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1School of Kinesiology, Faculty of Health Sciences, and 2Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada
Submitted 29 October 2004 ; accepted in final form 3 November 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-dihydrotestosterone (DHT) would show the intact male response. Fifty-four 8-wk-old male Sprague-Dawley rats were divided into intact, castrated, or castrated + DHT groups (n = 18/group). At 11 wk of age, 12 animals in each group undertook a 60-min bout of treadmill running at 30 m/min (2% incline) while the remaining 6 in each group remained sedentary. At 30 min or 24 h after exercise (n = 6/time point), blood and hearts were harvested for analysis. Serum T was undetectable in castrated and DHT-treated castrated rats, whereas serum DHT was significantly reduced in castrated animals only (
60% reduction) (P < 0.05). Although there were no differences in constitutive levels of Hsp70 protein, exercise significantly increased cardiac hsp70 mRNA and protein in intact and DHT-supplemented rats, but not in castrated animals (P < 0.05). To examine whether castration eliminated the ability to respond to stress, another six intact and six castrated animals were subjected to a 15-min period of hyperthermia (core temperature raised to 42°C) and killed 24 h later. As opposed to exercise, castrated animals subjected to heat shock exhibited increases in Hsp70 above nonshocked (i.e., sedentary) animals, similarly to intact males (P < 0.05). These data suggest that androgens, in addition to estrogen, play a role in the sexual dimorphism observed in the stress response to exercise but not heat shock.
heat shock protein 70; treadmill running; dihydrotestosterone; 
1-adrenergic receptor
Androgen effects on cardiovascular function and the role of the "male" sex hormones in cardiac sex differences are just now emerging (for review see Ref. 11). Although some studies have suggested a cardioprotective role for testosterone, others have not. For example, elimination of endogenous testosterone production by castration results in significantly reduced left ventricular function and cardiac contractile performance in postpubertal male rats (24). Function is restored with exogenous testosterone supplementation (25). Conversely, castration of male mice significantly reduces mortality and cardiac rupture after induced myocardial infarction (4). Nonetheless, these equivocal findings show that androgens play a much larger role in sex differences in cardiovascular function than can be accounted for by estrogen alone, as estrogen levels do not change with castration (9).
Therefore, we set out to determine how androgens influence the constitutive and exercise-induced expression of Hsp70 in cardiac tissue. It was hypothesized that removal of the primary production of endogenous testosterone by castration would reduce the accumulation of Hsp70 after exercise and that supplementation of castrated animals with testosterone's more active 5-reduced metabolite, 5-dihydrotestosterone (DHT), would return the intact male response. As previous research suggested modification of 1-adrenergic receptor expression in the hearts of castrated animals (7) and it was previously shown that a downstream signaling protein (PKA) is necessary for the exercise-induced increase in cardiac Hsp70 (17), it was further hypothesized that we would see a reduction in both cardiac 1-adrenergic receptor expression and density with castration that would return with DHT supplementation.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Use and treatment of laboratory animals was approved by the University of Western Ontario Council on Animal Care according to the guidelines of the Canadian Council on Animal Care. Adult male and similarly aged castrated male Sprague-Dawley rats (8 wk old) were obtained from Charles River Laboratories and housed in triplicate in standard shoebox rat cages. The vivarium was maintained at constant temperature and humidity with a 12:12-h light-dark cycle. All rats were fed LabDiet 5P00 standard rat chow and water ad libitum.
Hormone supplementation.
Upon arrival, 18 castrated rats were lightly anesthetized with pentobarbital sodium (25 mg/kg body wt), and a small incision was made at the nape of the neck. Subsequently, a 2.5-mg, 21-day, time-release pellet containing DHT (Innovative Research of America) was inserted under the skin 1 in. from the incision point with a 12-gauge trochar. DHT is a nonaromatizable metabolite of testosterone that has several times more affinity for the androgen receptor and is responsible for 95% of testosterone's anabolic effects. Moreover, it cannot be converted to estrogen. This is an important characteristic of the hormone, because estrogen has been shown to induce the expression of Hsp70 in adult male cardiac myocytes (10).
Acute exercise protocol.
At 10 wk of age, intact male, castrated, and castrated + DHT rats were randomly assigned to either a sedentary control group or an exercise group. Exercised rats were familiarized to treadmill running for 10 min at 5 and 3 days before the acute exercise bout (at 11 wk of age). Familiarization consisted of 2 min of running at 15 m/min, 4 min at 24 m/min, 2 min at 30 m/min, and 2 min at 15 m/min, all at a 2% incline. Rats were encouraged to run with a gentle air blast that blew on their hindquarters when they broke a photo sensor beam near the rear of the treadmill. Although the air blast was generally sufficient to keep the rats running, if they stopped on the grid at the back of the treadmill, they were further encouraged to run by the administration of a brief electric shock or air blast. Neither the familiarization nor the stimuli used to encourage running elicit a significant heat shock or stress response (unpublished results). Each exercise bout consisted of continuous treadmill running at 30 m/min for a period of 60 min (2% incline).
