1 Department of Animal Sciences, The Hebrew University of Jerusalem, P.O. Box
12, Rehovot 76100, Israel
2 Muscle Cell Biology Group, MRC Clinical Sciences Centre, Imperial College
School of Medicine, Hammersmith Hospital, Du Cane Road, London W12 0NN,
UK
3 Department of Zoology, The George S. Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv 69978, Israel
* Author for correspondence (e-mails: halevyo{at}agri.huji.ac.il ; ohalevy{at}caregroup.harvard.edu )
Accepted 8 January 2002
![]() |
Summary |
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Key words: Satellite cells, Laser irradiation, Apoptosis, Myofiber, Proliferation
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Introduction |
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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 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 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.
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Materials and Methods |
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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 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,-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.
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Results |
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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 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).
|
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.
|
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.
|
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).
|
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 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.
|
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).
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Discussion |
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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 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 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.
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Acknowledgments |
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References |
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