Although it has been demonstrated that exercise training has an antiapoptotic effect on postmitotic myocytes, the mechanisms responsible for this effect are still largely unclear. Because the antiapoptotic effect of exercise training in postmitotic myocytes could be possibly mediated by the upregulation of apoptotic suppressors, this study examined the effect of endurance training on endogenous apoptotic suppressors including X-chromosome-linked inhibitor of apoptosis protein (XIAP), apoptosis repressor with caspases recruitment domain protein (ARC), and FADD-like inhibitor protein (FLIP) in skeletal and cardiac muscles. Eight adult Sprague-Dawley rats were trained 5 days weekly for 8 wk on treadmill, and eight sedentary rats served as controls. Soleus and ventricle muscles were dissected 2 days after the last training session. The mRNA content of XIAP, ARC, and FLIP was estimated by RT-PCR with ribosomal 18S RNA used as an internal control. The protein expression of XIAP, ARC, FLIPS, and FLIPα was assessed by Western immunoblot. After training, mRNA content of ARC and FLIP was not different between the control and trained animals, whereas XIAP mRNA content was elevated by 22 and 14% in the trained soleus and cardiac muscles, respectively, relative to the control samples. No difference was found in the protein content of FLIPS and FLIPα between control and trained muscles, whereas XIAP and ARC protein content was increased by 18 and 38%, respectively, in the soleus muscle of trained animals. Furthermore, negative relationships were found between XIAP and apoptotic DNA fragmentation as well as ARC and caspase-3 activity. These findings are consistent with the hypothesis that the modulation of apoptotic suppressors is involved in training-induced attenuation of apoptosis in skeletal and cardiac muscles.
- cell death receptor
- apoptotic inhibitors
- X-chromosome-linked inhibitor of apoptosis protein
- FADD-like inhibitor protein
apoptosis is a highly organized and tightly coordinated biological process that plays a vital role in monitoring a variety of cellular events (e.g., tissue turnover). Aberrant regulation of apoptosis has been implicated in the pathogenesis of certain severe muscle-related diseases including Duchenne and facioscapulo human muscular dystrophy (33–35). The cellular decision resulting in the activation and subsequent execution of the apoptotic program is principally controlled under the simultaneous influence of both pro- and antiapoptotic signaling, which are primarily orchestrated by a specific cluster of apoptotic regulatory proteins (e.g., BCL-2 family and caspases). Among a number of proteins that have been identified in regulating the apoptotic machinery, a group of endogenous proteins referring to X-linked inhibitor of apoptosis protein (XIAP), apoptosis repressor with caspases recruitment domain protein (ARC), and Fas-associated death domain protein-like interleukin-1β-converting enzyme-like inhibitory protein (FLIP) constitute an assembly of elemental apoptotic suppressors in modulating the antiapoptotic signaling.
XIAP has been suggested to be a potent member of a protein family named inhibitor of apoptosis, which is fundamentally conserved among various species (7, 39). All known inhibitor of apoptosis proteins have been exhibited to include at least one baculovirus inhibitor of apoptosis repeat motif that is essential for their antiapoptotic activity through the inhibition on the initiator and effector caspases (6, 32, 39). Furthermore, ARC and FLIP are two other endogenous proteins abundantly expressed in muscle tissues that have been identified in constituting the antiapoptotic modulating mechanism (15, 19). Although it has been shown that the suppressive effect of ARC and FLIP on apoptosis is related to their negative effects on the selected caspases and is mostly associated with the death receptor-mediated apoptotic pathway (1, 15, 19), there have been recent data suggesting that the antiapoptotic function of ARC also may be mediated by influencing the mitochondria-mediated apoptosis pathway (e.g., interferes with the activation of proapoptotic Bax) (12). In addition, the suppressive action of FLIP in apoptosis may be dependent on the individual splice variant or protein isoform of FLIP (10, 15, 18).
