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 afterirradiation. These findings implicate regulation of these factors as part of the protective roleof LELI against apoptosis. Taken together, our findings are of critical importance inattempts to improve muscle regeneration following injury.Key words: Satellite cells, Laser irradiation, Apoptosis, Myofiber, Proliferation Top Summary Introduction Materials and Methods Results Discussion References IntroductionNormal skeletal muscle contains post-mitotic muscle fibers that themselves have verylimited remodeling capability. Remodeling is largely accomplished by myogenic cellsderived from quiescent mononuclear satellite cells, which lie beneath the basementmembrane of the muscle fiber, but outside of the plasmalemma of the multinucleated fiberitself (Mauro, 1961 ). Satellite cells are quiescent in normal adult mouse muscle (Schultz etal., 1978 ). They do, however, replicate in growing postnatal muscle, where they addmyonuclei to enlarging muscle fibers, and in regenerating adult muscle, where they giverise to new muscle fiber segments to replace those lost during muscle injury (reviewed inBischoff, 1994).Mechanisms of muscle fiber repair are of interest in understanding the restoration of muscleafter trauma or injury and during myopathic diseases. After severe injury, muscleregeneration is a slow process, during which scar tissue formation competes spatially withthe regenerating muscle fibers at the site of injury. Many approaches have been suggestedto enhance muscle restoration, including the use of muscle precursor cells, stem cells ormuscle fiber transplants (reviewed in Grounds, 1999 ). One of the disadvantages of celltransplantation methods is that most donor cells fail to survive (Rando et al., 1995 ; Fan etal., 1996 ; Beauchamp et al., 1997 ). Involvement of the host immune system in this rapiddeath has been suggested (reviewed in Smythe et al., 2000 ), but the precise mechanism ormechanisms underlying the phenomenon remain obscure.Low-energy laser irradiation (LELI) has been found to modulate various biologicalprocesses (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 regenerationand 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 studiedin 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 siteenhanced regeneration by two- and eightfold, respectively, relative to non-irradiatedcontrols (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 majorirradiation-responsive candidates (Weiss and Oron, 1992 ). This idea is supported bystudies of the effect of LELI on primary rat satellite cell cultures, revealing the induction ofcell cycle regulatory protein expression, increased satellite cell proliferation and inhibitionof cell differentiation (Ben-Dov et al., 1999 ). In the search for a mechanism by whichLELI manifests these cell responses, we very recently demonstrated that LELI specificallyactivates 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 phosphorylationof specific receptors (Shefer et al., 2001 ).Moreover, we have recently demonstrated a profound cardioprotective effect of LELI onchronic infarcted myocardium in rats and dogs, resulting in a 50 to 70% reduction in infarctsize 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 ofundamaged mitochondria and ATP content in the cardiomyocytes in the ischemic zone inLELI animals as compared with control non-irradiated ones (Oron et al., 2001 ). These dataled us to postulate that LELI enhances cell survival — a beneficial effect in terms of musclepreservation following injury or in myopathy. During normal myogenesis, as well as duringmuscular diseases (e.g., Duchenne muscular dystrophy), cells are known to self-destructwhen no longer needed or when damaged, by activating controlled machinery leading toprogrammed cell death or apoptosis (reviewed in Sandri and Carraro, 1999 ). Apoptosis is ahighly 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 shownapoptosis of replicating myoblasts upon deprivation of growth factors. The Bcl-2 familymembers are mainly involved at the mitochondrial level, and they play a key role in thechoice 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 andBak, can accelerate death and in some instances are sufficient to cause apoptosisindependently of other signals.In the present study, the effects of LELI on cell survival and proliferation were tested in asingle fiber system representing a three-dimensional organization of the surviving andrepaired segments of the fiber (Rosenblatt et al., 1995 ). This serves as a morecomprehensive model than standard tissue culture