BBA - Molecular Cell Research 1867 (2020) 118742

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BBA - Molecular Cell Research

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Review

Role of damage and management in muscle hypertrophy: Different T

behaviors of muscle stem cells in regeneration and hypertrophy
⁎
So-ichiro Fukadaa, , Takayuki Akimotob, Athanassia Sotiropoulosc
a
Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan
b
Faculty of Sport Sciences, Waseda University, Saitama, Japan
c
Institut National de la Santé et de la Recherche Médicale U1016, Institut Cochin, Paris, France

A R T I C LE I N FO A B S T R A C T

Keywords: Skeletal muscle is a dynamic tissue with two unique abilities; one is its excellent regenerative ability, due to the
Skeletal muscle activity of skeletal muscle–resident stem cells named muscle satellite cells (MuSCs); and the other is the
Muscle satellite cells adaptation of myofiber size in response to external stimulation, intrinsic factors, or physical activity, which is
Hypertrophy known as plasticity. Low physical activity and some disease conditions lead to the reduction of myofiber size,
Regeneration
called atrophy, whereas hypertrophy refers to the increase in myofiber size induced by high physical activity or
Damages
anabolic hormones/drugs. MuSCs are essential for generating new myofibers during regeneration and the in-
crease in new myonuclei during hypertrophy; however, there has been little investigation of the molecular
mechanisms underlying MuSC activation, proliferation, and differentiation during hypertrophy compared to
those of regeneration. One reason is that ‘degenerative damage’ to myofibers during muscle injury or upon
hypertrophy (especially overloaded muscle) is believed to trigger similar activation/proliferation of MuSCs.
However, evidence suggests that degenerative damage of myofibers is not necessary for MuSC activation/pro-
liferation during hypertrophy. When considering MuSC-based therapy for atrophy, including sarcopenia, it will
be indispensable to elucidate MuSC behaviors in muscles that exhibit non-degenerative damage, because de-
generated myofibers are not present in the atrophied muscles. In this review, we summarize recent findings
concerning the relationship between MuSCs and hypertrophy, and discuss what remains to be discovered to
inform the development and application of relevant treatments for muscle atrophy.

1. Introduction studies of regenerating muscle, the behaviors and molecular mechan-

Skeletal muscle is composed of terminally differentiated and mul-
tinucleated giant cells, called myofibers. In mammals, myofibers are
postmitotic and cannot give rise to new myofibers. Instead, skeletal
muscle possesses mononuclear stem cells, or muscle satellite cells
(MuSCs) [1], that are responsible for generating new myofibers [2,3].
Myofibers possess another ability, known as plasticity [4,5]. Myofibers
adjust their size in response to external stimulations, intrinsic factors, or
physical activity. Bedridden people, patients with cancer cachexia or
sepsis, and the elderly exhibit a loss of muscle mass (i.e., muscle
atrophy), whereas resistance training and some kinds of drugs leads to
an increase in muscle mass and function (i.e., muscle hypertrophy).
Regarding hypertrophy, many studies indicate that proliferating MuSCs
contribute to an increase in muscle size through fusion to the growing
myofiber and the addition of new myonuclei. However, compared to
isms underlying the activation and proliferation of MuSCs during hy-
pertrophy remains to be elucidated.
It is assumed that MuSCs in overloaded muscle respond to damaged
myofibers and repair them in a similar way to that observed during
regeneration [6]. It is well documented that eccentric muscle contrac-
tion damages skeletal muscles and the history of exercise-induced
muscle damage in humans has been summarized previously [7]. As
described by Damas et al. (2018), the term muscle ‘damage’ is con-
troversial and includes several physiological events, such as disturbance
of the myofibril structure, decreased stability of the sarcolemma, and
lethal damage to myofibers. In this review, we use the term degen-
erative damage to refer to damages causing myofiber death. There is
direct evidence demonstrating the existences of disrupted myofibril
structure, even in human studies [8], whereas such evidence regarding
degenerative damage of myofibers in human muscle is rare. Yu et al.

⁎
Corresponding author at: Project for Muscle Stem Cell Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita City, Osaka
565-0871, Japan.
E-mail address: fukada@phs.osaka-u.ac.jp (S.-i. Fukada).

https://doi.org/10.1016/j.bbamcr.2020.118742
Received 2 March 2020; Received in revised form 7 May 2020; Accepted 12 May 2020
Available online 14 May 2020
0167-4889/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).
S.-i. Fukada, et al.
(2002) reported that eccentric contraction did not cause degenerative
damage to myofibers in the soleus or vastus lateralis muscles of male
subjects (age: 18–39 years old) experiencing delayed onset muscle
soreness (DOMS) [9]. On the other hand, Crameri et al. (2004) reported
that one in eight male subjects (age: 25 ± 3 years) demonstrated signs
of degenerative damage to myofibers in the vastus lateralis muscle after
a single bout of high intensity exercise [10]. Because a muscle biopsy
only allows for the observation of a very small section of skeletal muscle
[7], the direct assessment of the degenerative damage to myofibers in
human muscle is difficult. Crameri et al. (2007) evidenced that artificial
electrical stimulation induced degenerative damage to myofibers that
could be detected histologically, whereas voluntary eccentric contrac-
tion did not induce such damage [11]. Using an electrical stimulation
model of human muscle regeneration, Mackey and Kjaer (2017) were
able to efficiently observe degenerative damage of myofibers [12].
Hence, there is little direct evidence to confirm that exercise induces
degenerative damage to myofibers.
Instead of histological studies, indirect assessments, including
magnetic resonance imaging (MRI), have generally been used to detect
degenerative damage, but an MRI also reflects edema (swelling caused
by excess fluid) in skeletal muscle. Increased levels of muscle proteins,
such as creatine kinase and myoglobin, in the blood are also widely
used as markers of degenerative damage [13]. However, these markers
might not accurately reflect the death of myofibers because myofibers
possess repair systems, independent of MuSC fusion, that seal the da-
maged sarcolemma [14,15]. In other words, it is possible that muscle
proteins leaked from the damaged sarcolemma during the membrane
repair process. There is no doubt that exercise can induce degenerative
damage of myofibers [16], but, to our knowledge, there is no direct
evidence disclosing that the degenerative damage of myofibers is re-
quired for MuSC activation/proliferation during hypertrophy. Darr and
Schultz (1987) observed BrdU+ (proliferating) MuSCs in overloaded rat
muscle without degenerative damage to myofibers [17]. Blood flow
restriction (BFR) alone, or in combination with exercise, results in
muscle hypertrophy and strength gain. Beyond the effects of training,
the degenerative damage of myofibers and subsequent MuSC-depen-
dent repair is one proposed mechanism. However, Loenneke et al.