Previously, our laboratory demonstrated that exercise training is capable of attenuating apoptosis in skeletal and cardiac muscles of adult rats, and our laboratory reported that the regulatory mechanisms responsible for the antiapoptotic effect of training comprise the alteration of Bcl-2, Bax, apoptotic protease-activating factor-1, heat shock proteing 70, and Mn superoxide dismutase (41). Nonetheless, whether apoptotic suppressor proteins are involved in causing the attenuation of apoptosis after exercise training remains unknown. Thus, in this study, we examined the response of the apoptotic suppressors (XIAP, ARC, and FLIP) to 8-wk endurance training in skeletal and cardiac muscles. We tested the hypothesis that the apoptotic suppressors are upregulated after exercise training, which is part of the cause for the antiapoptotic adaptation in skeletal muscle and heart.
Animals and treadmill training.
Adult male Sprague-Dawley rats aged 3 mo (Harlan, Indianapolis, IN) were studied. The rats were housed in pathogen-free conditions at ∼20°C. They were exposed to a reverse light condition of 12 h of light and 12 h of darkness each day and were fed rat chow and water ad libitum throughout the study period. Sixteen rats with similar body weights (range of 210–240 g) were randomly assigned to control (n = 8) or training (n = 8) groups. The animals were trained by running on a level motorized rodent treadmill (Columbus Instruments, Columbus, OH) 5 days weekly for 8 wk. The training intensity and duration were progressively increased, and we have shown that this training protocol stimulates moderate endurance training effect with an ∼25% increase in citrate synthase activity in rats (42, 43). For the first 4 wk, the speed of the treadmill and duration of the training sessions were gradually increased from a speed of 10 m/min for 10 min to a running speed of 28 m/min for 55 min by the end of week 4. For the next 4 wk, a 5-min warm-up session at a speed of 20 m/min was followed by the 55-min training session at a speed of 28 m/min. Control animals were handled daily and were subjected to the noise of the running treadmill by placing their cages next to the treadmill when the training animals ran on the treadmill. This procedure aimed to minimize the possible confounding effect of external factors (e.g., handling, treadmill noise, etc.). Training animals were killed 48 h after the last training session, and the control animals were euthanized at the same time as training animals. All animals were killed via CO2 inhalation followed by decapitation, at which time the soleus muscle and the apex cordis from the heart muscle were quickly removed and frozen immediately in liquid nitrogen and stored at −80°C until further analysis.
All experimental procedures carried approval from the Institutional Animal Use and Care Committee from 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 US Department of Health and Human Services.
Total RNA was obtained from soleus and ventricle muscles of control and trained animals with TriReagent (Molecular Research Center, Cincinnati, OH), which is based on the guanidine thiocyanate method. Briefly, frozen muscles were minced then mechanically homogenized on ice in 1 ml of ice-cold TriReagent. Total RNA was solubilized in RNase-free H2O and quantified in duplicate by measuring the optical density (OD) at 260 nm. Purity of RNA was determined by examining the 260-nm OD-to-280-nm OD ratio. Two micrograms of RNA were reverse transcribed with decamer primers and Superscript II RT in a total volume of 20 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 cDNA was then amplified by PCR using 100 ng of forward and reverse primers, ribosomal 18S primer pairs (Ambion, TX), 250 μM deoxyribonucleotide triphosphates (dNTPs), 1× 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 by restriction digestions. Preliminary experiments were conducted with each gene to assure that the number of cycles represented a linear portion for the PCR OD curve for the muscle samples. The cDNA 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 that were stained with ethidium bromide. Images were captured and the signals were quantified in arbitrary units as OD × band area using 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 samples loading and provide a semiquantitative measure of the relative changes in gene expression.
Protein preparation and Western immunoblot.
