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Am J Physiol Heart Circ Physiol 293: H3692-H3698, 2007. First published October 5, 2007; doi:10.1152/ajpheart.00827.2007
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Exercise-mediated regulation of Hsp70 expression following aerobic exercise training

C. W. James Melling,1 David B. Thorp,1 Kevin J. Milne,1 Matthew P. Krause,1 and Earl G. Noble1,2

1School of Kinesiology, Faculty of Health Sciences, and 2Lawson Health Research Institute, The University of Western Ontario, London, Ontario, Canada

Submitted 16 July 2007 ; accepted in final form 29 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
An issue central to understanding the biological benefits associated with regular exercise training is to elucidate the intracellular mechanisms governing exercise-conferred cardioprotection. Heat shock proteins (HSPs), most notably the inducible 70-kDa HSP family member Hsp70, are believed to participate in the protection of the myocardium during cardiovascular stress. Following acute exercise, activation of PKA mediates the suppression of an intermediary protein kinase, ERK1/2, which phosphorylates and suppresses the activation of the heat shock transcription factor 1 (HSF1). However, following exercise training, ERK1/2 has been reported to regulate the transcriptional activation of several genes involved in cell growth and proliferation and has been shown to be associated with training-mediated myocardial hypertrophy. The present project examined the transcriptional activation of hsp70 gene expression in acutely exercised (60 min at 30 m/min) naïve sedentary and aerobically trained (8 wk, low intensity) male Sprague-Dawley rats. Following acute exercise stress, no significant differences were demonstrated in the expression of myocardial Hsp70 mRNA and activation of PKA between sedentary and trained animals. However, trained animals elicited expression of the hsp70 gene (P < 0.05) in the presence of elevated ERK1/2 activation. Given the association of ERK1/2 and the suppression of hsp70 gene expression following acute exercise in naïve sedentary rats, these results suggest that training results in adaptations that allow for the simultaneous initiation of both proliferative and protective responses. While it is unclear what factors are associated with this training-related shift, increases in HSF1 DNA binding affinity (P < 0.05) and posttranscriptional modifications of the Hsp70 transcript are suggested.

protein kinase A; mitogen-activated protein kinase; protein phosphorylation; receptors; signal transduction

REGULAR PHYSICAL EXERCISE has been shown to reduce the incidence of cardiovascular disease in normal healthy adults (4, 17, 18, 26). While the intracellular mechanism(s) involved in this exercise-conferred cardioprotection has yet to be fully understood, several studies have indicated the involvement of a highly select group of proteins called heat shock proteins (HSPs). These proteins, most notably the inducible isoform of the 70-kDa HSP family Hsp70, function to ensure the proper folding and cellular transportation of newly formed polypeptides as well as the reassembly or degradation of damaged and abnormal proteins and suppression of cell death pathways (for reviews, see Refs. 1 and 2). Following acute exercise, upregulation of these proteins within the myocardium is at least partially responsible for a window of cardiovascular protection, whereby the heart is resistant to damage during an ischemia-reperfusion (I/R) insult (20, 27, 38).

Following acute exercise, transcriptional expression of the hsp70 gene is regulated primarily through heat shock transcription factor 1 (HSF1) (23). Upon activation, HSF1 undergoes a series of steps that includes trimerization, nuclear localization, and binding acquisition to the promotor region of the hsp70 gene's heat shock element (HSE) (32). Upon acquisition of HSF1-HSE DNA binding, an additional event(s) is necessary to initiate transcription of the hsp70 gene postexercise (24). Specifically, ERK1/2 would normally phosphorylate and suppress HSF1 transcriptional activation (9, 33) and Hsp70 expression (23), but exercise-induced activation of the downstream β-adrenergic receptor-mediated signaling kinase, PKA, inhibits ERK1/2 activation and therefore is permissive to increased Hsp70 expression (23).

