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J Appl Physiol 100: 451-456, 2006. First published October 13, 2005; doi:10.1152/japplphysiol.01022.2005
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Regulation of HSP25 expression and phosphorylation in functionally overloaded rat plantaris and soleus muscles

Kimberly A. Huey

Department of Kinesiology, University of Illinois, Urbana, Illinois

Submitted 23 August 2005 ; accepted in final form 6 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Functional overload (FO) is a powerful inducer of muscle hypertrophy and both oxidative and mechanical stress in muscle fibers. Heat shock protein 25 (HSP25) may protect against both of these stressors, and its expression can be regulated by changes in muscle loading and activation. The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation (pHSP25) in hypertrophying rat hindlimb muscle. HSP25 and pHSP25 levels were quantified in soluble and insoluble fractions of the soleus and plantaris to determine whether 3 or 7 days of FO increase translocation of HSP25 and/or pHSP25 to the insoluble fraction. p38 protein and phosphorylation (p-p38) was measured to determine its association with changes in pHSP25. HSP25 mRNA showed time-dependent increases in both the soleus and plantaris with FO. Three or seven days of FO increased HSP25 and pHSP25 in the soluble fraction in both muscles, with a greater response in the plantaris. In the insoluble fraction, HSP25 was increased after 3 or 7 days in both muscles, whereas pHSP25 was only increased in the 7-day plantaris. p38 and p-p38 increased in the plantaris at both time points. In the soleus, p-p38 only increased after 7 days. These results show that FO is associated with changes in HSP25 expression and phosphorylation and suggest its role in the remodeling that occurs during muscle hypertrophy. Increases in HSP25 in the insoluble fraction suggest that it may help to stabilize actin and/or other cytoskeletal proteins during the stress of muscle remodeling.

heat shock protein 25; functional overload

THE UPREGULATION OF HEAT shock proteins (HSP) in skeletal muscle has been shown to provide some protection against a variety of stressors, such as disuse atrophy (17, 22), oxidative stress (5, 22), increased calcium concentrations (28), and muscle damage (12, 13, 16). Furthermore, several observations suggest that the small HSPs, such as HSP25, play crucial roles in the function and integrity of skeletal muscles. HSP25 (homologous to human HSP27) is highly expressed in skeletal muscle, especially those composed primarily of fibers containing slow type I myosin heavy chain (MHC) (9, 11, 18). Importantly, studies have shown that chronic exposure to decreases or increases in muscle loading and activation is accompanied by changes in the expression of HSP25 (9, 11, 25). In other cell types, such as fibroblasts, HSP25 fulfills diverse functions, including chaperoning unfolded proteins, stabilizing the cytoskeleton, conferring resistance to oxidative stress, and inhibiting apoptosis (4). In skeletal muscle, however, its functions are less clear, and the physiological factors that regulate its expression and phosphorylation are poorly understood.

We have previously shown that atrophy of rat hindlimb muscles induced by spinal cord isolation is associated with significant decreases in HSP25 expression (9). In contrast, while functional muscle overload (FO) induces muscle hypertrophy, the associated changes in HSP25 expression are unknown. Furthermore, since FO is a powerful inducer of both oxidative and mechanical stress in muscle fibers, it is of interest to investigate the associated changes in HSP25, which may provide protection against both of these stressors (5, 13, 22). Data regarding the HSP response to chronic skeletal muscle overload are limited and are restricted to the larger HSPs. No studies to date have investigated changes in HSP25 expression in both the predominantly slow soleus and the faster plantaris muscles functionally overloaded by synergist ablation. However, acute increases in muscle loading and stretch are associated with significant increases in HSP25 (24, 26) and a shift from the soluble to the insoluble fraction, where it may potentially limit cytoskeletal disruption or assist in muscle repair (13).

