2. increased, whereas expression of the pro-apoptotic protein BAX was reduced. In culture,
these changes were accompanied by a reduction in the expression of p53 and the cyclin-
dependent kinase inhibitor p21, reflecting the small decrease in viable cells 24 hours after
irradiation. These findings implicate regulation of these factors as part of the protective role
of LELI against apoptosis. Taken together, our findings are of critical importance in
attempts to improve muscle regeneration following injury.
Key words: Satellite cells, Laser irradiation, Apoptosis, Myofiber, Proliferation
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
Introduction
Normal skeletal muscle contains post-mitotic muscle fibers that themselves have very
limited remodeling capability. Remodeling is largely accomplished by myogenic cells
derived from quiescent mononuclear satellite cells, which lie beneath the basement
membrane of the muscle fiber, but outside of the plasmalemma of the multinucleated fiber
itself (Mauro, 1961 ). Satellite cells are quiescent in normal adult mouse muscle (Schultz et
al., 1978 ). They do, however, replicate in growing postnatal muscle, where they add
myonuclei to enlarging muscle fibers, and in regenerating adult muscle, where they give
rise to new muscle fiber segments to replace those lost during muscle injury (reviewed in
Bischoff, 1994).
Mechanisms of muscle fiber repair are of interest in understanding the restoration of muscle
after trauma or injury and during myopathic diseases. After severe injury, muscle
regeneration is a slow process, during which scar tissue formation competes spatially with
the regenerating muscle fibers at the site of injury. Many approaches have been suggested
to enhance muscle restoration, including the use of muscle precursor cells, stem cells or
muscle fiber transplants (reviewed in Grounds, 1999 ). One of the disadvantages of cell
transplantation methods is that most donor cells fail to survive (Rando et al., 1995 ; Fan et
al., 1996 ; Beauchamp et al., 1997 ). Involvement of the host immune system in this rapid
death has been suggested (reviewed in Smythe et al., 2000 ), but the precise mechanism or
mechanisms underlying the phenomenon remain obscure.
Low-energy laser irradiation (LELI) has been found to modulate various biological
processes (reviewed in Conlan et al., 1996 ; Karu, 1999 ), such as increasing
3. mitochondrial respiration and ATP synthesis (Morimoto et al., 1994 ; Yu et al., 1997 ),
facilitating wound healing (Conlan et al., 1996 ) and promoting the process of regeneration
and angiogenesis (Weiss and Oron, 1992 ; Bibikova and Oron, 1993 ; Bibikova and Oron,
1994 ; Bibikova et al., 1994 ). The augmentation of regeneration by LELI has been studied
in various tissues, such as skin (Conlan et al., 1996 ), bone (Yaakobi et al., 1996 ), nerve
(Assia et al., 1989 ) and skeletal muscle (Weiss and Oron, 1992 ; Bibikova and Oron,
1993 ). In the skeletal muscle of rats and toads, He-Ne laser irradiation of the injured site
enhanced regeneration by two- and eightfold, respectively, relative to non-irradiated
controls (Weiss and Oron, 1992 ; Bibikova and Oron, 1993 ). The injured site in the LELI-
treated animals featured many young myofibers, suggesting that satellite cells are the major
irradiation-responsive candidates (Weiss and Oron, 1992 ). This idea is supported by
studies of the effect of LELI on primary rat satellite cell cultures, revealing the induction of
cell cycle regulatory protein expression, increased satellite cell proliferation and inhibition
of cell differentiation (Ben-Dov et al., 1999 ). In the search for a mechanism by which
LELI manifests these cell responses, we very recently demonstrated that LELI specifically
activates the mitogen-activated protein kinase/extracellular signal-regulated protein kinase
(MAPK/ERK) pathway, but not the stress pathways (i.e. p38 MAPK and the stress-
activated protein kinase/Jun N-terminal kinase (SAPK/JNK)), probably by phosphorylation
of specific receptors (Shefer et al., 2001 ).