Whereas in sedentary naïve subjects the expression of Hsp70 following acute exercise has been well characterized (23, 27, 30), sporadic acute exercise is unlikely to be timed such that it protects against a deleterious cardiovascular incident. Hence, regular exercise training is recommended as a more appropriate prophylactic. With chronic exercise training, however, adrenergic hormone release is diminished and proliferative pathways, including ERK1/2, are activated, initiating physiologically beneficial cardiac hypertrophy (37). PKA initiates this process by mediating the binding of β-adrenergic receptor kinase (β-ARK) and β-arrestins to the receptor, resulting in its internalization (10). The internalized receptor then acts as a molecular scaffold to bind and initiate the activation of ERK1/2 and other signaling components of cell growth (36). Controversy exists regarding the ability to elicit a robust exercise-mediated HSP response in trained individuals. In fact, trained rodents may exhibit diminished induction of Hsp70 expression following a subsequent bout of acute exercise (12). Given the positive role of the exercise-induced heat shock response following myocardial I/R (20, 27), activation of these proliferative pathways may result in a less robust response and reduced protection against stress.

The purpose of the present study was to examine the effect of an 8-wk treadmill running training program on the transcriptional expression of the hsp70 gene following acute exercise. Specifically, the binding of HSF1 to the hsp70 gene and the activation of the signaling molecules PKA and ERK1/2, reported to regulate its transcriptional activation, were examined. It was hypothesized that the myocardium from exercise-trained animals would demonstrate a reduced capacity to elicit Hsp70 expression, potentially hindering the ability of trained individuals to respond to additional stressors (i.e., myocardial infarction).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal characteristics. This study was approved by the University of Western Ontario Council on Animal Care and was performed in accordance with the guidelines of the Canadian Council on Animal Care. Adult (8 wk old) male Sprague-Dawley rats obtained from Charles River Laboratories (St. Constant, QC, Canada) were housed in standard rat cages for the duration of the training program (8 wk) at constant temperature and humidity with a 12:12-h light-dark cycle. Rats were fed and watered ad libitum.

Experimental protocol. Thirty-five animals were randomly assigned to one of three experimental groups: control animals (n = 10), acute-run sedentary (Acute-Sed) animals (n = 10), and trained animals (n = 15). The trained group was subdivided into an acute-run trained (Acute-Train) group (n = 10) and a trained-only (Train) group (n = 5). The Train group was used to assess the levels of Hsp70 mRNA and protein at the end of training for comparison with the Acute-Train animals. For the experimental exercise session, Acute-Sed and Acute-Train animals underwent 60 min of continuous running (30 m/min, 2% grade) on a motor-driven treadmill (24), whereas control animals were handled similarly to the exercised groups but did not undergo exercise. Prior to the acute exercise session, Acute-Train animals underwent an 8-wk, 21-session training protocol, which was intended to reflect the typical training regimen that might be employed by individuals intending to enhance their cardiovascular fitness. As shown in Fig. 1, the training program was a progressive protocol in which animals exercised five times over each 2-wk period with weekends off. For the first few sessions, animals were familiarized to the training, with an initial 15-m/min run lasting 15 min. Thereafter, runs were lengthened by 15 min every second session, such that by the sixth session, animals were running 15 m/min for 60 min, which was maintained for the next 2 wk (5 sessions). The exercise intensity was then increased to 18 m/min (for 60 min) for the following 2 wk (5 sessions). The last six sessions were all run at 21 m/min for 60 min. Acute-Sed animals were handled similarly to Acute-Train animals but did not undertake the training program. Acute-Sed and Acute-Train animals underwent the acute exercise protocol 3 days following the last training session of the Acute-Train group to allow sufficient time for the Acute-Train group to recuperate (31). The trained animals that did not undergo the acute exercise protocol were killed 3 days following the last training session for the measurement of Hsp70 protein and mRNA expression to assess the effects of training independent of the acute exercise session. Before and immediately after the acute exercise, colonic temperatures and blood samples (~1 ml from the tail) were taken. Animals were killed 30 min following acute exercise to examine HSF1-HSE DNA binding and ERK1/2 and PKA activation at a time point previously reported to show elevated levels of Hsp70 mRNA (24). Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (Somnotol, 65 mg/kg), and hearts were extirpated, weighed, and stored for subsequent analysis.