Regulation of HSP25 expression during hypertrophy is likely important; however, the phosphorylation state of HSP25 is potentially critical in modifying its function. HSP25 is phosphorylated by MAPKAPK2, a downstream kinase of p38 MAPK (p38) (23). Immediate increases in p38 phosphorylation (p-p38) were observed in overloaded soleus and plantaris muscles (3); however, it is unknown how p-p38 is impacted by longer periods of overload. Furthermore, the immediate phosphorylation and translocation of HSP25 to cytoskeletal proteins in response to injury suggest its ability to help maintain the integrity of proteins important for muscle structure (13). Taken together, the potential role of HSP25 phosphorylation (pHSP25) in maintaining muscle integrity and p38 activation by overload suggests the importance of pHSP25 during the muscle remodeling that occurs during hypertrophy. However, to date, no studies have investigated changes in pHSP25 during chronic muscle overload.

The primary purpose of the present study was to test the hypothesis that chronic FO increases HSP25 expression and phosphorylation in hypertrophying rat hindlimb muscle. We compared HSP25 mRNA, HSP25 protein, and pHSP25 in the soleus and plantaris, plantar flexors with different MHC profiles and mechanical properties. HSP25 and pHSP25 levels were quantified in both the soluble and insoluble fractions to test the hypothesis that the increased tension imposed on the muscles (and probable muscle damage) increases translocation of HSP25 and/or pHSP25 to the insoluble fraction. In addition, p38 protein and phosphorylation was measured to determine whether changes in pHSP25 were associated with changes in p38 and p-p38.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.   Young, female Sprague-Dawley rats, weighing 110–126 g (initial mean body weight of 118 ± 2 g), were assigned randomly to either a 3-day or 7-day unilateral FO group (n = 10/group for each time point). Rats were anesthetized (10 mg ketamine and/or 20 mg acepromazine/100 g) for all surgical procedures performed under aseptic conditions. Overload of the plantaris and soleus was performed via removal of the synergistic muscle, the gastrocnemius, in one leg only, as described in detail previously (2). The contralateral leg remained intact, and the contralateral plantaris and soleus represented the control (Con). It has been previously reported that unilateral FO does not alter MHC gene expression (6) in the contralateral limb, and within 1 day postsurgery the animals' locomotion and mobility were unaltered by the unilateral FO. In addition, a unilateral- overload animal will voluntarily run on an exercise wheel, on average, as much as the normal subjects, suggesting that the animals are not favoring their intact side (unpublished observations, K. M. Baldwin). Furthermore, to verify that HSP25 content and phosphorylation were not altered in the contralateral leg, we found no significant differences in HSP25 protein or phosphorylation between the contralateral muscles and muscles from a group of age-matched Con rats (n = 6). Values in the contralateral soleus were normalized to Con (1.0) and were 1.02 ± 0.05 and 0.99 ± 0.04 for HSP25 and pHSP25, respectively. Normalized values in the contralateral plantaris were 1.04 ± 0.04 and 1.04 ± 0.05 for HSP25 and pHSP25, respectively. Furthermore, in preliminary studies, we compared HSP25 expression and phosphorylation between Con and sham-operated animals (n = 4) and found no differences in either the soleus or plantaris values. Values in the sham-operated soleus were normalized to Con (1.0) and were 1.01 ± 0.02 and 1.04 ± 0.05 for HSP25 and pHSP25, respectively. Normalized values in the contralateral plantaris were 0.98 ± 0.04 and 1.03 ± 0.03 for HSP25 and pHSP25, respectively. We chose the unilateral model, since this design allows each animal to serve as its own control, having both non-FO and FO muscles, since several systemic inflammatory cytokines, such as TNF-{alpha} (19, 20), have been shown to phosphorylate HSP25 in vitro. After 3 or 7 days, the animals were euthanized, and the plantaris and soleus muscles were removed quickly, trimmed of excess fat and connective tissue, wet weighed, frozen in liquid nitrogen, and stored at –80°C until subsequent analysis. This study was approved by the Animal Use Committee at the University of Illinois, Urbana-Champaign, and followed the American Physiological Society Animal Care Guidelines.

mRNA analyses.   Muscle samples (50–60 mg) were homogenized in 1 ml of TRI Reagent (Molecular Research Center, Cincinnati, OH), and RNA was extracted according to the manufacturer's protocol. RNA concentration was determined with a spectrophotometer and stored at –80°C for subsequent RT-PCR.

