Satellite cell dynamics during skeletal muscle hypertrophy

Skeletal muscle is a dynamic tissue that adapts continuously to functional demands, a process that often requires resident muscle stem cells (MuSCs), commonly known as satellite cells [1–3]. MuSC were originally named satellite cells because they are located on the periphery of muscle fibers, just beneath the basal lamina and adjacent to the sarcolemma. First identified by Alexander Mauro and Bernard Katz in 1961, MuSCs are quiescent under homeostatic conditions [4,5]. In response to muscle injury, MuSCs become activated, proliferate, and differentiate into myoblasts; however, depending on the degree of damage, myoblasts can fuse to one another to form myotubes or fuse with damaged muscle fibers to restore muscle structure and function [6,7]. MuSCs are now recognized as the cellular basis for the remarkable regenerative capacity of skeletal muscle and are also thought to be necessary for maintaining muscle homeostasis throughout an organism's life. Secondary to muscle regeneration, MuSCs have also been shown to become activated, replicate, differentiate and then fuse to myofibers in response to a hypertrophic stimulus, resulting in an increase in myonuclear abundance [8–12]. In contrast with the well-characterized process of muscle regeneration, the dynamics of MuSCs behavior during muscle hypertrophy remain less explored. Recent studies, including work from our laboratory, have begun to elucidate the behavior of MuSCs during hypertrophy induced by mechanical overload (MOV) resulting from synergist ablation. In this review, we discuss the significance of MuSC dynamics during hypertrophy and highlight the molecular factors governing the division patterns. Finally, we review our recent discovery of a division-independent differentiation state exhibited by MuSCs, illuminating its implications for advancing our understanding of muscle growth.

Skeletal muscle plasticity is a unique feature of skeletal muscle that encompasses a capacity for hypertrophy as well as a remarkable regenerative ability [13]. Different models have been used to study skeletal muscle hypertrophy, including surgical induction of MOV on the plantaris muscle by tenotomy or synergist ablation [14]. One advantage of the MOV model of hypertrophy is that it provides a robust signal for MuSC activation with a well-defined time course for MuSC myofiber fusion [15]. Myonuclear accretion associated with hypertrophy is the result of MuSC fusion, as MuSC-depleted mice show no myonuclear accretion during hypertrophy [16,17]. Unexpectedly, we reported that in response to short-term (2 weeks) MOV, the increase in muscle mass is not affected by MuSC depletion [18]. The observation of effective short-term hypertrophy in the absence of MuSCs suggests there may be compensatory MuSCs-independent mechanisms of muscle growth. To this end, we found that resident myonuclei are able to further enhance their transcriptional output to compensate for the loss of myonuclear accretion [19]. Still, studies using other experimental methodologies and models, including genetic deletion of myomaker, have suggested that MuSC fusion is required for short-term MOV-induced hypertrophy [20,21]. Regardless, long-term hypertrophy (8 weeks) is attenuated by fibrosis in MuSC-depleted muscle [20–22]. In addition to myonuclear accretion as a result of myofiber fusion, we provide evidence to support a mechanism in which MuSCs communicate with fibroblasts to regulate extracellular matrix remodeling during hypertrophy [23]. Moreover, MuSC communication during the first 96 h of MOV is sufficient to allow for optimal long-term muscle hypertrophy [24].

While some aspects of MuSC dynamics in response to injury and mechanical loading are similar, there are key differences between regeneration and hypertrophy [16,17,25]. First, during muscle regeneration following injury, MuSCs proliferate between the remnants of the myofiber, referred to as ghost fibers, and the basal lamina, while during MOV-induced hypertrophy, myofibers remain intact such that MuSCs proliferate between the basal lamina and plasma membrane [16,17]. Proliferation-related genes, including downstream targets of Notch signaling and transcription factors (i.e. HeyL and Col5a1), also differ in MuSCs of regenerating and hypertrophying muscles [26]. Differences in fusion mechanisms also distinguish hypertrophy and regeneration. In the presence of intact fibers, MuSCs fuse to myofibers (heterotypic fusion). In contrast, in the early stage of muscle regeneration, MuSCs undergo myoblast-myoblast (homotypic) fusion [15]. Likewise, these different fusion mechanisms result in different myonuclear positioning, whereby regenerating myofiber nuclei are located in the center of the myofiber, while during hypertrophy, newly acquired myonuclei reside in the myofiber periphery [18,20]. Finally, the extent of inflammation is much greater during regeneration compared with hypertrophy [27,28]. These findings underscore the importance of distinguishing MuSC behavior in response to injury and MOV.

