Growth hormone (GH) supplementation at old age has been shown to improve body composition, although its effect on muscle performance is still debated. On the other hand, resistance training increases muscle mass and strength even when initiated at advanced age. In the present study, we investigated the effects of short-term GH supplementation and exercise training on physical performance and skeletal muscle apoptosis in aged rats. Old (28 mo) male Fischer 344 × Brown Norway rats were randomized to 4 wk of GH supplementation (300 μg subcutaneous, twice daily) or 4 wk of treadmill running or used as sedentary controls. Eight-month-old rats, sedentary or exercised, were used as young controls. Exercise training improved exercise capacity and muscle strength in old animals. In soleus muscle, age and exercise were not associated with significant changes in the extent of apoptosis. However, we detected an age-related increase of cleaved caspase-8 (+98%), cleaved caspase-3 (+136%), and apoptotic DNA fragmentation (+203%) in the extensor digitorum longus muscle of old sedentary rats, which was attenuated by exercise. GH administration neither ameliorated physical performance nor attenuated apoptosis in extensor digitorum longus and was associated with increased apoptosis in soleus muscle (+206% vs. old controls). Our findings indicate that a short-term program of exercise training started at advanced age reverses age-related skeletal muscle apoptosis and represents an effective strategy to improve physical performance. In contrast, short-term administration of GH late in life does not provide any protection against functional decline or muscle aging and may even accelerate apoptosis in slow-twitch muscles, such as the soleus.
- growth hormone
- muscle quality
- tumor necrosis factor-α
the aging process is characterized by a progressive and irreversible functional and structural decline of multiple physiological systems. In the case of skeletal muscle, advancing age has been associated with a progressive loss of muscle mass, strength, and quality, a phenomenon also known as sarcopenia (41). These age-related changes have been reported even among healthy, physically active subjects and are associated with several adverse outcomes, including frailty, disability, reduced quality of life, institutionalization, and mortality (26). Intriguingly, the decline of muscle strength and physical function at old age is accelerated relative to the loss of muscle mass, indicating that deterioration of muscle quality might play a prominent role in the disabling process (22, 35). Multiple etiological factors have been linked to the development of sarcopenia, including loss of α-type motoneurons (55), altered hormonal status [e.g., decline of growth hormone (GH) and testosterone levels] (50), increased production of catabolic cytokines (56), inadequate nutrition (17), and decreased physical activity (50).
The consequence of physical inactivity as observed by loss of muscle mass and strength at old age has been highlighted by several reports showing that exercise training counteracts the age-related muscle loss and functional decline even in frail older adults (3, 21, 27, 39). Furthermore, exercise training has recently been shown to attenuate skeletal muscle apoptosis in old rats (43). Notably, an age-related alteration of the apoptotic process has been identified as a potential mechanism in the pathogenesis of sarcopenia in both animals (15, 16, 37, 38, 42) and humans (33, 47, 58).
The GH-IGF-I axis plays a pivotal role in regulating body composition (49, 53). GH acts primarily via circulating and/or tissue-derived IGF-I and stimulates amino acid uptake and the synthesis of nucleic acids and proteins (7). In addition, GH reduces lipogenesis and promotes lipolysis (60). The functionality of the GH-IGF-I axis drops with advancing age (9), and GH replacement has been shown to improve body composition (i.e., decreases fat mass and increases lean body mass) in older adults (36, 52, 59). In contrast, the effects of GH supplementation on muscle strength and physical performance are controversial, with some studies reporting significant improvements (10, 48, 57), whereas others reporting marginal (6) or no effects (4, 28, 36, 52, 59). Despite the large number of studies conducted on GH supplementation, little is known about the mechanisms by which hormonal replacement improves skeletal muscle properties. GH supplementation was found to promote muscle satellite cell proliferation in adult rats (54). In addition, Sonntag et al. (45) reported that GH administration restored protein synthesis in skeletal muscle of old rats. Furthermore, Lange et al. (28) demonstrated that, in older men, GH administration induced an increased expression of myosin heavy chain type 2×, an isoform that is progressively less expressed with advancing age, thus shifting the myosin heavy chain pattern to a more juvenile profile. However, this effect was not accompanied by an increase in muscle strength (28). Interestingly, Dalla Libera et al. (11), using a rat model of experimental congestive heart failure (CHF), showed a significant reduction of skeletal muscle apoptosis assessed by terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling analysis after 2 wk of GH treatment. This was accompanied by reduced expression of proapoptotic mediators, such as Bax, caspase-9, and caspase-3, and increased expression of antiapoptotic Bcl-2 compared with untreated CHF animals. Mitochondrial cytochrome c release, a proapoptotic event occurring upstream of caspase activation, was also attenuated by GH administration. Finally, GH supplementation reduced serum levels of TNF-α and sphingosine, both of which are known to trigger apoptosis in skeletal myocytes.
