control of the kinase MSK1, downstream of p38 MAPK signalling⁽²⁷⁷⁾. Steinert et al. ⁽⁷⁴⁾ showed that TRIM28 was required for the accretion of myonuclei during hypertrophy of the plantaris after partial removal of the gastrocnemius muscle. Most recently, this group showed with satellite-cell-specific and tamoxifen-inducible TRIM28 knockout mice that satellite-cell-derived TRIM28 was required for the normal myonuclear accretion following mechanical loading and for myofibre regeneration following injury⁽²⁷⁸⁾. Knockdown of TRIM28 resulted in a fusion defect in primary myoblasts studied in vitro. A scheme outlining the signal transduction pathway indicated by these studies is shown in Fig. 6.

Regulation of the capacity for protein synthesis

Our earliest dietary studies of MPS in muscle identified ribosomal RNA as an important locus for regulation of proteostasis and growth of muscle⁽²⁴⁾ as discussed in ‘Proteostasis and turnover during skeletal muscle growth: the evidence base for the growth model’. The capacity for protein synthesis, that is, the translational apparatus, comprises ribosomes, tRNA and a number of translation regulatory proteins. Of the total cellular RNA, most is ribosomal RNA (>80%) and tRNA (10–15%). The transcription of ribosomal RNA from rDNA repeats, together with the transcription of the ∼80 ribosomal proteins (rPs) and associated regulatory factors by three classes of RNA polymerases (POL I, II and III), occurs in the nucleolus. It is the most intense, complex and energy-intensive transcriptional process in the cell^(279–282). Mammalian cells contain several hundred copies of near-identical rDNA repeats distributed across multiple chromosomes grouped within the nucleolus. Transcription involves RNA Polymerase I (Pol I) resulting in the 47S precursor rRNA, which is processed to mature 18S, 5.8S, 28S rRNAs and packaged with ∼80 rPs and 5S rRNA (transcribed by POL III), to form pre-ribosomal particles. These are exported into the cytoplasm where, after further processing and quality control, the mature functional 40S and 60S ribosomal particles are formed⁽²⁸⁰⁾. The nucleotide sequence of rRNA allows various loop structures to form and interact with the rPs and with tRNA molecules to form the functioning small and large ribosomal particles. The ∼80 rPs required are synthesised in the cytoplasm from a specific class of mRNA molecules and must be transported from the cytoplasm into the nucleolus for the assembly process together with up to 200 biogenesis factors. As for tRNA, although in the genetic code, the 21 proteinogenic amino acids are encoded by 61 nucleotide triplets, in fact >500 human tRNA genes have been identified⁽²⁸³⁾ and these are transcribed by Pol III⁽²⁸⁴⁾.

The transcription of the rRNA genes by POL I is the major rate-limiting step in ribosome biogenesis⁽²⁷⁹⁾, and is clearly an important target for regulation shown in the growth model (Fig. 1). This starts with the formation of a complex with upstream binding transcription factor (UBTF), selectivity factor 1 (SL1; also known as TIF-IB in mouse) and transcription initiation factor IA (TIF-IA, also known as RRN3), with other factors involved in translation. Overall regulation involves multiple signalling pathways^{(279,281,285–289)} . These include ERK, AMPK, mTORC1 and P70S6K1, which allows for control by hormones and mitogens (Fig. 5), nutrients, including amino acids⁽²⁹⁰⁾, and contractile activity. In addition, the Wnt/b-catenin/c-MYC mechanically sensitive signalling pathway involved in regulating cell growth is involved in virtually all aspects of ribosome formation and is highly expressed during skeletal muscle hypertrophy^(288,291) . Insulin may well be one important control factor. It has been shown to activate POL I in fibroblasts, hepatoma cells and adipocytes^(285,292) . Also insulin can regulate ribosome content in primary cultures of rat hepatocytes by accelerating the rate of transcription of rDNA, and by slowing the rate of ribosome degradation which occurs after polyribosomal disaggregation, when mRNA translation is inhibited⁽²⁹³⁾. Insulin action may be part of the mechanism of the postprandial increase in muscle ribosomes observed in our very early rat feeding studies⁽³¹⁾

