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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:481-489 (2007)
© 2007 The Gerontological Society of America

Age-Related Increase of Insoluble, Phosphorylated Small Heat Shock Proteins in Human Skeletal Muscle

Tetsuo Yamaguchi, Hideaki Arai, Naoki Katayama, Tomoji Ishikawa, Kiyoshi Kikumoto and Yoriko Atomi

1 Department of Life Sciences, The Graduate School of Arts and Sciences, The University of Tokyo, Japan.
2 Department of Orthopedic Surgery, Tokyo Kosei Nenkin Hospital, Japan.
3 Department of Health and Sports, Niigata University of Health and Welfare, Japan.
4 Department of Orthopedic Surgery, Tokyo Metropolitan Matsuzawa Hospital, Japan.

Address correspondence to Yoriko Atomi, PhD, Department of Life Sciences, University of Tokyo, 3-8-1 Meguroku, Tokyo, 153-8902, Japan. E-mail: atomi{at}idaten.c.u-tokyo.ac.jp


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Among mammalian heat shock proteins (Hsps), small Hsps (sHsps) are constitutively expressed in skeletal muscles. We investigated age-related changes of phosphorylation and cellular distribution of representative sHsps (Hsp27 and {alpha}B-crystallin) in human vastus lateralis muscle under resting conditions. We also examined upstream kinases which may be responsible for phosphorylation of sHsps, namely p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase-2, and extracellular signal-regulated kinase-1/2. The study groups consisted of nine young (15–38 years old) and nine aged (51–79 years old) patients who underwent orthopedic surgery. sHsps protein levels were higher in the insoluble fraction of aged muscles. The phosphorylated states of sHsps were enhanced in both the soluble and insoluble fraction of aged patients. The phosphorylated form of each upstream kinase was elevated in aged patients. Ubiquitinated proteins accumulated in the insoluble fractions of aged muscles. Aging mechanisms may affect the activation process of MAPKs, and the phosphorylation and accumulation of sHsps.

THE age-related loss of muscle mass and strength (termed sarcopenia) contributes to a higher risk of accidental falls and also is linked to several chronic afflictions including osteoporosis, arthritis, and insulin resistance in elderly persons (1). One of the characteristics of biological aging is the diminished ability to respond to various forms of stress. In the case of skeletal muscles, this includes decreased induction of heat shock proteins (Hsps) (2), lower activity of mitogen-activated protein kinases (MAPKs) following resistant training (3), and decreased responsiveness of phosphoinositide-3 kinase to insulin (4). The exact molecular mechanisms involved in the age-related changes of muscle are not fully understood. This is a clinically important issue in light of the growing number of older people in the developed world.

Hsps are important components of the cellular protective response against stresses. They are subdivided into several classes according to their molecular mass. Hsps with low molecular masses of 15–30 kd are called small Hsps (sHsps), and they commonly share a homologous sequence of about 80 amino acids called the {alpha}-crystallin domain (5). Among mammalian sHsps, Hsp27 and {alpha}B-crystallin show abundant constitutive expression in both skeletal and heart muscle (6). Importantly, mutation of the {alpha}B-crystallin gene causes a desmin-related myopathy (7). Furthermore, {alpha}B-crystallin shows specific muscle fiber-type expression (8). Most notably, it shows a strong staining pattern in Z-disks in longitudinal sections, indicating that it may have a role in establishing and maintaining muscle architecture (9). Several lines of evidence suggest that sHsps are associated with the cytoskeletal structure and modulate remodeling of the cytoskeletal network (10–15). sHsps also have an anti-apoptotic function in muscle cells (16,17). These findings suggest that sHsps may play key roles in muscle structure and further indicate their importance for muscle function. It was reported that the chaperone function of {alpha}-crystallin declines in aged human lenses (18); in contrast, little is known about age-related changes of sHsps, especially in human skeletal muscle.

