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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY
Laboratory of Muscle Biology and Sarcopenia, Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia
Submitted 21 March 2005 ; accepted in final form 20 May 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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muscle disuse; sarcopenia; programmed cell death
Apoptosis is an essential biological process that functions in maintaining the homeostasis of cell survival/death, tissue turnover, and other fundamental physiological events (22, 48). Recently, there has been consistent evidence showing that apoptosis is activated or accelerated in skeletal muscle during disuse and with normal aging (1–3, 5, 15, 23, 24, 34, 37, 38, 40, 45). Although the importance of apoptosis in muscle atrophy has yet to be resolved, these findings support the hypothesis that apoptosis may have a physiological role in regulating the process of muscle loss in response to muscle disuse. Indeed, it has been suggested that apoptosis can occur within a particular myonucleus without influencing the entire myofiber and this may function to mediate the elimination of individual myonuclei during muscle disuse to coordinate the size of the myonuclear pool (perhaps maintaining the nuclei-to-cytoplasm relationship) and therefore muscle atrophy (1, 24, 40). However, there also are findings showing that myonuclear number does not change with hindlimb suspension-induced atrophy in muscle of aged animals, and this suggests that the homeostatic balance of the myonuclear domain may be perturbed in aged skeletal muscle (19, 24). Furthermore, it has been exhibited that aging may influence the apoptotic response to unloading-induced muscle atrophy (24, 40). Altogether, aging presumably complicates a number of events (e.g., myonuclei homeostasis and apoptotic regulation) that have been proposed to be involved in mediating the process of atrophy.
Although the activation of apoptosis has been demonstrated in hindlimb-suspended soleus muscle (1, 24), a muscle that is primarily composed of fibers containing type I myosin heavy chains, it is unclear whether apoptosis is also involved in muscles composed of mixed fiber types (e.g., gastrocnemius, which contains both slow and fast myosin heavy chains), because these mixed-fiber muscles also atrophy during hindlimb suspension. Because global changes in skeletal muscle may not be just confined to the soleus muscle, it is important to determine whether differences in apoptotic responses to disuse may exist in muscle groups that are composed of predominated fast myosin-containing fibers compared with muscles that have predominately slow fibers (e.g., gastrocnemius vs. soleus). Moreover, there is a lack of data examining the influence of aging in suspension unloading-induced apoptosis. Therefore, this study examined the responses of apoptosis and apoptotic regulatory factors to 14 days of hindlimb suspension in young adult and aged gastrocnemius muscles. We tested the hypotheses that apoptosis is associated with the hindlimb suspension-induced mixed fiber-muscle loss and that those apoptotic responses to suspension are age dependent.
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METHODS |
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6-mo-old young adult and 30-mo-old aged Fischer 344 x Brown Norway rats (Harlan, Indianapolis, IN), a rodent strain that is approved to be an appropriate aging model by the National Institute on Aging. This rodent model has a low rate of pathologies including tumors (26, 49). This makes it appropriate to study the effects of aging without the complications of disease. The rats were housed in pathogen-free conditions at 20°C and were exposed to a reverse light condition of 12:12-h light-dark each day. They were fed rat chow and water ad libitum throughout the study period.
Hindlimb suspension procedure.