Based on these premises, we investigated the effects of 4 wk of GH supplementation on skeletal muscle mass and strength and apoptosis of myocytes in old rats. We also investigated whether 4 wk of treadmill running would have an effect on physical performance and muscle apoptosis similar to that observed with GH supplementation. Furthermore, we asked whether the two interventions act on the same or distinct apoptotic pathways. We focused on two specific pathways of apoptosis known to contribute to age-related muscle atrophy: the receptor-mediated pathway triggered by TNF-α (i.e., extrinsic pathway) (37, 38) and the pathway carried out by mitochondrial-derived, caspase-independent mediators [apoptosis-inducing factor (AIF) (16) and endonuclease G (EndoG) (19, 30)] (i.e., intrinsic pathway). To accomplish our aims, we conducted functional and biochemical analyses on a study designed to investigate the effect of GH supplementation and endurance exercise training on cardiac diastolic function.
To the best of our knowledge, this is the first study to investigate the effects of short-term GH supplementation and exercise training started late in life on skeletal muscle apoptosis in normally aged rats.
Fifty 8- and 28-mo-old male Fischer 344 × Brown Norway hybrid rats were purchased from National Institute on Aging colony at Harlan Industries (Indianapolis, IN). This strain of rat was chosen because of its increased longevity (median life span of 33.3 mo; maximum life span of 40 mo) and decreased cumulative lesion incidence compared with other strains (31). Rats were housed two per cage and maintained on a 12:12-h light-dark cycle, at constant temperature and humidity, in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care. Rats had ad libitum access to standard rat chow (Nestle Purina, St. Louis, MO) and tap water. Body weights of all rats were recorded biweekly. All procedures were in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and reviewed and approved by the Wake Forest University School of Medicine's animal care and use committee before commencement.
Rats were acclimatized to the one-lane rodent treadmill (Scientific Instruments, Stoelting, Woodale, IL) by walking at a speed of 20 cm/s, 10 min/day, for 1 wk. After the acclimatization period, young rats (10/group) were randomized to either a sedentary control (YC) or exercise group (YEX) and aged rats were randomized into one of three groups (10/group): sedentary control (OC), exercise (OEX), or recombinant porcine GH supplemented (Alpharma, Victoria, Australia). For this latter group (termed OGH), GH was administered twice daily (300 μg sc) for 4 wk. This supplementation regimen was chosen based on our data indicating that it is sufficient to increase plasma IGF-I levels in aged animals (24, 45). Analysis of plasma samples from rats supplemented with porcine GH for up to 17 mo revealed no antibodies against porcine GH (44). The 4-wk exercise training program consisted of running on the treadmill at a speed of either 36 cm/s (young) or 27 cm/s (old) for 25 min/day, 5 days/wk. To prevent avoidance and to ensure exercise training, rats received a light electrical shock (6 mA) if they sat at the base of the treadmill. Within 2 wk of training, the rates of exercise avoidance were minimal and electrical shock was no longer needed. The untrained rats, including those in the OC, OGH, and YC groups, remained familiarized to the treadmill by walking 10 min/day, once or twice per week, at a speed of 20 cm/s. The treadmill was set at 15° during walking, training, and testing.
Physical Performance Measures
After 4 wk of training, all rats underwent physical performance measures, including exercise tolerance, inclined plane, and grip strength.
The protocol for the exercise tolerance test consisted of walking at 20 cm/s for 3 min followed by 2-cm/s increases in speed every 2 min until the rat reached exhaustion. Time to exhaustion (seconds) was determined when the rat sat at the lower end of the treadmill, near a shock bar, for >5 s. During the period between the exercise tolerance testing and the other physical performance measures, exercise routines were continued in moderation (15 min/day).
This test is a measure of muscle tone and stamina. The rat was placed facing upward in one compartment on a 60° tilted 1-cm mesh screen. The time taken for the animal to fall onto two 7.6-cm foam pads was divided by the animal's weight and recorded with a maximum latency of 15 min.