sHsps are regulated by phosphorylation of specific serine residues. Phosphorylation of Hsp27 occurs at two sites in the rat (Ser-15 and Ser-85) and the mouse (Ser-15 and Ser-86) and at three sites in humans (Ser-15, Ser-78, and Ser-82). Phosphorylation is reportedly mediated by MAPK-activated protein kinase-2 (MK2) (19), MK3 (20), p38-regulated/activated protein kinase (21), or the {Delta} isoform of protein kinase C (22). Three serine residues in {alpha}B-crystallin (Ser-19, Ser-45, and Ser-59) are phosphorylated by three different protein kinases. Specifically, extracellular signal–regulated kinase-1/2 (ERK1/2), and MK2 targeting Ser-45 and Ser-59, respectively (23). The kinase responsible for phosphorylation of Ser-19 has not yet been identified. In unstressed cells, these sHsps are typically found in the soluble fraction as large oligomers of unphosphorylated monomers. Under conditions of stress, sHsps are phosphorylated and translocate from the soluble to the insoluble fraction of muscle homogenates (6,24,25). Phosphorylated sHsps might play a role in cytoskeletal reorganization by increasing cytoskeletal stability (24). In contrast, phosphorylated {alpha}B-crystallin has an anti-apoptotic function in cardiac muscle cells, but not in skeletal muscle cells (16,17). Phosphorylated sHsps have been implicated in several neurodegenerative states (Alexander's disease, Alzheimer's disease) and in normally aging patients. In those settings, significant levels of {alpha}B-crystallin and its phosphorylated form are observed in the brain, preferentially in insoluble fractions (26). Recent studies have implicated Hsp27 and phosphorylated {alpha}B-crystallin in the ubiquitin-proteasome machinery (27,28). Those findings suggest that sHsps and their phosphorylated forms may participate in degradation of cellular proteins. However, the physiological significance of sHsp phosphorylation remains to be clarified.

MAPK signaling cascades, including p38 MAPK, ERK1/2, and c-Jun NH2 terminal kinase (JNK) are activated in skeletal muscle in response to exercise (3). p38 MAPK is activated during myocyte fusion to form multinucleated myotubes during muscle differentiation (29). The ERK1/2 pathway has a direct effect on muscle cells, activating several myogenic transcription factors (30). In vivo, p38 MAPK activity in skeletal muscle was elevated by procatabolic states such as disuse atrophy, acute quadriplegic atrophy, and type 2 diabetes (6,31,32). In disused rat soleus muscle, phosphorylation of sHsps and MAPKs was observed (6). With regard to age-related changes in human skeletal muscle, MAPK signaling pathways are differentially activated under resting and exercise conditions in skeletal muscle from young and aged men (3). In the present investigation, we tested the hypothesis that sHsp and MAPK signaling pathways are modified in aged human muscle. We assessed changes in phosphorylation and cellular distribution of representative sHsps (Hsp27 and {alpha}B-crystallin) and modifications in phosphorylation of several key members of the MAPK signaling pathway, p38 MAPK, MK2, and ERK1/2.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Patients and Muscle Samples
The study group consisted of 18 patients undergoing scheduled orthopedic surgery. Each of the six age ranges consisted of three patients (age ranges were 15–20, 21–30, 31–40, 51–60, 61–70, and 71–80 years of age). We defined "young" patients as those ranging from 15 to 38 years old (9 patients) and "aged" as those ranging from 51 to 79 years old (9 patients). The physical characteristics of the patients are shown in Table 1. None of the patients had a previous record of muscular disease, arthritis, autoimmune disease, heart disease, cancer, or metabolic disorders. Muscle samples were obtained from the middle portion of the vastus lateralis (VL) muscle at the onset of the surgical procedures (anterior cruciate ligament reconstruction of the knee or hemi-hip arthroplasty for femoral neck fracture). The sampling site was not within the primary surgical area. Muscle biopsies (0.5–1.0 g) were immediately frozen in liquid nitrogen and stored for several days until analysis. The study was approved by the ethics committee of the University of Tokyo and conformed to the standards set by the Declaration of Helsinki (last modified in 2002). After patients had been fully informed of the goal of the experiments and of the risks involved in the procedure, written informed consent was obtained before admission to the study.


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  		Table 1. Physiological Characteristics of Patients.

