Regulation of Ribosome Biogenesis in Skeletal Muscle Hypertrophy

Abstract

The ribosome is the enzymatic macromolecular machine responsible for protein synthesis. The rates of protein synthesis are primarily dependent on translational efficiency and capacity. Ribosome biogenesis has emerged as an important regulator of skeletal muscle growth and maintenance by altering the translational capacity of the cell. Here, we provide evidence to support a central role for ribosome biogenesis in skeletal muscle growth during postnatal development and in response to resistance exercise training. Furthermore, we discuss the cellular signaling pathways regulating ribosome biogenesis, discuss how myonuclear accretion affects translational capacity, and explore future areas of investigation within the field.

Introduction

Cell growth is defined as an increase in cell mass (85). Cell growth is often conflated with cell proliferation because the two processes are typically coordinated to ensure cell size is maintained upon division (85). The uncoupling of cell growth from cell division can lead to an increase in cell size; this hypertrophic growth has been observed in differentiated, post-mitotic cells such as those of the liver, kidney, adipose, heart, and skeletal muscle (35, 45, 63, 72, 129). In particular, adult skeletal muscle cells are capable of dramatically increasing their size in response to increased mechanical loading, stretch, and growth factor signaling (1, 8, 10, 45, 128). A defining characteristic of skeletal muscle hypertrophy in both humans and rodents is a robust increase in the rate of protein synthesis (43, 138).

A primary determinant of skeletal muscle mass is the relationship between protein synthesis and protein degradation; a sustained, net increase in the rate of protein synthesis will ultimately lead to the accumulation of cellular protein resulting in muscle fiber hypertrophy (43, 44, 144). In general, the rate of protein synthesis is determined by two factors: translational efficiency and translational capacity. Translational efficiency is defined as protein synthesis per unit RNA, whereas translational capacity is determined by the total ribosomal content and often is expressed as the total RNA content per unit tissue (79, 83). Translational efficiency and capacity are coordinately regulated with the emerging notion (borrowed from the cardiac field) that the initial increase in translational efficiency observed following a hypertrophic stimulus is required for the subsequent increase in translational capacity, which allows for the sustained increase in protein synthesis necessary for skeletal muscle hypertrophy (53, 136). The purpose of this review is to provide a brief overview on ribosome biogenesis, its regulation during muscle growth, and the evidence demonstrating that ribosome biogenesis is an important mediator of skeletal muscle hypertrophy.

The Ribosome

The ribosome is a macromolecular machine responsible for the translation of mRNA into protein, i.e., protein synthesis. The mature ribosome (80S in eukaryotes) is composed of a small subunit (40S) and a large subunit (60S). Each subunit contains distinct ribosomal RNAs (rRNAs) and associated ribosomal proteins (r-proteins); the large 60S subunit is formed by 28S, 5.8S, and 5S rRNAs, and 47 r-proteins with the small 40S subunit composed of a 18S rRNA and 33 ribosomal proteins (7, 61).

Even though ribosome is composed of 80 r-proteins, it is the four rRNAs that confer the peptide bond formation activity (86, 96). The small subunit is responsible for the interaction between the mRNA codons and the tRNA anticodons, whereas the large subunit contains the rRNAs that interact to create the actual site of peptidyl transferase within the ribosome (86, 122). Although r-proteins are not strictly required for peptidyl transferase activity, they have been shown to be required for rRNA processing, maturation, assembly, and stabilization, as well as to affect translational fidelity (68, 95, 106, 145).

Although it may be argued that the ribosome is not in the stricto sensus an organelle, due to its lack of a membrane, it is a comparatively small organelle of 2.9 Å (61). Despite the relatively small size of the ribosome, ribosomes are found in high concentrations within the cell, from as little as several thousand per μm³, up to 14,000 ribosomes per μm³ in proliferating eukaryotic cells, occupying roughly 20% of the cell volume (21). Indeed, in any given cell, the rRNAs compose the vast majority of total cellular RNA (ranging from 70 to 90%), and thus any change in total cellular RNA content, as the result of ribosome biogenesis, is generally attributed to a change in rRNA abundance and considered to be indicative of an increase in the translational capacity of the cell (25, 29, 97, 134, 141). Given the abundance of rRNA, it is not surprising then that r-protein mRNAs make up a significant proportion of the total mRNA pool in many cell types, including in skeletal muscle tissue (4, 11, 135). Likewise, in growing yeast, ~50% of the total cellular RNA polymerase II transcription (protein-coding genes) is devoted to the transcription of r-protein mRNAs (see FIGURE 1) (134).

