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J Appl Physiol 100: 981-987, 2006. First published November 17, 2005; doi:10.1152/japplphysiol.00831.2005
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Slower skeletal muscle phenotypes are critical for constitutive expression of Hsp70 in overloaded rat plantaris muscle

David E. T. O'Neill,1 F. Kris Aubrey,1 David A. Zeldin,1 Robin N. Michel,1,3 and Earl G. Noble1,2

1School of Kinesiology, Faculty of Health Sciences, 2Lawson Health Research Institute, The University of Western Ontario, London, Ontario; and 3Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada

Submitted 12 July 2005 ; accepted in final form 16 November 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Heat shock protein 72 (Hsp70) is constitutively expressed in rat hindlimb muscles, reportedly in proportion to their content of type I myosin heavy chain. This distribution pattern has been suggested to result from the higher recruitment and activity of such muscles and/or a specific relationship between myosin phenotype and Hsp70 content. To differentiate between these possibilities, the fiber-specific distribution of Hsp70 was examined in male Sprague-Dawley rat plantaris under control conditions, following a fast-to-slow phenotypic shift in response to surgically induced overload (O) and in response to O when the phenotypic shift was prevented by 3,5,3'-triiodo-DL-thyronine administration. Constitutive expression of Hsp70 was restricted to type I and IIa fibers in plantaris from control rats, and this fiber-specific pattern of expression was maintained following O of up to 28 days, although Hsp70 content in the O muscle doubled. When O (for 40 days) of the plantaris was combined with 3,5,3'-triiodo-DL-thyronine administration, despite typical hypertrophy in the overloaded plantaris, prevention of the normal phenotypic transformation also blocked the increased expression of Hsp70 observed in euthyroid controls. Collectively, these data suggest that chronic changes in constitutive expression of Hsp70 with altered contractile activity appear critically dependent on fast-to-slow phenotypic remodeling.

compensatory overload; heat shock proteins; fiber types; thyroid hormone; fiber transformation

HEAT SHOCK OR STRESS PROTEINS (HSPs) are a set of proteins that are rapidly and preferentially synthesized by most organisms in response to protein-damaging stresses. They are also constitutively expressed in unstressed cells where they aid protein folding, facilitate intracellular trafficking, and may be involved in signaling processes (2, 6, 29). Although normally observed in low quantity in unstressed cells, the inducible isoform of the Hsp70 family is constitutively expressed at higher concentrations in the "slow" [e.g., high levels of slow myosin heavy chain (MHC) I] soleus muscle of the rat (12, 13, 22). In contrast, the fast rat plantaris (PLT) muscle displays lower levels of slow contractile proteins and Hsp70 (12, 13, 22). The bases for these muscle-specific differences in Hsp70 expression are not readily apparent. It has been postulated (13, 21, 23) that since muscles exhibiting high levels of MHC I tend to be more frequently recruited for ambulation and postural maintenance, it may be the constant stress of muscle loading that accounts for the elevated constitutive expression of Hsp70 in these tissues. A number of stressors associated with frequent contractile activity (including increased muscle fiber damage, protein turnover, oxidative stress, and temperature) could result in the induction of Hsp70 in muscle (19).

It is well established that muscles adapt to ongoing recruitment with a remodeling process, which results in the transcription and translation of isoforms of myofibrillar proteins metabolically suited for the imposed contractile demands (3). For instance, fast-to-slow type remodeling occurs following the increased contractile activity associated with compensatory overload, as evidenced by the increase in relative expression of MHC I (12, 27). This observation is noteworthy since expression of Hsp70 not only tends to parallel slower, more oxidative fiber-type gene expression in unstressed muscles (13, 22) but also during changes in expression of this gene program as a consequence of compensatory overload (12, 21, 23), clenbuterol-induced hypertrophy (24), or altered thyroid hormone status (12). More recently, it has been observed (22, 25) that the sudden and sustained increase of contractile activity in the developing rat is not accompanied by an immediate increase in Hsp70. Rather, increases in Hsp70 were correlated with developmentally regulated changes in slow phenotype expression. Hence, constitutive Hsp70 expression could be more closely aligned with fiber phenotype than fiber recruitment and loading.