in which to assess satellite cellproliferation. We show that LELI promotes the cell cycle entry and accumulation ofsatellite 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 handin hand with increasing Bcl-2 and decreasing BAX expression in both these fibers andmyogenic cultured cells. Top Summary Introduction Materials and Methods Results Discussion References Materials and MethodsAnimalsMale C57B1/10 mice were maintained in the Animal Unit at the Hammersmith Campus ofthe Imperial College School of Medicine. Care and treatment of the animals complied withthe 1986 Animals (Scientific procedures) Act.Single fiber culturesSingle 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 werecarefully 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 plasticPetri dish containing DMEM, and muscle fibers were separated from the whole muscle byrepeated gentle pipetting with a wide-mouth Pasteur pipette. Fibers were plated, one perwell, in a 24-well Primaria tissue culture pre-coated with 1 mg/ml growth-factor-reducedMatrigel (Becton Dickinson Labware). The fibers were incubated in serum-free DMEM orin DMEM containing 0.1% (v/v) horse serum (HS) in a water-saturated environment at 5%CO2 and 37°C.Cell culturei28 mouse myogenic cells (Irintchev et al., 1998 ; Wernig et al., 2000 ) were grown inDMEM supplemented with 20% (v/v) fetal calf serum. For experiments, cells were plated at7x104 cells per 60 mm dish for one night in 20% fetal-calf-serum-containing medium, thenstarved for 36 hours in serum-free DMEM (Ben-Dov et al., 1999 ).Laser irradiationSingle fibers or i28 cells were irradiated for 3 seconds through a grid composed of 1.8mmx1.8 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 tothe incubator for a further day and cells accumulating around the fiber were counted dailyfor 5 days. In the case of i28 cells, cells were irradiated 36 hours post starvation (Shefer etal., 2001 ). Control, non-irradiated fibers and cells were kept under the same conditions astheir treated counterparts — they were kept on the bench beside the irradiated cultures.Western blot analysisCells 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), clarifiedby centrifugation and normalized to protein content (BCA kit; Pierce). Proteins wereseparated 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 theappropriate primary antibodies. The membrane was then washed with TBS-T and incubatedfor 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. Thefollowing primary antibodies were used: polyclonal anti-Bcl-2 (1:500; Oncogene ResearchProducts), polyclonal anti-BAX (1:500; Santa Cruz Biotechnology), polyclonal anti-p21(1:1,000; PharMingen) and a monoclonal antibody against p53 (1:500, Santa CruzBiotechnology).Immunofluorescent stainingSingle fibers were fixed with fresh 4% (w/v) paraformaldehyde for 20 minutes. Fibers werepermeabilized 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-2antibodies. Samples were incubated with primary antibodies overnight at 4°C followed bythree 15 minutes washes in PBS. Antibodies were detected with biotinylated swine anti-rabbit IgG (1:500 dilution; Dako) followed by Alexa-594-conjugated streptavidin (1:500dilution; Molecular Probes). The stained samples were then mounted with fluorescentmount medium (Dako) and viewed with a fluorescent microscope. Nuclei were detectedwith 46-diamino-2-phenylindole (DAPI; Sigma-Aldrich).5-Bromo-2-deoxyuridine (BrdU) incorporationSingle fibers were cultured for 18 hours in DMEM containing 4 µM BrdU and thenimmunostained 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% ethanolfor another 10 minutes and rinsed in PBS. The cultures were incubated for 30 minutes atroom temperature in 2 M HCl, followed by washes in 50 mM NaCl, 100 mM Tris-HCl, pH7.4. 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-diaminobenzidinetetrahydrochloride (DAB; Sigma).Cell viability and apoptosis assaysCell viability was assessed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5,-