FIGURE 1.

RNA composition of the transcriptome

The total RNA pool within the cell is composed of ~80% ribosomal RNA (rRNA), with mRNA accounting for ~12% of the total RNA pool; a significant portion of mRNA encodes for ribosomal proteins. The remainder of the RNA content of the cell is composed of small noncoding RNAs such as transfer RNA (tRNA), long noncoding RNAs, and microRNAs.

Ribosome Biogenesis

Ribosome biogenesis is a complex, multistep process involving the de novo synthesis of ribosomes. It involves ribosomal DNA (rDNA) transcription with subsequent processing, maturation, and assembly of rRNAs and r-proteins. Ribosome biogenesis occurs primarily within a substructure within the nucleus, the nucleolus, either being synthesized in the nucleolar region or shuttled from other compartments into nucleolus. Ribosome biogenesis requires the activity of all three RNA polymerases I, II, and III (Pol I, Pol II, and Pol III). Pol I exclusively transcribes rDNA, whereas Pol II is responsible for the transcription of all mRNAs, including the r-protein mRNAs, with Pol III transcribing the 5S rRNA gene and small RNAs such a tRNAs (see FIGURE 1) (77).

The de novo synthesis of ribosomes is initiated by transcription of rDNA to produce the 47S pre-rRNA, which is considered the primary point of regulation in ribosome biogenesis (100). Transcription of rDNA by Pol I first requires the assembly of the pre-initiation complex (PIC) at the rDNA promoter (28, 109). The PIC is formed at the rDNA promoter by the upstream binding factor (UBF), selectivity factor 1 (SL-1) complex [formed by several proteins: the TATA-binding protein (TBP) and four TBP-associated factors (TAFs)], transcription initiation factor TIF-IA (encoded by Rrn3), and the RNA Pol I complex (28, 47, 50). The rDNA promoter contains two control sequences, the core promoter region (Core) and the upstream promoter element (UPE) (88). Specifically, UBF and SL-1 are responsible for DNA binding, with TIF-IA acting as bridge between these factors and Pol I (108); however, a recent study reported that TIF-IA has DNA binding activity (122a). As it will be discussed below, studies have provided clear evidence the PIC is a major signaling hub, allowing for the integration of different signaling pathways to affect ribosome biogenesis through modulation of rDNA transcription (67, 77). Following initiation, PIC components, UBF, and SL-1 complex, remain bound to rDNA promoter elements, whereas Pol I transcribes the rDNA until it encounters the termination elements (see FIGURE 2).

FIGURE 2.

Regulation of ribosome biogenesis during skeletal muscle hypertrophy

Relevant signaling transduction pathways to ribosome biogenesis are shown along with the pre-initiation complex (PIC) formed in the nucleolus, where active rRNA genes are being transcribed leading to processing, maturation, and assembly of ribosomes. This will result in increased number of ribosomes and increased translational capacity. Efficiency also contributes with translational capacity by promoting the specific translation of key proteins involved in ribosome biogenesis, such as the ribosomal proteins. The role of satellite cell by donating rDNA template is shown with a question mark to denote the need for further investigation.

Following synthesis of the 47S pre-rRNA, the long transcript is processed intro three mature rRNAs, 18S, 5.8S, and 28S, in a controlled manner by a series of cleavage enzymes (55). The processing involves the removal of external and internal transcribed spacers (ETS and ITS, respectively) from the pre-rRNA (56). In addition to these rRNAs, the mature ribosome requires a fourth rRNA, the 5S, which is transcribed by Pol III, outside the nucleolus (140). Altogether, these four rRNAs form the backbone of the mature ribosomes, along with 80 r-proteins.

