1 School of Kinesiology, Faculty of Health Sciences, 2 Lawson Health Research Institute, and 3 Department of Pharmacology and Toxicology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 3K7; 4 Faculty of Physical Education and Health, University of Toronto, Toronto, Ontario, Canada M5S 2W6; and 5 School of Dentistry, Oregon Health and Science University, Portland, Oregon 97201-3098
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ABSTRACT |
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Experiments involving chemical induction of the heat shock response in simple biological systems have generated the hypothesis that protein denaturation and consequential binding of heat shock transcription factor 1 (HSF1) to proximal heat shock elements (HSEs) on heat shock protein (hsp) genes are the result of oxidation and/or depletion of intracellular thiols. The purpose of the present investigation was to determine the role of redox signaling of HSF1 in the intact animal in response to physiological and pharmacological perturbations. Heat shock and exercise induced HSF1-HSE DNA binding in the rat myocardium (P < 0.001) in the absence of changes in reduced glutathione (GSH), the major nonprotein thiol in the cell. Ischemia-reperfusion, which decreased GSH content (P < 0.05), resulted in nonsignificant HSF1-HSE formation. This dissociation between physiological induction of HSF1 and changes in GSH was not gender dependent. Pharmacological ablation of GSH with L-buthionine-[S,R]-sulfoximine (BSO) treatment increased myocardial HSF1-HSE DNA binding in estrogen-naive animals (P = 0.007). Thus, although physiological induction of HSF1-HSE DNA binding is likely regulated by mediators of protein denaturation other than cellular redox status, the proposed signaling pathway may predominate with pharmacological oxidation and may represent a plausible and accessible strategy in the development of HSP-based therapies.
protein denaturation; exercise; glutathione; heat shock protein; ischemia-reperfusion
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INTRODUCTION |
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A HIGHLY CONSERVED, ubiquitous endogenous defense mechanism is the induction of heat shock proteins (HSPs) in response to proteotoxic stress. Aside from this protective feature, of particular interest in cardiovascular research, biologists have used the heat shock response as a model by which to study cellular sensing mechanisms.
In the unstressed cell, the products of the heat shock response, the HSPs themselves, are bound to inactive, monomeric heat shock transcription factors (HSF1; Refs. 1, 35, 44). Initiation of the response requires HSF1 trimerization for high-affinity binding with proximal promoter heat shock elements (HSEs) on hsp transcriptional units (28, 47, 52). HSPs serve to maintain intracellular components by assisting in the proper folding of nascent polypeptides and in the refolding of anative proteins, preventing aggregation and aiding in translocation and degradation of peptides (4, 8, 9, 12, 25). Stress-induced increases in intracellular levels of anative proteins require increased chaperoning activity for the maintenance of cellular homeostasis. Such demand for HSPs permits HSF1 trimerization and the acquisition of DNA binding competency and consequential upregulation of hsp gene expression. Indeed, heat shock and various other inducers of the response have been shown to increase cellular proteotoxicity (27, 36). Moreover, introduction of anative proteins into otherwise quiescent biological systems results in HSP induction (2, 20, 34). Thus protein denaturation is a key cellular signal by which the HSP response is regulated.
Observations of a wide variety of inducers of the heat shock response
converging to a single cellular event have led to the question of
whether this convergence occurs at the level of protein denaturation or
whether there is a more proximal merger. Russo et al. (42)
first observed that depletion of cellular reduced glutathione (GSH)
resulted in thermotolerance and concomitant synthesis of HSPs. Further
work continued this characterization using a variety of experimental
models and perturbations to manipulate intracellular redox status,
demonstrating that oxidation and depletion of nonprotein thiols results
in HSF1 activation, hsp gene transcription, and HSP
synthesis and, moreover, that maintaining a reducing cellular environment inhibits this response (14, 15, 17-19, 21, 22, 24, 29, 32, 43, 45, 51). This led to the hypothesis that protein
denaturation and consequential activation of HSF1 are the result of
anative protein modifications caused by oxidation and/or depletion of
intracellular nonprotein thiols (Fig. 1;
Ref. 51).