Figure 1
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  		Fig. 1. Outline of the experimental protocols used. Acute-run sedentary (Acute-Sed) and acute-run trained (Acute-Train) animals underwent a 60-min exercise session on a motorized treadmill at 30 m/min (2% grade) and were then killed 30 min following exercise for the analysis of 70-kDA heat shock protein (Hsp70) mRNA; heat shock transcriptional factor 1 (HSF1) and Hsp70 content; membrane β1-adrenergic receptor (β1-AR), β2-adrenergic receptor (β2-AR), and β-adrenergic receptor kinase (β-ARK) content; PKA and ERK1/2 activity; and HSF1-heat shock element (HSE) DNA binding. The control (Con) group did not undergo the exercise protocol but rested in standard rat cages. Acute-Train animals completed the same exercise protocol listed above but underwent 8 wk of exercise training. Animal colonic temperatures were taken immediately before and after the exercise protocol. Open bars indicate rest periods; solid bars indicate exercise periods; shaded bar indicates a 2-wk running period during which animals were acclimated with an increasing run time.

 
Cellular fractionation and protein quantification. Whole cell extracts and membrane fractions were prepared according to Locke et al. (19) and Carson et al. (6), respectively. Total protein concentrations were determined using the Bradford (5) protein method.

PKA activity assay. Total protein kinase activities were determined using the SignaTECT PKA assay system from Promega. This radioactive enzymatic assay involves the PKA-catalyzed transfer of 32P from [{gamma}-32P]ATP. In brief, 100 mg of heart tissue were homogenized in 5 volumes of extraction buffer [25 mM Tris·Cl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 10 mM β-mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF] and centrifuged at 14,000 g for 5 min at 4°C. Activity assays were performed in the presence of a PKA biotinylated peptide substrate for the activated reaction, whereas the control reaction was performed in its absence. All reactions were incubated at 30°C for 5 min, and 32P incorporation was measured by transferring the completed reaction onto a S-adenosylmethionine membrane (Promega). Results were then quantified utilizing a β-scintillation counter.

Serum lactate levels and plasma epinephrine. Blood samples obtained from the tail immediately before and after the acute exercise run were placed in tubes and centrifuged at 10,000 rpm for 4 min at 4°C. Serum lactate levels were then processed according to previously published protocols (25). For plasma epinephrine levels, blood samples were collected in tubes containing EGTA and glutathione and centrifuged at 2,000 rpm at 2°C. The plasma was then removed, measurements were determined using HPLC, and electrochemical detection was completed according to Green et al. (13).

RNA isolation and slot-blot analyses. Total heart RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform procedure (8). RNA samples were blotted onto a Zeta-Probe membrane (Bio-Rad) using a Schleicher & Schuell manifold. The membrane was cross linked and incubated at 50°C for 3 h in a prehybridization solution [5x SSC, 20 mM Na2HPO4 (pH 7.2), 7% SDS, 1x Denhardt's solution, and 100 µg/ml salmon sperm DNA]. The membrane was then incubated in the same solution with the addition of a 32P end-labeled oligonucleotide specific for inducible Hsp70 transcripts (5'-ATCTCCTTCATCTTGGTCAGCACCATGGAC-3') and exposed to Kodak X-OMAT MS film overnight at –70°C. For the standardization of values, blots were washed at 95°C in 0.5% SDS and 0.1x SSC and reprobed in hybridization solution containing a 32P end-labeled oligonucleotide specific for 28S rRNA.

