Id2 and p53 participate in apoptosis during unloading-induced muscle atrophy

Parco M. Siu and Stephen E. Alway

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


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptotic signaling was examined in the patagialis (PAT) muscles of young adult and old quail. One wing was loaded for 14 days to induce hypertrophy and then unloaded for 7 or 14 days to induce muscle atrophy. Although the nuclear Id2 protein content was not different between unloaded and control muscles in either age group, cytoplasmic Id2 protein content of unloaded muscles was higher than that in contralateral control muscles after 7 days of unloading in young quails. Nuclear and cytoplasmic p53 contents and the p53 nuclear index of the unloaded muscles were higher than those in control muscles after 7 days of unloading in young quails, whereas in aged quails, the p53 and Id2 contents and p53 nuclear index of the unloaded muscles were not altered by unloading. Immunofluorescent staining indicated that myonuclei and activated satellite cell nuclei contributed to the increased number of p53-positive nuclei. Conversely, unloading in either young adult or aged PAT muscles did not alter c-Myc protein content. Although Cu-Zn-SOD content was not different in unloaded and control muscles, Mn-SOD content increased in PAT muscles after 7 days of unloading in young quails, suggesting that unloading induced an oxidative disturbance in these muscles. Moderate correlational relationships existed among Id2, p53, c-Myc, SOD, apoptosis-regulatory factors, and TdT-mediated dUTP nick end labeling index. These data indicate that Id2 and p53 are involved in the apoptotic responses during unloading-induced muscle atrophy after hypertrophy in young adult birds. Furthermore, our data suggest that there is an aging-dependent regulation of Id2 and p53 during unloading of previously hypertrophied muscles.

inhibitor of DNA binding/differentiation protein; tumor suppressor gene; programmed cell death; aging


APOPTOSIS HAS BEEN DOCUMENTED under the experimental situations of skeletal muscle denervation and unloading (1, 3, 6, 25, 28). It also has been proposed that apoptosis may play a further physiological role in regulating unloading-induced muscle atrophy, especially in aging-associated muscle loss or sarcopenia (4, 15, 16, 33, 45, 50). To identify the possible role of apoptosis during muscle atrophy, it is imperative to understand the underlying regulatory mechanisms contributing to the activation of the apoptotic signaling pathway that results in unloading-induced apoptosis. A variety of cell cycle-regulatory proteins and transcription factors (e.g., Id2, p53, and c-Myc) have been demonstrated to have an essential role in regulating growth and apoptosis in different cell types (53, 56). Because these proteins could act as the mediators of the apoptotic signaling pathway, they may be involved in the regulation of apoptosis during muscle atrophy.

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., Id1–Id4). 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.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Japanese Coturnix quails were hatched and raised in pathogen-free conditions in the central animal care center at West Virginia University School of Medicine. The birds were housed at a room temperature of 22°C with a 12:12-h light-dark cycle, and they were provided with food and water ad libitum. Sixteen young adult birds aged 2 mo and sixteen 24-mo-old birds were examined in the present study. The lifespan of Japanese quails is ~26–28 mo, and they are both physically and sexually mature by age 1.5 mo (36, 43). All experimental procedures were approved by the Institutional Animal Use and Care Committee of the West Virginia University School of Medicine. The animal care standards were followed by adhering to the recommendations for the care of laboratory animals advocated by the American Association for Accreditation of Laboratory Animal Care and fully conformed to the American Physiological Society's "Guiding Principles for Research Involving Animals and Human Beings."

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·ml–1, 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.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Muscle mass loss. The declined degree of muscle hypertrophy in the experimental muscle relative to the contralateral control muscle was monitored throughout the experiment to evaluate the unloading-induced muscle loss in these previously hypertrophied muscles (50). A moderate extent of muscle hypertrophy (~35% in young adult and ~15% in aged quails) has consistently been demonstrated after the current 14-day stretch-loading procedure (2, 7, 51). In the young adult quails, the degree of hypertrophy was 15% after 7 days of unloading and had returned to the contralateral control levels after 14 days of unloading. In the aged quails, the extent of hypertrophy was 12% after 7 days of unloading, and the muscle mass of the experimental muscle was still 6% higher than that in the control muscle after 14 days of unloading (50).

