Skip to main content
Download PDF

Decreased number of satellite cells-derived myonuclei in both fast- and slow-twitch muscles in HeyL-KO mice during voluntary running exercise

Skeletal Muscle volume 14, Article number: 25 (2024) Cite this article

Abstract

Background

Skeletal muscles possess unique abilities known as adaptation or plasticity. When exposed to external stimuli, such as mechanical loading, both myofiber size and myonuclear number increase. Muscle stem cells, also known as muscle satellite cells (MuSCs), play vital roles in these changes. HeyL, a direct target of Notch signaling, is crucial for efficient muscle hypertrophy because it ensures MuSC proliferation in surgically overloaded muscles by inhibiting the premature differentiation. However, it remains unclear whether HeyL is essential for MuSC expansion in physiologically exercised muscles. Additionally, the influence of myofiber type on the requirement for HeyL in MuSCs within exercised muscles remains unclear.

Methods

We used a voluntary wheel running model and HeyL-knockout mice to investigate the impact of HeyL deficiency on MuSC-derived myonuclei, MuSC behavior, muscle weight, myofiber size, and myofiber type in the running mice.

Results

The number of new MuSC-derived myonuclei was significantly lower in both slow-twitch soleus and fast-twitch plantaris muscles from exercised HeyL-knockout mice than in control mice. However, expect for the frequency of Type IIb myofiber in plantaris muscle, exercised HeyL-knockout mice exhibited similar responses to control mice regarding myofiber size and type.

Conclusions

HeyL expression is crucial for MuSC expansion during physiological exercise in both slow and fast muscles. The frequency of Type IIb myofiber in plantaris muscle of HeyL-knockout mice was not significantly reduced compared to that of control mice. However, the absence of HeyL did not affect the increased size and frequency of Type IIa myofiber in plantaris muscles. In this model, no detectable changes in myofiber size or type were observed in the soleus muscles of either control or HeyL-knockout mice. These findings imply that the requirement for MuSCs in the wheel-running model is difficult to observe due to the relatively low degree of hypertrophy compared to surgically overloaded models.

Background

Skeletal muscles have a distinctive ability to adapt to internal and external physiological changes, known as plasticity. Skeletal muscles may weaken and decrease in size due to various diseases or disuses, a phenomenon termed as muscle atrophy. Conversely, increased mechanical loading triggers the remodeling of skeletal muscles, resulting in enhanced strength and mass, a process known as muscle hypertrophy [1]. Muscle-resident stem cells called muscle satellite cells (MuSCs) [2] respond to increased mechanical loads, proliferate, and subsequently supply new myonuclei to myofibers [3]. This process is vital for load-dependent muscle hypertrophy in resistance-training models [4,5,6].

Surgical overload models (resistance training models) have been widely used to analyze the contribution of MuSCs to myofibers in loaded muscles. These models have contributed to revealing mechanisms underlying MuSC proliferation, differentiation, and fusion [5, 7,8,9,10]. Myomaker, a fusogenic protein, is essential for MuSCs to fuse with myofibers in surgically loaded and regenerated muscles [5]. The overloaded models also shed light on the critical roles of macrophages and mesenchymal progenitors (FAPs) in MuSC proliferation and differentiation [7, 10, 11]. Furthermore, our previous study using a surgical overloaded model demonstrated that sustained expression of hairy/enhancer-of-split related to the YRPW motif-like protein (HeyL) is necessary for MuSC proliferation by suppressing MyoD expression in proliferating MuSCs [12]. HeyL, along with Hey1 and Hey2, belongs to the Hey family, which are direct targets of the canonical Notch signaling pathway, similar to the Hes family genes [13]. Hes1, Hey1, and HeyL are canonical and essential effector genes for the Notch-dependent inhibition of myogenic differentiation, including MyoD expression [14,15,16,17,18]. Although Hey1/HeyL double mutant mice show marked defects in skeletal muscle development and MuSC numbers, neither Hey1 nor HeyL single knockout mice exhibit substantial abnormalities [14]. However, HeyL-knockout mice display blunted muscle hypertrophy due to the premature differentiation of proliferating MuSCs in overloaded muscles [12, 19]. Whether similar mechanisms apply to MuSC behavior in slow-type muscles, as observed in fast-twitch muscles in our previous studies, and other physiological models, such as the voluntary wheel-running model, remain unclear.

Voluntary wheel running is a well-established model for endurance training. Plantar flexor muscles, including the soleus, plantaris, and gastrocnemius muscles, exhibit high responsiveness and adaptability to wheel running [20, 21]. In some cases, the addition of weight may be necessary to induce substantial muscle hypertrophy. However, Masschelein et al. observed an increased number of myonuclei contributed by MuSCs in mice running on a commercially available, non-weighted wheel [20]. In this study, using commercially available equipment, we aimed to investigate the role of HeyL in MuSC behavior in both slow and fast muscles of mice undergoing voluntary wheel running.