 
Preparation of Muscle Extracts
All chemicals and reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan) unless otherwise stated. A portion of frozen tissues (80–120 mg) was homogenized by using a glass type homogenizer, with 10 vol (vol/wt) of 80 mM HEPES-NaOH, pH 7.0, containing phosphatase and protease inhibitors (20 mM sodium fluoride, 100 nM okadaic acid, 3 mM sodium orthovanadate, 10 mM sodium ß glycerophosphate, trypsin inhibitor at 10 µg/mL, leupeptin at 2 µg/mL, aprotinin at 2 µg/mL, and 1 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged at 12,500 x g for 10 minutes at 4°C. The supernatant was used as the soluble fraction, and the pellet was washed twice with 300 µL of the above homogenizing buffer. The pellet was then solubilized with 300 µL of 2X Laemmli sample buffer with sonication. The insoluble portion was finally removed by centrifugation.

Immunochemical Reagents
Antibody against Hsp70 (SPA-810) was purchased from Stressgen (Ann Arbor, MI). Antibodies against Hsp27 (sc-1049), Hsc70 (sc-7298), p38 MAPK (sc-535), and phospho-ERK1/2 (sc-7383), which recognize p-Tyr-204, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against multiubiquitin (D071-3) was purchased from Medical Biological Labs (Nagoya, Japan). Antibody against {alpha}B-crystallin was raised in rabbits against C-terminal (SH)KPAVTAAPKK peptides of human {alpha}B-crystallin (33). Antibodies against ERK1/2 (no. 9102), MK2 (no. 3042), phospho-p38 MAPK (no. 4631S), which recognizes p-Thr-180/Tyr-192, and phospho-MK2 (no. 3044), which recognizes p-Thr-222, were purchased from Cell Signaling Technology (Beverly, MA). Phosphorylation site antibodies against Hsp27 (Ser-15, Ser-78, and Ser-82) and {alpha}B-crystallin (Ser-19, Ser-45, and Ser-59) were a generous gift from Drs. Kanefusa Kato and Hidenori Ito (Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Japan) (25).

Electrophoresis and Western Blot Analysis
Protein concentrations in soluble extracts of tissue and in sodium dodecyl sulfate (SDS)–solubilized insoluble fractions were determined with the Bio-Rad protein assay and detergent-compatible (DC) protein assay (Bio-Rad Laboratories, Hercules, CA), respectively, using bovine serum albumin as standard. Aliquots of soluble extracts of tissue (soluble fraction) (5 µg for Hsp27 and {alpha}B-crystallin, 5–15 µg for their phosphorylated forms; 5 µg for Hsp70; 10 µg for Hsc70; 5–10 µg for p38 MAPK, ERK1/2, and MK2, 10–15 µg for their phosphorylated forms) or SDS-solubilized insoluble fraction (15 µg for Hsp27 and {alpha}B-crystallin, 15–30 µg for their phosphorylated forms, 10 µg for Hsp70, 15 µg for Hsc70, 1 µg for multiubiquitin) were mixed with Laemmli sample buffer with ß-mercaptoethanol, and proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE; 10% or 12.5%). First, we determined the optimal amount of proteins for a single lane that is within the linear range with immunoblotting. Next, we loaded the same protein amount of all the samples on a single gel and transferred it to one single nitrocellulose membrane (Hybond enhanced chemiluminescence [ECL]; Amersham, Buckinghamshire, U.K.) using Towbin transfer buffer (34). In addition, we verified the quantification by staining the acrylamide gels with Coomassie Brilliant Blue dye. Therefore, the signals of all the bands in one blot are comparable for each antibody. The membrane was then blocked overnight in phosphate-buffered saline containing 2.5% skim milk, and incubated for 2 hours with the diluted primary antibodies (0.1 µg/mL for antibodies against {alpha}B-crystallin, 0.4 µg/mL for antibodies against Hsp27, 1.0 µg/mL for antibodies against Hsp70, 2.0 µg/mL for antibodies against Hsc70, and 0.5 µg/mL for antibodies to phosphopeptides of Hsp27 and {alpha}B-crystallin) and then for 1 hour with peroxidase-labeled secondary antibodies. The immunoreactive bands were detected on x-ray films by using an ECL kit (Amersham Biosciences, Piscataway, NJ). Developed immunoblots and Coomassie Brilliant Blue–stained gel bands were scanned into a computer using a charge-coupled device (CCD) camera (ATTO Corporation, Tokyo, Japan). Band intensities were quantified using NIH Image.