Relative RT-PCR was used to determine the expression of HSP25 mRNA relative to an 18S internal standard (Ambion, Austin, TX). One microgram of total RNA from each muscle sample was reverse-transcribed using Stratascript first-strand synthesis system (Stratagene, La Jolla, CA), according to the manufacturer's protocol.

One microliter of each RT reaction was amplified with PCR in a 25-µl reaction volume containing 1x PCR buffer, 0.2 mM dNTP, 100 nM HSP25 primers, 0.5 µM 18S primer-competimer mix, and 0.75 unit of DNA Taq polymerase (Invitrogen, Carlsbad, CA). Amplification parameters were as follows: a denaturing step at 95°C for 3 min, followed by 30 s at 95°C, 1 min at 60°C, 30 s at 72°C for 25 cycles, and a final elongation step for 5 min at 72°C. The HSP25 primer sequences were as follows: forward 5'-AGC CAT GTT CGT CCT GCC TTT CTT-3', reverse 5'-AGC GCC GCG TGC CCT TCT C-3' (Qiagen, Valencia, CA) and yielded a 388-bp fragment. In each PCR reaction, the 18S ribosomal RNA was coamplified with the target cDNA to serve as an internal standard and to allow correction for any differences in starting amounts of total RNA. Alternate 18S internal standards (Ambion) were used to yield a 324-bp product. 18S primers were mixed with competimers at an optimized ratio (1:3) that was experimentally determined based on the abundance of the target mRNA. Inclusion of 18S competimers was necessary to attenuate the 18S signal, allowing linear amplification in the same range as the coamplified target mRNA (relative RT-PCR kit protocol; Ambion).

PCR products were separated on 2% agarose gels, stained with ethidium bromide, and photographed under UV light. Band density was determined with ImageQuant software, and each HSP25 mRNA band was normalized to the corresponding 18S band. PCR conditions (cDNA dilutions, 18S competimer-primer mix, MgCl2 concentration, and annealing temperature) were set to optimal conditions so that both the target mRNA and 18S product yields were in the linear range of the semilog plot when the yield is expressed as a function of the number of cycles.

Protein analyses.   Plantaris or soleus (n = 10/time point) muscles from 3-day or 7-day FO animals were homogenized in 10 volumes of an ice-cold buffer containing 50 mM Tris·HCl (pH 7.8), 2 mM potassium phosphate, 2 mM EDTA, 2mM EGTA, 10% glycerol, 1% Triton X-100, 1 mM dithiothreitol, 3 mM benzamidine, 1 mM sodium orthovanadate, 10 mM leupeptin, 5 mg/ml aprotinin, and 1 mM 4-[(2-aminoethyl) benzenesulfonyl fluoride] using a motor-driven glass pestle. The homogenate was immediately centrifuged at 12,000 g for 20 min at 4°C. The supernate was removed as the detergent-soluble fraction. The detergent-insoluble fraction was obtained by resuspending the pellet in 10 mM Tris (pH 6.8), 2% SDS, and 2.5% beta-mercaptoethanol and homogenizing with a motor-driven glass pestle. The remaining insoluble fraction was removed by centrifugation. Protein concentrations were determined by using the Bio-Rad protein assay with BSA used for the standard curve. The samples were immediately saved in aliquots at –80°C for subsequent use in immunoblotting.

Western blot analysis was used to determine HSP25, pHSP25, p38, and p-p38 protein levels in Con, 3-day FO, and 7-day FO soleus and plantaris muscles. Thirty micrograms of detergent-soluble or insoluble proteins were boiled (5 min at 95°C) and separated by one-dimensional SDS-PAGE (15%) electrophoresis and transferred to nitrocellulose membranes (pore size: 45 µm). After protein transfer, the membranes were blocked for 1 h in Tris-buffered saline (TBS)-5% milk. Following blocking, the membranes were incubated overnight at 4°C with an anti-HSP25 (1:5,000), anti-pHSP25 (Ser82) (1:1,000) antibody, anti-p38 (1:1,000), or anti-p-p38 (1:1,000) in TBS-2.5% BSA. The HSP antibodies were purchased from Stressgen (Victoria, BC), and the p38 antibodies from Cell Signaling (Beverly, MA). Blots were washed three times in TBS-0.1% Tween and incubated with anti-rabbit secondary antibodies (Amersham Biosciences, Piscataway, NJ) at 1:5,000 for 1 h at room temperature. Blots then were washed in TBS-0.1% Tween, and the bound antibodies were detected with enhanced chemiluminescence (Amersham Biosciences).

