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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:1119-1129 (2006)
© 2006 The Gerontological Society of America

Age-Related Dystrophin–Glycoprotein Complex Structure and Function in the Rat Extensor Digitorum Longus and Soleus Muscle

Kevin M. Rice, Deborah L. Preston, David Neff, Michael Norton and Eric R. Blough

Departments of 1 Pharmacology, Physiology, and Toxicology, Joan C. Edwards School of Medicine, 2 Biological Sciences, and 3 Chemistry, Marshall University, Huntington, West Virginia.

Address correspondence to Eric Blough, PhD, Laboratory of Molecular Physiology, Suite 311, Science Building, Department of Biological Sciences, 1 John Marshall Drive, Marshall University, Huntington, WV 25755-1090. E-mail: blough{at}marshall.edu


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
This study tested the hypothesis that age-related changes in the dystrophin–glycoprotein complex (DGC) may precede age-associated alterations in muscle morphology and function. Compared to those in adult (6 month) rats, extensor digitorum longus (EDL) and soleus muscle mass was decreased in old (30 month) and very old (36 month) Fischer 344/NNiaHSD x Brown Norway/BiNia rats. The amount of dystrophin, ß-dystroglycan, and {alpha}-sarcoglycan increased with aging in the EDL and decreased with aging in the soleus. {alpha}-Dystroglycan levels were increased with aging in both muscles and displayed evidence of altered glycosylation. Immunostaining for the presence of antibody infiltration and dystrophin following increased muscle stretch suggested that the aging in the soleus was characterized by diminished membrane integrity. Together, these data suggest that aging is associated with alterations in EDL and soleus DGC protein content and localization. These results may implicate the DGC as playing a role in age-associated skeletal muscle remodeling.

AGE-RELATED muscle atrophy in humans and in the Fischer 344/NNiaHSD x Brown Norway/BiNia (F344/N x BN) rat model is associated with decreases in muscle mass, fiber cross-sectional area, muscle contractile function, increased muscle cell apoptosis, and a diminished ability of aged muscle to respond to a hypertrophic stimulus (1,2). Recently, our laboratory has demonstrated that aging in the F344/N x BN rat is characterized by alterations in the ability of the fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus to elicit load-induced alterations in mitogen-activated protein kinase (MAPK) phosphorylation, suggesting that muscle mechanotransduction may be altered with aging (3). The mechanism(s) responsible for these changes are not known. Similar alterations in muscle structure, function, and load-induced signaling have been observed in the muscular dystrophies (4,5). In dystrophic muscle, the progression of muscle dysfunction is mediated, in large part, by genetic defects of the dystrophin–glycoprotein complex (DGC). Known components of the DGC include dystrophin, the dystroglycans (DGs), sarcoglycans (SGs), integrins, caveolin-3, and others, with the expression of these proteins differing depending on muscle type (6). The DGC has been shown to be important in mediating interactions between the cytoskeleton, membrane, and extracellular matrix, and recent studies have suggested that the DGC plays a role in sensing mechanical forces imposed on sarcolemma (7–11). Although the proteins of specific compartments of the DGC are well described, little is known about their regulation in nondystrophic muscle. Moreover, to our knowledge, the effect of aging on the DGC has not been investigated.

In the mdx mouse and the human dystrophies, a "prenecrotic" phase of seemingly normal muscle functioning occurs prior to myofiber degeneration (12,13). On the basis of these findings and other studies demonstrating the integral role the DGC plays in mediating muscle structure and function, we hypothesized that age-related changes in the DGC may precede alterations in muscle morphology and function. The discovery of such a connection, if present, would be important as this information may provide insight regarding the mechanisms underlying age-associated muscle dysfunction.