SDS-PAGE and Western blot analysis. Whole cell extracts and membrane fractions were mixed with equal volumes of sample buffer (0.5 M Tris base, 13% glycerol, 0.5% SDS, 13% β-mercaptoethanol, and bromophenol blue) and separated according to their molecular weight on gels consisting of a 12% acrylamide separating gel overlaid by a 4% acrylamide stacking gel. A molecular weight standard (catalog no. 161-0373 Bio-Rad) was run concurrently on each gel for accurate determination of the proper molecular weight of the protein. After electrophoresis, proteins were transferred to nitrocellulose membranes and blocked in 3% milk blotto in Tris-buffered saline (TBS) for 2 h and then washed twice with 0.01% Tween 20 in TBS (TTBS) for 5 min each wash. Membranes were then incubated in primary antibody specific to either ERK1/2 (anti-ERK1/2 polyclonal antibody, 1:1,000, catalog no. 9102, Cell Signal Technology), β1-adrenergic receptor (anti-β1-adrenergic receptor polyclonal antibody, 1:500, SC-567, Santa Cruz Biotechnology), β2-adrenergic receptor (anti-β2-adrenergic receptor polyclonal antibody, 1:500, SC-567, Santa Cruz Biotechnology), β-ARK (anti-β-ARK polyclonal antibody, 1:500, SC-567, Santa Cruz Biotechnology), or Hsp70 (anti-Hsp70 polyclonal antibody, 1:5,000, SPA-812, Stressgen) in TTBS (2% BSA, catalog no. A-2153, Sigma). ERK1/2 activation was detected using antibodies raised against its phosphorylated form (anti-phospho-ERK polyclonal antibody, 1:1,000, catalog no. 9101, Cell Signal Technology). Following incubation, membranes were washed in TTBS and incubated with secondary antibody according to the manufacturer's instructions. Antibody detection was performed colorimetrically (Bio-Rad) for Hsp70 and via an enhanced chemiluminescence method (Amersham) for all other antibodies. To ensure equal protein loading, the Ponceau staining method was performed as described by Ping et al. (29). Briefly, membranes were incubated in Ponceau S solution (catalog no. 81463, Sigma-Aldrich) for 5 min immediately after antibody detection. The largest nonspecific band was subjected to densitometric analysis, and the average density was determined across the lanes. Antibody detection was then normalized to the ratio of Ponceau stain density in that lane and the average density of the Ponceau stain and represented as a percentage of standard.

HSF1-HSE DNA binding. For analyses of HSF1-HSE DNA binding, whole cell extracts (100 µg) were incubated in binding buffer (10% glycerol, 50 mM NaCl, 1.0 mM DTT, and 0.3 mg/ml BSA) consisting of 1.0 ng of a 32P-labeled self-complementary HSE oligonucleotide (5'-CTAGAAGCTTCTAGAAGCTTCTAG-3') and 1.0 µg of poly(dI·dC) for 30 min at room temperature. Samples were electrophoresed on 4% acrylamide gels at 200 V for 2–3 h. Gels were then dried and exposed to radiographic film. For standardization, HSF1-HSE binding interactions were represented as a percentage of the HSE oligonucleotide incubated with whole cell extracts from heat-treated animals (procedure reported in Ref. 19) that have been previously reported to initiate HSF1-HSE DNA binding interactions.

Statistical analyses. Blots were quantified using Scion Image Analysis software. Results are reported as mean ± SE, and values were compared using one-way ANOVA. Upon confirmation of a significant main effect, individual differences were determined with the use of a least-squares difference post hoc test. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal characteristics. Pre- and postexercise colonic temperatures and serum lactate levels are shown in Table 1, whereas heart-to-body weight ratios are shown in Table 2. Serum lactate levels were not significantly different between Acute-Sed and Acute-Train animals before and after the acute exercise, indicating that animals in both groups demonstrated similar and moderate metabolic stress during the acute exercise. Acute-Train animals demonstrated a significant increase (P < 0.05) in heart-to-body weight ratios compared with Acute-Sed and control animals, suggesting myocardial growth as a result of the training program.


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  		Table 1. Colonic temperatures and serum lactate concentrations following acute exercise

 

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  		Table 2. Body weights, heart weights, and heart-to-body weight ratios in control, Acute-Sed, and Acute-Train animals

 
Hsp70 protein content. Hsp70 protein levels were elevated only in the trained groups 30 min after acute exercise (P < 0.05; Fig. 2). To confirm that this elevation was due to the training program, a separate group of trained animals that did not undergo the acute exercise protocol was examined. This group demonstrated a similar increase in the expression of constitutive Hsp70 levels 3 days following the last training session in the absence of acute exercise (P < 0.01). Given that Hsp70 expression was not seen in Acute-Sed animals and the previously reported time period needed for the translation of the Hsp70 transcript (31), these results suggest that the training itself led to a significant elevation in constitutive levels of Hsp70 within the myocardium that was sustained until at least 3 days after the last training session.