diphenyltetrazolium bromide; Sigma-Aldrich) assay for mitochondrial activity. At theindicated times, MTT was added to the cells at a final concentration of 0.5 mg/ml for 2hours of incubation. For apoptotic measurements, nuclear DNA was stained by Hoechst33258 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. Cellswere scored as apoptotic only when they had pyknotic and/or fragmented nuclei(Stadelmann and Lassmann, 2000 ).StatisticsUnless otherwise indicated, raw data were analyzed using one- or two-way analysis ofvariance (ANOVA). In within-group tests, when data significantly deviated from thenormal distribution (Kolmogorof-Smirnov test for normality), a non-parametric t-test wascarried out. Alpha level was set to 0.05 in all experiments. Top Summary Introduction Materials and Methods Results Discussion References ResultsLELI promotes fiber-associated cells to enter the cell cycleOur experiments were aimed at investigating the effect of LELI on the accumulation ofcells near isolated fibers. Previous studies of ours have shown that LELI promoted cellcycle 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 fiberskept in serum-free medium for the entire experiment. Single myofibers were irradiated 19hours post plating, and the number of cells emanating from each fiber was monitored dailyfor 5 days following irradiation. Previously, it was shown that these cells are virtually allmyogenic 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 LELIfibers 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 immediateeffect of LELI on the cell cycle entrance of fiber-associated cells. Recently, we have shownthat 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 appliedimmediately 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). 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 conditionsWe 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 analysisdid 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 thesefour replicate experiments were pooled and averaged. The cumulative plot of the number ofcells per fiber clearly shows that as early as day 1 (i.e., 1 day post irradiation), the irradiatedfibers produced more cells than did the non-irradiated fibers (Fig. 2). The average numberof cells per fiber was twofold higher in the LELI fibers (P<0.001). A similar pattern ofdistribution was accompanied by an increase in the average number of cells per fiber, whichreached more than threefold the control group values, suggesting that LELI enables cellproliferation (Fig. 2, right panel; Fig. 1). However, overall proliferation was not pronouncedand cell number per fiber was even reduced on day 5 (Fig. 2, right panel), suggesting thatLELI is necessary but not sufficient to promote cell proliferation for long periods underserum-free conditions and that additional factors are required.
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 fibersTo test the relationship between LELI and serum, we conducted similar types of experimentbut now with fibers kept in low-serum-containing medium (0.1% HS). In general, on day 1fibers of both control and LELI groups already had relatively high numbers of adjacentcells, probably owing to the presence of the serum (Fig. 3A). However, in the case of thecontrol fibers, cell accumulation increased only slightly on day 2 and average cell numberper 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 amarked elevation in cell number per fiber, was seen (Fig. 3A, right panel), indicating theproliferation of most cells. This trend continued on subsequent days, and numerous fiberswith high numbers of adjacent cells were noted on day 5, despite the fact that the mediumwas never replaced during the experiment. A comparison of the relative changes of totalcell number (a pool of all experiments) in the LELI, HS and LELI+HS groups (Fig. 2, rightpanel, Fig. 3A, left and right panels, respectively) revealed that LELI and HS have asynergistic effect on cell proliferation (Fig. 3B) and that this effect is most pronounced ondays 3 and 5.
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 survivalThe results shown in Fig. 2 raised the possibility that under serum-starvation conditions,control fiber-associated cells remain quiescent. Another possibility is that under theseconditions cells die by apoptosis whereas those around LELI fibers survive. This is inagreement with previous demonstrations of the apoptotic effect of serum starvation in othercell 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 protectiveeffect of LELI under conditions of growth-factor deprivation, we tested the expression ofBAX, a death-promoting molecule, and Bcl-2, a survival protein, in single fibers that werenon-irradiated or had undergone LELI under serum-free conditions. Two fibers were platedin each 35 mm dish as far apart as possible, and only one half of the dish (with one of thefibers 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 ofthe three replications of this experiment, high levels of BAX were seen in the control fiberscompared with zero levels in the irradiated fibers (Fig. 4A, C). Conversely, high levels ofBcl-2 were seen in the irradiated fiber, in contrast to minimal levels of expression in thecontrol fiber (Fig. 4B,D).