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Although well supported in simple biological systems using chemical inducers of HSF1, this hypothesis has not been addressed in higher-order experimental systems, which limits its physiological relevance and potential applicability to HSP-based therapeutic research. Thus the purpose of the present investigation was to determine the role of redox signaling of HSF1 in the intact animal in response to physiological and pharmacological perturbations. Because GSH is the most prominent intracellular nonprotein thiol (46), changes in GSH levels should accurately reflect cellular thiol oxidation (21, 33, 46, 51). Because of the established cardioprotective potential of HSPs, the relevance of the current hypothesis to the regulation of the heat shock response in the heart was of particular interest.
Furthermore, we previously demonstrated (37, 38, 40) a gender-specific HSP response, with males demonstrating twofold greater levels of HSP70 than females after exercise. Removal of the ovaries, the major endogenous source of estrogen in females, resulted in increased induction of HSP70, similar to that observed for males. Exogenous replacement of estrogen in these animals reversed this effect. Because estrogen is an antioxidant compound (50), it was of interest to us to determine whether the ovarian hormone was mitigating HSP induction through this proposed redox signaling system. Thus the present experiments were conducted in estrogen-positive and estrogen-naive animal models.
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METHODS |
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The study was approved by the University of Western Ontario Council on Animal Care and was performed in accordance with the guiding principles of the Canadian Council on Animal Care. Eleven-week-old male, gonadally intact female, and ovariectomized female (major source of estrogen removed; ovariectomy performed at 8 wk) rats, purchased from Charles River, were housed two per cage in an environmentally controlled room with a 12:12 h dark-light cycle with food and water ad libitum.
Experimental procedures.
Animals were chosen at random for experimental treatment. Nonexertional
hyperthermia was induced as previously described (n = 4 males, 4 females; Ref. 48). Animals were lightly
anesthetized with Somnitol (30 mg/kg ip) and placed on heating pads.
Colonic temperatures were maintained between 41.5 and 42.0°C for 20 min, after which hearts were immediately extirpated. Exercise consisted of treadmill running at 30 m/min for 60 min. In the experiments reported in Fig. 3, animals were anesthetized immediately after exercise with Somnitol (60 mg/kg ip) and hearts were extirpated (n = 4 males, 4 females). Exercise experiments outlined
in Fig. 4 are those in which animals were decapitated to minimize the time between the end of the exercise bout and harvesting of tissues (n = 6 ovariectomized, 6 intact per treatment group).
Myocardial ischemia-reperfusion was performed with a modified
Langendorff procedure (31), with a 30-min zero-flow global
ischemic period followed by a 30-min period of reperfusion
(n = 4 males, 4 females). Tissues were immediately
frozen in liquid nitrogen and stored at 80°C for analysis.
Pharmacological manipulation of myocardial GSH.
Pharmacological manipulation of cardiac GSH content was achieved
by treatment with
L-buthionine-[S,R]-sulfoximine
(BSO), a specific inhibitor of -glutamylcysteine synthetase, the
rate-limiting enzyme in the synthesis of glutathione (33).
Rats were either treated with BSO (1 g/kg ip, dissolved in
physiological saline) or sham injected 24 and 3 h before
experimental treatment (n = 6 ovariectomized, 6 intact
per treatment group).
HSF1-HSE DNA binding. Gel mobility shift assays were performed with tissue samples homogenized in 15 vols of extraction buffer as per Locke et al. (30). Protein concentration was determined with a Bio-Rad assay modified for microplate analysis. One hundred micrograms of myocardial extracts were incubated with 1 ng of 32P-labeled self-complementary ideal HSE oligonucleotide (5'-CTAGAAGCTTCTAGAAGCTTCTAG-3') and separated on full-size nondenaturing polyacrylamide gels as previously outlined (30). Competition experiments were performed with 200-fold molar excess of cold HSE and Oct2A oligonucleotides, and supershift experiments were carried out with monoclonal anti-serum specific for HSF1 (Neomarkers).
GSH measurement.
Hearts (100 mg/ml) were homogenized in 10% perchloric acid with 15 µM -glutamylglutamate, which was used as HPLC standard. GSH content was assessed by HPLC as described by Farris and Reed (10) and Freeman et al. (16).
Statistical analysis. Quantitation of blots was carried out with Scion image analysis software (National Institutes of Health). HSF1-HSE oligonucleotide binding is reported as percentage of internal standard (100 µg of cardiac extract from heat-shocked male Sprague-Dawley rat; means ± SE). Group data were compared by analysis of variance among treatment groups. Pairwise comparisons were conducted with a Tukey post hoc test, where the minimum level of significance was assigned as P < 0.05.