Semiquantitative Reverse Transcription–Polymerase Chain Reaction
Total RNA was isolated from skeletal muscles of 3 young patients (17, 25, and 32 years old) and 3 aged patients (53, 60, and 78 years old) using RNeasy (QIAGEN, Valencia, CA). After DNase treatment, complementary DNAs were obtained by reverse transcription of 2 µg of total RNA (Ready-to-Go T-primed First-Stranded Kit; Amersham Biosciences). Primers were synthesized according to motif (35): Hsp27 sense, 5'-TCC CTG GAT GTC AAC CAC TT-3' and antisense, 5'-CAA AAG AAC ACA CAG GTG GC-3'; {alpha}B-crystallin sense, 5'-AGC TGG TTT GAC ACT GGA CT-3' and antisense, 5'-GCA ATT CAA GAA AGG GCA TC-3'; ß-actin sense, 5'-AAG ATG ACG CAG ATC ATG TTT GAG-3' and antisense, 5'-AGG AGG AGC AAT GAT CTT GAT CTT-3'. Polymerase chain reaction (PCR) conditions for each gene were optimized so that the numbers of cycles were within the exponential range. Conditions for PCR amplification of Hsp27, {alpha}B-crystallin, and ß-actin complementary DNA were as follows; 27 cycles at 94°C denaturation (30 s), 57°C annealing (30 s), and 72°C extension (30 s). At the beginning of all PCR amplifications, an initial denaturation step at 94°C for 3 minutes was performed. PCR was performed in triplicate. PCR products were analyzed by 1.5% agarose gel electrophoresis. The housekeeping gene ß-actin was used as a control template for normalizing relative changes of Hsp27 and {alpha}B-crystallin messenger RNA (mRNA) in reverse transcription (RT)–PCR.

Statistical Analysis
Data are expressed as means ± standard error of the mean (SEM); whole muscle results were analyzed using Student's t test. The statistical significance was established at p <.05.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Characteristics of Patients
Characteristics of patients are shown in Table 1. Body mass indices were not significantly different between young and aged patients. Body weights and heights were lower in aged patients than in young ones (p <.05).

Protein Levels of Hsps (Hsp27, {alpha}B-Crystallin, Hsp70, and Hsc70)
Following stresses such as heat shock, hypoxia-reoxygenation, or serum starvation, Hsp27 and {alpha}B-crystallin can translocate from the soluble cytosolic fraction to the insoluble nuclear and/or cytoskeletal fraction in various tissues and cells (36). Furthermore, inhibition of proteasomal activity leads to insolubilization of Hsp27 and {alpha}B-crystallin (25,36). To investigate possible changes in the distribution of sHsps with aging, muscle tissue samples from young and aged patients were homogenized, and the soluble cytoplasmic fractions and the insoluble (nuclei, cytoskeleton, myofibrils, membrane, aggregated proteins) (37–39) fractions were subjected to Western blot analysis. The expression levels of Hsp27 and {alpha}B-crystallin in the insoluble fraction of VL muscles were higher in aged patients (Hsp27: 2.8-fold, p <.001; {alpha}B-crystallin: 5.4-fold, p <.01). In contrast, there was no significant difference between young and aged patients in the soluble fraction (Figure 1A and B). To determine whether other Hsps were changed with aging, we examined the expression levels of Hsp70 and Hsc70, well-studied Hsps. Hsp70 is also known to translocate from the soluble fraction to the insoluble fraction following heat shock (40). Protein levels of Hsc70 in both the soluble and insoluble fractions were not significantly different between young and aged patients. The expression levels of Hsp70 in the insoluble fraction of VL muscles were higher in aged patients (1.5-fold, p <.05) (intensity graph not shown), whereas there was no significant difference in the soluble fraction. In both the soluble and insoluble fractions, there were no significant differences in the relative amounts of Hsp70 to Hsc70 between young and aged patients (soluble fraction: 1.1-fold, p =.79; insoluble fraction: 1.4-fold, p =.11) (Figure 1C). These results indicate that the levels of sHsps are enhanced in the insoluble fraction of aged VL muscle, whereas the changes of Hsp70 were less dramatic, indicating that changes associated with aging are more prominent for sHsps than for Hsp70.