Methods

Mice

C57BL/6J mice, 8–12 weeks of age, were purchased from Charles River Laboratories (Yokohama, Kanagawa, Japan). Dr. Kokubo generated HeyL-knockout (KO) mice as previously described [14]. All mice were housed in a controlled environment, at a maintained temperature of 24 ± 2 °C, humidity at 50% ± 10%, and subjected to a 12/12-h light/dark cycle. Sterilized standard chow (DC-8; Nihon Clean, Tokyo, Japan) and water were provided ad libitum.

Wheel running

The mice were housed individually in cages measuring 225 mm (width) × 345 mm (depth) × 210 mm (height). The cages were equipped with running wheels measuring 140 mm in diameter (MELQUEST, Toyama, Japan), and the mice were allowed to run voluntarily within the cages for a specified duration. The number of rotations was recorded using a CNT-10 (MELQUEST). Mice in the Non-exercise group were housed individually without access to the running wheels.

In vivo EdU labeling and detection

EdU (5- ethynyl − 2′-deoxyuridine; Thermo Fisher Scientific, #A10044) was dissolved in sterilized PBS at 10 mg/mL and stored at -20 °C. Alzet mini-osmotic pumps (Model 2002, Durect, Cupertino, CA, http://www.durect.com/) [22] were used to continuously administer EdU during wheel running or home cage control for 14 days. The pumps were filled with 200 µL of EdU solved in PBS (10 mg/mL), thus giving a delivery rate of 0.5 µL/h (120 µg EdU/day). EdU+ nuclei were detected using Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 647 dye (Thermo Fisher Scientific, #C10340). To identify EdU+ myonuclei, dystrophin staining (Abcam, #AB15277) was performed. Fluorescence was recorded using a BZ-X700 fluorescence microscope (Keyence; Osaka, Japan), and cell size was quantified using Hybrid Cell Count software (Keyence).

Muscle fixation

The isolated soleus and plantaris muscles were carefully positioned on the cork using kneaded Tragacanth Gum (Wako Pure Chemicals Industries, Osaka, Japan). Subsequently, the muscles were rapidly frozen by immersion in isopentane (Wako Pure Chemical Industries), which was pre-cooled with liquid nitrogen for 1 min. The muscles were then transferred onto dry ice and allowed to remain for 1 h to facilitate isopentane vaporization. Finally, the frozen muscles were stored in sealed containers at -80 °C.

Immunohistochemistry

Transverse cryosections (6 μm thick) were cut using a cryostat (Leica CM1860; Nussloch, Germany). After drying at room temperature for 30 min, the sections were fixed with 4% paraformaldehyde (PFA) for 10 min and washed with PBS in a coupling jar. For fiber type stainings, the sections were fixed with − 20ºC acetone for 10 min. After blocking with 5% skim milk/phosphate-buffered saline (PBS), the sections were reacted with primary antibodies, anti-Laminin α2 (Enzo, RRID: AB_2051764) and Dystrophin (Abcam, RRID: AB_301813) overnight at 4ºC. For myofiber type analyses, SC-71 (Mouse Myosin heavy chain Type-IIa, RRID: AB 2147165), BF-F3 (Mouse Myosin heavy chain Type-IIb, RRID: AB_2266724), and BA-D5 (Mouse Myosin heavy chain Type-I, RRID: AB_2235587) were purchased from Developmental Studies Hybridoma Bank (Iowa, IA, USA). The next day, the sections were washed three times with PBS and incubated with secondary antibodies. Signals were photographed using a BZ-X700 fluorescence microscope (Keyence). For all analyses, sections were obtained from the middle third of the plantaris or soleus muscles from control or KO mice. For the quantification of EdU + myonuclei or Pax7+ cells, the average number of EdU+ myonuclei or Pax7+ cells from two sections was used.

Measurement of CSA

Cross sections of soleus and plantaris muscles were stained with anti-Laminin α2 antibody, and fluorescence images of entire cross sections were recorded using BZ-X700 fluorescence microscope. For the analyses of Type-I and Type-IIa in soleus muscles and Type-IIb in plantaris muscles, three to four fields per sample were randomly selected to calculate the cross-sectional area. For the analyses of Type-IIa in plantaris muscles, all fibers were analyzed to calculate the cross-sectional area.

Single myofiber staining

Following a previously established protocol [23], single myofibers were isolated from the plantaris muscles of wild-type and HeyL-KO mice. The isolated myofibers were immediately fixed in 4% PFA/PBS solution and washed with PBS containing 0.1% Triton-X. To permeabilize the myofibers, they were treated with a solution containing 20 mM HEPES, 300 mM sucrose, 50 mM NaCl, 3 mM MgCl2, and 0.5% Triton X-100. After washing with PBS, the myofibers were treated with PBS containing 5% FCS and 0.01% Triton-X and reacted with anti-Pax7 (DSHB), MyoD (Abcam, RRID: AB_2890928), and Ki67 (Thermo Fisher Scientific, RRID: AB_10854564) antibodies overnight at 4ºC. The following day, the myofibers were washed and incubated with secondary antibodies for 1 h. After washing and encapsulation, the stained myofibers were observed under a BZ-X700 fluorescence microscope (Keyence).