Moreover, we have recently demonstrated a profound cardioprotective effect of LELI on
chronic infarcted myocardium in rats and dogs, resulting in a 50 to 70% reduction in infarct
size 4 to 6 weeks after chronic occlusion of the left descending coronary artery (Oron et al.,
2001 ). This phenomenon was partially due to significant elevation in the number of
undamaged mitochondria and ATP content in the cardiomyocytes in the ischemic zone in
LELI animals as compared with control non-irradiated ones (Oron et al., 2001 ). These data
led us to postulate that LELI enhances cell survival — a beneficial effect in terms of muscle
preservation following injury or in myopathy. During normal myogenesis, as well as during
muscular diseases (e.g., Duchenne muscular dystrophy), cells are known to self-destruct
when no longer needed or when damaged, by activating controlled machinery leading to
programmed cell death or apoptosis (reviewed in Sandri and Carraro, 1999 ). Apoptosis is a
highly orchestrated process in which cells `commit suicide' via degradation into membrane-
packaged fragments. Recent in vitro experiments on primary myoblasts (Chinni et al.,
1999 ) and the C2C12 myogenic cell line (Conejo and Lorenzo, 2001 ) have shown
apoptosis of replicating myoblasts upon deprivation of growth factors. The Bcl-2 family
members are mainly involved at the mitochondrial level, and they play a key role in the
choice between cell survival or death (reviewed in Gross et al., 1999 ). Factors such as Bcl-
2 and Bcl-xL prevent apoptosis, whereas pro-apoptotic proteins, exemplified by Bax and
Bak, can accelerate death and in some instances are sufficient to cause apoptosis
independently of other signals.
In the present study, the effects of LELI on cell survival and proliferation were tested in a
single fiber system representing a three-dimensional organization of the surviving and
repaired segments of the fiber (Rosenblatt et al., 1995 ). This serves as a more
comprehensive model than standard tissue culture in which to assess satellite cell
proliferation. We show that LELI promotes the cell cycle entry and accumulation of
satellite cells around fibers grown under serum starvation and that these effects are
4. synergistic with serum addition. The inhibitory effect of LELI on cell apoptosis goes hand
in hand with increasing Bcl-2 and decreasing BAX expression in both these fibers and
myogenic cultured cells.
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
Materials and Methods
Animals
Male C57B1/10 mice were maintained in the Animal Unit at the Hammersmith Campus of
the Imperial College School of Medicine. Care and treatment of the animals complied with
the 1986 Animals (Scientific procedures) Act.
Single fiber cultures
Single muscle fibers were prepared from freshly dissected extensor digitorum longus (EDL)
muscle of 21-day-old mice (Rosenblatt et al., 1995 ). The left and right EDL muscles were
carefully removed and incubated in a solution of 0.2% (w/v) type I collagenase (Sigma-
Aldrich) in DMEM at 35°C for 1 hour on a shaker. The muscle was transferred to a plastic
Petri dish containing DMEM, and muscle fibers were separated from the whole muscle by
repeated gentle pipetting with a wide-mouth Pasteur pipette. Fibers were plated, one per
well, in a 24-well Primaria tissue culture pre-coated with 1 mg/ml growth-factor-reduced
Matrigel (Becton Dickinson Labware). The fibers were incubated in serum-free DMEM or
in DMEM containing 0.1% (v/v) horse serum (HS) in a water-saturated environment at 5%
CO2 and 37°C.
Cell culture
i28 mouse myogenic cells (Irintchev et al., 1998 ; Wernig et al., 2000 ) were grown in
DMEM supplemented with 20% (v/v) fetal calf serum. For experiments, cells were plated at
7x104 cells per 60 mm dish for one night in 20% fetal-calf-serum-containing medium, then
starved for 36 hours in serum-free DMEM (Ben-Dov et al., 1999 ).
Laser irradiation
Single fibers or i28 cells were irradiated for 3 seconds through a grid composed of 1.8
mmx1.8 mm squares to ensure precise irradiation over the entire tissue-culture plate (Ben-
Dov et al., 1999 ) with a He-Ne laser (632.8 nm, 4.5 mW; 1.8 mm beam diameter; Ealing
5. Electro-Optics). Fibers were irradiated 18 hours after plating. Plates were then returned to
the incubator for a further day and cells accumulating around the fiber were counted daily
for 5 days. In the case of i28 cells, cells were irradiated 36 hours post starvation (Shefer et
al., 2001 ). Control, non-irradiated fibers and cells were kept under the same conditions as
their treated counterparts — they were kept on the bench beside the irradiated cultures.
