Laboratory of Muscle Biology and Sarcopenia, Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia
Submitted 12 October 2004 ; accepted in final form 9 December 2004
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ABSTRACT |
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inhibitor of DNA binding/differentiation protein; tumor suppressor gene; programmed cell death; aging
The inhibitor of DNA binding/differentiation (Id) protein family is structurally characterized by the helix-loop-helix (HLH) motif but lacks a basic amino acid domain necessary to bind DNA (810, 40, 47, 56). It has been suggested that the Id protein is one of the important regulators in cell proliferation, differentiation as well as apoptosis in various cell types (56). Id2 is one of the four members of this family (i.e., Id1Id4). It can promote Bax-mediated apoptosis independently of the HLH dimerization (17). Previous studies conducted at our laboratory have shown that Id2 is associated with the apoptosis-related skeletal muscle atrophy and age-associated sarcopenia (46). Although additional research is required to identify the entire molecular and cellular mechanisms by which Id2 may be involved in apoptosis regulation, it is reasonable to hypothesize that Id2 may be involved in the upstream regulatory mechanism for apoptosis during muscle atrophy on the basis of findings regarding the close relationship between Id2 and apoptosis.
Activation of tumor suppressor protein p53 has been shown to initiate cell growth arrest via p21Cip1/Waf1 mediation or the execution of Bax-associated apoptotic cell death in various cell types (53). It was recognized recently that p53 is one of the central modulators in cell cycling and apoptosis (53). Although most research in p53 has been conducted in active dividing cells, a few studies have attempted to investigate the response of p53 during disuse, laser irradiation, and ischemia in postmitotic skeletal myocytes (20, 28, 38, 42, 49). However, the possible role of p53 in regulating the apoptotic signaling pathway during unloading-induced muscle atrophy is still unclear. In addition, the proto-oncogenic transcription factor c-Myc also has been implicated in the initiation of apoptotic cell death, and its expression was found in skeletal myocytes during the postnatal development (52, 53). Nevertheless, whether c-Myc is involved in muscle unloading-induced apoptosis remains unknown. Accordingly, understanding the possible roles of Id2, p53, and c-Myc in mediating the apoptotic signaling pathway during muscle atrophy will provide insight into the mechanisms of the significant myocyte loss during muscle disuse and sarcopenia.
Recently, we demonstrated activation of apoptosis and apoptosis-regulatory factors to unloading-induced muscle atrophy after hypertrophy, and we found differential apoptotic responses to unloading after hypertrophy between young and aged quails (50). In the present study, we have investigated the regulatory mechanism of muscle atrophy-associated apoptosis during unloading using the same experimental model. We tested the hypothesis that Id2, p53, and c-Myc are activated as part of the apoptotic responses during unloading-induced muscle atrophy after hypertrophy.
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METHODS |
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Muscle overloading and unloading protocol.
The patagialis (PAT) muscle is flexed with the wing on the bird's back at rest, but it is stretched when the wing is extended. In our experimental stretch-overloading model, a tube containing 12% of the bird's body weight was placed over the left humeral-ulnar joint (7). This maintains the joint in extension throughout the period of stretch and induces stretch at the origin of the PAT muscle. Previous studies have shown that this stretch-overloading protocol results in moderate hypertrophy of the PAT muscles (i.e., 14-day stretch-loading induces
35% and
15% increases in the muscle mass of young adult and aged birds, respectively) (2, 51).
The left wing of animals was overloaded for 14 days and then the weight was removed. Seven days after the removal of weight, eight young and eight aged birds were killed by administration of an overdose of pentobarbital sodium. The remaining young and aged animals were killed 14 days after weight removal. The unstretched right PAT muscle served as the intra-animal control muscle for each bird. PAT muscles were dissected from the surrounding connective tissue, removed, weighed, frozen in isopentane cooled to the temperature of liquid nitrogen, and then stored at 80°C until used for analysis.