Statistics

Values were expressed as means ± standard deviation (S.D.). A two-sided unpaired Student’s t-test was used to conduct statistical comparisons between two groups. For comparisons involving more than two groups, one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test was employed. All statistical analyses were performed using the Prism 8 software. A p-value less than 0.05 was considered statistically. Additionally, Prism 10 software was used to generate all graphs.

Results

Decreased number of myonuclei derived from muscle satellite cells in exercised HeyL-KO mice

Voluntary wheel running is an exercise in which rodents engage in spontaneous running, driven by internal motivation, effectively loading their plantar flexor muscles. HeyL expression is specific to MuSCs in skeletal muscle [14, 24]. Because Hey1 compensates the function of HeyL, HeyL-KO did not exhibit a significant difference in MuSC number and EdU-uptake [12, 14, 15, 25]. However, HeyL-KO exhibited the decrease in MuSC proliferation in the surgically overloaded plantaris muscle [12]. To explore the role of HeyL in MuSC behavior in slow- and fast-twitch muscles of wheel-running mice, we analyzed two plantar flexor muscles, the soleus and plantaris muscles, which are representative slow- and fast-twitch muscles, respectively. Consistent with our previous study, we confirmed that MuSC number in the soleus or plantaris muscles of non-exercised HeyL-KO (KO: HeyL−/−) was comparable to that observed in non-exercised littermate controls (wild-type [WT]: HeyL+/+)(Fig. 1A). Then, HeyL-KO and littermate control mice were allowed free access to the wheel equipment for voluntary running (Fig. 1B). Two weeks after exercise, the total rotation number of HeyL-KO mice was comparable to that of control mice (Fig. 1C), indicating that the physical activity of HeyL-KO mice was not influenced by the loss of HeyL.

Using this model, we counted the number of MuSC-derived myonuclei in soleus and plantaris muscles by labeling MuSCs with EdU during exercise (Fig. 1B). The number of newly formed EdU-positive myonuclei in both the soleus and plantaris muscles was significantly reduced in HeyL-KO mice compared with controls (Fig. 1D and E). The number of EdU-positive MuSCs were also reduced in both the soleus and plantaris muscles from HeyL-KO mice (Fig. 1F and G). These findings demonstrate that HeyL plays a critical role in MuSCs in exercised muscles subjected to exercise, similar to observations in surgically overloaded muscles [12]. Furthermore, these results illustrated that HeyL is essential for MuSCs in both fast- and slow-twitch myofibers.

Fig. 1
figure 1

Reduced number of myonuclei derived from muscle satellite cells in exercised HeyL-KO mice. (A) Number of Pax7+ cells per 100 myofibers in sections of soleus or plantaris muscles from male non-exercised WT (HeyL+/+, n = 5) or KO (HeyL−/−, n = 5). (B) Experimental design for analyzing the effect of a 2-week wheel running exercise on the number of MuSC-derived myonuclei. Littermate control (WT: HeyL+/+) or HeyL-KO (KO: HeyL−/−) mice were randomly separated into two groups; home cage (-Ex: non-exercise group) or wheel running cage (+ Ex: exercise group). EdU was administrated through osmotic pumps during 2-week running. (C) Number of wheel rotations during 2 weeks in WT (n = 8) or KO (n = 7) mice. (D) Immunostaining of dystrophin (green) and EdU (white) in soleus muscle 2 weeks after wheel running. Arrowheads indicate EdU+ myonuclei. Scale bar, 25 μm. (E) Number of EdU+ myonuclei per 100 myofibers in sections of soleus or plantaris muscle from exercised WT (n = 8) or KO (n = 7) mice. (F) Immunostaining of Pax7 (red), Laminin α2 (LNα2, green), and EdU (white) in soleus muscle 2 weeks after wheel running. Arrows indicate Pax7+EdU+ cells. Red arrows or arrowheads indicate Pax7+EdU+ cells or EdU+ myonuclei, respectively. White arrow indicates Pax7+EdU− cell. Scale bar, 25 μm. (G) Number of Pax7+EdU+ cells per 100 myofibers in sections of soleus or plantaris muscle from exercised WT (n = 8) or KO (n = 6) mice

