A Role for Nitric Oxide in Muscle Repair: Nitric Oxide-mediated Activation of Muscle Satellite Cells

Abstract

Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of external lamina. Although their activation and recruitment to cycle enable muscle repair and adaptation, the activation signal is not known. Evidence is presented that nitric oxide (NO) mediates satellite cell activation, including morphological hypertrophy and decreased adhesion in the fiber-lamina complex. Activation in vivo occurred within 1 min after injury. Cell isolation and histology showed that pharmacological inhibition of nitric oxide synthase (NOS) activity prevented the immediate injury-induced myogenic cell release and delayed the hypertrophy of satellite cells in that muscle. Transient activation of satellite cells in contralateral muscles 10 min later suggested that a circulating factor may interact with NO-mediated signaling. Interestingly, satellite cell activation in muscles of mdx dystrophic mice and NOS-I knockout mice quantitatively resembled NOS-inhibited release of normal cells, in agreement with reports of displaced and reduced NOS expression in dystrophin-deficient mdx muscle and the complete loss of NOS-I expression in knockout mice. Brief NOS inhibition in normal and mdx mice during injury produced subtle alterations in subsequent repair, including apoptosis in myotube nuclei and myotube formation inside laminar sheaths. Longer NOS inhibition delayed and restricted the extent of repair and resulted in fiber branching. A model proposes the hypothesis that NO release mediates satellite cell activation, possibly via shear-induced rapid increases in NOS activity that produce "NO transients."

Full text

Molecular Biology of the Cell
Vol. 11, 1859–1874, May 2000
A Role for Nitric Oxide in Muscle Repair: Nitric
Oxide–mediated Activation of Muscle Satellite Cells
Judy E. Anderson*
Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba,
Canada R3E 0W3
Submitted September 3, 1999; Revised February 11, 2000; Accepted February 24, 2000
Monitoring Editor: Thomas D. Pollard
Muscle satellite cells are quiescent precursors interposed between myofibers and a sheath of
external lamina. Although their activation and recruitment to cycle enable muscle repair and
adaptation, the activation signal is not known. Evidence is presented that nitric oxide (NO)
mediates satellite cell activation, including morphological hypertrophy and decreased adhesion in
the fiber-lamina complex. Activation in vivo occurred within 1 min after injury. Cell isolation and
histology showed that pharmacological inhibition of nitric oxide synthase (NOS) activity pre-
vented the immediate injury-induced myogenic cell release and delayed the hypertrophy of
satellite cells in that muscle. Transient activation of satellite cells in contralateral muscles 10 min
later suggested that a circulating factor may interact with NO-mediated signaling. Interestingly,
satellite cell activation in muscles of mdx dystrophic mice and NOS-I knockout mice quantitatively
resembled NOS-inhibited release of normal cells, in agreement with reports of displaced and
reduced NOS expression in dystrophin-deficient mdx muscle and the complete loss of NOS-I
expression in knockout mice. Brief NOS inhibition in normal and mdx mice during injury
produced subtle alterations in subsequent repair, including apoptosis in myotube nuclei and
myotube formation inside laminar sheaths. Longer NOS inhibition delayed and restricted the
extent of repair and resulted in fiber branching. A model proposes the hypothesis that NO release
mediates satellite cell activation, possibly via shear-induced rapid increases in NOS activity that
produce “NO transients.”
INTRODUCTION
After muscle injury, satellite cells are activated and recruited
to cycle as precursors for new muscle formation. Between
injury and proliferation in vivo, satellite cells express imme-
diate early genes after 3–6 h (Weiss, 1994; Kami et al., 1995)
and muscle regulatory genes after 6 h (Grounds et al., 1992)
in concert with proliferating cell nuclear antigen (Chambers
and McDermott, 1996). The expression of these genes, re-
lease of growth factors such as bFGF, and DNA synthesis
24–30 h later are used to characterize muscle regeneration in
injured and dystrophic muscle (Grounds and McGeachie,
1989; Anderson et al., 1995; Floss et al., 1997, 1998). The
timing and sequence of events are specific to those of repair
(Megeney et al., 1996; Li et al., 1997; McIntosh et al., 1998) but
similar to those of development (Rudnicki and Jaenisch,
1995; Yun and Wold, 1996).
The fine structure of satellite cells, positioned intimately
between the fiber sarcolemma and the external lamina
(Mauro, 1961; Ishikawa, 1966), changes during their transi-
tion from quiescence to activation. Nuclei enlarge and be-
come euchromatic. The typical attenuated organelle-poor
cytoplasm expands, and organelles such as mitochondria
and rough endoplasmic reticulum hypertrophy (Schultz,
1976; Snow, 1977; Schultz et al., 1978, 1985). However, al-
though activation is recognized as essential to repair and
defined as precursor stimulation and recruitment to cycle
(Bischoff, 1990a), the initial signal, timing, and character of
activation are not known (Schultz and McCormick, 1994).
To date, the earliest indicator of satellite cell transforma-
tion during activation is the colocalization of hepatocyte
growth factor (also called scatter factor; HGF/SF) with its
receptor c-met shortly after injury in normal rat muscle
(Tatsumi et al., 1998). In normal and regenerating muscle,
satellite cells express c-met (Cornelison and Wold, 1997;
Tatsumi et al., 1998) and m-cadherin (Moore and Walsh,
1993; Irintchev et al., 1994; Rose et al., 1994). Although
HGF/SF also plays a role in differentiation (Gal-Levi et al.,
1998), it is the activating agent in extracts from crushed
muscle (Tatsumi et al., 1998). Thus, the shift of HGF/SF from
the periphery of the intact fiber to satellite cells means that
activation follows soon after muscle damage.
Other observations indicate that the activation signal is
transmitted along fibers from the site of direct injury. After
* Corresponding author. E-mail address: janders@ms.umanitoba.ca.
© 2000 by The American Society for Cell Biology 1859