The protein extraction method described by Rothermel et al. (30) was adopted with minor modification to obtain the total cytosolic protein fraction from the soleus and ventricle muscles. In brief, after removal of connective tissues, muscle was minced and homogenized on ice 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, 1 mM dithiothreitol). After centrifugation at 1,000 rpm for 1 min at 4°C to pellet the nuclei and cell debris, the supernatants were further centrifuged three times at 6,000 rpm for 5 min at 4°C to remove residual nuclei and stored as total cytosolic protein extract at −80°C after a protease inhibitor cocktail containing (in mM) 104 4-(2-aminoethyl)-benzenesulfonyl fluoride, 0.08 aprotinin, 2 leupeptin, 4 bestatin, 1.5 pepstatin A, and 1.4 E-64 (Sigma-Aldrich, St. Louis, MO) was added. The protein content of the extract was then measured in duplicate by detergent-compatible 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 (23). As a further measure to assure protein content, all protein samples were quantified in duplicate on a different occasion by bicinchoninic acid protein assay (Pierce, Rockford, IL) based on the biuret reaction and the bicinchoninic acid detection of cuprous cation (44).
Forty micrograms of protein were boiled for 5 min at 95°C in Laemmli buffer (161-0737, Bio-Rad) in the presence of 2-mercaptoethanol and were loaded on each lane of a 12% polyacrylamide gel and separated by SDS-PAGE at room temperature. The gels were blotted to nitrocellulose membranes (VWR, West Chester, PA) and stained with Ponceau S red (Sigma) 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 PBS with 0.05% Tween 20 at room temperature for 1 h and probed with 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, Santa Cruz Biotechnology, Santa Cruz, CA), or an anti-FLIPS/L mouse monoclonal antibody (1:100 dilution, sc-5276, Santa Cruz Biotechnology) diluted in PBS with 0.05% Tween 20 with 2% BSA. It is noted that we did not detect the FLIPL by using the anti-FLIPS/L mouse monoclonal antibody (sc-5276); therefore, an anti-FLIPα (i.e., FLIPL) rabbit polyclonal antibody (1:500 dilution, ab6144, Abcam, Cambridge, MA) was further used to detect the α-splice variant or long isoform of FLIP. Membrane was also probed with anti-beta tubulin rabbit polyclonal antibody (1:500 dilution, ab6046, Abcam) to examine the equality of protein loading. All primary antibody incubations were performed overnight at 4°C. Secondary antibodies were conjugated to horseradish peroxidase (1:3,000 dilution, Chemicon International, Temecula, CA), and signals were developed by 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 × band area by a one-dimensional image analysis system (Eastman Kodak) and recorded in arbitrary units. The predicted molecular sizes of the immunodetected proteins were verified by using prestained standard (LC5925, Invitrogen Life Technologies, Bethesda, MD).
Statistics were performed using the SPSS 10.0 software package. Independent t-test was used to examine differences between control and trained groups. 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 given as means ± SE.
mRNA content of XIAP, ARC, and FLIP.
Transcriptional expression of XIAP, ARC, and FLIP genes was analyzed by semiquantitative RT-PCR in soleus and cardiac muscles of trained and control animals. Inverted images of the representative ethidium bromide-stained gels for the PCR amplified products are shown in the insets of Fig. 1. After exercise training, we found that the XIAP mRNA content was elevated by 22% (P < 0.01) and 14% (P < 0.05) in soleus and cardiac muscles, respectively, compared with the control muscles (Fig. 1). The transcriptional expression of ARC and FLIP was similar in both soleus and cardiac samples obtained from trained and control animals (P > 0.05).
Protein content of XIAP, ARC, FLIPS, and FLIPα.
An immunoreactive band of ∼57 kDa corresponding to the predicted molecular mass of XIAP protein and ∼25 kDa immunoreactive band corresponding to ARC protein was detected in our immunoblots. According to the Western analysis, XIAP protein content in soleus muscle of the trained animals was 18% higher (P < 0.05) than that of the control animals, whereas the protein content of XIAP was similar in cardiac muscle obtained from the trained and control animals (Fig. 2). Although the ARC protein content was similar in the control and trained cardiac muscles, in the trained soleus muscle, the protein content of ARC was elevated by 38% (P < 0.05) relative to the control soleus muscle (Fig. 2). In contrast, no differences were found in FLIPS or FLIPα protein content between control and trained animals (P > 0.05).
Relationships of XIAP, ARC, FLIP, and apoptotic markers.