The animals were randomly assigned to a suspension group (young, n = 10; aged n = 10) or a control group (young, n = 10; aged, n = 8). The procedure of hindlimb suspension described by Morey-Holton and Globus (28) was adopted in the present study. In brief, an adhesive (tincture of benzoin) was applied to the tail and air-dried, and orthopedic tape was put along the proximal one-third of the tail. This practice distributed the load evenly and prevented excessive tension on a small area to the tail. The tape was then placed through a wire harness that was attached to a fish line swivel at the top of a specially designed National Aeronautics and Space Administration-approved hindlimb suspension cage. This provided the rats with 360° of movement around the cage, and the forelimbs maintained contact with a grid floor, allowing the animals to move and access food and water freely. Sterile gauze was wrapped around the orthopedic tape and was subsequently covered with a thermoplastic material, which formed a hardened cast (Vet-Lite; Veterinary Specialty Products, Boca Raton, FL). The distal tip of the tail was examined to verify that the procedure did not occlude the blood flow to the tail (i.e., tail remained pink). The suspension height was adjusted to prevent the animal’s hindlimb from touching any supportive surface, with care taken to maintain a suspension angle of 30° (20). The suspension height and animal behavior were monitored daily. Control animals were allowed to move unconstrained around the cages. After 14 days of suspension, animals were killed with an overdose of xylazine. The medial gastrocnemius muscles from the hindlimbs were excised, weighed, and frozen in isopentane cooled to the temperature of liquid nitrogen and stored at –80°C until used for analyses.
All experimental procedures were carried out with approval from the Institutional Animal Use and Care Committee of West Virginia University School of Medicine. The animal care standards were followed by adhering to the recommendations for the care of laboratory animals as advocated by the American Association for Accreditation of Laboratory Animal Care and following the policies and procedures detailed in the Guide for the Care and Use of Laboratory Animals as published by the U.S. Department of Health and Human Services and proclaimed in the Animal Welfare Act (PL89-544, PL91-979, and PL94-279).
RT-PCR. Total RNA was extracted from the medial gastrocnemius muscle of both suspended and control animals with TriReagent (Molecular Research Center, Cincinnati, OH), which is based on the guanidine thiocyanate method. Frozen muscle was mechanically homogenized on ice in 1 ml of ice-cold TriReagent. Total RNA were solubilized in RNase-free H2O and quantified in duplicate by measuring the optical density (OD) at 260 nm. Purity of RNA was assured by examining the ratio OD260/OD280. Ten micrograms of RNA were reverse transcribed with decamer primers and SuperScript II reverse transcriptase (RT) in a total volume of 30 µl according to standard methods (Invitrogen Life Technologies, Bethesda, MD). A control RT reaction was done in which the RT enzyme was omitted. The control RT reaction was PCR amplified to ensure that DNA did not contaminate the RNA. One microliter of complementary DNA (cDNA) was then amplified by PCR, using 100 ng of forward and reverse primers, ribosomal 18S primer pairs (Ambion, Austin, TX), 250 µM deoxyribonucleotide triphosphates, 1x PCR buffer, and 2.5 units of Taq DNA polymerase (USB, Cleveland, OH) in a final volume of 50 µl. PCR was performed using a programmed thermocycler (Biometra, Göttingen, Germany). The primer pairs were designed from sequences published in GenBank (Table 1), and PCR products were verified using restriction digestions. Preliminary experiments were conducted with each gene to ensure that the number of cycles represented a linear portion for the PCR OD curve for the muscle samples. The cDNAs from all muscle samples were amplified simultaneously using aliquots from the same PCR mixture. After the PCR amplification, 30 µl of each reaction were electrophoresed on 1.5% agarose gels stained with ethidium bromide. Images were captured, and the signals were quantified in arbitrary units as OD x band area using the Kodak image analysis system (Eastman Kodak, Rochester, NY). The size (number of base pairs) of each of the bands corresponded to the size of the processed mRNA. Ribosomal 18S primers were used as internal controls, while all RT-PCR signals were normalized to the 18S signal of the corresponding RT product to eliminate the measurement error from uneven sample loading and provide a semiquantitative measure of the relative changes in gene expression.