segmental damage, satellite cells proliferate and fuse to form
new myotubes both adjacent to the injury (Grounds and
McGeachie, 1987) and also at some distance from the injury
near the ends of fibers (Klein-Ogus and Harris, 1983; Schultz
et al., 1985; Bischoff, 1990b; Grounds et al., 1992; McIntosh et
al., 1994; McIntosh and Anderson, 1995). Satellite cells are
also activated without trauma and make DNA after exercise,
training, stretching, cold, compression, hypertrophy, sus-
pension, and denervation (Bischoff, 1986a,b; Darr and
Schultz, 1987; Appell et al., 1988, 1989; White and Esser, 1989,
1990b; Snow, 1990; Winchester et al., 1991; Buonanno et al.,
1992; Alway, 1997). Therefore, multiple signals initiate or
mediate activation. Nonetheless, it is clear that DNA syn-
thesis some 24–30 h after injury is a delayed index of pre-
vious and completed satellite cell activation.
A novel insight linking biophysical shear, muscle struc-
ture, fiber hypercontraction in injury, and the rapid shift of
HGF/SF to its receptor suggested the idea that nitric oxide
(NO) release from fibers may mediate satellite cell activa-
tion. NO is a very small, freely diffusible, and ubiquitous
molecule produced constitutively at high levels in muscle by
neuronal nitric oxide synthase (NOS-I
␮
) (Nakane et al., 1993;
Kobzik et al., 1994; Silvagno et al., 1996). NOS-I
␮
is com-
plexed at its N terminus to
␣
1-syntrophin, which, in turn, is
linked to the dystrophin cytoskeleton, especially in fast-
twitch fibers; in dystrophic muscles without dystrophin,
NOS-I
␮
is reduced and displaced to the cytoplasm (Bren-
man et al., 1995, 1996; Chang et al., 1996; Chao et al., 1996;
Grozdanovic et al., 1996; Wakayama et al., 1997; Hemler,
1999). NO is also made constitutively by NOS-III and by
inducible NOS-II activity and transduces signals in vascular
endothelium and smooth muscle, brain, and liver. NO is the
subject of exciting new ideas of pathophysiology (Kanner et
al., 1991; Lowenstein and Snyder, 1992; Palmer, 1993; Lo-
wenstein et al., 1994; Schmidt and Walter, 1994; Garthwaite
and Boulton, 1995; Wang et al., 1995; Kro¨ncke et al., 1997;
Gossrau, 1998; Reid, 1998). Critical controls on NO action are
imposed by the biophysical properties of a tissue. Impor-
tantly, NO release is also regulated by mechanical forces
such as shear, which is produced by pressure in a structure
when its layers shift laterally across one another (Rubanyi et
al., 1986; Nathan and Xie, 1994; Busse and Fleming, 1998;
Chien et al., 1998). Gradients and contours of NO concentra-
tion signal to nearby cells (Lancaster, 1994, 1997), whereas
hemoglobin heme acts as a huge sink to neutralize NO
(Beckman and Koppenol, 1996).
Because satellite cells are intimately contoured to fibers
and often stay attached to the external lamina as the sarco-
lemma buckles after injury (Schultz and McCormick, 1994),
they are ideally positioned to be “first responders” to a
shear-induced release of NO from the subjacent NOS-I
␮
.In
the present experiments, the release of myogenic cells from
single crush-injured muscles was used as an index of the
collective processes in muscle. It was reasoned that activa-
tion would increase the harvest of myogenic cells from a
single muscle by reducing their adhesion to fibers and lam-
ina and would also affect subsequent muscle repair. Exper-
iments were carried out in normal mice pretreated to inhibit
or augment NOS activity, and one tibialis anterior (TA)
muscle was subjected to crush injury 30 min later. Cell yields
from injured and undamaged muscles were determined for
30 min immediately after injury, and longer-term repair was
also examined. Muscle in mdx mice lacks subsarcolemmal
NOS-I
␮
and shows rapid repair and precursor cycling
(McIntosh et al., 1994; McIntosh and Anderson, 1995; Per-
nitsky and Anderson, 1996), whereas NOS-I knockout mice
have complete loss of NOS-I expression (Huang et al., 1993).
Therefore, the effects of low or absent NOS expression (sim-
ilar in outcome to NOS inhibition) on cell yield in mdx and
NOS-I knockout mice, and on mdx satellite cells and muscle
repair, were examined. The rapid activation of satellite cells
by injury, shown by increased myogenic cell release and
morphological changes, was delayed by NOS inhibition in-
duced pharmacologically by N
␻
-nitro-l-arginine methyl es-
ter and by primary and secondary defects in NOS-I gene
expression. Activation was transiently observed with a
slower time course in intact contralateral muscles, and NOS
inhibition negatively affected muscle regeneration.
MATERIALS AND METHODS
In all experiments, 6- to 8-wk-old male normal mice (C57BL/6 and
B6129SF; Jackson Laboratories, Bar Harbor, ME), mdx mutant mice
(C57BL/10 ScSn; Central Animal Care Services, University of Mani-
toba), and NOS-I knockout mice (B6129S-Nos1
tm1Plh
; Jackson Labo-
ratories) were treated double blind and in accord with the guide-
lines of the Canadian Council on Animal Care (reference No. R-99-
003). Studies were designed to determine the effects of manipulating
NOS activity on the number and myogenic nature of cells isolated
from muscles with and without injury and to examine the longer-
term effects of NOS inhibition.
NOS activity was influenced by an intraperitoneal injection (80–
100
␮
l by Hamilton syringe) exactly 30 min before crush injury to
the right TA muscle of mice rested for at least 1 week after transport
from the breeding facility. Mice were injected with saline or saline
containing one of three drug treatments as follows: the NOS inhib-
itor N
␻
-nitro-l-arginine methyl ester (l-NAME; 7.5, 10, or 15 mg/
kg), the NO donor l-arginine (l-Arg; 225 mg/kg), or combined
l-NAME (7.5 mg/kg) plus l-Arg. Fifteen minutes later, animals
were anesthetized (intraperitoneal ketamine:xylazine). The crush
injury was delivered to the right TA muscle (RTA) with the use of
a hemostat clamp closed for 3 s (McIntosh et al., 1994). Skin was held
closed or sutured for longer recovery (see below). The time-course
study from 0 to 30 min after injury was completed in 1 d for each
treatment group, treatments were coded, and each set of experi-
ments was carried out by the same individual(s).
The time course of treatment effects was determined at two in-
tervals: during the early response 0, 5, 10, and 30 min after injury,
and over the longer term after6dofrecovery. Short-term experi-
ments were repeated at least twice. The animals used in longer-term
experiments were maintained on either plain drinking water or
water containing fresh l-NAME at 12.5 mg/100 ml (30
mg䡠kg
⫺1
䡠d
⫺1
), based on an intake of 6–7 ml/d per mouse (McIntosh
et al., 1994). In the longer-term-repair studies, there were four nor-
mal mice in each saline- and l-NAME–treated group. An additional
two normal and two mdx mice were injected once before injury with
l-NAME and given plain water for 6 d.
Tissues were harvested rapidly within 1–2 min after cervical
dislocation under anesthesia. Whole muscles were carefully dis-
sected from animals in the following order: RTA, left TA (LTA), left
extensor digitorum longus (LEDL), left soleus (LSOL); and right
soleus (RSOL); TAs and RSOL were then weighed. Muscles were
used to determine cell yield or embedded for cryosectioning (7
␮
m
thick) to examine morphology.
Cell yield was determined immediately after tissue collection.
Satellite cells from RTA, LTA (representative fast-twitch muscles),
and RSOL (a representative slow-twitch muscle) were isolated by
standard procedures (Allen et al., 1998) modified for brevity and to
collect only the cells available for harvest after a short digestion.
J.E. Anderson
Molecular Biology of the Cell1860

