Non-regenerative myogenesis in adult skeletal muscles: myofiber death-independent muscle satellite cell expansion

Abstract

Muscle regeneration processes triggered by injuries following myofiber death have been extensively investigated owing to the availability of suitable in vivo animal models, including disease models. During muscle regeneration, skeletal muscle-resident stem cells, also known as muscle satellite cells (MuSCs), exit the quiescent state, become activated, and proliferate to generate new myofibers. These MuSC behaviors have also been observed in resistance- and endurance-training models. In these models, MuSC behaviors are thought to follow the same mechanisms as myogenesis observed during regeneration (regenerative myogenesis), as certain animal training models induce myofiber death; moreover, myofiber damage markers have been detected in human training samples. Nevertheless, accumulating evidence suggests that MuSCs can become activated, proliferate, and differentiate in the absence of overt signs of myofiber death. Considering the therapeutic potential of MuSCs for muscle atrophy, injury-independent myogenesis (non-regenerative myogenesis) must be investigated, as apparent and frequent myofiber death does not occur in many atrophic conditions. In this review, we introduce the concept and evidence for “non-regenerative myogenesis” in adult skeletal muscle and highlight its characteristics and the remaining research questions.

Background

Muscle satellite cells (MuSCs) are skeletal muscle-specific stem cells that are committed exclusively to the myogenic lineage [1, 2]. Under steady-state conditions, MuSCs remain quiescent in the G0 phase of the cell cycle [3]. However, upon muscle injury following myofiber death, MuSCs are rapidly activated and enter the proliferative phase, at which stage they are referred to as myoblasts, and express the myoblast determination protein 1 (MyoD). Subsequently, Myogenin expression in the myoblasts induces their withdrawal from the cell cycle, enabling cell fusion [4, 5]. Fusion events between myoblast–myoblast and myoblast–myotube (nascent myofibers) increase the number of nuclei in growing myofibers [6, 7]. This process eventually results in the reconstruction of mature myofibers. Myofiber death is a hallmark of muscular dystrophies, and repeated myofiber degeneration results in the reduced regenerative ability of MuSCs. Myofiber death also occurs due to tissue trauma. Therefore, enhancing muscle regeneration is a promising therapeutic strategy for treating muscular dystrophy [8, 9] and for promoting recovery in humans, including athletes.

Unnecessary activation, proliferation, or differentiation of MuSCs leads to depletion of their pool and impairs their regenerative capacity; therefore, they should not be activated or proliferate during the contraction and relaxation of skeletal muscle in daily life. To maintain the MuSC pool, several active signaling pathways, including Notch [10–14], Calcitonin receptor (CalcR) [15–17], and M/N-cadherin [18, 19] signaling, are involved in MuSCs. The Notch-Rbp-J-Hey1/HeyL/Hes1 axis is essential for maintaining an undifferentiated state (MyoD suppression) [10, 11, 13], and the Notch–Collagen V-CalcR-PKA-YAP axis contributes to the maintenance of quiescence (G0 state) [15, 17]. M/N-cadherin-α/β-catenin is necessary for anchoring MuSC to myofibers [19]. When myofibers are damaged or MuSCs detach from them, these maintenance signals are rapidly declined [20]. Therefore, it is reasonable to assume that the condition of myofibers (intact or damaged), as well as their presence or absence, significantly impacts the MuSC behavior.