(2014) questioned whether the degenerative damage of myofibers is a
necessary effect of training, because available evidence does not sup-
port the hypothesis that BFR in combination with low-intensity exercise
increases the incidence of degenerative damage to myofibers [18].
Recently, we showed that MuSCs proliferate on living myofibers in
mouse models of tenotomy and overload [19]. These results imply that
the degenerative damage of myofibers is not necessary for MuSC acti-
vation and proliferation in overloaded muscle. In addition, it is assumed
that the environments for the activation and proliferation of MuSCs in
regenerating and overloaded muscle are completely different [19].
When considering appropriate therapy for diseases resulting in muscle
atrophy, the elucidation of the mechanisms underlying MuSC activa-
tion/proliferation in non-injured muscle is essential, because they do
not accompany the degenerative damage of myofibers. In this review,
the molecular mechanisms regulating MuSC quiescence in steady-state
conditions are updated and recent findings regarding the relationship
between MuSCs and hypertrophy, especially whether the degenerative
damage of myofibers is necessary for the activation/proliferation of
MuSCs, are summarized. Finally, we will discuss how to apply these
findings to the treatment of muscular atrophy.
2. Muscle stem cells under steady-state conditions
MuSCs are mononuclear cells located beneath the basal lamina of
mature skeletal muscle fibers [1]. In steady-state conditions, MuSCs are
maintained in a quiescent and undifferentiated state. However, when
myofibers are damaged and degeneration occurs, MuSCs start to ex-
press myoblast determination protein 1 (MyoD) and subsequently enter
the cell cycle [20]. There is no doubt that MuSCs are indispensable in
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BBA - Molecular Cell Research 1867 (2020) 118742
the production of new myofibers during muscle regeneration [2,3].
Although some investigations indicate that MuSCs exhibit multipotent
differentiation abilities (for example, MuSCs differentiate into fibro-
blastic cells and brown adipocytes in certain disease conditions, ge-
netically modified mice, aged mice, and in vitro) [21–24], this ability,
in normal MuSCs, is limited to the myogenic lineage [25,26]. Based on
previous knowledge, MuSCs are more primed for myogenic lineage by
retaining transcripts of the myogenic determination genes, MyoD and
myogenic factor 5 (Myf5), without subsequent translation thereof
[27,28]. The expression of Myf5 is also reported in quiescent MuSCs
[29]. In skeletal muscle, muscle-resident mesenchymal progenitors,
also known as fibro-adipogenic progenitors (FAPs), are considered the
major source of fibrosis and fat accumulation in both humans and mice
[22,25,26,30,31].
In the past decade, molecular mechanisms underlying the quiescent
and undifferentiated state of MuSCs have been discovered [32–35]. The
canonical Notch signaling pathway is essential in sustaining the un-
differentiated state of MuSCs via direct target genes, namely Hey1,
HeyL, Hes1, collagen V, and mir-708 [36–42]. Intriguingly, collagen V
operates as a surrogate ligand for calcitonin receptor (CalcR), which
represents another signaling pathway for maintaining MuSC quiescence
[36,43–45]. Recently, we revealed that the CalcR-protein kinase A
(PKA) axis regulates Hippo pathway members, large tumor-suppressor
kinases 1/2 (Lats1/2), and Yes-associated protein 1 (Yap1) to sustain
quiescence in MuSCs [46]. The quiescent MuSC-specific microRNA,
mir-489, is encoded by intron 4 of the mouse Calcr gene and is also
important for maintaining quiescence by targeting the oncogene Dek
[47].
Endothelial cells are thought to be important in maintaining MuSC
quiescence because MuSCs are approximal to endothelial cells [48].
Verma et al. (2018) also exhibited that MuSCs and endothelial cells are
in close proximity and a positive relationship exists between the
quiescent state and distance between the latter and former. They also
evidenced that Notch ligand Dll4, expressed in endothelial cells, in-
duces quiescence during MuSC self-renewal [49]. It is predicted that
myofibers also supply Notch ligands to activate canonical Notch sig-
naling in MuSCs [34]. The relationship between myofiber- and en-
dothelial cell–derived Notch ligands needs to be clarified to decipher
the role of Notch signaling in MuSC regulation. Myofibers play another
role in MuSC maintenance by establishing direct cell contact with
MuSCs through N- and M-cadherin [50]. Additionally, myofiber-de-
rived Wnt4 is reported to maintain MuSCs quiescence by inhibiting
Yap1 transcription [51]. Moreover, Yap1 activity can also be regulated
by cell-cell contact between myofibers and MuSCs, which suggests that
MuSCs can employ at least three pathways to regulate Yap1 activity in
order to sustain quiescence (Fig. 1).
Mesenchymal progenitors are another cell type thought to
Fig. 1. Molecular mechanisms underlying quiescence in MuSCs.
Three putative molecular pathways regulate Yap1 activity in quiescent MuSCs.
Fzd (Frizzled), Ror1/2, and Vangl1/2 are putative receptor and co-receptors for
Wnt4.
S.-i. Fukada, et al.
contribute to the maintenance of MuSCs in normal muscle tissue, be-
cause the depletion of mesenchymal progenitors resulted in the loss of
MuSC pool in a mouse model [52]. Compared to cell-autonomous and
other niche cells' regulation of MuSCs, the molecular mechanisms un-
derlying the role of mesenchymal progenitors in the maintenance of
MuSCs are unknown. The identification of these factors will lead to a
more in-depth understanding of MuSC quiescence, which will con-
tribute to the explanation of MuSC behaviors in regenerating and
overloaded muscle.