In the present study, we examined the effects of aging on the relative content of several proteins belonging to the DGC in the EDL and soleus of the F344/N x BN rat. Using probability of survival curves generated by the National Institute on Aging, we selected animal ages to correspond roughly to humans in their third (young adult), sixth (old), and eighth (very old) decade of life (1). This latter time point is of particular interest considering that age-related dysfunction in humans is accelerated in this interval and because this age group represents one of the fastest growing segments of the aging population in the United States (14,15). Our data suggest that aging alters the regulation of DGC proteins and that this regulation is different between the EDL and soleus of the F344/N x BN rat. Further, although it is likely that dissimilar mechanism(s) underlie the muscle dysfunction seen in aging and the muscular dystrophies, our findings are consistent with the hypothesis that alterations in DGC may be associated with age-related muscle remodeling.


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Animal care and procedures were conducted in accordance with the Animal Use Review Board of Marshall University using the criteria outlined by the American Association of Laboratory Animal Care (AAALAC) as proclaimed in the Animal Welfare Act (PL89-544, PL91-979, and PL94-279). Adult (6 months, n = 12), old (30 months, n = 12), and very old (36 months, n = 12) male F344/N x BN rats were obtained from the National Institute on Aging. Rats were housed two per cage in an AAALAC–approved vivarium. Housing conditions consisted of a 12-h light/dark cycle, with temperature maintained at 22 ± 2°C. Animals were provided food and water ad libitum. Rats were allowed to recover from shipment for at least 2 weeks before experimentation began. During this time the animals were carefully observed and weighed weekly. None of the animals exhibited signs of failure to thrive, such as precipitous weight loss, disinterest in the environment, or unexpected gait alterations.

Materials
Antibodies used were dystrophin (dys-2) from Novacastro (Ontario, Canada); {alpha}-DG (VIA4-1) from Upstate (Charlottesville, VA); ß-DG (sc16165) from Santa Cruz Biotechnology (Santa Cruz, CA); {alpha}-SG (VP-A105) from Vector Laboratories (Burlingame, CA); and rat immunoglobulin G (IgG) antibody (T6392) from Molecular Probes (Eugene, OR). Rabbit and mouse IgG secondary antibodies were purchased from Cell Signaling Technology (Beverly, MA). Enhanced chemiluminescence (ECL) western blotting detection reagent was from Amersham Biosciences (Piscataway, NJ). Restore western blot stripping buffer was obtained from Pierce (Rockford, IL), and 3T3 and L6 cell lysates were from Santa Cruz Biotechnology. All other chemicals were purchased from Sigma (St. Louis, MO).

Determination of DGC Protein Content
Animals were anesthetized with a ketamine/xylazine (4:1) cocktail (50 mg/kg ip) and supplemented as necessary for reflexive response (6). EDL and soleus muscles were isolated and quickly removed, blotted dry, trimmed of visible fat and tendon projections, weighed, and immediately frozen in liquid nitrogen. Tissues were stored at –80°C until use. Prior to biochemical processing, muscles were cut into thirds with the middle segment reserved for histological analysis. Muscle segments to be used for immunoblotting were pulverized in liquid nitrogen using a mortar and pestle and were washed three times with ice-cold phosphate-buffered saline (PBS). Proteins were extracted by incubating samples on ice in modified RIPA Buffer (50 mM Tris, 150 mM NaCl, 1% Nonidet-P40, 0.25% Na-deoxycholate, 1 mM EDTA, aprotinin at 1 µg/mL, leupeptin at 1 µg/mL, pepstatin at 1 µg/mL, 1 mM phenylmethylsulphonylfluoride, 1 mM Na3VO4, 1 mM NaF) at 10 µL/mg and centrifuging for 10 minutes at 10,000 g. After removing the supernatant, pellets were re-extracted twice and then washed three times with ice-cold PBS. Membrane proteins were obtained by resuspending the pellets (1 mL/g) in a buffer containing 1% deoxycholate, 1% Nonidet-P40, 10 mM NaPO4, 140 mM NaCl, and 2 mM EDTA supplemented with100 mM NaF, 1 mM Na3VO4, 2 mM phenylmethylsulphonylfluoride, aprotinin at 1 µg/mL, leupeptin at 1 µg/mL, and pepstatin at 1 µg/mL. After collecting the supernatant by centrifugation for 10 minutes at 13,000 g, protein concentration was determined in triplicate via the Bradford method (Pierce) using bovine serum albumin as a standard. Samples were boiled and analyzed using 7.5% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. To allow direct comparisons to be made between age groups, samples from different age groups were run on the same gel. Following electrophoresis, protein was transferred onto Hybond nitrocellulose membranes (Amersham Biosciences) using standard conditions (16). To confirm successful transfer of protein and equal loading of lanes, which occurred in all cases, the membranes were stained with Ponceau S (data not shown). Membranes were blocked in Tris-buffered saline (TBS) with 0.5% Tween-20 (TBST) and 5% milk for 1 hour at room temperature, washed (TBST, 3 x 5 minutes), and incubated in primary antibody overnight at 4°C or 1 hour at room temperature as outlined by the antibody manufacturer. After extensive washing (TBST, 3 x 5 minutes), membranes were incubated in horseradish peroxidase (HRP)–linked secondary antibodies for 1 hour at room temperature and rewashed (TBST, 3 x 5 minutes). Proteins were visualized by ECL western blotting detection reagent (Amersham Biosciences) and quantified by densitometry using a flatbed scanner (Epson Perfection 3200 PHOTO) and Imaging software (AlphaEaseFC; Alpha Innotech Corporation, San Leandro, CA) Exposure time was adjusted to keep the integrated optical densities within a linear and nonsaturated range. Specificity of the bands was assessed using Dual Color molecular-weight markers, a biotinylated protein ladder, and NIH 3T3 or L6 cell lysates as positive controls. Immunoblots were stripped with Restore western blot stripping buffer as detailed by the manufacturer. After verifying the absence of residual antibody binding by interrogating the membrane with the ECL reagent, membranes were washed and reprobed. To minimize potential experimental error associated with membrane stripping, the order of antibody incubations was randomized between experiments.