Quantification of the bands was performed by using ImageQuant Analysis software. All of the necessary controls for Western blots were performed to ensure antibody specificity. To ensure uniformity, only bands within the same blot were used to compare the relative amounts of each protein among groups. Consequently, Con, 3-day, and 7-day samples were run on every blot. The intensity of phosphorylated bands for either HSP25 or p38 was first normalized to the intensity of the corresponding non-phosphorylated bands, and the ratio was normalized to the contralateral Con.

Statistical analyses.   All data are presented as means ± SE. Differences in the mRNA or protein response in either the soleus or plantaris to 3 or 7 days of FO compared with the contralateral Con were determined by using a one-way ANOVA. If significant differences were found, the Bonferroni post hoc test was used to determine the source of the difference. All analyses were performed with Graphpad Prism 4.0. Significance level was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Changes in body and muscle weights.   The initial mean body weights for both groups was 118 ± 2 g and increased to 128 ± 1 and 143 ± 2 g, respectively, in the 3- and 7-day FO groups. Mean absolute plantaris weights after 3 and 7 days of FO were 138 and 210% of Con values, respectively (Table 1). Mean absolute soleus weights after 3 and 7 days of FO were 141 and 176% of Con values, respectively. The mean plantaris weights expressed relative to body weight were 137 and 210% of Con values after 3 and 7 days of FO, respectively. The mean soleus weights expressed relative to body weight were 143 and 180% of Con values after 3 and 7 days of FO, respectively.


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  		Table 1. Absolute and relative (to body weight) muscle weights in 3-day control, 7-day control, 3-day functional overload, and 7-day functional overload groups

 
Changes in HSP25 mRNA expression and protein content.   HSP25 mRNA expression relative to 18S showed significant time-dependent increases in both the soleus and plantaris with FO (Fig. 1). In the soleus, HSP25 expression increased 1.4- and 2.0-fold over control values after 3 and 7 days of FO, respectively. Similarly, in the plantaris, HSP25 mRNA levels increased 1.5- and 1.9-fold over Con after 3 and 7 days, respectively. These increases in mRNA were accompanied by significant increases at the protein level in both the soleus and plantaris (Figs. 2 and 3). However, despite similar increases in HSP25 mRNA relative to Con values, FO of the plantaris resulted in a greater increase relative to Con values in soluble HSP25 protein compared with the soleus (Fig. 2A). In the soluble fraction, FO of the plantaris resulted in 2.7- and 2.4-fold increases in HSP25 protein after 3 and 7 days, respectively. In the soleus, FO resulted in 1.4- and 1.5-fold increases after 3 and 7 days, respectively. In both the plantaris and soleus, relative HSP25 protein content did not significantly increase between 3 and 7 days of FO.


Figure 1
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  		Fig. 1. Average values for heat shock protein 25 (HSP25) mRNA in the control (Con), 3-day (3d) functional muscle overload (FO), and 7-day (7d) FO plantaris (plt) and soleus. Values are means ± SE. OD, optical density. Significantly different from *Con and {dagger}3d at P < 0.05.

 

Figure 2
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  		Fig. 2. Time course of the average relative changes (% of Con) in HSP25 protein (A) and phosphorylated HSP25 (pHSP25; B) in the soluble fraction in the plantaris and soleus muscles after 3d or 7d of FO. In both graphs, the Con value is set at 100, as indicated by the solid line. Values are means ± SE. Representative Western blots from Con, 3d FO, and 7d FO soleus muscles are shown. The intensity of phosphorylated bands was first normalized to the intensity of the corresponding nonphosphorylated bands, and the ratio was normalized to the contralateral Con. Significantly different from *Con and {dagger}3d at P < 0.05.