To address these possibilities, we sought to 1) characterize the fiber type-specific expression of Hsp70 during the phenotypic transformation to a sustained increase in the frequency of contractile activity and 2) determine whether contractile activity or phenotypic remodeling is responsible for elevated Hsp70 content following this adaptation. It was hypothesized that Hsp70 content would only be altered in concert with changes in the slower muscle phenotype as exemplified by altered levels of MHC I.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Experimental outline.   All procedures involving animals in the following experiments were approved by The University of Western Ontario Animal Care Committee and were performed in accordance with the guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats (250 g; Charles River Laboratories) were used in these experiments, and anesthesia was induced with pentobarbital sodium at 65 mg/kg ip, unless otherwise indicated. Temperature was measured using a Yellow Springs Instruments telethermometer inserted 5 cm into the animal’s rectum. At euthanasia, tissues were rapidly harvested and immediately frozen in liquid nitrogen and stored at –80°C for subsequent analyses. Blood was also collected for determination of serum 3,5,3'-triiodo-DL-thyronine concentration using a commercially available kit (Diagnostic Products RIA kit).

Compensatory overload.   Rat PLT muscle was subjected to compensatory overload following removal of the gastrocnemius and soleus of each hindlimb (8). Animals were randomly assigned to control and overload groups. Overload muscles were harvested at 3 days and 1, 2, and 4 wk after synergistic ablation. These times were chosen so as to follow the time course of fiber-specific Hsp70 expression while separating the potential short-term effects of muscle damage, inflammation, and rapid growth (1, 9) from that observed at 4 wk, when a stable muscle phenotype has been established (9). Shorter term groups underwent surgery 2.5 wk later than the longer term groups, thereby allowing all muscles, including those from control animals, to be harvested at a similar age and body weight. Midsections of the PLT were mounted on a cork, quickly frozen in isopentane precooled with liquid nitrogen (for histochemical analysis), and subsequently stored as above until processing.

Hyperthyroidism and increased muscle use.   In this model, PLT muscle was subjected to compensatory overload after removal of the ipsilateral gastrocnemius of one hindlimb only. The other hindlimb, which had been subjected to a similar hormonal milieu, served as a control (10). The day after surgery, animals were randomly assigned to thyroid hormone (T3; 300 µg/kg 3,5,3'-triiodo-DL-thyronine dissolved in 0.6 mM NaOH ip) and sham-injected (0.6 mM NaOH carrier solution) groups and received injections every second day over 40 days. This treatment period was the same as that employed by Ianuzzo et al. (10), and it also ensured that phenotypic changes in the overloaded muscle had stabilized before assessment (9). Although the molecular basis by which T3 alters muscle phenotype is not fully established, the combination of overload and T3 treatments enabled a selective increase in the contractile activity of the PLT while preventing the normal overload-induced changes in slow gene expression (10). At the end of the experimental period, both overloaded and contralateral control PLT muscles were harvested.

To address the possibility that T3 treatment had a direct and irreversible effect on Hsp70 induction, hyperthyroid rats were subjected to heat shock (H), an established inducer of Hsp70. Rats were injected every second day with either T3 or carrier solutions for 20 days [a point at which T3-induced changes in skeletal muscle have stabilized (18)]. Half the animals from each injection (T3H and H from hyperthyroid and control groups, respectively) group were lightly anesthetized with pentobarbital sodium (35 mg/kg ip) and wrapped in a heating blanket until rectal temperature reached 41.5°C, where it was maintained for 15 min. Twenty-four hours later, animals were fully anesthetized and PLT muscles were harvested.

Gel electrophoresis.   Muscle samples weighing 50–100 mg were homogenized in 19 volumes of 15 mM Tris (pH 7.5), 600 mM NaCl containing 1/200th volume each of 0.2 M phenylmethylsulfonyl fluoride in ethanol, 0.2 mg/ml pepstatin A in ethanol, and 0.1 mg/ml leupeptin in double-distilled H2O as protease inhibitors. During homogenization, samples were kept on ice and then centrifuged at 5,000 g for 10 min at 4°C, and the supernatant was stored at –80°C until analyses were performed. Proteins were quantified by the Lowry method (16), and one-dimensional SDS-PAGE was performed on muscle homogenates as described previously (12), except that the separating gel was 12% polyacrylamide.