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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Fig. 1. A: nuclear Id2 protein content. Id2 is a member of the inhibitor of DNA binding/differentiation (Id) protein family. Data are expressed as optical density (OD) x resulting band area in arbitrary units x 107. Representative blots for Id2 in control and experimental muscles isolated from young adult and aged animals are shown below the graph. The data are presented as means ± SE. Young, young adult quails; Aged, aged quails; 7d unloading, 7 days of unloading after 14 days of muscle loading; 14d unloading, 14 days of unloading after 14 days of muscle loading. B: cytoplasmic Id2 protein content. Data are expressed as OD x resulting band area and expressed in arbitrary units x 107. Representative blots for Id2 in control and experimental muscles isolated from young adult and aged animals are shown below the graph. Data are means ± SE. *P < 0.05, significantly different from corresponding intra-animal control muscles. The main effects of time and interaction (age x time) on cytoplasmic Id2 protein content in these animals were analyzed using 2 x 2 ANOVA.

 
Furthermore, the cytoplasmic Id2 protein content of the experimental muscle was 150 ± 81% (P < 0.05) higher than that in the control muscle after 7 days of unloading in young quails (Fig. 1B). In aged quails, although the Id2 protein content of the experimental muscle appeared to be higher than that of the control muscle after 7 days of unloading and seemed to be lower than that in the control muscle after 14 days of unloading, these data did not reach statistical significance (P > 0.05). For the cytoplasmic Id2 protein content, ANOVA indicated that the main effects of time [F(1,56) = 6.132; P < 0.05] and interaction (age x time) [F(1,56) = 5.204; P < 0.05] existed in these animals (Fig. 1B).

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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Fig. 2. A: nuclear p53 protein content. Data are expressed as OD x resulting band area in arbitrary units x 104. Representative blots for p53 in control and experimental muscles isolated from young adult and aged animals are shown below the graph. Data are means ± SE. *P < 0.05, significantly different from corresponding intra-animal control muscles. B: cytoplasmic p53 protein content. Data are expressed as OD x resulting band area in arbitrary units x 104. Representative blots for p53 in control and experimental muscles isolated from young adult and aged animals are shown below the graph. Data are means ± SE. *P < 0.05, significantly different from corresponding intra-animal control muscles. The main effects of time and age on cytoplasmic p53 protein content in these animals were analyzed using 2 x 2 ANOVA.

 
The main effects of time [F(1,56) = 4.486, P < 0.05] and age [F(1,56) = 12.304, P < 0.01] were found in the cytoplasmic p53 protein content (Fig. 2B). The cytoplasmic p53 protein content of the experimental muscle was 85 ± 27% (P < 0.05) higher than that in 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. 2B). In the aged quails, the changes in cytoplasmic p53 protein content were not significantly different between the experimental and intra-animal control muscles after 7 or 14 days of unloading (P > 0.05) (Fig. 2B).

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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Fig. 3. Nuclear c-Myc protein content. Data are expressed as OD x resulting band area in arbitrary units x 106. Representative blots for c-Myc in control and experimental muscles isolated from young adult and aged animals are shown below the graph. Data are means ± SE.

 
Cu-Zn-SOD and Mn-SOD protein contents. An immunoreactive band of ~20 kDa was detected. This corresponded to the predicted molecular mass of the SOD protein. There was no difference in the protein content of Cu-Zn-SOD between the experimental and control muscles in either young or aged quails (Fig. 4A).



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Fig. 4. A: Cu-Zn-SOD protein content. Data are expressed as OD x resulting band area in arbitrary units x 108. Representative blots for Cu-Zn-SOD in control and experimental muscles isolated from young adult and aged animals are shown below the graph. Data are means ± SE. B: Mn-SOD protein content. Data are expressed as OD x resulting band area in arbitrary units x 108. Representative blots for Mn-SOD in control and experimental muscles isolated from young adult and aged animals are shown below the graph. Data are means ± SE. *P < 0.05, significantly different from corresponding intra-animal control muscles. The main effects of age on Mn-SOD protein content in these animals were analyzed using 2 x 2 ANOVA.