Briefly, connective tissue was removed, and muscles were minced to
a slurry in PBS and digested for 1 h (37°C) in 1 ml of 0.125% protease
XIV (Sigma, St. Louis, MO) with inversion every 15 min. Samples
were triturated for 1 min, and enzyme action was stopped by
adding 10 ml of growth medium (DMEM containing 15% FBS, 1%
antimycotic, 0.5% gentamicin, and 2% chick embryo extract; Life
Technologies/BRL, Grand Island, NY). Cells were pelleted by cen-
trifugation (1500 ⫻ g for 4 min), and the supernatant was discarded.
Cells were resuspended in 15 ml of warm PBS, filtered through
Nitex gauze, and centrifuged (1500 ⫻ g for 4 min). The pellet was
resuspended in 500
␮
l of sterile PBS. A 100-
␮
l aliquot of cell sus-
pension was diluted in 10 ml of isotone for Coulter counting. The
number of cells isolated per muscle (cell yield) was calculated and
plotted over time. In three preliminary experiments, cells were
counted with the use of a hemocytometer to ensure that they were
nucleated cells and not isolated myonuclei or red blood cells.
To characterize the cell yield from each muscle, remaining cells
were plated on 35-mm Petri dishes precoated with polylysine and
fibronectin and cultured in growth medium for 1–5 d under 95%:5%
CO
2
:O
2
at 37°C. Some cultures were incubated for the final 30 min
with bromodeoxyuridine (BrdU; 1 mg in 2 ml of medium) to label
DNA synthesis. After washing in PBS, cells were fixed (10 min) in
1% paraformaldehyde in PBS and blocked (10% horse serum plus
1% BSA in PBS) before routine immunostaining (Tatsumi et al., 1998)
with the use of antibodies against BrdU (diluted 1:1000; Sigma) or
c-met receptor protein (diluted 1:400; Santa Cruz Laboratories,
Santa Cruz, CA). Negative control slides were incubated in blocking
solution without primary antibody. Appropriate peroxidase-conju-
gated secondary antibodies (diluted 1:250–1:400) and 3,3⬘-diamino-
benzidine tetrahydrochloride/nickel chloride visualization (Dimen-
sion Laboratories, Mississauga, Ontario, Canada) were used to
determine the relative myogenicity (c-met
⫹
staining) and level of
proliferation (BrdU
⫹
staining).
In the same experiments (n ⫽ 8 animals, repeated twice), the
LSOL and LEDL were embedded for cryosectioning to monitor the
effects of treatment or remote injury on tissue histology, as visual-
ized by fresh hematoxylin and eosin (H&E) staining and immuno-
staining for c-met and m-cadherin (see below).
In the longer-term study of NOS inhibition during repair, saline-
and l-NAME–treated normal mice recovered for 6 d. Two mice per
group were injected 2 h before being killed with BrdU (1.6 mg
intraperitoneal in 0.4 ml of saline), and sections were immuno-
stained with the use of anti-BrdU antibodies as described above.
A separate experiment was conducted to study the immediate
effects of injury on muscle and satellite cell histology. LTA and RTA
were collected immediately at 0 and 10 min after crush from normal
mice after saline or l-NAME pretreatment (total n ⫽ 4). Muscles
were bisected longitudinally, and half of each muscle was frozen in
Tissue Tek optimal cutting temperature (Miles Scientific, Elkhart,
IN) for cryosectioning. Sections were stained with H&E or immu-
nostained with the use of primary antibodies to c-met (1:400),
HGF/SF (1:1000; R&D Systems, Minneapolis, MN), or developmen-
tal myosin heavy chain (devMHC) (1:250; Novocastra Laboratories,
Newcastle-upon-Tyne, United Kingdom) as reported (Pernitsky et
al., 1996; Tatsumi et al., 1998) or against m-cadherin (1:50; Santa Cruz
Laboratories). The other half of each muscle was fixed in 2.5%
glutaraldehyde in 0.1 M cacodylate buffer, pH 7.35, postfixed in
osmium tetroxide, and embedded in methacrylate resin. Sections
(0.5
␮
m thick) were collected on glass slides and stained with
toluidine blue. The inhibition of NOS enzyme activity by l-NAME
treatment 30 min before crush was confirmed with the use of
NADPH-diaphorase enzyme histochemistry with jejunal epithelium
as the positive control, according to Beesley (1995).
Sections and cultures were viewed on an Olympus (Tokyo, Japan)
microscope equipped with epifluorescence and phase-contrast op-
tics. Observations were based on systematic viewing of two to four
longitudinal sections per muscle (separated by ⬎100
␮
m). In the
case of muscle regenerating from crush injury, observations (with-
out knowledge of treatment group) were made in preset fields of
muscle from the central crush region, the adjacent regenerating
region, and the surviving region, as reported (McIntosh et al., 1994).
Representative photographs of muscle fibers and satellite cells were
taken on ⬎700 frames of ASA 400 Fuji (Tokyo, Japan) Sensia slide
film. Where stated, the number of satellite cells observed in each
category, group, or condition was estimated from photographed
slides rather than from direct counts made during observations
under oil immersion. Selected slides were scanned (Olympus ES-10
film scanner), formatted into plates with little or no enlargement,
and printed (Freehand 8.0, Macromedia, San Francisco, CA).
RESULTS
Effects of NOS Manipulation in Normal Muscle
The myogenic nature of cells isolated from muscles in the 0-
to 30-min time course was confirmed by counting the pro-
portion of c-met
⫹
cells 12–24 h after plating. Myogenic cells
formed the large majority of cells isolated from the normal
LTA (83–94%) and RTA (86–92%) muscles (n ⫽ 997 cells).
After 24 h in culture, cells were typically round or elongated,
and 10–25% had nuclei that were intensely positive for BrdU
incorporation. After 4–5 d in culture, dark c-met staining
was present in single cells and in small multinucleated myo-
tubes. Cultured cells from different treatments, recovery
times, and muscles were identical in appearance despite
differences in cell yield (see below).
Muscle weight as a proportion of body weight (Figure 1,
A–D) was used to monitor edema secondary to tissue dam-
age. The weight of muscles dissected from saline-treated
normal mice showed a 10–15% increase in RTA over LTA
that began immediately after injury. During l-NAME treat-
ment, RTA weight increased only at 10 min relative to LTA
weight, whereas l-Arg and combined l-NAME plus l-Arg
treatment produced little or no change in muscle weight
profile. Because the profile of RTA weight differed over time
and among the four normal treatment groups, cell yield was
expressed as cells per muscle, based on the assumption that
LTA and RTA in one normal animal have similar-sized
populations of myogenic precursors. Other observations
made during tissue collection suggested that RTA hemor-
rhage at the crush site in the l-NAME–treated animals ap-
peared later and at 30 min was subjectively more pro-
nounced than in the other three groups.
Cell yield in the time course 0–30 min after injury in
normal mice changed dramatically with treatment and dif-
fered between LTA and RTA (and RSOL) muscles (Figure 1,
E–H). After saline treatment, the LTA released 2.0 ⫻ 10
5
cells
at 0 min (herein referred to as basal LTA level). In marked
contrast, the crushed RTA from the same mouse yielded
twofold more cells at 0 min. The RTA cell yield decreased
briefly from 5–10 min and then increased again. Surpris-
ingly, at 10 min the LTA yield doubled over the basal yield
(LTA at 0 min) and then declined below the basal yield by 30
min. The yield from RSOL (an uninjured slow-twitch muscle
ipsilateral to RTA and included for comparison with fast-
twitch TA) was lower than the yield from LTA on a per
muscle basis (although it was twofold to sevenfold higher
when expressed as cells per milligram) and did not change
during the 30-min time course. The data compiled from
three repeat experiments on normal mice treated with saline
(including C57BL/6 and B6129SF mice) are presented as the
ratio of cell yield in RTA/LTA (mean ⫾ SEM) in Figure 2
NO and Satellite Cell Activation
Vol. 11, May 2000 1861

and demonstrate the consistent large immediate increase in
cell yield at 0 min.
l-NAME treatment (7.5 mg/kg) substantially changed the
time course of cell yield, preventing the initial injury-in-
duced increase in RTA yield (Figure 1F) and delaying the
increased cell yield until 10 min after injury. The yields from
LTA and RSOL were lower at 0 min than in the saline-
treated mice (15 and 50%, respectively). Notably, 30% fewer
cells were isolated at 0 min from RTA than LTA. By 10 min,
yields from RTA and LTA were higher (3.5 and 2-fold,
respectively) than at 0 min. By 30 min, cell yield from both
RTA and LTA had decreased once again. This time course
was consistent, as shown by a plot of relative cell yield
(RTA/LTA ratio, mean ⫾ SEM) from three experiments on
normal muscle (Figure 2), demonstrating the prevention of
immediate cell release from RTA relative to LTA at 0 min.
l-NAME treatment at flanking doses indicated that the de-
layed peak RTA yield could be shorter (3 mg/kg) or longer
(10 or 12.5 mg/kg) than 10 min after injury (one experiment
at each dose) without the high RTA yield at 0 min. Interest-
ingly, although peak cell yield in RTA was delayed (not
reduced) by l-NAME, the peak cell yield in LTA (at 10 min)
was reduced but not delayed after l-NAME treatment.
In the time course of cells isolated from mice treated with
l-Arg, the basal yield in LTA was similar to that after saline,
Figure 1. Representative graphs from one experiment each on normal control (C57BL/6) (A–H) and mdx mice (I–L). Panels show the time
course of changes in muscle weight to body weight (mg/g) (A–D, I, and J) and cell yield (cells/muscle ⫻ 10
5
) (E–H, K, and L) in three muscles
(RTA [⽧], LTA [f], and RSOL [Œ]) for groups of mice treated 30 min before injury with saline (A, E, I, and K), l-NAME (B, F, J, and L), l-Arg
(C and G), or l-NAME plus l-Arg (D and H). Data from the same animals are represented for muscle weight and cell yield. In normal mice,
the immediate increase in RTA yield in saline-treated animals was absent after l-NAME treatment, and the transient increase in LTA yield
at 10 min was reduced by l-NAME treatment. In mdx mice, there was no immediate increase in RTA yield above the LTA basal level after
injury, whereas at 10 min RTA yield was increased. l-NAME treatment in mdx mice prevented the increased RTA yield at 10 min.
Figure 2. Time course of cell yield (cells/muscle) expressed as the
ratio of RTA to LTA (mean ⫾ SEM) for normal mice (C57BL/6 and
B6129SF, three experiments, ⽧), normal mice treated with l-NAME
(C57BL/6, three experiments, Œ) and “NOS mutant” mice including
mdx and B6129S-Nos1
tm1Plh
(NOS-I knockout mice, three experi-
ments, f). Satellite cell activation (cell yield ratio of RTA to LTA) in
normal mice begins at 0 min and is significantly greater than in mice
with NOS inhibition as a result of pharmacological treatment (by
l-NAME), a primary gene defect (NOS-I knockout mice), or second-
ary to dystrophin deficiency (mdx mice).
J.E. Anderson
Molecular Biology of the Cell1862