Protein extraction and subcellular fractionation. Cytoplasmic and nuclear protein extracts were obtained from PAT muscles using the method described by Rothermel et al. (46). Briefly, 50 mg of muscle were homogenized in ice-cold lysis buffer (10 mM NaCl, 1.5 mM MgCl2, 20 mM HEPES, pH 7.4, 20% glycerol, 0.1% Triton X-100, and 1 mM dithiothreitol). The cytoplasmic protein fraction contained in the supernatants was obtained after centrifugation at 1,000 rpm for 1 min at 4°C. The nuclei in the remaining pellet were resuspended in 360 µl of lysis buffer and 49.8 µl of 5 M NaCl. The nuclear pellet was incubated on a rocker for 1 h at 4°C and centrifuged at 14,000 rpm for 15 min at 4°C, and the nuclear protein fraction was collected from the supernate. A protease inhibitor cocktail (P8340; Sigma-Aldrich, St. Louis, MO) was added to the cytoplasmic and nuclear protein extracts, and then the total protein contents of the extracts were quantified in duplicate using bicinchoninic acid reagents (Pierce Biotechnology, Rockford, IL) and bovine serum albumin standards. The purity of the extracted fractions was verified by immunoblotting the extracted fractions with an anti-histone H2B and an anti-Cu-Zn-SOD rabbit polyclonal antibody (50). The cytoplasmic fraction protein was later used for immunoblotting of superoxide dismutase (SOD; Cu-Zn-SOD and Mn-SOD), while c-Myc was determined in the nuclear fraction protein. Protein contents of Id2 and p53 were measured in both the cytoplasmic and nuclear fraction proteins.
Western immunoblot analysis. Protein expression of transcription repressor Id2, tumor suppressor p53, proto-oncoprotein c-Myc, and antioxidant enzymes (Cu-Zn-SOD and Mn-SOD) was determined in the PAT muscles of experimental (left) and intra-animal control (right) wings. Forty micrograms of soluble protein were boiled for 5 min at 95°C in Laemmli buffer, loaded onto each lane of a 12% polyacrylamide gel, and separated by performing SDS-PAGE. The gels were blotted onto nitrocellulose membranes (Bio-Rad, Hercules, CA) and stained with Ponceau red S (Sigma Chemical, St. Louis, MO) to confirm equal loading and transferring of proteins to the membrane in each lane. Another approach used in verifying similar loading between the lanes was to load gels in duplicate, with one gel stained with Coomassie blue. The membranes were then blocked in 5% nonfat milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T) and probed with anti-Id2 rabbit polyclonal antibody (1:200 dilution, sc-489; Santa Cruz Biotechnology, Santa Cruz, CA), anti-p53 mouse monoclonal antibody (1:200 dilution, sc-99; Santa Cruz Biotechnology), anti-c-Myc mouse monoclonal antibody (1:250 dilution, 631206; BD Pharmingen, San Diego, CA), anti-SOD-1 rabbit polyclonal antibody (1:500 dilution, sc-11407; Santa Cruz Biotechnology), or anti-Mn-SOD goat antibody (1:2,000 dilution, A300449A; Bethyl Lab, Montgomery, TX) diluted in PBS-T with 2% BSA. Whole cell lysate of actively dividing human 293T cells was included as a positive control for probing Id2 and p53. Primary antibody incubation was performed at 4°C overnight. Secondary antibodies were conjugated to horseradish peroxidase (HRP; Chemicon, Temecula, CA), and the signals were developed using West Pico chemiluminescent substrate (34080; Pierce Biotechnology, Rockford, IL). The signals were visualized by exposing the membranes to X-ray films (BioMax MS-1; Eastman Kodak, Rochester, NY), and digital records of the films were captured using a Kodak 290 camera. The resulting bands were quantified as optical density (OD) x band area using a one-dimensional image analysis system (Eastman Kodak, Rochester, NY) and expressed in arbitrary units. The sizes of the immunodetected proteins were verified using a prestained standard (LC5925; Invitrogen/Life Technologies, Bethesda, MD).