Muscle Loading Protocol
To determine if alterations in DGC content were associated with changes in the ability of muscles to resist mechanical insult, rats were anesthetized with a ketamine/xylazine (4:1) cocktail (50 mg/kg ip) and supplemented as necessary for reflexive response. In a sterile environment, the dorsal surface of the hind limb was shaved and cleaned, and the soleus and EDL muscles were isolated using blunt dissection. Calipers were used to determine the in situ resting muscle length of each muscle at 90° ankle flexion. After the muscle was removed from the animal, a suture (4.0) was tied to the proximal and distal tendons and the muscles were mounted in a custom-designed incubation chamber at the predetermined in situ resting length. Dissection and mounting procedures were performed rapidly and with care to prevent stretching or tearing of the muscle. Soleus or EDL muscles from the same animal were incubated in tandem. After 15 minutes of equilibration, muscles (n = 4/age group) were subjected to a controlled increase in muscle length equal to 135% of the in situ muscle length for 15 minutes or maintained at the in situ muscle length for 15 minutes. All muscle incubations were performed in oxygenated (95% O2, 5% CO2) Krebs-Ringer bicarbonate buffer (137 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM KH2PO4, 1 mM MgSO4, 24 mM NaHCO3, and 11 mM glucose containing 0.025 tubocurarine chloride) thermostatically maintained at 20°C (optimal for maintaining the stability of muscles in vitro) for the duration of the experimental period (17). After incubation or exposure to lengthening, muscles were weighed and immediately frozen in liquid nitrogen.

