Aging and lifelong calorie restriction result in adaptations of skeletal muscle apoptosis repressor, apoptosis-inducing factor, X-linked inhibitor of apoptosis, caspase-3, and caspase-12

Free Radical Biology & Medicine, Vol. 36, No. 1, pp. 27 – 39, 2004 Copyright D 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter

doi:10.1016/j.freeradbiomed.2003.10.003

Original Contribution AGING AND LIFELONG CALORIE RESTRICTION RESULT IN ADAPTATIONS OF SKELETAL MUSCLE APOPTOSIS REPRESSOR, APOPTOSIS-INDUCING FACTOR, X-LINKED INHIBITOR OF APOPTOSIS, CASPASE-3, AND CASPASE-12 Amie J. Dirks1 and Christiaan Leeuwenburgh Biochemistry of Aging Laboratory, University of Florida, Gainesville, FL 32611, USA (Received 5 June 2003; Revised 18 September 2003; Accepted 3 October 2003)

Abstract—The mechanisms of apoptosis in the loss of myocytes in skeletal muscle with age and the role of mitochondrial and sarcoplasmic reticulum-mediated pathways of apoptosis are unknown. Moreover, it is unknown whether lifelong calorie restriction prevents apoptosis in skeletal muscle and reverses age-related alterations in apoptosis signaling. We investigated key apoptotic regulatory proteins in the gastrocnemius muscle of 12 and 26 month old ad libitum fed and 26 month old calorie-restricted male Fischer-344 rats. We found that apoptosis increased with age and that calorie-restricted rats showed less apoptosis compared with their age-matched cohorts. Moreover, pro- and cleaved caspase-3 levels increased significantly with age and calorie-restricted rats had significantly lower levels than the aged ad libitum group. Neither age nor calorie restriction had any effect on muscle caspase-3 enzyme activity, but the levels of Xlinked inhibitor of apoptosis, particularly an inhibitor of caspase-3, increased with age and were reduced significantly in the 26 month old calorie-restricted cohort. The apoptotic inhibitor apoptosis repressor with a caspase recruitment domain (ARC), which inhibits cytochrome c release, underwent an age-associated decline in the cytosol but increased with calorie restriction. In contrast, mitochondrial ARC levels increased with age and were lower in calorie-restricted rats than in age-matched controls, suggesting a translocation of this protein to attenuate oxidative stress. The translocation of ARC may explain the reduction in cytosolic cytochrome c levels observed with age and calorie restriction. Moreover, we found a striking f350% increase in the expression of procaspase-12 (caspase located at the sarcoplasmic reticulum) with age which was significantly lower in the 26 month old calorie-restricted group. The total protein level of apoptosis-inducing factor in the plantaris muscle increased with age and was reduced in calorie-restricted rats compared with age-matched controls, but there were no significant changes in this pro-apoptotic protein in the isolated nuclei. Calorie restriction is able to lower the apoptotic potential in aged skeletal muscle by altering several key apoptotic proteins toward cellular survival, thereby reducing the potential for sarcopenia. D 2003 Elsevier Inc. All rights reserved. Keywords—Mitochondria, Sarcoplasmic reticulum, Aging, Oxidative stress, Apoptosis-inducing factor, Cytochrome c, Free radicals

and the possible mechanisms involved, although evidence suggests that apoptosis may play a role during muscle aging [2,3], muscular dystrophy [4], muscle denervation [5], and unloading [6,7]. By age 80 it is estimated that humans generally lose f30 – 40% of skeletal muscle fibers, particularly from muscles containing type II fibers such as the vastus lateralis muscle [1]. Studies using rodents show between a f20 and 50% loss in muscle fibers depending on the specific fiber type studied [8,9]. Several investigators have attempted to elucidate the factors involved in skeletal muscle apoptosis by employing various experimental paradigms [6,10,11] and link

INTRODUCTION

Skeletal muscle atrophy and the loss of myofibers contribute to sarcopenia, a condition associated with normal aging [1]. However, relatively little is known regarding the relevance of apoptosis to skeletal muscle homeostasis Address correspondence to: Christiaan Leeuwenburgh, Ph.D., Biochemistry of Aging Laboratory, University of Florida, 25 FLG, P.O. Box 118206, Gainesville, FL 32611, USA. Fax: +1-352-3920316. E-mail: [email protected]. 1 Current address: School of Pharmacy, Wingate University, Wingate, NC 28174, USA. E-mail: [email protected]. 27

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bind to caspase-3 and inhibit its protease activity. Recently, it has been shown that increased intracellular Ca2+ concentrations can significantly contribute to increased susceptibility to apoptosis via activation of caspase-12, a caspase localized on the cytoplasmic side of the endoplasmic reticulum (sarcoplasmic reticulum in skeletal muscle) [37,38]. Efficiency of calcium storage alters with age and this may explain the increase in resting intracellular Ca2+ levels in senescent animals [13,39]. Because calorie restriction reverses many age-related alterations in skeletal muscle, including mitochondrial dysfunction [23,24], and attenuates myofiber loss [24,27], we hypothesized that calorie restriction would also attenuate the rate of apoptosis and would effect the ratio of anti- to pro-apoptotic proteins toward survival. We studied the effects of lifelong calorie restriction on apoptosis, caspases (caspase-3, -9, and -12), and apoptotic regulatory proteins (ARC, XIAP, Apaf-1, cytochrome c, and AIF) in the gastrocnemius and plantaris muscles. We report that lifelong calorie restriction attenuates apoptosis in aging skeletal muscle and reverses age-related alterations in the mitochondria-mediated and sarcoplasmic reticulum-mediated apoptosis pathways. The decrease in apoptosis and reduced susceptibility to apoptosis may be a contributing mechanism through which calorie restriction reduces the age-associated skeletal muscle atrophy, loss of muscle fibers, and impaired muscle function [24,26,27] indicative of sarcopenia.