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Furthermore, to estimate the release/accumulation of mitochondria-housed apoptotic factors including cytochrome c, apoptosis-inducing factor (AIF), and Smac/DIABLO (second mitochondria-derived activator of caspase) to the cytosol, a nuclei-free, mitochondria-free cytosolic protein fraction was prepared as described by Rokhlin et al. (31), and the protein contents of these mitochondrial apoptotic factors were measured in this mitochondria-free cytosolic fraction as described below. Muscle was dissected from the connective tissues and minced in ice-cold extraction buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 0.1 mM PMSF, pH 7.4) in the presence of protease inhibitor cocktail. After a gentle homogenization with a Teflon pestle motorized with an electronic stirrer, homogenates were centrifuged at 800 g for 10 min at 4°C to pellet the nuclei and cell debris. The supernatants were then spun twice at 16,000 g for 20 min at 4°C to pellet the mitochondria, and the final supernatants were collected as nuclei-free, mitochondria-free cytosolic protein fractions. The above-described subcellular protein fractionation procedures have been routinely used in our laboratory to obtain high-purity protein fractions as assessed by immunoblotting the fractions with an anti-histone H2B (a nuclear protein), an anti-CuZnSOD (a cytosolic isoform of superoxide dismutase), and an anti-MnSOD (a mitochondrial isoform of superoxide dismutase) antibody (38, 40).
The protein contents of the protein extracts were quantified in duplicate by DC protein assay (Bio-Rad, Hercules, CA) based on the reaction of protein with an alkaline copper tartrate solution and Folin reagent, which was similar to Lowry assay (27). As a further means to confirm the protein contents, all the protein samples were measured in duplicate on a different occasion by BCA protein assay (Pierce, Rockford, IL) based on the biuret reaction and the bicinchoninic acid detection of cuprous cation (41).
Apoptotic cell death ELISA. Cell death detection ELISA kit (Roche Applied Science, Indianapolis, IN) was used to quantitatively determine the apoptotic DNA fragmentation by measuring the cytosolic histone-associated mono- and oligonucleosomes. The high levels of sensitivity and specificity in detecting apoptosis with the use of this kit have been demonstrated by assaying cellular samples in response to the known apoptogenic chemical (e.g., camptothecin) as reported by the manufacturer (Roche Applied Science). Briefly, the extracted nuclei-free cytosolic fraction of gastrocnemius muscle was used as an antigen source in a sandwich ELISA with a primary anti-histone mouse monoclonal antibody coated to the microtiter plate and a second anti-DNA mouse monoclonal antibody coupled to peroxidase. The amount of peroxidase retained in the immunocomplex was determined photometrically by incubating with 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) as a substrate for 20 min at 20°C. The change in color was measured at a wavelength of 405 nm by using a Dynex MRX plate reader controlled through personal computer software (Revelation, Dynatech Laboratories, CA). Measurements were performed with control and suspended samples analyzed on the same microtiter plate in the same setting. The OD405 reading was then normalized to milligrams of protein used in the assay and presented as an apoptotic index.
Fluorometric caspase activity assay. The total cytosolic protein fraction (50 µl) without protease inhibitor of the muscles was incubated in 50 µl of assay buffer (50 mM PIPES, 0.1 mM EDTA, 10% glycerol, and 10 mM DTT, pH 7.2) with 50 µM of the fluorogenic 7-amino-4-trifluoromethyl coumarin (AFC)-conjugated substrate (Ac-DEVD-AFC for caspase-3, Ac-LEHD-AFC for caspase-9; Alexis, San Diego, CA) at 37°C for 2 h. Caspase-specific inhibitor Z-VAD-FMK (Calbiochem, La Jolla, CA) was used as a control to validate the specificity of caspase. The change in fluorescence was measured on a spectrofluorometer (CytoFluor; Applied Biosystems, Foster City, CA) with an excitation wavelength of 390/20 nm and an emission wavelength of 530/25 nm before and after the 2-h incubation. Caspase activity was estimated as the change in arbitrary fluorescence units normalized to milligrams of protein used in the assay. Control and suspended samples were run on the same microplate in the same setting.
Western immunoblot analyses.