3. Muscle hypertrophy
Muscle hypertrophy, in response to intrinsic and extrinsic factors, is
a characteristic of skeletal muscle plasticity that is even observed in
Drosophila (fruit flies) [53]. This means that hypertrophy is a capacity
that is evolutionarily conserved in skeletal muscle, like its regenerative
ability [54]. In order to identify the mechanisms underlying the in-
crease in myofiber size, many studies have focused on the roles of
MuSCs in several kinds of animal models. However, whether the de-
generative damage of myofibers is necessary for MuSC activation/
proliferation during muscle hypertrophy has not been sufficiently de-
bated, and the molecular mechanisms regulating MuSC activation,
proliferation, and differentiation have not been identified. In the fol-
lowing subsections, animal models used to investigate the role of
MuSCs in muscle hypertrophy are introduced, and thereafter we discuss
whether degenerative damage to myofibers is required for MuSC acti-
vation and proliferation.
3.1. Animal models of muscle hypertrophy
3.1.1. Surgical procedures
In rodent studies, synergistic ablation (SA) or tenotomy is widely
used to induce muscle hypertrophy (Fig. 2). In both cases, reduced
gastrocnemius/soleus muscle tension is often observed by targeting the
plantaris muscle. The plantaris muscle receives all the tension following
surgery, resulting in overload hypertrophy. The difference between SA
and tenotomy is whether or not the synergistic muscles (gastrocnemius
and soleus muscles) are removed. In tenotomy, only the Achilles tendon
is cut, whereas in SA, the gastrocnemius and soleus muscles are par-
tially removed. The increase in plantaris muscle weight is attenuated
2–3 weeks after tenotomy, relative to SA, but the muscle weight con-
tinues to increase for a longer period in the SA model [55]. Even for a
short period, the ratio of muscle weight is also higher in the SA model
than that of the tenotomy model. However, the increase in muscle
weight, or myofiber size, is considered to be a result of edema-induced
muscle swelling, especially in the early phase of muscle hypertrophy,
which also occurs during human resistance training [13]. It is therefore
important to pay attention to the effects of edema-induced muscle
Fig. 2. Two surgical models for inducing muscle hypertrophy in plantaris.
Difference between the surgery of synergistic ablation and tenotomy.
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BBA - Molecular Cell Research 1867 (2020) 118742
swelling, especially in the early phase shortly after surgery.
Although both surgical procedures are equally invasive and may be
attributed to the degenerative damage of myofibers, the SA model has a
higher risk of degenerative damage compared to the tenotomy model
(see Section 3.3). Both models are commonly used for the analyses of
MuSCs behaviors.
3.1.2. Voluntary weightlifting exercise
The surgical procedures described above have a risk of causing
degeneration or inflammation in muscles. Klitgaard et al. (1988) de-
veloped a non-invasive, voluntary weightlifting rat model, which evi-
denced a significant increase in plantar flexor muscle weight and
strength after 36 weeks of training [56]. Because this model requires
constant human intervention, it has not been widely used in the re-
search field. Considering this shortfall, Cui et al. (2020) developed a
novel mouse model of voluntary weightlifting that does not involve
human handling during the training [57]. In order to access food, mice
are required to push a weighted cage which leads to voluntary hindlimb
plantar flexion movement against shoulder-loaded resistance. Com-
pared to surgical models, the degree of muscle hypertrophy in the study
is modest, but highly consistent with that observed in humans
(8%–12%) [58]. This type of model is suitable for the study of MuSCs
behaviors, on a physiological level, in muscle hypertrophy during re-
sistance training.
3.1.3. Running equipment
Wheel running is an endurance training model that also leads to
muscle hypertrophy. Rodents are internally motivated to run sponta-
neously. In our analyses, the average running distance of a mouse is
6–10 km per night (unpublished data). A combined model, including
increasing load and wheel running, was also developed to observe more
remarkable adaptations of skeletal muscle [59,60]. The effect of vo-
luntary wheel running alone on soleus wet mass was well observed, and
the wet mass of additional muscles (plantaris, vastus lateralis, and ex-
tensor carpi radialis longus and brevis) in rats increased in combination
with load [61]. The plantaris and soleus muscles are both responsive to
wheel running, but the soleus muscle does not undergo a shift in
myofiber type. The plantaris muscle, consisting mainly of fast type
myofibers (30–40% Type IIa and 60–70% Type IIb + IIx), exhibits a
shift toward slow-twitch oxidative myofibers (increased ratio of Type
IIa and decreased ratio of Type IIb + IIx) following weighted wheel
running as well as tenotomy in mouse models [60]. Both the soleus and
plantaris muscles of mice and rats have been used extensively in the
analyses of MuSC behaviors in this model [62,63]. In mice, BrdU pulse
experiments have detected the presence of BrdU+ cells between 3 and
14 days after the start of unweighted wheel running [63]. Basically,
many studies have indicated that degenerative damage to myofibers is
the factor that induces MuSC proliferation [64].
Treadmill running is a forced exercise model for endurance training
in rodents. Because an eccentric contraction produces greater muscle
hypertrophy than a concentric contraction, downhill running is a
widely used method of eccentric training in both human and rodents.
Although muscles in humans and rodents differ due to varied gaits, one
advantage of the treadmill experiment is that it replicates the training
in humans, compared to the spontaneous wheel running model.
However, close attention must be paid to the negative effects of
treadmill running because alterations in neuroendocrine tissues and
immune responses, commonly associated with chronic stress, were
observed in male Sprague-Dawley rats [65]. In addition, many studies
indicated that downhill running injures the deep slow muscles of the
antigravity groups [66]. For example, Darr and Schultz (1987) showed
that downhill treadmill running induced myofiber degeneration that
was histologically characterized by the infiltration of mononuclear
cells, including macrophages, and necrotic myofibers in the rat soleus
muscle [17]. Moreover, the degenerative damage of myofibers was
even observed in non-eccentric exercise training. McCormick and
S.-i. Fukada, et al.
Thomas (1992) demonstrated that 10 weeks of progressive treadmill
running induced MuSC proliferation with double the amount of de-
generative damage or repaired myofibers (defined morphologically
using central nuclei or necrotic fibers) in the rat soleus muscle [67].
Umnova and Seene (1991) reported that the number of MuSCs in-
creased by 2.5-fold in the quadriceps femoris of male rats with localized
degenerative damage [68]. The frequency of the degenerative damage
of myofibers depends on the intensity, duration, and frequency of ex-
ercise, and many studies that employed the treadmill model reported
that MuSC activation/proliferation results from degenerative damage to
myofibers. Therefore, increased physical activity can induce degen-
erative damage to myofibers. Except for the report by Darr and Schultz
(1987) [17], there has been little discussion on the need for degen-
erative damage in MuSC activation/proliferation, whereas whether
damage, in the broadest sense of the word, is required for muscle hy-
pertrophy itself is a highly debated issue [69,70].