It is widely accepted that exercises beyond a certain threshold, including resistance or endurance training, induce the activation, proliferation, and differentiation of MuSCs [21]. Exercise-induced muscle damage (EIMD) is considered the primary mechanism underlying MuSC activation and proliferation. EIMD in humans frequently occurs following unaccustomed exercise, particularly when the activity involves high intensity (e.g., near-maximal or maximal voluntary contraction) and a large volume of eccentric contractions (ECCs) [22], such as downhill running [23] or plyometric exercise (e.g., repeated drop jumps) [24]. Muscle injury markers in the bloodstream such as elevated creatine kinase (CK), myoglobin, C-reactive protein (CRP), and interleukin-6 (IL-6) have been reported in humans following ECC exercise [25]. ECCs induced by neuromuscular electrical stimulation (NMES) are widely used as a model of EIMD in humans [26] and rodents [27]. However, as Clarkson and Hubal noted, direct assessment of damage in human muscle biopsies is difficult because only a small portion of the muscle is available [28]. Therefore, in human studies, indirect assessment of myofiber death has mainly been employed. Muscle damage, including disturbance of the myofibril structure and decreased stability of the sarcolemma, was detected in the direct assessment; however, this damage does not necessarily cause myofiber death, and direct evidence of myofiber death is limited [26]. Collectively, it is difficult to accurately estimate the extent of myofiber death caused by exercise, and it is unclear whether myofiber damage or death is necessary for exercise-induced MuSC activation and proliferation. If MuSCs can proliferate in the presence of mature myofibers and independently of myofiber death, elucidating the mechanisms underlying such myofiber death-independent MuSC behaviors is crucial for the feasibility of MuSC-mediated restoration of atrophied muscles, such as sarcopenia, because active myofiber death, frequently observed in muscular dystrophies, does not occur in many types of muscle atrophy.

MuSC proliferation has been demonstrated in exercise-loaded mouse models in which myofiber death was scarcely observed [29–32]. In addition, we recently demonstrated that the suppression of CalcR signaling, a quiescence signal, accompanied by administration of IL-6, a representative exerkine, can induce MuSC proliferation throughout the body in sedentary mice [33] (Fig. 1A). These findings suggest that an exercise-dependent, but myofiber-damage- and myofiber-death-independent, mechanism drives MuSC proliferation, which may represent the primary pathway underlying exercise-induced MuSC activation and proliferation [34, 35]. This review outlines the activation and proliferation mechanisms of MuSCs independent of muscle injury, termed as ‘Non-regenerative myogenesis’, covering its definition, animal models, relevance to muscle atrophy treatment, and key unresolved issues.

Fig. 1.

Molecular mechanisms underlying non-regenerative myogenesis. A Proliferation of MuSCs in MuSC-specific CalcR conditional knockout mice induced by the exerkine IL-6 in non-increased mechanical loaded muscles (TA and EDL). WT MuSCs remain in a quiescent state in TA and EDL of exercised mice. B Behavior of MuSCs in mechanically loaded versus regenerating muscle. Cartoons illustrate distinct cellular and molecular characteristics associated with each process. All images were sourced from https://scidraw.io/ and modified, except for mice (A), blood vessels (A), and macrophages (B). CalcR, calcitonin receptor; WT, wild type; Mø, macrophage

Evidence of myofiber death-independent MuSC proliferation in adult skeletal muscle

In 1987, Darr and Schultz reported MuSC proliferation in mechanically overloaded muscles with little evidence of myofiber damage [36]. Recent studies have also provided evidence of injury-independent MuSC proliferation [29–32, 37]. For example, we observed active MuSC proliferation and MuSC–myofiber fusion in surgically overloaded muscles. Notably, we isolated living myofibers from the overloaded muscles in numbers comparable to those from control muscles and observed proliferating MuSC clusters on living myofiber [29]. Even minimal injury precludes the isolation of living myofibers, indicating that the majority of myofibers in this overloaded muscle remain intact. Similar MuSC proliferation and MuSC–myofiber fusion were detected, even under conditions with a lower risk of myofiber death, such as in voluntary wheel-running mice [31]. Fessard et al. also demonstrated that NMES induces muscle hypertrophy, accompanied by an increased number of Pax7⁺ cells and myonuclei without overt signs of muscle damage and regeneration [32]. Collectively, these results suggest that myofiber death-independent MuSC proliferation occurs in mechanically loaded muscles induced by different types of exercise (Fig. 1A).

However, the effects of local myofiber death cannot be completely excluded in these exercise models. In addition, these animal models may induce myofiber tears and disturbances to the myofibril structure, which do not cause myofiber death. Therefore, for definitive evidence of non-regenerative myogenesis, it is crucial to recapitulate MuSC proliferation and differentiation in sedentary mice via exercise-related factors. Recently, we successfully induced MuSC proliferation and MuSC–myofiber fusion in sedentary CalcR-cKO mice by injecting exercise serum or recombinant IL-6 [33] (Fig. 1A). CalcR expression is strictly restricted to the quiescent state in MuSCs, and its downregulation occurs under non-injured conditions, such as aging. Elevated IL-6 levels induced by exercise are also considered not to be associated with muscle injury [38–40]. Furthermore, CalcR downregulation has been observed in both a wheel-running and mouse model of resistance training (tenotomy; a mild mechanical overload model) [30]. IL-6, a well-investigated exerkine (factors released into the blood in response to exercise), is also involved in MuSC proliferation in the overload model [41, 42]. These results strongly support the presence of non-regenerative myogenesis as a major mechanism driving MuSC proliferation in loaded muscles during various types of exercise (Fig. 1).