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RESULTS |
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HSF1-HSE DNA binding.
To establish a gel mobility shift assay specific for HSF1 and the HSE
oligonucleotide, several control experiments were performed (Fig.
2). No mobility shift was observed for
lanes loaded with the labeled HSE alone or for cardiac extracts from
control hearts (see following sections). Heat shock and exercise
treatments resulted in retarded migration of the labeled
oligonucleotide, indicating the presence of a factor in the cardiac
extracts with HSE binding competency. Competitive experiments with a
200-fold molar excess of unlabeled HSE resulted in a loss of the
mobility shift, whereas competition with a control oligonucleotide,
Oct2A, did not, indicating specificity of the cellular binding factor
for the HSE oligonucleotide. Addition of antibodies specific for HSF1
resulted in a supershift, indicating that the factor in extracts from
heat-shocked and exercised animals responsible for retarding migration
of the labeled HSE was HSF1. Thus the present gel mobility shift assay
is specific for HSF1-HSE DNA binding.
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Dissociation of HSF1 activation and changes in GSH with
physiological stimuli.
Heat shock and exercise markedly induced cardiac HSF1-HSE
oligonucleotide binding in males and females (P < 0.001; Fig. 3). However, neither heat
shock nor exercise was accompanied by alterations in cardiac GSH levels
in either males or females. Analysis of mixed cysteinyl glutathione,
the mixed disulfide between glutathione and cysteine, also indicated
low levels of oxidation with these perturbations (data not shown).
Ischemia-reperfusion, a well-established model of oxidative
stress, diminished myocardial GSH content (and increased cysteinyl
glutathione; P < 0.05). However, only minimal, statistically nonsignificant HSF1-HSE binding was observed in these
experiments. Thus, in intact animal models of both genders, heat shock
and exercise, physiological inducers of HSF1 DNA binding, were not
associated with alterations in cardiac GSH and physiological depletion
of GSH was not accompanied by induction of HSF1.
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Pharmacological depletion of GSH induces cardiac HSF1-HSE DNA
binding.
Decreasing myocardial GSH levels via BSO treatment resulted in
significantly greater levels of HSF1-HSE binding relative to sham
treatment in ovariectomized animals (P = 0.007; Fig.
4). In line with previous work with
simple biological systems, these findings represent an important
positive control for the present work and indicate the potential
for redox-mediated induction of cardiac HSF1. To investigate a
possible additive effect of chemical and physiological stimuli on
induction of HSF1, animals were administered BSO before exercise.
However, such treatment did not potentiate the response. BSO treatment
in intact females, which also resulted in decreased GSH content
(P = 0.008), had no effect on HSF1-HSE binding.
Thus pharmacological depletion of GSH activated myocardial HSF1-HSE
oligonucleotide binding in estrogen-naive but not estrogen-positive animals.
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Constitutive HSF1-HSE DNA binding is dependent on method of
euthanasia.
The first series of experiments, those illustrated in Fig. 3, were
performed with animals killed by cardiac extirpation after anesthetization. Extracts from control animals subjected to such treatment demonstrated no HSF1-HSE binding. Because GSH levels are
transient, to ensure that GSH measurement was not hindered by the time
required for this process, subsequent experiments employed decapitation
as the method of euthanasia (Fig. 4). Myocardial extracts from these
control animals demonstrated constitutive HSF1-HSE
oligonucleotide binding. To more discriminately determine the
effect of different euthanizing techniques on the activation state of
HSF1, animals were killed by one of three methods commonly employed by
animal researchers: 1) cardiac extirpation after
anesthetization (extirpation); 2) exsanguination before
extirpation (exsanguination); and 3) decapitation.
Extirpation of the heart after pentobarbital treatment produced no DNA
binding (Fig. 5, lane 1).
Exsanguination before extirpation resulted in a low level of
extract-oligonucleotide interaction (Fig. 5, lane 2).
Cardiac extracts from decapitated animals demonstrated a consistent and
high degree of HSE oligonucleotide binding (Fig. 5, lane 3)
comparable to that observed after heat shock (Fig. 5, lane
4; Table 1). Moreover,
extract-oligonucleotide binding affinity was similar between
decapitation and heat shock conditions as extract-oligonucleotide
binding reaction temperatures of 25, 30, and 37°C resulted in a
proportionate decrease in signal (data not shown). Because we
previously documented (37, 38, 40) gender-specific,
hormone-mediated HSP induction, these experiments were performed on
male, intact female, and ovariectomized female rats. However, neither
gender nor hormonal status appeared to influence this pattern of
response.