3.1.4. Hormones and chemicals
Androgens are a group of anabolic steroid hormone and generally
refer to male sex hormones and substances with similar physiological
effects. Natural male sex hormones include testosterone, dehy-
drotestosterone (DHT), dehydroepiandrosterone (DHEA), and andros-
tenedione. It is well known that androgens have muscle strengthening
effects. However, testosterone sensitivity not equally expressed in all
muscles. In rodents, the perineal muscles (the levator ani (LA) and
bulbocavernosus muscle) are very sensitive to testosterone [71]. Using
myofiber-specific Cre mice (HSA-Cre), Chambon et al. (2010) reported
that the myofiber-androgen receptor (AR) transduces androgen-de-
pendent postnatal fiber hypertrophy in the perineal muscles but not in
limb muscles. In addition, they also identified that myofiber-AR is es-
sential to generate maximum force of fast- and intermediate-twitch leg
muscles by controlling the myofibrillar organization of androgen-in-
duced hypertrophic myofibers [72]. Regarding MuSCs, Sinha-Hikim
et al. (2003) indicated a correlation between the number of MuSCs and
serum testosterone concentrations in humans who were administered
weekly testosterone enanthate injections for 20 weeks [73]. Nnodim
(2001) reported that testosterone is responsible for denervation-in-
duced MuSC proliferation in the LA muscle because testosterone
treatment restored MuSC function in the denervated LA of castrated rats
[74]. Therefore, MuSCs are possible candidates for targeted therapy
with testosterone. An informative review regarding the regulation of
satellite cell function with androgens is summarized by Chen et al.
(2005) [75]. When regarding MuSC quiescence, the mechanism indu-
cing MuSC activation/proliferation can either be a consequence of
muscle hypertrophy, or the direct effect of androgens on quiescent
MuSCs to activate them. A study that investigated the effects of the
anabolic steroid, nandrolone decanoate, indicated that the use of this
steroid did not lead to myofiber damage in normal mice, although it
worsened symptoms of muscular dystrophy in mdx mice [76]. Thus,
MuSC activation/proliferation in an anabolic steroid model seems to be
independent of degenerative damage to myofibers.
The anabolic effects of beta2-agonists on skeletal muscle are also
well established and involve changes in protein kinetics which results in
muscle hypertrophy. Interestingly, the beta2-agonist, clenbuterol, in-
creased DNA and protein concentrations in denervation-induced muscle
atrophy, but had no impact on the DNA content in normal rat soleus
muscles [77], which suggests that the effects of clenbuterol on MuSCs
are limited to atrophied muscles.
In the following subsections, the proliferation of MuSCs in surgical
overload and testosterone models are discussed.
3.2. Roles of muscle stem cells in muscle hypertrophy
3.2.1. Surgical procedure
In order to sustain the cytoplasm-to-myonucleus ratio (known as the
myonuclear domain theory), the supply of new MuSC-derived
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BBA - Molecular Cell Research 1867 (2020) 118742
myonuclei is required for hypertrophy. In a SA-induced model of
muscle hypertrophy, both McCarthy et al. (2011) and Egner et al.
(2016) commonly observed that MuSC-depleted mice showed no in-
crease in the number of myonuclei during hypertrophy [78,79]. Goh
et al. (2017) also detected that the number of myonuclei is reduced
when MuSCs cannot fuse with myofibers due to a lack of myomaker, a
fusion protein [80]. Although a handful of studies have demonstrated
that other stem cells can contribute to myofiber regeneration under
physiological conditions [81,82], there is no doubt that MuSCs are the
most essential type of cell for supplying new myonuclei during over-
load-induced muscle hypertrophy. Whereas, McCarthy et al. (2011)
established a relatively short-term SA model and reported that myofi-
bers lacking MuSCs grew similar to control myofibers, thereby con-
cluding that the addition of MuSC-derived myonuclei is not required for
muscle hypertrophy [78]. On the contrary, after employing the same
strategy, Egner et al. (2016) argued that MuSCs are critical for efficient
muscle hypertrophy [79]. Thus, whether MuSCs are required for muscle
hypertrophy is a highly debated issue. Recently, independent studies
have established different animal models, examining animals of dif-
ferent ages and for different time periods, and indicated that the loss of
MuSCs, or the impairment of their fusion capacity or proliferation, leads
to blunted muscle hypertrophy [19,55,80,83,84]. In the first 2–3 weeks
following overload, the contribution of MuSCs might be minor or
blunted by some experimental conditions, including edema or the age
of the mice [84]. However, in the late stages of overload, the efficiency
of muscle hypertrophy can be blunted due to the myonuclear domain
being too large or the loss of MuSC paracrine factors [19,55,85]. Gen-
erally, both MuSCs-independent (e.g., mammalian target of rapamycin-
(mTOR-) dependent protein synthesis) and MuSC-dependent (e.g.,
myonuclear accretion) factors are required for efficient muscle hyper-
trophy (Fig. 3).
3.2.2. Androgen
Sinha-Hikim et al. (2002) indicated that testosterone-induced
myofiber hypertrophy is associated with a dose-dependent increase in
myonuclear number [86], as well as an increased number of MuSCs
after testosterone administration in humans [73]. Using a mouse model,
it was demonstrated that the administration of testosterone increased
myonuclear number as a consequence of MuSC proliferation, because
such an increment was not observed in MuSC-depleted mice [87]. In
fact, an increase in MuSC numbers was observed in the soleus (22%
increase compared to the sham muscle) and plantaris muscle (29%) of
Fig. 3. Loss of MuSC ability results in blunted muscle hypertrophy.
Both mTOR-dependent and MuSC-dependent mechanisms are critical for ef-
fective muscle hypertrophy. The effect of MuSC might be obscured by some
experimental condition, but an increase in myonuclei is also important for ef-
ficient muscle hypertrophy.
S.-i. Fukada, et al.
6-month-old control mice harboring MuSCs, but was not the case in
MuSC-depleted mice three weeks after testosterone administration.