Full size image

Increased number of MyoD-positive proliferating MuSCs in HeyL-KO mice

We previously reported that MuSCs proliferate without significant MyoD expression in surgically overloaded muscles [12, 26]. HeyL is involved in the suppression of MyoD, as the loss of HeyL leads to increased MyoD expression in proliferating MuSCs, eventually resulting in a reduced number of new MuSC-derived myonuclei and biased myogenic differentiation, rather than proliferation [12]. Therefore, the same mechanism was observed for the reduced number of EdU+ myonuclei in running-exercise HeyL-KO mice (Fig. 1E). To verify this, we isolated single myofibers from the plantaris muscles of exercised control and HeyL-KO mice and stained them with anti-MyoD and Pax7 antibodies (Fig. 2A). Because the extent of exercise affected the results, we analyzed mice that exercised more than 50,000 wheel-rotations for 7 days (Fig. 2B). As expected, the number of MyoD+ MuSCs was significantly higher in HeyL-KO mice than in control mice (Fig. 2C–E). Further, MuSC number on single myofiber from HeyL-KO mice was significantly lower than that of control mice (Fig. 2F). While, the frequency of Ki67+ activated/proliferating cells was not significantly different between control and HeyL-KO mice (Fig. 2G). These findings suggest that HeyL does not directly influence the proliferation of MuSCs; rather, it indirectly affects MuSC expansion in exercised muscles by inhibiting MyoD-dependent premature differentiation. Our previous study demonstrated that untrained/sedentary HeyL-KO mice exhibited a modestly higher percentage of MyoD-positive MuSCs compared to wild-type (WT) mice (2.4% vs. 12.4%) [14]. In exercised mice, the frequency of MyoD-positive cells increased dramatically, reaching approximately 60% in Ki67+ MuSCs. This finding suggests that HeyL is necessary to suppress muscle differentiation during the exercise-induced proliferation of MuSCs, a phenomenon also observed in overloaded muscle [12].

Fig. 2
figure 2

Increased MyoD expression in proliferating HeyL-KO MuSCs. (A) Experimental scheme for analyzing the effect of HeyL-KO on MyoD expression in MuSCs on isolated myofibers from plantaris muscles 7 days after voluntary wheel running. (B) Number of wheel rotations during 7 days in WT (n = 5) or KO (n = 7) mice. (C and D) Immunostaining of Pax7 (red), MyoD (green), and Ki67 (white) in MuSCs on isolated myofibers at 7 days after wheel running from WT (C) or KO (D) mice. Nuclei were counterstained with DAPI. Scale bar, 100 μm. (E) Percentage of MyoD+ cells in Pax7+ cells (left) or Pax7+Ki67+ (right) cells on isolated myofibers from exercised WT (n = 4) or KO (n = 5) mice. (F) Number of Pax7+ cells on isolated myofibers from exercised WT (n = 4) or KO (n = 5) mice. (G) Percentage of Ki67+ cells in Pax7+ cells on isolated myofibers from exercised WT (n = 4) or KO (n = 5) mice

Full size image

Increase in muscle weight by voluntary wheel running in HeyL-KO mice

To investigate the impact of HeyL deficiency on muscle hypertrophy, male HeyL-KO and littermate control (WT) mice were randomly assigned to two groups. One group had free access to the wheel equipment and ran voluntarily for 4 weeks, while the other remained in cages without the wheel equipment (Fig. 3A). Consistent with the results observed in female mice after two weeks of exercise (Fig. 1C), the total number of rotations over four weeks in male HeyL-KO mice was comparable to that in control mice (Fig. 3B). After euthanasia, muscle weights of these exercised (+ Ex) or non-exercised (-Ex) mice were analyzed. In control mice, the plantaris and soleus muscle weights per body weight significantly increased after 4 weeks of voluntary running compared to those in the (-Ex) group (Fig. 3C). Although there was no significant difference in the plantaris muscle between (-Ex) and (+ Ex) HeyL-KO mice, the soleus muscle weight per body weight was significantly increased in HeyL-KO mice, similar to the observations in WT mice (Fig. 3C). Mechanical loading in the EDL muscles did not increase with this voluntary wheel-running exercise [20], therefore, EDL muscle weights remained unchanged by exercise in both HeyL-KO and control mice (Fig. 3C).

Fig. 3
figure 3

Muscle response to exercise in HeyL-KO mice. (A) Experimental scheme for analyzing the effect of 4-week wheel running on muscle weight and myofiber types. Littermate male control (WT: HeyL+/+) or HeyL-KO (KO: HeyL−/−) mice were randomly separated into two groups; home cage (-Ex: non-exercise group) or wheel running cage (+ Ex: exercise group). (B) Number of wheel rotations during 4 weeks in WT (n = 5) or KO (n = 4) mice.(C) Normalized muscle weight [muscle weight/body weight (BW)] from WT or KO mice. N.S.: not significant

Full size image

Size or type of myofiber in exercised HeyL-KO mice

Wheel running exercise induces a transition in myofiber types from fast-to slow-twitch fibers [27]. Therefore, we analyzed the myofiber type composition along with myofiber size (CSA: cross-sectional area) of the plantaris and soleus muscles of control and KO mice in Fig. 3 (Fig. 4A). In the plantaris muscles, the CSA of Type IIa increased or tended to increase in both control and KO mice after the running exercise, whereas the CSA of Type IIb remained unchanged by exercise (Fig. 4B). Concerning myofiber-type composition, the proportion of Type IIa increased, whereas that of Type IIb decreased in WT mice (Fig. 4C), which was consistent with published data [27]. In KO mice, the proportion of Type IIa was increased, but that of Type IIb was not significantly decreased (Fig. 4C). In the soleus muscles, no significant differences were observed in myofiber type and size between the control and KO mice (Fig. 4D and E). These results suggest that the absence of HeyL did not dramatically impact myofiber sizes and types of plantaris and soleus muscles in exercised mice.

