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,1
* Biomolecular Characterization Team and the
Bioarchitecht Research Group, RIKEN, Wako, Saitama, Japan
1Correspondence: The Biomolecular Characterization Team and the Bioarchitect Research Group, RIKEN, 2–1 Hirosawa, Wako, Saitama 351-0198, Japan. E-mail: morishim{at}postman.riken.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apoptosis • Bcl-xL • caspase-12 • IGF-II • myoblast
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Previous studies established experimental conditions for in vitro fusion of myoblast cell lines by lowering the concentration of mitogen (3)
. Hence, myoblast cell lines (e.g., murine C2C12 cells) have been used to study muscle differentiation and have contributed to the analysis of myogenesis regulatory genes (4)
. Differentiated myoblast cultures may also provide material for tissue repair (5)
, and muscle tissue produced in vitro is promising for muscle compensation after age- and disease-related muscle loss (6
, 7)
. Since differentiated myofibers circumvent the possible dangers of tumorigenesis that are anticipated for proliferating cells, they have an advantage over myoblasts for tissue repair (8)
. In addition to these applications, myoblast cultures are useful for the study of insulin uptake and resistance (9
, 10)
, because skeletal muscle is the primary tissue responsible for glucose use in the postprandial state (11)
. Skeletal muscle is thus a major focus of research that aims to overcome the insulin resistance that occurs in diabetes.
In vitro, myoblasts alter their protein expression pattern, and their morphology changes significantly during myogenesis at the time that myoblasts begin to express muscle-specific structural proteins and fuse into multinucleated myotubes (3
, 12)
. However, myotubes formed in vitro do not always transform into myofibers that spontaneously contract (13)
. Furthermore, myofibers formed in vitro are smaller than those found in vivo (14)
. These observations suggest that factors critical for successful myogenesis are still missing in the established conditions for in vitro myogenesis. We recently refined the conditions for in vitro myogenesis during a study of myogenesis-associated apoptosis (15)
.
Apoptosis is an active process of cell suicide that is essential for successful organogenesis during development and normal physiological homeostasis throughout adulthood. The central executioner molecules of apoptosis are a family of cysteine proteases called caspases (16)
. Apoptosis is often associated with cell differentiation (17
, 18)
, and myoblast apoptosis is an example of developmental apoptosis in which a subpopulation of myoblasts undergoes apoptosis around the time that myoblast fusion occurs as differentiation begins. Although myoblast apoptosis was reported in the literature >50 yr ago (17)
, the cause of apoptosis and the mechanism that initiates caspase activity during differentiation were largely unknown. Thus, we have been focusing on identifying the trigger of caspase activation and the role of apoptosis during myoblast differentiation.
Our earlier work found that caspase-12, the endoplasmic reticulum (ER) stress-specific caspase, was extensively activated in differentiating myoblasts in vivo and in vitro (15)
. ER stress is defined as the cell state in which unfolded or misfolded proteins accumulate in the ER (19)
. Because it is likely that cells experience ER stress under pathological conditions, ER stress and caspase-12 are often discussed from the pathological viewpoint. Indeed, they are important topics in research on misfolded protein-related diseases, such as Alzheimer's (20)
and prion disease (21)
. However, our earlier observation suggests that ER stress is present, under physiological conditions, during the early stages of myoblast differentiation. Not only apoptotic cells, but also differentiating myoblasts, showed induction of two ER stress-specific proteins (15)
: CHOP, a transcription factor known to be up-regulated by ER stress (22
, 23)
; and BiP, an ER-specific molecular chaperone (reviewed in ref. 24
). Stress signaling during myogenesis appears to be mediated by activating transcription factor 6 (ATF6) (15)
. Remarkably, inhibition of ATF6 activation blocks apoptosis and myotube formation in culture models of myogenesis, suggesting that ER stress or ER stress signaling is required for these processes (15)
.