apoptosis to sarcopenia [2,3]. Moreover, the pathology of sarcopenia is characterized by mitochondrial DNA damage, DNA deletions, increases in intracellular calcium levels, and mitochondrial electron transport abnormalities [12 – 14]. Because calorie restriction has been shown to improve calcium homeostasis, increase mean and maximum life span in mammals [15 – 18], attenuate oxidative damage to proteins, lipids, and DNA [17,19 –21], reduce oxidant production [21,22], reduce mitochondrion dysfunction [23,24], and prevent muscle contractile dysfunction [25,26] and loss of muscle fibers [24,27], we further investigated the ability of lifelong caloric restriction to attenuate apoptosis and the adaptations in apoptotic signaling pathways in skeletal muscle. The mitochondria and the endoplasmic reticulum play a significant part in the cell death program by activating caspases (cysteine-dependent, aspartate-specific proteases) [13,28,29]. Mitochondrial dysfunction can lead to mitochondrial cytochrome c release [30 – 32]. In the cytoplasm, cytochrome c, Apaf-1, caspase-9, and dATP form an apoptosome, which can activate caspase-3, a key cell death protease. Procaspase-3 is the full-length form of the cysteine protease, which is cleaved by an initiator caspase (such as caspase-2, -8, -9, or -12) to produce the more active subunit. Furthermore, there are mechanisms of apoptosis that do not require activation of caspases, such as the release of apoptosis inducing factor (AIF) from mitochondria [33], which can induce large-scale DNA fragmentation and apoptosis following translocation to the nucleus. What is more, specific inhibitors of apoptosis may have a role in preventing the loss of cells in postmitotic tissues, such as skeletal muscle. For example, cytochrome c release is regulated, in part, by the Bcl-2 family of proteins and also by apoptosis repressor with a caspase recruitment domain (ARC) [34,35]. ARC is an endogenous inhibitor that is thought to function as an inhibitor of caspase-2 and -8 activation and plays a role in the mitochondria-mediated pathway by preventing cytochrome c release from mitochondria. Furthermore, caspase-9 and -3 activity can be regulated by inhibitors of apoptosis (IAPs) [36]. Currently, several mammalian IAPs have been discovered, one example being X-linked inhibitor-of-apoptosis protein (XIAP), which has not been investigated in muscles. XIAP can

MATERIALS AND METHODS

Animals and experimental design Ad libitum-fed and calorie restricted male Fischer 344 rats were obtained from the National Institute of Aging colony (Harlan Sprague Dawley, Indianapolis, IN, USA) several weeks before being sacrificed. We used 12-month ad libitum-fed (12AD, n = 11); 26-month ad libitum-fed (26AD, n = 8), and 26-month calorie-restricted (26CR, n = 9) animals. Calorie restriction was started at 3.5 months of age (10% restriction), increased to 25% restriction at 3.75 months, and maintained at 40% restriction from 4 months throughout the lifespan. All rats were housed individually at an ambient temperature of 18 –22jC and in a light-

Table 1. Body Weight, Muscle Mass, Body Weight-to-Muscle Mass Ratio of Male Fischer 344 Rats 12AD Body weight (g) Gastrocnemius mass (mg) Muscle mass/body weight (mg/g) Plantaris mass Muscle mass/body weight (mg/g)

410.7 1.36 3.32 0.32 0.78

F F F F F

11.93 0.03 0.07 0.01 0.02

26AD 384.4 1.03 2.70 0.25 0.66

Male Fischer 344 rats (12AD, n = 11; 26AD, n = 8; 26CR, n = 9).

F 16.17 F 0.05* F0.18* F 0.02* F0.05*

26CR

p

265.9 F 6.30§ 0.93 F 0.05 3.48 F 0.26§ 0.22 F 0.02 0.83F 0.05§

§ p < 0.0001 *p < 0.0001 *p = 0.0089, §p = 0.0501 *p = 0.0065 *p = 0.040, §p = 0.0504

Lifelong caloric restriction alters apoptosis signaling in muscle

controlled environment (12-h light/dark cycle). After 1 week of acclimation, the animals were randomly sacrificed (two per day) with an intraperitoneal injection of sodium pentobarbital (Abbot Laboratories, IL, USA, 5 mg/100 g body) and the gastrocnemius and plantaris muscles were excised. These muscles where chosen because they show similar and significant atrophy with age (Table 1). The plantaris and gastrocnemius muscles both consist of a mix of Type II and Type I fibers, but they are predominately Type II and contain subtypes (i.e., Types IIa, IIx, and IIb). Mitochondria and cytosolic proteins were isolated from one gastrocnemius muscle and used for functional respiratory measurements [21] and for determination of levels of apoptosis regulatory proteins. The other gastrocnemius muscle was frozen in liquid nitrogen and stored at
80jC also to determine apoptosis regulatory proteins. The plantaris muscles were frozen in liquid nitrogen and stored at
80jC to determine nuclear and total levels of AIF. All protocols received local institutional animal care and use committee approval. Isolation of mitochondrial and cytosolic protein fractions For analysis of total levels of protein content the tissues were phosphate-buffered saline homogenized in phosphate-buffered saline (PBS) with a dilution of 1:25 (0.2 M sodium phosphate monobasic, 0.2 M sodium phosphate dibasic, 5 mM EDTA, pH 7.4) using a Potter– Elvehjem glass homogenizer. Total tissue homogenate was centrifuged at 500g for 10 min and the supernatant was used for biochemical analysis. Mitochondrial and cytosolic protein fractions were isolated as previously described from the gastrocnemius muscle [21,22]. Briefly, the right gastrocnemius muscle was homogenized in isolation buffer (0.225 M mannitol, 0.075 M sucrose, 0.2% bovine serum albumin (BSA), 1 mM EDTA, pH 7.4) with a dilution of 1:25 using a Potter– Elvehjem glass homogenizer. Sample was subsequently centrifuged at 1000g for 10 min. The supernatant was centrifuged at 14,000g for 10 min. The supernatant was stored at
80jC for analysis of parameters in the cytosolic fraction. The mitochondrial pellet was resuspended in 5 ml of isolation buffer and centrifuged at 14,000g for 10 min. The final mitochondrial pellet was resuspended in 0.5 ml of buffer. Isolation of nuclear extracts Nuclear extracts were isolated from plantaris muscle using the protocol described by Blough et al. [40]. Briefly, 100 mg plantaris muscle was homogenized in 35 ml of Buffer 1 (10 mM Hepes, pH 7.5, 10 mM MgCl2, 5 mM KCl, 0.1 mM EDTA, pH 8.0, 0.1% Triton X-100, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 2 Ag/ml aprotinin, and 2 Ag/ml leupeptin) and centrifuged for 5 min at 3000g at 4jC. The resulting pellet was