Protein expression of B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), X-linked inhibitor of apoptosis protein (XIAP), apoptosis repressor with caspase recruitment domain (ARC), and Fas-associated death domain protein-like interleukin-1
-converting enzyme-like inhibitory protein (FLIP) was determined in the total cytosolic protein fraction, whereas AIF was measured in the nuclear fraction.
Sixty micrograms of protein were boiled for 5 min at 95°C in Laemmli buffer, loaded on each lane of a 12% polyacrylamide gel, and separated by SDS-PAGE. The gels were blotted to nitrocellulose membranes (VWR, West Chester, PA) and stained with Ponceau S red (Sigma Chemical, St. Louis, MO) to verify equal loading and transferring of proteins to the membrane in each lane. As another approach to validate similar loading between the lanes, gels were loaded 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) at room temperature for 1 h and probed with the following primary antibodies diluted in PBS-T with 2% BSA: anti-Bcl-2 mouse monoclonal antibody (1:100 dilution, sc-7382), anti-Bax rabbit polyclonal antibody (1:200 dilution, sc-6236), anti-hILP/XIAP mouse monoclonal antibody (1:250 dilution, 610762; BD Biosciences, San Jose, CA), anti-ARC rabbit polyclonal antibody (1:200 dilution, sc-11435), anti-FLIP
rabbit polyclonal antibody (1:500 dilution, ab6144; Abcam, Cambridge, MA), or anti-AIF mouse monoclonal antibody (1:500 dilution, sc-13116HRP). Bcl-2, Bax, ARC, and AIF antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All primary antibody incubations were performed overnight at 4°C. Secondary antibodies were conjugated to horseradish peroxidase (Chemicon International, Temecula, CA), and signals were developed using West Pico chemiluminescent substrate (Pierce) or ECL detection kit (Amersham Biosciences, Piscataway, NJ). The signals were then visualized by exposing the membranes to X-ray films (BioMax MS-1, Eastman Kodak), and digital records of the films were captured with a Kodak 290 camera. Resulting bands were quantified as OD x band area with the use of a one-dimensional image analysis system (Eastman Kodak) and recorded in arbitrary units. The molecular sizes of the immunodetected proteins were verified using prestained standard (LC5925; Invitrogen Life Technologies).
Estimation of mitochondrial cytochrome c, Smac/DIABLO, and AIF release/accumulation. Cytochrome c, AIF, and Smac/DIABLO are apoptotic factors normally confined to mitochondria, and their release into the cytosol has been demonstrated during the activation of apoptosis (44). In the present study, the steady-state protein abundance, as a function of release and degradation, of these apoptotic factors was determined in the mitochondria-free cytosolic fraction to provide an estimated index for the release/accumulation of these mitochondrial proteins to the cytosol. This interpretation of this estimated apoptotic "release" assumes that degradation of the apoptotic factors remains constant, but this was not measured. The release and/or accumulation of Smac/DIABLO and AIF into the cytosol was estimated by measuring their protein contents in the extracted mitochondria-free cytosolic protein fraction by immunoblotting with an anti-Smac/DIABLO mouse monoclonal antibody (1:500 dilution, 612244; BD Biosciences) and an anti-AIF monoclonal mouse antibody. Moreover, a cytochrome c ELISA kit (MBL International, Woburn, MA) was used to assess the protein content of cytochrome c in the mitochondria-free cytosol fraction to evaluate the release/accumulation of the mitochondrial cytochrome c into the cytosol. Following the manufacturer’s protocol, we used 60 µl of the extracted mitochondria-free cytosolic fraction as an antigen source in a sandwich ELISA with a horseradish peroxidase-conjugated anti-cytochrome c polyclonal antibody in microwell strips coated with an anti-cytochrome c antibody. After being washed, the peroxidase retained in the immunocomplex was detected by incubating with a chromogenic substrate, tetramethylbenzidine/hydrogen peroxide, followed by addition of an acid solution to terminate the enzyme reaction and stabilize the developed color. The change in color was monitored at a wavelength of 450 nm using a Dynex MRX plate reader. Measurements were performed with the control and suspended samples analyzed on the same microplate, and the cytochrome c content was expressed as OD450 per milligrams of protein.
