Making Sense of MYC in Skeletal Muscle: Location, Duration, and Magnitude

The A-to-Z of M-Y-C in muscle

In skelelal muscle, the most voluminous cells are relatively enormous muscle fibers that are syncytial and generally considered non-replicative. Because of muscle fiber resistance to replication, skeletal muscle is less cancer-prone than other tissues (1) and is used as a model organ for the study of tumor-combatting strategies (2). c-MYC (MYC) is a powerful oncogene and Yamanaka factor that can drive tumor growth in mononuclear cells (3). MYC levels are typically low or undetectable in healthy adult skeletal muscle tissue, but MYC transcript and protein is upregulated in muscle fiber nuclei (myonuclei) during a hypertrophic stimulus (4-6). Higher-than-baseline levels of MYC can be sustained in murine skeletal muscle for up to 14 days during mechanical overload (MOV) without overt signs of tumor development or pathology (7). MYC induction in muscle fibers is thought to drive skeletal muscle hypertrophy, potentially by stimulating ribosome biogenesis and protein synthesis (8), but mechanistic evidence has been elusive. Recent work provides fundamental insights on the role of MYC in skeletal muscle using Cre-mediated murine models of constitutive (chronic) MYC overexpression and knockout (9). This work dovetails with studies using a muscle fiber-specific doxycycline-mediated (pulsatile) model of MYC induction to understand how this oncogene affects adult skeletal muscle (4, 10, 11). In the context of these recent studies and the broader body of literature on MYC in muscle, we ask: is there a level of MYC induction that is “just right” for inducing favorable muscle adaptations?

Chronically high MYC levels in muscle fibers is expectedly bad

Foundational work defining the role of mTORC1 – another central driver of tumor development – with respect to skeletal muscle mass regulation showed that mTORC1 is essential to loading-induced skeletal muscle growth (12, 13). On balance, sustained mTORC1 induction leads to muscle pathology and atrophy in mice (14-16). Similar to what occurs with chronic mTORC1 activation, MYC overexpression using a muscle fiber-specific tamoxifen-inducible and constitutive Cre/LoxP model causes muscle pathology in adult mice. This pathology is characterized by disruptions to muscle fiber structure and function, atrophy of fast-twitch and glycolytic myosin heavy chain type 2B- and 2X-enriched muscles, and body weight loss (17). The pathological response is observed after 4 days of chronic MYC overexpression in the muscles studied, and is exacerbated by 10 days. Another study induced MYC for two weeks in the primarily fast-twitch tibialis anterior muscle of mice using AAV and did not report atrophy or pathology, but histology was not presented (8). Persistent MYC levels with AAV may be lower and less deleterious than with a genetic Cre-mediated approach. A growing body of evidence suggests that hyperactive mTORC1 is a cause of age-related sarcopenia (15, 18-21). To our knowledge, MYC is not elevated in skeletal muscle with aging. We are not aware of any naturally-occuring conditions where chronic MYC elevation in muscle explains a deleterious etiology. Preliminary evidence suggests a loss of MYC may accelerate normal muscle aging (22, 23), whereas suppression of mTORC1 likely does not (24).

Considering the muscle-specific and temporal aspects of MYC

Following a bout of resistance exercise in human skeletal muscle, MYC transcript in the Type 1 and 2a-enriched vastus lateralis muscle is strongly induced and peaks at 3 hours, remains elevated at 8 hours, then returns to baseline at 24 hours (25). The muscle MYC mRNA response to a bout of exercise in rodents and humans may become blunted as training status progresses (26, 27); this suggests MYC could be most important for the early training response. Alternatively, MYC transcriptional dynamics after a bout of exercise may “phase shift” and peak at a different time in the trained versus untrained state, which has been shown to occur with other transcription factors in mouse muscle after endurance exercise when well-trained (28). More experimentation is needed to corroborate such a “phase shift” with resistance training. MYC protein is induced in human skeletal muscle following resistance exercise (29-32). In the untrained state, MYC is the most influential transcription factor governing global gene expression after a bout of resistance exercise in humans, specifically in the 8-24 hour recovery window (10) coinciding with a pulse of ribosome biogenesis (25). We reason that this transient response after exercise is important for MYC to function properly in skeletal muscle fibers and contribute positively to muscle hypertrophic remodeling.

In contrast to constitutive muscle fiber-specific MYC induction, and in an effort to replicate the “pulsatile” nature of MYC after resistance exercise in skeletal muscle, we developed a muscle fiber-specific doxycycline-inducible “Tet-ON” MYC mouse (4). When doxycline is delivered in drinking water, MYC protein in muscle is induced by 12 hours then returns to baseline within 24 hours after discontinuing doxycycline (33). A 12-hour pulse of MYC in murine skeletal muscle imitates major aspects of myonuclear gene expression during MOV of the fast-twitch plantaris muscle (4) and elicits signs of epigenetic reprogramming in the oxidative soleus muscle (33). This pulsatile model was used to determine whether repeated transient MYC is sufficient to induce skeletal muscle fiber hypertrophy.