Unlike protein-coding genes, there are hundreds of copies of rDNA genes found within the genome; on average, humans have ~300 copies, ranging from as low as 60 copies up to 1,590 copies (40, 101). The rDNA copies are arranged in tandem and located on five different chromosomes (101). These rDNA clusters form chromosomal loops known as nucleolus organizer regions, which interact through hundreds of nucleolar-specific proteins to form the nucleolus, thereby facilitating transcription of the 47S pre-rRNA (111, 123). In addition to pre-rRNA synthesis, the nucleolus is the site where pre-rRNA processing and ribosome assembly occurs. Following translation in the cytoplasm, r-proteins are imported into the nucleolus, where they associate with their respective ribosomal subunit; thus ribosome maturation takes place in both the nucleolus and nucleoplasm, whereas the final steps occurs in the cytoplasm (55, 88).

Ribosome Biogenesis During Postnatal Skeletal Muscle Hypertrophy

The significant increase in skeletal muscle mass after birth in mammals occurs primarily through muscle fiber hypertrophy given that fiber number appears to be established during early postnatal development (71, 139). The roughly sevenfold increase in fiber size during postnatal development is accompanied by a fivefold increase in myonuclear number as the result of satellite cell fusion (114, 139). The increase in muscle DNA content during this rapid period of growth is paralleled by a similar increase in total RNA and protein content, suggesting that increased myonuclear accretion promotes growth by yielding increased translational capacity (60, 81). In fact, the concentration of ribosomes in skeletal muscle, based on total RNA per milligram of tissue, was found to be the greatest at early stages of postnatal development; however, although the concentration of ribosomes progressively decreases during skeletal muscle growth and maturation, the total translation capacity of the muscle (as assessed by total RNA content) is much higher in adulthood (see FIGURE 3) (36, 37, 60, 102, 146). The importance of ribosome biogenesis to skeletal muscle growth during postnatal development is supported by studies in which diet is manipulated during this period. Young and colleagues found that pups placed on a low protein diet showed a decrease in skeletal muscle total RNA concentration and blunted growth (146). Similarly, nutritional restoration in undernourished pups restored muscle mass, which was associated with an increase in UBF levels and translational capacity (36).

FIGURE 3.

RNA concentration vs. RNA content

The concept of RNA concentration vs. RNA content is important for understanding the role of translational capacity in the regulation of skeletal muscle hypertrophy. A: postnatal skeletal muscle growth is characterized by a high concentration of total RNA immediately after birth. During postnatal development, both skeletal muscle mass and RNA content are significantly increased; however, the concentration of total RNA decreases because the increase in mass is greater than the increase in the total amount of RNA. B: different hypothetical scenarios are presented to show how the relationship between muscle mass, RNA content, and RNA concentration can change with hypertrophy and atrophy: i, in growing muscle, ribosome biogenesis results in an increase translational capacity (total RNA content) but a modest decrease in RNA concentration because growth outpaces rRNA production; ii, there is muscle growth but in the absence of ribosome biogenesis such that translational capacity remains unchanged but with a significant decrease in RNA concentration; iii, during muscle atrophy, translational capacity remains unchanged such that RNA concentration is significantly increased; iv, during muscle atrophy, there is a significant loss of translational capacity such that RNA concentration is also significantly reduced.

Ribosome Biogenesis During Adult Skeletal Muscle Hypertrophy

In Vitro Studies

In vitro experiments provided the first evidence linking ribosome biogenesis to skeletal muscle hypertrophy. Pioneering work from Nader and colleagues showed the treatment of myotubes with fetal bovine serum (FBS; a serum enriched with growth factors) activated mTOR signaling and cyclin-dependent kinase 4 (CDK4) activity, which, in turn, promoted UBF activity and rDNA transcription leading to higher rRNA abundance and myotube hypertrophy (91).

More recent studies using the specific Pol I inhibitor CX-5461 have provided further evidence supporting a requirement for ribosome biogenesis, i.e., increased translational capacity in muscle hypertrophy (27, 51, 118, 133). The findings of these studies showed inhibition of Pol I activity by CX-5461 significantly blunted serum-stimulated myotube hypertrophy, which was associated with a diminished increase in 47S pre-rRNA expression, total RNA abundance, and protein synthesis (118, 133). Contrary to these findings, Crossland and coworkers found IGF-1-induced myotube hypertrophy still occurred independent of ribosome biogenesis (20).

Studies in cell culture have also demonstrated a novel role for mTOR protein in the regulation of ribosome biogenesis. The well-established role of mTOR in cell growth is most often attributed to its cytoplasmic role in regulating translational efficiency, von Walden and colleagues reported that mTOR associates with rDNA promoter in muscle cells and is involved in rRNA transcription, confirming an earlier study in non-muscle cells (91, 127, 133). This novel finding shows that mTOR, independent of its well-known cell signaling function, is able to promote cell growth through direct regulation of ribosome biogenesis.