However, the increase in myofiber cross-sectional area was not affected
by the depletion of MuSCs at least three weeks after testosterone
treatment [87]. Like the overload model, a longer period of study or
muscle force measurement is required to conclude whether MuSCs are
necessary for testosterone-induced muscle hypertrophy. In addition, the
role of testosterone might be age-dependent, because it was reported
that SA-induced muscle hypertrophy is dependent on MuSC accretion in
young mice (below 4 months old), which is not the case in mature mice
(over 4 months old) [84].
3.3. Degenerative damage to myofibers and increased myofiber size are not
necessary for muscle stem cell activation/proliferation
As described above, exercise-induced muscle hypertrophy is fre-
quently accompanied by degenerative damage to myofibers in animal
models, and such injuries may also be observed in animal models of
overload. Therefore, it is commonly believed that degenerative damage
to myofibers induces MuSC activation/proliferation in these models of
muscle hypertrophy. Thus, whether degenerative damage is necessary
for MuSC activation/proliferation has not been debated to a great ex-
tent. However, if overload-induced muscle hypertrophy accompanies
severe and extensive degenerative damage to myofibers, MuSC-de-
pleted mice should exhibit more severe phenotypes because MuSCs are
indispensable to the repair of damaged myofibers. This means that the
area of degenerative damage is limited in the overload models. In fact,
Peterson group evidenced that approximately 30% of myofibers stained
positive for embryonic myosin heavy chain (eMyHC; detects newly
generated myofibers) 14 days after SA [78], whereas 0.1–1.4% or
0.2–18.4% of myofibers were positive for eMyHC 10 days after te-
notomy in 2- or 4-month-old C57BL/6 mice, respectively [84]. Ad-
ditionally, we previously implemented a tenotomy model to induce
hypertrophy and demonstrated increased MuSC numbers with little
degenerative damage to myofibers. Furthermore, almost all analyzed
myofibers contained Ki67+ MuSCs [19]. Generally, the majority of
MuSCs become activated and proliferate in overloaded live myofibers,
regardless of the extent of degenerative damage, which means that
degenerative damage to myofibers does not seem to be necessary for
MuSC activation and proliferation.
It is also important to distinguish whether muscle hypertrophy al-
ways follows an increase in myonuclear number because this will reveal
the relevance between increased myofiber size and MuSC quiescence.
Inducible activation of Akt (also known as protein kinase B; PKB) sti-
mulates myofiber hypertrophy without an increase in myonuclear
number [88]. Importantly, the force generated by skeletal muscle was
also increased by Akt activation. Furthermore, new MuSC-derived
myonuclei were not observed in JunB-induced muscle hypertrophy or
myostatin-null mice [89,90]. These results suggest that signaling
pathways in myofiber growth, and increased myofiber size and strength
do not affect MuSC quiescence and mobilization in the growing myo-
fibers.
Conversely, a considerable amount of data indicates that MuSC
behaviors, independent of myofiber hypertrophy, are affected by ex-
ercise or mechanical load [91]. In a human study using muscle biopsies
of endurance-trained cyclists, an increase in myonuclear number, in the
absence of hypertrophy or injury, was observed in response to an in-
tensified training load [91–93]. Similarly, a rat treadmill running and
unweighted wheel running model reported an increase in the number of
MuSC-derived myonuclei, a lower incidence of histological disruption,
and unchanged myofiber diameter [62,94]. It is also noteworthy that
MuSC-dependent myonuclear accretion occurred in the absence of hy-
pertrophy in aged mice subjected to SA surgery [95]. Moreover, a
human study in the elderly reported that MuSC proliferation and
muscle hypertrophy occur independently of one another [96]. There-
fore, MuSC activation and proliferation do not seem consequential of
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BBA - Molecular Cell Research 1867 (2020) 118742
increased myofiber size or degenerative damage. Alternatively, the two
events are unrelated, and the signaling pathways that regulate both
active and quiescent states stimulate MuSC activation and proliferation
and occur independently of myofiber growth. Questions remain re-
garding how exercise and overload induce MuSC proliferation and fu-
sion independently of hypertrophy. We discuss this point in Section 4.1.
4. Comparison of muscle stem cell behaviors between muscle
hypertrophy and regeneration
During muscle regeneration, MuSCs proliferate beneath the retained
basal lamina, called ‘ghost fibers’ [97]. We have previously evidenced
that reduced myogenic regeneration occurs where ghost fibers are di-
minished (caused by injecting a fig protease, ficin, in combination with
cardiotoxin), which indicates that the retained basal lamina is critical
for MuSCs behaviors during muscle regeneration [98]. On the other
hand, it is assumed that MuSCs proliferate between the basal lamina
and plasma membranes of myofibers in muscles experiencing hyper-
trophy and minimal degeneration (tenotomy model). This means the
environments for MuSC activation/proliferation and the kinetics of
MuSC behavior are completely different in regenerating and overloaded
muscle (Fig. 4) [19]. In regenerating muscle, a stepwise process of
MuSC activation, proliferation, and differentiation proceeds, whereas
MuSC proliferation and differentiation occur simultaneously, for rela-
tively long periods, in tenotomy-overloaded muscle [19]. In this sec-
tion, the behaviors of MuSCs are compared between regenerating and
overloaded muscle (Fig. 4).
4.1. Activation
Several models, including injury by freezing and barium chloride,
notexin, and cardiotoxin injection, are used to observe muscle re-
generation. Detailed analyses of multiple injury models are summarized
by Hardy et al. (2016) [99]. Perhaps MuSCs start to express MyoD
quickly in all regenerating muscles, and subsequently enter the cell
cycle. In our previously established cardiotoxin model of muscle re-
generation, we observed that MuSCs first start to express MyoD, and at
least 50% of MuSCs are Ki67+ one day after injury [100]. In re-
generating muscle, the components of the MuSC niche, including
myofibers, disappear. Therefore, it is conceivable that the loss of the
MuSC niche leads to reduced quiescence signaling, which is partially
responsible for the activation of MuSCs. In contrast, the MuSC niche is
relatively, or completely, retained in overloaded muscle. In our ana-
lyses, MuSCs start to express Ki67 two days after tenotomy [19], in-
dicating that MuSC activation occurs later in overloaded muscle than in
regenerating muscle. The factor that induces MuSC activation in over-
loaded muscle is unknown, but some potential mechanisms have been
identified. One possibility is that MuSCs perceive mechanical cues in a
direct manner. Another possibility is that non-MuSCs, including myo-
fibers, sense the mechanical stress and subsequently release the acti-
vation factor which acts on MuSCs or disrupts their quiescent state.