Definition and characteristics of non-regenerative myogenesis in adult skeletal muscle

Non-regenerative myogenesis follows the mechanism of MuSC activation, proliferation, and differentiation without myofiber death or tears in adult skeletal muscles. During the first step, MuSCs exit the quiescent state, become activated, and proliferate on intact myofibers. A key feature of this process is that MuSCs fuse with myofibers and the resulting MuSC-derived myonuclei are positioned peripherally within the myofibers (Fig. 1B). This is in contrast to regenerated myofibers, in which the myonuclei are centrally located [43] (Fig. 1B). Peripheral myonuclei resulting from MuSC–myofiber fusion are also observed during postnatal muscle growth. White et al. reported no net gain in the myofiber number in the EDL between postnatal day 7 (P7) and P56 mice [44]. However, the number of MuSC-derived myonuclei increased until P21, indicating that MuSC proliferation and fusion occurred without new myofiber generation between P7 and P21 in the mouse model. Importantly, almost all myonuclei were in peripheral positions within the myofibers [12], suggesting that the stage of increased myonuclei during postnatal development is similar to that of non-regenerative myogenesis in adult skeletal muscles.

Some MuSC-specific gene knockout mice also exhibited non-regenerative myogenesis. For example, loss of Notch signaling-related genes in MuSC induces MuSC-myofiber fusion, with MuSC-derived nuclei positioned peripherally within the myofiber [11, 12], although some MuSC can fuse with myofibers without undergoing cell cycling [10]. Furthermore, in the mid-to-late stages of regeneration, MuSCs fuse with the regenerating myofibers, where MuSC-derived myonuclei are positioned peripherally [45, 46]. Collectively, when MuSCs fuse with myofibers that have already undergone a certain degree of differentiation or regeneration, MuSC-derived myonuclei are consistently located peripherally.

In contrast, gene deletions specific to MuSCs, such as those on the G-protein G₁₂-G₁₃ [47] or N-cadherin (more in M/N-cadherin) [18], have been shown to increase the number of myofibers with central nuclei. It remains unclear whether these molecules are necessary for maintaining the peripheral position of MuSC-derived nuclei or if the appearance of centrally nucleated myofibers, similar to regenerated myofibers, results from fusion between MuSCs. However, in the absence of specific gene deletions, the nuclei derived from MuSCs were positioned peripherally in the exercise models.

In summary, non-regenerative myogenesis in adult skeletal muscle can be defined as the process of generating peripheral myonuclei supplied by MuSCs through the following mechanisms (Fig. 1B):

1. Myofiber death- and injury-independent but exercise-dependent mechanisms inducing the activation and proliferation of MuSCs on intact and living myofibers.

2. MuSC–myofiber fusion occurring to supply new peripheral myonuclei.

Exercise/training models and non-regenerative myogenesis

While intense loading facilitates MuSC proliferation, it also increases the risk of myofiber death. Conversely, weak loading decreases the risk of myofiber death, but also decreases the frequency of MuSC proliferation. An experimental system that induces MuSC activation and proliferation, while completely avoiding myofiber death, has not yet been established. As it is necessary to confirm the markers related to muscle injury and regeneration in each experiment, we describe three models in which non-regenerative myogenesis is thought to occur.