Figure 01
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  		Figure 1. Correlation of age and levels of heat shock proteins (Hsps) (Hsp27, {alpha}B-crystallin, Hsp70, and Hsc70). The protein levels of Hsp27 and {alpha}B-crystallin were determined from vastus lateralis muscle samples using Western blot analysis with antibodies against Hsp27 (A), {alpha}B-crystallin (B), or Hsp70 and Hsc70 (C). C, Relative amount of Hsp70 to Hsc70 was calculated. Study groups consisted of 9 young (15–38 years) and 9 aged (51–79 years) patients. Data are expressed as means ± standard error of the mean. *p <.01, **p <.001 for young versus aged patients

 
Levels of mRNAs for Hsp27 and {alpha}B-Crystallin
To determine whether the age-related changes in sHsps levels are attributable to transcriptional modification, we used semiquantitative RT–PCR to measure the levels of mRNAs for Hsp27 and {alpha}B-crystallin in VL muscles. The levels of mRNAs for Hsp27 and {alpha}B-crystallin were not significantly different between young and aged patients (Figure 2). This finding indicates that the changes of protein levels of sHsps with aging are associated with post-transcriptional regulation rather than transcriptional regulation.


Figure 02
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  		Figure 2. Effect of age on levels of messenger RNAs (mRNAs) for heat shock protein 27 (Hsp27) and {alpha}B-crystallin. Study groups consisted of 3 young (17, 25, and 32 years) and 3 aged (53, 60, and 78 years) patients. Total RNA was extracted and reverse-transcribed. Complementary DNA (cDNA) levels of Hsp27 and {alpha}B-crystallin mRNA in vastus lateralis muscle were determined by semiquantitative reverse transcription–polymerase chain reaction and normalized to ß-actin cDNA levels. Data are expressed as means ± standard error of the mean

 
Phosphorylation States of Hsp27 and {alpha}B-Crystallin
sHsps are posttranslationally modified by phosphorylation at several serine residues following stress stimuli such as heat shock (19,23). It is possible that aging also affects the phosphorylation of sHsps in skeletal muscles. To test this hypothesis, we measured the phosphorylation states of sHsps using phosphospecific antibodies against Hsp27 and {alpha}B-crystallin. In soluble fractions, phosphorylation of Hsp27 was significantly enhanced in aged patients compared to young, i.e., 22-fold higher at Ser-15 (p <.01), 6.7-fold higher at Ser-78 (p <.001), and 2.2-fold higher at Ser-82 (p <.01) (Figure 3A–C). Furthermore, phosphorylation of {alpha}B-crystallin was significantly elevated in the soluble fraction in aged patients compared to young patients: Ser-19 (3.3-fold, p <.001), Ser-45 (25-fold, p <.01), and Ser-59 (3.7-fold, p <.01) (Figure 3D–F). In the insoluble fractions from aged patients, we observed enhanced phosphorylation of Hsp27 at Ser-15 (2.6-fold, p <.001), Ser-78 (15-fold, p <.01), and Ser-82 (15-fold, p <.001) (Figure 3A–C). Elevated phosphorylation of insoluble {alpha}B-crystallin was seen at Ser-19 (3.1-fold), Ser-45 (16-fold), and Ser-59 (11-fold) (each, p <.01; Figure 3D–F). These results suggest that phosphorylation of Hsp27 and {alpha}B-crystallin is enhanced in both soluble and insoluble fractions from elderly persons.