Overload disrupts the extracellular matrix (ECM) and might initiate
MuSC activation by releasing cytokines that were retained in the ECM
structure, or by reducing the amount of ligands available for quiescence
signaling. In fact, the remodeling or disruption of the ECM (caused by
the increased expression of some kinds of matrix metalloprotease
(MMP), tissue inhibitor of metalloproteinase (TIMP), and collagens)
was observed three days after SA in rats [101]. However, because this
model includes the degenerative damage of myofibers, it is unclear to
what extent overload alone alters ECM remodeling without the added
effects of degenerative damage to myofibers.
Even if myofibers evade cell death, the effect of a damaged sarco-
lemma on MuSC activation cannot be ignored. In a murine cancer ca-
chexia model, the structures of the sarcolemma and ECM were per-
turbed by circulating cachectic factors, which led to MuSC activation
[102]. In addition, an increase in the myonuclear number was detected
S.-i. Fukada, et al.
Fig. 4. Behaviors of muscle stem cell in regenerating or overloaded muscle.
The upper cartoon summarizes of MuSC behaviors and time course during regeneration processes. The lower cartoon shows the kinetics and behaviors of MuSCs in
overloaded muscle.
in the absence of muscle injury in patients with rheumatoid arthritis
(RA), a chronic inflammatory disease [103]. These results suggest that
damage to the structure of the sarcolemma or ECM triggers MuSC ac-
tivation, even in healthy muscle hypertrophy. It could also be specu-
lated that inflammation, including edema, directly induces MuSC acti-
vation or indirectly disrupts MuSC quiescence by perturbing the
structure of the sarcolemma or ECM. In order to determine the
minimum factors required for inducing MuSCs activation in hyper-
trophy, the identification of factors, or genes, that activate MuSCs and
induce their proliferation in sedentary mice would be a challenging, yet
fruitful experiment.
4.2. Proliferation
Two to three days after injury, MyoD+ MuSCs proliferate vigorously
and begin to express myogenin to promote MuSC fusion. We previously
reported that the majority of MuSCs proliferated in the absence of
MyoD protein expression in overloaded muscle [19]. Notably, gene-
expression analyses indicated that Hey1 expression was decreased in
proliferating MuSCs to the same extent as that observed in the injury
model, whereas HeyL expression was sustained in MuSCs in overloaded
muscle. Besides HeyL, Notch target genes are highly expressed in
MuSCs in overloaded muscle compared to that of regenerating muscle
[19]. Some studies have evidenced that the number of MyoD+ cells
increased in response to eccentric contraction in humans and a rat SA
model [104,105]. However, compared to Pax7+ cells, the number of
MyoD+ cells was minimal (< 10% or 30% of Pax7+ cells) [104], and
MyoD binding activity was not increased following exercise [105].
Single cell RNA-sequence analyses will elucidate the fate of pro-
liferating MyoD− MuSCs, and more detailed analyses are required to
conclude the existence of this MuSC population in overloaded human
muscle.
To study the proliferation of stem cells, including MuSCs, two
models, namely symmetric and asymmetric cell division, have been
proposed. In regenerating muscles, MuSCs proliferate either symme-
trically or asymmetrically both in vivo and in vitro [106–108]. Re-
cently, Evano et al. (2020) indicated that 5 days after notexin (a kind of
snake toxin that differs from cardiotoxin) injection, about a quarter of
cell divisions were asymmetric and gave rise to Pax7+ and myogenin+
6
BBA - Molecular Cell Research 1867 (2020) 118742
cells, whereas the frequency of asymmetric division was 4.3% 3 days
after injection [106]. Moreover, most MuSCs had proliferated on
myotubes and nascent myofibers 5 days after notexin injection.
Therefore, one can speculate that the presence of myofibers affects cell
division. In muscle hypertrophy, MuSCs proliferate on existing myofi-
bers, suggesting that asymmetric division frequently occurs to generate
Pax7+MyoD− cells and myogenin+ differentiated cells in growing
myofibers.
In vitro, there are many cytokines that promote MuSC proliferation.
Hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF),
and insulin-like growth factor (IGF) are candidates for MuSC pro-
liferation in muscle regeneration [109–111], but there is no direct
evidence indicating the physiological importance of these cytokines for
MuSC proliferation in vivo. Unexpectedly, the depletion of the HGF
receptor, c-Met, did not affect MuSC proliferation in regenerating
muscle [112]. It could be speculated that the redundant roles of other
cytokines might obscure the effect of the loss of HGF signaling in re-
generating muscle. On the other hand, Zhu et al. (2016) indicated that
signal transducer and activator of transcription 3 (Stat3) is physiolo-
gically critical for MuSC proliferation in regenerating muscle [113].
Therefore, the cytokines regulating Stat3 activation (e.g., the inter-
leukin-6 (IL-6) family) might be essential for MuSC proliferation. Ev-
erything considered, it could be predicted that several cytokines, in-
cluding the activator of Stat3, play redundant roles in MuSC
proliferation during regeneration.
In overloaded muscle, IL-6 deficiency abrogated MuSC proliferation
and myonuclear accretion through decreased Stat3 activation, which
indicates the importance of IL-6 in MuSCs behavior in overloaded
muscle [114]. Guerci et al. (2012) also found that myofiber-specific
depletion of serum response factor (SRF), a transcription factor, blunts
overload-induced hypertrophy and reduces MuSC proliferation due to
decreased IL-6 levels [115]. Interestingly, SRF in myofibers induces IL-6
expression in response to overload, which leads to MuSC proliferation
[115]. These results suggest that SRF plays a role in the translation of
mechanical stimuli into paracrine signals and regulates MuSC pro-
liferation through IL-6. However, the molecular mechanism underlying
the transduction of mechanical stimuli in order to activate SRF in
overloaded muscle is still unknown. Whereas SRF activity is reduced in
atrophied muscle due to the altered subcellular localization of the
S.-i. Fukada, et al.