Immunoblotting.   Proteins were electrophoretically transferred (for a total of 200 V/h) to 0.2-µm pore size nitrocellulose membranes in 20% methanol-1.44% glycine buffer (pH 8.3) containing 0.1% SDS and 0.3% Tris·HCl. Transfer buffer temperature was maintained below 15°C by placing the transfer apparatus at 4°C. Membranes were then blocked overnight in a 5% skim milk powder solution in Tris-buffered saline (500 mM CaCl, 20 mM Tris·HCl pH 7.5), washed twice (5 min each) in Tris-buffered saline containing 0.5% Tween 20 (TTBS), and then reacted with primary antibodies specific for MHC I (anti-MHC I monoclonal antibody, 10D10; 1:750) or Hsp70 (anti-Hsp70 polyclonal antibody, StressGen SPA-812; 1:3,000) in TTBS with 2% skim milk powder for 2 h. After two washes of 5 min each in TTBS, membranes were reacted with secondary antibodies [goat-anti-mouse and goat-anti-rabbit alkaline-phosphatase (Bio-Rad) conjugated for the MHC I and Hsp70 blots, respectively] at 1:1,000 dilutions in TTBS with 2% skim milk powder for 2 h. After washes of 5 min each in TTBS and Tris-buffered saline, blots were transferred to 100 ml of bicarbonate buffer (100 mM NaCO3, 1 mM MgCl2) containing 1 ml each of 3% (wt/vol) p-nitro blue tetrazolium chloride p-toluidine salt in 70% (wt/vol) N,N-dimethylformamide and 1.5% (wt/vol) 5-bromo-4-chloro-3-indolyl phosphate in 100% N,N-dimethylformamide to visualize the protein bands. After color development (2–10 min), blots were rinsed in two changes of a large volume of water and placed between filter paper to dry. Blots were scanned and quantified using the Scion Image (National Institutes of Health) analysis system. Data for both Hsp70 and MHC I were then corrected relative to a standard of pooled soleus (obtained from 8- to 11-wk-old male rats) and normalized to the appropriate control, set as 100%. Individual gels contained samples from each experimental group used in the analysis.

Immunohistochemistry.   Serial tissue cross sections (10 µm) from the muscle midbelly were cut at –20°C using a cryostat (Reichart-Jung, Heidelberg, Germany), adhered to microscope slides and placed within a humidified chamber. Hsp70 was detected using methods described previously (4). Briefly, tissues were fixed using cold neutral buffered formalin (pH 7.4) for 2 min and rinsed in (2 x 10 min for all washes unless otherwise noted) washes with cold 25 mM PBS (pH 7.6). Tissues were then permeablized with 0.3% Triton-X in PBS for 10 min, followed by PBS washes. Tissues were incubated for 1 h in a blocking solution consisting of 5% goat serum (Sigma, St. Louis, MO) in a carrier solution consisting of 0.5% bovine serum albumin in PBS. Sections were subsequently incubated for 4 h at 4°C in carrier solution with a Hsp70 inducible-specific polyclonal antibody (1:200; SPA-812, StressGen, Victoria, BC, Canada). Sections were rinsed with PBS (3 x 10 min) and incubated at 4°C in peroxidase-conjugated goat anti-rabbit immunoglobin G (1:500). After another PBS rinse, the bound antibodies were visualized using diaminobenzidine as a chromogen (4). For serial sections stained for detection of MHC isoforms, tissues were treated at room temperature and underwent a similar procedure with the following exceptions: tissues were not fixed or permeablized; tissues were incubated for 1 h in working dilutions of mouse or rat monoclonal antibodies raised against MHC I (BA-F8), IIa (SC-71), IIx, and IIb (212F), all isoforms except IIx (BF-35) and IIb (BF-F3); and tissues were incubated with a goat anti-mouse secondary at room temperature. Tissues were mounted with Permount (Fisher, Toronto, Canada).