Recent work has focused on control of the production of rPs. For these proteins control is exerted mainly at the translational stage through a common cis-regulatory element, the 5′ terminal oligo-pyrimidine (5′TOP) motif. These TOP mRNAs are transcribed by POL II, and comprise all 80 ribosomal proteins and a number of translation regulatory factors, and other proteins indirectly connected to the translational machinery accounting to >200 in total. Crucially, their translation is controlled by mTORC1^(294–296). Stoichiometric considerations require rapid transport of ribosomal proteins into the nucleus during POL I–III-mediated ribosome biogenesis⁽²⁸⁰⁾, and this seems to be achieved by maintaining a cytoplasmic store of stabilised 5′TOP mRNAs. One likely mechanism involves the multimeric La-related protein 1, (LARP1)⁽²⁹⁷⁾. This binds through two regions of the protein to each end of the 5′TOPmRNA molecule regulating their stability and initially inhibiting their translation. Subsequent activation of their translation is a primary role of mTORC1^{(207,295,297–299)} , and a likely model is shown in Fig. 7 (see Jia et al., 2021⁽²⁹⁷⁾). This model predicts that LARP1 acts to inhibit translation and maintain an accessible store of 5′TOPmRNAs when mTORC1 is inactive. It also acts to enable efficient translation when required after mTORC1 activation by maintaining the mRNA in a circular conformation.

5′TOP mRNAs are a small fraction (e.g. 4%⁽²⁹⁵⁾) of the many thousand mRNA transcripts in the cell and during cell growth the entire cellular transcriptome must be translated to enable expansion of the cellular proteome. All nuclear-encoded eukaryotic mRNAs contain a 7-methyl guanosine cap and their translation involves the binding of eIF4E to the cap as shown in Fig. 7 regardless of the subsequent oligonucleotide sequence^(300,301) . Whilst it is usually stated that translation of 5′-TOP mRNAs is more sensitive to mTORC1-directed activation than Cap-dependent translation⁽²⁰⁷⁾, the extent of any difference, if any, during active growth is not known. Furthermore, other non-mTOR kinases are known to activate cap-dependent translation such as cyclin-dependent kinase 1 (CDK1)⁽³⁰²⁾, which is essential for myoblast proliferation, muscle regeneration and muscle fibre hypertrophy⁽³⁰³⁾. Detailed accounts of the regulation of translation have been described for muscle by Kimball and Lang⁽³⁰⁴⁾ and in general by Proud⁽¹⁴²⁾.

Another post-translational regulatory mechanism important for muscle growth regulation involves modification of the polyA mRNA tail by Cytoplasmic Polyadenylation Element Binding protein 1 (CPEB1). This factor binds to the 3′ untranslated regions (UTRs) at the end of mRNA transcripts and recruits cytoplasmic polyA polymerase GLD2 to elongate the polyA tail to maintain mRNA stability. The stability of mRNAs is positively correlated with translational output and CPEB1 can consequently regulate cellular function by post-transcriptionally controlling the translation of its targeted transcripts. Zeng et al. ⁽³⁰³⁾ have shown in muscle stem cells that phosphorylation-dependent CPEB1 promoted MYOD1 protein synthesis by binding to the 3′ UTR of MYOD1mRNA, increasing its translation and thereby increasing SC activation and proliferation. According to Zeng et al., CPEB1 in muscle stem cells acts as a key regulator to reprogram their translational landscape, directing SC activation and subsequent proliferation⁽³⁰³⁾.

Finally, recent careful examination by Hornbergers group of the absolute dependency of work-induced hypertrophy on mTORC1 through its regulation of protein synthesis, has raised some unexpected findings⁽²¹³⁾. They created skeletal muscle-specific and inducible raptor knockout mice, to eliminate signalling by mTORC1 (the raptor containing mTOR as distinct from the rictor-containing mTORC2 complex). These were unable to undergo any overload-induced hypertrophy as observed in control mice. It might have been assumed that this meant an inability of mTORC1 to direct the overload-induced increase in protein synthesis (e.g. as shown in Fig. 5). However, the raptor KO mice exhibited no reduction in the overload-induced increased protein synthesis rate observed in control mice (measured as the amount of puromycin-labelled peptides 30 min after the in vivo injection of puromycin). Clearly, the complete inhibition of overload-induced muscle growth implies no expansion of the proteome so that the observed increase in protein synthesis was quite inconsistent and difficult to explain apart from either incomplete translation/processing or a matching increase in autophagy due to the lack of mTORC1 signalling.

Evidence for limitation of muscle growth through a ‘bag full’ signal

The concept that, during muscle growth, its volume and consequent mass was limited by the connective tissue ECM in terms of the epi-, peri- and endomysial sheaths was a speculative idea at the core of a ‘bag’ theory of muscle growth^(5,6) . It allowed muscle growth to be conveniently described in terms of ‘bag’ enlargement, through ECM remodelling, and ‘bag’ filling in terms of myofibre protein deposition. It provided a potential mechanism for the stability of muscle mass throughout the life cycle and prior to the onset of sarcopenia. Specifically during the diurnal cycle of post-absorptive losses and postprandial gains of body protein⁽⁶⁾, losses and gains of muscle could be identified as ‘bag’ emptying and refilling until the ‘bag-full’ signal would terminate myofibre protein deposition.