Forelimb grip strength was determined by an automated grip strength meter (Columbus Instruments, Columbus, OH). The experimenter grasped the rat by the tail and suspended it above a grip ring. After ∼3 s, the animal was gently lowered toward the grip ring and allowed to grasp the ring with its forepaws. The experimenter then quickly lowered the remainder of the animal's body to a horizontal position and tugged the animal's tail until its grasp of the ring was broken. The mean force in grams was determined with a computerized electronic pull strain gauge that is fitted directly to the grasping ring and was divided by body weight. Average measurements from three successful trials were taken as the final outcome. Successful trials were defined as those in which the animal grasped the ring with both forepaws and pulled the ring without jerking.
Preparation of Cytosolic Tissue Extracts
Three days after the grip strength procedure, rats were killed by rapid decapitation. Blood was collected for determination of IGF-I levels in the serum, and hindlimb muscles were removed, trimmed of adipose tissue and tendons, and weighed. Immediately after dissection, muscles were snap-frozen in liquid nitrogen and subsequently stored at −80°C. Muscles chosen for this study were the soleus, predominately composed of type I, slow-twitch fibers, and extensor digitorum longus (EDL), a predominately type II, fast-twitch muscle.
The left soleus and EDL muscles were pulverized under liquid nitrogen with a porcelain mortar and pestle. The powder was suspended in 500 μl of ice-cold lysis buffer (250 mM sucrose, 10 mM Tris·HCl, 1 mM EDTA, 0.01% protease inhibitor cocktail, pH 6.5), vortexed for 15 s, and centrifuged at 12,000 g at 4°C for 10 min to pellet cellular debris, intact nuclei, and mitochondria. The supernatant was collected, aliquoted, and kept at −80°C until biochemical analyses. Protein concentration was determined with the method developed by Bradford (5).
Western Blot Analysis
Prior to loading samples were boiled at 95°C for 5 min in Laemmli buffer (62.5 mM Tris·HCl, 2% SDS, 25% glycerol, 0.01% bromophenol blue, pH 6.8; Bio-Rad, Hercules, CA) with 5% β-mercaptoethanol. Proteins were separated by using 10% and 15% pre-cast Tris·HCl gels (Bio-Rad). For TNF receptor 1 (TNF-R1) expression, 50 μg of protein were applied to each lane, whereas 100 μg were used for caspase-8, cleaved caspase-3, EndoG, and AIF expression. Separated proteins were transferred to polyvinylidene difloride membranes (Immobilon P, 0.45 μm; Millipore, Billerica, MA) using a semidry blotter (Bio-Rad). Transfer efficiency was verified by staining the gels with GelCode blue stain reagent (Pierce Biotechnology, Rockford, IL) and the membranes with Ponceau S (Sigma-Aldrich, St. Louis, MO). Ponceau S staining was also used as a loading control. For TNF-R1 experiments, membranes were blocked in StartingBlock Tris-buffered saline (TBS) blocking buffer with 0.05% Tween 20 (Pierce Biotechnology) for 1 h at room temperature, washed in TBS for 5 min, and incubated overnight in rabbit polyclonal antibody to TNF-R1 (Abcam, Cambridge, MA), 1:1,000, at 4°C. The next morning, membranes were washed four times in TBS with 0.05% Tween 20 and subsequently incubated with alkaline phosphatase-conjugated secondary antibody (Sigma-Aldrich), 1:30,000, at room temperature for 1 h. Membranes were then washed three times in TBS with 0.05% Tween 20, rinsed in TBS, and washed once in Tris·HCl (100 mM, pH 9.5). Finally, the DuoLux chemiluminescent/fluorescent substrate for alkaline phosphatase (Vector Laboratories, Burlingame, CA) was applied, and the chemiluminescent signal was captured with an Alpha Innotech Fluorchem SP imager (Alpha Innotech, San Leandro, CA). The digital images were then analyzed by AlphaEase FC software (Alpha Innotech). Spot density of the target band was normalized to that of the most prominent band on the corresponding Ponceau S-stained membrane and expressed in arbitrary optical density units. For the analysis of caspase-8, cleaved-caspase-3, EndoG, and AIF, a Vectastain ABC-AmP immunodetection kit (Vector Laboratories) was used, according to the manufacturer's instructions. The following primary antibodies and relative dilutions were used: rabbit monoclonal antibody to cleaved-caspase-3 (Cell Signaling Technology, Beverly, MA), 1:1,000; rabbit monoclonal antibody to caspase-8 (Abcam), 1:500; rabbit polyclonal antibody to EndoG (Abcam), 1:1,000; rabbit polyclonal antibody to AIF (BD PharMingen, San Diego, CA), 1:500. Generation of the chemiluminescent signal, digital acquisition, and densitometry analysis were performed as described above.