The fiber-type composition of each muscle (n = 4–5/time point) was determined as follows (4): fibers were randomly chosen from within five distinct areas of each tissue midbelly section; all fibers (n > 400/section) were identified with the aid of a microscope linked to a computer-based image-analysis system; fibers were classified according to their staining profile, and fiber proportions were calculated for each muscle (I, IIa, IIx, fibers expressing MHC I, IIa, or IIx, respectively; I/IIa, IIa/IIx, fibers coexpressing MHC I and IIa or IIa and IIx, respectively; IIx/IIb, fibers expressing MHC IIb alone or coexpressing MHC IIb and IIx). After fiber-type classification of MHC isoform, a staining intensity score was derived from the average pixel density for encircled fibers on images of sections incubated with anti-Hsp70. Intensity of Hsp70 was reported in arbitrary linear (grayscale) units that were the result of subtracting background (primary antibody excluded) from positive (primary antibody included) serial sections. Microscope settings and conditions were held constant during capture of images of background and Hsp70 stains.

Statistical analyses.   All data are expressed as means ± SE. Body weight, serum T3, heart weight, and heart weight-to-body weight ratios were compared by unpaired t-test. Comparisons among time points within a fiber type or among fiber types within a time point, during the adaptation to overload, were made using a one-way ANOVA. With muscle overload or heating and T3 protocols, muscle weight and protein data were compared using a two-way ANOVA. On determination of significant interactions, pairwise post hoc comparisons employing Tukey's test were used to determine differences between experimental groups. When tests of significance were made between specific groups, the Bonferroni correction for multiple comparisons was used. Differences were considered significant at P values of <0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Adaptation to compensatory overload.   Overload induced significant hypertrophy and remodeling in the PLT muscle (Table 1). By 4 wk of overload, all fiber types had adapted to the increased load by displaying significantly higher CSAs, with type I and IIa fibers nearly doubling in size. This hypertrophy was accompanied by a significant increase in muscle mass relative to body weight. Overload also resulted in remodeling of the muscle toward a slower phenotype. This shift was evident by the first week of adaptation in which the de novo expression of fibers coexpressing types I and IIa myosin could be observed. These fibers likely represent the shifting in phenotype from fast-to-slow as by 4 wk of overload a significant reduction in fast MHC IIx- and IIb-expressing fibers could be contrasted against the increase in the number of slow-type MHC I (and presence of type I/IIa) expressing fibers.


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  		Table 1. Effects of compensatory overload on plantaris muscle characteristics and relative Hsp70 content

 
Effect of increased muscle use on fiber-type expression of Hsp70.   Unstressed rat PLT muscle displayed fiber type-specific expression of Hsp70. In particular, Hsp70 was detected in MHC I- and IIa-expressing fibers in control muscle (Fig. 1, A and B). In contrast, fibers not expressing either of these myosin isoforms (types IIx, IIx/IIb) had undetectable levels of Hsp70. During the adaptation to overload, the tendency to increase staining intensity (or Hsp70 content) in the same fibers, which display constitutive expression, did not achieve statistical significance (P > 0.05). Despite hypertrophy in all fiber types and significant remodeling within the muscle, the fiber-specific profile of Hsp70 was unchanged throughout the time course of adaptation to compensatory overload. From 2 wk on, the increase in the relative content of Hsp70 in the overloaded muscle (as assessed by Western blot) had stabilized and was likely due, in part, to the increased proportion of type I and I/IIa fibers observed at these times (Table 1).