Figure 2
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  		Fig. 2. Effect of training on exercise-induced myocardial Hsp70 protein content. Hsp70 content was measured in 100 µg of cardiac tissue using SDS-PAGE and transferred onto a nitrocellulose membrane, where it was detected using antisera specific to Hsp70. Quantitative analysis of Hsp70 protein content in myocardial tissue was represented as a percentage of a recombinant Hsp70 standard, respectively. A representative blot is shown in the inset. Data are means ± SE of 5–10 animals/group. *Significantly different from control treatment (P < 0.05).

 
Plasma epinephrine, PKA, and ERK1/2 activity levels. After acute exercise, there was a significant increase in plasma epinephrine concentrations in both Acute-Sed (P < 0.001) and Acute-Train (P < 0.001) animals (Fig. 3A). This exercise-induced increase in plasma epinephrine was significantly lower in Acute-Train animals compared with Acute-Sed animals (P < 0.01). In conjunction with a rise in plasma epinephrine, a significant elevation in PKA activity in both Acute-Train (P < 0.05) and Acute-Sed (P < 0.05) animals was observed compared with control animals (Fig. 3B). However, there were no significant differences between the two exercised groups. ERK1/2 phosphorylation/activation was significantly elevated in Acute-Train animals compared with control and Acute-Sed animals (P < 0.01; Fig. 3C).


Figure 3
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  		Fig. 3. Plasma epinephrine responses to acute exercise in sedentary and trained animals and myocardial PKA levels and ERK1/2 activation following training. Blood samples were obtained immediately before (Pre-EX) and after acute exercise (Post-EX) for measurements of plasma epinephrine (A) levels. PKA activity (B) was analyzed 30 min postexercise using a commercially available kit. ERK1/2 phosphorylation (C) was measured in 50 µg of cardiac tissue using SDS-PAGE and transferred onto a nitrocellulose membrane, where it was detected using antisera specific to phosphorylated (p-)ERK1/2 and ERK1/2. Quantitative analysis of phosphorylation in myocardial tissue was represented as a percentage of its nonphosphorylated form. Representative blots are shown in the insets. Data are means ± SE of 10 animals/group. *Significantly different from control treatment or preexercise values (P < 0.05); {alpha}significantly different from postexercise values of sedentary animals (P < 0.05).

 
Membrane β-ARK and β1- and β2-adrenergic receptor levels. Acute-Train animals demonstrated a significant elevation in the expression of membrane β-ARK (P < 0.01; Fig. 4A), whereas no significant differences were observed in β1- and β2-adrenergic receptor levels within the myocardial membrane (P = 0.786 and P = 0.831, respectively; Fig. 4, B and C).


Figure 4
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  		Fig. 4. Exercise-induced membrane localization of myocardial β-ARK, β1-AR, and β2-AR. Membrane localization of β-ARK (A), β1-AR (B), and β2-AR (C) was measured in 50 µg of cardiac tissue using SDS-PAGE and transferred onto a nitrocellulose membrane, where it was detected using antisera specific to β-ARK, β1-AR, and β2-AR, repectively. Quantitative analysis of phosphorylation in myocardial tissue was represented as a percentage of control. Representative blots are shown in the insets. Data are means ± SE of 10 animals/group. *Significantly different from control treatment (P < 0.05).