Wheel-running models

Wheel-running is a moderate-intensity, long-duration exercise, typically classified as an endurance-style activity. Mice with free access to running wheels voluntarily run several kilometers per day over multiple hours [48]. This voluntary activity induces aerobic and endurance adaptations, including improved oxidative capacity in skeletal muscle, and is widely used as a rodent model of moderate-intensity endurance exercise in humans [49]. MuSC proliferation can be detected in the plantar flexor muscles (plantaris and soleus), but not in the dorsiflexion muscles (tibialis anterior and extensor digitorum longus) ³⁰⁾. Although the extent of MuSC proliferation in wheel-running models is lower than that observed in surgically overloaded models, the risk of myofiber death in wheel-running models is lower. In addition, we observed non-regenerative myogenesis in both slow- (soleus) and fast-twitch (plantaris) muscles in a wheel-running model. In exercise science research, other wheel-running models increase exercise load using weights [50]. Although this additional load allows for more efficient observation of MuSC proliferation, myonuclear accretion, and muscle hypertrophy; however, it may simultaneously increase the risk of myofiber damage. Therefore, it is necessary to verify the extent of muscle damage.

Surgical Overloaded Models

A group of muscles contracts and relaxes synergistically. For example, the plantar flexor muscles, comprising the plantaris, soleus, and gastrocnemius, function cooperatively to support foot movements. When the Achilles tendon, which connects the soleus and gastrocnemius muscles to the bone, is removed, the plantaris muscle alone perceives the entire mechanical load [51, 52]. This procedure results in an increased loading on the plantaris muscle, leading to MuSC activation and proliferation. Notably, a substantial number of viable myofibers have been isolated from overloaded plantaris muscle [29], indicating that myofiber death in this overload model is relatively rare. However, we cannot completely exclude the possibility of local myofiber death in surgically overloaded muscles.

In surgical models, several different approaches can be applied to the plantar flexor muscles, such as preserving only the plantaris (PLA) [53], preserving both the PLA and the soleus [54, 55], cutting only the tendon [29, 56], or removing the tendon along with part of the muscle [57–59]. To minimize the incidence of myofiber death, removal of the tendon alone is recommended ⁴⁴⁾. Moreover, a technique that selectively excises only the gastrocnemius muscle tendon while preserving both the plantaris and soleus tendons can further reduce the risk of myofiber death in rat models [58]. Nevertheless, preserving the soleus tendon in mouse models is technically challenging, highlighting the need for simpler and more reliable approaches.

Isometric neuromuscular electrical stimulation (NMES) training

Contractile activity can be generated by repeated application of electrical stimuli. This method is commonly referred to as NMES in humans and has been employed to enhance athletic performance and support rehabilitation [60]. NMES is an excellent method for investigating both adaptive and maladaptive responses to exercise-induced stress as it allows for precise control over stimulation parameters such as contraction mode, intensity, and volume. As mentioned above, excessive ECCs induced by NMES have frequently been used as models of EIMD [26, 27]. In contrast, isometric NMES, which involves muscle contractions without changes in muscle length, is less likely to cause muscle damage [61]. Fessard et al. demonstrated that low-intensity isometric NMES training leads to MuSC proliferation and myonuclear accretion in fast-twitch myofibers in mice [32]. These changes were not accompanied by an increase in IgG-positive fibers, embryonic myosin heavy chain expression, or centrally located myonuclei, suggesting that NMES using isometric contractions represents a promising training modality for inducing non-regenerative myogenesis.

Mechanotransducers/mechanosensors and non-regenerative myogenesis

Cells sense mechanical cues from their microenvironment through mechanosensitive structures, including cell-surface receptors such as integrins, the actin–myosin cytoskeleton, and other intracellular proteins [62, 63]. These mechanical signals are transmitted to the nucleus, where they modulate chromatin organization and the activity of mechanosensitive transcriptional regulators, ultimately leading to context-dependent changes in gene expression [63–65]. As MuSC activation and proliferation occur without myofiber death and tears, the most plausible initiators disrupting the quiescence state in MuSC are mechanical cues as they activate mechanotransducers and mechanosensors, including YAP/TAZ, PIEZO1/2, and MRTFA/B [64, 66–68]. As an indirect mechanism promoting MuSC proliferation, we previously demonstrated that the activation of YAP/TAZ in mesenchymal progenitors (also known as FAPs: fibro/adipogenic progenitors) [69] is critical for MuSC expansion in surgically overloaded muscles [30]. In our single-cell RNA sequencing (scRNA-seq) analyses, YAP and TAZ were detected, whereas PIEZO1/2 and MRTFA/B were not detected in intact mesenchymal progenitors (Fig. 2) [37], suggesting that YAP/TAZ predominantly functions as mechanotransducers in mesenchymal progenitors.