Figure 1
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  		Fig. 1. Immunohistochemical detection of various distinct myosin heavy chain (MHC) isoforms and heat shock protein (Hsp70) in Sprague-Dawley rat plantaris muscle during adaptation to overload. Serial sections were stained with monoclonal antibodies raised against MHC types and a polyclonal antibody raised against Hsp70. Representative samples are from 2-wk exposure to overload condition. A: Hsp70; B: MHC I (BA-F8); C: MHC IIa (SC-71); D: MHC IIx and IIb (212F); E: all MHC isoforms except IIx (BF-35); F: IIb (BF-F3). G: effect of overload on reactivity to anti-Hsp70 in Sprague-Dawley rat plantaris muscle. After fiber-type classification using MHC isoform-specific monoclonal antibodies on serial sections was performed, a staining intensity score was derived from the average image pixel density for encircled fibers on sections incubated with anti-Hsp70 and a goat-anti-rabbit secondary antibody. Sections were visualized with diaminobenzidine as a chromogen. Intensity is reported in arbitrary linear units that were the result of subtracting staining scores of sections incubated according to all steps of the immunohistochemical procedure with the exception of inclusion of the primary antibody (background) from a score derived from a serial section that underwent the same procedure with the inclusion of the primary antibody. Con, control group; 3 day, 1, 2, and 4 wk, length of exposure to overload condition; I, IIa, and IIx, fibers expressing type I, IIa, or IIx MHC, respectively; I/IIa, IIa/IIx, fibers coexpressing type I and IIa or IIa and IIx MHC, respectively; IIx/IIb, fibers expressing MHC IIb alone or coexpressing MHC IIb and IIx. {alpha}Fiber types under this symbol were not significantly different from background but were significantly different from groups without the symbol at all corresponding time points.

 
Evidence of altered thyroid state and altered phenotype.   The T3 treatment protocols resulted in elevated serum T3 concentrations (Table 2). In addition, an increase in heart weight, heart weight-to-body weight ratio, and body temperature in T3-treated animals is reflective of hyperthyroidism (7). MHC I expression was used to assess the effects of thyroid hormone as the full complement of expressed contractile protein isoforms (or phenotype) are affected in conjunction with MHC I in these established models (4, 5). Accumulation of MHC I with compensatory overload was blocked by the administration of T3 (10, 28). Muscle overload resulted in weight gain in both sham and hyperthyroid PLT. When normalized to body weight or expressed as percentage of growth, sham and hyperthyroid appear to have undergone similar degrees of hypertrophy (~67 vs. 69% increase in muscle mass, respectively; Table 3).


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  		Table 2. Evidence of altered thyroid state in rats in overload and heating experiments

 

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  		Table 3. Evidence of altered phenotype and increased muscle weight in rat skeletal muscles undergoing increased contractile activity and/or altered thyroid state

 
Effect of hyperthyroidism and increased muscle use on Hsp70 expression.   Overload was associated with significantly higher levels of Hsp70 (Fig. 2). However, although overload-induced hypertrophy was still evident in hyperthyroid rats (as discussed above; Table 3), when the transition to a slow phenotype was blocked (hyperthyroid) so were increased Hsp70 levels.


Figure 2
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  		Fig. 2. Effect of 3,5,3'-triiodothyronine (T3) treatment and overload on Hsp70 protein expression in rat plantaris muscle. Relative protein content was determined from scanned images of immunoblots that were reacted with a polyclonal antibody for Hsp70 and visualized with a goat anti-rabbit alkaline phosphatase-conjugated secondary antibody. S, sham injected. Control and overload muscles were taken from the right and left legs, respectively, within the same animal. Values are expressed as a percentage of S control mean ± SE (n = 10 per group). *Significantly different from all other experimental groups (P < 0.05).

 
To determine whether the suppression of the normal overload-induced increase in Hsp70 was a direct and irreversible effect of T3, hyperthyroid rats were heat shocked. To our knowledge, a direct repressive effect of T3 on the Hsp70 promoter has not been reported, and although selective repression of Hsp70 by T3 remains a possibility, the normal heat shock-induced increase in Hsp70 in hyperthyroid rats was not attenuated (non-heat shocked control, 100.0 ± 6.7%; heat shocked control, 177.0 ± 28.2%; non-heat shocked, T3 treated, 97.7 ± 7.27%; and heat shocked, T3 treated, 160.2 ± 11.2%).


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The principal findings of the present investigation are that 1) in the rat PLT, constitutive Hsp70 expression is not restricted to slower, more oxidative type I fibers but also to relatively faster IIa fiber counterparts both at rest and after functional overload and 2) this fiber-specific pattern of expression is related more to fiber phenotype than compensatory growth or increased levels of motor unit recruitment.