It is important to highlight, however, that studies in cell culture may have inherent limitations that preclude its translation to in vivo conditions, especially in an adult organism where the number of neighboring myoblasts (or satellite cells) near myofibers are very low. Even though most of these studies have attempted to remove myoblasts following cell differentiation via cytosine arabinoside (AraC) treatment, this does not remove all myoblasts, and the role of myoblast fusion into the existing myotubes cannot be completely ruled out. In particular, the use of CX-5461 likely decreases ribosome biogenesis in myoblasts as well, reducing proliferation and, consequently, myoblast fusion. Despite the mechanism, in vitro studies have demonstrated that a growth stimulus promotes ribosome biogenesis and translational capacity, leading to myotube hypertrophy. We positioned that the role of myonuclei accretion in ribosome biogenesis is better answered via in vivo studies that have been depleted of satellite cells, which will be discussed in Influence of Satellite Cells and Myonuclear Accretion on Ribosome Biogenesis.

Rodents Models of Muscle Hypertrophy

Since its original description over 50 years ago, the synergist ablation model has been shown to be a useful tool to study the molecular and cellular mechanisms underlying skeletal muscle hypertrophy (42). An early cell-free study hinted at the possibility that the increase in total RNA observed in hypertrophied muscle may be responsible for the higher rate of protein synthesis associated with muscle growth (52). Since then, numerous studies using synergist ablation have firmly established the increase in total RNA per milligram of tissue in response to a hypertrophic stimulus (2, 16, 45, 46, 48, 54, 64, 117). It is only more recently, however, that studies have begun to uncover the mechanisms regulating ribosome biogenesis that lead to the increase in rRNA with mechanical loading in rodents and humans.

The Nader laboratory has been at the forefront of understanding the role of ribosome biogenesis in skeletal muscle hypertrophy. Von Walden and colleagues provided the first evidence showing an increase in 47S pre-rRNA expression in response to a hypertrophic stimulus induced by synergist ablation (132). The increase in 47S pre-rRNA expression preceded fiber hypertrophy and was associated with higher UBF binding at the rDNA promoter as well as c-Myc, a major driver of cell growth, and Pol I itself (132). The enrichment of Pol I regulon factors at the rDNA promoter, along with evidence of chromatin remodeling, further suggested the intriguing possibility that, during hypertrophy, additional copies of rDNA become open, available for transcription (132). Later studies have confirmed these initial findings, showing an increase in ribosome biogenesis, as assessed by 47S pre-rRNA expression, following synergist ablation (49, 64, 65). More recently, Nakada and coworkers provided compelling evidence showing a strong correlation between the magnitude of hypertrophy and translational capacity but not markers of translational efficiency (93).

Resistance Exercise Training Studies

Although rodent studies have shown a clear relationship between skeletal muscle hypertrophy and ribosome biogenesis, it has only been very recently that studies were undertaken to determine whether ribosome biogenesis occurs in humans following resistance exercise and its potential role in promoting skeletal muscle hypertrophy.

Several recent studies have now investigated the acute and chronic effects of resistance exercise on 47S pre-rRNA levels (30–33, 38, 92, 104, 119). Based on these studies, summarized in Table 1, the increase in 47S pre-rRNA expression following a single resistance exercise bout occurs only after several hours. In particular, 47S pre-rRNA expression was increased above baseline levels at 4 h and continues to increase until 24 h, remaining elevated at 48 h (FIGURE 4A) (32, 92, 119). Analysis of pre-rRNA expression at earlier time points, e.g., at 1, 2, and 3 h, showed no change in pre-rRNA levels (30, 33, 38). Although pre-rRNA expression is not increased at these earlier time points, activation of key events, such as increased TIF-IA phosphorylation at an ERK-dependent site, suggests that, following an acute bout of resistance exercise, Pol I regulon factors become primed to promote PIC formation and subsequent rDNA transcription (30, 32).

Table 1.