Fig. 4
figure 4

Myofiber size and types in running HeyL-KO mice. (A) Representative images of staining for Type-IIa (plantaris), Type-IIb (plantaris), and Type-I (soleus) with anti-laminin staining (green) in male WT or KO mice. Scale bar: 100 μm. (B) Cross sectional area (CSA) of Type-IIa or -IIb myofibers in plantaris muscle (PLA) from male WT (-Ex; n = 5, +Ex; n = 5) or KO (-Ex; n = 5, +Ex; n = 4) mice. (C) The frequency of Type-IIa or -IIb myofibers in plantaris (PLA) muscle from male WT (-Ex; n = 5, +Ex; n = 5) or KO (-Ex; n = 5, +Ex; n = 4) mice. (D) CSA of Type-I or -IIa myofibers in soleus muscle from male WT (-Ex; n = 5, +Ex; n = 5) or KO (-Ex; n = 5, +Ex; n = 4) mice. (E) The frequency of Type-I or -IIa myofibers in soleus muscle from male WT (-Ex; n = 5, +Ex; n = 5) or KO (-Ex; n = 5, +Ex; n = 4) mice

Full size image

Discussion

In this study, we found that proliferating MuSCs in exercising mice required HeyL to suppress MyoD expression, similar to observations in surgically overloaded HeyL-KO mice [12]. Surgically overloaded models have been employed for resistance training, whereas wheel-running has been utilized for endurance training. Each training type elicited a distinct response. Endurance training diminishes the activity of glycolytic enzymes but enhances oxidative enzyme activity and mitochondrial density, leading to a shift from fast-type to slow-type myofibers [28]. In contrast, resistance training decreases mitochondrial density and has a minimal effect on enzymatic activity [28]. Therefore, endurance training facilitates aerobic processes, whereas resistance training increases anaerobic responses to muscle strength. Accordingly, the present study demonstrated that the requirement of HeyL for MuSC proliferation in exercised muscles is similar in endurance and resistance training, despite inducing distinct biological responses in myofibers.

MuSCs have properties distinct from those of slow- and fast-twitch myofibers [29]. Motohashi et al. reported that MuSCs derived from the soleus exhibited higher self-renewal and lower differentiation abilities than those derived from the EDL [29]. The present study demonstrated reduced myonuclear accretion in both fast- and slow-twitch muscles in HeyL-KO mice, suggesting that the requirement for HeyL and the proliferation mechanism of MuSCs are likely to be common in both muscle types, fast- and slow-switch muscles. Notably, de Lima et al. demonstrated that HeyL suppresses myomaker expression during muscle development [30]. Given that the myomaker is essential for myogenesis in both slow and fast myofibers, it is not surprising that HeyL is also critical for MuSC proliferation in both types of exercised myofibers. In contrast to Myomaker-mutant mice, HeyL-KO mice did not exhibit any developmental defects, suggesting the presence of a compensatory pathway that mitigates the loss of HeyL during development. The precise mechanism remains unidentified; however, Hey1, which plays a redundant role in maintaining quiescence in MuSCs, is a potential candidate involved in developmental processes. Among the Hes and Hey family genes, HeyL transcripts are highly expressed in MuSCs and are the most sensitive to induction by Notch ligands in mice [14]. Hes1 and Hey1 are predominantly expressed and induced by Notch ligands [31]. Although HeyL primarily functions in loaded murine MuSCs, other Hes and Hey genes may play similar roles in loaded human MuSCs.

The requirement for MuSCs and MuSC-derived myonuclei has been highly debated. In surgically overloaded muscles, blunted muscle hypertrophy has been observed in mice with defective MuSC proliferation and fusion, at least in long-term designed experiments [6]. Our group also demonstrated low efficiency of muscle hypertrophy in HeyL-null mice [12]. The present data demonstrated that the requirement of HeyL for the increased number of MuSC-derived myonuclei in our wheel-running model. However, we could not observe the effect associated with a reduction in newly formed myonuclei in this model. Compared to the surgically overloaded models, the increased ratios of muscle weight and myofiber size in running mice were very mild. The increase in the number of MuSC-derived myonuclei in running mice was lower than that in surgically overloaded mice. In addition, owing to the decreased motivation of mice, the running distance was decreased in the wheel-running exercise [20], suggesting that loading to the muscle is also decreased during long-term exercise. Considering the blunted muscle hypertrophy in MuSC-depleted or fusion-defective mice using other running models [32, 33], our model seems insufficient to observe the effect of the reduced number of MuSC-derived myonuclei on the efficiency of muscle hypertrophy. Thus, there may be a threshold for the rate of hypertrophy required to detect the effects of additional MuSC-derived myonuclei.

Conclusions

HeyL is required for MuSC expansion and increases the myonuclei number by suppressing MyoD expression in a wheel-running model similar to the surgical overload model.