 

Figure 3
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  		Fig. 3. Time course of the average relative changes (% of Con) in HSP25 protein (A) and pHSP25 (B) in the insoluble fraction in the plantaris and soleus muscles after 3d or 7d of FO. In both graphs, the Con value is set at 100, as indicated by the solid line. Values are means ± SE. Representative Western blots from Con, 3d FO, and 7d FO soleus muscles are shown. The intensity of phosphorylated bands was first normalized to the intensity of the corresponding nonphosphorylated bands, and the ratio was normalized to the contralateral Con. Significantly different from *Con and {dagger}3d at P < 0.05.

 
In the insoluble fraction, HSP25 protein levels were dramatically increased relative to Con in both the plantaris and soleus (Fig. 3A). In the plantaris, there was a time-dependent increase in HSP25 with significantly greater HSP25 protein after 7 days compared with 3 days of FO (3.0- and 4.2-fold increases, respectively). In contrast, in the soleus, 5.0- and 4.8-fold increases in HSP25 were observed after both 3 and 7 days, respectively.

Changes in phosphorylation HSP25 state.   In the soluble fraction, pHSP25 was significantly increased after 3 and 7 days in both the plantaris and soleus (Fig. 2B). In the plantaris, pHSP25 levels increased 3.1-fold over Con after 3 days of FO and remained elevated after 7 days. In the soleus, pHSP25 increased 1.5-fold over Con after 3 days of FO and continued to significantly increase between 3 and 7 days to a level 1.9-fold greater than Con values.

Despite the significant increase in HSP25 protein in the insoluble fraction in both muscles after either 3 or 7 days, the phosphorylation state of HSP25 was either unchanged or less than Con, with the exception of the 7-day plantaris (Fig. 3B). In the plantaris, pHSP25 levels were significantly lower than Con after 3 days, but after 7 days pHSP25 was significantly increased 2.0-fold over Con values. In the soleus, pHSP25 levels were unchanged from Con values after 3 days before significantly decreasing to 69% of Con and 3-day levels.

Changes in p38 protein and phosphorylation state.   FO of the plantaris was associated with significant increases in p38 protein expression and phosphorylation after both 3 and 7 days of FO relative to Con values (Fig. 4). Furthermore, after 7 days, the p-p38 levels in the plantaris were significantly greater than both Con and 3-day values (Fig. 4B).


Figure 4
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  		Fig. 4. Time course of the average relative changes (% of Con) in p38 protein (A) and phosphorylated p38 (p-p38; B) in the soluble fraction in the plantaris and soleus muscles after 3d or 7d of FO. In both graphs, the Con value is set at 100, as indicated by the solid line. Values are means ± SE. Representative Western blots from Con, 3d FO, and 7d FO plantaris muscles are shown. The intensity of phosphorylated bands was first normalized to the intensity of the corresponding nonphosphorylated bands, and the ratio was normalized to the contralateral Con. Significantly different from *control and {dagger}3d at P < 0.05.

 
In the soleus, p38 protein levels were not significantly different from Con after either 3 or 7 days of FO (Fig. 4A). Phosphorylation of p38 was unchanged after 3 days before increasing 7.3-fold over Con levels after 7 days (Fig. 4B).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results provide the first evidence that chronic muscle overload modulates both the time-dependent expression and phosphorylation state of HSP25 during hypertrophy of two rat plantar flexors with differing fiber-type and contractile profiles.

Changes in HSP25 mRNA levels.   The increases in HSP25 mRNA suggest that increases in gene transcription contribute to the observed elevations in HSP25 protein in both the plantaris and soleus. We observed 139 and 152% increases in HSP25 mRNA relative to Con in the soleus and plantaris, respectively, after 3 days of FO. These results are consistent with previous findings in the human biceps brachii in which one bout of high-force eccentric contractions was associated with a 135% increase in HSP25 mRNA 48 h postexercise (25). With continued FO, there was a significant increase in HSP25 mRNA between 3 and 7 days in the soleus or plantaris; however, no corresponding changes were observed at the protein level. One possibility is that, during this time, the increases in major muscle proteins (e.g., contractile, cytoskeletal) necessary for the significant muscle hypertrophy required near maximal utilization of the muscle's protein synthetic machinery.