ER stress had never been considered a factor in establishing the conditions for in vitro differentiation of myoblasts. In the present study, we treated C2C12 myoblasts with the ER stress inducers tunicamycin and thapsigargin and examined whether increased ER stress during myogenesis enhanced myoblast differentiation. We found that the ER stress inducers enhanced apoptosis during myoblast fusion and that surviving cells were highly resistant to the apoptotic stimuli. Remarkably, the surviving cells efficiently formed contracting myofibers that are otherwise rarely found in culture systems of myoblast cell lines. The positive effects of ER stress are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture
C2C12 cells (RIKEN Cell Bank, Tsukuba, Ibaraki, Japan) were placed on gelatin-coated dishes and cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA, USA), supplemented with 20% (v/v) fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen) at 37°C with 5% CO2 [growth medium (GM)]. Cells were pretreated with ER stress inducers or remained untreated before transfer to differentiation culture. Note that, for cultures, the same batch of cells were seeded on culture plates and grown to subconfluence, and plates for either pretreatment or control cultures were randomly selected. To pretreat cells, GM was replaced with fresh medium that contained either 2 µg/ml tunicamycin or 1 µM thapsigargin, and cells were incubated for 0.5–6 h at 37°C with 5% CO2. Control cells were incubated in fresh medium that did not contain ER stress inducers. Differentiation was induced in differentiation medium (DM) with 2% horse serum (Invitrogen) instead of 20% fetal bovine serum and 1 µg/ml insulin (Sigma-Aldrich). The differentiation medium was replaced at 24 h intervals.
Antibodies
The primary antibodies used for immunoblotting were anti-alpha-actinin (Sigma-Aldrich), anti-Bcl-xL (BD Biosciences, San Jose, CA, USA), anti-cathepsin B (Upstate Biotechnology, Charlottesville, VA, USA), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Chemicon, Temecula, CA, USA), anti-horse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), anti-IGF-II (R&D Systems, Minneapolis, MN, USA), and anti-myosin (Sigma-Aldrich).
Western blot analysis
C2C12 cells were lysed in sample buffer (62.5 mM Tris-HCl pH 6.8, 6 M urea, 10% glycerol, 2% SDS, 0.003% bromphenol blue, and 5% mercaptoethanol). To break DNA, samples were sonicated on ice for 20 s and incubated for 15 min at 65°C immediately before loading on an SDS-polyacrylamide gel. Samples that were not immediately analyzed by gel electrophoresis were stored at –20°C. To analyze secreted proteins, culture media were centrifuged at 400 g for 10 min to remove cells. Cleared media were treated by the addition of one-fifth volume of 6x sample buffer (300 mM Tris-HCl pH6.8, 60% glycerol, 12% SDS, 0.6% bromphenol blue, and 0.6 M DTT) and boiled for 5 min. DTT was omitted from the buffer for a more sensitive analysis of procathepsin B. Lysates or media were separated on an SDS-polyacrylamide gel (12 or 14% acrylamide) and transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA). After blocking, the membrane was incubated with primary antibodies and then with a secondary horseradish peroxidase-conjugated anti-IgG antibody (Jackson ImmunoResearch Laboratories). Signals were detected using ECL-plus reagents (GE Healthcare Bio-Science, Piscataway, NJ, USA). GAPDH was used as a loading control for cell lysates, and horse IgG was used for culture media samples.
Quantitation of apoptosis
Apoptotic cells that were floating or loosely attached were recovered from 5–15 10-cm culture plates. Media was suctioned and centrifuged at 1000 g for 5 min at 4°C. Living cells were recovered by scraping the plates and centrifuging in microtubes under the same conditions. Cell pellet volumes (typically 5–40 µl) were measured by comparison to a series of standard tubes containing 1–50 µl water in 1 µl increments. Cell pellets were treated by addition of sample buffer (2x pellet volume to adjust cell density to the same value) to denature the proteins, and a portion of each denatured sample was subjected to SDS-polyacrylamide gel electrophoresis. Equal volumes of each sample were loaded on a single gel. The accuracy of the volumetric measurement was confirmed by Western blot analysis of GAPDH in the myoblast cells the band intensities of which should be comparable across lanes, because cell density and loading volume of each sample was the same.