Western blot analysis
Cells were lysed in NP-40 lysis buffer as described in Ben-Dov et al. (Ben-Dov et al.,
1999 ). Cell extracts were sonicated using an ultrasonic cell disrupter (Microson), clarified
by centrifugation and normalized to protein content (BCA kit; Pierce). Proteins were
separated by SDS-PAGE and transferred to nitrocellulose filters (Schleicher and Schuell).
Membranes were blocked with TBS-T (50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 0.5%
(v/v) Tween 20) containing 3% (w/v) BSA and incubated overnight at 4°C with the
appropriate primary antibodies. The membrane was then washed with TBS-T and incubated
for 1 hour with horseradish-peroxidase goat anti-mouse or horseradish-peroxidase goat anti-
rabbit IgG (Zymed). Proteins were visualized by enhanced chemiluminescence (Pierce).
Densitometric analysis was performed on bands using the Image Pro Plus 3.0 software. The
following primary antibodies were used: polyclonal anti-Bcl-2 (1:500; Oncogene Research
Products), polyclonal anti-BAX (1:500; Santa Cruz Biotechnology), polyclonal anti-p21
(1:1,000; PharMingen) and a monoclonal antibody against p53 (1:500, Santa Cruz
Biotechnology).
Immunofluorescent staining
Single fibers were fixed with fresh 4% (w/v) paraformaldehyde for 20 minutes. Fibers were
permeabilized with 0.1% (v/v) Triton in PBS/TBS for 15 minutes and blocked with 3%
BSA and 5% carrageenin (0.37% w/v) in PBS for 3 hours for the BAX and Bcl-2
antibodies. Samples were incubated with primary antibodies overnight at 4°C followed by
three 15 minutes washes in PBS. Antibodies were detected with biotinylated swine anti-
rabbit IgG (1:500 dilution; Dako) followed by Alexa-594-conjugated streptavidin (1:500
dilution; Molecular Probes). The stained samples were then mounted with fluorescent
mount medium (Dako) and viewed with a fluorescent microscope. Nuclei were detected
with 4'6'-diamino-2-phenylindole (DAPI; Sigma-Aldrich).
5-Bromo-2'-deoxyuridine (BrdU) incorporation
Single fibers were cultured for 18 hours in DMEM containing 4 µM BrdU and then
immunostained with mouse anti-BrdU antibody as described (Kaufman and Foster, 1988 ).
In brief, cultures were fixed in 4% paraformaldehyde for 10 minutes, then in 95% ethanol
for another 10 minutes and rinsed in PBS. The cultures were incubated for 30 minutes at
room temperature in 2 M HCl, followed by washes in 50 mM NaCl, 100 mM Tris-HCl, pH
7.4. Incubation of cells with mouse anti-BrdU antibody (diluted 1:20; G3G4;
Developmental Studies Hybridoma Bank, University of Iowa; contributed by D. Kaufman)
for 1 hour was followed by incubation with streptavidin-peroxidase (diluted 1:250; Dako)
for 30 minutes. Peroxidase activity was visualized with 3,3'-diaminobenzidine
tetrahydrochloride (DAB; Sigma).
Cell viability and apoptosis assays
Cell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,-
6. diphenyltetrazolium bromide; Sigma-Aldrich) assay for mitochondrial activity. At the
indicated times, MTT was added to the cells at a final concentration of 0.5 mg/ml for 2
hours of incubation. For apoptotic measurements, nuclear DNA was stained by Hoechst
33258 dye (final concentration of 5 µg/ml; Sigma-Aldrich). Cells were then fixed with 2%
paraformaldehyde, washed in PBS and visualized under a fluorescence microscope. Cells
were scored as apoptotic only when they had pyknotic and/or fragmented nuclei
(Stadelmann and Lassmann, 2000 ).
Statistics
Unless otherwise indicated, raw data were analyzed using one- or two-way analysis of
variance (ANOVA). In within-group tests, when data significantly deviated from the
normal distribution (Kolmogorof-Smirnov test for normality), a non-parametric t-test was
carried out. Alpha level was set to 0.05 in all experiments.
Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
Results
LELI promotes fiber-associated cells to enter the cell cycle
Our experiments were aimed at investigating the effect of LELI on the accumulation of
cells near isolated fibers. Previous studies of ours have shown that LELI promoted cell
cycle entry of cultured cells kept under serum-free conditions (Ben-Dov et al., 1999 ;
Shefer et al., 2001 ). Therefore, in the first set of experiments we used isolated single fibers
kept in serum-free medium for the entire experiment. Single myofibers were irradiated 19
hours post plating, and the number of cells emanating from each fiber was monitored daily
for 5 days following irradiation. Previously, it was shown that these cells are virtually all
myogenic and when grown in growth medium have the ability to re-enter the cell cycle
(Rosenblatt et al., 1995 ). At zero time, virtually all the cells were still attached to the fibers
(data not shown); 1 day post irradiation, the average number of cells adjacent to the LELI
fibers was approximately twofold higher than that in controls (see further on).
The rapid increase in the number of cells near the irradiated fibers suggested an immediate
effect of LELI on the cell cycle entrance of fiber-associated cells. Recently, we have shown
that LELI activates quiescent satellite cells and increases their proliferation (Ben-Dov et al.,
1999 ; Shefer et al., 2001 ). To evaluate the state of cells in the S-phase, BrdU was applied
immediately after irradiation, and fibers were fixed and immunostained for BrdU after 1
7. day. BrdU incorporation could be clearly seen in cells that were still attached to the LELI-
treated fibers (Fig. 1B); this was not the case in the control non-irradiated fibers (Fig. 1A).
Fig. 1. LELI promotes cell cycle entry of cells
attached to single fibers. BrdU was added to control
non-irradiated (A) or LELI (B) fibers immediately
after irradiation for 1 day, after which they were fixed
and examined for BrdU incorporation by
immunohistochemistry. Most of the cells in the non-
View larger version (75K): irradiated fibers were negative for BrdU, whereas
[in this window] some were positive (brown) in the LELI fibers.
[in a new window] Arrows indicate some of the BrdU-positive cells. Bar,
30 µM.
LELI increases the number of fiber-adjacent cells under serum-free conditions
We next analyzed the kinetics of cell accumulation near irradiated and non-irradiated fibers.
Four replicates of the serum-starvation experiments were carried out, and statistical analysis
did not reveal any significant difference in the counts of cells adjacent to fibers (Kruskal-
Wallis ANOVA median test; R (4, 12)=3.00; P=0.0626; n.s.). Therefore, the results of these
four replicate experiments were pooled and averaged. The cumulative plot of the number of
cells per fiber clearly shows that as early as day 1 (i.e., 1 day post irradiation), the irradiated
fibers produced more cells than did the non-irradiated fibers (Fig. 2). The average number
of cells per fiber was twofold higher in the LELI fibers (P<0.001). A similar pattern of
distribution was accompanied by an increase in the average number of cells per fiber, which
reached more than threefold the control group values, suggesting that LELI enables cell
proliferation (Fig. 2, right panel; Fig. 1). However, overall proliferation was not pronounced
and cell number per fiber was even reduced on day 5 (Fig. 2, right panel), suggesting that
LELI is necessary but not sufficient to promote cell proliferation for long periods under
serum-free conditions and that additional factors are required.
8. Fig. 2. LELI enhances the number of satellite cells emanating
from single fibers. Single myofibers prepared from EDL muscle
of 21-day-old mice were non-irradiated (left panels) or irradiated
with He-Ne laser (right panels) and kept in serum-free medium
for the entire experiment. Data are expressed as the cumulative
rank of data from single fibers of four independent experiments
(n=200). Results are ranked according to the number of cells that
accumulated around each single fiber on the indicated days.
Individual ranks are expressed as percentage of total rank to
normalize the data for sample size, plotted on the vertical axis.
Superimposed on each curve as an H symbol is the mean value±1
View larger s.e.m. for each treatment. Median value is indicated by dotted
version (24K): lines. In most cases, the mean and median values are close,
[in this window] therefore most samples are not distant from a Gaussian
[in a new distribution. The population distribution of LELI cells is
window] significantly different from control cells on all days (Kruskal-
Wallis ANOVA median test; Rao R (4,99)=9.65; P<0.001).