During regeneration, MuSC division is intrinsically coupled to differentiation, and MuSC division occurs through two distinct modes: symmetric division, yielding either two MuSCs (self-renewal) or two differentiating cells, and asymmetric division, resulting in one stem cell and one differentiating cell [29]. The mode of division is regulated by a myriad of intrinsic and extrinsic signals. The ‘intrinsic’ mechanism involves the uneven distribution of cellular factors determining cell fate. During this process, certain proteins such as MyoD, SCA1, p38α/β, pERK, PKCλ, and Par3 preferentially segregate into the differentiating daughter MuSCs. Symmetric division, on the other hand, evenly distributes these proteins between both daughter cells [30,31]. Emerging evidence indicates that cytokinesis remnants, such as the midbody, might not segregate evenly between daughter cells. Instead, these factors may be passed down in an asymmetric manner, possibly acting as a dynamic signaling center that steers the course of MuSC growth and fate [31,32]. Investigations using Drosophila melanogaster male germline stem cells have underscored the non-equivalent inheritance of specific histone variants during asymmetric cell division, indicating a potential role for epigenetic mechanisms in regulating stem cell fate determination [33,34]. However, recent findings by Evano et al. [29] challenges the notion that asymmetric cell fate decisions in MuSCs are dictated by the inheritance of parental and newly synthesized histone pools, suggesting that epigenetic modification of histones may not be a determinant of MuSC fate.

The second mechanism of asymmetric division involves the asymmetrical positioning of daughter cells in response to external cues, known as the ‘extrinsic’ mechanism [35]. This process results in one daughter cell remaining within the MuSC niche, maintaining its stem cell characteristics, while the other daughter cell is positioned away from the niche, begins to undergo differentiation [36]. The MuSC niche play a crucial role in maintaining stemness by providing a microenvironment rich in signaling molecules which interact with MuSCs and initiate downstream transcriptional networks that define their identity [2,35,37]. Therefore, even if the division itself is intrinsically symmetric, this mechanism achieves an asymmetric outcome. For example, compromised Notch signaling leads to the depletion of muscle progenitors in mouse embryos [36,38–42]. Here, the Notch ligand Delta-like1 in committed cells is thought to signal to neighboring MuSCs, preserving the stem cell pool [36,38,41,42]. Another niche factor, the ligand Wnt7a can promote the symmetric expansion of MuSCs but not their differentiated progeny through the non-canonical Wnt signaling pathway [43,44]. This pathway induces a polarized distribution of Vangl2, a Planar Cell Polarity effector, within MuSCs. This spatial arrangement likely assists MuSCs in detecting the uneven distribution of signaling molecules in their microenvironment, thereby preserving their stem cell properties [43,44].

Using SNAP-tagged histone H3-reporter mice and clonogenic tracing, Evano et al. [29] demonstrated the dynamic capability of MuSCs to alternate between symmetric and asymmetric cell divisions. Their findings propose the involvement of intrinsic mechanism or external signaling between sister MuSCs in regulating this transition [29]. MuSCs, primarily quiescent, pose challenges for direct observation due to their infrequent divisions. However, during regeneration, a brief period of heightened MuSC activity (i.e. MuSC self-renewal and differentiation) allows for easier monitoring [45]. Intriguingly, observations at single-cell resolution in vivo reveal that MuSCs predominantly undergo self-renewing symmetric divisions at 3 days post-injury, transitioning to both symmetric and asymmetric divisions by day 5 [29]. The shift from symmetric to asymmetric division during this timeframe, coincides with reduced proliferative capacity [29,45,46]. The adoption of symmetric division likely serves to replenish the MuSC pool depleted during regeneration, while asymmetric division maintains the MuSC pool during homeostasis [47]. Recent ex vivo studies tracking individual cells across successive divisions also revealed the ability of MuSCs to switch from asymmetric to symmetric divisions; however, the implications of this intriguing switch in MuSC division behavior remains unclear [29].