Immunocytochemistry
C2C12 cells grown on 3.5 cm or 6 cm culture dishes (Nalge-Nunc, Rochester, NY, USA) were fixed and permeabilized in methanol at –20°C for 2 min. The fixed and permeabilized cells were blocked in PBS containing 3% BSA (Jackson ImmunoResearch Laboratories) and incubated overnight at 4°C with primary antibodies in blocking solution. Immunoreactivity was detected with a combination of biotin-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) and Alexa-conjugated streptavidin (Molecular Probes, Eugene, OR, USA). Cell nuclei were stained with DAPI (Sigma-Aldrich).
Time-lapse images
Cells were placed on a gelatin-coated culture dish and observed with an IX70 microscope (Olympus, Shinjuku, Tokyo, Japan) equipped with an ORCA cooled charge-coupled device camera (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan). Time-lapse images were acquired with IPLab Software (Scanalytics, Rockville, MD, USA) and edited in a QuickTime Player (Apple Computer, Cupertino, CA, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Live and apoptotic cells can be distinguished under a light microscope, based on their morphology. Apoptotic cells are rounder and much smaller than live cells and readily detach from the plate (Fig. 1
). Ten to 15% of C2C12 cells undergo apoptosis during the first 24 h of incubation in DM. After 24 h, the percentage of small round cells gradually decreased, while apoptosis continued to occur in a subpopulation of myoblast cells as well as in partially differentiated cells that expressed myosin (15)
. On the third day and thereafter, myosin was detected by immunocytochemistry (15)
. Although apoptosis in differentiating C2C12 cells continued to be observed after a week in DM, apoptosis in differentiating muscle tissues of embryonic mice is a transient event, observed for only a day or so after the onset of myotube formation at embryonic day 13.5 (25)
. We hypothesized that insufficient ER stress in vitro was at least partly responsible for those differences in the duration of the apoptotic period. Because ER stress has not been considered as a factor in establishing the conditions for in vitro differentiation of myoblasts, we conceived the idea of treating myoblasts with ER stressors to increase the degree of ER stress in vitro, and we predicted that would enhance apoptosis and myotube formation.
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We transiently exposed C2C12 cells to ER stress inducers in GM before transferring them to DM. ER stress inducers were used at doses that induce apoptosis in 40–50% of growing C2C12 cells after treatment with inducers for 24 h in GM (26)
. In preliminary experiments, myoblasts treated for 6 h with tunicamycin or thapsigargin recovered from ER stress after they were transferred to stressor-free GM (data not shown). However, when cells were pretreated for only 1 h and transferred to DM without stressors, a dramatic increase in apoptosis was observed. On day 1 in DM, 20–40% of the pretreated cells showed an apoptotic morphology (i.e., small and round), which corresponded to a two- to threefold increase in apoptosis (Fig. 1)
(15)
. Extensive caspase-12 activation was detected in the apoptotic cells, as in the case of untreated cultures (data not shown), indicating that the augmentation of apoptosis was due to further activation of the ER stress pathway (20
, 26)
. Although apoptosis was enhanced on days 1 and 2, apoptotic cells were rarely observed in the pretreated culture from day 3 onward (see the next section).
One of our concerns about microscopic quantification of apoptosis was that the number of apoptotic cells could be overestimated due to fragmentation of a dead cell, which might be interpreted as several apoptotic cells. Furthermore, fused myoblasts sometimes underwent apoptosis. Although this was infrequent, it made counting dead cells difficult. To analyze apoptosis in a more consistent manner, we collected cells that were floating or loosely attached to the plate by suctioning off the medium and centrifuging it, and we used the volume of the cell pellet as an index of apoptosis (see Materials and Methods).