Cell Death ELISA for Quantification of DNA Fragmentation
Apoptotic DNA fragmentation was quantified by measuring the amount of cytosolic mono- and oligonucleosomes using an ELISA kit (cell death detection ELISA; Roche Diagnostics, Mannheim, Germany), following the manufacturer's instructions. Absorbance was measured at 405 nm with a Synergy HT multidetection microplate reader (BioTek, Winooski, VT) and reported as arbitrary optical density units per milligram of protein.
Relative Quantification of TNF-α Gene Expression in Skeletal Muscle by Quantitative PCR
To determine the relative gene expression of TNF-α in soleus and EDL muscles, quantitative PCR (Q-PCR) analysis was performed. Total RNA was isolated with TriReagent (Sigma-Aldrich). Briefly, ∼50 mg of tissue were homogenized in 1 ml of TriReagent using a motorized mortar and pestle. The homogenate was cleared by centrifugation, and RNA was isolated from the supernatant according to the manufacturer's instructions. Total RNA was then dissolved in diethylpyrocarbonate (DEPC)-treated water and quantified spectrophotometrically. To remove possible contaminating DNA, DNase digestion was performed using the TURBO DNA-free kit from Ambion (Foster City, CA). All steps were performed as per the manufacturer's directions. After DNase treatment, RNA concentrations and purity were determined spectrophotometrically (i.e., 260 nm-to-280 nm ratio). First-strand cDNA synthesis was achieved from 2 μg of RNA using the cMaster RTplusPCR system kit (Eppendorf, Hamburg, Germany). Briefly, total RNA, 1 μl of 0.5 μg oligo(dT) primer, 1 μl of 50 ng/μl random hexamers, 1 μl of 10 mM dNTP mix, and DEPC water were mixed and heated to 65°C for 5 min. The samples were then placed on ice, and 4 μl of 25 mM RT-PCR buffer, 1 μl of 0.75 U/μl RT enzyme, 0.5 μl of RNase inhibitor, and DEPC water were added. The samples were heated to 42°C for 1 h, and the reaction was terminated by heating to 85°C for 5 min. Q-PCR was performed using the ABI 7500 real-time PCR system (ABI, Foster City, CA). TaqMan Universal PCR Master Mix (2×) (Roche, Branchburg, NJ), as well as 0.2 μM primers and TaqMan probe mix (ABI) were used for each 25-μl reaction. Amplification of TNF-α (NM_012675) and β-actin (endogenous control, NM_031144) was achieved with the following PCR cycling conditions: enzyme activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and anneal/extend at 60°C for 1 min. All samples were examined in triplicate, with the YC group used as a calibrator. For both genes, negative controls (i.e., no template and no reverse transcriptase) were also included and run in triplicate.
Determination of Serum IGF-I
IGF-I (Bachem, Torrance, CA) was radiolabeled with the lactoperoxidase, glucose oxidase method and purified on a Sep-Pak silica cartridge (Waters, Milford, MA). Serum was extracted in acid-ethanol (13), and IGF-I was measured by radioimmunoassay as previously described (46). The intra-assay coefficient of variance for this assay was 9%.
All data are reported as means ± SE. Statistical analysis was performed with GraphPrism 4.0.3 software (GraphPad Software, San Diego, CA). Differences between experimental groups were explored with two-way ANOVA with age and exercise as factors, followed by Bonferroni's posttest when appropriate. To identify statistical differences among OC, OEX, and OGH groups, one-way ANOVA, with Tukey's post hoc test when needed, was also employed. Pearson's test was used to explore correlations between variables. For all tests, significance was accepted at P < 0.05.