Immunofluorescence cytochemistry. Nuclear protein contents of p53 and c-Myc were further estimated by immunofluorescent staining. In brief, frozen, 10-µm-thick muscle cross sections from the middle belly of the experimental and control PAT muscles were cut in a freezing cryostat at 20°C and placed onto the same glass slide to control for processing differences (e.g., incubation time, temperature). The midregion of PAT muscle was used to prepare the muscle section on the basis of the previous finding that the hypertrophied effect of stretch loading is greatest in the proximal and distal regions of quail anterior latissimus dorsi muscle (7). The tissue sections were air dried at room temperature, fixed in ice-cold methanol-acetone (1:1) for 10 min, permeabilized with 0.2% Triton X-100 in 0.1% sodium citrate at 4°C for 5 min, and blocked in 1.5% goat serum in PBS. All incubations were performed at room temperature for 30 min. After being washed in PBS, sections were incubated with anti-p53 mouse monoclonal antibody (1:20 dilution, sc-99; Santa Cruz Biotechnology) or anti-c-Myc mouse monoclonal antibody (1:20 dilution, 631206; BD Pharmingen, San Diego, CA), followed by anti-mouse IgG Cy3 conjugate F(ab')2 fragment incubation (1:200 dilution, C2181; Sigma Chemical). Negative control experiments were done by omitting the p53 or c-Myc antibodies on the tissue sections. To visualize the labeled nuclei under the basal lamina of PAT muscles and thereby identify the muscle-originated nuclei (e.g., myonuclei or satellite cell nuclei), the tissue sections were then incubated with anti-chick laminin mouse monoclonal antibody (20 µg·ml1, clone 31-2; D. M. Fambrough, The Johns Hopkins University, Baltimore, MD), followed by anti-mouse IgG biotin-conjugated antibody (Vector Laboratories, Burlingame, CA) and then by Fluorescein Avidin DCS incubation (1:200 dilution, A2011; Vector Laboratories). The sections were finally mounted with 4',6-diamidino-2-phenylindole (DAPI) mounting medium (Vectashield mounting medium; Vector Laboratories). The p53- or c-Myc- and DAPI-positive nuclei and laminin staining were examined under a fluorescence microscope (Biological Research Microscope Eclipse E800, Nikon, NY). Images were obtained using a SPOT RT camera and SPOT RT software (Diagnostic Instruments, Sterling Heights, MI) was used to stack the images of p53- or c-Myc-, DAPI-positive nuclei, and laminin staining. The numbers of p53- or c-Myc-positive nuclei and DAPI-positive nuclei were counted from six random nonoverlapping fields under x40 magnification. Only the labeled nuclei that were under the laminin staining were counted to exclude any nonmuscle nuclei (e.g., from fibroblasts). It is noted that c-Myc-immunopositive nuclei were not detected in the muscle sections obtained from all groups of the birds. The p53-positive nuclei were expressed as a p53 nuclear index, which was calculated from the number of immunopositive nuclei divided by the total number of nuclei (i.e., DAPI-positive nuclei) x 100.
To determine the mitotic property of the p53-positively labeled nuclei (i.e., mitotic or postmitotic), bromodeoxyuridine (BrdU) labeling on the muscle sections after the p53 labeling procedure was performed in a separate set of measurements. Briefly, after p53 and Cy3 labeling, the tissue sections were incubated with anti-BrdU mouse monoclonal biotinylated antibody (1:5 dilution, 75512L; BD Pharmingen, San Diego, CA), followed by incubation with fluorescein avidin DCS (1:100 dilution, A2011; Vector Laboratories), and finally mounted with DAPI mounting medium. The p53-, DAPI-, and BrdU-positive nuclei were examined under a fluorescence microscope, and the captured images were stacked using SPOT RT software as described above.
Statistical analysis. Statistical analyses were performed using the SPSS version 10.0 software package. ANOVA (2 x 2) was performed to examine the main effects of time (7 and 14 days of unloading), age (young and aged), and interaction (time x age) on the measured variables in all groups of animals. Student-Newman-Keuls post hoc analysis was used to examine differences between experimental and contralateral control PAT muscles. Relationships between given variables were examined by computing the Pearson product-moment correlation coefficient (r). Statistical significance was accepted at P < 0.05. All data are expressed as means ± SE.
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RESULTS |
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TUNEL index. The TdT-mediated dUTP nick end labeling (TUNEL) index was used to estimate the extent of apoptosis, which has been described previously in this group of birds (50). In these animals, the TUNEL index in unloaded muscles was 246% (P < 0.01) greater than that in the control muscle after 7 days of unloading, but it had returned to control levels by 14 days of unloading in young quail. The TUNEL index in experimental muscles of aged birds was 91% (P < 0.001) and 44% (P < 0.01) greater than that in the control muscle after 7 days and 14 days of unloading, respectively.
Id2 protein content.
An immunoreactive band of 15 kDa corresponding to the predicted molecular mass of Id2 protein was detected in both the cytoplasmic and nuclear protein fractions of all muscles. In our immunoblots, nuclear Id2 protein content was not different between the experimental and control muscles after 7 and 14 days of unloading in both young adult and aged quails (Fig. 1A).
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p53 protein content.