The analysis of apoptosis based on the cell pellet volume demonstrated that apoptosis decreased gradually under standard differentiation conditions (Fig. 2
). This is consistent with our microscopic observation. In contrast, in pretreated cells, apoptosis was significant on day 1, quickly decreased on day 2, and reached background levels by day 3 (Fig. 2)
. Apoptosis of pretreated cells on days 1 and 2 was enhanced severalfold compared to apoptosis under standard conditions. The initial enhancement, and subsequent sharp fall, of apoptosis was reproduced in five independent experiments. This result suggests that, as expected, pretreatment with ER stress inducers increases ER stress induced apoptosis at the transition between proliferation and differentiation. Interestingly, apoptosis in pretreated cultures was nearly absent on day 3, while apoptosis under standard conditions persisted after day 3 in the differentiation cultures (Fig. 2)
. It is likely that enhanced ER stress signals expedited apoptosis that would otherwise persist for more than a week. In other words, apoptosis-prone cells in the differentiation culture were efficiently eliminated by day 3.
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In addition to augmenting apoptosis, pretreatment with tunicamycin or thapsigargin significantly improved cell fusion efficiency, resulting in differentiating cells that were morphologically distinct from those that were not pretreated. The pretreated myotubes increased in size for 
2 wk, and they fused during that time. They eventually became as large as in vivo mouse myofibers (1–3 mm long and 0.2–0.5 mm wide) and were more than 5- to 10-fold larger than the control cells (Fig. 3
A). The control cultures fused until about day 7, after which the myotubes did not grow significantly. Rather, they began to atrophy. Anti-myosin or anti-alpha-actinin staining of the syncytial cells revealed a cross-striated structure in nearly every myofiber in the pretreated cultures, demonstrating improved sarcomere formation (Fig. 3B
, TUN). In contrast, we did not observe cross-striation in the untreated cultures from days 7–12 (Fig. 3B, UT
, and data not shown). Furthermore, the number of nuclei in the pretreated syncytial cells was generally considerably higher than in untreated cells (Fig. 4
). Medium-sized myofibers, the cell contours of which could be clearly followed, contained more than 250 nuclei (Fig. 4)
. Myotubes in the control cultures contained 40 nuclei at most, which is comparable to values reported in the literature (e.g., 27
). These results indicate that pretreatment enhanced myofiber formation in terms of size, sarcomere formation, and fusion efficiency.
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Consistent with the formation of sarcomeres, myotubes began spontaneously contracting on day 7 (1–2 cycles/sec; see Supplemental Movie 1 online), indicating that they were functional myofibers. Fifty or more myofibers were reproducibly formed in each 3.5 cm dish using 10 different batches of cells, and most of the myofibers continued contracting until day 25 (Table 1
). Control cells (without pretreatment) formed a few myofibers that began to contract at about day 9 (see Supplemental Movie 2 online) but stopped by day 15. This suggests that additional ER stress enhances the effect of reduced mitogen concentration in provoking myofiber formation. ER stress seems to be unique in causing efficient formation of myofibers, since other apoptotic stimuli (e.g., etoposide) can increase myoblast apoptosis to a level comparable to that induced by ER stressors, but we could not determine any conditions in which such stimuli resulted in enhanced myofiber formation (data not shown).
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We achieved comparable improvement of myofiber formation and enhanced apoptosis by pretreating C2C12 cell cultures with tunicamycin or thapsigargin for 0.5–6 h (data not shown). This suggests that the effect of pretreatment is not simply proportional to ER damage (i.e., the number of misfolded proteins) and leads to the question of how comparable levels of apoptosis were achieved with such short pretreatment with ER stress inducers.
Apoptosis and survival after pretreatment with ER stress inducers are regulated by autocrine factors
To elucidate how ER stressor pretreatment of myoblasts regulates apoptosis and cell survival during the initial stage of differentiation, especially on days 1–2, we focused on a growth factor that holds the key to survival of differentiating myoblasts. Previous studies have revealed that insulin-like growth factor II (IGF-II) secreted in the medium regulates myoblast differentiation (28)
. IGF-II acts as an autocrine survival factor that binds to the insulin-like growth factor I (IGF-I) receptor and indirectly activates an Akt-dependent antiapoptotic pathway during the transition from proliferation to differentiation (29
, 30)
. IGF-II is induced on myoblast differentiation within 1 h of transferring cells to DM (31)
.