Constitutive expression of Hsp70 is fiber specific.   The constitutive expression of Hsp70 in slow-type skeletal muscle has been suggested to be related to the frequent loading of these muscles (7, 13, 21). Although ongoing contractile activity has been associated with increased levels of Hsp70 (12, 20–23, 25) the critical factor(s) linking these two observations have not been clear. To first characterize the adaptation to sustained contractile activity, the fiber-specific expression of Hsp70 during compensatory overload was examined. Using an objective, quantitative immunohistochemical approach, a distinct pattern of expression was observed during this remodeling process. Expression of Hsp70 was restricted to the smallest-caliber and thus most-recruited fiber types (types expressing MHC I and IIa) at all time points (3 days to 4 wk), including control conditions (day 0). These data are thus similar to those reported by Neufer et al. (20) in control rabbit tibialis anterior muscle but in contrast to the observations of Ogata et al. (22) who reported that Hsp70 was found only in type I fibers in the rat PLT. To determine whether differences in the present study vs. that of Ogata et al. (22) were a function of the rat species (Wistar vs. Sprague-Dawley) or Hsp70 antibody [monoclonal (StressGen SPA-810) vs. polyclonal (SPA-812)] employed, additional experiments were performed. Imunohistochemistry was conducted on cross sections of PLT muscle from groups of 8-wk-old male Wistar and Sprague-Dawley rats (n = 5 per strain) using the monoclonal Hsp70 antibody (StressGen SPA-810) employed by Ogata et al. (22). In our hands, this monoclonal antibody also revealed constitutive expression of Hsp70 in both type I and IIa fibers in each rat strain (Fig. 3). The reason(s) for the discrepancy between these data and those of Ogata et al. (22) remain unexplained. However, in agreement with previous observations in rabbit tibialis anterior (20), the present results, employing two different rat models and Hsp70-specific antibodies, clearly demonstrate that Hsp70 is not only present in fibers expressing both MHC I and/or IIa but actually tends to exhibit higher levels in type IIa fibers (Fig. 1B).


Figure 3
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  		Fig. 3. Immunohistochemical detection of Hsp70 in both type I and IIa fibers in plantaris of Sprague-Dawley and Wistar rats. Serial sections were stained with monoclonal antibodies raised against MHC types and Hsp70. Representative samples from a group of n = 5. A: sections of plantaris from male Wistar rat. B: sections of plantaris from male Sprague-Dawley rat. Left: Hsp70 (StressGen, SPA-810). Middle: MHC I (BA-D5). Right: MHC IIa (SC-71).

 
As noted above, Hsp70 has been implicated in protein turnover (2, 19). With the model used in the present investigation, however, all fiber types (I, IIa, IIx, IIx/IIb, or IIb) underwent hypertrophy, presumably in response to the recruitment stress of overload; yet detectable levels of Hsp70 were not required in some fibers (i.e., IIx and IIb) to facilitate this adaptation. Moreover, it has been proposed that, in response to contractile-induced remodeling, Hsp70 initially assisted slow fiber types and later fast fiber types during the shift to a slower phenotype (20). However, in response to compensatory overload, the apparent remodeling of IIb and/or IIb/IIx fibers did not result in expression of Hsp70 (Fig. 1B). Thus these data support previous suggestions (22, 24, 25) that Hsp70 appears to play another yet-to-be-determined role in skeletal muscle in response to contractile activity with normal recruitment patterns.

A reasonable interpretation of the data would be that a threshold level of contractile activity is required for the induction of Hsp70 (17) and that the necessary recruitment was only achieved by those fibers expressing MHC I and IIa. Indeed, these fibers underwent the greatest degree of hypertrophy. However, the extent to which the shift from fast to slow phenotype or maintenance of a slow phenotype, in the presence of contractile activity, contributed to the overall change in Hsp70 content was unclear. Because earlier observations suggest that muscle phenotype could be important (12, 13, 21, 22, 24, 25), we sought to test this hypothesis by separating the effect of increased contractile activity from phenotypic transformation.