Body weight was significantly higher in old than in young animals (age effect: P < 0.0001; Table 1). Exercise training did not induce changes in body weight in young rats, whereas it was associated with a significant reduction in aged rats (P < 0.05). On the contrary, OGH rats exhibited a significant increase in body weight compared with the OEX group (P < 0.01). The difference in body weight observed between the two interventions is likely the result of water retention associated with GH administration (14) combined with the decreased body weight in the OEX rats. Wet muscle weight for soleus was increased in the old rats compared with their younger counterparts (age effect: P < 0.05), regardless of the treatment condition (Table 1). However, when soleus weight was normalized to the body weight (MW-to-BW ratio or MW/BW), the old animals had a significantly lower ratio than their younger counterparts (age effect: P < 0.05), indicative of sarcopenia. Neither exercise nor GH supplementation induced significant changes in soleus weight or MW/BW. Wet muscle weight for EDL did not change across groups (Table 1). However, expression of EDL wet weight relative to body weight revealed an age-related decrease (age effect: P < 0.01), again indicating that substantial muscle loss had taken place with advancing age. Similar to that for the soleus, no changes in EDL weight or MW/BW were detected with either exercise or GH administration.
Physical Performance Measures
Four weeks after initiation of the experimental protocol, all rats underwent an exercise tolerance test, as well as inclined plane and grip strength procedures, to quantify physical performance. We found that in young rats exercise tolerance was higher than that in old animals (age effect: P < 0.0001; Fig. 1A). YEX and OEX rats performed significantly better than their respective sedentary counterparts (treatment effect: P < 0.0001; Fig. 1A). In contrast, OGH animals did not experience any improvement, with their performance being poorer than that of the OEX rats (P < 0.0001) and comparable to that of the OC group. The grip strength performance in YEX animals was significantly better than that of OEX rats (age effect: P < 0.0001; Fig. 1B). However, exercise training significantly improved grip strength in OEX (P < 0.05) but not in YEX rats. In contrast, OGH rats had a poorer performance than the OC (P < 0.05) and OEX groups (P < 0.001), even without correction for body weight (data not shown). Finally, YEX animals showed a better performance in the inclined plane test than OEX rats (age effect: P < 0.0001; Fig. 1C). However, no changes were apparent with either exercise or GH supplementation.
Serum levels of IGF-I were reduced in OC and OEX rats compared with their younger counterparts (age effect: P < 0.01; Fig. 2). Exercise training did not induce significant changes in either age group. In contrast, OGH rats displayed increased levels of IGF-I compared with both OC and OEX animals (P < 0.001), indicating that our supplementation regimen effectively restored systemic IGF-I levels.
Expression of TNF-R1, Caspase-8, Cleaved Caspase-3, EndoG, and AIF
Western blot analysis of TNF-R1 expression in soleus muscle did not reveal any significant changes across age or treatment groups (Fig. 3A). In contrast, in EDL muscle, advanced age was associated with an increased expression of TNF-R1 (P < 0.01; Fig. 3B) that was reversed by both exercise training and GH administration (P < 0.01).
Procaspase-8 expression did not change across groups in either soleus or EDL muscle (data not shown). No significant differences were observed in cleaved caspase-8 content among groups for soleus muscle (Fig. 4A). In contrast, expression of cleaved caspase-8 in EDL was increased in OC compared with YC rats (P < 0.05; Fig. 4B). Exercise training and GH supplementation had a significant protective effect (P < 0.05).
In soleus muscle, cleaved caspase-3 content did not significantly differ across groups (Fig. 5A). In the EDL muscle, aging was associated with an increased content of cleaved caspase-3 (P < 0.05; Fig. 5B) that was attenuated by exercise training (P < 0.05) but not by GH supplementation.
AIF and EndoG.
Neither age nor treatments had any significant effect on cytosolic levels of AIF and EndoG (Table 2).
Apoptotic Cell Death
In soleus muscle, the extent of the apoptotic DNA fragmentation, as quantified by cell death ELISA, was unchanged regardless of either age or physical exercise (Fig. 6A). However, OGH animals displayed increased levels of DNA fragmentation compared with OC rats (P < 0.05). In contrast, in EDL, advanced age was associated with increased apoptotic DNA fragmentation (P < 0.01; Fig. 6B), with a protective effect of exercise training (P < 0.01) and no changes with GH supplementation.
Q-PCR for TNF-α Expression
Expression of TNF-α in myocytes as determined by Q-PCR did not differ significantly among groups in either soleus or EDL muscles (Table 3).
Results from the present study indicate that short-term GH supplementation initiated late in life does not improve physical performance or muscle mass and strength. Furthermore, age-related skeletal muscle apoptosis was not attenuated by the hormonal intervention. GH supplementation also resulted in increased DNA fragmentation (indicative of apoptosis) in soleus muscle. In striking contrast, the short-term program of physical exercise improved exercise capacity and muscle strength at old age and reversed the age-associated increase in skeletal muscle apoptosis in EDL.