 
A main effect of age [F(1,56) = 43.476; P < 0.01] was found in Mn-SOD protein content (Fig. 4B). Mn-SOD protein content of the experimental muscle was 13 ± 5% (P < 0.05) higher than that of the control muscle after 7 days of unloading, but it was not different between the experimental and control muscles after 14 days of unloading in young quails (Fig. 4B). No significant differences in Mn-SOD protein content were found between the experimental and intra-animal control muscles from aged quails after 7 or 14 days of unloading (P > 0.05).

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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Fig. 5. A: p53-Positive nucleus under the laminin staining. Immunofluorescent labeling on p53, laminin, and 4',6-diamidino-2-phenylindole (DAPI) was used to identify the p53-positively labeled nuclei under the laminin staining as an estimate of the nuclear p53 index. p53 (red) stained with secondary Cy3 labeling, laminin (green) stained with secondary fluorescein labeling, and nuclei (blue) labeled using DAPI stain. This image was obtained using x100 magnification. The large and small boxed insets show a p53-positive nucleus under the laminin staining. Bar, 10 µm. B: p53 nuclear index. The number of nuclei positively labeled by p53 relative to the total number of nuclei estimated using immunofluorescent staining was expressed as the p53 nuclear index. Data are means ± SE. *P < 0.05, significantly different from corresponding intra-animal control muscles. The main effects of age on p53 nuclear index in these animals were analyzed using 2 x 2 ANOVA.

 
Our double-immunofluorescence analysis of p53 and BrdU demonstrated that both the BrdU-positive (Fig. 6A) and BrdU-negative (Fig. 6B) labeled p53-positive nuclei existed in the experimental muscles of young quail after 7 days of unloading and in those of aged quails after 7 and 14 days of unloading, indicating that both the dividing and nondividing cell nuclei contributed to the p53-immunopositive nuclei in these experimental muscles. In addition, although the nuclear c-Myc protein was detected in our immunoblot analysis, nuclei that labeled positively for c-Myc were not found in the PAT muscles of either age group.



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Fig. 6. A: nuclei labeled positive by p53 and bromodeoxyuridine (BrdU). Immunofluorescent labeling of p53, BrdU, and DAPI was used to identify the mitotic property of the nuclei labeled positively by p53. ad: nuclei labeled positively by both p53 and BrdU are shown. a: nuclei labeled by DAPI staining. b: p53-immunopositive nucleus with secondary labeling by Cy3. c: BrdU-immunopositive nucleus with secondary labeling by fluorescein. d: merged images showing p53, BrdU, and DAPI labeling. Arrow indicates a nucleus labeled positively by p53 and BrdU. These images were obtained at x100 magnification. Bar, 10 µm. B: nucleus labeled positively by p53 but negatively by BrdU. BrdU-immunonegative, p53-positive nucleus labeling is shown. a: nuclei labeled by DAPI staining. b: p53-immunopositive nucleus with secondary labeling by Cy3. c: merged image showing p53 and DAPI labeling. Arrow indicates nucleus labeled positively by p53 but immunonegatively by BrdU. These images were obtained at x100 magnification. Bar, 10 µm.

 
Relationships of Id2, p53, c-Myc, SOD, apoptosis-regulatory factors, and apoptosis. The relationships of Id2, p53, c-Myc, SOD, TUNEL index, and apoptosis-regulatory factors [Bcl-2, Bax, and apoptosis-inducing factor (AIF)] were analyzed by examining the corresponding Pearson's correlation coefficient (r2). We have previously shown evidence for apoptosis as indicated by the increase in TUNEL-positive nuclei and the changes in apoptosis-regulatory factors, including Bcl-2, Bax, and AIF, consistent with apoptosis (50).

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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Fig. 7. Correlation analyses of cytoplasmic Id2 protein content. Scatterplots show the relationships between the cytoplasmic protein content of Id2 and Bcl-2 protein content (A), TdT-mediated dUTP nick end labeling (TUNEL) index (B), Bax protein content (C), and the cytoplasmic protein content of p53 (D). The control and experimental muscles of both young adult and aged quails of all groups were collapsed and treated as a single, pooled group (n = 64). r, Pearson product-moment correlation coefficient; Y7, young adult quails after 7 days of unloading; Y14, young adult quails after 14 days of unloading; A7, aged quails after 7 days of unloading; A14, aged quails after 14 days of unloading.