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resuspended in 500 Al of Buffer 2 (20 mM Hepes, pH 7.9, 25% glycerol, 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, pH 8.0, 0.5 mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, 2 Ag/ml aprotinin, and 2 Ag/ml leupeptin) and centrifuged for 5 min at 3000g at 4jC. The supernatant was transferred to a 5000 nominal molecular weight limit Ultrafree Filter Unit (4 ml; Millipore, Bedford, MA, USA) and centrifuged for 30 min at 4500g at 4jC. The concentrated sample was used to determine the concentration of AIF (see later section). Determination of cytosolic mono- and oligonucleosomes Endogenous endonucleases activated during apoptosis cleave double-stranded DNA in the linker region between nucleosomes to generate mono- and oligonucleosomes of 180 bp or multiples. The apoptotic DNA fragmentation was quantified by measuring the amount of cytosolic mono- and oligonucleosomes using a Cell Death ELISA kit (Roche Molecular Biochemicals, Germany) with the peroxidase substrate ABTS [3,41]. Results were reported as arbitrary OD units normalized to milligram of protein. Determination of the content of caspase-3, caspase-9, caspase-12, Apaf-1, XIAP, ARC, and AIF We determined the levels of apoptotic signaling proteins using Western blot analysis. Equal amounts of protein from the different groups were loaded on the same gel to avoid any potential intergel variation and standardized to the 12 month old ad libitum-fed animals. Proteins were separated using a 4 – 12% SDS – polyacrylamide gradient gel (BMA Bioproducts, Rockland, ME, USA) under denaturing conditions and transferred to nitrocellulose membranes for Western analysis. Nitrocellulose membranes were blocked overnight using a blocking solution containing 5.0% powdered milk. Membranes were incubated with the polyclonal primary antibodies with an appropriate dilution: mouse monoclonal caspase3 (BD Biosciences, San Diego, CA, USA); mouse monoclonal caspase-9 (US Biological, Swampscott, MA, USA); rabbit polyclonal caspase-12 (Oncogene, Boston, MA, USA); rabbit polyclonal Apaf-1 (Biovision, Palo Alto, CA, USA); rabbit polyclonal ARC (Imgenex, San Diego, CA, USA); mouse monoclonal XIAP (MBL, Watertown, MA, USA); and rabbit polyclonal AIF (Alpha Diagnostic International, San Antonio, TX, USA). Membranes were incubated for 90 minutes in anti-rabbit or anti-mouse Ig horseradish peroxidase (Amersham Life Science, Arlington Heights, IL, USA) with an appropriate dilution. Specific protein bands were visualized using ECL reagent (Amersham – Pharmacia, UK). Blots were analyzed using the Apple-J program downloaded from the NIH web site. Values were expressed as arbitrary OD

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units calculated by multiplying the area of each band by its optical density and normalized to the 12-month ad libitum-fed animals. Ponceau staining (Pierce Biochemicals, Rockford, IL, USA) of the nitrocellulose membrane was used to assure equal loading of protein.

Sunnyvale, CA, USA) at excitation: 380 nm, emission 460 nm for AMC. Values were expressed as arbitrary units of OD per milligram of protein. Protein concentration was measured using the Bradford method [42]. Statistical analysis

Cytosolic cytochrome c Cytochrome c was quantified in the cytosolic fraction of the gastrocnemius using a cytochrome c ELISA kit (R&D Systems, Minneapolis, MN, USA). A microplate reader was used for analysis. Data were reported as nanomoles per milligram of cytosolic protein.

Analyses were performed in duplicate and the mean was used for statistical analysis. Independent t tests were run using Graph-pad Prism (San Diego, CA, USA). Statistical comparisons were made between the 12AD and 26AD and between the 26AD and 26CR rats. A p value < 0.05 was considered statistically significant. All data were expressed as means F SEM.

Caspase-3 activity Caspase-3 activity was measured in the cytosolic fraction of the gastrocnemius using a fluorometric activity assay kit (Roche Molecular Biochemicals, Pleasanton, CA, USA) following the manufacturer’s protocol. The substrate Ac-DEVD-AMC is cleaved proteolytically by caspase-3. Fluorescence was determined using a Spectra Max Fluorescent Microplate Reader (Molecular Devices,

RESULTS

Body weight, muscle weight, and muscle weight-to-body weight ratio Body weight was not significantly different between 12AD and 26AD rats (Table 1), however calorie restric-

Fig. 1. We determined procaspase-3, cleaved caspase-3, and X-linked inhibitor of apoptosis (XIAP) protein content, as well as enzymatic activity of caspase-3 in the gastrocnemius muscle of 12 month adult ad libitum-fed (12AD, n = 11), 26 month old ad libitum-fed (26AD, n = 8), and 26 month old calorie-restricted (26CR, n = 9) male Fischer 344 rats. Proaspase-3, cleaved caspase-3, and XIAP content was determined by Western analysis, and results (means F SEM) are reported as percentages of that of the 12AD group (see Materials and Methods). Caspase-3 activity was determined by a fluorometric assay (see Materials and Methods) and results were expressed as OD per milligram of cytosolic protein. (A) Procaspase-3: *p = 0.015, 12AD vs. 26 AD (100.0 F 22.3% vs. 169.0 F 1.9%), and **p < 0.0001, 26AD vs. 26CR (77.2 F 5.5%). (B) Cleaved caspase-3: *p = 0.0001, 12AD vs. 26AD (100.0 F 11.7% vs. 229.0 F 27.4%), and **p < 0.0001, 26AD vs. 26CR (103.8% F 8.2%). (C) Caspase-3 activity: nonsignificant, 12AD vs. 26AD (arbitrary OD/mg cytosolic protein), and nonsignificant, 26AD vs. 26CR. (D) XIAP: *p = 0.0015, 12AD vs. 26AD (100.0 F 2.5% vs. 129.9 F 7.7%), and **p = 0.0045, 26AD vs. 26CR (99.1 F 5.73%).