then increased and stayed high until 30 min. The RTA yield
increased sharply at 5 min and also stayed high. RSOL
counts were unchanged.
Combined treatment with l-NAME and l-Arg increased
the yield in LTA and RTA by 50% at 0 min compared with
saline-treated mice. Although LTA yield gradually de-
creased over 30 min, RTA yield at 5 min was the highest
yield observed in a normal TA (6.0 ⫻ 10
5
cells/muscle) and
decreased again by 10 min. The RSOL yield after combined
treatment showed the only changes of any group of normal
RSOL muscles. A sharp 80% increase between 0 and 5 min
after RTA injury was followed by a decrease to the level at
0 min.
Effects of NOS Inhibition in mdx Dystrophic Muscle
versus NOS-I Knockout Muscle
The myogenic proportions of cells isolated from mdx mus-
cles were very high in LTA and RTA (95 and 96% respec-
tively, 295 cells counted) and likely included both satellite
cells from fibers and myoblasts from the interstitium of
dystrophic muscles. Muscle weight as a proportion of body
weight had a different profile in mdx and normal mice (Fig-
ure 1I). RTA weight increased later (after 5 min) and was
maintained for 30 min in saline-treated mdx mice, whereas
l-NAME abolished the increase in RTA weight for 30 min
(Figure 1J). During tissue collection, mdx RTA muscles were
subjectively less hemorrhagic after l-NAME than after saline
treatment.
The time course of cell yield from saline-treated mdx mice
(Figure 1K) showed five major distinctions from that in
saline-treated normal mice and more closely resembled the
profile of the normal muscle yield after NOS inhibition.
First, the basal level of LTA yield was ⬃30% more in mdx
mice than in normal mice. In mdx mice, RTA yield did not
show an immediate increase at time 0. Instead, counts for
LTA and RTA were similar. Over 10 min, the RTA yield
doubled and then leveled off somewhat. The cell yield in
LTA did not change over time, whereas RSOL yields de-
creased by half from 0 to 10 min.
The cell yields in LTA and RTA from l-NAME–treated
mdx mice at 0 min were similar to the yields from saline-
treated mdx mice and again higher than yields from normal
mice (Figure 1L). LTA yield increased by 50% at 10 min and
returned to basal yield by 30 min (as in normal mice after
l-NAME). RTA yield increased slowly during the 30-min
time course. After l-NAME, the cell yield from mdx RSOL
did not change over time after l-NAME. Thus, NOS inhibi-
tion in mdx mice increased cell yield in uninjured LTA. NOS
inhibition also decreased and further delayed the peak of
myogenic cells isolated from the injured mdx RTA muscle
compared with saline treatment in mdx mice.
NOS-I knockout mice showed a time course of cell yield
from LTA, RTA, and RSOL that was very similar to that in
mdx mice (summarized in Figure 2; three experiments,
pooled data from mdx and NOS-I knockout mice). The time
course of cell yield in mdx and NOS knockout mice, ex-
pressed as the ratio of RTA/LTA yields, showed no differ-
ence from the profile of cell yield in l-NAME–treated nor-
mal muscle. The immediate increase in cell yield in RTA of
normal mice was absent in RTA muscle of both mdx and
NOS-I knockout mice.
Effects of NOS Inhibition on Early Muscle and
Satellite Cell Responses to Injury
All the RTAs collected immediately after injury showed a
crush site at 0 min that was very similar to uncrushed
muscle (Figure 3; n ⫽ 4). After only 10 min, however, overt
microscopic damage was present in the crushed region of all
RTA sections of both saline- and l-NAME–treated mice,
including transverse bands of fiber hypercontraction, delta
lesions, and empty or disrupted external lamina directly at
the crush site.
Histology and immunostaining showed that normal rapid
changes in satellite cell size and position were consistently
delayed and restricted after l-NAME treatment (Figure 4;
n ⫽ 10–12, except n ⫽ 2 per group for resin sections).
NADPH-diaphorase staining experiments on sections from
the same mice confirmed that pretreatment with l-NAME
inhibited NOS activity, detected as a thin outline located just
inside the sarcolemma of all muscle fibers from saline-in-
jected animals. The identity, position, and configuration of
⬃300 satellite cells observed on fibers were confirmed by
m-cadherin and c-met staining. M-cadherin was interposed
between fibers and all satellite cells observed in undamaged
muscle, and typically surrounded large satellite cells on
fibers in saline-treated RTA at 0 and 10 min (Figure 4A). At
10 min, large m-cadherin
⫹
cells were very often observed on
the empty external lamina sheaths present after fiber retrac-
tion (Figure 4B). Interestingly, satellite cells were easily vis-
ible on nearly every fiber by H&E staining at 0 min at the
RTA fiber periphery (Figure 4C) and were often prominent
in the RSOL, LEDL, LTA, and RTA at 10 min. They con-
tained large vesicular nuclei and many crimson cytoplasmic
granules, likely mitochondria (Figure 4D). After saline treat-
ment, c-met staining of LTA at time 0 consistently showed
typical attenuated satellite cells very close to fibers. How-
ever, RTA at 0 min showed many large satellite cells (⬃50 of
65 satellite cells, and a higher proportion in the crushed
regions) that were c-met
⫹
and HGF/SF
⫹
(Figure 4E) and
that had a higher ratio of cytoplasm to nucleus than satellite
cells in the contralateral LTA. Those enlarged satellite cells
often bulged from the fiber contour even at a distance from
the crush region in RTAs. The same features were more
pronounced at 10 min after crush in RTAs (Figure 4F),
although 15–20% of satellite cells were small and attenuated
and positive for m-cadherin or c-met in their location on
some undamaged fibers at the edge of the crushed region.
In contrast, in l-NAME–treated mice, the large majority of
satellite cells (85% of ⬎70 satellite cells identified with either
m-cadherin
⫹
or c-met
⫹
staining) were thin and attenuated
in both the RTA and LTA at 0 min (Figure 4I), were not
prominent by H&E staining (Figure 4J), or were c-met
⫹
but
did not stain for HGF/SF (Figure 4N). However, by 10 min
there were typically large m-cadherin
⫹
satellite cells on
many fibers in every section (Figure 4M), and c-met and
HGF/SF were colocalized in at least 70% of satellite cells
(25–30 were clearly observed per longitudinal section) bulg-
ing from fibers (Figure 4O) or at the external lamina. These
features of cell enlargement and c-met/HGF colocalization
were also less frequent after l-NAME treatment than saline
treatment in observations made at the surviving ends of the
RTA fibers not directly injured by the crush.
Resin sections showed details of more than 50 cells in the
satellite position on fibers, later confirmed by electron mi-
NO and Satellite Cell Activation
Vol. 11, May 2000 1863