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DISCUSSION |
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A major deficiency in HSP research is a lack of understanding of the regulation of these transcriptional units in complex biological systems. Although the response is highly conserved evolutionarily, there have been reports of interorganismal and, indeed, interspecies variations (11, 47). Our understanding of the mechanisms of the HSP response is derived largely from in vitro and cell culture experiments. Validating these hypotheses in higher-order experimental models tremendously increases the physiological significance of such work and is a prerequisite to harnessing the protective potential of these critical cellular components.
The present study was undertaken to investigate a hypothesis, well developed and well supported in simple biological systems with chemical inducers of the heat shock response, in whole animal models. The possibility that this highly complex endogenous defense mechanism may be signaled by a single early cellular event, which is relatively easy to manipulate, is an attractive postulate for potential applications in cardiac pathology and preventive medicine, particularly as redox-related compounds such as BSO have been tested in clinical trials.
If the hypothesized signaling pathway is involved in HSF1 acquiring DNA binding competency in response to physiological inducers, then such perturbations should cause thiol depletion. Although heat shock and exercise resulted in marked HSF1-HSE DNA binding, neither stressor was accompanied by decreases in cardiac GSH content in either males or females. It should be noted that such treatments may have caused some transient cellular oxidation but because of the time course selected and/or the speed at which glutathione reductase catalyzes its reaction these alterations were not detected. However, analysis of cysteinyl glutathione, the mixed disulfide between cysteine and glutathione, a more stable marker of cellular oxidation, revealed a similar pattern of low-level oxidation after heat shock and exercise. Moreover, in an effort to minimize the time interval between the completion of exercise and the harvesting of hearts, GSH data from animals killed by decapitation also revealed a lack of change in GSH content. Thus exercise-induced activation of HSF1 is not likely mediated through cellular oxidation. Furthermore, ischemia-reperfusion, which was effective in decreasing GSH levels (and increasing cysteinyl glutathione levels), resulted in only faintly detectable and statistically nonsignificant HSF1-HSE DNA binding, findings inconsistent with the present hypothesis. Thus, although the data presented do not completely rule out the possibility of changes in redox status and association with activation of HSF1, in response to physiological stimuli induction of myocardial HSF1 is likely regulated predominantly by mechanisms other than redox signaling.
If thiol oxidation can signal HSF1 DNA binding in the whole animal, then pharmacological depletion of GSH should induce the response. Indeed, depletion of myocardial GSH with BSO treatment resulted in increased HSF1-HSE DNA binding in ovariectomized (estrogen naive) animals. These findings indicate that alterations in cardiac redox status can signal activation of HSF1 in response to pharmacological oxidation. Such findings, however, were not observed for intact females (estrogen positive) despite the effectiveness of BSO in depleting GSH in these animals. Such discrepancy may be due to the indirect antioxidant cell membrane stabilizing activity of estrogen (50), particularly as denaturation of membrane-bound proteins has been shown as a key regulator of HSP induction in response to nonthermal stress (13, 43). Alternatively, the effect of BSO on HSF1-HSE binding observed in ovariectomized animals may be related to effects of the compound other than those related to oxidation. Thus estrogen may attenuate BSO-induced activation of HSF1 through such nonspecific mechanisms.
The rationale for including estrogen-naive and estrogen-positive models in the present series of experiments is derived from our previous findings of gender-specific, hormone-mediated HSP regulation (37, 38, 40). Gender has been a discounted factor in biomedical research, and it is only beginning to become apparent how great an impact sex and hormonal status have on biological function. The disparate effects of pharmacological induction of HSF1 between estrogen-naive and estrogen-positive animals serves as yet another example of this and may indicate that exploitation of this signaling system in cardiac therapeutics may also be dependent on these factors.