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 theirassociated cells undergo apoptosis in a p53-dependent manner. p53 and its downstreamprotein p21 are involved in growth arrest or apoptosis following DNA damage or cellularstress (El-Diery et al., 1993; Ko and Privas, 1996; Levine, 1997 ). Moreover, p53 has beenshown to induce apoptosis by upregulating the BAX gene (Miyashita and Reed, 1995 ). Toanalyze the modulations in expression of these proteins as a consequence of LELI, weemployed a myogenic cell culture that had been previously shown to be driven into the cellcycle 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 wereor were not irradiated for 3 seconds (Shefer et al., 2001 ). Cells were left in DMEM andharvested 24 and 48 hours post irradiation, and equal amounts of protein wereelectrophoresed and analyzed for p53, p21, BAX and Bcl-2. Densitometry analysis revealedthat at zero time, cells expressed p53 and Bcl-2 proteins, whereas low or undetectable levelsof BAX and p21, respectively, were seen (Fig. 4E,F). The relatively high expression of p53at 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 capableof re-entering the cell cycle upon LELI or serum stimulation (Shefer et al., 2001 ). p53, aswell as BAX and p21 protein levels, were higher in the control non-irradiated cells 24 hourspost-irradiation than in the LELI cells (Fig. 4E,F). Conversely, expression of Bcl-2 wasfurther increased in LELI cells, while remaining undetectable in the control non-irradiatedcells, just as with the LELI fibers (Fig. 4D). However, after 48 hours, the differencebetween 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 redoxactivity 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 overallnumber of viable cells declined in all serum-free cultures. However, the reduction wasmuch more pronounced in the control non-irradiated cells (Fig. 5A), with a sharp decreasein cell viability between zero and 24 hours. After 48 hours, only 30% of the non-irradiatedcells 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, andapoptotic nuclei were scored by morphological criteria (Fig. 5B). The number of cellsdesignated apoptotic by these criteria, 24 hours post irradiation, showed an approximatelythreefold increase in the non-LELI cultures, compared with only a 1.5-fold increase afterLELI (Fig. 5C). This trend persisted for only the next 24 hours, reaching approximately30% apoptotic cells in the LELI group and nearly 50% in the control group. The differencebetween 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 notimprove the state of the cells, and they underwent the same level of apoptosis as did cellsthat had been subjected to LELI only once (data not shown). Discussion
TopPrevious demonstrations of the stimulatory effect of LELI on cell Summaryproliferation have been reported in primary mass cultures of Introductionsatellite cells and myogenic cell lines (Ben-Dov et al., 1999 ; Materials and MethodsShefer et al., 2001 ). However, such cultures do not closely mimic Results Discussionthe cell biology of myogenic cells within skeletal muscle, Referencesparticularly with respect to the condition of myogenic cells in thecell cycle. Myoblasts in tissue culture, even under low nutrient andgrowth factor conditions, do not accurately reproduce the G0 status of the dormant satellitecell. Accordingly, we extended the application of LELI to isolated single fibers, comprisingthe contractile muscle fiber and its associated satellite cells. It was important for ourpurposes that these latter cells are in G0 at the time of isolation and during the entirepreparation (Rosenblatt et al., 1995 ), thus giving the closest available approximation oftheir condition in vivo.Using this model system, we show that LELI promotes accumulation of satellite cells nearisolated single fibers grown under serum-free conditions and that the addition of even traceamounts of serum augments this effect synergistically. Such accumulation appears to reflectboth promotion in mitosis of myogenic cells and, at least under serum-free conditions, theirpreservation from apoptosis by LELI. Moreover, LELI aids the survival of fibers underthese pro-apoptotic conditions.The effect of LELI on fiber-adjacent cell proliferationIn 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 progressionof quiescent satellite cells on freshly isolated muscle fibers (Fig. 1). At the time ofirradiation, these cells are in contact with the muscle fiber and have not divided (data notshown). In fact, the number of cells that accumulated around non-irradiated fibers over theentire time period (Fig. 2) was about equal to the number of satellite cells residing on theaverage EDL muscle fiber (Beauchamp et al., 2000 ), and therefore, proliferation need nothave been involved (Fig. 1C). In contrast, proliferation has to have occurred to account forthe 20 or more cells that accumulated around the LELI fibers during the 2 days afterirradiation. Thus, LELI drives quiescent satellite cells into at least one round of proliferationunder 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: underserum-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 cellnumber, 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 cellsinto 