Changes in HSP25 protein and phosphorylation state.   The significant increases in HSP25 in the primarily slow soleus and the primarily fast plantaris further support the role of HSP25 in muscle adaptation during either chronic increases or decreases in muscle activation and loading. In addition, these results extend previous work demonstrating increases in HSP25 in response to acute muscle overload induced by a single bout of high-intensity resistance training in humans (24–26). Although HSP25 significantly increased in both muscles, the response in both the soluble and insoluble fractions was muscle specific. In the soluble fraction, the increases in HSP25 were significantly greater in the plantaris compared with the soleus, despite similar relative hypertrophic responses. One potential reason for this observed difference is the higher basal expression level of HSP25 in muscles composed of predominantly slow MHC compared with muscles with predominantly fast MHC profiles (9, 11, 18). Thus the more robust response in the plantaris may be mediated in part by the lower values in unstressed muscles. Furthermore, since HSP25 may provide protection against increased oxidative and mechanical stress, the greater response in the plantaris may be indicative of the greater relative stress imposed on the plantaris compared with the soleus, which is a highly oxidative antigravity muscle normally adapted to tonic activation.

Interestingly, the relative increase in HSP25 in the insoluble fraction was greater than the response in the soluble fraction, particularly in the soleus. These differences may be explained in part by the lower relative levels of HSP25 in the insoluble fraction in unstressed muscle cells, as observed in the present study and previous work (13). Thus the exposure to chronic stress imposed by FO is associated with a relatively greater response compared with Con levels. The increase in HSP25 in the insoluble fraction may be due to several factors, such as muscle damage or accumulation of damaged proteins. For example, acute lengthening contractions of the rat extensor digitorum longus were associated with an immediate translocation of HSP25 from the soluble to insoluble fractions (13). In addition, the accumulation of insoluble damaged protein complexes induced by proteosome inhibition is associated with translocation of HSP25 to the insoluble fraction in cardiac myoblasts (27). Thus the relatively greater response in the soleus compared with the plantaris after 3 days of FO may suggest increased muscle damage and/or protein turnover in the soleus during the initial muscle remodeling process.

This is the first study to investigate the phosphorylation state of HSP25 in chronically overloaded rat slow and fast hindlimb muscles. The importance of pHSP25 in maintaining the integrity of nonmuscle cells during periods of stress has been demonstrated by phosphorylation-dependent stabilization of the actin cytoskeleton (7, 10, 14, 15, 21). In muscle cells, however, it remains unclear if phosphorylation-dependent actin or cytoskeletal protein stabilization occurs within the contractile apparatus. While high-force eccentric contractions increase HSP25 translocation to regions of greatest muscle damage (13), the role of phosphorylation and subsequent functions of HSP25 are poorly understood. From a functional standpoint, the substantial increase in total pHSP25 in the insoluble fraction that we observed could contribute to muscle remodeling during hypertrophy. However, the corresponding increase in nonphosphorylated HSP25 resulted in no significant change in the ratio of phosphorylated to nonphosphorylated HSP25, with the exception of day 7 in the plantaris. Clearly, future studies are necessary to establish the role of pHSP25 during muscle remodeling.

Changes in p38 expression and phosphorylation state.   Previously, Carlson et al. (3) reported that p-p38 peaked after 1 h of FO and remained significantly elevated until 24 h of FO in both the soleus and plantaris. With continued FO, we found that p-p38 remained significantly elevated relative to Con in the plantaris after 3 days and continued to increase up to 7 days of FO. In the soleus, however, p-p38 levels returned to Con levels after 3 days before significantly increasing after 7 days of FO.