We have previously found relationships between XIAP, ARC, and FLIP with apoptotic markers including caspase-3 activity and apoptotic DNA fragmentation in the muscles of these animals (41). In this study, we found a negative r value between XIAP protein content and apoptotic DNA fragmentation (expressed as apoptotic index) in the control and trained soleus muscles (r = −0.647, P = 0.007, n = 16), and when the soleus and ventricle muscles of control and trained animals were treated as a single group (r = −0.512, P = 0.003, n = 32). The ARC mRNA content was negatively correlated with caspase-3 activity when the soleus and ventricle samples of the control and trained animals were pooled as a single group (r = −0.415, P = 0.018, n = 32). Individually, the correlation between ARC mRNA and caspase-3 activity in the ventricle muscles of control and trained animals was r = −0.624 (P = 0.01, n = 16), and in the trained ventricle muscles this relationship was r = −0.720 (P = 0.044, n = 8).
Our laboratory has previously demonstrated that 8 wk of moderate endurance training attenuates apoptosis in skeletal and cardiac muscles of young adult rats (41). Here, we extend our laboratory's prior findings by showing that endogenous apoptotic suppressors (XIAP and ARC) appear to be involved in mediating the antiapoptotic effect of exercise training in postmitotic myocytes. We demonstrated that the mRNA and protein content of XIAP and the protein content of ARC were elevated in the soleus muscle while XIAP mRNA content was upregulated in the ventricle muscle of rats after 8 wk of endurance training. Moreover, negative relationships existed between XIAP and apoptotic DNA fragmentation as well as ARC and caspase-3 activity. Overall, our data are in agreement with the hypothesis that modulation of apoptotic suppressors (e.g., XIAP and ARC) is involved in explaining the antiapoptotic effect of exercise training in skeletal and cardiac muscles.
The suppressive characteristic of XIAP in apoptosis has been well examined in mitotic cell lineages (6). However, only a few studies have investigated XIAP in postmitotic myocytes. With a focus on mitochondrial encephalomyopathic human skeletal muscle, Ikezoe and colleagues (14) reported that sarcoplasmic expression of XIAP was evident concomitant with the expressions of Bax and apoptotic protease-activating factor-1, cytochrome c release, and caspase-3 activation, but no significant increase in DNA fragmentation as estimated by light/electron microscopy-facilitated TUNEL. Accordingly, they concluded that XIAP might be involved in suspending the apoptotic process in mitochondrial encephalomyopathies. In addition, XIAP expression has been shown to increase in the skeletal muscle of the aged rats compared with the young adult rats, even though acceleration of apoptosis in these aged muscles was still observed (i.e., increases in apoptotic DNA fragmentation and pro-/cleaved caspase-3 protein content) (9). Although it appears that XIAP responds adaptively to mitochondrial encephalomyopathy and aging in skeletal muscle, in cardiomyocytes, it has been demonstrated that hypoxic or ischemic injury is related to the decline in XIAP (24, 40). Altogether, the exact role of XIAP in postmitotic myocytes is still not clear and is worth investigating in future studies. Nonetheless, it is generally agreed that XIAP has antiapoptotic properties (6), and our present data showed that endurance training increases XIAP expression in skeletal and cardiac muscles, which is consistent with the training-induced antiapoptotic response that our laboratory reported (41).