Pre-rRNA synthesis analysis following acute and chronic resistance exercise in humans

Reference	Time Point Post-RE	Biopsy Site	Subjects	47S Change
30	1 h and chronic	VL	Young men	↔1 h, ↑ chronic
92	4 h	Biceps brachii	Young men and women	↑4 h
32	2, 24, and 48 h	VL	Young men trained	↔2, ↑24 and 48 h
119	24 h	VL	Young and elder men and women	↑24 h
38	3 h and chronic	VL	Young men	↔3 h and chronic
33	2 and 4 h	VL	Elder men	↔2 and 4 h

Note that only the control groups are included. Experimental groups that received different nutritional interventions or non-resistance training protocol were not included. VL, vastus lateralis; RE, resistance exercise.

FIGURE 4.

Resistance exercise increases the translational capacity of the muscle

Based on the findings from human studies, a working model is presented showing the change in translational capacity following a single bout vs. multiple bouts of resistance exercise. A: a single bout of resistance exercise results in a significant increase in ribosome biogenesis, as assessed by 47S pre-rRNA expression, starting 4 h postexercise and maintained until at least 48 h with no detectable change in translational capacity of the muscle. B: in response to multiple bouts of resistance exercise, there is a progressive increase in the translational capacity of the muscle.

Although a single bout of resistance exercise increased 47S pre-rRNA levels, it does not appear to be sufficient to increase the abundance of mature rRNA significantly (32, 99). The reason for this disconnect between 47S pre-rRNA and rRNA levels remains to be determined, but one possible explanation is the inability to precisely measure minor changes in the rRNA, given it makes up ~85% of the total RNA pool of the cell. However, cumulative bouts of resistance exercise eventually lead to the accumulation of mature rRNAs, leading to increased concentration of total RNA (13, 30, 104, 118) (FIGURE 4B). This increase in translation capacity following resistance exercise in humans was significantly correlated with the change in muscle mass (12, 30, 104). The importance of ribosome biogenesis in human skeletal muscle hypertrophy is further underscored by studies showing the increase in muscle size in extreme responders to resistance exercise training was associated with a greater change in total RNA concentration, a finding consistent with an earlier rodent study (84, 93, 118).

Total RNA content of muscle has been shown to be correlated with the in vitro rate of protein synthesis (137), indicating a possible mechanism in vivo. Moreover, given that the magnitude of rRNA accumulation following chronic resistance training has been associated with muscle growth in vivo (12, 30, 93, 118), the increase in translational capacity should result in a higher basal rate of protein synthesis. In support of this view, several studies have found that muscle protein synthesis was increased at resting conditions following resistance exercise training (6, 62, 87, 104, 142). Moreover, the change in the basal rate of protein synthesis was recently shown to be significantly correlated with the degree of muscle hypertrophy (104). Although the magnitude of the change in the basal rate of protein synthesis is relatively minor, it may have a greater impact on growth than the relatively transitory increase in translational efficiency, given it occurs over a much longer period of time. These findings, together with previous results showing an association between total RNA and rRNA levels with muscle growth, provide compelling evidence that translational capacity is a determining factor driving skeletal muscle growth induced by resistance exercise training (12, 30).

Regulation of rDNA Transcription During Skeletal Muscle Hypertrophy

Studies investigating cell growth and proliferation have revealed the intimate connection between the cell cycle and ribosome biogenesis. An important challenge in skeletal muscle research is to verify whether the signaling pathways shown to regulate ribosome biogenesis during cell growth are conserved in adult skeletal muscle. Although there has been considerable effort to describe the signaling pathways involved in mediating the hypertrophic response of skeletal muscle to resistance exercise, most of these studies have focused on the regulation of translational efficiency. Although these studies have been designed to capture changes in cell signaling thought to regulate translational efficiency, i.e., early time points, we have, when appropriate, inferred the observed changes in cell signaling may also regulate ribosome biogenesis.

In particular, UBF is a central player in the regulation of rDNA transcription given that it possesses DNA-binding activity via several high mobility group (HMG) box domains, which receive input from different signaling pathways regulated by hormonal, nutritional, and cellular energy (67, 108). Of relevance for skeletal muscle, growth factors, such as IGF-1, and amino acids, such as leucine, may regulated UBF activity (67).