Synergistic effect of LELI and serum on cell accumulation near the fibers
To test the relationship between LELI and serum, we conducted similar types of experiment
but now with fibers kept in low-serum-containing medium (0.1% HS). In general, on day 1
fibers of both control and LELI groups already had relatively high numbers of adjacent
cells, probably owing to the presence of the serum (Fig. 3A). However, in the case of the
control fibers, cell accumulation increased only slightly on day 2 and average cell number
per fiber remained almost the same during subsequent days (Fig. 3A, left panel). In contrast,
in the LELI fibers, a noticeable shift in the cumulative curve on day 2, accompanied by a
marked elevation in cell number per fiber, was seen (Fig. 3A, right panel), indicating the
proliferation of most cells. This trend continued on subsequent days, and numerous fibers
with high numbers of adjacent cells were noted on day 5, despite the fact that the medium
was never replaced during the experiment. A comparison of the relative changes of total
cell number (a pool of all experiments) in the LELI, HS and LELI+HS groups (Fig. 2, right
panel, Fig. 3A, left and right panels, respectively) revealed that LELI and HS have a
synergistic effect on cell proliferation (Fig. 3B) and that this effect is most pronounced on
days 3 and 5.
9. Fig. 3. Analysis of the number of satellite cells emanating from single
fibers in the presence of serum. (A) Data are expressed as the
cumulative rank of the data from single fibers grown in DMEM
containing 0.1% HS and either non-irradiated (left panel) or LELI
(right panel) (n=200). Curves were plotted as described in Fig. 2. The
population distribution of LELI cells is significantly different from
control cells on all days (Kruskal-Wallis ANOVA median test; Rao R
(4,99)=9.65; P<0.001). (B) A synergistic effect of LELI and serum on
cell accumulation around the fibers. Data are presented as the
percentage change of the total cell number that was pooled from all
experiments in each treatment relative to day 1. White bars, non-
View larger irradiated fibers were grown in 0.1% HS-DMEM. Black bars, fibers
version were irradiated and kept in serum-free DMEM. Hatched bars, fibers
(21K): were subjected to LELI and kept in DMEM-0.1% HS.
[in this
window]
[in a new
window]
Effects of LELI on cell survival
The results shown in Fig. 2 raised the possibility that under serum-starvation conditions,
control fiber-associated cells remain quiescent. Another possibility is that under these
conditions cells die by apoptosis whereas those around LELI fibers survive. This is in
agreement with previous demonstrations of the apoptotic effect of serum starvation in other
cell systems (Kulkarni and McCulloch, 1994 ; Hasan et al., 1999 ) as well as in myoblasts
(Chinni et al., 1999 ; Conejo and Lorenzo, 2001 ). To further investigate the protective
effect of LELI under conditions of growth-factor deprivation, we tested the expression of
BAX, a death-promoting molecule, and Bcl-2, a survival protein, in single fibers that were
non-irradiated or had undergone LELI under serum-free conditions. Two fibers were plated
in each 35 mm dish as far apart as possible, and only one half of the dish (with one of the
fibers in it) was irradiated, and the other half served as a control. One day post irradiation,
both fibers were fixed and reacted with antibodies against either Bcl-2 or BAX. In each of
the three replications of this experiment, high levels of BAX were seen in the control fibers
compared with zero levels in the irradiated fibers (Fig. 4A, C). Conversely, high levels of
Bcl-2 were seen in the irradiated fiber, in contrast to minimal levels of expression in the
control fiber (Fig. 4B,D).
10. Fig. 4. Effect of LELI on anti- and pro-apoptotic proteins. Two
fibers were plated on each Petri dish: one was irradiated and the
other served as a control. One day post irradiation, fibers were fixed
and stained for BAX (A, C) or Bcl-2 (B, D) and counter-stained
with DAPI. Bar, 30 µM. (E) Western blot analysis of Bcl-2, BAX,
p53 and p21 in control and irradiated i28 myogenic cells on various
days post-irradiation. Equal quantities of proteins were loaded as
evidenced by staining for -tubulin. (F) Densitometric analysis of
protein expression levels normalized to that of -tubulin. Results are
averages of two independent repeats.
View larger
version (21K):
[in this
window]
[in a new
window]
The upregulation of BAX in fibers kept under serum-free conditions suggested that their
associated cells undergo apoptosis in a p53-dependent manner. p53 and its downstream
protein p21 are involved in growth arrest or apoptosis following DNA damage or cellular
stress (El-Diery et al., 1993; Ko and Privas, 1996; Levine, 1997 ). Moreover, p53 has been
shown to induce apoptosis by upregulating the BAX gene (Miyashita and Reed, 1995 ). To
analyze the modulations in expression of these proteins as a consequence of LELI, we
employed a myogenic cell culture that had been previously shown to be driven into the cell
cycle and to proliferate in response to LELI (Ben-Dov et al., 1999 ; Shefer et al., 2001 ).