Lifelong caloric restriction alters apoptosis signaling in muscle

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pattern is observed with cleaved caspase-3 (Fig. 1B); the level of this active subunit was significantly higher in 26AD rats than in 12AD rats (p = 0.0001) and the level in 26CR rats was comparable to that seen in 12AD rats, but significantly less than that in 26AD rats (p < 0.0001).

Fig. 2. Apoptosis determined by the quantification of DNA fragmentation (mono- and oligonucleosomes) in the gastrocnemius muscle of 12AD, 26AD, and 26CR male Fischer 344 rats using a quantitative ELISA (see Materials and Methods). Results (means F SEM) are reported as arbitrary OD units per milligram of cytosolic protein. Significantly larger amounts of mono- and oligonucleosomes were observed in the 26AD group when compared with the 12AD group (1.70 F 0.12 vs. 1.21 F 0.07, *p = 0.0008). Calorie restriction attenuated the age-associated increase in the levels of mono- and oligonucleosomes compared with the age-matched ad libitum-fed animals (26CR, 1.37 F 0.06, **p = 0.01).

tion resulted in a significant reduction in body weight in the 26CR rats compared with the 26AD rats ( p < 0.0001). Although the body weights of the 12AD and 26AD rats did not differ, the mass of the gastrocnemius muscle was 24% less (p < 0.0001) and that of the plantaris muscle mass was 22% less (p = 0.0065) in the 26AD rats than in the 12AD rats. Conversely, while the body weight of the 26CR rats was less than that of 26AD rats ( p < 0.0001) there was no difference in the plantaris muscle weight. Gross muscle weight data in the different groups of animals are misleading as body mass differs significantly among groups. After we divided muscle weight by mean body weight the muscle weight per unit body weight was significantly reduced in both the gastrocnemius ( p = 0.0089) and plantaris (p = 0.040) muscles in the old 26AD rats compared with the adult 12AD rats. In striking contrast, the muscle weight per unit body weight increased significantly in the gastrocnemius (p = 0.0501) and plantaris (p = 0.0504) muscles in the calorie-restricted animals. Procaspase-3 levels, cleaved caspase-3 levels, caspase-3 protease activity, and XIAP levels with age and calorie restriction The level of procaspase-3 was significantly higher in 26AD rats than in 12AD rats ( p = 0.015) (Fig. 1A). Moreover, the level of procaspase-3 in 26CR rats was significantly less than that in 26AD rats ( p < 0.0001) and comparable to that in the 12AD rats. A very similar

Fig. 3. Cytosolic (A) and mitochondrial (B) ARC content was determined in the gastrocnemius muscle of 12AD, 26AD, and 26CR male Fischer 344 rats. ARC was determined by Western analysis and results (means F SEM) are reported as percentages of that of 12AD group. There were no significant differences in total ARC levels with age or with calorie restriction (100.0 F 6.20, 90.0 F 18.91, and 98.0 F 13.53%). Cytosolic ARC protein content (A) tended to decrease in the 26AD group when compared with the 12AD group (p = 0.07, 12AD vs. 26AD, 100.0 F 15.7% vs. 68.4 F 10.4%). The calorie-restricted animals had a significantly increased level of cytosolic ARC when compared with the age-matched ad libitum-fed controls (**p = 0.015, 26AD vs. 26CR, 145.3 F 26.7%). (B) Mitochondrial ARC protein content increased significantly in the 26AD group when compared with the 12 AD group (*p = 0.008, 12AD vs. 26AD, 100.0 F 6.2 vs. 147.3 F 13.2%). The calorie-restricted animals had a significantly decreased level of mitochondrial ARC when compared with the age-matched ad libitum fed controls (**p = 0.03, 26AD vs. 26CR, 116.5 F 6.2).

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Although differences in the amounts of procaspase-3 and caspase-3 were observed, there were no differences among the three groups with respect to caspase-3 protease activity (Fig. 1C). The cytosolic content of XIAP (Fig. 1D) showed a pattern similar to that observed with caspase-3 in that the level in 26AD rats was significantly greater than that in 12AD rats (p = 0.0015) or in 26CR rats (p = 0.0045). Cell death with age and calorie restriction It is well established that caspase-3 is able to cleave endonuclease inhibitors and therefore activate caspasedependent DNase (CAD), which is responsible for cleavage of double-stranded DNA between nucleosomes forming 180 bp mononucleosome or multiple oligonucleosome fragments [3,43]. DNA fragmentation was quantified in the gastrocnemius muscle of the 12 AD, 26AD, and 26CR rats (Fig. 2). The 26AD rats had significantly greater levels of cytosolic mono- and oligonucleosomes compared with the 12AD rats (1.70 F 0.12 vs. 1.21 F 0.07, p = 0.0008). Calorie restriction attenuated the age-associated rise in the levels of monoand oligonucleosomes (26CR: 1.37 F 0.06, p = 0.01).