To examine whether pretreatment with the ER stressors affects IGF-II protein levels, we analyzed the conditioned media prepared after culturing cells in fresh DM for 24 h. IGF-II reached a level detectable by immunoblotting on day 2 in the conditioned medium of control cultures (CCM), whereas it was not detectable until day 4 in the conditioned media of tunicamycin- (TUNCM) or thapsigargin-treated (TGCM) cultures (Fig. 5
A). This suggests that lower levels of IGF-II are partially responsible for enhanced apoptosis during the first 2 days of culture in DM. It is possible that IGF-II is an important regulator of apoptosis during myogenesis that is linked to the ER stress response. Consistent with this idea, adding recombinant IGF-II to the medium nearly completely prevented apoptosis of differentiating C2C12 cells on day 1, with or without pretreatment (Fig. 5B
). Consistent with the pattern for extracellular IGF-II, levels of intracellular IGF-II precursor in pretreated cells were lower than those in control cells on days 1- 3 (Fig. 5C
), suggesting that the decrease in extracellular IGF-II results, in part, from a decrease in its synthesis.
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Previous studies have shown that the level of extracellular IGF-II is modulated as a result of its internalization by the IGF-II/mannose-6-phosphate (Man-6-P) receptor (32)
. Ligands of this receptor, including procathepsin B (33)
, are proteins that have been modified with Man-6-P. Although Man-6-P-modified procathepsin B levels were constant in CCM from days 1–3, levels of the modified protein were much lower in pretreated cultures on day 1 (Fig. 5D
). Only the unglycosylated form of procathepsin B was detected in TUNCM cultures, whereas relatively little of the glycosylated or unglycosylated forms were detected in TGCM cultures. The decrease in extracellular Man-6-P-modified proteins, including procathepsin B, may facilitate the association of IGF-II and its receptor. These results suggest that pretreatment of myoblasts with ER stress inducers reduces IGF-II synthesis and enhances internalization of extracellular IGF-II by the IGF-II/Man-6-P receptor.
Higher resistance of the surviving pretreated cells
Analysis of myoblast apoptosis after pretreatment with ER stressors at the early stage of differentiation has shown that for 2 days after pretreatment, apoptosis is enhanced, after which the rate of apoptosis declines significantly (Fig. 2)
. Enhanced apoptosis in pretreated cultures can be explained by a delayed appearance of IGF-II protein in the medium. However, this cannot account for the cessation of apoptosis from day 3 on. It is reasonable to assume that cell survival depends on the vulnerability of the cell to ER stress and that myoblasts that did not undergo apoptosis within 2 days of pretreatment were somehow resistant to ER stress. To confirm that the surviving cells were resistant to ER stress induced apoptosis, we examined the vulnerability of living myoblasts at an early stage of differentiation. The percentage of dead cells in myoblasts cultured without pretreatment gradually decreased over a week. From day 3 on, 5% or fewer cells (assessed by microscopic observation) underwent apoptosis. On day 3, we treated differentiating C2C12 cells with the apoptotic inducers tunicamycin or thapsigargin at doses that would induce 40% apoptosis (26)
.
We observed that growing and differentiating myoblasts were hypersensitive to ER stressors when treated in DM. For instance tunicamycin (2 µg/ml), which would normally induce apoptosis in 30–40% of C2C12 cells in GM, completely killed the cells in DM. This lethal effect of ER stressors is likely due to the reduction of mitogen concentration in DM. Therefore, we cultured myoblasts in DM for 2 days and then treated them with ER stressors in GM. Control myoblasts cultured in DM for 2 days showed slightly more resistance to ER stress inducers than proliferating cells grown in GM. Twenty to 30% of the cells cultured in DM underwent apoptosis (Fig. 6
). In contrast, pretreated cells cultured in DM for 2 days were highly resistant to the ER stress inducers (Fig. 6)
. Interestingly, the pretreated cells were also resistant to apoptotic stimuli that were not associated with ER stress. For instance, on day 3, differentiating cells were also resistant to staurosporine (a nonselective protein kinase inhibitor) and etoposide (a topoisomerase II inhibitor; Fig. 6
). These results indicate that the surviving pretreated cells acquired a general resistance to apoptosis that was not limited to ER stress induced apoptosis.