Statistical analyses. Statistical analyses were performed using the SPSS 10.0 software package. ANOVA was performed to examine the main effects of suspension, age, and interaction (suspension x age) on the measured variables. ANOVA followed by Tukey’s honestly significant difference post hoc analysis was used to examine differences between groups. All data are given as means ± SE. Statistical significance was accepted at P < 0.05.
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RESULTS |
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30% in both young adult and aged rats (young: 993 vs. 699 mg; aged: 761 vs. 520 mg), whereas the muscle weight normalized to the animal bodyweight was reduced by 11 and 19% in young adult and aged rats, respectively (young: 2.7 vs. 2.4 mg/g; aged: 1.4 vs. 1.1 mg/g) (Table 2). The interpretation of the normalized data after hindlimb suspension is limited, because the decrease in body weight with hindlimb unloading (Table 2) may reflect, in part, losses in connective tissue and bone mass and/or changes in visceral weight and body composition as well as changes in muscle weight following hindlimb suspension (13, 50). Nevertheless, muscle weight normalized to body weight provides a gauge that suggests a preferential loss of muscle relative to the total loss of body weight following hindlimb suspension and preferential loss of muscle weight relative to body weight with aging.
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DISCUSSION |
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Age-specific apoptotic responses to hindlimb suspension in gastrocnemius muscle. Apoptosis has been implicated to have a physiological role in regulating the process of skeletal muscle wasting. This idea was primarily based on the consistent observations that apoptosis is activated or accelerated under various muscle atrophic conditions including hindlimb suspension-mediated hypokinesia, muscle unloading, muscle denervation, muscle dystrophy, neuromuscular disorders, strenuous physical exercise, and aging-associated sarcopenia (1, 2, 15, 24, 30, 32–35, 38, 40, 45). With a focus on the muscle disuse mediated by hindlimb suspension, Allen et al. (1) first demonstrated that the number of myonuclei exhibiting double-stranded DNA fragmentation and/or morphologically abnormal myonuclei was significantly increased in the soleus muscles of young rats after 14 days of suspension. Accordingly, apoptosis was suggested to have a role in the elimination of myonuclei during hindlimb suspension-induced muscle atrophy (1). Recently, the findings of apoptosis in hindlimb suspension-mediated muscle disuse have been expanded by Leeuwenburgh et al. (24), who showed that soleus muscle from aged animals also demonstrates activation of apoptosis in response to 14 days of hindlimb suspension. Furthermore, using the technique of immunohistochemistry, they demonstrated that apoptosis in soleus muscle is partly mediated by the subsarcolemmal mitochondria through endonuclease G (a mitochondrial caspase-independent apoptogenic factor) translocation to the myonucleus in response to hindlimb suspension, and these results suggest that the underlying pathways involved in apoptosis may be distinct in young and old muscles (24). In this study we extended these observations by showing that in addition to slow-twitch plantar flexor soleus muscle, the mixed-fiber (but primarily fast-twitch containing) plantar flexor gastrocnemius muscle also demonstrates activation of apoptosis after acute hindlimb suspension. Together, these data suggest that apoptosis is a global program involved in regulation of decreased muscle mass in both fast and slow myosin-containing fibers. The activation of apoptosis is indicated by the elevation of apoptotic DNA fragmentation and changes of apoptotic regulatory factors including increases in mitochondrial cytochrome c release/accumulation and proapoptotic Bax after suspension-mediated unloading. These findings show that apoptosis in muscle loss induced by hindlimb suspension in muscle groups is not limited to only muscles containing only slow myosin heavy chains.