Moreover, our current findings illustrate that HeyL is essential for MuSC proliferation in both fast- and slow-twitch muscles. However, the absence of HeyL did not affect myofiber size or type in the wheel-running model.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

CSA:

cross-sectional area

EdU:

5-ethynyl-2′-deoxyuridine

Ex:

exercise

FAP:

Fibro/adipogenic progenitor

HeyL:

hairy/enhancer-of-split related to YRPW motif-like

KO:

knockout

MuSC:

muscle satellite cell

MyoD:

myogenic differentiation 1

Pax7:

paired box 7

PFA:

paraformaldehyde

WT:

wild-type

References

  1. Bamman MM, Roberts BM, Adams GR. Molecular Regulation of Exercise-Induced muscle Fiber hypertrophy. Cold Spring Harb Perspect Med. 2018;8:a029751. https://www.ncbi.nlm.nih.gov/pubmed/28490543.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol. 1961;9:493–5. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=13768451.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Schiaffino S, Bormioli SP, Aloisi M. The fate of newly formed satellite cells during compensatory muscle hypertrophy. Virchows Arch B Cell Pathol. 1976;21:113–8. https://www.ncbi.nlm.nih.gov/pubmed/822576.

    Article  CAS  PubMed  Google Scholar 

  4. Egner IM, Bruusgaard JC, Gundersen K. Satellite cell depletion prevents fiber hypertrophy in skeletal muscle. Development. 2016;143:2898–906. http://www.ncbi.nlm.nih.gov/pubmed/27531949.

    Article  CAS  PubMed  Google Scholar 

  5. Goh Q, Millay DP. Requirement of myomaker-mediated stem cell fusion for skeletal muscle hypertrophy. Elife. 2017;6:e20007. http://www.ncbi.nlm.nih.gov/pubmed/28186492.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Fukada SI, Ito N. Regulation of muscle hypertrophy: involvement of the akt-independent pathway and satellite cells in muscle hypertrophy. Exp Cell Res. 2021;409:112907. https://www.ncbi.nlm.nih.gov/pubmed/34793776.

    Article  CAS  PubMed  Google Scholar 

  7. Noviello C, Kobon K, Delivry L, Guilbert T, Britto F, Julienne F, Maire P, Randrianarison-Huetz V, Sotiropoulos A. RhoA within myofibers controls satellite cell microenvironment to allow hypertrophic growth. iScience. 2022;25:103616. https://www.ncbi.nlm.nih.gov/pubmed/35106464.

    Article  CAS  PubMed  Google Scholar 

  8. Guerci A, Lahoute C, Hebrard S, Collard L, Graindorge D, Favier M, Cagnard N, Batonnet-Pichon S, Precigout G, Garcia L, Tuil D, Daegelen D, Sotiropoulos A. Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 2012;15:25–37. http://www.ncbi.nlm.nih.gov/pubmed/22225874.

    Article  CAS  PubMed  Google Scholar 

  9. Serrano AL, Baeza-Raja B, Perdiguero E, Jardi M, Munoz-Canoves P. Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy. Cell Metab. 2008;7:33–44. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=18177723.

    Article  CAS  PubMed  Google Scholar 

  10. Kaneshige A, Kaji T, Zhang L, Saito H, Nakamura A, Kurosawa T, Ikemoto-Uezumi M, Tsujikawa K, Seno S, Hori M, Saito Y, Matozaki T, Maehara K, Ohkawa Y, Potente M, Watanabe S, Braun T, Uezumi A, Fukada SI. Relayed signaling between mesenchymal progenitors and muscle stem cells ensures adaptive stem cell response to increased mechanical load, Cell Stem Cell 29 (2022) 265–280 e266. https://www.ncbi.nlm.nih.gov/pubmed/34856120

  11. Zhang L, Saito H, Higashimoto T, Kaji T, Nakamura A, Iwamori K, Nagano R, Motooka D, Okuzaki D, Uezumi A, Seno S, Fukada SI. Regulation of muscle hypertrophy through granulin: relayed communication among mesenchymal progenitors, macrophages, and satellite cells. Cell Rep. 2024;43:114052. https://www.ncbi.nlm.nih.gov/pubmed/38573860.

    Article  CAS  PubMed  Google Scholar 

  12. Fukuda S, Kaneshige A, Kaji T, Noguchi YT, Takemoto Y, Zhang L, Tsujikawa K, Kokubo H, Uezumi A, Maehara K, Harada A, Ohkawa Y, Fukada SI. Sustained expression of HeyL is critical for the proliferation of muscle stem cells in overloaded muscle. Elife. 2019;8:e48284. http://www.ncbi.nlm.nih.gov/pubmed/31545169.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Fischer A, Gessler M. Delta-Notch–and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors, Nucleic Acids Res 35 (2007) 4583–4596. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17586813

  14. Fukada S, Yamaguchi M, Kokubo H, Ogawa R, Uezumi A, Yoneda T, Matev MM, Motohashi N, Ito T, Zolkiewska A, Johnson RL, Saga Y, Miyagoe-Suzuki Y, Tsujikawa K, Takeda S. Yamamoto, Hesr1 and Hesr3 are essential to generate undifferentiated quiescent satellite cells and to maintain satellite cell numbers. Development. 2011;138:4609–19. http://www.ncbi.nlm.nih.gov/pubmed/21989910.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Noguchi YT, Nakamura M, Hino N, Nogami J, Tsuji S, Sato T, Zhang L, Tsujikawa K, Tanaka T, Izawa K, Okada Y, Doi T, Kokubo H, Harada A, Uezumi A, Gessler M, Ohkawa Y, Fukada SI. Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells: HeyL requires Hes1 to bind diverse DNA sites. Development. 2019;146:dev163618. http://www.ncbi.nlm.nih.gov/pubmed/30745427.