An immunoreactive band of 53 kDa corresponding to the predicted molecular mass of p53 protein was detected in both the cytoplasmic and nuclear protein fractions of all muscles. The nuclear p53 protein content of the experimental muscle was 126 ± 71% (P < 0.05) greater than that of the control muscle after 7 days of unloading, but no difference was found between the experimental and control muscles after 14 days of unloading in young quails (Fig. 2A). In the aged quails, we did not find any significant changes in the nuclear p53 protein content of the experimental muscle compared with the intra-animal control muscle after 7 or 14 days of unloading (P > 0.05) (Fig. 2A).
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c-Myc protein content.
An immunoreactive band of 62 kDa was detected on immunoblots, which corresponded to the predicted molecular mass after SDS-PAGE fractionation of the c-Myc protein. c-Myc protein content was similar in experimental and intra-animal control muscles in both young and aged quails after 7 or 14 days of unloading (P > 0.05, Fig. 3).
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p53 positively labeled nuclei and p53 nuclear index. The number of p53-immunopositive nuclei relative to the total nuclei was expressed as a p53 nuclear index. An example of immunopositive staining for p53 under the laminin staining in an experimental PAT muscle from a young quail after 7 days of unloading is shown in Fig. 5A. ANOVA showed a main effect of age [F(1,56) = 4.437; P < 0.05] in the p53 nuclear index. The p53 nuclear index of the experimental muscle was 87% (P < 0.05) greater than that of the control muscle after 7 days of unloading, but no difference was found between the experimental and control muscles after 14 days of unloading in young quails (P > 0.05) (Fig. 5B). The p53 nuclear indices of the experimental muscles tended to be greater than those in the control muscles after 7 or 14 days of unloading in aged quails; however, these changes did not reach statistical significance.
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When the experimental and control PAT muscles of all groups were collapsed and treated as a single group (n = 64), cytoplasmic Id2 protein content was negatively correlated with the Bcl-2 protein content (r2 = 0.287; P < 0.05) (Fig. 7A) and positively correlated with the TUNEL index (r2 = 0.426; P < 0.001) (Fig. 7B), Bax protein content (r2 = 0.261; P < 0.05) (Fig. 7C), and cytoplasmic p53 protein content (r2 = 0.289; P < 0.05) (Fig. 7D).
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DISCUSSION |
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Id2 in muscle remodeling. Id proteins are known to play a role in promoting cell proliferation and act as negative regulators of cell differentiation in numerous cell lineages (810, 47, 56). However, there is evidence suggesting that Id proteins also may have a role in modulating apoptosis (4, 6, 17, 19, 40). Florio et al. (17) demonstrated that Id2 can initiate Bax-associated apoptosis and that the apoptogenic properties of Id2 reside in the NH2-terminal region independent of HLH dimerization. Consistent with these reports, previous studies conducted at our laboratory have illustrated the dual roles of Id2 in promoting cell proliferation and apoptotic cell death during muscle hypertrophy and atrophy, respectively, in skeletal myocytes in vivo (4, 6). In the present study, we have extended our previous findings by demonstrating that the cytoplasmic Id2 protein content, but not the nuclear Id2 protein content, was elevated in the young adult unloaded muscle relative to the control muscle after 7 days of unloading after hypertrophy. In addition, we have found that the cytoplasmic protein content of Id2 correlated positively with the proapoptotic markers (e.g., Bax, AIF, p53, and TUNEL index) and negatively with the antiapoptotic marker (e.g., Bcl-2). Notably, the nuclear protein content of Id2 was unchanged in response to unloading after hypertrophy and was negatively correlated with the proapoptotic factors, including Bax and p53. We interpret these findings to indicate that the cytoplasmic Id2 is associated with proapoptotic events, whereas the nuclear Id2 may be related to the antiapoptotic affair and probably to the promotion of cell proliferation. This speculation is supported by data showing that the apoptogenic ability of Id2 is associated with Bax, which is a cytoplasmic protein (17).