Fig. 2.

Expression of mechanosensor and mechanotransducer genes. Gene expression pattern of Yap1, Taz1, Piezo1, Piezo2, Mrtfa, and Mrtfb in 13 cell fractions of sham (intact) and overloaded muscle (ope) using data set GSE232257

Our scRNA-seq analyses also showed that quiescent MuSCs expressed YAP/TAZ and Piezo1 transcripts (Fig. 2) [37]. In the context of YAP, CalcR and Wnt4a signaling inhibit YAP activation [17, 70]. Therefore, a mechanism that downregulates CalcR and Wnt4a signaling is required before YAP functions, suggesting that in MuSCs, YAP does not serve as the primary mechanotransducer of MuSC activation in response to mechanical loads. Nonetheless, it is conceivable that the amount of YAP within the nucleus of MuSCs contributes to the threshold for the initiation of MuSC proliferation. Although YAP activation alone is insufficient to induce MuSC proliferation in CalcR-deficient MuSCs [17], exercise-dependent factors increase nuclear YAP levels in CalcR-deficient MuSCs, resulting in proliferation and an increased number of MuSCs [33].

TAZ is reportedly necessary for MuSC proliferation in an exercise model [71]; however, whether it functions as a mechanotransducer in response to mechanical loading remains unclear. Ma et al. reported that Piezo1 affected the morphology of quiescent MuSCs and that its pharmacological activation primed them toward a transitionally activated state [72]. Therefore, Piezo1 may act as a primary mechanical sensor and initiator, inducing the transition of MuSCs from quiescent to activated states in response to mechanical loading during non-regenerative myogenesis. Hirano et al. showed that another mechanosensitive ion channel, TRPM7, is critical for MuSC activation and proliferation through Mg2⁺ influx and is involved early responses, including quiescent projection retraction and AP-1 (Fos and c-Jun) induction [73]. In summary, although the mechanism by which MuSCs directly sense mechanical cues and set the threshold for exercise intensity- and duration-dependent activation and proliferation is unclear, mechanosensitive ion channels including Piezo1 and TRPM7 are a potential primary mechanosensor for MuSCs that responds to exercise-induced mechanical loading (Fig. 1B).

Characteristics of MuSC behaviors in non-regenerative myogenesis

In our previous review in Skeletal Muscle (2022) [35], we described the distinct behavior of MuSCs in muscle regeneration and hypertrophy. In this review, we briefly highlight MuSC behavior in non-regenerative myogenesis by incorporating recent findings.

Activation

The downregulation of CalcR expression in MuSCs has been observed in both surgical overload and wheel-running models [30, 33], suggesting that reduced CalcR expression is a common event, regardless of the type of exercise. Furthermore, as decreased CalcR expression is inversely correlated with Ki67 expression [30], the suppression of CalcR represents a crucial event during the transition from a quiescent state to an active state in MuSCs during non-regenerative myogenesis. The mechanism by which CalcR expression is regulated remains unclear. However, in the overloaded muscles of mice lacking mesenchymal progenitors (but not in mesenchymal progenitor-specific YAP/TAZ conditional double-knockout mice), CalcR expression was found to unchanged [30], suggesting that mesenchymal progenitors may contribute to the regulation of CalcR expression.

Proliferation

A defining feature of proliferating MuSCs during non-regenerative myogenesis is their localization between the basal lamina and sarcolemma of myofibers [29] (Fig. 1B). As both the basal lamina and myofibers serve as niches for MuSCs, it is plausible that proliferating MuSCs in non-regenerative myogenesis retain stem cell-like features compared with myoblasts observed during regenerative processes. Indeed, the expression levels of Pax7, Myf5, and Notch-related genes, which are highly expressed in quiescent MuSCs, were retained in proliferating MuSCs during non-regenerative myogenesis in a surgically overloaded model [29]. Notably, we observed low or undetectable levels of MyoD protein in proliferating MuSCs in both the surgical overload and wheel-running models [29] (Fig. 1B). Mechanistically, HeyL, a Notch target gene, suppresses MyoD expression in proliferating MuSCs in both models, as evidenced by an increased proportion of MyoD⁺ proliferating MuSCs in HeyL-KO mice subjected to surgical overload or wheel-running [29, 31].