 
The cytoplasmic protein content of p53 was negatively correlated with the Bcl-2 protein content (r2 = –0.278; P < 0.05) (Fig. 8A), while it was positively correlated with the TUNEL index (r2 = 0.421; P < 0.001) (Fig. 8B), Bax protein (r2 = 0.390; P < 0.001) (Fig. 8C), and mRNA contents (r2 = 0.273; P < 0.05) (Fig. 8D). The nuclear protein content of p53 was positively correlated with the mRNA content of Bax (r2 = 0.393; P < 0.001) (Fig. 9A) and the TUNEL index (r2 = 0.249; P < 0.05) (Fig. 9B). In agreement with the suggestion that nuclear p53 is related to apoptosis, the p53 nuclear index as estimated using immunocytochemistry was positively correlated with the TUNEL index (r2 = 0.501; P < 0.001) (Fig. 9C) and cytoplasmic AIF protein content (r2 = 0.280; P < 0.05) (Fig. 9D).



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Fig. 8. Correlation analyses of cytoplasmic p53 protein content. Scatterplots show the relationships between the cytoplasmic protein content of p53 and Bcl-2 protein content (A), TUNEL index (B), and protein and mRNA content of Bax (C and D). The control and experimental muscles from both young adult and aged quails of all groups were collapsed and treated as a single, pooled group (n = 64).

 


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Fig. 9. Correlation analyses of nuclear p53 protein content and p53 nuclear index. Scatterplots show the relationships between the nuclear protein content of p53 and Bax mRNA content (A) and the TUNEL index (B), as well as the relationships between the p53 nuclear index and TUNEL index (C) and the cytoplasmic apoptosis-inducing factor (AIF) protein content (D). The control and experimental muscles of both young adult and aged quails of all groups were collapsed and treated as a single, pooled group (n = 64).

 
c-Myc protein content was negatively correlated with the TUNEL index (r2 = –0.309; P < 0.05) (Fig. 10A), nuclear p53 protein content (r2 = –0.291; P < 0.05) (Fig. 10B), and the p53 nuclear index (r2 = –0.311; P < 0.05) (Fig. 10C).



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Fig. 10. Correlation analyses of nuclear c-Myc protein content. Scatterplots show the relationships between the nuclear protein content of c-Myc and TUNEL index (A), the nuclear protein content of p53 (B), and the p53 nuclear index (C). The control and experimental muscles from both young adult and aged quails of all groups were collapsed and treated as a single, pooled group (n = 64).

 
Moreover, a positive correlation between the Mn-SOD protein content and the Bcl-2 protein content (r2 = 0.372; P < 0.01) (Fig. 11A), while negative correlations existed between the protein content of Mn-SOD and the TUNEL index (r2 = –0.438; P < 0.001) (Fig. 11B) as well as Bax protein content (r2 = –0.314; P < 0.05) (Fig. 11C). Also, the protein content of Cu-Zn-SOD was negatively correlated with the nuclear protein content of AIF (r2 = –0.250; P < 0.05) (Fig. 11D).



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Fig. 11. Correlation analyses of SOD protein content. AC: scatterplots showing the relationships between the protein content of Mn-SOD and Bcl-2 protein content (A), TUNEL index (B), and the protein content of Bax (C). D: scatterplots showing the relationship between the Cu-Zn-SOD protein content and the nuclear protein content of AIF. The control and experimental muscles of both young adult and aged quails of all groups were collapsed and treated as a single, pooled group (n = 64).