    Article  CAS  PubMed  Google Scholar 

  16. Lahmann I, Brohl D, Zyrianova T, Isomura A, Czajkowski MT, Kapoor V, Griger J, Ruffault PL, Mademtzoglou D, Zammit PS, Wunderlich T, Spuler S, Kuhn R, Preibisch S, Wolf J, Kageyama R, Birchmeier C. Oscillations of MyoD and Hes1 proteins regulate the maintenance of activated muscle stem cells. Genes Dev. 2019;33:524–35. http://www.ncbi.nlm.nih.gov/pubmed/30862660.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhang Y, Lahmann I, Baum K, Shimojo H, Mourikis P, Wolf J, Kageyama R, Birchmeier C. Oscillations of Delta-like1 regulate the balance between differentiation and maintenance of muscle stem cells. Nat Commun. 2021;12:1318. https://www.ncbi.nlm.nih.gov/pubmed/33637744.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gioftsidi S, Relaix F, Mourikis P. The notch signaling network in muscle stem cells during development, homeostasis, and disease. Skelet Muscle. 2022;12:9. https://www.ncbi.nlm.nih.gov/pubmed/35459219.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ato S, Fukada SI, Kokubo H, Ogasawara R. Implication of satellite cell behaviors in capillary growth via VEGF expression-independent mechanism in response to mechanical loading in HeyL-null mice. Am J Physiol Cell Physiol 322 (2022). https://www.ncbi.nlm.nih.gov/pubmed/35020502

  20. Masschelein E, D’Hulst G, Zvick J, Hinte L, Soro-Arnaiz I, Gorski T, von Meyenn F, Bar-Nur O, De Bock K. Exercise promotes satellite cell contribution to myofibers in a load-dependent manner. Skelet Muscle. 2020;10:21. https://www.ncbi.nlm.nih.gov/pubmed/32646489.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. D’Hulst G, Palmer AS, Masschelein E, Bar-Nur O, De Bock K. Voluntary Resistance running as a model to induce mTOR activation in mouse skeletal muscle. Front Physiol. 2019;10:1271. https://www.ncbi.nlm.nih.gov/pubmed/31636571.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Ikemoto-Uezumi M, Uezumi A, Tsuchida K, Fukada S, Yamamoto H, Yamamoto N, Shiomi K, Hashimoto N. Pro-insulin-like Growth Factor-II ameliorates Age-Related Inefficient Regenerative response by orchestrating self-reinforcement mechanism of muscle regeneration. Stem Cells. 2015;33:2456–68. http://www.ncbi.nlm.nih.gov/pubmed/25917344.

    Article  CAS  PubMed  Google Scholar 

  23. Rosenblatt JD, Lunt AI, Parry DJ, Partridge TA. Culturing satellite cells from living single muscle fiber explants. Vitro Cell Dev Biol Anim. 1995;31:773–9. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=8564066.

    Article  CAS  Google Scholar 

  24. Fukada S, Uezumi A, Ikemoto M, Masuda S, Segawa M, Tanimura N, Yamamoto H, Miyagoe-Suzuki Y, Takeda S. Molecular signature of quiescent satellite cells in adult skeletal muscle. Stem Cells. 2007;25:2448–59. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17600112.

    Article  CAS  PubMed  Google Scholar 

  25. Yamaguchi M, Murakami S, Yoneda T, Nakamura M, Zhang L, Uezumi A, Fukuda S, Kokubo H, Tsujikawa K, Fukada S. Evidence of Notch-Hesr-Nrf2 Axis in muscle stem cells, but absence of Nrf2 has no effect on their quiescent and undifferentiated state. PLoS ONE. 2015;10:e0138517. http://www.ncbi.nlm.nih.gov/pubmed/26418810.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Fukada SI, Nakamura A. Exercise/Resistance training and muscle stem cells. Endocrinol Metab (Seoul). 2021;36:737–44. https://www.ncbi.nlm.nih.gov/pubmed/34372625.

    Article  CAS  PubMed  Google Scholar 

  27. Jackson JR, Kirby TJ, Fry CS, Cooper RL, McCarthy JJ, Peterson CA, Dupont-Versteegden EE. Reduced voluntary running performance is associated with impaired coordination as a result of muscle satellite cell depletion in adult mice. Skelet Muscle. 2015;5:41. https://www.ncbi.nlm.nih.gov/pubmed/26579218.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Tanaka H, Swensen T. Impact of resistance training on endurance performance. A new form of cross-training? Sports Med. 1998;25:191–200. https://www.ncbi.nlm.nih.gov/pubmed/9554029.