 
Hsp70 mRNA, HSF1-HSE DNA binding, and HSF1 content. Compared with control animals, both Acute-Sed (P < 0.05) and Acute-Train (P < 0.05) animals demonstrated a significant increase in Hsp70 mRNA following acute exercise (Fig. 5A). This increase in Hsp70 mRNA was not significantly different between the two groups, suggesting that training did not alter the ability of acute exercise to elevate the expression of the hsp70 gene in the myocardium. Following exercise, Acute-Sed (P < 0.05; Fig. 5B, lanes 4 and 5) and Acute-Train (P < 0.001; Fig. 5B, lanes 6 and 7) animals demonstrated a significant increase in HSF1-HSE DNA binding compared with control animals (Fig. 5B, lanes 2 and 3). Within the exercised groups, there was a significantly greater increase in HSF1-HSE DNA binding in Acute-Train (P < 0.05; Fig. 5B, lanes 6 and 7) animals compared with Acute-Sed (Fig. 5B, lanes 4 and 5) animals. Myocardial HSF1 levels were not significantly different between the groups, suggesting that the increased binding of HSF1 to the hsp70 gene was not due to an elevation in myocardial HSF1 content (P = 0.76; data not shown).


Figure 5
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  		Fig. 5. Effect of training on exercise-induced myocardial Hsp70 mRNA levels and HSF1-HSE DNA binding. Hsp70 mRNA (A) was measured using ~5 µg of total RNA incubated with an oligonucleotide probe specific for Hsp70 mRNA. The membrane was then analyzed, stripped, and incubated with 28S rRNA oligonucleotide probe to enable standardization. Quantitative analysis of Hsp70 mRNA and Hsp70 protein content in myocardial tissue was represented as a percentage of 28S rRNA. Gel mobility shift analysis (B) of exercise-induced HSF1-HSE DNA binding was performed using 100 µg of cardiac tissue incubated with a complimentary HSE oligonucleotide. The solid arrowhead indicates HSF1-HSE DNA binding. HS, heat shock. Representative blots of 5–10 animals/group are shown in the insets. *Significantly different from control treatment (P < 0.05); {alpha}significantly different from Acute-Sed animals (P < 0.05).

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Exercise is an important intervention in maintaining cardiovascular health (4, 17, 18, 26). One mechanism that may partially account for the prophylactic effect of exercise is an increase in myocardial HSPs (3). For example, following acute exercise, increases in Hsp70 have been found to protect the myocardium against I/R injury (27). However, while exercise training results in an elevation in constitutive levels of this protective protein in skeletal muscle (31), the muscle's ability to respond to additional exercise stress after exercise training is equivocal (12). The present data suggest that there is a significant elevation in constitutive Hsp70 levels in the myocardium of trained animals. Despite this elevation in constitutive Hsp70 expression, trained animals still exhibit the capacity to respond to an additional exercise stress and subsequently induce the transcriptional expression of the hsp70 gene.

β-Adrenergic receptor-mediated signaling is an important regulator of Hsp70 induction following a single bout of acute exercise (28). Specifically, the downstream β-adrenergic receptor-mediated protein kinase PKA regulates the exercise-induced elevation in Hsp70 mRNA through the activation of HSF1 (24) via inhibition of ERK1/2 (23). Following exercise training, an attenuation of sympathetic output to the myocardium, which is associated with the typical training-induced bradycardia at rest and during submaximal exercise, is observed (7). Indeed, in the present study, postexercise plasma epinephrine levels were attenuated in the group that had exercise trained. One consequence of this attenuated sympathetic drive could be reduced PKA activation, enabling increased ERK1/2 activation and reduced initiation of the heat shock response (23). However, in the present study, trained animals exhibited similar exercise-induced activation of PKA as their sedentary counterparts. Given that β-adrenergic receptor content at the myocardial membrane was unaltered as a result of training, these results would suggest that alterations in β-adrenergic receptor signaling occurred (i.e., enhanced receptor binding affinity) to maintain PKA activation following acute exercise. Consistent with these data are reports of heightened sensitivity of the β-adrenergic receptor to sympathetic stimuli following exercise training, with both enhanced receptor binding affinity and PKA activation (11, 22).