Growing i28 cells were switched to serum-free DMEM for 36 hours, after which they were
or were not irradiated for 3 seconds (Shefer et al., 2001 ). Cells were left in DMEM and
harvested 24 and 48 hours post irradiation, and equal amounts of protein were
electrophoresed and analyzed for p53, p21, BAX and Bcl-2. Densitometry analysis revealed
that at zero time, cells expressed p53 and Bcl-2 proteins, whereas low or undetectable levels
of BAX and p21, respectively, were seen (Fig. 4E,F). The relatively high expression of p53
at this time point was due to cells being in cell cycle arrest (El-Dairy et al., 1993). Still,
these cells had not undergone apoptosis, because they were previously shown to be capable
of re-entering the cell cycle upon LELI or serum stimulation (Shefer et al., 2001 ). p53, as
well as BAX and p21 protein levels, were higher in the control non-irradiated cells 24 hours
post-irradiation than in the LELI cells (Fig. 4E,F). Conversely, expression of Bcl-2 was
further increased in LELI cells, while remaining undetectable in the control non-irradiated
cells, just as with the LELI fibers (Fig. 4D). However, after 48 hours, the difference
between the LELI and control cells with respect to protein expression was less pronounced,
except for p21 expression, which remained lower in the LELI cells relative to the controls.
Cell viability was determined by MTT assay, which detects alterations in cellular redox
activity in mitochondria. Cells were treated as described in Fig. 4, and the number of viable
11. cells was measured after MTT addition 24 and 48 hours post irradiation. The overall
number of viable cells declined in all serum-free cultures. However, the reduction was
much more pronounced in the control non-irradiated cells (Fig. 5A), with a sharp decrease
in cell viability between zero and 24 hours. After 48 hours, only 30% of the non-irradiated
cells remained viable, in contrast to approximately 60% viability in the LELI cells.
Fig. 5. Survival of irradiated or non-irradiated i28
cells as measured by MTT assay of mitochondrial
function (A). (B) Hoechst staining for DNA nuclei,
24 hours post irradiation. Note the
fragmented/apoptotic nuclei versus the intact ones in
control and laser-treated cells, respectively. (C)
Apoptotic nuclei were counted and data are presented
as a percentage of total nuclei. Each graph represents
mean±s.e.m of three replicates in which more than
1,000 cells were individually examined.
View larger version (68K):
[in this window]
[in a new window]
In a parallel experiment, nuclei were cytochemically labeled with Hoechst stain, and
apoptotic nuclei were scored by morphological criteria (Fig. 5B). The number of cells
designated apoptotic by these criteria, 24 hours post irradiation, showed an approximately
threefold increase in the non-LELI cultures, compared with only a 1.5-fold increase after
LELI (Fig. 5C). This trend persisted for only the next 24 hours, reaching approximately
30% apoptotic cells in the LELI group and nearly 50% in the control group. The difference
between the LELI and control cells became smaller after 48 hours, suggesting an evident,
albeit transitory, effect of LELI on apoptosis. Repeated irradiation after 24 hours did not
improve the state of the cells, and they underwent the same level of apoptosis as did cells
that had been subjected to LELI only once (data not shown).
Discussion
12. Top
Previous demonstrations of the stimulatory effect of LELI on cell Summary
proliferation have been reported in primary mass cultures of Introduction
satellite cells and myogenic cell lines (Ben-Dov et al., 1999 ; Materials and Methods
Shefer et al., 2001 ). However, such cultures do not closely mimic Results
Discussion
the cell biology of myogenic cells within skeletal muscle, References
particularly with respect to the condition of myogenic cells in the
cell cycle. Myoblasts in tissue culture, even under low nutrient and
growth factor conditions, do not accurately reproduce the G0 status of the dormant satellite
cell. Accordingly, we extended the application of LELI to isolated single fibers, comprising
the contractile muscle fiber and its associated satellite cells. It was important for our
purposes that these latter cells are in G0 at the time of isolation and during the entire
preparation (Rosenblatt et al., 1995 ), thus giving the closest available approximation of
their condition in vivo.
Using this model system, we show that LELI promotes accumulation of satellite cells near
isolated single fibers grown under serum-free conditions and that the addition of even trace
amounts of serum augments this effect synergistically. Such accumulation appears to reflect
both promotion in mitosis of myogenic cells and, at least under serum-free conditions, their
preservation from apoptosis by LELI. Moreover, LELI aids the survival of fibers under
these pro-apoptotic conditions.