UBF is considered to be the master regulator of rDNA transcription because of its direct contact with the promoter regions of rDNA to form active nucleolar organizer regions, in addition to its central role in the recruitment and stabilization of SL1 (24, 98, 121). UBF receives input via phosphorylation from MAPK and mTOR signaling pathways as well as cell cycle regulators. There are several known serine phosphorylation sites (Ser388, Ser484, and Ser637) within the UBF protein, with Ser388 and Ser484 phosphorylation required for upregulation of rDNA transcription (130, 131). Along with phosphorylation status, the total level of UBF protein has been shown to influence Pol I recruitment and rDNA transcription rates (110, 131). In addition to UBF, TIF-IA is also required for PIC formation and rDNA transcription, and also receives cues from different signaling pathways (14, 82, 113, 147). As mentioned before, TIF-IA participates in the PIC formation by recruitment of the Pol I complex, acting as a bridge between the SL-1 and Pol I complex via protein-protein interaction (9, 14, 82, 103, 147).

MAPK Signaling and Cell Cycle Regulators

The MAPK signaling pathway regulates ribosome biogenesis at multiple levels by 1) promoting translation of specific cell cycle proteins such as cyclin D1, 2) enhancing the stabilization of c-Myc protein, and 3) phosphorylation of PIC proteins, UBF, and TIF-IA (69, 73, 115, 120, 148). Cyclin D1 expression is primarily regulated at the level of translation by eukaryotic translation initiation factor 4E (eIF4E). eIF4E promotes cyclin D1 translation following MNK1 (MAP kinase interacting serine/threonine kinase 1) activation of eIF4E via Ser209 phosphorylation (73, 107, 126). Once translated, cyclin D1 binds and activates CDK4, which allows CDK4 to directly phosphorylate UBF, thereby promoting rDNA transcription (5, 66, 131). MAPK signaling has also been shown to promote ribosome biogenesis through phosphorylation of TIF-IA at Ser649 via RSK1 (ribosomal protein S6 kinase polypeptide 1) (148).

Numerous studies have investigated activation of the MAPK signaling pathway in response to exercise. In particular, kinases such as ERK 1/2, p38, MNK1, and p90RSK have been shown to be highly phosphorylated immediately after exercise as well as a few hours postexercise (22, 30, 32, 39, 75, 143). Following resistance exercise, there is an increase in both the expression and the MAPK phosphorylation of eIF4E (30, 32, 78, 142). Thus these studies suggest that resistance exercise induces activation of the MNK1-eIF4E-cyclin D1 axis, leading to UBF phosphorylation and rDNA transcription. Similarly, RSK1 phosphorylation of TIF-IA Ser649 has been reported 1–3 h following resistance exercise but not at 24 or 48 h (30, 32, 38).

PI3K-AKT-mTOR Pathway

Growth factors have been shown to be potent stimulators of ribosome biogenesis (58, 77). Growth factors, such as IGF-1, affect muscle cells via both the PI3K-AKT-mTOR and MAPK signaling pathways (112). This in part could be attributed to Akt activity, since Akt has been shown to be a potent stimulator of ribosome biogenesis in many cells types via growth factors; however, Akt regulation of ribosome biogenesis in skeletal muscle in response to a hypertrophic stimulus has yet to be investigated (17, 23). mTOR signaling, on the other hand, is known to regulate ribosome biogenesis through multiple mechanisms. mTOR promotes the translation of the r-proteins and other accessory proteins through a 5′-TOP (5′-terminal oligopyrimidine tract) mechanism (94, 125). Furthermore, mTOR was shown to be associated with rDNA promoter (127, 133). mTOR may regulate the formation of transcriptionally engaged PIC via TIF-IA activation and nucleolar chromatin structures (19, 70, 133).

A number of studies in rodents and muscle cells have shown that mTOR plays a role in muscle growth via ribosome biogenesis (74, 99, 133). However, the mechanisms through which mTOR promotes ribosome biogenesis is not fully clear, since rapamycin-sensitive but apparent mTOR-independent mechanism may exist (48, 99). More studies are required to elucidate how mTOR affects ribosome biogenesis and muscle growth, and whether the different complexes, mTORC1 and mTORC2, have roles in regulating the translational capacity of skeletal muscle.