Although the number of macrophages is limited compared with that in the regeneration process, it increases in overloaded muscles [37] (Fig. 1B). YAP/TAZ in mesenchymal progenitors and Rho signaling in myofibers have been proposed to recruit monocyte/Mø via CCL2/7 and CCL3/CX3CL1, respectively, independent of myofiber death [37, 74]. The genetic or pharmacological depletion of mesenchymal progenitors or macrophages reduced the number of proliferating MuSC in surgically overloaded muscles [30, 37, 74], indicating that these cell types are involved in MuSC proliferation during non-regenerative myogenesis. Both mesenchymal progenitors and macrophages express soluble factors that promote the proliferation of MuSCs. Additionally, we demonstrated that Granulin released from macrophages contributes to the suppression of MyoD in surgically overloaded muscles [37] (Fig. 1B). As proliferating MuSCs on myofibers constantly carry the risk of fusion, we hypothesize that MyoD expression in these proliferating MuSCs could be suppressed through multiple mechanisms. In summary, mesenchymal progenitors and macrophages induce MuSC proliferation, similar to the regeneration process. However, because proliferation must occur on myofibers, a mechanism that suppresses fusion with myofibers appears to be a characteristic feature of MuSC proliferation during non-regenerative myogenesis.

Differentiation

As described above, MuSCs fuse with myofibers during nonregenerative myogenesis. As Myogenin⁺ MuSCs are detected in surgically overloaded muscles, Myogenin expression is required for MuSC fusion with myofibers [30] (Fig. 1B). The fusogenic protein Myomaker is also essential for MuSC–myofiber fusion, as evidenced by the loss of MuSC-derived myonuclei in Myomaker-KO mice in both surgically overloaded and wheel-running models [75, 76]. Collectively, these findings indicate that terminal myogenic differentiation programs are shared between regenerative and nonregenerative myogenesis (Fig. 1B).

During regenerative myogenesis, it remains unclear whether a subset of MuSCs differentiates without undergoing cell division. In contrast, during non-regenerative myogenesis, approximately half of MuSCs fuse with myofibers without cell cycling (Fig. 1B). Ismaeel et al. found that the number of MuSC-derived myonuclei labeled with doxycycline-dependent H2B expression was greater than that of EdU⁺ MuSC-derived myonuclei [77]. Similarly, MuSCs lacking Rbp-J fuse with myofibers without cell division [10]. Given the heterogeneous levels of Dll4, a Notch ligand, on myofibers [78], these results suggest that MuSCs with low Notch signaling preferentially fuse with myofibers without cell division. This type of differentiation may be specific to non-regenerative myogenesis (Fig. 1B), and further underscores the importance of Notch signaling in regulating MuSC proliferation during non-regenerative myogenesis.

Necessity of studying non-regenerative myogenesis

In skeletal muscle research, numerous types of diseases, including inherited muscular dystrophies, sarcopenia, bed rest-induced muscle loss, cancer cachexia, trauma, autoimmune diseases, and secondary impairments caused by diabetes and kidney dysfunction, must be overcome and treated. These diseases can be broadly classified into two categories: those associated with myofiber death followed by regeneration processes, and those without evident signs of myofiber death or regeneration, characterized by a decline in myofiber size, known as muscle atrophy.

MuSC transplantation and regeneration-enhancing therapies are useful for treating muscular dystrophy. However, under atrophic conditions, such regenerative processes do not occur frequently, indicating that approaches aimed solely at enhancing muscle regeneration are not suitable for treating muscle atrophy. The number of myonuclei is positively correlated with myofiber size in mice and humans [79]. The supply of new myonuclei through MuSCs may represent a therapeutic approach for muscular atrophy. Similarly, under non-damaging conditions, if MuSC proliferation can be induced and genes efficiently introduced, it may be possible to improve or compensate for dysfunctional myonuclei. To achieve these goals, the molecular mechanisms underlying non-regenerative myogenesis must be elucidated.