Low-dose doxycycline was delivered to muscle-specific “Tet-ON” MYC mice in drinking water for 48 hours, then removed for 5 days. The procedure was repeated 5 times over one month. The rationale for this approach was: 1) Yamanaka factors (Oct3/4, Sox2, Klf4, and Myc) have been induced in muscle fibers using a similar doxycycline model for 2.5-8.5 days without deleterious effects; this strategy improved muscle healing after injury (34), and 2) 48 hours of induction approximates muscle fiber MYC exposure with resistance exercise training a muscle group twice per week - a standard practice for gymgoers. Brief pulsatile MYC over four weeks resulted in whole muscle and muscle fiber hypertrophy of the soleus (10) without overt signs of pathology such as abnormally small or large muscle fibers, apparent immune cell infiltration, or central myonuclei, with maintained body weight (10). The murine soleus muscle is comprised of type 1 and type 2A fibers - the primary myosin fiber types found in human skeletal muscle – making it the hindlimb muscle most analagous to human ambulatory muscles according to fiber type content and distribution. Hypertrophy of the soleus muscle also occurs after 10 days of chronic Cre-mediated MYC induction (9). MYC gene expression is more rapid and exaggerated in the soleus than the type 2B- and 2X-enriched plantaris in response to MOV in rodents (35). The transcriptome response to a genetically-driven MYC pulse is also more pronounced in the soleus (33) than the plantaris (4) despite similar MYC protein levels (10), consistent with the muscle-specific hypertrophic response to genetic MYC induction (9, 10). The soleus may therefore be unique in its responsiveness and/or tolerance to MYC. It is worth noting that the unique hypertrophic response to sustained MYC induction in the soleus is also observed with chronic mTORC1 activation in mice (16). Whole muscle hypertrophy of Type 2B- and 2X-containing hindlimb muscles (e.g. plantaris, gastrocnemius, tibialis anterior) was not observed with MYC pulses, but there was also no atrophy or signs of pathology. Tuning MYC duration and/or amount may be required to reveal beneficial affects in Type 2B- and 2X-enriched muscles of rodents and capture the “exercise effect”.

The transcriptomic response to MYC in muscle suggests less is more

We sought to gain a better understanding of acute versus chronic MYC induction in muscle at the molecular level. An analysis of published RNA-sequencing from acute (overnight) (4, 33) versus chronic (4-10 day) MYC induction (9) shows massive downregulation of muscle development genes and upregulation of mitochondrial genes favoring the chronic condition (adj. p<0.05, Figure 1, Supplemental Table 1). Furthermore, known signs of muscle fiber damage (36) – α-actinin (Actn2), cell division cycle 42 (Cdc42), filamin C (Flnc), heat shock protein beta 1 (Hspb1), neonatal myosin heavy chain (Myh8) and Myh10, as well as dysferlin (Dysf) – were strongly induced only with chronic MYC (all adj. p<0.05, Supplemental Table 2). Loss of muscle identity and possible metabolic crisis with constitutive MYC may override the pro-hypertrophic ribosome-related gene response characterizing acute and chronic MYC induction (4, 8-10, 33), with the most negative effects in Type 2B- and 2X-containing muscles. These data collectively provide evidence for the beneficial versus pathological roles of MYC in muscle based on the duration and/or magnitude of induction.

Figure 1.

Analysis of RNA-sequencing from an acute pulse of MYC in the plantaris (4) and soleus (10) (12 hours of doxycycline, 12 hour chase, differentially expressed genes (DEGs) with adjusted p<0.05 combined for both muscles, derived from supplemental tables of original manuscripts) versus constitutive MYC induction in the gastrocnemius (9) (4- and 10-day overexpression data, differentially expressed genes with adjusted p<0.05 combined for both time points). Transcript-level quantification data were obtained from Ham et al. (GEO accession: GSE28001). For the Ham et al. data, transcript abundances from Salmon quantification files were imported using tximport (v1.34.0), and gene-level differential expression analysis was performed using DESeq2 (v1.46.0) in R (v4.4.3). Genes were analyzed by timepoint (4d vs. 10d) and filtered into up- or downregulated gene sets based on the direction of log₂ fold change, then combined to make a list of “chronic” MYC genes. Ontology Biological Process (GO:BP) enrichment analysis was performed on the chronic DEG sets using the enrichGO() function from clusterProfiler (v4.14.6), with gene annotation from org.Mm.eg.db (v3.20.0). Analyses used a minimum gene set size of five and an adjusted p of 0.05, tested against the background of all expressed genes. The top 15 enriched terms from each set were visualized using ggplot2 (v3.5.1), with bar length corresponding to gene count and fill color representing enrichment significance (−log₁₀ adjusted p-value). All analyses for GO:BP, GO:Cellular Component (CC), and GO: Molecular Function (MF) are in Supplemental Table 1, and DEGs used for analyses are in Supplemental Table 2.