The effect of LELI on fiber-adjacent cell proliferation
In accordance with our previous observations in mass cell cultures (Ben-Dov et al., 1999 ;
Shefer et al., 2001 ), we found that LELI enhanced the activation and cell cycle progression
of quiescent satellite cells on freshly isolated muscle fibers (Fig. 1). At the time of
irradiation, these cells are in contact with the muscle fiber and have not divided (data not
shown). In fact, the number of cells that accumulated around non-irradiated fibers over the
entire time period (Fig. 2) was about equal to the number of satellite cells residing on the
average EDL muscle fiber (Beauchamp et al., 2000 ), and therefore, proliferation need not
have been involved (Fig. 1C). In contrast, proliferation has to have occurred to account for
the 20 or more cells that accumulated around the LELI fibers during the 2 days after
irradiation. Thus, LELI drives quiescent satellite cells into at least one round of proliferation
under the low-serum conditions in which they do not otherwise show any such response.
However, this cell accumulation near the irradiated fibers was limited and transient: under
serum-free conditions, no further net increase in cell number occurred after the second day,
and by day 5 a slight reduction was noted. Interestingly, a more rapid reduction in cell
number, with clear signs of apoptosis, was evident in the mass cell cultures (Fig. 4B, Fig.
5). In previous studies of mass cultures under low-growth conditions, LELI stimulated cells
into the cell cycle up to 24 hours post irradiation (Shefer et al., 2001 ) but beyond 48 hours,
most cells did not incorporate BrdU (data not shown). The relatively prolonged effect of
LELI on the proliferation of cells associated with isolated fibers might be attributed to
factors secreted by the fibers in response to irradiation. However, additional LELI 24 hours
after the initial exposure did not alter the course of events in either cell culture or fibers
(data not shown), suggesting, as an alternative explanation, that factors other than those
secreted from the fibers per se are required to preserve the initial survival effect of LELI.
Indeed, a synergistic effect of LELI and serum on cell proliferation was observed in
13. cultures that were kept at serum concentrations as low as 0.1% (Fig. 3B). Notably, even
under those conditions, the fact that the cells were uniformly stimulated into one or two
extra divisions implies that the proliferative effect of LELI involves only a transient release
from the tightly controlled mechanisms of cell cycle regulation.
LELI enhances Bcl-2 and decreases BAX expression
Our demonstration that proliferation of satellite cells on myofibers is stimulated by LELI
confirms and extends previous observations on mass cell cultures (Ben-Dov et al., 1999 ;
Shefer et al., 2001 ). However, a novel finding in the present study was the enhanced
survival of both the isolated fibers and fiber-associated myogenic cells as a consequence of
LELI. This was clearly seen under serum-free conditions, where the surviving cells and the
myofibers in LELI cultures contained demonstrably higher levels of the anti-apoptotic
protein Bcl-2 and lower amounts of the pro-apoptotic protein BAX relative to control
fibers. This effect was also seen in cultures that contained one irradiated and one non-
irradiated fiber in the same culture well, suggesting that this effect was direct and not
mediated by diffusible factors. Similarly, LELI diminished the apoptotic rate of serum-
starved bulk cultures of myogenic cells, again accompanied by low levels of BAX and high
levels of Bcl-2 proteins.
Our observations fit with the widely accepted view that BAX overexpression promotes cell
death in response to apoptotic stimuli, whereas Bcl-2 inhibits it (Sentman et al., 1991 ;
Strasser et al., 1991 ; Oltvai et al., 1993 ). BAX and Bcl-2 can form heterodimers, and
overexpression of one antagonizes the effect of the other (Oltvai et al., 1993 ). Our finding
of a clear reciprocal pattern of Bcl-2 and BAX expression in control versus LELI fibers and
cultured cells strongly implicates regulation of these factors as part of the protective role of
LELI against apoptosis. In this same vein, we have recently reported that LELI specifically
activates the MAPK/ERK signaling pathway but not the SAPK/JNK signaling pathway
(Shefer et al., 2001 ). The former is a key pathway promoting cell survival in response to
growth factors (Xia et al., 1995 ; Gardner and Johnson, 1996 ; Shimamura et al., 1999 ),
whereas targets for the latter (i.e. c-Jun, BAX) have been shown to play a role in apoptosis
after growthfactor deprivation (Bossy-Wetzel et al., 1997 ; Miller et al., 1997 ). This
explanation is also in line with our finding of lower levels of p53, p21 and BAX in LELI
versus untreated cells 24 hours after irradiation, because it has been shown that activated
p53 mediates growth arrest and apoptosis by activating the expression of a number of
cellular genes, such as p21 and BAX (El-Diery et al., 1993; Miyashita et al., 1994 ). This
inhibition may involve activation of the MAPK/ERK signaling pathway by LELI (Shefer et
al., 2001 ), as this pathway has been shown to protect against p53-dependent apoptosis
(Anderson and Tolkovsky, 1999).