 
When the muscle samples were collapsed and analyzed as different subgroups (e.g., all control muscles), the cytoplasmic protein content of Id2 generally was positively correlated with the TUNEL index, Bax protein content, and cytoplasmic and nuclear protein contents of AIF as well as nuclear p53 protein content (P < 0.05) (Table 1), whereas the nuclear protein content of Id2 was negatively correlated with the Bax mRNA content and cytoplasmic p53 protein content (P < 0.05) (Table 1). The cytoplasmic protein content of p53 was positively correlated with the TUNEL index, Bax protein, and mRNA contents but negatively correlated with the protein contents of Bcl-2 and Mn-SOD (P < 0.05) (Table 1). Positive correlations existed between the nuclear protein content of p53 and TUNEL index, Bax mRNA content, and cytoplasmic protein content of AIF (P < 0.05) (Table 1). In addition, the p53 nuclear index was positively correlated with the TUNEL index, Bax, and cytoplasmic Id2 protein contents, whereas a negative correlation existed between the p53 nuclear index and nuclear c-Myc and Cu-Zn-SOD protein contents. The nuclear protein content of c-Myc was negatively correlated with the TUNEL index, cytoplasmic, and nuclear protein contents of p53, whereas c-Myc was positively correlated with the nuclear protein content of Id2 and Cu-Zn-SOD protein content.


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Table 1. Correlation of Id2, p53, and c-Myc

 
In the subgroups of muscles samples, the protein content of Mn-SOD was positively correlated with the Bcl-2 protein and mRNA contents but negatively correlated with the TUNEL index, Bax protein, and mRNA contents, as well as the nuclear protein content of AIF (P < 0.05) (Table 2). Furthermore, the protein content of Cu-Zn-SOD was positively correlated with the Bcl-2 protein and mRNA content, whereas it was negatively correlated with the TUNEL index, Bax mRNA content, and nuclear protein content of AIF (P < 0.05) (Table 2).


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Table 2. Correlation of SOD

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis has been implicated as having a physiological role in mediating unloading-induced muscle atrophy (1, 6, 50). However, the underlying cellular and molecular regulatory mechanism contributing to the activation of the subsequent apoptotic signaling pathway that results in unloading-induced apoptosis is not currently well understood. In the present study, we have shown that Id2 and p53 may be involved in the regulation of unloading-induced apoptosis after muscle hypertrophy. We have demonstrated that the subcellular fractionated protein content of Id2 and p53 is moderately correlated with apoptosis estimated on the basis of TUNEL index and apoptosis-regulatory factors, including Bcl-2, Bax, and AIF. Our immunocytochemical findings indicate that both the mitotic and postmitotic cell nuclei (presumably activated myogenic satellite cells and existing myocytes) are responsible for the elevated nuclear p53 protein content in the unloaded muscle relative to control muscle. Nonetheless, we previously have shown that the activated satellite cell nuclei (both fused and unfused satellite cell nuclei), but not the existing myonuclei, are eliminated via apoptotic mechanisms during unloading after hypertrophy on the basis of the observations that almost all nuclei that labeled positively on the basis of the TUNEL index were BrdU immunopositive (50). In this study, we report negative relationships between c-Myc and apoptotic factors in muscle samples. In addition, we found an aging-specific response of Id2 and p53 to unloading after hypertrophy. This finding may partly explain our previously reported findings of age-related differences in muscle adaptation during unloading after hypertrophy (50). Taken together, our findings are consistent with the hypothesis that Id2 and p53 may have a role in mediating the homeostasis of apoptosis-regulatory factors and apoptosis during unloading-induced muscle atrophy after hypertrophy in young adult skeletal muscle. Moreover, additional research is needed to further identify the influence of aging on the regulatory mechanisms contributing to the activation of apoptosis during unloading after hypertrophy.

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-{kappa}{beta} 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 21–28 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.


    ACKNOWLEDGMENTS
 
This study was supported by National Institute on Aging Grant R01 AG-021530.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. E. Alway, Division of Exercise Physiology, West Virginia Univ. School of Medicine, Robert C. Byrd Health Science Center, Morgantown, WV 26506-9227 (E-mail: salway{at}hsc.wvu.edu)

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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Allen DL, Linderman JK, Roy RR, Bigbee AJ, Grindeland RE, Mukku V, and Edgerton VR. Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting. Am J Physiol Cell Physiol 273: C579–C587, 1997.[Abstract/Free Full Text]

2. Alway SE. Attenuation of Ca2+-activated ATPase and shortening velocity in hypertrophied fast twitch skeletal muscle from aged Japanese quail. Exp Gerontol 37: 665–678, 2002.[CrossRef][ISI][Medline]