The role of MuSCs in muscle growth and regeneration has been traditionally understood to involve both symmetric and asymmetric divisions prior to differentiation (Figure 1A) [29,48,49]. This paradigm posits that the differentiation of MuSCs is inherently tied to their division patterns. While it remains to be determined if MuSCs undergo symmetric and/or asymmetric division upon activation during hypertrophy, recent studies have provided evidence that MuSCs may be able to differentiate directly without prior division (Figure 1B) [50–52]. Our laboratory has provided further evidence to support this alternative MuSC behavior. By employing scRNA-seq, a novel mouse model for tracking MuSC-derived myonuclei, and in vivo DNA replication labeling, we have identified a distinct subpopulation of MuSCs. These MuSCs demonstrate the ability to differentiate and fuse directly to adult muscle fibers in response to a hypertrophic stimulus without undergoing cellular division [15].

While ongoing studies are focused on better understanding the underlying mechanism and functional significance of direct MuSC fusion during hypertrophy, evidence from some recent studies offer some intriguing possibilities. Work by Wang and colleagues found the genetic depletion of mitochondrial Complex IV (C-IV) in MuSCs caused their direct fusion to myofibers. Furthermore, the direct fusion of MuSCs upon C-IV depletion was prevented when the mice were treated with the antioxidant N-acetylcysteine [52]. This finding suggests that elevated ROS, as a result of mitochondrial dysfunction, can initiate a fusogenic program in MuSCs. The authors proposed that such a mechanism might provide a way for muscle to maintain a healthy pool of MuSCs [52]. While speculative, the direct fusion of MuSCs in response to a hypertrophic stimulus might represent a similar situation. In support of a ROS mechanism, our scRNA-seq analysis of MuSCs found lower expression of Nrf2, a master regulator of antioxidant genes, in MuSCs in the direct differentiation trajectory; however, this proposed mechanism remains to be tested [15]. Alternatively, there may be a subset of quiescent MuSCs which are potentially primed for differentiation, positioning them as early responders upon activation. This subpopulation may be characterized by the down-regulation of Notch 1 expression [15]. Notably, previous studies have shown the genetic inhibition of Notch signaling spontaneously induces direct MuSC fusion which leads to the depletion of the MuSC pool [38,50,53].

Similarly, Eliazer et al. found that Delta-like 4 (Dll4), a Notch receptor ligand, is unevenly distributed along the periphery of the muscle fiber [54]. This spatial variability could limit MuSC Notch activation in regions with low Dll4. As a result, MuSCs in these areas, despite expressing Notch receptors, may experience reduced Notch signaling, leading to direct MuSC fusion.

A final potential mechanism regulating direct MuSC fusion triggered by MOV-induced hypertrophy might involve alterations in MuSC extrinsic cues including growth factors and inflammatory cytokines [55,56]. These environmental cues could potentially impose a selective pressure on MuSCs, which might lead to heterogeneous responses within the MuSC population. Thus, hypothetically, some MuSCs could undergo immediate differentiation and fuse with existing myofibers, potentially contributing directly to muscle growth, while others might prioritize proliferation to replenish the MuSC pool. Further research is warranted to determine the mechanism regulating the direct differentiation and fusion of MuSC with existing myofibers, and to evaluate their implications for muscle growth. While the Pax7^rtTA; TRE-H2B-GFP mouse has played a key role in discovering the direct fusion of MuSCs during hypertrophy, a major challenge moving forward will be figuring out how to perform loss- and gain-of-function studies in a specific subpopulation of Pax7+ MuSCs. The development of such tools will allow us to determine the mechanism regulate the direct fusion of MuSCs and the functional importance of such MuSC-derived myonuclei to muscle hypertrophy.

• During muscle regeneration, MuSCs can undergo symmetric or asymmetric division, with the choice between these modes being influenced by both intrinsic and extrinsic factors.

• Typically, MuSCs undergo cell division prior to differentiation; however, a novel finding demonstrates that MuSCs are capable of directly differentiating in response to growth stimuli without first undergoing cell division.

• The focus of future studies will be to determine the mechanism regulating the direct fusion of MuSCs during muscle hypertrophy and the functional implications.

The authors declare that there are no competing interests associated with the manuscript.

This work was supported by National Institutes of Health grants from the National Institute on Aging (AG069909) to J.J.M.

Open access for this article was enabled by the participation of University of Kent in an all-inclusive Read & Publish agreement with Portland Press and the Biochemical Society under a transformative agreement with JISC.

MOV

mechanical overload

MuSC

muscle stem cell