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Effect of Age on Skeletal Muscle Proteolysis in Extensor Digitorum Longus Muscles of B6C3F1 Mice

^a Division of Geriatric Medicine, University of Michigan, and Veteran Affairs Ann Arbor Health Care System, Ann Arbor

Thomas H. Reynolds IV, Department of Pharmacology, 1300 Jefferson Park Avenue, University of Virginia Health System, P.O. Box 800735, Charlottesville, VA 22908-0735 E-mail: thr2n{at}virginia.edu.

Decision Editor: Edward Masoro, PhD

	Abstract

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The purpose of this study was to determine if age-related muscle atrophy is associated with an increased rate of protein degradation in extensor digitorum longus (EDL) muscles from young (YG; 2–4 months), middle-aged (MA; 12–17 months), and aged (AG; 22–24 months) B6C3F1 mice. EDL muscles from AG mice weighed less than EDL muscles from MA mice (p = .01). EDL muscles from MA mice weighed more than EDL muscles from YG mice (p = .02). The rate of protein degradation, as assessed by tyrosine release during in vitro incubations, was higher in EDL muscles from AG mice than it was in those from MA mice (p = .03). The rate of protein degradation was higher in EDL muscles from YG mice than it was in those from MA mice (p = .04). An inverse relationship existed between muscle mass and protein degradation (r = -.67; p = .0001). We conclude that skeletal muscle protein degradation rates decrease with maturation and increase with advancing age.

AGING is associated with a progressive loss of muscle mass in both humans ⁽¹⁾ and rodents ⁽²⁾. Because muscle mass is dependent on the balance between protein synthesis and degradation rates, muscle atrophy can result from a reduction in the rate of protein synthesis, an increase in the rate of protein degradation, or both. The rate of skeletal muscle protein synthesis appears to decline with advancing age in both humans ⁽³⁾ and rodents ⁽⁴⁾⁽⁵⁾, although some studies in rodents report no effect of aging on protein synthesis rates ⁽⁶⁾⁽⁷⁾. The effect of aging on the rate of skeletal muscle protein degradation has yet to be established. Studies have reported an increase ⁽⁸⁾, decrease ^(9)(10), or no change ⁽¹¹⁾ in the rate of skeletal muscle protein degradation with advancing age. The inconsistent findings regarding the effect of age on skeletal muscle protein degradation may be due to differences in methodology ^(8)(10)(12), the age of the animals studied ⁽¹³⁾, and whether the muscle studied exhibited age-related atrophy ⁽²⁾.

Previous studies that have reported a decline in the rate of skeletal muscle protein degradation with advancing age have utilized in vivo methodology ^(9)(10)(14). There are a variety of methods to assess in vivo protein degradation rates, but they are all limited by technical problems ⁽¹⁵⁾. In order to avoid the technical difficulties associated with in vivo protein degradation measurements, some investigators have utilized an in vitro technique. This procedure involves the intact isolation of thin rodent muscles (e.g., rat epitrochlearis or mouse extensor digitorum longus muscles) and subsequent incubation in well-oxygenated media. The rate of protein degradation is measured by the release of amino acids from the muscle into the incubation media. A major advantage of the in vitro protein degradation assessment method is the ability to prevent reutilization of amino acids with cycloheximide ^(16)(17)(18), a protein synthesis inhibitor that completely blocks protein synthesis without altering protein degradation rates in isolated rodent skeletal muscle ⁽¹⁹⁾. Investigators using this in vitro technique have documented that protein degradation rates are elevated in several rodent models of muscle atrophy ^(16)(17)(18), indicating that proteolysis may play a role in regulating muscle size.

The purpose of the present study was to determine if age-related muscle atrophy is associated with an increased rate of protein degradation. This hypothesis was tested by assessing in vitro protein degradation rates in the extensor digitorum longus (EDL) muscle from young (YG; 2–4 months, n = 4), middle-aged (MA; 12–17 months, n = 7), and aged (AG; 22–24 months, n = 7) B6C3F1 male mice. The mouse EDL muscle was utilized because it has previously been shown to undergo age-related atrophy ^(20)(21) and is an appropriate muscle for in vitro studies ⁽¹⁵⁾.

	Methods

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Animals and Housing
Specific pathogen-free male B6C3F1 mice (YG, 2–4 months; MA, 12–17 months; AG, 22–24 months) were obtained from the National Institute on Aging breeding colony maintained by Harlan Sprague Dawley (Indianapolis, IN). The median length of life and maximal life span of this male B6C3F1 colony is 35 months and 44 months, respectively. Upon arrival, mice were housed in a temperature-controlled specific pathogen-free animal room maintained on a 12 hour light–12 hour dark cycle at the Unit for Laboratory Animal Medicine at the University of Michigan. The animals were ad libitum fed Lab Diet 5001 rodent chow (Richmond, IN) and water. All animal care and surgery was in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals (DHEW Publication No. 85-23). All experimental protocols were approved by the University of Michigan Committee for the Use and Care of Animals.