croscopy as satellite cells and containing nuclei. Large sat-
ellite cells were present only at time 0 in the injured region
of RTAs from saline-treated mice (Figure 4G). They were
demarcated from the subjacent fibers, contained large vesic-
ular nuclei with prominent nucleoli, and had many dark
cytoplasmic granules identical to typical mitochondria be-
tween fibrils and at the fiber periphery (Figure 4G). By 10
min in RTA from a saline-treated mouse, the large satellite
cells were often present and were observed lifting from
fibers (Figure 4H). In comparison, nuclei and cells in the
satellite position of LTA and in the RTA from an l-NAME–
treated mouse at time 0 were typically thin and nearly
agranular and their nuclei could seldom be distinguished
from internal myonuclei because the cells were in tight
apposition to fibers (Figure 4K). At 10 min after injury, many
myonuclei inside fibers had a folded nuclear membrane on
the aspect adjacent to hypercontracted fibrils (Figure 4L) and
were easily distinguished from the smoothly contoured nu-
clei of enlarged satellite cells at the fiber periphery (Figure
4P).
Effects of NOS Inhibition on Longer-Term Repair in
Normal Muscle
After6dofrecovery from injury, normal mice treated with
saline injection and plain drinking water (n ⫽ 4) had RTAs
with small characteristic central necrotic crush sites flanked
by small myotubes (Figure 5, A–C). In systematic observa-
tions of adjacent and surviving regions (McIntosh et al.,
1994) of four different sections per muscle, adjacent regions
contained many mononuclear cells and capillaries between
the long myotubes. Surviving tissue at the ends of RTA
contained fibers interspersed or continuous with new myo-
tubes. Many mononuclear cells (more than half of 20–30
satellite cells clearly identified per section) stained for both
c-met and HGF/SF, whereas myotubes did not stain for
either protein.
In contrast, muscle regeneration was reduced by exposure
to l-NAME during the 6 d of repair (n ⫽ 4) (Figure 5, D–F).
Outside a large central crush site, persistent necrotic fiber
segments contained macrophages and some calcified fiber
segments that were infrequent in RTAs of saline-injected
mice. Many mononuclear cells surrounded the thin baso-
philic (immature) myotubes, which, in addition, were seen
at much lower density per field compared with myotubes in
similar RTA fields from saline-treated mice. New myotubes
were also infrequent among surviving fibers at the ends of
RTA after l-NAME treatment. The prevalence and size of
new myotubes were confirmed by devMHC-positive immu-
nostaining.
Interestingly, a single injection of l-NAME before injury
had also produced subtle effects on muscle regeneration
after6d(n⫽ 2) (Figure 6). RTAs had small remnant crush
lesions, mononuclear cells in the adjacent regions, and num-
bers and size of myotubes similar to normal regenerating
muscle, and many mononuclear cell nuclei were BrdU
⫹
(Figure 6G). However, in regions of surviving segments,
large cells in the satellite cell position (m-cadherin
⫹
) had
granular cytoplasm and were connected directly with long,
thin myotubes while still resident on fibers within the exter-
Figure 3. Representative effects of crush injury in
normal muscle at 0 min (A and B) and 10 min (C–E)
after injury and after saline (A, B, and E) or l-NAME
(C and D) pretreatment. (A) LTA section shows nor-
mal undamaged muscle. (B) RTA section at 0 min
after crush injury. (C) At low magnification, a dark
band of hypercontraction in fiber segments (to the
left) and extravasated blood cells between fibers ap-
pear across the muscle belly at 10 min in a saline-
treated animal. Fibers are thin and retracted to the
right of the hypercontracted region. (D) Two delta
lesions in a fiber after l-NAME treatment and 10 min
after crush. (E) Higher-magnification view of muscle
10 min after injury showing extravasated blood cells
between hypercontracted and retracted fiber seg-
ments and segments with early sarcomere disrup-
tion. Original magnification in A, B, D, and E ⫻132;
in C, ⫻33.
J.E. Anderson
Molecular Biology of the Cell1864

nal lamina. This feature was observed at least once in every
⫻40 field containing surviving fiber segments in the region
adjacent to the crush site. A small number (estimated at
5–10%) of myotubes appeared to be incompletely fused
blocks of eosinophilic (Figure 6F) or devMHC
⫹
cells, espe-
cially notable with phase-contrast optics. New, small
devMHC
⫹
myotubes were continuous with larger myotubes
formed since the injury (Figure 6H) or were located among
mononuclear cells adjacent to the injury site. Four m-cad-
herin
⫹
satellite cells were seen on new myotubes (Figure
6L). Satellite cells were always very intensely stained in the
spindle fiber complexes (Figure 6M), and in undamaged
EDL or SOL from the same mice, satellite cells were very
prominent and intensely eosinophilic on many fibers (Figure
6, J and K).
Effects of NOS Inhibition on Longer-Term Repair in
Dystrophic Muscle
A single l-NAME injection also produced subtle changes in
regenerating mdx muscles during6d(n⫽ 2) (Figure 7). In
regenerating muscles of l-NAME–treated mdx mice, many
large new myotubes extended from a small necrotic crush
site through the adjacent region and between the fiber seg-
ments that survived the injury (Figure 7A). More large myo-
tubes were present compared with normal regenerating
muscle, as reported previously for mdx mice (McIntosh et al.,
1994; McIntosh and Anderson, 1995), and many satellite
cells, elongated mononuclear cells, and new myotubes were
m-cadherin
⫹
(Figure 7C). In one field, a binucleate satellite
cell was lifted off the fiber sarcolemma (Figure 7D).
DevMHC was expressed by new myotubes (Figure 7E), and
Figure 4. Satellite cell changes in vivo are delayed by NOS inhibition in normal mice treated with saline (A–H) or l-NAME (I–P). (A)
M-cadherin outlines a large satellite cell at 0 min after injury. (B) Large m-cadherin
⫹
satellite cell on the external lamina 10 min after injury.
(C) H&E-stained satellite cells (arrows) in low-magnification RTA fibers at 0 min. (D) At high magnification, hypertrophic satellite cells on
fibers in RSOL at 10 min. (E and F) Large satellite cell shows colocalized (yellow) staining for HGF/SF (Texas Red) and c-met (FITC) at 0 min
(E) and 10 min (F). (G) Two resin sections (stained with toluidine blue) show large satellite cells (between arrowheads) at 0 min in RTA. (H)
At 10 min in RTA, satellite cells (arrows) with granulated cytoplasm and euchromatic nuclei are partially lifting off adjacent fibers. (I) After
l-NAME treatment, m-cadherin stains an attenuated satellite cell at 0 min in RTA. (J) Satellite cells are not prominent by H&E staining of RTA
at 0 min. (K) Thin strips of cytoplasm and a contoured nucleus are probable satellite cells (at arrowhead) at the fiber periphery in resin
sections. (L) At high magnification, a myonucleus in a resin section from RTA 10 min after injury shows a folded upper membrane near the
contracted fibrils. (M) Ten minutes after injury, large m-cadherin
⫹
satellite cells are adjacent to an unstained fiber. (N) At 0 min after injury,
c-met (FITC) in satellite cells is not colocalized with HGF/SF (red). (O) A large satellite cell at 10 min after injury shows colocalization (yellow)
of c-met (FITC) and HGF/SF (red) fluorescence. (P) A hypertrophic satellite cell (between arrows) is partly separated from an RTA fiber 10
min after injury. Original magnification, ⫻330 except in C and J (⫻132).
NO and Satellite Cell Activation
Vol. 11, May 2000 1865

Figure 5. l-NAME treatment for 6 d reduces normal muscle regeneration. (A) At low magnification (H&E), normal muscle repair after saline
pretreatment includes a small necrotic crushed region (to the right), a region of adjacent mononuclear cells and myotubes (arrows), and
surviving fiber segments (to the left). (B) New myotubes in the adjacent region contain many central nuclei and eosinophilic sarcoplasm after
6 d of regeneration. (C) New myotubes (arrows) are also present among surviving fibers. (D) After continuous l-NAME treatment for 6 d,
the necrotic area (to the left) and the adjacent region of mononuclear cells are enlarged, and a few myotubes (arrow) are present. (E) Among
mononuclear cells in the adjacent region, new myotubes (arrows) are thin and contain immature, basophilic cytoplasm. (F) Very few
myotubes are found between surviving fiber segments at the ends of the RTA. Original magnification in A and D, ⫻13; in B, C, E, and F, ⫻132.
Figure 6. A single l-NAME injection before injury affects myogenic repair in normal muscle. (A) At low magnification (H&E), the RTA 6 d
after injury shows a large necrotic region (to the right), an adjacent area of mononuclear cells and small new myotubes (arrows), and
surviving fiber segments (to the left). (B) At high magnification, a myotube (arrows) extends between mononuclear cells and a fiber segment.
(C) A very thin intensely eosinophilic myotube originates immediately beside a surviving fiber segment. (D) At higher magnification, the
same myotube has formed from the satellite cell position, apparently inside the external lamina. (E) An eosinophilic satellite cell (arrow) is
elongated into a thin myotube. (F) A column of apparently unfused centrally nucleated cells with granular cytoplasm makes up a myotube.
(G) A BrdU
⫹
nucleus adjacent to a new myotube. (H and I) Thin new myotube segments are positive for devMHC (Texas Red fluorescence)
whether they extend from a larger myotube (H) or are located among mononuclear cells near the crush (I). (J) A crimson satellite cell (arrow)
on an EDL fiber. (K) A large satellite cell (arrow) with crimson cytoplasm on a SOL fiber. (L) M-cadherin is present between a satellite cell
(arrow) and a small new myotube (arrowheads). (M) M-cadherin staining is intense on satellite cells located on the four intrafusal muscle
fibers in a spindle complex. Original magnification in A, ⫻13; in C, ⫻33; in B, E, and M, ⫻132; in D and F–L, ⫻330.
J.E. Anderson
Molecular Biology of the Cell1866