Because the phosphorylation of p38 may occur in response to several growth factors (29), the dramatic increase in p-p38 between 3 and 7 days is likely associated with activation of pathways contributing to muscle hypertrophy. The increased muscle weights after 3 days were likely due to edema and inflammation (1). However, with continued FO, inflammation is reduced and muscle hypertrophy is stimulated by the activation of growth-promoting pathways. In the plantaris, this increase in p-p38 was associated with a significant increase in pHSP25 between 3 and 7 days in the insoluble, but not soluble, fractions (Figs. 2B and 3B). In contrast, between 3 and 7 days in the soleus, the dramatic increase in p-p38 was associated with a significant increase in pHSP25 in the soluble, but not the insoluble, fractions. The variable associations between p-p38 and pHSP25 between fractions and muscles could be due to p-p38-dependent phosphorylation of other cellular targets besides MAPKAPK2. For example, in cardiac muscle cells, p-p38 activation of myocyte enhancer factor-2 may contribute to hypertrophy (8).

Summary and conclusions.   We have presented novel results demonstrating changes in HSP25 expression and phosphorylation during the initial week of overload of both the soleus and plantaris muscles. Significant increases in both the expression and phosphorylation of HSP25 suggest its role in the muscle remodeling that occurs during muscle hypertrophy. Furthermore, these data extend our previous work showing modulation of HSP25 expression and phosphorylation during chronic muscle disuse and suggest that activation and/or loading levels are critical factors in determining the phosphorylation levels of HSP25 in skeletal muscle.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant RO3 AR-049855 to K. A. Huey.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. A. Huey, Dept. of Kinesiology, Univ. of Illinois, Urbana-Champaign, 120 Freer Hall, 906 S. Goodwin Ave., Urbana, IL 61801 (e-mail: khuey{at}uiuc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Armstrong RB, Marum P, Tullson P, and Saubert CW. Acute hypertrophic response of skeletal muscle to removal of synergists. J Appl Physiol 46: 835–842, 1979.[Abstract/Free Full Text]
  2. Baldwin KM, Valdez V, Herrick RE, MacIntosh AM, and Roy RR. Biochemical properties of overloaded fast-twitch skeletal muscle. J Appl Physiol 52: 467–472, 1982.[Abstract/Free Full Text]
  3. Carlson CJ, Fan Z, Gordon SE, and Booth FW. Time course of the MAPK and PI3-kinase response within 24 h of skeletal muscle overload. J Appl Physiol 91: 2079–2087, 2001.[Abstract/Free Full Text]
  4. Concannon CG, Gorman AM, and Samali A. On the role of Hsp27 in regulating apoptosis. Apoptosis 8: 61–70, 2003.[CrossRef][Web of Science][Medline]
  5. Escobedo J, Pucci AM, and Koh TJ. HSP25 protects skeletal muscle cells against oxidative stress. Free Radic Biol Med 37: 1455–1462, 2004.[CrossRef][Web of Science][Medline]
  6. Giger JM, Haddad F, Qin AX, and Baldwin KM. Functional overload increases beta-MHC promoter activity in rodent fast muscle via the proximal MCAT (betae3) site. Am J Physiol Cell Physiol 282: C518–C527, 2002.[Abstract/Free Full Text]
  7. Guay J, Lambert H, Gingras-Breton G, Lavoie JN, Huot J, and Landry J. Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27. J Cell Sci 110: 357–368, 1997.[Abstract]
  8. Han J and Molkentin JD. Regulation of MEF2 by p38 MAPK and its implication in cardiomyocyte biology. Trends Cardiovasc Med 10: 19–22, 2000.[CrossRef][Web of Science][Medline]
  9. Huey KA, Thresher JS, Brophy CM, and Roy RR. Inactivity-induced modulation of Hsp20 and Hsp25 content in rat hindlimb muscles. Muscle Nerve 30: 95–101, 2004.[CrossRef][Web of Science][Medline]
  10. Huot J, Houle F, Marceau F, and Landry J. Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80: 383–392, 1997.[Abstract/Free Full Text]