Muscle Preparation and Incubation
Animals in the postprandial state were anesthetized with 5 mg/100 g of body weight of sodium pentobarbital. Both EDL muscles were dissected out, blotted on gauze, weighed, and transferred to 25-ml Erlenmeyer flasks (1 muscle/flask) containing 3 ml of Krebs Henseleit buffer (0.160M NaCl, 0.0046M KCl, 0.0012M KH₂PO₄, 0.0025M NaHCO₃, 0.0025M CaCl₂, and 0.0012M MgSO₄), 0.1% bovine serum albumin, 10mM glucose, 200µM valine, 170µM leucine, and 100µM isoleucine. Following a 30-minute preincubation period at 37°C in a shaking water bath, muscles were transferred to a second set of 25 ml Erlenmeyer flasks (1 muscle/flask) containing fresh media. They were incubated for a 2-hour experimental period at 37°C in the flaccid state (no tension) with shaking. In order to prevent the reutilization of amino acids released from EDL muscles during incubations, 0.5mM cycloheximide (protein synthesis inhibitor) was added to the media ^(16)(17). This concentration of cycloheximide inhibits 95% of protein synthesis without altering protein degradation in isolated rodent skeletal muscle ⁽¹⁹⁾. All flasks were pregassed with 95% O₂ and 5% CO₂ for 3 minutes immediately prior to both incubations as well as every 20 minutes (15 seconds) during the experimental incubation period. Following the 2-hour experimental period, the incubation media were collected and stored at -20°C until further analysis. The muscles were frozen with metal tongs cooled to the temperature of liquid N₂ and stored at -80°C. The validity of this in vitro technique has been described in detail by Lecker and colleagues ⁽¹⁵⁾ and Tawa and Goldberg ⁽²²⁾.

Assessment of Protein Degradation Rate
The rate of protein degradation was assessed by measuring tyrosine levels in the media samples by fluorometry as described by Price and colleagues ⁽¹⁶⁾. Briefly, tricarboxylic acid (TCA; final concentration, 5%) was added to the media samples to precipitate proteins. Following centrifugation (800 x g, 10 min, 4°C), a 0.750-ml aliquot of the supernatant was transferred to a tube containing 0.750 ml of 5% TCA, 0.750 ml of 1% nitrosonapthol (wt/vol ethanol), and 0.750 ml of 17.5% nitric acid containing 0.5% NaN₂ (wt/vol). The samples were then incubated at 55°C for 30 minutes and allowed to cool; then 7.5 ml of dichloroethane was added. Following vigorous mixing, the samples were centrifuged (800 x g, 10 min, 4°C) and the content of tyrosine in the supernatants was assessed by fluorometry (450-nm excitation; 550-nm emission). Tyrosine release values for each EDL muscle are expressed as (nanomoles per milligram) per 2 hours.

Statistical Analysis
The effects of age on the rate of protein degradation, muscle weight, and body weight were analyzed by using a 1 x 3 analysis of variance. When the F ratio was significant, a Fisher least significant difference post hoc test was utilized to locate the significant differences between groups. Regression analysis was utilized to assess the relationship between muscle weight and the rate of protein degradation. Data are expressed as means ± standard error of the means, and the level of statistical significance was set at p .05.

	Results

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B6C3F1 Mice Body Weights and EDL Muscle Weights
The body weights of the YG mice were 18% and 10% less than those of the MA mice and AG mice, respectively (YG, 29.3 ± 0.8, vs MA, 34.7 ± 1.5, vs AG, 32.4 ± 1.6 g); however, these differences were not statistically significant (p = .10). EDL muscles from AG mice weighed significantly less (13%) than EDL muscles (Fig. 1) from MA mice (AG, 13.8 ± 0.58, vs MA, 15.6 ± 0.42 mg; p = .01) but not from YG mice (AG, 13.8 ± 0.58, vs YG, 13.7 ± 0.36 mg; p = .96). EDL muscles from MA mice weighed significantly more (14%) than muscles from YG mice (MA, 15.6 ± 0.42, vs YG, 13.7 ± 0.36 mg; p = .03). The muscle weight:body weight ratios were not significantly different among YG, MA, and AG mice (YG, 0.486 ± 0.017, vs MA, 0.458 ± 0.034, vs AG, 0.427 ± 0.021; p = .393).

View larger version (10K): [in this window] [in a new window]   Figure 1. Effect of age on muscle mass in extensor digitorum longus (EDL) muscles isolated from B6C3F1 mice. *Significantly different from the middle-aged group (p .05).

View larger version (12K): [in this window] [in a new window]   Figure 2. Effect of age on the rates of protein degradation in extensor digitorum longus muscles isolated from B6C3F1 mice. *Significantly different from the middle-aged group (p .05).