BrdU
⫹
nuclei were found in nearby mononuclear cells and
in some muscle precursor cells close to surviving fiber seg-
ments and new myotubes (Figure 7F). Three fields of regen-
erating muscle (in the two animals) also contained small
collections of intensely BrdU
⫹
nuclear fragments in myo-
tubes (Figure 7G). As in normal mice treated once with
l-NAME, satellite cells (outlined by m-cadherin) were ob-
served in continuity with the new myotubes that were an-
chored inside external lamina sheaths on remnant fiber seg-
ments (roughly 5% of new myotubes). Satellite cells in mdx
LTA were very large and c-met
⫹
(Figure 7H), as were sat-
ellite cells in NOS-I knockout LTAs, although their extensive
cytoplasm was not as granulated or as distinct from fiber
sarcoplasm as in normal undamaged muscles after l-NAME
treatment (Figure 7I; compare with Figure 6, J and K).
DISCUSSION
The present results show that satellite cell activation oc-
curs immediately upon muscle injury, is mediated by NO
release, is briefly transmitted to distant muscles, and is
prevented under pharmacological and genetic conditions
that reduce the activity or expression of NOS-I. Time-
course studies of myogenic cell yield and morphology
showed two aspects of activation, namely altered adhe-
sion and morphological changes. Before identifying
HGF/SF as an activator of satellite cells, the nature of
activation was elusive because it was studied with later
markers, such as regulatory gene expression or DNA
synthesis. The present demonstration in satellite cells of a
rapid shift by HGF/SF to its “mitogenic and motogenic”
receptor (Rong et al., 1994) upon activation confirms a
previous report (Tatsumi et al., 1998). The disposition of
satellite cells positive for both c-met and its ligand be-
tween fiber and laminar sheath suggested that the phys-
ical signal of injury was rapidly transduced from a fiber to
activate its satellite cells. In vivo studies on myogenesis
after injury demonstrated that pharmacological inhibition
of NOS activity was detrimental to the outcome of muscle
regeneration. Interestingly, two mutants with decreased
or absent NOS-I expression showed enhanced activation
in situations in which normal muscle is quiescent and
showed very effective repair after an imposed injury.
Together, these acute and chronic experiments strongly
Figure 7. A single treatment with l-NAME 30 min before injury affects dystrophic muscle regeneration. (A) Low magnification (H&E)
shows a large crush region (just to the left), an adjacent region of new myotubes (arrows), and surviving fiber segments (to the right). (B)
Many large new myotubes adjacent to the crush. (C) Elongated mononuclear cells and myotubes are m-cadherin
⫹
. (D) An elongated crimson
cell is binucleate and located in the satellite position on a surviving fiber segment. (E) A new myotube extends from a surviving segment and
contains devMHC (Texas Red fluorescence). (F) A BrdU
⫹
nucleus next to new myotubes with unstained central nuclei. (G) A new myotube
contains apoptotic BrdU
⫹
nuclear fragments. (H) Large c-met
⫹
satellite cells (FITC) on HGF/SF
⫹
fibers (Texas Red) in LTA. (I) A large
satellite cell (arrow) with granular cytoplasm (H&E) on an LEDL fiber has less prominent margins than in undamaged normal muscles (see
Figure 5, J and K). Original magnification in A, ⫻13; in B, ⫻130; in C–I, ⫻330.
NO and Satellite Cell Activation
Vol. 11, May 2000 1867

indicate a pivotal role for NO in transducing activation,
satellite cell adhesion, and subsequent repair processes.
For the first time, the nature and possible impact of injury-
induced activation were expressly addressed. Data showed
that reduced NOS activity, by means of inhibition with
l-NAME in normal muscle, from complete genetic loss of
NOS-I expression (in NOS-I knockout mice), or secondary to
dystrophin deficiency in mdx muscle, prevented the imme-
diate increase in myogenic cells isolated from injured mus-
cle. Rapid changes in the nuclear profile, cytoplasmic gran-
ularity, and ratio of cytoplasm to nucleus were consistent
with the known hypertrophic alterations of satellite cells as
they become activated and were also inhibited by l-NAME.
That NOS inhibition thereby delayed and restricted injury-
induced satellite cell activation, defined by changes in ad-
hesion, cell yield, morphology, and expression in cells of
two satellite cell markers, c-met and m-cadherin. Studies of
noninjured normal muscles showed a surprising, albeit
short-lived, increase in LTA cell yield after 10 min, coinci-
dent with hypertrophy of a large proportion of those satel-
lite cells, and suggest that a circulating factor can at least
transiently activate satellite cells in intact distant muscles. In
regenerating muscle, longer-term NOS inhibition (by
l-NAME in drinking water) delayed removal of debris, de-
creased the formation of new myotubes, and confined them
closer than usual to the site of injury. Although more subtle
changes in repair resulted from a single event of NOS inhi-
bition at the time of injury, the appearance of fiber duplica-
tion inside the persistent external lamina on damaged fibers
appeared to divert repair toward fiber branching. The apo-
ptotic nuclear fragmentation (Blandino and Strano, 1997;
Evan and Littlewood, 1998) that was present in regenerating
muscles after briefly perturbing activation could reduce the
number of nuclei in new fibers and potentially affect myo-
tube domains (J. Kong and J.E. Anderson, unpublished ob-
servations) and the stability of repair. These data can now
stimulate more focused examination of activation and the
potential applications of NO manipulation.
A model is presented for the hypothesis that NO release,
which is exquisitely responsive to shear in other systems
(Traub and Berk, 1998; Dimmeler et al., 1999), mediates
satellite cell activation by a similar mechanism (Figure 8).
This working hypothesis broadens the field of NO signaling
in muscle (reviewed by Grozdanovic and Baumgarten,
1999). The time course of satellite cell release and the onset
of organelle hypertrophy were very rapid, occurring by
35–45 s after injury (the time from injury and death to
collection and freezing of tissue was ⬍2 min). To date, there
are no other reports showing such rapid transduction of
morphological or adhesion changes after injury or in repair.
Normal cyclic loading of muscle produces pulsatile NO
release (Tidball et al., 1998) by the rapid diffusion of NO
down its concentration gradient and may maintain satellite
cell quiescence. Thus, a large release of NO would move as
a wave front across the narrow clefts between a fiber and its
satellite cells. The subsequent lapse in pulsatile or bolus NO
release would constitute the second phase of a powerful
signal, a “nitric oxide transient” in physiological terms. Te-
leologically, the external lamina wrapping fibers may pro-
vide the potential for satellite cells to respond to shear
between the sarcolemma and the lamina. Satellite cells hug
fibers across an even 15 nm cleft without obvious junctional
complexes, and they associate closely with external lamina
(Bischoff, 1990a; Schultz and McCormick, 1994). Thus, satel-
lite cells have the ideal topography to detect a rapid peak of
NO release from underlying fibers after shear and also to be
kept quiescent by normally continuous small pulses of NO
from the fiber. The speed of the NO-mediated signal for
activation suggests that the signal, such as mechanical shear
forces, acts on constitutive NOS-I, because the response time
is too short to induce expression or increase activity (McCall
et al., 1991; Rubinstein et al., 1998). The effects of l-NAME on
edema (and hemorrhage) were congruent with the known
effects of NO on vascular tone (Busse and Fleming, 1998).
Therefore, the time course of cell yield after injury and its
change by NOS inhibition suggest that a large NO release
mediates or directly signals activation. The transient decline
in RTA yield at 10 min in saline-treated normal mice sug-
gests that other signals are then needed to maintain or
complete activation. The nature of those additional signals
was suggested by the brief, delayed increase in LTA yield at
10 min in both saline- and l-NAME–treated normal mice.
Because HGF/SF is released from crushed muscle and acti-
vates muscle precursors in vivo and in vitro (Tatsumi et al.,
1998), HGF/SF or other factors may become activated them-
selves and circulate from RTA to initiate activation of satel-
lite cells located outside the damaged muscle. Without in-
jury or reinforcement in LTA, normal fibers would repress
activation and their satellite cells would return to quies-
cence, whereas satellite cells in RTA would receive the sec-
ondary circulating signal on top of the damage-induced
local changes and would complete the activation sequence.
Combined treatment with a NO donor and a NOS inhibitor
partly reversed the effects of NOS inhibition on RTA yield
and prevented the temporary increase in LTA yield. There-
fore, additional signals involved in fully activating precur-
sor cells likely include both NO-mediated and NO-indepen-
dent mechanisms.
NO-mediated satellite cell activation may account for re-
cent findings by L. Hall-Martin, J. Morgan, and T.A. Par-
tridge (personal communication). An isolated single intact
normal fiber and attached satellite cells (⬃10) was injected
into mdx muscle. Results showed a 5000-fold more efficient
production of dystrophin-positive fiber segments in mdx
mice than myoblast transfer with the use of 5 ⫻ 10
6
cells,
with equally wide-ranging dispersal. Although shear, pro-
duced by layers that shift laterally against each other, would
be strong during segmental retraction within the external
lamina, it would be very intense during fiber injection. Com-
pared with myoblast transfer and according to the model,
injection could maximize shear-induced satellite cell activa-
tion and supply crushed muscle extract containing HGF
directly to the site of implantation. This hypothesis, there-
fore, can integrate diverse topics of NO physiology, mechan-
ical force transduction, cell signaling, dystrophy, and repair.
In that context, the gain of three magnitudes in repair out-
come with the use of fiber injection reveals the huge poten-
tial for NO manipulation of satellite cell activation to dra-
matically improve muscle repair in health and disease.
Transient precursor proliferation in denervation and persis-
tent proliferation after trauma or segmental disease can be
explained by applying the idea of NO-mediated, shear-in-
duced satellite cell activation upon total synchronized nerve
and fiber depolarization and then loss of membrane poten-
J.E. Anderson
Molecular Biology of the Cell1868