Histological Imaging and Immunolabeling
To study morphology, tissue segments obtained from the muscle midbelly were serially sectioned (8 µm) using an IEC Minotome cryostat and collected on polylysine (Sigma)-coated slides. After fixing in acetone (–20°C for 2 minutes), sections were stained with hematoxylin and eosin, mounted, and coverslipped for the evaluation of muscle morphology. Immunostaining for dystrophin, rat IgG, {alpha}-DG, ß-DG, and {alpha}-SG was visualized by immunofluorescence as outlined by the antibody manufacturer. Briefly, sections were incubated for 30 minutes in a blocking solution (5% bovine serum albumin and PBS containing 0.5% Tween 20, pH 7.5) and then incubated with specific antisera diluted in PBS-T (antidystrophin, 1:100; {alpha}-DG, 1:100; ß-DG, 1:100; {alpha}-SG, 1:100; rat-IgG, 1:100) for 1 hour at 37°C in a humidified chamber. After washing in 5% bovine serum albumin and PBS containing 0.5% Tween 20 (3 x 5 minutes), sections were incubated with the appropriate FITC- or Texas Red–labeled secondary antibody (1:200) if appropriate, for 1 hour at 37°C in a humidified chamber. Specimens were mounted and visualized by epifluorescence using an Olympus fluorescence microscope (Melville, NY) fitted with a x40 objective or with a Nikon Diaphot microscope for confocal analysis. Confocal imaging was performed using a Bio-Rad (Hercules, CA) MRC 1024 scanning system using the 488 nm line from a krypton/argon laser for fluorophore excitation. Emissions were collected through either 515-nm long pass or 522/532 nm band pass filters. To allow approximate quantitative comparisons, all imaging parameters (pixel dwell time, optical and electronic filters, confocal iris, laser power, and detector gain and offset) were kept constant between samples.

Data Analysis
Results are presented as mean ± standard error of the mean. Data were analyzed by using SigmaStat 3.0 (Systat Software, Inc., Point Richmond, CA). A one-way analysis of variance (ANOVA) on ranks was performed for overall comparison with the Student–Newman–Keuls post hoc test used to determine differences between groups. The level of significance accepted a priori was p ≤.05.


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Aging Affects the Dystrophin Content of the EDL and Soleus Differently
EDL muscle mass was decreased by 16.3% (182 ± 6 mg vs 152 ± 8 mg) and 34.7% (182 ± 6 mg vs 119 ± 9 mg) at 30 and 36 months, respectively (p <.05), compared to that in young adults (6 months). Similarly, soleus muscle mass was reduced by 17.8% (179 ± 10 mg vs 147 ± 8 mg) and 38.1% (179 ± 10 mg vs 110 ± 12 mg) in the 30- and 36-month groups, respectively (p <.05).

Immunoblots of membrane preparations from adult, old, and very old EDL and soleus muscles were examined for the relative abundance of dystrophin. An immunoreactive band of ~427 kd corresponding to the predicted molecular mass of the dystrophin protein (Figure 1A) was observed as previously described (10). Densitometric scanning revealed that the dystrophin content was ~15.8 ± 4.6% higher in 36-month muscles than in the 6-month EDL (p <.05) (Figure 1A). Conversely, in the soleus, dystrophin was ~6.3 ± 0.8% and ~20.4 ± 1.8% lower in the 30- and 36-month animals, respectively (p <.05) (Figure 1A). To assess the subcellular distribution of dystrophin, muscle cross-sections were examined by using immunohistochemical analysis (Figure 1B). Immunofluorescent labeling with antibodies against the COOH terminus of dystrophin in the 6-month EDL and soleus muscles showed uniformly arranged intact fibers, whereas in aging muscle fibers were smaller in size compared with the normal fibers and were pulled apart from each other in some areas (Figure 1). In 6-month animals, EDL and soleus muscle fiber dystrophin immunoreactivity appeared uniform and continuous in nature. With aging, particularly in the 36-month soleus, dystrophin immunoreactivity often appeared discontinuous and was at times completely absent along the sarcolemma of both EDL and soleus fibers (Figure 1B, Panels B and D). Together, these data suggest that dystrophin content and localization are altered with aging and that aging alters these parameters differently in the fast-twitch EDL and the slow-twitch soleus.


Figure 01
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  		Figure 1. A, Dystrophin content is increased in the extensor digitorum longus (EDL) but diminished in the soleus with aging. Dystrophin levels were determined by immunoblotting. Data are presented as means ± standard error. Insets: Representative blots for dystrophin. * Significantly different from 6-month values (p <.05); n = 5 for all groups. B, Immunolabeling of dystrophin in 6-month (A and C) and 36-month (B and D) EDL (A and B) and soleus (C and D) muscles. Note areas of diminished or missing dystrophin immunoreactivity along the sarcolemma of 36-month EDL and soleus muscles (arrowheads). Bar = 10 µm