The antiapoptotic effect of XIAP and ARC has been initially shown to be related to their inhibitory action on selected caspases (1, 6, 19, 39). Although, in the present study, we found that the expression of XIAP or ARC increased in soleus and cardiac muscles after training, we did not observe any influence in the enzyme activity of caspase-3, an essential effector caspase, from endurance training in these muscles (41). Because the present animals were killed and the muscle samples were obtained 48 h after the last training session, one of the possible reasons may be the limitation of the time point being selected. The findings show that ARC interacts with selected caspases (e.g., caspase-2 and -8) but does not interact with other caspases, including caspase-1, -3, and -9 (19). Thus it was not surprising that caspase-3 activity did not change in our study despite increases in ARC levels. Furthermore, it is worth noting that the muscle samples examined in our study were obtained from healthy young adult rats, and we would expect a relatively low level of apoptotic activity (e.g., effector caspase-3 activation) in these postmitotic tissues. This may also preclude us from acquiring evidence that shows a direct role of increases in ARC or XIAP levels to suppress caspase-3 activity after training. Moreover, there have been data suggesting that both ARC and XIAP can demonstrate their antiapoptotic properties by influencing multiple pathways involving the apoptotic factors other than caspases (12, 36, 37, 46). In addition to the findings showing that ARC can exhibit its antiapoptotic action by interfering with the activation of Bax rather than via the caspases (12), XIAP may also inhibit apoptosis through an alternative caspase-independent mechanism that depends on the activation of JNK1 and/or neutralization of Smac/DIABLO (36, 37, 46). Although we cannot rule out the possibility that our observed changes in XIAP may have involved a caspase-dependent mechanism, it seems more likely that the exercise training-induced responses of XIAP and ARC may have contributed to the antiapoptotic signaling through a caspase-independent mechanism. Nevertheless, this possibility warrants further investigation.
Considering that the aging-associated physiological and functional impairments (e.g., sarcopenia) inevitably occur in all humans (31), active research has been undertaken with the goal of finding measures that can counteract the deteriorating effect of normal aging and thereby ameliorate the quality of life in the aged populations. Physical exercise training is an attractive strategy because it is a relatively economical and natural regimen that does not introduce any drug- or chemical-induced side effects and can be readily applied to a large scale of populations. Resistance training has been suggested to be effective in alleviating the adverse outcomes with aging (e.g., falling due to frailty of musculoskeletal system) by preserving a considerable amount of muscle mass and muscular strength (13, 17). Although age-associated muscle and strength losses are still inevitable even in masters athletes who endurance train for many years (2), there is a lack of research that has investigated whether endurance-type exercise training might reduce or delay the onset of sarcopenic muscle loss with aging. Clearly, appropriate endurance training causes a number of beneficial cellular adaptations (e.g., improved antioxidant defense capacity and stress protein-mediated protective mechanisms) and physiological changes (e.g., improved cardiovascular functional capacity and decreased sympathetic neural tone) (5, 11, 16, 20, 22, 25, 29) that could be considered consistent with a favorable environment for the maintenance of muscle tissue. Because there has been evidence demonstrating that the loss of postmitotic tissues with aging may be attributed partly to the accelerated apoptosis (3, 4, 8, 9, 21, 26–28, 38, 45) and the present study and our previous findings showed that endurance training causes the antiapoptotic cellular responses in adult postmitotic skeletal and cardiac muscles (41), it is appealing to hypothesize that endurance training may slow the apoptotic processes leading to sarcopenia. Providing that the process of aging-related postmitotic myocytes loss occurs gradually and progressively from midlife to later stages of one's lifespan, the antiapoptotic adaptations from life-long endurance training may be capable of decreasing or delaying the apoptosis-associated loss of postmitotic myocytes. Nonetheless, additional research is needed to examine whether exercise training also causes the similar apoptotic responses in postmitotic tissues from aged individuals.
In conclusion, the present data support the hypothesis that modulation of apoptotic suppressors is involved in mediating the antiapoptotic effect of exercise training on postmitotic myocytes. We have demonstrated that the apoptotic suppressors (XIAP and ARC) are upregulated after endurance training while negative relationships exist between XIAP and apoptotic DNA fragmentation as well as ARC and caspase-3 activity in these animals. Although the cellular and molecular mechanisms responsible for the endurance training-induced attenuation in apoptosis are still unclear, our findings suggest that apoptotic suppressors contribute, at least in part, to the regulatory mechanisms for the antiapoptotic adaptation from endurance training in postmitotic myocytes. Because exercise training is potentially a valuable regimen in counteracting the loss of postmitotic myocytes with aging, additional research in apoptosis and exercise training is warranted to fully understand the underlying mechanisms of the antiapoptotic adaptation from exercise training.
This study was supported by National Institute on Aging Grant R01 AG-021530.
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