    Article  CAS  PubMed  Google Scholar 

  29. Motohashi N, Uezumi A, Asakura A, Ikemoto-Uezumi M, Mori S, Mizunoe Y, Takashima R, Miyagoe-Suzuki Y, Takeda S, Shigemoto K. Tbx1 regulates inherited metabolic and myogenic abilities of progenitor cells derived from slow- and fast-type muscle. Cell Death Differ. 2019;26:1024–36. https://www.ncbi.nlm.nih.gov/pubmed/30154444.

    Article  CAS  PubMed  Google Scholar 

  30. Esteves de Lima J, Blavet C, Bonnin MA, Hirsinger E, Havis E, Relaix F, Duprez D. TMEM8C-mediated fusion is regionalized and regulated by NOTCH signalling during foetal myogenesis. Development 149 (2022). https://www.ncbi.nlm.nih.gov/pubmed/35005776

  31. Sakai H, Fukuda S, Nakamura M, Uezumi A, Noguchi YT, Sato T, Morita M, Yamada H, Tsuchida K, Tajbakhsh S, Fukada SI. Notch ligands regulate the muscle stem-like state ex vivo but are not sufficient for retaining regenerative capacity. PLoS ONE. 2017;12:e0177516. http://www.ncbi.nlm.nih.gov/pubmed/28498863.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Englund DA, Murach KA, Dungan CM, Figueiredo VC, Vechetti IJ Jr., Dupont-Versteegden EE, McCarthy JJ, Peterson CA. Depletion of resident muscle stem cells negatively impacts running volume, physical function and muscle hypertrophy in response to lifelong physical activity. Am J Physiol Cell Physiol 318 (2020). https://www.ncbi.nlm.nih.gov/pubmed/32320286

  33. Goh Q, Song T, Petrany MJ, Cramer AA, Sun C, Sadayappan S, Lee SJ, Millay DP. Myonuclear accretion is a determinant of exercise-induced remodeling in skeletal muscle. Elife 8 (2019). https://www.ncbi.nlm.nih.gov/pubmed/31012848

Download references

Acknowledgements

We sincerely thank all lab members for their technical support. We thank Editage (www.

editage.com) for English language editing.

Funding

SF was supported by a Grant-in-Aid for Scientific Research (B, 22H03466), Challenging Research (Exploratory, 20K21757), an Intramural Research Grant for Neurological and Psychiatric Disorders of NCNP (2–6), the Takeda Science Foundation, the Astellas Foundation for Research on Metabolic Disorders, and the Association Francaise contre les Myopathies (AFM; 23574). This research was partially supported by the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under grant numbers JP23ama121054, JP23ama121052, JP24ama121054, and JP24ama121052.

Author information

Author notes

  1. Kanako Iwamori, Manami Kubota and Lidan Zhang contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Stem Cell Regeneration and Adaptation, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, 565-0871, Osaka, Japan

    Kanako Iwamori, Manami Kubota, Lidan Zhang, Kazuki Kodama, Atsushi Kubo & So-ichiro Fukada

  2. Center for Medical Epigenetics, School of Basic Medical Sciences, Chongqing Medical University, Chongqing, 40016, China

    Lidan Zhang

  3. Department of Cardiovascular Physiology and Medicine, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minamiku, Hiroshima, 734-8551, Japan

    Hiroki Kokubo

  4. Faculty of Sport Sciences, Waseda University, 2-579-15 Mikajima, Tokorozawa, 359-1192, Saitama, Japan

    Takayuki Akimoto

Authors
  1. Kanako Iwamori
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Manami Kubota
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Lidan Zhang
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Kazuki Kodama
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Atsushi Kubo
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Hiroki Kokubo
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Takayuki Akimoto
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. So-ichiro Fukada
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

KI, MK, LZ, and KK. performed the experiments and analyzed the data. HK provided the critical materials (HeyL-KO mice) for this study. TK and SF designed wheel-running experiments. SF designed the experiments, interpreted data, assembled input data, and wrote the manuscript. AK supported KI, and KK performed the experiments and wrote the manuscript. All authors discussed the results and implications, and commented on the manuscript. All the authors have read and approved the final version of the manuscript.

Corresponding author

Correspondence to So-ichiro Fukada.

Ethics declarations

Ethics approval

All animal experiments were performed in accordance with guidelines approved by the Experimental Animal Care and Use Committee of Osaka University (approval number: 30 − 15, R02-1-10).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Iwamori, K., Kubota, M., Zhang, L. et al. Decreased number of satellite cells-derived myonuclei in both fast- and slow-twitch muscles in HeyL-KO mice during voluntary running exercise. Skeletal Muscle 14, 25 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13395-024-00357-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13395-024-00357-z

Keywords

Skeletal Muscle

ISSN: 2044-5040

Contact us