We found elevation of the antiapoptotic Bcl-2 protein in the suspended muscle relative to the control muscle in both young and aged animals. These data are consistent with observations of other investigators who reported increases in the antiapoptotic Bcl-2 during denervation-induced muscle wasting (21, 38, 46). Nevertheless, the increase in Bcl-2 is opposite to the general pattern of proapoptotic signaling in unloaded muscles, including increases in apoptotic DNA fragmentation, mitochondrial cytochrome c release/accumulation, and Bax levels in suspended vs. control muscles. We speculate that the elevation of Bcl-2 may be an adaptive or compensatory change that is invoked in response to suspension in the gastrocnemius muscle in an attempt to reduce the apoptotic loss of myonuclei and, consequently, muscle mass.
Moreover, in this study we found that the release/accumulation of mitochondrial AIF (a caspase-independent apoptogenic factor) in the cytosol is observed in the aged gastrocnemius muscle after hindlimb unloading, but this change was not found in the young muscle. These AIF findings are in accordance with the endonuclease G data that have been recently reported in suspended soleus muscles of aged rats (24). Together, these data support the concept that different apoptotic mechanisms are responsible for the activation of apoptosis in young and aged skeletal muscles. As suggested in the previous study (24), caspase-dependent and -independent apoptotic mechanisms may contribute, to a different degree, to the suspension-induced activation of apoptosis in an age-specific manner, and the present findings appear to be in agreement with this proposition. However, we did not find any significant changes of caspase-3 or caspase-9 mRNA abundances and protease activities with suspension unloading in the gastrocnemius muscles from either young or aged animals. Nonetheless, our data did not allow us to exclude the possibility that some caspase-related changes may have occurred in the muscles from young and aged animals during suspension at the time points that were not examined in this study. In line with these age-related findings, we also observed previously (37, 40) that the apoptotic signaling components (e.g., Bax, Bcl-2, AIF, Id2, and p53) respond to unloading differently in the hypertrophied muscles from young and aged birds. In contrast to the young muscle, we previously reported antiapoptotic changes, including increases in Bcl-2, decreases in Bax and nuclear AIF, and unchanged Id2 and p53 abundances, in aged hypertrophied muscle after 14 days of subsequent removal of load, although TUNEL-indicated apoptosis was still evident in these aged unloaded muscles. Nevertheless, we cannot rule out the possibility that these findings may be unique to the nature of the model (i.e., muscle loss from hypertrophied state back to the resting state) and/or species (i.e., bird). However, it was clearly demonstrated that aging can influence the apoptotic response to unloading, at least in this quail hypertrophied muscle unloading model. Aging also has been demonstrated to affect the homeostatic regulation of myonuclear domain (19, 24). Nevertheless, it remains largely unclear whether the age-dependent alteration of myonuclear domain regulation or the age-specific apoptotic signaling has a physiologically important role during muscle disuse. Future research is needed to resolve these novel but relatively unresolved age-related responses.
Clarification of the changes in apoptotic signaling accompany with the aging-associated sarcopenic muscle loss.