Total, cytosolic, and mitochondrial ARC levels in the gastrocnemius muscle with age and calorie restriction We found that there was a trend for cytosolic ARC levels to be lower in the 26AD rats when compared with the 12AD group (Fig. 3A), but this was not statistically significant (p = 0.07). Calorie-restricted rats showed a significant increase in the levels of cytosolic ARC when compared with the age-matched ad libitum-fed group ( p = 0.015). ARC is known to translocate to the mitochondria under oxidative stress, which occurs extensively in skeletal muscle with age. Therefore, we measured ARC content in the mitochondria (Fig. 3B) and found that the levels were significantly increased by 47% in the 26AD rats as compared with the 12AD rats ( p = 0.008). The age-associated rise in ARC content was attenuated by CR ( p = 0.03). Moreover, there were no significant differences in total tissue gastrocnemius ARC levels with age or with calorie restriction (100.0 F 6.20%, 12AD; 90.0 F 18.91%, 26AD; and 98.0 F 13.53%, 26CR). These data suggest that ARC may translocate from the cytosol to the mitochondria in response to oxidative stress with age and may be in part responsible for the

Fig. 4. Components of the apoptosome (caspase-9, Apaf-1, and cytochrome c) were determined in the gastrocnemius muscle of 12AD, 26AD, and 26CR male Fischer 344 rats. Apaf-1, procaspase-9, and cleaved caspase-9 were determined by Western analysis and results (means F SEM) are reported as percentages of that of the 12AD group (see Materials and methods). Cytochrome c concentration was determined using an ELISA (see Materials and Methods) and results were expressed as nanograms of cytochrome c per milligram of cytosolic protein. (A) Cytochrome c: *p = 0.03, 12AD vs. 26AD (233.7 F 15.6 vs. 181.0 F 21.3), and nonsignificant, 26AD vs. 26CR (165.8 F 18.3). (B) Apaf-1: *p = 0.03, 12AD vs. 26AD (100.1 F 5.7% vs. 120.8 F 7.8%), and **p = 0.001, 26AD vs. 26CR (75.2 F 8.4%). (C) Procaspase-9: nonsignificant, 12AD vs. 26AD (100.0 F 6.0, 90.5 F 6.3), and nonsignificant, 26AD vs. 26CR (115.0 F 12.4%). (D) Cleaved caspase-9: p = 0.052, nonsignificant, 12AD vs. 26AD (100.0 F 4.0% vs. 91.2 F 0.6%) and nonsignificant, 26AD vs. 26CR (100.2 F 21.3%).

Lifelong caloric restriction alters apoptosis signaling in muscle

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attenuated the age-associated rise in the levels of Apaf-1 ( p = 0.001). There was no change in the levels of procaspase-9 in the 26AD rats when compared with the 12AD group (Fig. 4C). Moreover, calorie restriction did not affect the levels of procaspase-9 when compared with the age-matched ad libitum-fed group. Furthermore, there was no significant change in cleaved caspase-9 with age and calorie restriction had no significant effect on the content of cleaved caspase-9 (Fig. 4D).

Fig. 5. Procaspase-12 and cleaved caspase-12 were determined in the gastrocnemius muscle of 12AD, 26AD, and 26CR male Fischer 344 rats. Caspase-12 was determined by Western analysis and results (means F SEM) are reported as percentages of that of the 12AD group (see Materials and Methods). (A) Caspase-12: *p = 0.0002, 12AD vs. 26AD (100.0 F12.3% vs. 356.4 F 43.1%) and **p = 0.006, 26AD vs. 26CR (186.0 F 29.6%). (B) Cleaved caspase-12; nonsignificant 12AD vs. 26AD (100.0 F 13.6, 96.6 F 8.5), and **p = 0.04, 26AD vs. 26CR (74.46 F 7.23%).

attenuated cytochrome c release from the mitochondria (see next section). Apaf-1 content, procaspase-9 levels, cleaved caspase-9, and cytochrome c levels with age and calorie restriction There was a significant decrease ( p = 0.03) in the levels of cytosolic cytochrome c in the 26AD rats when compared with the 12AD group (Fig. 4A). Calorie restriction had no effect on the levels of cytosolic cytochrome c when compared with the age-matched ad libitum-fed group. Contents of other components of the apoptosome (caspase-9 and Apaf-1) were also determined. There was a significant increase ( p = 0.03) in levels of Apaf-1 in the 26AD rats when compared with the 12AD group (Fig. 4B). Moreover, calorie restriction

Fig. 6. Total and nuclear apoptosis-inducing factor (AIF) levels were determined in the plantaris muscle of 12AD, 26AD, and 26CR male Fischer 344 rats. Plantaris muscle was homogenized (see Materials and Methods) and used to determine total AIF content. Nuclear extracts were isolated from the plantaris muscle using differential centrifugation (see Materials and Methods). AIF was determined by Western analysis and results (mean F SEM) are reported as percentages of that of the 12AD group (see Materials and Methods). (A) Total AIF: *p = 0.002, 12AD vs. 26AD (100.0 F 6.7% vs. 155.5 F 10.5%) and **p = 0.015, 26AD vs. 26CR (109.2 F 14.35%). (B) Nuclear AIF: nonsignificant, 12AD vs. 26AD (100 F 11.42, 81.5 F 9.75), and nonsignificant, 26AD vs. 26CR (65.3 F 9.6%).

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Procaspase-12 and cleaved caspase-12 with age and calorie restriction We quantified caspase-12 in the gastrocnemius muscles of the 12AD, 26AD, and 26CR rats. There was a striking 350% increase ( p = 0.0002) in the content of procaspase-12 with age in the gastrocnemius muscle compared with the 12AD group (Fig. 5A). Calorie restriction significantly attenuated the age-associated rise in the levels of procaspase-12 ( p = 0.006). Cleaved caspase-12 (Fig. 5B) showed a similar pattern, although the content of the active subunit was not significantly elevated with age, but cleaved caspase-12 was significantly lower in the 26CR rats compared with the 26AD rats ( p = 0.04). Apoptosis-inducing factor with age and calorie restriction We determined the content of AIF in the plantaris muscle, because this muscle fiber type is very similar to the gastrocnemius and isolation of nuclei required an extraction procedure different from that used for the other assays. Total AIF (Fig. 6A) was significantly elevated in aged skeletal muscle ( p = 0.002) and total AIF was lower with calorie restriction compared with age-matched controls ( p = 0.015). In contrast, when we evaluated the levels of AIF in isolated nuclei of the plantaris muscle we observed no changes in content with either age or calorie restriction, suggesting rapid degradation of AIFtargeted nuclei (Fig. 6B).