3. Alway SE, Degens H, Krishnamurthy G, and Chaudhrai A. Denervation stimulates apoptosis but not Id2 expression in hindlimb muscles of aged rats. J Gerontol A Biol Sci Med Sci 58: 687–697, 2003.[ISI][Medline]

4. Alway SE, Degens H, Krishnamurthy G, and Smith CA. Potential role for Id myogenic repressors in apoptosis and attenuation of hypertrophy in muscles of aged rats. Am J Physiol Cell Physiol 283: C66–C76, 2002.[Abstract/Free Full Text]

5. Alway SE, Degens H, Lowe DA, and Krishnamurthy G. Increased myogenic repressor Id mRNA and protein levels in hindlimb muscles of aged rats. Am J Physiol Regul Integr Comp Physiol 282: R411–R422, 2002.[Abstract/Free Full Text]

6. Alway SE, Martyn JK, Ouyang J, Chaudhrai A, and Murlasits ZS. Id2 expression during apoptosis and satellite cell activation in unloaded and loaded quail skeletal muscles. Am J Physiol Regul Integr Comp Physiol 284: R540–R549, 2003.[Abstract/Free Full Text]

7. Alway SE, Winchester PK, Davis ME, and Gonyea WJ. Regionalized adaptations and muscle fiber proliferation in stretch-induced enlargement. J Appl Physiol 66: 771–781, 1989.[Abstract/Free Full Text]

8. Barone MV, Pepperkok R, Peverali FA, and Philipson L. Id proteins control growth induction in mammalian cells. Proc Natl Acad Sci USA 91: 4985–4988, 1994.[Abstract/Free Full Text]

9. Benezra R, Davis RL, Lassar A, Tapscott S, Thayer M, Lockshon D, and Weintraub H. Id: a negative regulator of helix-loop-helix DNA binding proteins: control of terminal myogenic differentiation. Ann NY Acad Sci 599: 1–11, 1990.[ISI]

10. Benezra R, Davis RL, Lockshon D, Turner DL, and Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61: 49–59, 1990.[CrossRef][ISI][Medline]

11. Borriello A, Roberto R, Della Ragione F, and Iolascon A. Proliferate and survive: cell division cycle and apoptosis in human neuroblastoma. Haematologica 87: 196–214, 2002.[ISI][Medline]

12. Charge SB and Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol Rev 84: 209–238, 2004.[Abstract/Free Full Text]

13. Chipuk JE and Green DR. Cytoplasmic p53: Bax and forward. Cell Cycle 3: 429–431, 2004.[ISI][Medline]

14. Chipuk JE, Kuwana T, Bouchier-Hayes L, Droin NM, Newmeyer DD, Schuler M, and Green DR. Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303: 1010–1014, 2004.[Abstract/Free Full Text]

15. Dirks A and Leeuwenburgh C. Apoptosis in skeletal muscle with aging. Am J Physiol Regul Integr Comp Physiol 282: R519–R527, 2002.[Abstract/Free Full Text]

16. Dirks AJ and Leeuwenburgh C. Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12. Free Radic Biol Med 36: 27–39, 2004.[CrossRef][ISI][Medline]

17. Florio M, Hernandez MC, Yang H, Shu HK, Cleveland JL, and Israel MA. Id2 promotes apoptosis by a novel mechanism independent of dimerization to basic helix-loop-helix factors. Mol Cell Biol 18: 5435–5444, 1998.[Abstract/Free Full Text]

18. Garrido C, Gurbuxani S, Ravagnan L, and Kroemer G. Heat shock proteins: endogenous modulators of apoptotic cell death. Biochem Biophys Res Commun 286: 433–442, 2001.[CrossRef][ISI][Medline]

19. Gleichmann M, Buchheim G, El-Bizri H, Yokota Y, Klockgether T, Kügler S, Bähr M, Weller M, and Schulz JB. Identification of inhibitor-of-differentiation 2 (Id2) as a modulator of neuronal apoptosis. J Neurochem 80: 755–762, 2002.[CrossRef][ISI][Medline]

20. Hatoko M, Tanaka A, Kuwahara M, Yurugi S, Iioka H, and Niitsuma K. Difference of molecular response to ischemia-reperfusion of rat skeletal muscle as a function of ischemic time: study of the expression of p53, p21WAF-1, Bax protein, and apoptosis. Ann Plast Surg 48: 68–74, 2002.[CrossRef][ISI][Medline]