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 ofLELI on the proliferation of cells associated with isolated fibers might be attributed tofactors secreted by the fibers in response to irradiation. However, additional LELI 24 hoursafter 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 thosesecreted 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, evenunder those conditions, the fact that the cells were uniformly stimulated into one or twoextra divisions implies that the proliferative effect of LELI involves only a transient releasefrom the tightly controlled mechanisms of cell cycle regulation.LELI enhances Bcl-2 and decreases BAX expressionOur demonstration that proliferation of satellite cells on myofibers is stimulated by LELIconfirms 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 enhancedsurvival of both the isolated fibers and fiber-associated myogenic cells as a consequence ofLELI. This was clearly seen under serum-free conditions, where the surviving cells and themyofibers in LELI cultures contained demonstrably higher levels of the anti-apoptoticprotein Bcl-2 and lower amounts of the pro-apoptotic protein BAX relative to controlfibers. 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 notmediated 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 highlevels of Bcl-2 proteins.Our observations fit with the widely accepted view that BAX overexpression promotes celldeath 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, andoverexpression of one antagonizes the effect of the other (Oltvai et al., 1993 ). Our findingof a clear reciprocal pattern of Bcl-2 and BAX expression in control versus LELI fibers andcultured cells strongly implicates regulation of these factors as part of the protective role ofLELI against apoptosis. In this same vein, we have recently reported that LELI specificallyactivates 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 togrowth 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 apoptosisafter growthfactor deprivation (Bossy-Wetzel et al., 1997 ; Miller et al., 1997 ). Thisexplanation is also in line with our finding of lower levels of p53, p21 and BAX in LELIversus untreated cells 24 hours after irradiation, because it has been shown that activatedp53 mediates growth arrest and apoptosis by activating the expression of a number ofcellular genes, such as p21 and BAX (El-Diery et al., 1993; Miyashita et al., 1994 ). Thisinhibition may involve activation of the MAPK/ERK signaling pathway by LELI (Shefer etal., 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 increasein 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 itsdownstream genes. The protective effect of Bcl-2 upregulation in response to LELI in bothfibers 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-mediatedcell survival and p53 regulation, we cannot rule out other mechanisms by which LELI mayprevent 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 aconstant 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 hasbeen reported to prevent mitochondrial dysfunction by regulating proton flux (Shimizu etal., 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 directlyinduces upregulation of Bcl-2 at the post-transcriptional level.Implications of LELI in muscle repairOur 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 entranceof quiescent satellite cells into the cell cycle, acting synergistically with serum elements toenhance their proliferation and survival. Moreover, the fact that neither LELIsenhancement of proliferation nor its protective effects against apoptosis were maintained fora long period argues against any mechanism involving cell transformation. These findingsare of the utmost importance in attempts to improve muscle regeneration following injury.Immediately after muscle injury (e.g., partial excision), the traumatized area also suffersischemic injury and a lack of nutrients and oxygen supply. The latter is renewed as newblood vessels form in the injured area. Nevertheless, during the initial phase, apoptosis maytake place in the injured fibers and satellite cells (U.O., unpublished). Apoptosis ofmyonuclei has also been shown in skeletal muscle during hindlimb unloading-inducedatrophy (Allen et al., 1997 ). Results of the present study suggest that LELI could reducethis process. Indeed, the above-mentioned phenomenon was demonstrated in single fibersas well as in cultured cells in low-serum and serum-free media, which may mimic theinitial, post-traumatic phase. Moreover, we have reported a profound cardio-protectiveeffect of LELI for cardiomyocytes in the infracted heart (Oron et al., 2001 ), furthersuggesting that LELI enhances cell survival. Taken together, we believe that LELI, as arelatively non-invasive technique that enhances both cell survival and proliferation, mightprovide an effective means of ameliorating the long-term consequences of muscle injury. Acknowledgmentsi28 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 EuropeanBiochemical Societies and European Molecular Biology Organization. L. Heslop issupported by Biotechnology BIO4 CT 95-0284 and the MRC Clinical Sciences Centre andJ.G. Gross by the MRC Clinical Sciences Centre.
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