View larger version (19K): [in this window] [in a new window]   Figure 3. Relationship between muscle mass and the rate of protein degradation in extensor digitorum longus (EDL) muscles isolated from young, middle-aged, and aged B6C3F1 mice.

	Discussion

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Several muscle wasting conditions ^(16)(17)(18), including muscle unweighting ⁽²³⁾ and denervation atrophy ⁽¹⁷⁾, have been associated with an increased rate of protein degradation. Because muscle fiber denervation and physical inactivity (i.e., muscle unweighting) are both thought to contribute to age-related muscle atrophy, it is reasonable to hypothesize that protein degradation rates would be elevated with advancing age. In support of this theory, the present study demonstrates that the rate of protein degradation is higher with advancing age. Our results are supported by evidence from other studies reporting that aged skeletal muscle exhibits greater proteolytic activity ⁽⁸⁾ and has an enhanced expression of proteins involved in skeletal muscle proteolysis ⁽⁴⁾. Although other studies have reported a decline in the rate of protein degradation with advancing age, these studies did not actually analyze the data with statistical methods; instead they reported observations. Therefore, it is difficult to interpret or compare the data from these studies with those of other studies ^(9)(10)(14). Furthermore, studies that have reported a decline in protein degradation rates with advancing age have actually examined maturation rather than aging ⁽¹³⁾. In the present study we observed that the rate of protein degradation in muscles from 2- to 4-month-old mice was lower compared with that in muscles from 12- to 17-month-old mice. This difference may be due to the fact that the 2- to 4-month-old mice were not completely mature and therefore may represent a developmental rather than an age-related change. In contrast to the unsubstantiated belief that the rate of skeletal muscle protein degradation declines with advancing age ^(5)(24), our results suggest that the rate of protein degradation declines with maturation and increases with advancing age. In support of our hypothesis that protein degradation plays a role in regulating muscle size, we observed a strong inverse relationship between muscle mass and the rate of protein degradation in EDL muscles from YG, MA, and AG mice (see Fig. 3). The increased rate of protein degradation in EDL muscle from AG mice was accompanied by a decrease in EDL muscle mass when compared with MA mice. Likewise, a decreased rate of protein degradation in EDL muscles from MA mice was accompanied by an increase in EDL muscle mass when compared with YG mice. Although these data suggest that proteolysis may play a role in regulating muscle mass, the singular effect of maturation or aging cannot be delineated because the respective changes in protein degradation rates may, in part, be a function of another factor that regulates muscle mass (i.e., physical activity). However, the present inverse relationship between the rate of protein degradation and muscle mass is supported by evidence demonstrating that a primary mediator of muscle atrophy during several metabolic diseases and following disuse and muscle denervation is accelerated proteolysis (for reviews see 15 and 18).

Although the mechanism responsible for the greater skeletal muscle proteolysis in aged mice is unknown, one potential mechanism is increased oxidative damage to skeletal muscle protein with advancing age ⁽²⁵⁾. In this scenario, increased protein degradation could represent an appropriate adaptive response by aging myocytes to extrude oxidatively damaged proteins from the cell. This hypothesis is supported by the finding that pro-oxidants increase ubiquitin-proteasome activity ^(26)(27) and reduce myosin heavy chain expression in rodent skeletal muscle ⁽²⁶⁾. In addition, glucocorticods have been shown to produce oxidative damage and muscle atrophy ⁽²⁸⁾ as well to as stimulate ubiquitin and proteasome subunit gene expression ⁽²⁹⁾. This finding is of particular interest because old rats appear to be more susceptible to muscle atrophy induced by glucocorticoid treatment than adult rats ⁽¹²⁾.

In summary, the present study demonstrates that skeletal muscle protein degradation decreases with maturity and increases with advancing age. It appears that the changes in protein degradation may play a role in regulating muscle mass as evidenced by a significant inverse relationship between EDL muscle mass and protein degradation rates. Although these data suggest that proteolysis may play a role in the age-related muscle atrophy, this cannot be established because the increase in protein degradation with advancing age may be due to another factor and not aging alone. Future studies will be required to determine the extent to which skeletal muscle proteolysis is responsible for age-related muscle atrophy as well as to identify the mechanisms responsible for the increase in skeletal muscle protein degradation.

	Acknowledgments

 
T. H. Reynolds was supported by an Institutional National Research Service Award (T32 AG00114: "Multidisciplinary Research Training in Aging"), M. A. Supiano was supported by National Institutes of Health (NIH) Grant AG00924, and D. R. Dengel was supported by NIH Grant KO1 AG000723. This work was also supported by the University of Michigan Geriatrics Center and the Ann Arbor Veteran Affairs—Geriatric Research, Education, and Clinical Center.

The authors are indebted to S. Russell Price, PhD, for assistance with the protein degradation assay.

Received September 21, 2001

Accepted January 24, 2002

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