There has been recent evidence indicating that apoptosis may play a considerable role in mediating the progression of sarcopenia (2, 3, 15, 16, 23, 24, 29, 30, 42). Given that the etiology of sarcopenia is still largely unknown, active investigations are ongoing in several laboratories with the aim of fully understanding this unavoidable and detrimental process with aging. Although there has been evidence showing apoptosis in sarcopenic skeletal muscle, the apoptotic signaling pathways responsible for the activation of apoptosis and the importance of apoptotic contributions to sarcopenia have not been fully unraveled. Several pieces of data indicate that certain BCL-2 family proteins, caspases, and other apoptotic regulators may be responsive to aging (2, 3, 15, 16, 24). Previous investigations conducted in our laboratory have shown that proapoptotic Bax and selective caspase activities are upregulated in the gastrocnemius and plantaris muscles of 30-mo-old Fischer 344 x Brown Norway rats, whereas antiapoptotic Bcl-2 is downregulated in aged plantaris muscle compared with the young adult muscles (2, 3). It is noted that these age-related apoptotic changes, to some extent, are not in agreement with the aging data reported by Leeuwenburgh’s group (15, 16), who did not find any age-related changes in Bax, Bcl-2, and caspase-3 activity. However, these discrepancies are reasonably explained by the differences in the age and the strain of the animals being examined in the different studies and the approach of measurement being adopted. In both of the studies reporting unchanged Bax, Bcl-2, or caspase activity (15, 16), 24-mo-old Fischer 344 rats were used as the aged model. It is likely that the different responses between our study using Fischer 344 x Brown Norway rats and that of other groups who examined Fischer 344 rats (15, 16) can be accounted for by the differences in the ages and species of rats used in these studies. Data from Leeuwenburgh’s group showing that caspase-3 activity tends to increase in the soleus muscle of 32-mo-old Fischer 344 x Brown Norway rats relative to young animals (P = 0.052) further support the above explanation (24). Moreover, unchanged Bax and Bcl-2 levels were reported according to the results of an ELISA analysis performed on the mitochondrial fraction of muscle lysates. Compared with the total protein lysate, this may also contribute to the contradictory findings on Bax and Bcl-2, because these BCL-2 family proteins have been demonstrated to localize also in the subcellular compartments other than the mitochondria (e.g., endoplasmic reticulum) (8, 51).
In the present study, we provide additional information showing that aging favors the proapoptotic tendencies in the gastrocnemius muscle by upregulating the content of Bax mRNA and protein, Apaf-1 mRNA, AIF mRNA, caspase-3 activity, caspase-9 mRNA and activity, XIAP mRNA and protein, and the release/accumulation of mitochondrial cytochrome c and Smac/DIABLO. Notably, we found that the Bcl-2 content is upregulated with aging in gastrocnemius muscles. The reported downregulated Bcl-2 in the plantaris muscle from 30-mo-old Fischer 344 x Brown Norway rats (3) and unchanged Bcl-2 in the gastrocnemius muscle from 24-mo-old Fischer 344 rats (15), in conjunction with our current Bcl-2 observation, suggests that muscle group (gastrocnemius vs. plantaris) and species and/or age of rats may be important factors that influence the response of antiapoptotic Bcl-2 to aging.
In this study, we have demonstrated that the activation of apoptosis, as denoted by elevated apoptotic DNA fragmentation and changes of cytochrome c and BCL-2 family proteins, is evident in a mixed-fibered (gastrocnemius) muscle from both young adult and aged rats in response to acute hindlimb suspension. We report increases in the protein expression of proapoptotic Bax, release/accumulation of mitochondrial apoptogenic factor cytochrome c, and the extent of apoptotic DNA fragmentation in young adult and aged gastrocnemius muscles after 14 days of hindlimb suspension. These findings further clarify the role of apoptosis in skeletal muscle atrophy during hindlimb suspension by showing that the apoptotic program is activated not only in slow-twitch plantar flexor muscle (e.g., soleus) but also in mixed-fiber plantar flexor muscle (e.g., gastrocnemius muscle). Another notable finding in the present study is that only the suspended aged gastrocnemius muscle exhibited the release/accumulation of mitochondrial AIF (a caspase-independent apoptogenic factor) to the cytosol, whereas this was not observed in the suspended young adult muscle. Along with the recent findings demonstrating that endonuclease G (another mitochondrial caspase-independent apoptogenic factor) is involved in hindlimb suspension-induced muscle atrophy exclusively in the aged soleus muscle (24), our AIF findings support the hypothesis that the mechanisms responsible for the activation of apoptosis during hindlimb suspension may be age dependent. Nonetheless, more investigations are required to fully understand the complicated influence of aging in the apoptotic regulation under the situation of muscle disuse.