tial. Interestingly, intense m-cadherin
⫹
satellite cells in mus-
cle spindles suggest that high shear responsiveness may
accompany the spindle function as a length-tension recep-
tor. There is also a potential for NO interaction with m-
cadherin in mediating loss of adhesion and normal quies-
cence during activation. The ratio of RTA/LTA of ⬍1at0
min during NOS inhibition or decreased NOS-I expression
(Figure 2) suggests that reduced NO after injury may medi-
ate an increase in satellite cell adhesion to the fiber-lamina
complex.
Until now, satellite cell activation was defined structurally
as cytoplasmic and organelle hypertrophy and dynamically
as recruitment to cycle. The close adherence of satellite cells
to parent fibers must decrease during activation for satellite
cells to move through the external lamina to form new
fibers. Therefore the loss-of-adhesion feature was tested as a
simple index of activation. The ability to isolate myogenic
cells after brief standard digestion was a conservative esti-
mate of available satellite cells and not an estimate of total
myogenic cells. (Additional myogenic cells are found in the
material collected on the Nitex filter during cell isolations.)
NO is known to modulate leukocyte and platelet adhesion
(Kubes et al., 1991; de Graaf et al., 1992), and m-cadherin
mediates muscle precursor adhesion to fibers. So it is also
Figure 8. A model for the process of
shear-induced, NO-mediated events that
activate satellite cells after skeletal muscle
injury. (A) In undamaged muscle with
normal contraction and relaxation, thin
quiescent satellite cells are demarcated by
m-cadherin and contain few organelles.
They are interposed between the overlying
external lamina and the sarcolemma of a
subjacent fiber and are subject to pulsatile
NO released from NOS-I
␮
that is anchored
to syntrophin. Normally, NO diffuses cy-
lindrically out from the fiber to act on cells
and enzymes in the interstitium or is neu-
tralized by red cell hemoglobin in the ves-
sels that wrap each fiber. (B) After sar-
colemmal injury, depolarization is not
followed by repolarization. A single large
contraction produces intense shear be-
tween the fiber membrane and the external
lamina. Shear induces a bolus release of
NO that diffuses down its concentration
gradient through the satellite cells hugging
the fiber. (C) Satellite cells become acti-
vated and begin to enlarge as organelles
such as mitochondria hypertrophy.
HGF/SF from the damaged fiber is acti-
vated and shifts to the c-met receptor on
satellite cells. Fibrils hypercontract and
damaged segments retract within the ex-
ternal lamina, maintaining shear and NO
release and activating satellite cells along
the fiber length. The adhesiveness of m-
cadherin decreases, and the damaged fiber
releases proteins, including HGF/SF, to
the interstitium. A released factor, such as
HGF/SF, enters the circulation and can
transiently activate distant satellite cells on
undamaged muscles, although normal
pulsatile NO release will mostly attenuate
that response. Capillaries dilate and blood
cells extravasate into the interstitium. (D)
Fiber segments fully retract and satellite
cells become motile precursors as HGF/SF
binds to c-met. The external lamina re-
mains as a scaffold for the satellite cells,
now surrounded by less adhesive m-cad-
herin. The precursors may leave the fiber
as the sequential expression of early imme-
diate genes, muscle regulatory genes, pro-
liferating cell nuclear antigen, and later
DNA synthesis begin before proliferation.
NO and Satellite Cell Activation
Vol. 11, May 2000 1869