Moreover, we have previously demonstrated that 6 mo of GH treatment in aged rats (34–37 mo of age), although it reduced fat mass and increased lean mass, had only a marginal effect on physical performance (8). The lack of a positive effect of long-term GH administration on muscle mass at old age has also been reported in human studies. For example, Hennessey et al. (25) did not detect any improvement in either fiber cross-sectional area or fiber number in frail older adults after 6 mo of GH supplementation. In addition, Taaffe et al. (51) reported that 10 wk of GH administration failed to enhance the muscle fiber hypertrophic response achieved through resistance training in older men.
With respect to skeletal muscle apoptosis, we found that GH administration reduced the expression of TNF-R1 and cleaved caspase-8 in the EDL muscle, both of which were elevated in aged rats (Figs. 3B and 4B). However, content of cleaved caspase-3, one of the main executioners of the apoptotic program, and apoptotic DNA fragmentation were not reduced by the hormonal intervention (Figs. 5B and 6B). In other words, the protective effects of GH supplementation on the early steps of the extrinsic pathway of apoptosis (i.e., TNF-R1 expression and procaspase-8 cleavage) did not translate into an effective mitigation of the actual apoptotic events (i.e., cleavage of caspase-3 and DNA fragmentation) in the EDL muscle. It is unclear whether these findings can be attributed to the short duration of the hormonal intervention (4 wk) or to a real ineffectiveness of the GH supplementation to counteract apoptosis. Dalla Libera et al. (11) recently demonstrated that GH administration for 2 wk is associated with decreased skeletal muscle apoptosis in a rodent model of experimental CHF. The inconsistency between our findings and those from Dalla Libera et al. may be related to the substantial difference of the experimental models used (i.e., normal aging vs. CHF). Furthermore, in the case of CHF, GH has been shown to improve hemodynamics and myocardial mass (18, 20, 29), and this might have contributed to the reduction of skeletal muscle apoptosis observed by Dalla Libera et al.
Surprisingly, in our study, GH administration was associated with increased apoptotic DNA fragmentation in the soleus muscle (Fig. 6A). This phenomenon seems to occur independently of caspase activation, since the content of cleaved caspase-8 and caspase-3 was not significantly modified by the hormonal intervention. Likewise, mitochondrial caspase-independent AIF and EndoG were not significantly elevated after GH administration. Therefore, based on the present knowledge of the apoptotic pathways involved in muscle atrophy and on our biochemical measurements, a definitive mechanistic explanation of the proapoptotic effects of GH supplementation on soleus muscle cannot be provided at this time. The exact mechanisms underlying this phenomenon, as well as its apparent muscle selectivity, deserve further investigation.
In sharp contrast to the substantial ineffectiveness of GH supplementation to improve physical performance, we found that a short-term program of physical exercise started at advanced age was associated with improved exercise capacity and muscle strength, as assessed by grip strength test (Fig. 1). The lack of a parallel increase in muscle mass after exercise training is likely the result of the short duration of the intervention. In this context, it has been well documented that strength gain precedes muscle hypertrophy (2), as a result of an early improvement of neural drive (1, 34). In contrast to the OEX rats, we did not observe any sizeable increase in muscle strength in the YEX animals, which may be attributed to the insufficient intensity of the exercise training in this age group.
In agreement with a previous study from our laboratory (37), we did not find significant changes in TNF-R1 expression, cleaved caspase-8 and -3 content, and apoptotic DNA fragmentation with age in soleus muscle, nor were these parameters affected by the exercise training (Figs. 3A–6A). Thus our findings confirm the hypothesis that type I muscle fibers, such as the soleus muscle, are less prone to age-associated apoptosis than type II fibers (37) and therefore less likely to be affected by short-term exercise training. However, we observed a significant attenuation of the age-related increase in apoptosis in EDL after exercise training (Fig. 6B). Previous studies have shown that the apoptotic potential is elevated in type II, fast-twitch muscle fibers at advanced age and that myocytes can be rescued by interventions such as life-long calorie restriction and endurance exercise (37, 43). In our study, expression of TNF-R1 and content of cleaved caspase-8 and -3, as well as the extent of apoptotic DNA fragmentation, were increased in EDL of OC rats compared with their younger counterparts (Figs. 3B–5B). The same parameters were significantly attenuated by our short-term exercise training program, indicating a protective effect of treadmill running on the TNF-α-driven pathway of apoptosis in EDL. The antiapoptotic effect of physical exercise in EDL observed in our study is in keeping with a recent study by Song et al. (43), who reported a marked reduction of apoptotic DNA fragmentation and cleaved caspase-3 content in the white gastrocnemius muscle (predominantly comprised of type II fibers) of old rats subjected to 12 wk of treadmill training.