DISCUSSION

Loss of skeletal muscle mass and fiber number (sarcopenia) is prevalent in old age. Calorie restriction has been shown to attenuate the loss in muscle fibers with age, which partly contributes to the pathogenesis of sarcopenia [24,27]. Aiken and co-workers [24,27] showed that calorie restriction was able to attenuate muscle fiber loss with age by counting epitrochlearus midbelly muscle fibers. However, the mechanism through which calorie restriction attenuates muscle fiber loss is unknown and has been investigated in this study. For the first time we present data that are consistent with the hypothesis that lifelong calorie restriction reduces the extent of apoptosis in skeletal muscle and reverses many of the age-associated changes observed in apoptotic signaling. Caspase-3 plays an important role in mediating cell death in that many of the apoptotic signaling pathways, such as the mitochondria-mediated, receptormediated, and sarcoplasmic reticulum-mediated pathways, converge at these points in the caspase cascade [30,31,38]. With age the levels of several caspases,

which are endoproteases, were significantly increased. We found that procaspase-3, cleaved caspase-3, and procaspase-12 were increased with age, suggesting that apoptotic signaling and the susceptibility for apoptosis are elevated in aged skeletal muscle. Moreover, the ageassociated rise in procaspase-3 and cleaved caspase-3 as well as in cleaved caspase-12 was attenuated by calorie restriction. Therefore, these data suggest that activation of these proteolytic caspases may be partly responsible for the initiation of muscle protein degradation and the eventual loss of muscle nuclei, muscle weight, and possibly muscle fibers. However, it still remains unclear how classic apoptosis (cell death) actually occurs in muscle fibers with age [2,3,44]. It is feasible that apoptosis in multinucleated cells (such as myocytes) may initiate a multicomplex process of proteolytic activity, resulting mostly in local atrophy and finally death of the myocyte [44]. Indeed, Aiken and co-workers [27,28] showed that specific muscle fibers harboring mitochondrial deletions often displayed atrophy, cellular splitting (invagination of the plasma membrane), and increased steady-state levels of oxidative nucleic damage. The splitting of an electron transport abnormal fiber resembles cell invagination similar to that seen in the early phase of apoptosis [30,45]. We show that calorie restriction appears to reduce apoptosis and alter key proteins determining the apoptotic potential, a significant finding, but recognize that not all mechanisms through which this occurs are elucidated. A potential limitation to this study is the lack of direct evidence that the nuclei undergoing apoptosis with age are all indeed myocyte nuclei. Quantification of the apoptotic index remains rather difficult in vivo and therefore quantitative ELISAs have been developed to detect and quantify cytosolic mononucleosomes and oligonucleosomes. Further, apoptosis is a fairly rapid process in cell culture, but it is unknown how fast this process occurs in vivo, specifically in multinucleated muscle cells. The work of Allen et al. [6] provides an example of this point. They found that only a small fraction of the total number of apoptotic nuclei in skeletal muscle could be attributed to myocyte apoptosis. In addition, because apoptosis is a transient phenomenon and there is evidence of satellite cell proliferation, it is very difficult to determine the actual rate. Therefore, in this current study we focused on several indices of apoptosis, alterations in apoptotic signaling proteins, the activation of several caspases, and inhibitors of apoptosis. It is possible that the ‘‘apoptotic program’’ has evolved differently in muscle fibers, with a main objective to regulate number of nuclei within muscle fibers. As calorie restriction generally delays proliferation and age-associated cellular damage, a greater turnover of individual muscle nuclei (due to oxidative

Lifelong caloric restriction alters apoptosis signaling in muscle

damage with age) could be reduced by the effects of calorie restriction. Many cell types contain IAPs that bind to cleaved caspase-3 and -9, suppressing their protease activity [36,46]. In this study we found higher levels of cleaved caspase-3 with age: however, the protease activity measured with our assay was not altered. In addition, we found that XIAP, the most potent of the IAPs [36], was elevated with age, in conjunction with cleaved caspase-3 levels, possibly suggesting a role for XIAP in the suppression of caspase-3 activity. Previous data from our laboratory and those of others showed that the addition of cytochrome c to skeletal muscle cytosolic fractions was unable to activate caspase-3, suggesting that skeletal muscle contains specific inhibitors [3,47]. We show that lifelong calorie restriction attenuates the age-associated increases in the levels of procaspase-3, cleaved caspase-3, and XIAP, but has no effect on procaspase-9 or cleaved caspase-9. These data suggest that calorie restriction may suppress various apoptotic stimuli and therefore caspase-3 activation, which may be partly regulated by XIAP. Expression of XIAP is under the control of the transcription factor NF-kB [48]. Because calorie restriction reduces mitochondrial production of hydrogen peroxide [49], a known activator of NF-kB, this may be the mechanism through which calorie restriction results in lower XIAP levels. Although the content of XIAP was highest in the 26AD group, the level of apoptosis was also highest. XIAP inhibits apoptosis by binding to and inhibiting the activity of caspase-3. Therefore, it is possible that the apoptosis seen in the 26AD group may be caspase-3 independent. A variety of new apoptosis independent pathways have emerged, one such pathway was investigated in this study and could be of greater importance in targeting neighboring nuclei to undergo nuclear fragmentation. AIF may be the critical determinant in the nuclearto-cytosolic domain ratio and therefore a main contributor to local fiber atrophy, such as reported by Lee et al. [24], who suggested that regions with mitochondrial deletions undergo atrophy to a much greater extent. Moreover, it is also possible that during an acute apoptotic stimulus the mitochondria release Smac/Diablo, a protein that relieves the inhibition of caspase-3 by XIAP [50]. We did not assess the role of Smac/Diablo in the current study. Hence, we believe that it is possible to have an environment with elevated XIAP in conjunction with elevated levels of apoptosis. Unlike most other caspases that localize to the cytosol, caspase-12 is found on the cytoplasmic side of the endoplasmic reticulum (ER), the site of protein assembly and secretion, as well as calcium homeostasis [37,38]. Yuan and colleagues have recently identified this novel