In the above series of experiments, an unexpected observation was made. To minimize the time required for harvesting of tissues, animals were euthanized by decapitation. Cardiac extracts from control animals subjected to this treatment demonstrated significant HSF1-HSE oligonucleotide binding. Control animals killed by extirpation demonstrated no such response. To more discriminately investigate the influence of method of euthanasia on induction of HSF1 DNA binding, animals were euthanized by three techniques commonly employed by animal researchers. Decapitation consistently resulted in dramatic HSF1-HSE DNA binding relative to exsanguination and extirpation. This phenomenon, although beyond the scope of the present work, may be the result of hormonal signaling of the stress response (3, 11, 39, 49), because decapitation results in dramatic increases in plasma catecholamines (41) and tissue levels of second messenger activity (26). Relevant to the present question, there were no differences in myocardial GSH levels between decapitated animals and those killed by extirpation, despite the marked HSF1-HSE binding observed with the former treatment. Such observations provide an additional nonpharmacological model of HSF1 induction that is likely regulated by mechanisms other than those mediated by cellular redox state.
This proposed relationship between redox state and induction of HSF1
was of particular interest to us because we previously reported
(37, 38, 40) a gender-specific, hormone-mediated HSP
response. After exercise, male rodents demonstrated higher HSP70 levels
than females and estrogen administration to estrogen-naive animals
resulted in an attenuated, femalelike response. Estrogen has been
characterized as an antioxidant (50). Thus, if cellular oxidation was involved in signaling the HSP response, then estrogen may
mitigate HSP signaling through this cascade. However, exercise-induced activation of HSF1 was not associated with changes in GSH levels, and
moreover, there were no differences in cardiac GSH levels among male,
intact female, and ovariectomized animals with exercise. These
observations are consistent with our previous reports (37, 38) indicating that estrogen-mediated attenuation of HSP
induction is likely conferred through physicochemical membrane
stabilization. Antioxidant compounds with structural and
membrane-stabilizing properties similar to those of 17-estradiol,
the major endogenous estrogen in mammalian systems, mitigated HSP
induction in a fashion similar to that of the hormone. Therefore, the
mechanism by which estrogen attenuates HSP induction is not likely
related to cellular redox state but rather to indirect antioxidant
stabilization of cellular membranes.
The present investigation was undertaken to test the hypothesis that intracellular thiol oxidation serves as a proximal signal for HSF1-HSE DNA binding in the whole animal. This issue was previously addressed indirectly in the intact animal. Ethanol treatment in rats resulted in cellular oxidation and increased HSP70 content in the central nervous system (5, 6). Ito et al. (23) demonstrated induction of hsp32 mRNA in rat liver and kidney after depletion of GSH with BSO. It should be noted, however, that these studies assessed distal parameters in the heat shock response. The present hypothesis specifically requires HSF1-HSE DNA binding as the critical outcome of protein denaturation. Because proximal promoter elements, other than HSEs, have been shown to mediate HSP synthesis with nondenaturing stimuli (7), the alterations in HSP and hsp mRNA levels in the above studies may not have been HSF1-HSE mediated. Thus this is the first study to directly investigate the relationship between redox state and HSF1-HSE DNA binding in the intact animal.
Clearly, activation of HSF1 is dependent on protein denaturation. The present results indicate that the proximal events in this process may be stress specific as physiological induction is not likely related to redox signaling but perhaps is a consequence of thermal unfolding, mechanical disruption, and proteolytic and/or proteasome activity. However, activation of the response with pharmacological oxidation, in both simple and complex biological systems, may be mediated through the current hypothesized signaling cascade. Transgenic and gene transfection approaches have clearly established the cardioprotective potential of the heat shock response. However, such perturbations must overcome significant obstacles before they are employed in therapeutics. Although the present study does not support the hypothesis that cellular oxidation is a proximal signal common to all inducers of HSF1, exploiting this signaling system may represent a plausible and accessible strategy in the development of HSP-based therapies.
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ACKNOWLEDGEMENTS |
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This research was made possible by a Natural Sciences and Engineering Research Council of Canada postgraduate scholarship to Z. Paroo, an operating grant and a Career Investigator Award from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Ontario, respectively, to M. Karmazyn, a Natural Sciences and Engineering Research Council of Canada and Heart and Stroke Foundation of Canada operating grant (NA-4445) to E. G. Noble, and the St. Joseph's Hospital Foundation Doris May Anderson Fund.
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FOOTNOTES |
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Address for reprint requests and other correspondence: E. Noble, Rm. 2160C Thames Hall, Faculty of Health Sciences, 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.
March 27, 2002;10.1152/ajpcell.00051.2002
Received 31 January 2002; accepted in final form 23 March 2002.
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