Thus, it is possible that LELI overcomes p53-dependent apoptosis by inhibiting the increase
in p53 induced by cellular stresses, such as growth-factor deprivation (Blandino et al.,
1995 ; Hasan et al., 1999 ; Honda et al., 2000 ), with subsequent induction of its
downstream genes. The protective effect of Bcl-2 upregulation in response to LELI in both
fibers and cell cultures could also be mediated by the suppression of p53 expression
(Miyashita et al., 1994 ). At the same time, despite the correlation between LELI-mediated
cell survival and p53 regulation, we cannot rule out other mechanisms by which LELI may
prevent apoptosis. For instance, LELI has been shown to elevate ATP content and preserve
14. mitochondrial structure in infarcted heart (Oron et al., 2001 ) and liver (Passarella et al.,
1984 ; Yu et al., 1997 ) of rats. Continuous ATP synthesis is necessary to maintain a
constant H+ efflux and membrane potential, which is deregulated following the death signal
(Vander Heiden et al., 1997 ) (reviewed in Gross et al., 1999 ). Interestingly, Bcl-2 has
been reported to prevent mitochondrial dysfunction by regulating proton flux (Shimizu et
al., 1998). A recent report of direct upregulation of Bcl-2 by IL-7 in immature thymocytes
(Von Freeden-Jeffry et al., 1997 ) also leaves open the possibility that LELI directly
induces upregulation of Bcl-2 at the post-transcriptional level.
Implications of LELI in muscle repair
Our current study on single fibers and our previous studies in vivo and in cell cultures
(Weiss and Oron, 1992 ; Ben Dov et al., 1999 ) suggest that LELI potentiates the entrance
of quiescent satellite cells into the cell cycle, acting synergistically with serum elements to
enhance their proliferation and survival. Moreover, the fact that neither LELI's
enhancement of proliferation nor its protective effects against apoptosis were maintained for
a long period argues against any mechanism involving cell transformation. These findings
are of the utmost importance in attempts to improve muscle regeneration following injury.
Immediately after muscle injury (e.g., partial excision), the traumatized area also suffers
ischemic injury and a lack of nutrients and oxygen supply. The latter is renewed as new
blood vessels form in the injured area. Nevertheless, during the initial phase, apoptosis may
take place in the injured fibers and satellite cells (U.O., unpublished). Apoptosis of
myonuclei has also been shown in skeletal muscle during hindlimb unloading-induced
atrophy (Allen et al., 1997 ). Results of the present study suggest that LELI could reduce
this process. Indeed, the above-mentioned phenomenon was demonstrated in single fibers
as well as in cultured cells in low-serum and serum-free media, which may mimic the
initial, post-traumatic phase. Moreover, we have reported a profound cardio-protective
effect of LELI for cardiomyocytes in the infracted heart (Oron et al., 2001 ), further
suggesting that LELI enhances cell survival. Taken together, we believe that LELI, as a
relatively non-invasive technique that enhances both cell survival and proliferation, might
provide an effective means of ameliorating the long-term consequences of muscle injury.
Acknowledgments
i28 cells were a generous gift from A. Wernig and A. Irintchev (University of Bonn, Bonn,
Germany). The work was supported by the European Community (EC) Biomed grant
(BMH4-97-2767). G. Shefer was supported by fellowships from the Federation of European
Biochemical Societies and European Molecular Biology Organization. L. Heslop is
supported by Biotechnology BIO4 CT 95-0284 and the MRC Clinical Sciences Centre and
J.G. Gross by the MRC Clinical Sciences Centre.
15. Top
Summary
Introduction
Materials and Methods
Results
Discussion
References
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