Remained question/future study

Even in daily life, muscles contract and relax randomly, during which MuSCs are retained in a quiescent state. However, exercise above a certain threshold drives MuSCs out of quiescence and into proliferation. In non-regenerative myogenesis, it is critical to elucidate the mechanism involved in determining this threshold, including factors and cell types. YAP/TAZ activity in mesenchymal progenitors represents one mechanism that contributes to this regulation [30]. In the broader context of mechanosensing and mechanotransduction, the involvement of factors beyond YAP/TAZ should be examined. Future studies are needed to clarify how these factors, including YAP/TAZ, are integrated to establish a threshold.

CalcR expression is reduced in proliferating MuSCs during non-regenerative myogenesis regardless of the type of exercise or training [30, 33]. YAP/TAZ in mesenchymal progenitors was not required for the reduced expression of CalcR, suggesting that a YAP/TAZ-independent mechanism regulates CalcR expression in loaded muscles. The mechanism regulating CalcR expression is an important issue that requires clarification in the context of the threshold for MSC activation.

Another remaining question concerns the differences in MuSC behavior between slow- and fast-twitch myofibers. In fast- and slow-twitch myofibers, differences exist not only in the myosin heavy chain, but also in MyoD and Myogenin expression [80, 81]. In addition, the number of MuSCs in the soleus muscle is greater than that in the fast-twitch muscles, suggesting that the myofiber type influences MuSC proliferation and differentiation during postnatal development. In this context, MuSC behavior may be differentially regulated by slow- and fast-twitch myofibers. In wheel-running mice, the number of MuSC-derived myonuclei in the soleus muscle is greater than that in the plantaris muscle ³⁰⁾. Future research interest includes determining whether this difference arises from the initial MuSC number, the intensity of mechanical loading, or distinct mechanisms regulating MuSC behavior.

Another important question is whether the mechanism of MuSC proliferation is consistent across different types of exercise. For example, regarding myokines, whose circulating levels increase in a movement-dependent manner, it is well-established that their responses differ between resistance and endurance training [82]. This indicates that the response of skeletal muscles may vary depending on the exercise modality, particularly the intensity and duration of the mechanical load imposed. Accordingly, the mechanisms underlying MuSC proliferation, especially those involving non-cell-autonomous factors, may differ depending on the type of exercise.

Similar to the regeneration process, during non-regenerative myogenesis, most proliferating MuSCs differentiate by fusing with myofibers, and a subset of MuSCs is thought to contribute to maintaining the MuSC pool (self-renewal). Elucidating this molecular mechanism and understanding the self-renewal mechanism of MuSCs remain unresolved challenges in MuSC biology. Advancements in each field of research, regenerative and non-regenerative myogenesis, are expected to provide reciprocal insights.

Abbreviations

Calcr

Calcitonin receptor

Dll4

Delta-like canonical Notch ligand 4

ECCs

Eccentric contractions

EIMD

Exercise-induced muscle damage

GDF

Growth differentiation factor

Hes1

Hairy and Enhancer of split 1

Hey1

Hairy/enhancer-of-split related with YRPW motif protein 1

HeyL

Hairy/enhancer-of-split related with YRPW motif-like protein

IL-6

Interleukin-6

MuSCs

Muscle satellite cells

Myf5

Myogenic factor 5

MyoD

Myoblast determination 1

NMES

Neuromuscular electrical stimulation

Pax7

Paired box 7

Pdgfra

Platelet-derived growth factor receptor alpha

PKA

Protein kinase A

Rbp-J

Recombination signal binding protein for immunoglobulin kappa J region

YAP

Yes-associated protein 1

TAZ

Transcriptional coactivator with PDZ-binding motif

Authors’ contributions

S.F. and A.K.: writing the original draft and editing. T.Y.: writing the original draft on EIMD. T.A.: writing and editing on surgical overloaded models. The authors read and approved the final manuscript.

Funding

S. F. was funded by a Grant-in-Aid for Scientific Research (B) (19H04000, 22H03466), a Grant-in-Aid for Scientific Research (A)(25H01098), a Grant-in-Aid for Challenging Exploratory Research (20K21757), Association Francaise contre les Myopathies (AFM, #23574), and AMED-Crest (25gm2110002s).

Data availability

Not applicable.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.