35

cell death role for caspase-12 abundant in skeletal muscle [37,38,51,52]. We further investigated what role the sarcoplasmic reticulum may play in inducing apoptosis in skeletal muscle. We found a dramatic f350% increase in the expression of procaspase-12 with age, which may suggest that aged skeletal muscle is characterized by severe ER stress. The causes of age-related ER stress and imbalances in calcium regulation in skeletal muscle are not clear. Cellular oxidative and nitrosative stress causes damage to proteins of the ER membrane, such as the Ca2+ ATPase pump, causing the cytosolic levels of calcium to rise [53,54]. Another possible mechanism of calcium dyshomeostasis may be the inability of the mitochondria to sequester cytosolic calcium due to agerelated mitochondrial dysfunction [3,55]. Despite the significant increase in procaspase-12, the cleaved form of caspase-12 was not significantly altered. Recent reports suggest that most procaspases contain 1 – 10% of the proteolytic activity found in the active enzymes [51,52]. Moreover, studies on caspase-12 are unclear if the pro- or cleaved form exhibits the functional protease activity (J. Yuan, personal communication), which is in contrast to many other caspases [37,38,51,52]. Hence, the increased levels of procaspase-12 suggest an increased ER stressinduced programmed cell death—a novel mitochondrial and Apaf-1-independent pathway. Furthermore, lifelong caloric restriction was effective in reducing the levels of both the pro- and cleaved capases-12, suggesting a better calcium homeostasis. Several studies have reported that intracellular calcium handling is drastically improved following periods of caloric restriction [25,26]. These data suggest that this new caspase-12-mediated pathway of apoptosis may play a key role in sarcopenia and is attenuated by calorie restriction. Moreover, in muscle fibers where high levels of calcium are the norm as opposed to other cell types caspase-12 could have other functions. Mitochondrial dysfunction can trigger apoptosis via the release of pro-apoptotic proteins, such as cytochrome c, into the cytosol [30,56]. This initiates the activation of a caspase cascade, which includes activation of caspase-9 and cleavage of caspase-3. We did not find higher levels of cytochrome c in the cytosol of the old animals and caspase-9 was not activated. We suggest that these findings may be the result of several adaptive responses; including increased mitochondrial ARC levels and an increase in the Bcl-2/Bax ratio [3,30], both known to stabilize the mitochondrial membrane by controlling the release of pro-apoptotic proteins. In response to mitochondrial stress, ARC has been shown to translocate to and stabilize the mitochondrial membrane and prevent cytochrome c release [57]. Studies show that ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-de-

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rived H9C2 cells [57]. We show that in skeletal muscle cytosolic levels of ARC decrease with age while mitochondrial levels increase. These changes in localization occurred without a change in the total cellular content of ARC, thereby suggesting that ARC may be translocating from the cytosol to the mitochondria. Hence, calorie restriction results in less mitochondrial oxidant stress [17,22,49] and therefore less translocation of ARC to the mitochondria. Another adaptive response that may preserve mitochondrial integrity in aged skeletal muscle may involve alterations in Bcl-2 family proteins, as these are known to play an important role in regulating the release of cytochrome c [34]. Previously, we showed that the Bcl-2/Bax ratio in subsarcolemmal mitochondria tends to increase with age in skeletal muscle and brain cortices, the result of which may be inhibition of mitochondrial release of pro-apoptotic proteins [3,55]. Taken together, the adaptive responses in ARC and the Bcl-2/ Bax ratio may be responsible for the increased stability of the functional mitochondria in the old animals. Moreover, the increased availability of cytosolic ARC in the

calorie-restricted animals is likely beneficial under stressful conditions by increasing the stability of the mitochondrial membrane and thereby preventing cytochrome c release. These age-associated adaptive responses along the mitochondria-mediated signaling pathway may be an attempt to protect the muscle fiber or regions in the muscle fiber from undergoing apoptosis (Fig. 7). Skeletal muscle of aged rats exhibits specific muscle regions, which undergo atrophy (Fig. 7), contain cytochrome c oxidase negative fibers, and overexpress succinate dehydrogenase activity (COX
/SDH+) [8,9,27]. Mitochondria contain AIF, which, on apoptotic stimuli, can be released and translocated to the nucleus where it induces DNA fragmentation in a caspase-independent pathway [33]. The total cellular levels of AIF were significantly increased with age, suggesting an increased potential for apoptosis. Moreover, this age-associated rise in AIF in the plantaris muscle was attenuated by calorie restriction, demonstrating another possible mechanism through which calorie restriction may reduce sarcopenia, specifically the loss in skeletal muscle nuclei [27].

Fig. 7. Proposed scenario of nuclear and cytosolic degradation of a muscle fiber, potentially leading to the death of an entire myocyte. (A) Oxidative stress in mitochondria could lead to the release of cytochrome c and apoptosis inducing factor (AIF) in specific myocyte regions activating various caspases and destroying nuclei, respectively. A reduced nuclei-to-cytosoli ratio could initiate sarcopenia and evidence has accumulated to support a role for this modulation of myonuclear number during muscle remodeling in response to aging. Moreover sarcoplasmic reticulum (SR) stress mediated by intracellular calcium, calpain, and caspase-7 could lead to activation of caspase-12, located on the cytoplasmic site of the SR. With age, this scenario could be responsible for a substantial loss of muscle mass and eventually loss of entire muscle fibers, thus affecting skeletal muscle function. (B) SR stress and oxidants, such as hydrogen peroxide, are signals to initiate cellular activation of apoptotic pathways, such as the mitochondrial and sarcoplasmic reticulum-mediated pathways. Adaptive responses, such as translocation of ARC, upregulation of Bcl-2, and increases in XIAP expression are all able to slow down the process of sarcopenia.