 
Alterations in Membrane Dystrophin Content Are Associated With Changes in Soleus Membrane Stability
The DGC is thought to play a pivotal role in stabilizing the muscle cell membrane (4). The loss of sarcolemmal integrity allows influx of molecules into muscle fibers. To assess whether age-associated alterations in dystrophin are associated with alterations in membrane leakiness, we examined tissue sections obtained from adult, old, and very old EDL and soleus muscles for the presence of IgG (150 kd), a serum protein normally excluded from entry into the muscle fiber. In EDL and soleus muscles obtained from 6-month animals, no evidence of IgG immunoreactivity was demonstrated. In contrast, with aging, positive staining with anti-rat IgG antibodies was observed in the endo- and perimysium of both the EDL and soleus (Figure 2). Intracellular staining was diffusely distributed across the myofiber cytoplasm, although sometimes it appeared to be localized to the fiber periphery in the 36-month EDL. Rat IgG staining was even more pronounced in the 36-month soleus with gross morphological analysis revealing visibly higher uptake, and in some (albeit rare) sections, rat IgG infiltration appeared to occur in the cytoplasm of clustered myofibers (Figure 2D).


Figure 02
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  		Figure 2. Membrane damage with aging. Loss of membrane integrity is indicated by intracellular staining of rat immunoglobulin G (IgG) in 6-month (A and C) and 36-month (B and D) extensor digitorum longus (EDL) (A and B) and soleus (C and D) muscles. Arrows: Rat IgG-positive myofibrils. Bar = 25 µm

 
To further understand the role of altered dystrophin regulation with aging, we investigated the extent of fiber damage elicited by muscle lengthening. In adult EDL and soleus muscles, lengthening did not appear to induce muscle fiber damage as determined by qualitative inspection of muscle morphology following immunofluorescent staining for dystrophin (Figure 3). Similar to what has been reported by others examining muscle damage (18,19), we observed an increased propensity towards muscle fiber disruption with aging in both muscle types. The amount of damage induced by muscle lengthening appeared to be greater in the old soleus than the EDL and was characterized by a pronounced focal loss of dystrophin. Together, these data suggest that age-related alterations in DGC proteins are related to changes in the ability of aged muscle to resist mechanical injury.


Figure 03
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  		Figure 3. Membrane damage with aging and increased mechanical loading. Immunolabeling of dystrophin in 6-, 30-, and 36-month extensor digitorum longus (EDL) (A–F) and soleus (G–L) muscles following ex vivo incubation at resting (control A–C; G–I) or stretched (stretch D–F; J–L) conditions. Note increased susceptibility of load-induced dystrophin disruption with aging in both the EDL and soleus. Bar = 50 µm

 
Aging Alters DGC Protein Content Differently in the Fast-Twitch EDL and Slow-Twitch Soleus
Because alterations in the amount of dystrophin may influence the composition of the DGC, we examined whether aging may affect the amount of membrane-associated DG and SG. Immunoblot analysis using an antibody to a glycosylated epitope showed that the EDL content of {alpha}-DG in the 30- and 36-month animals was ~80.3 ± 9.0% and ~168 ± 9.6% higher, respectively (p <.05) (Figure 4). Relative to 6-month animals, {alpha}-DG content in the 30- and 36-month soleus was ~34.3 ± 2.5% and ~32.1 ± 1.36% higher, respectively (p <.05) (Figure 4). Further, we show a prominent increase in the amount of smaller isoforms of {alpha}-DG expressed in skeletal muscle with aging. These alterations in {alpha}-DG mobility are similar to previous findings examining the regulation of {alpha}-DG expression following muscle denervation, and are thought to be associated with modifications in protein glycosylation (20). Immunohistochemical comparisons between adult and very old EDL and soleus cross-sections revealed that aging was associated with alterations in the amount of {alpha}-DG appropriately targeted to the cell membrane (Figure 4B, Panels B and D). Assuming that there has been no epitope masking, our immunoblotting and immunohistological observations suggest that there is an alteration in the glycosylated epitopes of {alpha}-DG with aging.