Phenotypic transformation is critical for increased expression of Hsp70 following 40 days of compensatory overload.   Despite a dramatic increase in contractile stress in overloaded muscle, 40 days of T3 treatment was able to suppress the shift toward a slower phenotype with increased Hsp70 (Table 3; Fig. 2). These data are similar to those recently reported by others (21) and suggest that interrupting signaling pathways, which are normally activated following contractile activity, results in specific consequences for Hsp70 expression. For example, signals leading to muscle hypertrophy may not require Hsp70 expression (it is never required in certain fiber types; see Fig. 1B), since overloaded muscles in the T3 group underwent a similar degree of hypertrophy as controls with no increase in Hsp70 expression (Table 3). Similar observations were made when muscle hypertrophy was induced with 4 wk of clenbuterol treatment (24). In the latter case, soleus hypertrophied by 16%, yet Hsp70 content was actually reduced by 52% (24). Also, in the developing diaphragm, the sudden onset of contractile activity coupled with a high rate of growth does not result in an increase in Hsp70 expression until myosin has begun to shift to a slower phenotype (25). However, in the present investigation, we cannot exclude that Hsp70 may have been upregulated early in the hypertrophic process and returned to control levels by 40 days. Indeed, in young animals, it has been observed that after only 2 wk of overload, T3 treatment reduced but did not completely suppress an overload-associated increase in Hsp70 (21). Nonetheless, these data suggest that slower phenotype remodeling may be a critical factor linking sustained contractile activity to elevated constitutive levels of Hsp70.

It should be noted that, under certain circumstances, changes in contractile activity can alter Hsp70 expression independent of phenotypic remodeling. For example, exercise training is known to elevate Hsp70, yet this change is not consistently accompanied by a parallel change in MHC I expression (11). In addition, Ornatsky and coworkers (26) reported an increase in Hsp70 following chronic low-frequency stimulation and no change in MHC I expression. However, chronic low-frequency stimulation eventually upregulates slow-type gene expression, and the discordant responses may represent differences in timing rather than regulation. The discrepancy between observations in the above studies and those of the present investigation may also be a consequence of multiple regulatory pathways influencing Hsp70 expression. For example, exercise may increase Hsp70 expression by directly activating the typical stress response pathway involving HSF1 (14). This pathway is also likely responsible for the elevated Hsp70 in PLT following heat shock (Fig. 3). However, other regulatory pathways must also be involved in elevated constitutive expression of Hsp70, since this protein is found at high levels in the unstressed rodent soleus without concomitant HSF1 activation (15). Since Hsp70 is known to modulate various transcription factors and mitogenic pathways (6), perhaps Hsp70 is a necessary component of the pathway(s) responsible for induction of the slower fiber phenotype(s).

In summary, we note fiber type-specific (type I and IIa fibers) expression of Hsp70 under control conditions as well as during adaptation to increased contractile activity. The mechanism underlying this restricted expression appears to be related to fast-to-slow phenotypic remodelling rather than a response to contractile activity per se. Indeed, by 40 days of overload at least, slower phenotype expression was critical to the increase in Hsp70 in muscles undergoing chronic contractile activity. Although some work in this regard has been initiated (21, 23, 24) future research should strive to elucidate the molecular signals and physiological importance underlying this intriguing relationship.


   GRANTS
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 ABSTRACT
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 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported by Natural Sciences and Engineering Research Council of Canada (E. G. Noble and R. N. Michel), the Canadian Institutes of Health Research, ALS and MDAC Granting Partnership (to R. N. Michel), and the Heart and Stroke Foundation of Ontario (T5036) (to E. G. Noble).


   ACKNOWLEDGMENTS
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 ABSTRACT
 METHODS
 RESULTS
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REFERENCES
 
Monoclonal antibodies raised against specific isoforms of the MHC were generous gifts from Dr. S. Schiaffino, University of Padua, Padua, Italy, and Dr. D. J. Parry, University of Ottawa, Ottawa, Canada (BA-F8, SC-71, BF-F3, and BF-35), Dr. P.A. Merrifield, University of Western Ontario, London, Canada (212F, and 10D10), and Dr. C. T. Putman, University of Alberta (BA-5D). We gratefully acknowledge Dr. B. G. Atkinson, The University of Western Ontario, and Dr. S. E. Dunn, Stanford University, for assistance with immunohistochemical analyses.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Noble, Thames Hall Rm. 2160C, School of Kinesiology, The Univ. of Western Ontario, London, ON, Canada N6A 3K7 (e-mail: enoble{at}uwo.ca)

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
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