Lifelong caloric restriction alters apoptosis signaling in muscle

However, no changes were observed when we determined the protein levels of AIF in the isolated nuclei with age or calorie restriction. Under normal physiological conditions the nuclei of muscle cells undergo a turnover process, in which nuclei are degraded and replaced by satellite cells which differentiate into skeletal muscle nuclei [7]. This modulation of myonuclear number to maintain a constant nuclear-to-cytoplasm ratio appears central to muscle remodeling in response to injury, aging, adaptation, and disease [7,44]. Hence, these data would suggest that nuclei containing AIF might be degraded rapidly and be difficult to detect. The exact time course in which an apoptotic nucleus is degraded is unclear, but will certainly affect atrophy in specific fibers and/or specific regions of muscle fibers. Indeed, Aiken and co-workers [8,9,27] showed that the accumulation of electron transport system abnormalities was fiber specific and could be directly linked to specific regions undergoing severe sarcopenia. The vastus lateralis muscle (Type 2 fiber) of 36 month old male Fischer 344  Brown Norway hybrid rats showed a high degree of atrophy and loss of nuclei, and stained positive for cytochrome c oxidase-negative fibers [8,9,27]. Degradation of multiple nuclei in skeletal muscle may not immediately equate to cell death of the entire myocyte. Therefore, further studies need to examine nuclei undergoing apoptosis in the regions and simultaneously determine the levels of AIF. It is likely that apoptosis in multinucleated cells may initiate a multicomplex process of proteolytic activity, partly resulting in atrophy and cell death of the myocyte (Fig. 7) [58,59]. In summary, this study shows that the apoptotic potential is increased in aged skeletal muscle and that is reduced in calorie-restricted rats compared with their agematched controls. The pro-apoptotic proteins procaspase3, cleaved caspase-3, procaspase-12, Apaf-1, and AIF levels, are all elevated with age (Table 2). These alterations could also explain reports in the literature demonstrating impaired muscle repair after damage in old humans and rodents [60 – 62]. Moreover, slowing the aging process by lifelong calorie restriction appears to attenuate potential apoptotic stimuli, such as mitochondrial oxidant production (hydrogen peroxide) and DNA damage (8-oxo-7,8-dihydro-2V-deoxyguanosine) reported in Ref. [49]. In this study the levels of procaspase-3, cleaved caspase-3, procaspase-12, cleaved caspase-12, XIAP, AIF, and Apaf-1 were reduced significantly with calorie restriction (Table 2) and this reduced the potential for skeletal muscle apoptosis which may result in a reduction in the incidence and severity of sarcopenia through the preservation of muscle mass and muscle function and a slowing of myofiber loss with age. However, the relationship between body weight and muscle mass is complicated by the fact that adiposity

37

Table 2. Overview of Changes in Apoptosis and Apoptotic Regulatory Proteins in Skeletal Muscle with Aging and Calorie Restriction (12AD vs. 26AD and 26CR vs. 26AD) Aging

Calorie restriction

Gastrocnemius muscle Apoptosis Caspase-3 (procaspase)b Caspase-3 (cleaved caspase)b Caspase-3 (activity)c XIAPc ARCb ARCc ARCd Cytochrome cc Apaf-1b Caspase-9 (procaspase)b Caspase-9 (cleaved caspase)b Caspase-12 (procaspase)b Caspase-12 (cleaved caspase)b

za z z ! z ! # z # z ! ! z !

# # # ! # ! z # ! # ! ! # #

Plantaris muscle Apoptosis inducing factorb Apoptosis inducing factore

z !

# !

a

z, Increase; #, decrease; !, no change. Total tissue. Cytosolic. d Mitochondrial. e Nuclear. b c

differs with both age and calorie restriction. After muscle weight is divided by mean body weight the muscle weight per unit body weight is reduced in old animals compared with adult animals, but is increased in calorie-restricted animals (Table 1). These data show an inverse relationship between relative muscle mass (mg/g) and apoptosis and, in turn, support the conclusion that calorie restriction may attenuate sarcopenia by reducing skeletal muscle apoptosis. These data also validate the model chosen and provide evidence, albeit circumstantial, that calorie restriction may prevent sarcopenia by reducing apoptosis. Although our data provide an example of the beneficial effects of calorie restriction on both apoptosis and muscle mass, the relationship between muscle mass and body weight is complex, because it is dependent on both skeletal size and the load muscles have to bear (i.e., body weight). Moreover, we used 12 month old rats (as adults) because skeletal muscle is fully developed (size and mass reach a maximum) and after this age there seems to be a gradual decline in muscle mass and fiber number reflecting sarcopenia. However, more studies need to be conducted using animals of different ages to determine exactly what period of calorie restriction is sufficient to influence apoptosis and the signaling transduction cascade of apoptosis. For example, additional experimental work should include a 12 month old cohort of calorierestricted animals and a cohort of restricted animals older than 26 months of age. The latter cohort could provide even better insight into whether calorie restriction delays

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the age-related change or alters the process of aging in skeletal muscle. Calorie-restricted rats at an age at which they demonstrate a percentage survival comparable to that of the 26AD group could be selected. Although calorie restriction lowers the apoptotic potential (apoptotic markers) compared with age-matched controls, future studies need to be conducted that would allow us to draw stronger conclusions about the level of apoptosis in skeletal muscle as it relates to mortality. The biochemical information obtained from this study could potentially permit the development of physiological or genetic interventions that may attenuate the loss of skeletal muscle myocytes and sarcopenia indicative of advancing age. Acknowledgments—We thank Colin Selman and Tracey Phillips for critical reading and editing of the manuscript and Manish Patel for technical assistance. This research was supported by Grants AG-1799401 and AG-10485-08 from the National Institute of Aging, National Institutes of Health.

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