In contrast, the proliferating and antiapoptotic properties of the nuclear Id2 may be due to the indirect effects of Id2 on transcriptional regulation in nuclei. On the basis of the fact that Id2 can suppress the transcription of genes that regulate cell differentiation by sequestering the E proteins (e.g., E12, E47, E2-2) and preventing the heterodimer formation of E proteins with the basic HLH transcription factors (9, 10, 35, 56), it is reasonable to speculate that Id2 may be capable of influencing the transcription of the genes associated with apoptosis and proliferation. Indeed, Ling et al. (34) have demonstrated that Id1 can promote cell proliferation by inducing antiapoptotic factor expression via the activation of the NF- pathway in prostate cancer cells (34). In addition, Id2 has been implicated to explain the elevated level of an apoptosis inhibitor, survivin, and the deficiency of procaspase-8 in neuroblastoma cells (11). Moreover, it has been shown that Id2 can abrogate the growth-suppressive functions of retinoblastoma protein (pRb), p16, p21, and tumor suppressor proteins by interacting with pRb (29, 30, 37, 40, 47, 56). Nevertheless, we are aware that our data do not permit us to conclude whether the nuclear Id2 is directly related to the increased expression of the proliferation-related genes and/or the decreased proapoptotic gene expression during muscle remodeling. Although we have demonstrated that the cytoplasmic Id2 is associated with apoptosis during unloading after hypertrophy, additional research is required to examine whether the nuclear Id2 is directly related to cell proliferation in response to the hypertrophic stimulus. Nevertheless, the data in the present study suggest that the subcellular fractionated protein expression (cytoplasmic vs. nuclear protein content) maybe an important factor in determining the physiological roles of Id2.
p53 during unloading-induced apoptosis after hypertrophy. It has commonly been recognized that p53 plays a critical role in regulating cell cycling, survival, and programmed cell death in various mitotic cell lineages (53). However, there is a relative paucity of data on the role of p53 in skeletal myocytes. Although a skeletal myocyte is biologically different from other mitotic cell types because it is terminally differentiated and postmitotic (21), it is reasonable to hypothesize that p53 may have a role in regulating muscle atrophy. In support of this idea, p53 has been shown to be one of the central modulators in apoptosis in a tissue-nonspecific manner (53). Furthermore, p53 protein levels increase in atrophied rat skeletal muscles after a 14-day spaceflight (42). However, a consensus regarding the role of p53 in muscle remodeling is still lacking, because p53 has been reported to be unchanged in skeletal muscles during atrophy induced by nerve injury, steroid, or alcohol (28, 32, 38).
In the present study, we have demonstrated that both the nuclear and cytoplasmic fractions of p53 are elevated in the unloaded muscle relative to the control muscle after 7 days of unloading after hypertrophy in young adult animals. We also have shown a positive correction of p53 protein content and proapoptotic markers (Bax, AIF, and TUNEL index) and a negative correlation of p53 with antiapoptotic marker Bcl-2. Our findings are in accord with the documented apoptogenic properties of p53 in numerous cell types (13, 14, 44, 53, 54). In the nuclei, p53 has been suggested to mediate apoptotic cell death by elevating the transcriptional expression of several proapoptotic genes (e.g., Bax, PUMA, and Noxa) (24, 41, 48). Thus it was not surprising that the nuclear fraction of p53 protein content increased concomitant with the TUNEL index and the Bax-to-Bcl-2 ratio during unloading-induced muscle atrophy after hypertrophy (50). In further support of the elevation of nuclear p53 protein content measured using immunoblot analysis, we found that the number of p53 positively labeled nuclei assessed using fluorescence immunocytochemistry increased with unloading and that these labeled nuclei were muscle originated, because only those nuclei under the laminin staining were included. Although we were not able to determine the precise identity of these p53-labeled nuclei (i.e., satellite cell or myocyte nuclei, because both of them reside under the basal lamina), our double-immunofluorescence staining of p53 and BrdU indicated that both the mitotic and postmitotic cell nuclei were involved in explaining the increased p53-labeled nuclei after unloading. On the basis of the fact that nearly all non-muscle cell populations are mitotic (e.g., fibroblasts, pericytes, and other blood vessel-associated cells), while the myocyte is the abundant postmitotic cell type in the skeletal muscles, it is reasonable to conclude that our observed labeling of p53-immunopositive but BrdU-immunonegative nuclei originated from the existing myocyte nuclei. We acknowledge that these labeled p53-positive/BrdU-negative nuclei might belong to those quiescent mitotic cells that had not been dividing during the whole experimental period of 2128 days, although this is unlikely. However, it is unlikely for quiescent cells to express substantially elevated levels of p53. Nonetheless, we interpret these findings as indicating that both the activated satellite cell nuclei and the existing myonuclei contribute to the increased level of p53 during the unloading-induced atrophy. This observation also implies that the cellular changes (e.g., in p53) occurring in both satellite cells and myocytes are involved in regulating the apoptosis of activated satellite cell nuclei during unloading after hypertrophy, even though we previously demonstrated that only the activated satellite cell nuclei (both fused and unfused nuclei), not the existing myonuclei, undergo apoptosis during unloading after hypertrophy (50). Although the exact mechanisms for cell-cell interaction (e.g., cell adhesion, cytoskeletal reorganization, and fusion) between myogenic satellite cells and myocytes during muscle remodeling are not completely understood (12), it is reasonable to speculate that the cellular changes occurring in myocytes can be a factor influencing the response of satellite cells during unloading in previously hypertrophied muscle, based on the fact that muscle satellite cells are localized and function intimately with the myofibers.