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We next examined whether Bcl-xL, a potent anti-apoptotic protein (34)
, was involved in the increased resistance of differentiating myoblasts. Bcl-xL exerts its anti-apoptotic effect on a wide range of apoptotic stimuli, including ER stress. We previously reported that, in C2C12 cells, Bcl-xL binds to proapoptotic BH3-only proteins, such as Bim, and inhibits BH3-only proteins from triggering caspase-12 (35)
. We compared levels of Bcl-xL in proliferating and differentiating cells, separating the latter into surviving and apoptotic populations (see Materials and Methods). Figure 7
A shows that the level of Bcl-xL in surviving cells on days 1–7 was almost the same as that in the proliferating cells, irrespective of pretreatment. Thus, it is difficult to explain higher resistance of the pretreated cells in terms of the level of Bcl-xL. On the other hand, the level of Bcl-xL in apoptotic cells was lower than that in proliferating cells and surviving cells (Fig. 7A
). This result is consistent with the idea that low levels of Bcl-xL are at least partly responsible for vulnerability of apoptotic cells. Differences in the level of Bcl-xL in surviving and apoptotic cells were observed specifically in differentiating C2C12 cells. In the case of C2C12 cells grown in GM and treated with ER stressors, the level of Bcl-xL in apoptotic cells differed only negligibly from that in living cells (Fig. 7B
). This result suggests that negative selection, based on Bcl-xL level, operates specifically during myoblast differentiation. It is possible that decreased Bcl-xL is an important determinant of apoptosis during myoblast differentiation (see Discussion).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Taken together, these results support the idea that ER stress has a beneficial, and possibly essential, role in successful myogenesis. The positive effects of ER stress include elimination of vulnerable cells, thereby excluding these cells from myotube formation and augmenting resistance to apoptosis. Alternatively, but not mutually exclusive, it is possible that treatment with stressors changes the cell population and enhances its potential for differentiation.
Examination of the vulnerability of differentiating cells has shown that C2C12 cells acquire apoptotic resistance after partially undergoing differentiation (Fig. 6)
. The level of resistance of the cell population might be related to the extent of stress it has experienced. For instance, pretreated differentiating cells are more resistant than untreated cells, which in turn, are more resistant than proliferating cells. Cell cultures contained at least two types of living cells during myogenesis: stress-resistant cells that formed large myofibers and stress-sensitive cells that survived standard differentiation conditions but were eliminated by apoptosis after pretreatment (Fig. 8
). It is likely that resistant cells are competent to efficiently fuse with one another and form large myofibers. In the case of standard differentiation culture (i.e., no pretreatment), however, cell fusion included both resistant and sensitive cells, and the efficiency of sensitive cell fusion may be lower than that of resistant cells. Alternatively, but not mutually exclusive, stress-sensitive cells may inhibit further fusion if they are included in multinucleated myotubes (Fig. 8)
.
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It would make sense if muscles formed from resistant myoblasts, because muscle contraction inevitably occurs under stressful conditions. Ca2+ is a key regulator of muscle contraction (reviewed in ref. 36
). Ca2+ released from the sarcoplasmic reticulum binds to troponin that transmits information via structural changes throughout actin-tropomyosin filaments, activating myosin ATPase activity and muscle contraction. Since fluctuations in Ca2+concentration can induce ER stress (37)
, resistance of myofibers to ER stress is critical for repeated contraction. Thus, the system that eliminates apoptosis-prone myoblasts from terminal differentiation may have appeared during vertebrate evolution due to selective pressure.
On the basis of the present study, we propose that appropriate stress and stress induced apoptosis control the quality of differentiating myoblasts so that only apoptosis-resistant cells go through terminal differentiation to form muscle tissue. Quality control by apoptosis during myoblast differentiation would be a novel role for apoptosis in the sense that apoptosis controls the quality rather than the number of cells. Although the major role of apoptosis during development has been thought to be removal of excess cells (38)
, it would be interesting to examine whether the concept that we propose for apoptosis in myogensis is applicable to organogenesis systems other than muscle tissue.