21. Hawke TJ and Garry DJ. Myogenic satellite cells: physiology to molecular biology. J Appl Physiol 91: 534–551, 2001.[Abstract/Free Full Text]

22. Huang C, Ma WY, Maxiner A, Sun Y, and Dong Z. p38 Kinase mediates UV-induced phosphorylation of p53 protein at serine 389. J Biol Chem 274: 12229–12235, 1999.[Abstract/Free Full Text]

23. Hussain SP, Amstad P, He P, Robles A, Lupold S, Kaneko I, Ichimiya M, Sengupta S, Mechanic L, Okamura S, Hofseth LJ, Moake M, Nagashima M, Forrester KS, and Harris CC. p53-induced up-regulation of MnSOD and GPx but not catalase increases oxidative stress and apoptosis. Cancer Res 64: 2350–2356, 2004.[Abstract/Free Full Text]

24. Jeffers JR, Parganas E, Lee Y, Yang C, Wang JL, Brennan J, MacLean KH, Han J, Chittenden T, Ihle JN, McKinnon PJ, Cleveland JL, and Zambetti GP. Puma is an essential mediator of p53-dependent and -independent apoptotic pathways. Cancer Cell 4: 321–328, 2003.[CrossRef][ISI][Medline]

25. Jejurikar SS, Marcelo CL, and Kuzon WM Jr. Skeletal muscle denervation increases satellite cell susceptibility to apoptosis. Plast Reconstr Surg 110: 160–168, 2002.[ISI][Medline]

26. Ji LL. Antioxidants and oxidative stress in exercise. Proc Soc Exp Biol Med 222: 283–292, 1999.[Abstract/Free Full Text]

27. Ji LL. Exercise-induced modulation of antioxidant defense. Ann NY Acad Sci 959: 82–92, 2002.[Abstract/Free Full Text]

28. Jin H, Wu Z, Tian T, and Gu Y. Apoptosis in atrophic skeletal muscle induced by brachial plexus injury in rats. J Trauma 50: 31–35, 2001.[ISI][Medline]

29. Lasorella A, Iavarone A, and Israel MA. Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins. Mol Cell Biol 16: 2570–2578, 1996.[Abstract]

30. Lasorella A, Noseda M, Beyna M, Yokota Y, and Iavarone A. Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407: 592–598, 2000.[CrossRef][ISI][Medline]

31. Lawler JM, Song W, and Demaree SR. Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle. Free Radic Biol Med 35: 9–16, 2003.[CrossRef][ISI][Medline]

32. Lee MJ, Lee JS, and Lee MC. Apoptosis of skeletal muscle on steroid-induced myopathy in rats. J Korean Med Sci 16: 467–474, 2001.[ISI][Medline]

33. Leeuwenburgh C. Role of apoptosis in sarcopenia. J Gerontol A Biol Sci Med Sci 58: 999–1001, 2003.[ISI][Medline]

34. Ling MT, Wang X, Ouyang XS, Xu K, Tsao SW, and Wong YC. Id-1 expression promotes cell survival through activation of NF-{kappa}B signalling pathway in prostate cancer cells. Oncogene 22: 4498–4508, 2003.[CrossRef][ISI][Medline]

35. Liu CJ, Ding B, Wang H, and Lengyel P. The MyoD-inducible p204 protein overcomes the inhibition of myoblast differentiation by Id proteins. Mol Cell Biol 22: 2893–2905, 2002.[Abstract/Free Full Text]

36. Marks HL. Long-term selection for body weight in Japanese quail under different environments. Poult Sci 75: 1198–1203, 1996.[ISI][Medline]

37. Matsumura ME, Lobe DR, and McNamara CA. Contribution of the helix-loop-helix factor Id2 to regulation of vascular smooth muscle cell proliferation. J Biol Chem 277: 7293–7297, 2002.[Abstract/Free Full Text]

38. Nakahara T, Hashimoto K, Hirano M, Koll M, Martin CR, and Preedy VR. Acute and chronic effects of alcohol exposure on skeletal muscle c-myc, p53, and Bcl-2 mRNA expression. Am J Physiol Endocrinol Metab 285: E1273–E1281, 2003.[Abstract/Free Full Text]