possible that changes in adhesion during activation, and the
m-cadherin molecule itself in repair, may be affected by NO.
The present data also suggest that specifically manipulating
satellite cell activation via changes in NOS-I
␮
activity or
shear, rather than giving systemic alkali dietary supple-
ments to stimulate bone formation and indirectly stimulate
muscle fibers (Landauer and Burke, 1998), could directly
prevent muscle atrophy in microgravity.
The marked difference in activation time course between
normal and mdx muscle is entirely congruent with the dif-
ferent locations of NOS-I
␮
in the two types of muscle, as is
the similarity between RTA/LTA yield ratios in muscles
from mdx, NOS-I knockout, and l-NAME–treated normal
mice. NOS-I
␮
is subsarcolemmal and in mdx muscle is re-
duced and displaced to the cytosol as a result of the absence
of dystrophin. Mdx muscle pathology was recently reported
to be independent of NOS-I perturbation (Chao et al., 1998;
Crosbie et al., 1998). The authors hypothesized that dis-
placed NOS-I contributed free radical NO damage to the
sarcoplasm of fibers and would exacerbate dystrophy. How-
ever, this idea was rejected, because total removal of NO by
NOS-I knockout in mdx mice did not reduce dystrophy. An
alternative explanation derives from the present data. Cyto-
plasmic NOS-I in mdx muscle would act as a diffuse areal
source of NO rather than the nearby linear source, typically
subjacent and parallel to satellite cells found in normal mus-
cle fibers. The normally steep NO gradient across the cleft
between fiber and satellite cell, therefore, would become
more shallow and diffuse more slowly, and the small NO
transient would be manifest as an attenuated responsiveness
to shear forces. If normal pulsatile NO acts to maintain
quiescence, a smaller gradient in dystrophy from the pulsa-
tile NO of cytoplasmic origin could release mdx satellite cells
from what is normally full quiescence and account for the
greater proliferative activity and larger satellite cells in mdx
muscle and primary cultures (McIntosh and Anderson, 1995;
Pernitsky and Anderson, 1996; Moor et al., 2000). Rapid
repair by mdx muscle is consistent with the notion that mdx
satellite cells are partly activated or on “standby.” Likewise,
it would follow that acute injury would not necessarily
augment immediate activation, as reported here in cell yield
studies for mdx and NOS-I knockout mice. By that reason-
ing, repair after imposed injury in the NOS-I ⫻ mdx double
mutant should be less effective and/or delayed compared
with mdx muscle repair. Dystrophy in that double mutant
may be more severe than in mdx mice if it were assessed in
younger mice (⬍12 mo) before the index of repair (central
nucleation) has reached its theoretical plateau. In addition,
because human fibers are larger than mdx fibers, cytoplasmic
NOS-I in human fibers would serve as an even smaller
nonlinear NO source than in mdx muscle. The resulting very
shallow gradient or physiological NO transient across satel-
lite cells could partly account for the severity of Duchenne
dystrophy, almost as if the standby activation (like a “hair
trigger”) contributes to overly enthusiastic successive repair
events and early senescence (Decary et al., 1996, 1997). It is
now clear that satellite cell activation needs to be considered
separately from dystrophy.
Three observations are consistent with the cytosolic loca-
tion of NOS-I
␮
and the standing activation of mdx satellite
cells. Hypertrophic c-met
⫹
satellite cells are typical in mdx
muscles without injury (Anderson, 1998; this study). Adult
mdx muscle yields high-density myoblast cultures that rap-
idly begin to proliferate (Pernitsky and Anderson, 1996;
Moor et al., 2000), and mdx muscle is more effective than
normal in myogenic repair (Zacharias and Anderson 1991;
McIntosh et al., 1994; McIntosh and Anderson, 1995; Per-
nitsky et al., 1996). The normal quiescence of mdx satellite
cells should be restored along with subsarcolemmal NOS-I
after mini-dystrophin gene transfer (Decrouy et al., 1998).
The general inhibition of the delayed activation seen in
injured and intact mdx muscles after l-NAME suggests that
activation could still be modulated pharmacologically via
NO, possibly in combination with deflazacort (glucocorti-
coid) treatment to improve repair (Anderson et al., 1996,
2000). Other studies of longer-term l-NAME exposure of
regenerating mdx and normal muscle in vivo showed a high
prevalence of branched myotubes (J.E. Anderson, unpub-
lished data), emphasizing the distinct role of NO in fusion
(Lee et al., 1994) separate from activation. Indeed, deflazacort
itself may affect activation, because another glucocorticoid,
dexamethasone, is a specific inhibitor of inducible NOS-II
(McCall et al., 1991). Experiments are in progress to deter-
mine whether l-Arg can further augment mdx muscle satel-
lite cell activation in vivo, as suggested by preliminary ex-
periments on single-fiber cultures in vitro (O. Pilipowicz and
J.E. Anderson, unpublished data). Other data demonstrated
that regenerating muscle in NOS-I knockout mice (n ⫽ 2)
had extensive myotube formation during 6 d after injury,
similar to mdx mice (McIntosh et al., 1994; McIntosh and
Anderson, 1995). Interestingly, NOS-I knockout mice also
had modest focal myopathy (segmental muscle fiber dam-
age and inflammation) in TA and diaphragm. That myop-
athy was not present in the control strain (B6129SF) and may
relate to the absence of NOS-I expression in the nervous
system or a constitutive heightening of satellite cell activa-
tion. Together, the mdx and NOS-I knockout experiments
suggest that increased satellite cell activation from reduced
or absent NOS expression may benefit myogenesis in the
short term (and through a few cycles) by facilitating stand-
ing activation and precursor recruitment to cycle. However,
in the longer term, that standby activation may be detrimen-
tal, such that mdx dystrophy may be reduced by increasing
local (not systemic) pulsatile NO release in intact muscle
fibers and the bolus of NO that activates satellite cells after
fiber injury. We can now test whether human muscular
dystrophy is more severe than mdx dystrophy as a result of
even greater attenuation of the typical NO gradient through
the larger fibers, overrecruitment of satellite cells, and accel-
erated precursor senescence.
NO has a broad impact on glucose uptake, insulin resis-
tance, exercise, blood flow, and contractility (Balon and Na-
dler, 1994; Shen et al., 1995, 1997; Joyner and Dietz, 1997;
Kapur et al., 1997; Chen et al., 1998; Young and Leighton,
1998). NO also mediates denervation responses, inflamma-
tory myopathy, aging, and neuromuscular transmission
(Tews et al., 1997a,b; Capanni et al., 1998; Ribera et al., 1998;
Tews and Goebel, 1998). Therefore, the collective effects of
NO have a significant impact on muscle pathophysiology.
Although one study of rat muscle after crush injury showed
that l-NAME could prevent traumatic shock by inhibiting
NOS-II and NOS-III, no change in NOS-I was reported, and
satellite cells and repair were not examined (Rubinstein et
al., 1998).
J.E. Anderson
Molecular Biology of the Cell1870

A surprise from the present experiments was the obser-
vation of short-lived satellite cell activation in normal un-
damaged fast-twitch muscle, according to the dual criteria of
hypertrophy in vivo and loss of adherence in cell yield
studies. The consistency of the findings was emphasized by
comparison with cell yield studies in the slow SOL muscle
that expresses less NOS-I
␮
(Kobzik et al., 1994). Although
the idea that circulating HGF/SF can activate satellite cells
in intact muscle needs to be tested, it recalls a report that
serum collected after partial hepatectomy-induced shear
will stimulate proliferation of liver cells (Wang and Lautt,
1999), which stain intensely for c-met (J.E. Anderson, un-
published observations). Distant activation may also involve
NO interactions, possibly with a converting enzyme that
activates HGF or other factors (Lowenstein et al., 1994;
Miyazawa et al., 1996). The complex pharmacology of NOS
(Nathan and Xie, 1994; Reid, 1998) suggests that careful
trials to sustain the activation in undamaged normal muscle
and attenuate it in dystrophic muscle are needed. However,
the data for uninjured muscles suggest that manipulation of
NO (through combinations of increased and decreased NO)
holds a tantalizing potential to prevent or treat muscle atro-
phy (as from disuse, age, or zero gravity) and to promote
hypertrophy and new fiber growth (as in meat production,
athletic training, and animal racing) in otherwise healthy
muscle. They also suggest that activation may require an
initiating step (e.g., injury-induced NO release) and then a
second step to be fully maintained or completed. That sec-
ond phase of activation could involve factors activated by
NO or released by damaged muscle (such as HGF/SF in
crushed muscle extract) to act directly on injured muscle
fibers and indirectly and transiently on uninjured muscles.
These ideas are being tested in the isolated fiber culture
model with the use of DNA synthesis as the marker of
completed activation. Therapies that apply such findings by
effectively and specifically manipulating NO levels will be
extremely complex, given tissue interactions, NOS isoforms,
NO diffusion and inactivation, and the variable cause and
progression of atrophy and muscle diseases. Interestingly, in
situ hybridization experiments show that satellite cells
themselves express NOS-I
␮
(J.E. Anderson, unpublished ob-
servations). This suggests that satellite cells may direct (in an
autocrine manner) their own activation by shear or other
stimuli in addition to receiving paracrine signals from fibers.
The present results address for the first time the initial
steps of satellite cell activation. A single exposure to NOS
inhibition had subtle effects on myotube formation that echo
NO-stimulated myoblast fusion in vitro. Longer NOS inhi-
bition reduced the effectiveness of repair and restricted its
distribution, in agreement with the idea that shear-induced
responses become attenuated longitudinally away from the
injury. The significant negative effect of pharmacological
NOS inhibition on myogenic repair reported here was fur-
ther extended by the recent preliminary studies on repair in
NOS-I knockout mice (n ⫽ 2) and during longer-term NOS
inhibition in mdx mice (n ⫽ 22). Bearing in mind that l-
NAME nonspecifically inhibits all NOS activity, including
vascular smooth muscle and endothelial responsiveness,
and will have a broad impact, 3 wk of systemic l-NAME
treatment appeared to increase the severity of dystrophy in
diaphragm, SOL, EDL, and TA in young mdx animals.
A model proposes the hypotheses that NO mediates rapid
satellite cell activation, including hypertrophy and altered
adhesion inside the fiber-lamina complex, and that distant
muscle precursors may be transiently activated by circulat-
ing factors released from injured muscle. Although the
model certainly needs exploration at many levels, perhaps
the largest insights are the rapidity of activation and the
notion that immediate satellite cell responses to muscle in-
jury may be contributed by the physical character of the
external lamina and mechanical shear. The signaling mech-
anism underlying NOS-I activity in response to shear can
also be determined and may involve Akt/PKB-dependent
phosphorylation of NOS-I, as reported recently for NOS-III
(Dimmeler et al., 1999). With these signals better defined,
new strategies to promote and regulate the action of satellite
cells in disease and repair can be devised.
ACKNOWLEDGMENTS
The technical expertise of Cinthya Vargas and graphics by Jerry
Kostur and Jay Anderson are gratefully acknowledged. The labora-
tory is supported by grants from the Muscular Dystrophy Associ-
ation (USA) and the Heart and Stroke Foundation of Canada.
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