We detected a reduction of Bcl-xL in apoptotic cells (Fig. 7A
) compared to resistant cells, which may be related to myoblast elimination by apoptosis. This result can be interpreted in at least two ways: 1) the C2C12 cultures were already heterogeneous in terms of Bcl-xL level during proliferation, or 2) heterogeneity was generated during differentiation. Unfortunately, we could not detect variation in Bcl-xL expression levels among individual C2C12 cells, either proliferating or differentiating, although we attempted to do so with immunocytochemistry (data not shown). We analyzed single clones derived from the C2C12 culture to examine if isolated clones also became heterogeneous. Cells in the single clones partitioned into cells undergoing apoptosis or differentiation (data not shown). This result favors the idea that the C2C12 cells were initially homogeneous in terms of vulnerability and Bcl-xL expression but became divided during differentiation into cells with a tendency to undergo apoptosis or with a greater tendency to form myotubes. Since Bcl-xL has an ability to enhance cell cycle arrest on the one hand and regulates apoptosis on the other hand (39)
, it seems reasonable to eliminate cells with reduced levels of Bcl-xL. The mechanism underlying this observation will be interesting to study in the future.
We previously showed that ER stress is extensively generated in developing muscle tissues in vivo (15)
. The present results imply that exposure of myoblasts to ER stress inducers for a short time can mimic transient stress in vivo in a more precise manner than treating myoblasts under established differentiation conditions. The pretreatment is quite simple, involving only the addition of ER stress inducers before transferring cells to DM, yet its effect is significant and reproducible. There have been several attempts to induce myoblast fusion by imitating in vivo conditions, including use of mechanical and electrical stimulation (7)
. Compared to these methods, transient exposure to ER stressors may more directly affect gene regulation and/or protein expression necessary for myofiber formation. In this study, we have identified IGF-II, procathepsin B, and Bcl-xL as candidate critical factors that are regulated by ER stress during myoblast differentiation. It is likely that the combined action of these factors makes the difference between cell survival and death, thereby eliminating apoptosis-prone cells from the process of myotube formation. It is not yet clear whether other, unidentified factors are also involved in that decision process.
It is likely that a transient increase in ER stress causes alterations in intracellular signaling and/or gene and protein expression. An immediate effect of ER stress on cells is rapid induction of a process known as the unfolded protein response (UPR), which occurs much earlier than caspase activation (35)
. The UPR involves ER stress-specific intracellular signaling, up-regulation of protein folding and degradation pathways in the ER, and inhibiting protein synthesis in the cytosol (40
, 41)
. It has been established that the UPR alters transcription (42)
and protein expression (43)
patterns involved in cellular self-defense, although all of the target proteins of the UPR have not been identified. Identification of factors critical for initiation of apoptosis and acquisition of resistance during myofiber formation will also be a topic of future studies.
Conditions that enable myoblasts to undergo efficient myofiber formation are of great interest from a therapeutic standpoint, because they are important for enhancing muscle growth after injury or decreasing the loss of muscle mass due to disease or aging. The ability to induce myoblasts to undergo efficient myofiber formation may be applicable to myofiber transplantation in patients with muscle damage. In addition, developing methods to prevent loss of muscle mass during space travel will become increasingly important. Thus, the present results not only reveal a benefit of ER stress in muscle development, but they bring us closer to developing improved treatments for muscle damage and loss.
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ACKNOWLEDGMENTS |
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Received for publication January 9, 2007. Accepted for publication March 8, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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impairs insulin signaling and insulin stimulation of glucose uptake in C2C12 muscle cells. Am. J. Physiol. 276,E845-E849This article has been cited by other articles:
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  M. Kitamura Endoplasmic reticulum stress and unfolded protein response in renal pathophysiology: Janus faces Am J Physiol Renal Physiol, August 1, 2008; 295(2): F323 - F334. [Abstract] [Full Text] [PDF]
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, UT; 
, TUN; 
, TG.