A matter of place and time after loss of MYC in muscle

MYC is induced in skeletal muscle during development but declines into adulthood (35). Viral induction of MYC in muscle during the early phase of postnatal development in chickens causes enhanced muscle growth, and signs of pathology were not noted on histology (37). One might speculate that a loss of MYC in muscle during postnatal development would reduce muscle mass in early adulthood, but this does not appear to be the case in mice (9, 22). Interestingly, systemic post-developmental loss of MYC in mice causes premature aging associated with muscle atrophy beginning around 10 months of age (22). Loss of muscle strength and/or function with global MYC knockout – at least in male mice – tended to manifest earlier in life (22), which tracked with some observations from muscle-specific loss of MYC (9). Overt signs of premature aging (22) with muscle fiber-specific loss of MYC from birth is not apparent by 5-7 months (9), but these mice were not analyzed later in life. These results collectively suggest that MYC loss could contribute to aspects of muscle maintenance, either directly or indirectly, as age progresses.

Experiments to test the requirement of muscle fiber MYC for MOV-induced skeletal muscle hypertrophy yielded suprising results. MYC was deemed dispensible for loading-induced hypertrophy in adult mice when using the muscle fiber-specific MYC knockout model (9). Other isoforms of the MYC gene, N-MYC and L-MYC, were not compensatorily elevated after muscle fiber MYC knockout throughout postnatal development; however, these data were not reported for MOV (9). The role of MYC in Pax7+ muscle stem cells (satellite cells) has been tested during MOV using an inducible satellite cell-specific MYC knockout (9). MYC knockout in satellite cells impaired proliferation, which is expected given MYC’s major role in mononuclear cell growth and prior confirmatory findings in myoblasts (37). Perhaps unexpectedly, MYC knockout in satellite cells blocked hypertrophy after short-term MOV (14 days) and even caused atrophy relative to controls after 28 days of MOV in male mice. Muscle hypertrophy over 7-14 days of MOV proceeds in the absence of satellite cells (38-40), whereas disruption to satellite cell fusion via Myomaker deletion during MOV of similar duration prevents muscle hypertrophy (41). One recent study showed that interrupting satellite cell fusion during short-term MOV via satellite cell-specific loss of TRIM28 caused abnormal dystrophin expression in muscle fibres akin to some myopathies (42). Furthermore, satellite cell fusion in the setting of myopathy can be deleterious (43), highlighting how the fusion process disturbs the muscle fiber membrane. Perhaps satellite cells that initiate fusion processes but ultimately do not fuse can compromise sarcolemmal integrity, thereby triggering adverse outcomes such as atrophy and/or fibrosis (9, 41). Beyond fusion, proper satellite cell communication with other cell types – including the muscle fiber – is vital to the role of these cells in hypertrophic adaptation (44-47). We speculate that dysfunctional satellite cells could conversely have negative consequences in muscle due to dysregulated intercellular communication. While satellite cells are required for sustained muscle hypertrophy (40, 46, 48, 49), at least in the short-term and in the context of non-regenerative adult muscle adaptation, dysfunctional satellite cells may be more deleterious than having no satellite cells at all.

Perspectives and summary – a proposed “Goldilocks” zone for MYC in muscle

Recent studies and the overall literature suggest that the magnitude and duration of MYC induction, as well as the fiber type and/or function of the muscle where MYC induction is occurring, are critical determinants of the cellular response. Perhaps there is a “Goldilocks zone” for MYC levels in the muscle fibre. The “right” amount is beneficial, but too much or not enough (duration and/or magnitude) is detrimental - similar to how mTORC1 exerts its function in skeletal muscle. This “just right” amount likely differs across muscles and fibre types. Our “Goldilocks” hypothesis is reinforced by satellite cell experiments that explore the role of MYC in muscle injury repair: pronounced induction as well as complete knockout of MYC in satellite cells both impair muscle regeneration (9). Cre-mediated mouse models create constitutive expression that can make it challenging to replicate the transient nature of the molecular response to a bout of exercise. A more temporally-resolved approach which affects the duration and magnitude of gene expression, such as that provided by the Tet-ON system, could prove very important for investigating the effects of MYC and other criticial exercise-responsive genes in skeletal muscle.

Supplementary Material

DOI: 10.6084/m9.figshare.29572910