Figure 04
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  		Figure 4. A, {alpha}-Dystroglycan ({alpha}-DG) content is increased in the extensor digitorum longus (EDL) and soleus with aging. Levels were determined by immunoblotting. Data are presented as means ± standard error. Insets: Representative blots for {alpha}-DG. * Significantly different from 6-month values (p <.05); n = 5 for all groups. B, Immunolabeling of {alpha}-DG in 6-month (A and C) and 36-month (B and D) EDL (A and B) and soleus (C and D) muscles. Note alterations in membrane {alpha}-DG immunoreactivity in the 36-month EDL and soleus muscles (arrowheads). Bar = 25 µm

 
The muscle content of ß-DG was differentially regulated in the fast-twitch EDL and the slow-twitch soleus with aging. Compared to the 6-month EDL, ß-DG was ~64 ± 2.6% and ~128 ± 5.6% higher in the 30- and 36-month muscles (p <.05) (Figure 5). Conversely, in the soleus, the amount of ß-DG was ~26.6 ± 3.2% lower in 36-month muscles with aging (p <.05). Supporting the immunoblotting findings, immunofluorescence experiments demonstrated that the amount of membrane-associated ß-DG was altered with aging, particularly in the EDL. In contrast to the smooth and well-defined membrane bordering each myofiber in the 6-month EDL, the ß-DG immunoreactivity in 36-month EDL muscles was less uniform and was characterized by punctuated patches of high intensity immunoreactive signal (Figure 5B, Panel B).


Figure 05
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  		Figure 5. A, ß-Dystroglycan (ß-DG) content is increased in the extensor digitorum longus (EDL) but not the soleus with aging. Levels were determined by immunoblotting. Data are presented as means ± standard error. Insets: Representative blots for ß-DG. * Significantly different from 6-month values (p <.05); n = 5 for all groups. B, Immunolabeling of ß-DG in 6-month (A and C) and 36-month (B and D) EDL (A and B) and soleus (C and D) muscles. ß-DG immunoreactive signal is increased and characterized by diffuse membrane reactivity in the 36-month EDL (arrowheads). Bar = 50 µm

 
The muscle content of {alpha}-SG with aging was regulated similarly to that of ß-DG. With aging the EDL content of {alpha}-SG was unchanged in 30-month rats but was found to be ~63.7 ± 9.4% higher in 36-month animals (p <.05) (Figure 6). Conversely, in the soleus {alpha}-SG protein was ~31.1 ± 2.8% and ~15.9 ± 2.6% lower in 30- and 36-month animals, respectively (p <.05) (Figure 6). Consistent with our immunoblotting findings, immunofluorescence experiments showed increased {alpha}-SG membrane immunoreactivity in EDL muscles obtained from 36-month animals. With aging {alpha}-SG appears microscopically as a broad band at the sarcolemma with occasional wispy extensions into the proximal sarcoplasm. Compared to that in 6-month soleus muscles, membrane {alpha}-SG immunofluorescence was lower in the aged soleus (Figure 6B, Panel B).


Figure 06
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  		Figure 6. A, {alpha}-Sarcoglycan ({alpha}-SG) content is increased in the extensor digitorum longus (EDL) but not in the soleus with aging. Levels were determined by immunoblotting. Data are presented as means ± standard error. Insets: Representative blots for {alpha}-SG. * Significantly different from 6-month values (p <.05); n = 5 for all groups. B, Immunolabeling of {alpha}-SG in 6-month (A and C) and 36-month (B and D) EDL (A and B) and soleus (C and D) muscles. In the 36-month EDL muscles, {alpha}-SG membrane immunoreactivity is asymmetrical and jagged in appearance (arrowheads). Bar = 10 µm

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Previous investigations have indicated that aging in the F344/N x BN rat model is associated with decreases in muscle mass, contractile function, a diminished ability of aged animals to respond to a hypertrophic stimulus, and alterations in muscle mechanosensitivity (1). Here, we extend these findings by showing that aging in this model is characterized by alterations in DGC-related protein content and localization. Moreover, we demonstrate that DGC-related alterations with aging are regulated differently between fast- and slow-twitch muscle types.