  11. Inaguma Y, Goto S, Shinohara H, Hasegawa K, Ohshima K, and Kato K. Physiological and pathological changes in levels of the two small stress proteins, HSP27 and alpha B crystallin, in rat hindlimb muscles. J Biochem (Tokyo) 114: 378–384, 1993.[Abstract/Free Full Text]
  12. Koh TJ. Do small heat shock proteins protect skeletal muscle from injury? Exerc Sport Sci Rev 30: 117–121, 2002.[CrossRef][Web of Science][Medline]
  13. Koh TJ and Escobedo J. Cytoskeletal disruption and small heat shock protein translocation immediately after lengthening contractions. Am J Physiol Cell Physiol 286: C713–C722, 2004.[Abstract/Free Full Text]
  14. Landry J and Huot J. Modulation of actin dynamics during stress and physiological stimulation by a signaling pathway involving p38 MAP kinase and heat-shock protein 27. Biochem Cell Biol 73: 703–707, 1995.[Web of Science][Medline]
  15. Lavoie JN, Gingras-Breton G, Tanguay RM, and Landry J. Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock. HSP27 stabilization of the microfilament organization. J Biol Chem 268: 3420–3429, 1993.[Abstract/Free Full Text]
  16. Liu Y and Steinacker JM. Changes in skeletal muscle heat shock proteins: pathological significance. Front Biosci 6: D12–D25, 2001.[Web of Science][Medline]
  17. Naito H, Powers SK, Demirel HA, Sugiura T, Dodd SL, and Aoki J. Heat stress attenuates skeletal muscle atrophy in hindlimb-unweighted rats. J Appl Physiol 88: 359–363, 2000.[Abstract/Free Full Text]
  18. Neufer PD and Benjamin IJ. Differential expression of B-crystallin and Hsp27 in skeletal muscle during continuous contractile activity. Relationship to myogenic regulatory factors. J Biol Chem 271: 24089–24095, 1996.[Abstract/Free Full Text]
  19. Park KJ, Gaynor RB, and Kwak YT. Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation. J Biol Chem 278: 35272–35278, 2003.[Abstract/Free Full Text]
  20. Rogalla T, Ehrnsperger M, Preville X, Kotlyarov A, Lutsch G, Ducasse C, Paul C, Wieske M, Arrigo AP, Buchner J, and Gaestel M. Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation. J Biol Chem 274: 18947–18956, 1999.[Abstract/Free Full Text]
  21. Schneider GB, Hamano H, and Cooper LF. In vivo evaluation of hsp27 as an inhibitor of actin polymerization: hsp27 limits actin stress fiber and focal adhesion formation after heat shock. J Cell Physiol 177: 575–584, 1998.[CrossRef][Web of Science][Medline]
  22. Selsby JT and Dodd SL. Heat treatment reduces oxidative stress and protects muscle mass during immobilization. Am J Physiol Regul Integr Comp Physiol 289: R134–R139, 2005.[Abstract/Free Full Text]
  23. Stokoe D, Engel K, Campbell DG, Cohen P, and Gaestel M. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett 313: 307–313, 1992.[CrossRef][Web of Science][Medline]
  24. Thompson HS, Clarkson PM, and Scordilis SP. The repeated bout effect and heat shock proteins: intramuscular HSP27 and HSP70 expression following two bouts of eccentric exercise in humans. Acta Physiol Scand 174: 47–56, 2002.[CrossRef][Web of Science][Medline]
  25. Thompson HS, Maynard EB, Morales ER, and Scordilis SP. Exercise-induced HSP27, HSP70 and MAPK responses in human skeletal muscle. Acta Physiol Scand 178: 61–72, 2003.[CrossRef][Web of Science][Medline]
  26. Thompson HS, Scordilis SP, Clarkson PM, and Lohrer WA. A single bout of eccentric exercise increases HSP27 and HSC/HSP70 in human skeletal muscle. Acta Physiol Scand 171: 187–193, 2001.[CrossRef][Web of Science][Medline]
  27. Verschuure P, Croes Y, van den IPR, Quinlan RA, de Jong WW, and Boelens WC. Translocation of small heat shock proteins to the actin cytoskeleton upon proteasomal inhibition. J Mol Cell Cardiol 34: 117–128, 2002.[CrossRef][Web of Science][Medline]
  28. Welch WJ. Mammalian stress response: cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol Rev 72: 1063–1081, 1992.[Free Full Text]
  29. Widmann C, Gibson S, Jarpe MB, and Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 79: 143–180, 1999.[Abstract/Free Full Text]



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