Two of the best-known downstream targets of mTORC1, 4E-BP1 and p70S6K, affect translation initiation and elongation. 4E-BP1 is directly involved in promoting the translation of TOP 5′ mRNA such as r-proteins, which are required for 47S pre-rRNA processing and ribosome assembly (125). As for p70S6K, it does not appear to be necessary for rDNA transcription of 47S pre-rRNA but may instead may be involved in rRNA processing, maturation, assembly, and ribosome export (18, 74, 133). So, although it has been clearly established that mTOR activation following resistance exercise enhances translational efficiency, it remains to be determined whether the acute activation of mTOR postexercise promotes its association with, or activity of, rDNA promoters (3, 26, 30, 75, 105, 124).

Combined, these data demonstrated that a bout of resistance exercise promotes signaling transduction pathways associated with PIC formation and transcription of rDNA, along with increased translation of ribosomal proteins (see FIGURE 2). Interestingly, a few upstream kinases and proteins can be upregulated shortly after a bout of resistance exercise, such as phosphorylation of TIF-IA, and levels of cyclin D1, although increased pre-RNA synthesis is not yet observed. It is possible that these effects are priming PIC formation at the rDNA promoter region to activate pre-rRNA synthesis at a later time point.

Influence of Satellite Cells and Myonuclear Accretion on Ribosome Biogenesis

Determining the role of satellite cells in adult skeletal muscle hypertrophy has been an area of intense interest for the skeletal muscle community for some time (76, 89). Satellite cells are thought to be required for hypertrophy through fusion with existing myofibers; this fusion event leads to an increase in the number of myonuclei, which is considered to be necessary for maintaining the myonuclear domain within the growing fiber. Indeed, each nucleus added to the myofiber results in the myofiber possessing a greater number of rDNA copies for transcription, which, in theory, would increase the capacity of the cell to promote ribosome biogenesis. Although this is a reasonable hypothesis, the myofiber contains hundreds of nuclei, with each nucleus having ~300 copies of rDNA, as previously mentioned. Furthermore, most of the rDNA copies are not available for transcription at a given time, i.e., the rDNA copies exist in both active and inactive states, suggesting that the fiber has a sufficient reserve of rDNA copies available for transcription (57, 110). In mice, the increase in rDNA transcription occurs significantly earlier (~3–5 days post-synergistic ablation) than satellite cell fusion (7–10 days), indicating that the addition of nuclei is not required for substantial increase in 47S pre-rRNA expression (65, 132). Similarly in humans, pre-47S pre-RNA expression appears to be maximally stimulated within 1 day following a single session of resistance exercise (Table 1), whereas myonuclear accretion can only be observed following months of repeated bouts of resistance training (116).

To determine the necessity of myonuclear accretion for skeletal muscle hypertrophy, we developed the Pax7-DTA mouse model, which allows for the conditional ablation of satellite cells in adult skeletal muscle (80). Using the Pax7-DTA mouse, we demonstrated that significant muscle growth can occur in the absence of satellite cell fusion in adult mice (65, 80, 90). These findings provide evidence that myonuclear addition via satellite cell fusion is not required for muscle hypertrophy and that resident myonuclei are capable of supporting the transcriptional demand necessary for muscle growth. In support of this idea, Tyler and colleagues used metabolic labeling to show that resident myonuclei in satellite cell-depleted muscle are capable of enhanced transcription during hypertrophy (65). The enhanced transcription in satellite cell-depleted muscle was paralleled by a significant increase in 47S pre-rRNA expression, indicating the resident myonuclei were able to compensate by further upregulating ribosome biogenesis compared with muscle with satellite cells (65). Older subjects have been reported to have a higher number of myonuclei than their young counterparts (59) but yet show blunted ribosome biogenesis in response to a hypertrophic stimulus (12, 64, 119). These findings support the notion that it is not necessarily the number of myonuclei per fiber that is important for hypertrophy but rather the ability of resident myonuclei to activate rDNA transcription, thereby promoting ribosome biogenesis and the subsequent increase in translational capacity of the myofiber.