Aging Alters the Regulation of DGC Proteins Differently in Fast- and Slow-Twitch Muscles
Aging in the EDL is associated with increases in the amount of dystrophin, ß-DG, and {alpha}-SG. Conversely, in the soleus the muscle content of these proteins is diminished with aging (Figures 1, 5, and 6). The exact function of the DGC is not completely determined, but it has been reported to play a role in regulating the structural stability of the muscle cell membrane. In the presence of diminished dystrophin or alterations in DGC composition, it is thought that the muscle membrane is destabilized (21,22). This membrane destabilization, in turn, has been associated with fiber damage, membrane leakage, and muscle atrophy (23). Our findings that aging in the F344/N x BN rat is associated with changes in DGC protein levels (Figures 1, 4–6GoGo), increases in soleus muscle membrane permeability (Figure 2), and increases in EDL and soleus susceptibility to lengthening-induced damage (Figure 3) are consistent with this premise. The factor(s) regulating the expression of DGC proteins with aging are unknown. In adult animals, the DGC appears to be regulated in part by muscle load and innervation status. For example, Chopard and colleagues (24) reported that the muscle content of DGC proteins was increased following 6 weeks of rat hind-limb unweighting. Muscle denervation appears to have a similar effect, demonstrating increases in dystrophin content in both the EDL and soleus (25,26). This latter finding may have particular relevance to aging, as it is generally accepted that fast-twitch muscles tend to transition towards the slow-twitch phenotype through the process of denervation and selective re-innervation by slow motor neurons. As such, it is possible that these increases in EDL DGC protein levels we observe with aging could be due to the EDL undergoing conversion to a slower phenotype. In contrast to what is seen in response to unweighting or denervation, the muscle content of dystrophin, ß-DG, and {alpha}-SG is reduced significantly following tentomy of the rat gastrocnemius (10). Whether age-related changes in the state of muscle loading, innervation status, or activity level are involved in the etiology of age-associated DGC alterations remains unclear. Irrespective of the mechanism, our data are consistent with the notion that aging affects the regulation of dystrophin, ß-DG, and {alpha}-SG differently in fast- and slow-twitch muscle types.

EDL and Soleus Muscle {alpha}-DG Content Is Increased With Aging
DG is encoded by a single gene as a precursor that is cleaved by an as yet unidentified protease into two polypeptides, the extracellular matrix receptor {alpha}-DG and the transmembrane dystrophin-binding protein ß-DG (27). Of these, {alpha}-DG is thought to serve as a link between the basement membrane and the muscle cell surface and ultimately the intracellular cytoskeleton, forming a superstructure that maintains the integrity of the cell surface (28). Unlike dystrophin, {alpha}-SG, or ß-DG, the age-associated regulation of {alpha}-DG is similar between muscle types. In both the EDL and soleus, aging is associated with higher {alpha}-DG levels (Figure 4). These changes in {alpha}-DG appear to be among the first DGC alterations to occur with increases in {alpha}-DG occurring at 30 months in both muscle types. Posttranslational N-linked and extensive O-linked glycosylation cause {alpha}-DG to migrate on sodium dodecyl sulfate–polyacrylamide gel electrophoresis as a broad band (20). We show that the {alpha}-DG band tends to broaden with aging in both the EDL and soleus (Figure 4). These data suggest that the posttranslational processing of {alpha}-DG is altered with aging. Furthermore, it appears that the extent of these alterations in processing is different across muscle types. For example, in the soleus the {alpha}-DG band appears to migrate faster with increasing age, whereas in the EDL, this effect is much lower (Figure 4). To our knowledge, these findings have not been shown before. The physiological significance of these age-associated alterations is unknown; however, it is interesting to note that these changes in {alpha}-DG precede the rapid loss of EDL and soleus muscle mass we observe between the 30- and 36-month rats. The proper glycosylation of {alpha}-DG is required for laminin binding activity (29). Indeed, muscle denervation and a number of muscle disorders (including Fukuyama muscular dystrophy, Walker–Warburg syndrome, Muscle-eye-brain disease, Congenital muscular dystrophy 1C, and limb-girdle muscular dystrophy 2I) are associated with marked alterations in the glycosylation of {alpha}-DG (20,30,31). The improper glycosylation of {alpha}-DG in these disorders is not fully understood; however, there is evidence suggesting that these alterations may be caused by aberrant or missing glycosyltransferase activity (32,33). Whether changes in glycosyltransferase activity or degree of muscle innervation are occurring in the aging F344 x BN skeletal muscle is not clear.