Figure 03
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  		Figure 3. Effect of age on phosphorylation states of heat shock protein 27 (Hsp27) and {alpha}B-crystallin. Phosphorylation states of Hsp27 and {alpha}B-crystallin were determined from vastus lateralis muscle samples using Western blot analysis with phosphorylation site antibodies against Ser-15, Ser-78, or Ser-82 peptide in Hsp27 (A–C) and with phosphorylation site antibodies against Ser-19, Ser-45, or Ser-59 peptide in {alpha}B-crystallin (D–F). Study groups consisted of 9 young (15–38 years) and 9 aged (51–79 years) patients. Data are expressed as means ± standard error of the mean. *p <.01, **p <.001 for young versus aged patients

 
The ratio of phosphorylated forms to total protein was higher in the soluble fraction of aged patients for both Hsp27 and {alpha}B-crystallin. Figure 4A shows significant differences for Hsp27 (Ser-15, p <.02; Ser-78, p <.01), and Figure 4B displays differences for {alpha}B-crystallin (Ser-19, p <.01; Ser-45, p <.02; Ser-59, p <.02). Although phosphorylation states of Hsp27 at Ser-82 increased 1.5-fold in aged patients (Figure 4A), this increase did not reach statistical significance (p =.13). We did not calculate these values for the insoluble fraction because the expression level of sHsps in young patients was too low for evaluation (Figure 1A and B; insoluble fraction).


Figure 04
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  		Figure 4. Relative amounts of phosphorylated forms to total protein in the soluble fraction. A, Relative amounts of phosphorylated forms (
Figure 3A–C; each, soluble fraction) to total heat shock protein (Hsp)27 (
Figure 1A; soluble fraction) were calculated. B, Relative amounts of phosphorylated forms (
Figure 3D–F; each, soluble fraction) to total {alpha}B-crystallin protein (
Figure 1B; soluble fraction) were calculated. Data are expressed as means ± standard error of the mean. *p <.02, **p <.01 for young (15–38 years) versus aged (51–79 years) patients

 
Activities of p38 MAPK, MK2, and ERK1/2
Phosphorylation of Ser-15, Ser-78, and Ser-82 in Hsp27 and Ser-59 in {alpha}B-crystallin is catalyzed by MK2, downstream of p38 MAPK (19,23). Thus, we analyzed the phosphorylation states of p38 MAPK and MK2 in extracts of VL muscle. In comparisons of young and aged patients, expression levels of p38 MAPK protein were not significantly different. However, the relative amounts of phosphorylated forms of p38 MAPK were 4.0-fold enhanced in aged patients (p <.01; Figure 5A). Although the expression levels of MK2 protein were lower in aged patients (p <.001; Figure 5B), the relative amounts of phosphorylated forms of MK2 were 16-fold higher in aged patients (p <.01; Figure 5B).


Figure 05
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  		Figure 5. Effect of age on phosphorylation states of p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase-2 (MK-2), and extracellular signal–regulated kinase-1/2 (ERK1/2). Study groups consisted of 9 young (15–38 years) and 9 aged (51–79 years) patients. Phosphorylation states of p38 MAPK, MK2, and ERK1/2 were determined from vastus lateralis muscle samples using Western blot analysis. A, Protein extracts from soluble fractions were analyzed with antibodies against p38 MAPK and its phosphorylated form. Data show intensities relative to intensities of total p38 MAPK. B, Protein extracts from soluble fractions were analyzed with antibodies against MK2 and its phosphorylated form. Data show intensities relative to intensities of total MK2. C, Protein extracts from soluble fractions were analyzed with antibodies against ERK1/2 and its phosphorylated form. Plots show intensities relative to intensities of total ERK1/2. Data are expressed as means ± standard error of the mean. *p <.01, **p <.001 for young versus aged patients

 
Phosphorylation of one of the sites in {alpha}B-crystallin, Ser-45, is mediated by ERK1/2, downstream of MAPK/ERK kinase (23). Therefore, the activation (phosphorylation) states of ERK1/2 in extract of VL muscles were also examined. There was no significant difference in the expression levels of ERK1/2 between young and aged patients. In contrast, phosphorylated forms of ERK1/2 were more abundant in aged patients (4.2-fold, p <.01; Figure 5C). These results indicate that all of the upstream kinases responsible for the phosphorylation of sHsps (that is, p38 MAPK, MK2, and ERK1/2) are phosphorylated (activated) in aged skeletal muscle.