Recently, it has been suggested that p53 can also directly mediate the apoptotic cascades by translocating to the mitochondria via a transcription-independent mechanism (14, 44). Consistent with this suggestion, we have demonstrated that the cytoplasmic p53 protein content is elevated in young adult muscles that were unloaded for 7 days. Although our data do not allow us to fully identify the physiological role of p53 during unloading-induced apoptosis, we speculate that the increased cytoplasmic p53 may interact with Bax or other cytosolic apoptogenic factors and activate apoptosis during unloading. Nonetheless, further study is required to reveal other possible p53-related mechanisms involved in regulating the unloading-induced apoptosis in skeletal muscle, perhaps by regulation of apoptosis by p53 phosphorylation (22, 55).
Oxidative stress may be an important factor involved in regulating sarcopenia, and increased activity may in part offset oxidative stress (26, 27). On the other hand, mechanical unloading can lead to disruption of antioxidant-oxidant status by increasing Cu-Zn-SOD and reactive oxygen species in skeletal muscles (31). Furthermore, p53 can induce an imbalance in antioxidant enzymes and oxidative stress by inappropriately upregulating Mn-SOD and glutathione peroxidase (23). In this study, we observed that both the p53 and Mn-SOD protein content was increased in young adult muscles unloaded for 7 days. We interpret this finding to indicate that unloading resulted in an oxidative disturbance in the atrophied muscles.
Intriguingly, in the aged muscles, we did not find any significant differences in Id2 and p53 between the experimental and intra-animal control muscles after 7 or 14 days of unloading. Although these proteins appeared to increase with unloading (Fig. 2), the magnitude of the responses was more variable in old animals than in young animals. Assuming that p53 and the cytoplasmic Id2 are proapoptotic, our observations suggest that aging muscle has invoked a strategy that attempts to reduce the tendency toward apoptosis in unloaded muscles compared with the muscles of young adult animals. Nevertheless, these findings are consistent with the idea that the regulatory mechanisms contributing to the activation of apoptosis during unloading may be different between the aged and young adult muscles. In addition, these data are in agreement with our previous observations of increased mRNA and protein levels of Bcl-2, decreased protein level of Bax, and decreased nuclear AIF protein level in muscles of aged birds that were unloaded for 14 days compared with the young animals (50). These findings underscore the possibility that the mechanisms regulating apoptotic consequences during unloading-induced muscle loss may be aging specific, which warrants further investigation.
In conclusion, we have provided evidence suggesting that transcriptional repressor protein Id2 and tumor suppressor protein p53 are related to the unloading-induced apoptosis after hypertrophy in young adult quail skeletal muscles. We have demonstrated that both the nuclear and cytoplasmic protein contents of p53 significantly increased in the unloaded muscle relative to the control muscle after 7 days of unloading after hypertrophy. Although the nuclear protein content of Id2 did not respond to unloading, we found that the cytoplasmic protein content of Id2 markedly increased in the unloaded side compared with the control side after 7 days of unloading after hypertrophy in young adult birds. We also have shown that correlations exist among Id2, p53, TUNEL index, and the apoptosis-regulatory factors, including Bcl-2, Bax, and AIF. However, our observed correlation relationships among Id2, p53, and apoptosis were moderately varied in strength (positive correlations ranged from 0.26 to 0.88, and negative correlations ranged from 0.25 to 0.85). Although Id2 and p53 appear to have a role in pathways leading to unloading-induced atrophy, these findings suggest that the unloading-induced apoptosis may not be explained fully by the responses of Id2 and p53. Other cellular modulators (e.g., heat shock proteins) (18) that were not examined in this study may also play a role in regulating apoptosis-induced atrophy. Although it has been suggested that c-Myc may have an apoptogenic role, especially during tumor genesis (39), our data do not support the hypothesis that c-Myc has a proapoptotic role during unloading-induced apoptosis after muscle hypertrophy. The inverse relationships between c-Myc and proapoptotic factors implicate more complex mechanisms, and additional research is needed to understand the physiological function of c-Myc during muscle remodeling.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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