transcriptional co-activator called myocardin-related transcription
factor (Mrtf) [116]. Interestingly, disuse atrophy induced the nuclear
accumulation of G-protein, which reduces SRF activity by inhibiting the
nuclear accumulation of Mrtf [116]. Because Mrtf is involved in cardiac
hypertrophy [117], it could be speculated that overload-dependent
hypertrophy in muscles also requires the nuclear location of Mrtf,
which is controlled by mechanical cue-induced actin dynamics for the
regulation of SRF transcriptional activity. In line with this hypothesis,
mechanical strain applied to myoblasts in culture was sufficient to in-
duce rapid actin polymerization and nuclear accumulation of Mrtf
[118]. Another mechanism that bridges the gap between SRF activity
and mechanical cues is the activity of Yap/Taz, two molecules that are
well known mechanosensors [119]. In mammary epithelial and breast
cancer cells, SRF interacts directly with Yap1 [120], suggesting that
mechanical stimulation of the Yap1-SRF axis is important for overload-
induced hypertrophy in muscles. Collectively, growth factors important
in MuSC proliferation might be limited in overloaded muscle, and IL-6
family-mediated Stat3 activation might be a common mechanism for
MuSC proliferation in both regenerating and overloaded muscle.
However, the cellular source and expression mechanisms of cytokines
common to MuSC proliferation in overloaded muscle are likely to differ
from those of regenerating muscle.
Another potential factor for MuSC proliferation is Silent mating type
information regulation 2 homolog 1 (Sirt1)-dependent factors in myo-
fibers. Sirt1 is a sensitive modulator of metabolic processes, and reg-
ulates fat and glucose metabolism [121]. An increase in the expression
level of Sirt1 in mRNA and proteins was observed 2 weeks after SA
surgery in a rat model [122]. In sedentary mice, the overexpression of
Sirt1 in adult myofibers accompanied an increase in myonuclear
number while the MuSC pool size was sustained [123], suggesting that
MuSCs are self-renewed and supplied myonuclei by the increased ex-
pression of Sirt1. These results also suggest that myofiber-derived fac-
tors promote proliferation of MuSCs regardless of degenerative damage
to myofibers.
4.3. Fusion
In the regeneration processes, mononuclear cells first fuse with each
other, and then with the multinuclear myotubes. The fusion proteins,
myomaker and myomixer/myomerger/minion, are involved in the fu-
sion processes. For myoblast-myoblast fusion, myomaker is required
bilaterally (i.e., on both fusing cells) and myomixer/myomerger/
minion is required unilaterally (i.e., on only one cell) [124–129]. In
overloaded muscle, mononuclear cell-myofiber fusion occurs and
MuSC-expressed myomaker is critical for this type of fusion [80],
however it is not clear whether myomaker is also required on the
myofiber membrane [128]. Different fusion mechanisms might exist
between myoblast-myoblast fusion and MuSC-myofiber fusion.
SRF, in the MuSCs, has been identified as a master regulator of
MuSC fusion (in both fusion partners) and is required for the efficient
fusion of MuSCs to growing myofibers (in overloaded muscle) and with
each other in order to form newly regenerated myofibers [83]. Inter-
leukin 4 (IL-4) is reported to promote the fusion of myoblasts to nascent
myotubes [130]. Guerci et al. (2012) evidenced that IL-4, derived from
myofibers, is critical for the fusion of MuSCs to myofibers in overloaded
muscle [115]. Collectively, these results imply that the critical cell fu-
sion mechanisms of regenerating and overloaded muscles are relatively
similar.
It is important to note, in a murine model of injury, the myonuclei in
regenerated myofibers were centrally located, but new MuSC-derived
myonuclei in overloaded muscle are observed in peripheral locations
[19,78,80]. It would be interesting to investigate the functional dif-
ference between central and peripheral nuclei derived from MuSCs.
7
BBA - Molecular Cell Research 1867 (2020) 118742
4.4. Macrophages and infiltration
During the regeneration processes, masses of inflammatory cells
invade degenerated myofibers [131]. Within 12 h of muscle injury,
neutrophils infiltrate, and then major immune cells are switched to
macrophages [131]. The importance of inflammatory cells, especially
macrophages, is well established [132–134]. The depletion of macro-
phages results in delayed or impaired regeneration [133,134]. One
important function of macrophages is the removal of degenerated
myofibers and debris. Like remodeling in other tissues, the contribu-
tions of different macrophage subtypes are spatially and temporally
coordinated. In the early stages, pro-inflammatory (M1) macrophages,
which secrete pro-inflammatory cytokines, play an essential role by
removing the debris. M2 macrophages, which exhibit an anti-in-
flammatory phenotype, support the myogenic differentiation processes,
and macrophage skewing from the M1 toward the M2 phenotype is
important during muscle regeneration [135]. Supposing that hyper-
trophy does not accompany the death of myofibers, macrophages re-
sponsible for debris removal will not be needed. This means that,
compared to regenerating muscle, a limited subtype of macrophages
might contribute to muscle hypertrophy. Little is known regarding the
contribution of macrophages to hypertrophy in overloaded muscle,
whereas the contribution of macrophages to the recovery from muscle
atrophy and the correlation between the number of macrophages and
increased myofiber size in human-endurance exercise training, but not
resistance training, has been reported [136,137].
In a murine study, treatment with the specific COX-2 inhibitor, NS-
398, blunted the increase in mass and protein content without affecting
the Akt pathway in overloaded muscles compared to that of control
mice [138], suggesting that the inflammatory response is also necessary
for muscle hypertrophy. However, this treatment might also affect
MuSC fusion processes because COX-2 regulates IL-4 expression in
overloaded myofibers [115]. Further study of macrophage depletion
will reveal the importance of macrophages in MuSC behaviors in
muscle hypertrophy.
5. What remains to be discovered and possible applications in the
treatment of muscular disorders
Muscular disorders can simply be classified into two categories:
diseases with degenerative damage to myofibers, such as muscular
dystrophy, and diseases without such degenerative damage, such as
muscle atrophy. Muscular dystrophy patients exhibit the progressive
loss of myofibers due to reduced regenerative ability, which is one
potential factor for explaining the differences in pathological outcomes
observed even when using animal models with the same kind of mu-
tation. For example, there is a dramatic difference among mouse strains
[139] as C57BL/6 mice exhibit excellent muscle regenerative ability,
whereas DBA/2 mice demonstrated a progressive loss of muscle weight
in a model of Duchenne Muscular Dystrophy (DMD) [140–143]. These
results suggest that the augmentation of muscle regenerative ability
could provide a treatment option for muscular dystrophies. In this case,
the investigation of muscle regeneration processes, especially the
identification of potent growth-promoting factors for MuSCs, could lead
to the development of ‘regenerative medicine’ for treating muscular
diseases accompanied by degenerative damage to myofibers.