Levels of Polyubiquitinated Proteins
Next, we attempted to elucidate the mechanisms of age-related activation of MAPKs in human skeletal muscle. One of the most prominent features of aging in various tissue and cells is the alteration in proteasome function (39). In aged rat skeletal muscle, there is a decline in the rate of protein turnover as well as in proteasome function (41). Also, the proteasome inhibitor MG-132 activates MAPKs in C2C12 mouse myoblasts (42). It is possible that one of the mechanisms of MAPK activation in aged human skeletal muscle as seen in our study is the result of the proteasome dysfunction. To determine whether proteins accumulate in aged human skeletal muscle, we assessed the levels of polyubiquitinated proteins in extracts of the insoluble fraction from VL muscles. As shown in Figure 6, the levels of polyubiquitinated proteins were 3.3-fold higher in aged patients (p <.001).


Figure 06
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  		Figure 6. Effect of age on levels of polyubiquitinated proteins. Extracts of insoluble fractions were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequent Western blot analysis with antibodies against polyubiquitin. Study groups consisted of 9 young (15–38 years) and 9 aged (51–79 years) patients. Data are expressed as means ± standard error of the mean. *p <.001 for young versus aged patients

 

   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
In this report, we demonstrated that the insoluble fraction of aged muscle contains elevated levels of sHsps. In contrast, no difference was observed in the soluble fraction. We also showed that, in both the soluble and insoluble fractions of VL muscle, phosphorylation of sHsps is enhanced in aged patients. Further, the phosphorylated (activated) forms of p38 MAPK, MK2, and ERK1/2 are elevated in aged patients. These results suggest that age-related changes in the levels of phosphorylated Hsp27 and {alpha}B-crystallin may be attributable to changes in the activities of p38 MAPK, MK2, and ERK1/2. As far as we know, these results are the first to report age-related changes of sHsps and MAPKs under resting conditions in human skeletal muscle.

sHsps and Aging
Our analysis of muscle revealed an age-dependent increase in the levels of Hsp27 and {alpha}B-crystallin proteins in the insoluble fraction, but not in the soluble fraction. Hsp27 and {alpha}B-crystallin have the protective functions in contraction-induced damages in skeletal muscle and in ischemia-induced impairment in heart muscle (24,43). Because sHsps are known to protect the cytoskeleton, increase of sHsps in the insoluble fraction may help to limit cytoskeletal disruption or aid in repair of injured structures in aged skeletal muscle. Aging is associated with an accumulation of proteins modified by oxidation and glycation (18,39). In skeletal muscle cells, the proteasome is the major proteolytic complex responsible for the selective degradation of oxidized proteins (41) and myofibrillar proteins during muscle atrophy (44). Several lines of evidence suggest that proteasome function decreases in skeletal muscles with aging. Moreover, when ubiquitinated, oxidized, and misfolded protein aggregates accumulates, they can inhibit proteasome activity, leading to their enhanced accumulation in the insoluble fraction (18,39,41). We found an accumulation of polyubiquitinated proteins in aged muscle. Hsps are known to function as molecular chaperones to maintain the correct folding of other proteins. To carry out that function, sHsps recognize and bind to misfolded or modified proteins. Because chaperone activity may decrease with aging (18), some sHsps may accumulate as insoluble protein complexes in aged skeletal muscle.

Phosphorylation is the most well-studied posttranslational modification of sHsps. Our results showed that, in both the soluble and insoluble fractions, phosphorylation of both Hsp27 and {alpha}B-crystallin was higher in aged muscles. Clemen and colleagues (45) reported that Hsp27 Ser-82 and Ser-15 as well as {alpha}B-crystallin Ser-59 and Ser-45 are the major serine phosphorylation isoforms in normal and diseased human skeletal muscle. However, their study did not provide details concerning normal muscle tissues of patients. Although mechanisms are uncertain, some aspects of aged muscle may be similar to those of myopathied muscle. Because Hsp27 is a phosphorylation-dependent regulator of actin cytoskeleton (24), it is possible that enhanced phosphorylation of sHsps in aged muscle modifies skeletal muscle structure and/or function. With aging, oxidative stress produces severe disruption of the microfilament cytoskeleton or modification of actin (46), in which case phosphorylated sHsps may reorganize the cytoskeleton in aged muscle cells. A recent study reported that phosphorylated {alpha}B-crystallin interacts with an F-box–containing protein that is a component of the ubiquitin ligase complex (28). Binding of phosphorylated {alpha}B-crystallin stimulated the ubiquitination of insoluble protein. Phosphorylated sHsps may induce the ubiquitination of insoluble proteins such as misfolded and oxidized aggregated protein in aging muscle cells.