Studies of DG function suggest that the binding of the DC complex to extracellular matrix proteins involves both "outside-in" and "inside-out" signaling (34). It is likely that this signaling plays an important part in regulating muscle viability. For example, inhibition of {alpha}-DG binding to laminin-2 has been found to be associated with diminished phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3ß, and induction of apoptosis in muscle cell cultures (35). Recent studies using the F344 x BN rat have suggested that the load-induced regulation of Akt and glycogen synthase kinase-3ß is altered in 30-month compared to adult animals (36,37). Similarly, Siu and colleagues (38) demonstrated increased muscle apoptosis in muscles from 30- versus 6-month F344 x BN rats. Whether these events are due to age-associated alterations in {alpha}-DG binding to laminin-2 alone or are in concert with other factors awaits further clarification.

Muscle Content of ß-DG and {alpha}-SG Is Regulated Differently in the Soleus and EDL With Aging
ß-DG is a single-pass transmembrane protein that binds with dystrophin to stabilize the cell membrane. We show that the muscle content of ß-DG and {alpha}-SG increases significantly with aging in the EDL, whereas in the soleus the levels of these proteins diminish with increasing age (Figures 5 and 6). This latter finding in particular supports our earlier supposition that soleus muscle membrane integrity is compromised with aging (Figure 2). Although the exact physiological ramifications of these changes are unclear, recent data suggest that, in addition to its structural role, ß-DG may also be involved in cell signaling. We have recently demonstrated that the ability of the EDL and soleus to activate MAPK signaling is altered with aging in the F344/N x BN (3). Specifically, in 36-month animals, EDL stretch failed to induce c-Jun NH2-terminal kinase (JNK)–MAPK phosphorylation, whereas in the soleus, muscle stretch was unable to induce the phosphorylation of p-38 MAPK. Whether alterations in ß-DG expression are directly responsible for these changes remains to be investigated.

The SGs are a family of homologous transmembrane proteins with single membrane spanning domains (39,40). It is thought that the SGs function primarily to stabilize the association of DGs with dystrophin and thus act to reinforce the overall structure of the membrane (41). A deficiency of {alpha}-SG results in the disruption of the entire SG complex and leads to limb-girdle muscle dystrophy type 2D (42,43). Using {alpha}-SG mutant mice, Danieli-Betto and coworkers (44) showed that {alpha}-SG deficiency is associated with fiber degeneration and increased variability of muscle fiber diameter. We and others have demonstrated similar findings of muscle fiber variability in aging muscle and in the aging F344/N x BN soleus (1,45). Patel and coworkers (46), using mice deficient in {alpha}-SG, have suggested that diminished {alpha}-SG is associated with increased muscle stiffness. Previous data have suggested that muscle stiffness increases with aging in rat soleus muscle (47,48). Whether altered {alpha}-SG levels alone or in conjunction with other factors is responsible for such changes is unknown.

Summary
We show that the expression and localization of DGC proteins are altered in the EDL and soleus of aged F344/N x BN rats. The extent to which the combined changes in tissue content and action of DGC participants plays a role in aging skeletal musculature cannot be accurately assessed in the absence of extensive studies to evaluate experimental manipulation of these proteins. Nevertheless, in view of the known role DGC proteins play in the dystrophies, it may be reasonable to speculate that these changes contribute to or are the result of age-associated muscle dysfunction.


   Acknowledgments
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 Abstract
 Materials and Methods
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 Discussion
 References
 
This study was supported by National Institute on Aging grants AG-20370, AG-027103 to E. R. Blough, and by NSF EPSCoR to Marshall University.


   Footnotes
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Decision Editor: Huber R. Warner, PhD

Received January 10, 2006

Accepted April 11, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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