Apoptosis has been shown to occur in disuse atrophy of slow myosin-containing muscles (i.e., soleus muscle), where muscle loss is severe (1, 24). In the present study, we have shown that hindlimb suspension activates apoptotic signaling in the gastrocnemius muscle, which is less severely affected by disuse than slow muscles and also predominately contains type II myosin heavy chains. The suspension-induced elevations of BCL-2 family and mitochondrial cytochrome c release/accumulation and the responses of Apaf-1, Smac/DIABLO, XIAP, ARC, and FLIP in gastrocnemius muscle as shown in this study have not been examined in the soleus muscle; however, this warrants additional investigations to fully understand apoptotic signal transduction in atrophying soleus muscle. Such data will be useful to determine whether apoptotic responses follow similar general patterns in muscles of different muscle types undergoing different degrees of muscle loss during muscle disuse. Notably, according to the findings in the models of rat hindlimb suspension and limb immobilization, disuse muscle atrophy has been attributed to the alteration of protein turnover comprising reduction of protein synthesis and increased rate of protein degradation or proteolysis in skeletal muscle (7, 47). Prolonged bed rest, another ground-based model that mimics spaceflight-induced muscle wasting, also has been shown to result in decreases in skeletal muscle protein synthesis, which contributes to muscle mass loss, and resistance exercise throughout the period of bed rest can prevent the reduction of protein synthesis (17, 18). Although the rate of muscle protein synthesis and degradation has not been assessed in the present study, it is reasonable to assume that protein degradation exceeded protein synthesis in the suspended muscle, leading to reduction in muscle protein abundance and therefore muscle mass. Nevertheless, it is not known whether the proapoptotic response to hindlimb suspension in the atrophying soleus or gastrocnemius muscles as reported by the present investigators and by others (1, 24) may have interacted with either the decrease in protein synthesis or the increase in proteolysis, or both, and this needs to be resolved in subsequent research.
The animal model of hindlimb suspension has been developed and approved to be an appropriate ground-based model to simulate the deleterious effects of the zero gravitational environment during spaceflight. Hindlimb suspension permits the investigation of skeletal muscle adaptations under non-weight-bearing conditions. The findings of the present study provide data that help to elucidate cellular and molecular mechanisms responsible for muscle wasting during muscle unweighting in rodents. Further investigations are needed to determine whether apoptosis also occurs in fast and slow muscles of humans after spaceflight and other unloading-induced muscle wasting situations (e.g., bed rest), as is the case in rats during hindlimb suspension.
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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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 K. M. Rice, D. L. Preston, D. Neff, M. Norton, and E. R. Blough Age-Related Dystrophin-Glycoprotein Complex Structure and Function in the Rat Extensor Digitorum Longus and Soleus Muscle J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2006; 61(11): 1119 - 1129. [Abstract] [Full Text] [PDF]
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J. Quadrilatero and J. W. E. Rush Increased DNA fragmentation and altered apoptotic protein levels in skeletal muscle of spontaneously hypertensive rats J Appl Physiol, October 1, 2006; 101(4): 1149 - 1161. [Abstract] [Full Text] [PDF]
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E. E. Dupont-Versteegden, J. D. Fluckey, M. Knox, D. Gaddy, and C. A. Peterson Effect of flywheel-based resistance exercise on processes contributing to muscle atrophy during unloading in adult rats J Appl Physiol, July 1, 2006; 101(1): 202 - 212. [Abstract] [Full Text] [PDF]
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K. Csukly, A. Ascah, J. Matas, P. F. Gardiner, E. Fontaine, and Y. Burelle Muscle denervation promotes opening of the permeability transition pore and increases the expression of cyclophilin D J. Physiol., July 1, 2006; 574(1): 319 - 327. [Abstract] [Full Text] [PDF]
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P. M. Siu, E. E. Pistilli, Z. Murlasits, and S. E. Alway Hindlimb unloading increases muscle content of cytosolic but not nuclear Id2 and p53 proteins in young adult and aged rats J Appl Physiol, March 1, 2006; 100(3): 907 - 916. [Abstract] [Full Text] [PDF]
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