MAPKs and Aging
Muscle samples were obtained from patients with fractures and ligament injuries. Although we cannot rule out the possibility that the injury or surgical anesthesia causes some activation of MAPKs, our observations of p38 MAPK and ERK1/2 activation were in agreement with the results of biopsy samples from healthy individuals in resting conditions reported by Williamson and colleagues (3). Their work, focused on the ERK pathway, showed that ERK1/2, p90 ribosomal S6-kinase, and MAPK-interacting kinase 1 were activated in resting elderly men compared to young men. Our focus was mainly on the p38 MAPK–MK2–sHsps pathway, and the data demonstrated activation of p38 MAPK, MK2, and sHsps. Our study demonstrates that phosphorylated forms of p38 MAPK and ERK1/2 are present at higher levels in aged patients. This activation could be attributable to downregulation of MAPK phosphatases (MKPs), to activation of upstream MAPKs, or to both. As for MKPs, Williamson and colleagues (3) reported that MKP1 was increased in aged skeletal muscle. Because the effects of MKPs on MAPKs activity are still controversial (47), their role in skeletal muscle aging remains to be clarified. Aging is associated with increased oxidative damage and/or inflammatory activity. Inflammatory activity increases circulating levels of proinflammatory cytokines, such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1 (48). Oxidative stress or proinflammatory cytokines activate both p38 MAPK and ERK1/2 (3,49,50); these findings support our results. MAPKs are also activated by cell stress induced by proteasomal inhibition. Our finding that polyubiquitinated proteins accumulate in aged muscles may be indicative of proteasomal deficiency. The proteasome inhibitor MG-132 activates MAPKs in U373 MG human glioma cells and C2C12 mouse myoblasts (25,42); these findings could explain the higher level of phosphorylated MAPKs under resting conditions in aged skeletal muscles. Taken together, it appears that aging activates various signaling pathways that control proliferation, differentiation, and stress responses in skeletal muscles.

Our results showed an age-related increase in the activities of MAPKs as well as increased accumulation and phosphorylation of sHsps. Similar observations were made in suspended rat hind limbs (a model of soleus muscle disuse) (6). The biochemical similarities between aged and disused muscle suggests that the underlying mechanisms contributing to aging and atrophy may share common molecular pathways. MAPKs, especially p38 MAPK, were activated in pathological conditions of muscle, such as acute quadriplegic myopathy and type 2 diabetes (31,32). Thus, MAPK activities may play a key role in muscle atrophy. If so, regulating MAPK activities might slow atrophic changes in aging muscle. Additional studies are needed to develop novel therapeutic strategies for treatment of aging skeletal muscle.

Summary
In the present investigation we tested the hypothesis that human muscle undergoes age-dependent modifications in the phosphorylation of sHsps and in the activation of MAPK signaling pathways. Our data documented the accumulation of sHsps as well as the enhanced phosphorylation of sHsps and upstream kinases during muscle aging. These changes in sHsps and signaling pathways may be a response to the aging of muscle or may actually contribute to the underlying mechanism of aging in human skeletal muscle. This information may lead to the development of novel therapeutic strategies for the treatment of aging skeletal muscle.


   Acknowledgments
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 Abstract
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 Results
 Discussion
 References
 
This study was partially supported by the Ministry of Education, Science, Sports and Culture, grant-in-aid for Scientific Research (A no. 17200039), and the Japan Science Society (Sasagawa Scientific Research Grant).

We thank Drs. Kanefusa Kato and Hidenori Ito of the Aichi Human Service Center and Dr. Syunichiro Kubota, University of Tokyo, Japan, for providing antibodies.


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Decision Editor: Huber R. Warner, PhD

Received June 3, 2006

Accepted January 26, 2007


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