39. Nilsson JA and Cleveland JL. Myc pathways provoking cell suicide and cancer. Oncogene 22: 9007–9021, 2003.[CrossRef][ISI][Medline]

40. Norton JD and Atherton GT. Coupling of cell growth control and apoptosis functions of Id proteins. Mol Cell Biol 18: 2371–2381, 1998.[Abstract/Free Full Text]

41. Oda E, Ohki R, Murasawa H, Nemoto J, Shibue T, Yamashita T, Tokino T, Taniguchi T, and Tanaka N. Noxa, a BH3-only member of the Bcl-2 family and candidate mediator of p53-induced apoptosis. Science 288: 1053–1058, 2000.[Abstract/Free Full Text]

42. Ohnishi T, Takahashi A, Wang X, Ohnishi K, Ohira Y, and Nagaoka S. Accumulation of a tumor suppressor p53 protein in rat muscle during a space flight. Mutat Res 430: 271–274, 1999.[ISI][Medline]

43. Ottinger MA. Quail and other short-lived birds. Exp Gerontol 36: 859–868, 2001.[CrossRef][ISI][Medline]

44. Perfettini JL, Kroemer RT, and Kroemer G. Fatal liaisons of p53 with Bax and Bak. Nat Cell Biol 6: 386–388, 2004.[CrossRef][ISI][Medline]

45. Pollack M, Phaneuf S, Dirks A, and Leeuwenburgh C. The role of apoptosis in the normal aging brain, skeletal muscle, and heart. Ann NY Acad Sci 959: 93–107, 2002.[Abstract/Free Full Text]

46. Rothermel B, Vega RB, Yang J, Wu H, Bassel-Duby R, and Williams RS. A protein encoded within the Down syndrome critical region is enriched in striated muscles and inhibits calcineurin signaling. J Biol Chem 275: 8719–8725, 2000.[Abstract/Free Full Text]

47. Ruzinova MB and Benezra R. Id proteins in development, cell cycle and cancer. Trends Cell Biol 13: 410–418, 2003.[CrossRef][ISI][Medline]

48. Schuler M and Green DR. Mechanisms of p53-dependent apoptosis. Biochem Soc Trans 29: 684–688, 2001.[CrossRef][ISI][Medline]

49. Shefer G, Partridge TA, Heslop L, Gross JG, Oron U, and Halevy O. Low-energy laser irradiation promotes the survival and cell cycle entry of skeletal muscle satellite cells. J Cell Sci 115: 1461–1469, 2002.[Abstract/Free Full Text]

50. Siu PM, Pistilli EE, Butler DC, and Alway SE. Aging influences the cellular and molecular responses of apoptosis to skeletal muscle unloading. Am J Physiol Cell Physiol In press: 2004.

51. Summers PJ, Ashmore CR, Lee YB, and Ellis S. Stretch-induced growth in chicken wing muscles: role of soluble growth-promoting factors. J Cell Physiol 125: 288–294, 1985.[CrossRef][ISI][Medline]

52. Veal EA and Jackson MJ. C-myc is expressed in mouse skeletal muscle nuclei during post-natal maturation. Int J Biochem Cell Biol 30: 811–821, 1998.[CrossRef][ISI][Medline]

53. Vermeulen K, Berneman ZN, and Van Bockstaele DR. Cell cycle and apoptosis. Cell Prolif 36: 165–175, 2003.[CrossRef][ISI][Medline]

54. Wu X and Deng Y. Bax and BH3-domain-only proteins in p53-mediated apoptosis. Front Biosci 7: d151–d156, 2002.[ISI][Medline]

55. Yeh PY, Chuang SE, Yeh KH, Song YC, and Cheng AL. Nuclear extracellular signal-regulated kinase 2 phosphorylates p53 at Thr55 in response to doxorubicin. Biochem Biophys Res Commun 284: 880–886, 2001.[CrossRef][ISI][Medline]

56. Yokota Y and Mori S. Role of Id family proteins in growth control. J Cell Physiol 190: 21–28, 2002.[CrossRef][ISI][Medline]