Some aspects of our study deserve further discussion. First, our study did not include the combination of GH supplementation and exercise training. However, in this initial investigation, we were interested in comparing the effects of treadmill running and GH administration per se on physical performance and muscle apoptosis. In future studies, it will be important to investigate whether the combination of exercise training and GH supplementation at advanced age retains the ability to reduce apoptosis in fast-twitch muscles and prevents the observed proapoptotic effect of GH administration on slow-twitch muscles.
Our PCR data (Table 3) do not support previous reports of increased expression of TNF-α in aged skeletal muscle (23, 37). However, our findings are in agreement with a recent study by Pistilli et al. (38), in which the authors also did not detect an age-related increase of TNF-α expression in skeletal muscle of Fischer 344 × Brown Norway rats. These observations, together with the well-documented age-related increase in plasma TNF-α levels (40), suggest that systemic rather than muscle-derived TNF-α may be responsible for the proapoptotic signaling observed in our study. Furthermore, although a significant correlation was found between the content of cleaved caspase-8 and -3 (Pearson's r = 0.71, P < 0.05; data not shown), we cannot rule out the possibility that pathways other than those initiated by caspase-8 (e.g., caspase-9) might have contributed to the cleavage of caspase-3. In addition, because of the limited tissue available, it was not possible to assess the levels of adaptor proteins involved in the constitution of the death-inducing signaling complex (DISC). Binding of TNF-α to TNF-R1 recruits adaptor proteins such as Fas-associated death domain and TNF receptor-associated death domain, giving rise to the DISC, which in turn recruits and activates procaspase-8 (12). In our study, considering that the expression levels of both TNF-R1 and cleaved caspase-8 were increased in EDL muscle of old animals, it is conceivable that the formation of the DISC had indeed occurred and that this complex was downregulated by physical exercise and GH supplementation. Finally, in contrast to previous studies (16, 42), we did not detect an age-related increase of the cytosolic content of AIF in our experimental model (Table 2). It should however be considered that mitochondrial release of AIF and EndoG represents the first step in the caspase-independent apoptotic process, whereas the actual DNA fragmentation takes place only after their translocation to the nucleus (12). Nuclear content of AIF and EndoG was not assessed in this study, and the possibility exists that nuclear translocation of these mediators was enhanced in old animals, regardless of their cytosolic levels.
Perspectives and Significance
Results from our study indicate that a short-term program of exercise training started at advanced age reverses the age-related skeletal muscle apoptosis and represents an effective strategy to improve physical performance and muscle strength. On the other hand, short-term administration of GH late in life does not attenuate functional decline and muscle aging and may even accelerate apoptosis in type I, slow-twitch muscles, such as the soleus. From these findings and with the consideration that GH supplementation, especially in older subjects, is often accompanied by several adverse reactions (e.g., carpal tunnel syndrome, fluid retention, joint pain, impaired glucose tolerance, and, possibly, increased risk of cancer) (32), GH administration may not be an advisable strategy to improve body composition and functionality in healthy older individuals.
This research was supported by grants to L. Groban (Dennis Jahnigen Career Development and Paul Beeson Award, National Institute on Aging Grant K08 AG-026764-02), C. S. Carter (National Institute on Aging Grant R01 AG-024526-02), and C. Leeuwenburgh (National Institute on Aging Grants R01 AG-17994 and AG-21042). E. Marzetti and S. E. Wohlgemuth are supported by the University of Florida Institute on Aging and Claude D. Pepper Older Americans Independence Center (National Institute on Aging Grant 1P30 AG-028740).
The authors thank Colleen Bennett, MS, for performing daily GH injections over the course of the study. The Q-PCR analysis was performed in the laboratory of Dr. Wronski (Department of Physiological Sciences, University of Florida).
↵* E. Marzetti and L. Groban contributed equally to this work.
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- Copyright © 2008 the American Physiological Society