The demonstration of the ‘bag-full’ phenomenon with studies of postprandial MPS in adults^(305–308) confirmed a regulatory signal which limited protein deposition in the myofibre. However, its mechanism in terms of the signalling examined in those studies was by no means resolved. In fact, MPS fell whilst intracellular leucine concentration remained elevated and mTORC1 signalling was still active^(306,308) , at least those signals associated with the initiation of protein synthesis (p70S6K1, and 4EBP1). However, eEF2 phosphorylation does seem to increase coincident with the fall in MPS⁽³⁰⁰⁾, and this indicates that the elongation phase of protein synthesis was inhibited, an effect observed in meal-fed rats in which the increased MPS in response to a meal was transient returning to baseline coincident with an increase in eEF2 phosphorylation⁽³⁰⁹⁾. Classically, eEF2 phosphorylation increases in response to AMPK signalling associated with energy stress, such as muscle contraction, to protect ATP and PCr levels since it is the translational phase of MPS which is so energy dependent⁽¹⁴²⁾. In meal-fed rats, post-meal supplementation with either leucine or carbohydrate prevented eEF2 phosphorylation and extended the increase in MPS⁽³¹⁰⁾. However, in human muscle the ‘bag-full’ return to baseline of MPS occurs with no indication of inhibitory AMPK signalling or energy stress in terms of muscle ATP or phosphorylcreatine concentrations⁽³⁰⁸⁾. This means that the triggering of the response was unexplained. Nevertheless, it represents the only observed response which could be involved in mediating the ‘muscle-full’ inhibition of MPS.

It was originally assumed that anabolic/catabolic signals associated with changes in muscle cell volume would be important for the ‘bag-filling-bag-full’ signalling concept⁽³¹¹⁾. As extensively discussed elsewhere⁽⁵⁾, it is highly likely that myofibre volume changes occur during the feeding–fasting cycle, with evidence for linkage between cell volume, mTOR and two different anion channels associated with cell swelling⁽³¹²⁾ and shrinkage⁽³¹³⁾. However, after reviewing the available evidence a satisfactory mechanism linking volume change to the inhibition of MPS was not immediately obvious.

There is a current discussion about the nature of volume changes within muscle during the increases in fibre cross-sectional area consequent to some types of resistance exercise in adult human muscle, although this relates to the phenomenon of sarcoplasmic hypertrophy⁽³¹⁴⁾. This is a disproportionate expansion of the sarcoplasm relative to myofibril protein accretion observed in some studies of muscle growth during resistance training⁽³¹⁵⁾. Potential explanations of this phenomenon offered by this group include that it is a transient phenomenon which results in cell swelling which triggers SC activation and fusion allowing subsequent myofibrillar accretion in the new MND restoring the usual balance between sarcoplasmic and myofibrillar volumes. While such data add detail to what may well occur during the bursts of post-natal muscle growth in children and adolescents it offers little insight into the molecular mechanism linking changes in overall myofibre volume to proteostasis control.

Taken together, the above indicates that, while the ‘bag-full’ termination of postprandial protein deposition in muscle has clearly been shown to occur, its mechanism remains to be identified. As for bag enlargement, this is an inevitable accompaniment to muscle growth. We know that increased muscle collagen synthesis is an early response to stretch-overload-induced muscle growth in fowl⁽⁷⁸⁾, and is observed in adult muscle stimulated by various intense exercise regimes which induce muscle growth^(48,137) . As shown in Fig. 4, ECM remodelling is part of satellite cell activation and myogenesis, and can be expected to accompany the muscle hypertrophy in response to maximal muscle contractions shown in Fig. 6. During the rapid developmental muscle growth in young rodents, it would be predicted that there is continuous remodelling of the ECM. In accord with this, in our rat muscle and bone growth studies^(80,81) we observed active proteoglycan synthesis in muscle during rapid growth which was reduced as growth slowed with parallel changes in muscle IGF-1^{(80,81,84,85)} . Much more detailed studies of muscle ECM turnover during its rapid post-natal growth are needed.