Although our studies using the Pax7-DTA mouse have provided convincing evidence to show adult (>4 mo of age) skeletal muscle is capable of hypertrophic growth independent of satellite cells, more recent analysis has revealed an age-dependent requirement for satellite cells for muscle hypertrophy (90). When satellite cells were ablated in still maturing 2-mo-old mice, the muscle was unable to hypertrophy in response to 10 days of synergist ablation (90). Given actively growing mice have a high translational capacity (as measured by total RNA per milligram of tissue), we hypothesize that, during postnatal growth, the transcriptional output is maximized, and thus the additional transcription demand induced by mechanical overload cannot occur without satellite cell fusion (FIGURE 5). Thus myonuclei accretion promotes a transcriptional capacity reserve that can be further utilized in a fully grown animal. These findings, together with the well-established need for satellite cells during postnatal muscle hypertrophy, support the concept of a continuum in the requirement for satellite cells that progressively diminishes from postnatal development to adulthood. More research is necessary to confirm the concept of an age-dependent requirement for satellite cells during skeletal muscle and the underlying mechanism.

FIGURE 5.

Age-dependent requirement for satellite cells for skeletal muscle hypertrophy

A theoretical model is proposed in which there is a strict requirement for satellite cells, i.e., myonuclear accretion during postnatal growth to meet the high transcription demand necessary for muscle hypertrophy. As the muscle reaches adulthood and the rate of growth levels off, the dependence on satellite cells for hypertrophic growth disappears as resident myonuclei have sufficient reserve capacity to meet the transcriptional output required for hypertrophic growth.

Future Directions and Conclusions

Ribosome biogenesis, as stated before, appears to be largely regulated at rDNA transcription. Because of that, and because most of the current knowledge accumulated from the recent studies in skeletal muscle have focused on this step, the present review has largely also focused at this cellular event. However, the possibility that other steps governing ribosome biogenesis are also altered by exercise and during growth is promising and should receive attention. In addition, to better understand the mechanisms of rDNA transcription following an anabolic stimulus, such as mechanical overload or resistance exercise training, there are other avenues of research regarding ribosome biogenesis and translational capacity, described below, that have yet to be fully explored.

Ribophagy

Ribophagy is the autophagic process that results in the degradation of ribosomes. Whether ribophagy is altered following a bout of resistance exercise or mechanical overload induced by synergist ablation remains to be fully investiagted. A change in ribosome turnover could have a significant impact on the translational capacity of the cell and its response to an anabolic stimulus. Furthermore, determining whether ribophagy is affected by age may provide insight into why older individuals have comparatively more ribosomes than younger individuals.

rRNA Processing

Although most of the focus of recent studies has been on the initiation of ribosome biogenesis, i.e., rDNA transcription, there is a paucity of data regarding the regulation pre-rRNA processing and maturation. We anticipate that resistance exercise and/or mechanical overload may also increase the machinery involved in rRNA processing, given that genes encoding rRNA processing factors have been reported to be upregulated in rodent and human skeletal muscle during hypertrophic growth (15, 32).

rDNA Copy Number

The number of rDNA copies varies considerably within the population (40, 41). Humans have on average ~300 copies of rDNA per genome, ranging from 60 up to 1,590 copies (101). Thus, within the population, it is possible to find subjects with 25 times the number of rDNA copies of another individual. An unanswered question is whether such a dramatic difference in rDNA copy has an impact on ribosome biogenesis in response to a hypertrophic stimulus (FIGURE 4). Are individuals with a higher rDNA copy number more responsive to anabolic stimulus compared with persons with a lower number of rDNA copies?

In addition to a possible impact on anabolic response, it may be that rDNA copy number has an influence on the requirement for myonuclear accretion during muscle growth. For instance, an individual with a low rDNA copy number may benefit from myonuclear accretion as a way to increase the number of rDNA copies available for transcription; alternatively, a person with a high rDNA copy number might not require myonuclear accretion because their myofibers already possess a sufficient number of rDNA copies to support ribosome biogenesis. This may also help explain some of the controversy regarding the requirements for satellite cell and muscle hypertrophy found in the literature. Although this is a provocative idea, it remains highly speculative and requires further investigation.

Over the past few years, there has been considerable progress in better understanding the role of ribosome biogenesis in skeletal muscle mass regulation. Findings from numerous studies support an important role for an increase in translational capacity as a central regulator of muscle hypertrophy; however, there are still open questions to be answered. Future studies will need to use loss- and gain-of-functions strategies to provide definitive evidence for the necessity and sufficiency, respectively, of ribosome biogenesis for skeletal muscle hypertrophy. If such studies provide confirmatory evidence, it will support ribosome biogenesis as a therapeutic target to prevent and/or restore the loss of skeletal muscle mass associated with various wasting conditions, aging, and cancer cachexia.