Despite a similar exercise-induced activation of PKA in sedentary and trained animals, ERK1/2 activation was significantly elevated in trained animals following acute exercise. Contrary to our previously reported data demonstrating a negative influence of PKA on ERK1/2 activation following acute exercise (23), exercise training must influence an intermediate step between these signaling systems allowing for their simultaneous activation. Interestingly, during chronic catecholamine stimulation of the β-adrenergic receptor, such as would occur during the onset of repetitive exercise training, PKA mediates the binding of β-ARK and β-arrestins to the receptor, resulting in its internalization (10). Once internalized, the receptor then acts as a molecular scaffold to bind and initiate the activation of ERK1/2 and other related signaling components associated with cellular growth (36). One plausible reason for such an adaptation is that ERK1/2 activation could initiate favorable training-induced myocardial growth (37) without diminishing PKA signaling to other exercise sensitive systems. Consistent with our results, it has been shown that β-adrenergic-stimulated myocardial hypertrophy occurs in conjunction with a 2.8-fold increase in β-ARK expression (15). While the present study does not demonstrate a significant reduction of either the β1- or β2 -adrenergic receptor in trained animals, there was a significant elevation in the training-induced expression of membrane β-ARK levels. Furthermore, we demonstrated a significant increase in heart-to-body weight ratios in trained animals compared with the other groups. An increase in heart-to-body weight ratio has been used as a marker to indicate myocardial hypertrophy (16).

Following acute exercise, there appears to be a delicate balance that exists between proliferative stimuli emanating from ERK1/2 activation and induction of hsp70 gene expression (23). However, upon exercise training, these two responses coexist, thereby allowing the heart to exhibit ERK-mediated responses associated with training while maintaining induction of the cardioprotective heat shock response following acute exercise. As such, these results indicate that training must initiate alternative mechanisms to elicit expression of the hsp70 gene. One such mechanism might involve a training-induced enhancement of HSF1 DNA binding affinity to the hsp70 gene. Indeed, trained animals in the present study demonstrated a significant increase in HSF1-HSE DNA binding interactions compared with sedentary animals following acute exercise. Elevated basal levels of hsp70 gene transcription as a result of enhanced HSF1-HSE DNA binding could maintain the normal elevation of Hsp70 mRNA following acute exercise, even in the presence of training-related increases in ERK1/2 activation (34). Alterations in the transcriptional regulation of the hsp70 gene have also been reported following heat shock treatment. For instance, 30 days of heat acclimatization did not alter the heat shock-induced expression of the hsp70 gene despite an increase in baseline levels of Hsp70, which might normally be expected to inhibit transcriptional activation (21). It is worth noting that in addition to the novel mechanism reported herein of enhanced HSF1-HSE DNA binding, posttranscriptional mechanisms may also play a role in the expression of the hsp70 gene following training. Factors such as increased stability of Hsp70 mRNA and the rate by which the Hsp70 transcript undergoes translational synthesis have been reported to influence the expression of Hsp70 (35). Finally, transcriptional cofactors associated with HSF1 may influence transcriptional activation of the hsp70 gene in trained animals. Indeed, several coactivators have been reported to play essential roles in the transcriptional activation of the HSF1 molecule following heat shock (14). While it is presently unclear as to the role of these cofactors following acute exercise, it is plausible that enhanced activation of the HSF1 molecule is due to the upregulation of these cofactors following training.

The primary finding of the present investigation is that 8 wk of mild to moderate exercise training does not alter the activation of transcriptional expression of the heat shock response following acute exercise. Exercise training enhances the myocardial constitutive content of Hsp70 and does not diminish the capacity to robustly respond to an additional stress with further increases in this protein. This occurs despite an exercise-induced activation of ERK1/2, which has been shown to suppress the transcriptional activation of the hsp70 gene (23). This work also suggests a novel manner (increases in HSF1-HSE DNA binding) by which the normal regulation associated with hsp70 gene expression following an unaccustomed exercise bout is altered with exercise training. Importantly, this allows the myocardium to favorably adapt to exercise through physiological cardiac hypertrophy while maintaining a vigorous response to potential stressors.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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REFERENCES
 
This work was supported by National Science and Engineering Research Council of Canada Grant 8170-05 RGPIN and Heart and Stroke Foundation of Ontario Grant T-5036 (to E. G. Noble) and by Ontario Graduate Scholarships for Science and Technology (to C. W. J. Melling).


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. G. Noble, School of Kinesiology, Faculty of Health Sciences, and Lawson Health Research Institute, The Univ. of Western Ontario, London, Ontario, 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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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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