Conclusions

This review has examined a model for post-natal muscle growth developed in experimental animals, in which its size and strength is controlled by mechanotransduction when nutrition provision is adequate. Mechanotransduction derives from both bone length growth-induced stretching and muscle force development against gravity, which generates a growth capacity through ECM remodelling and myogenesis mediated by satellite cell activation. Nutritional adequacy involves dietary protein and other key nutrients which regulates bone length growth and initiates appropriate signalling and provides substrates for protein deposition in muscle which fills the growth capacity.

As with all work with animals as models for the human condition, they show us what can occur and the evidence for the model shown in Fig. 1 as an explanation of rodent muscle growth is currently very strong. Is such a model relevant to better understanding childhood growth? The key implication of the growth model for childhood is the concept of a growth capacity for skeletal muscle, especially for the appendicular muscles, which is set by mechanotransductive forces. These forces can derive from either an increase in stature or increased demands for muscle work, but when they are minimal, which may be quite often in preschool children, dietary stimulation of muscle growth is likely to be very limited. As recently argued, the evidence from the timing of the stages of the pubertal growth spurt is consistent with this growth model explaining the phenomenology of muscle growth as a function of height growth in childhood⁽⁵⁾.

The inability of dietary protein alone to stimulate muscle growth in childhood in excess of the growth which accompanies either height growth or in response to excessive physical demands is one explanation of the ‘Early Protein Hypothesis’⁽³¹⁶⁾. This postulates that dietary protein in excess of the intake required for lean tissue growth, a demand which may be quite low after the first year of life, increases plasma insulin and IGF-1 concentrations. This response mediates adipogenic activity and associated weight gain, and the programming of obesity in later childhood. This implies that the endocrine action of IGF-1 has no influence on muscle growth during stasis periods for bone length growth.

Similarly, during adult maintenance the phenotypic muscle mass can be maintained over a wide range of dietary protein intakes, with no evidence that the phenotypic lean body mass is a function of dietary protein intake, apart from that which varies with BMI and weight gain⁽³¹⁷⁾. This is consistent with the ‘bag’ filling and enlargement concepts referred to above.

However, obvious gaps remain in the knowledge base. One is the phenomenon of saltatory, (discontinuous with leaps), growth and stasis in human height^(318,319) and animal length⁽³²⁰⁾. Although this is not accepted by all, when considered in the context of the mechanotransduction of muscle growth, saltatory growth implies that passive stretch forces associated with a saltation could be much greater than those implied by a continuous but very slow rate of human long bone growth. This raises the possibility of fibre damage occurring in response to saltations, a phenomenon which could explain the anecdotal ‘growing pains’ reported in young children⁽³²¹⁾ and lambs⁽³²⁰⁾. This means that those experimental ‘supraphysiological’ models of muscle hypertrophy such as the weighted wing^(21,76,78) may be more relevant to developmental growth than often accepted.

Another gap in the mechanistic knowledge base relates to the fact that some of the animal hypertrophy work discussed here does not completely discriminate between events in the myofibre as opposed to within SCs. This is important in relation to the different requirements of the growth response: acquiring new myonuclei, expanding the myofibre proteome, and connective tissue remodelling to allow expansion of the myonuclear domain within the increasing myofibre CSA. Our early studies, (Fig. 2), showed that, in the adult phase of physiological muscle growth in the male rat, considerable expansion of the myofibre proteome occurs with only a modest amount of SC activation and myogenesis as indicated by DNA accumulation. Nevertheless, connective tissue remodelling can be assumed to be a requirement at this time, as with all circumstances of growth when the myofibre CSA increases. Such remodelling could be achieved through activation of interstitial fibrogenic cells including fibro/adipocyte progenitors (FAPs)^(170,322) . It would be of great interest to map the changes in these stromal cells to better understand their involvement in muscle growth.

Finally, in the context of proteostasis within the growth model shown in Fig. 1, this review has focused only on the anabolic components. Proteolysis, including lysosomal autophagy, the ubiquitin–proteasome system and calpains, together with other systems known to mediate proteolysis in muscle by poorly understood mechanisms, has not been discussed here, but have been recently reviewed elsewhere⁽⁸⁾. The ‘turnover’ component of muscle proteolysis was largely mysterious when the authors work commenced five decades ago, and notwithstanding attempts to provide molecular explanations⁽¹⁰³⁾, this remains the case today. It will be of great interest to see how this develops.

Acknowledgements

Much of the authors animal work described here was achieved by students, post-doctoral assistants and longer term collaborators identified here in their published work, of whom I want to particularly acknowledge Dr Peter Bates and Dr Zainal H. A. Yahya. I also thank Troy Hornberger for his sharing of his research findings.