Considering the treatment of muscle atrophy, promoting muscle
regeneration is not an adequate strategy because cycles of myofiber
degeneration and regeneration are not observed. Although the loss of
MuSCs did not affect myonuclear numbers during aging, changes in
myonuclear shape and decreased numbers of myonuclei in the extensor
digitorum longus (EDL) or tibialis anterior (TA) muscle, but not in the
plantaris muscle, in response to aging have been reported [144–146].
These results imply that MuSC myonuclear turnover does not occur
frequently in sedentary mice. The question remains whether myo-
nuclear number reduction is responsible for age-associated muscle
S.-i. Fukada, et al.
atrophy, especially in humans [147].
When the quality and function is reduced in old myonuclei or
atrophied myofibers, the supplementation of new MuSC-derived myo-
nuclei is a potential therapeutic strategy for muscle atrophy. Given that
MuSC behaviors and regulatory mechanisms in overloaded muscle
differ from those in regenerating muscle, we should learn from the
molecular mechanisms underlying MuSC activation/proliferation/fu-
sion in overloaded muscle in order to accomplish ‘myonuclear replen-
ishment therapy’ using MuSCs. Accordingly, Taylor-Weiner et al.
(2020) used an in vitro culture system and showed that myonuclear
proteins, including transcriptional factors, can propagate to different
myonuclei in the same myofiber [148]. Furthermore, they evidenced
that muscle hypertrophy increased the transport of high molecular
weight nuclear proteins, while atrophy restricted the transport of
smaller nuclear proteins. If propagation occurs similarly in vivo, studies
on the individual myonuclear level (myonucleus analyses) are required
to understand alterations in myonuclear functions and the effect of
‘diseased myonuclei’ on other myonuclei during disease progression. In
order to consider ‘myonuclear replenishment therapy’ using MuSCs, it is
also necessary to investigate whether MuSC-derived myonuclei affect
‘diseased myonuclei’ or vice versa.
Using genetically MuSC-depleted sedentary mice, it was demon-
strated that the role of MuSCs in the onset of sarcopenia is limited
[145,146]. This suggests that the loss of MuSCs might not be re-
sponsible for the onset and progression of sarcopenia. However, Eng-
lund et al. (2020) indicated that lifelong physical activity induces the
loss of MuSCs which blunts myofiber hypertrophy [149]. Furthermore,
the inhibitory roles of MuSCs and MuSC-derived myonuclei in the de-
generation of the neuromuscular junction during aging have also been
reported [150]. The extent to which decreased MuSC pool size and
function are responsible for the onset and progression of human sar-
copenia is still unknown. However, if we can control MuSC behaviors
and renew myonuclei in atrophic muscles, regardless of the contribu-
tion of MuSCs to human sarcopenia, MuSCs might be a great potential
target for the treatment of muscular atrophy, including sarcopenia, as
well as muscular dystrophy.
6. Future directions
Notably, MuSCs are maintained in randomly contracting or relaxing
myofibers, meaning that MuSCs might always perceive these move-
ments evoked by myofibers. Although MuSC quiescence should be
sustained in daily life, both overload and strength exercise induce MuSC
proliferation, meaning that MuSCs have a physiological contraction
threshold in order to avoid accidental activation/proliferation. The
molecules and pathways that regulate the impact of daily myofiber
contractions, and which molecules disrupt the quiescent state are still
unknown. Such investigations will help us to identify the mechanisms
underlying MuSC activation/proliferation in overloaded muscle, which
may lead to MuSC-based therapy for atrophied or aging muscle.
The theory of muscle memory is also a fascinating issue in MuSC
biology. There are signs that previously trained muscles reacquire some
parameters at an accelerated rate if they are resubmitted to exercise,
even after prolonged detraining [151–153]. Bruusgaard et al. (2010)
indicated that SA surgery lead to an increase in the myonuclear number
and was subsequently sustained in denervation-induced atrophied
myofibers, which supports the muscle memory requirement as myo-
nuclei are able to sustain certain parameters [154]. However, Dungan
et al. (2019) indicated the number of myonuclei gained during training
(a combination of load with wheel running) was lost after detraining
[60]. Thus, the muscle memory requirement regarding sustained
amount of myonuclei is highly debated [155]. The physiological and
memory roles of myonuclei in daily life should be elucidated in the near
future as this may provide us with a new sense of appreciation re-
garding the value of MuSCs in the development of new treatments for
muscular disease.
8
BBA - Molecular Cell Research 1867 (2020) 118742
7. Conclusion
The importance of MuSCs in repairing degenerated myofibers is
unquestionable, but whether they are required for muscle hypertrophy
is debatable. In our opinion, the supplementation of new MuSC-derived
myonuclei is critical for efficient muscle hypertrophy, and MuSC acti-
vation/proliferation is independent of mTOR/Akt signaling in myofi-
bers [88,115]. Mouse models (e.g., a beta-adrenergic agonist, Akt-tg
mice, JunB, and myostatin-null) have evidenced muscle hypertrophy
without MuSC accretion which indicates that muscle hypertrophy is not
always accompanied by increased myonuclear number. Therefore,
MuSC activation/proliferation does not occur as a consequence of an
increase in myofiber size, whereas the abovementioned results indicate
that active signaling pathways exist and induce MuSC activation/pro-
liferation in overloaded uninjured muscle. Perhaps, given that MuSCs
demonstrate different behaviors and kinetics in overloaded and re-
generating muscle, overloaded muscle may require overload-specific
molecules and events.
Finally, our conclusion is that degenerative damage to myofibers is
not necessary for both MuSC behaviors and hypertrophy in overloaded
muscle.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We thank Katherine Ono for proofreading the paper. This study is
supported by JSPS KAKENHI grant, Grant-in-Aid for Scientific Research
(B) (19H04000 to S. F.) and Intramural Research Grant for Neurological
and Psychiatric Disorders of NCNP (28-6 to S. F.).
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