Animals were weighed, and rectal temperature was measured immediately before the exercise bout with a thermometer inserted 5 cm into the rectum. Rectal temperature was subsequently measured at 30 min and immediately after the run (60 min). Peak temperature was determined to be the greater of either the 30 min or 60 min temperature measure. Control rats were handled similarly to their exercising counterparts without being placed on the treadmill. All animals completed the full hour of their respective exercise bouts.
Weight-matched exercised castrated group.
Castrated animals generally exhibited lower body weights and peak exercise temperatures than either intact or castrated animals supplemented with DHT. To ensure that the results observed in the present study were not due to differences in weight and temperature and consequently reduced exercise stress, a separate group of castrated animals (n = 6) were housed until their body weights approximated those seen in the intact and DHT-supplemented groups. These animals then went through the familiarization and exercise protocol as outlined above and were killed 24 h after the exercise bout for analysis of Hsp70 protein expression.
Heat shock protocol.
Animals were lightly anesthetized with pentobarbital sodium (35 mg/kg) and wrapped in a heating blanket for whole body heating. Rectal temperature was measured as noted above for the duration of the experiment. Animals were heated until rectal temperature reached 42°C and then maintained at this temperature for 15 min. At the end of the 15 min, the animals were removed from the heating source and allowed to recover at room temperature. They were monitored and given water until they were completely self-sufficient, at which point they were returned to their normal cages. These animals were killed 24 h after the heating protocol for analysis of Hsp70 expression.
Tissue collection.
At 30 min and 24 h after the acute exercise bout (n = 6/time point), animals were weighed, and tissues were subsequently collected while they were under pentobarbital sodium anesthesia (65 mg/kg). Thirty minutes and 24 h after exercise were chosen to correspond with the time points at which hsp70 mRNA and Hsp70 protein are elevated, respectively (17). Blood was collected from the abdominal aorta immediately before removal of the heart (within 10 s). The left ventricle of the heart was harvested, rapidly frozen in liquid nitrogen, and subsequently stored at –70°C until further analysis. Whole blood was centrifuged at 10,000 g for 10 min, after which the serum was transferred to microcentrifuge tubes and stored at –20°C for later analysis.
Cardiac membrane isolation.
Cardiac membranes were isolated for the measurement of 1-adrenergic receptor binding with a modified version of the method outlined by Persad et al. (22) and Wang et al. (29). Briefly, 200 mg of ventricular cardiac tissue was homogenized in 50 mM Tris·HCl, pH 7.4 (15 ml/g tissue). The resulting homogenate was centrifuged at 1,000 g for 10 min, and the pellet was discarded. The supernatant was centrifuged at 48,000 g for 25 min. The pellet was resuspended in the Tris·HCl buffer and centrifuged again at the same speed. The final pellet was resuspended in 50 mM Tris·HCl, pH 7.4, containing 25 mM sucrose and 10 mM histidine, and stored at –70°C until further analysis.
1-Adrenergic receptor antagonist binding.
1-Adrenergic receptor binding was accomplished with a modified version of the method described by Persad et al. (22). Briefly, 0.1 mg/ml of the isolated cardiac membranes from sedentary intact, castrated, and DHT-treated castrated animals (n = 6/group) were incubated with 200 pM 125I-labeled cyanopindolol ([125I]CYP; 2,000 Ci/mmol, Amersham Biosciences) for 60 min at 37°C in the presence or absence of 50 µM atenolol (a 1-adrenergic receptor-specific antagonist). This concentration of [125I]CYP was used because preliminary saturation binding assays showed that there were no differences in affinity (Kd) between treatment groups and 200 pM [125I]CYP was sufficient to saturate the membrane-bound receptors. Saturation assays were run in triplicate in a total volume of 250 µl/well, using a 96-well GF/C Multiscreen filter plate (Millipore, Bedford, MA), and terminated by rapid filtration through GF/C filters. The filters were washed three times with ice-cold buffer and then dried for at least 2 h at 60°C before being read in a Beckman 5500B gamma counter. 1-Adrenergic receptor density was determined by subtracting the nonspecific binding (displaced by 50 µM atenolol) from the mean total [125I]CYP bound for each sample.
Circulating hormone levels.
Serum testosterone and DHT were measured according to manufacturers' guidelines with commercially available enzyme immunoassay (EIA) (ICN Pharmaceuticals, Orangeburg, NY) and RIA (Diagnostic Systems Laboratories, Webster, TX) kits, respectively. The EIA for testosterone and RIA for DHT have detection limits of 0.06 ng/ml (0.86% cross-reactivity with DHT) and 4 pg/ml (0.02% cross-reactivity with testosterone), respectively.
hsp70 gene expression.
Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction (5) and slot blotted as previously described (20).
Western blotting.
Approximately 70 mg of tissue was cut from the frozen muscle samples, immediately homogenized in 15 volumes of extraction buffer (600 mM NaCl, 15 mM Tris base, pH 7.5), and centrifuged at 16,000 g for 20 min to collect the supernatant as the myocardial extract. Sample homogenates were then stored at –70°C until determination of total protein concentration and electrophoresis. Determination of total protein concentration was accomplished with the Bradford protein assay (1). Equal amounts of protein were run on 10% [for detection of heat shock transcription factor (HSF)1] or 12% (for all other proteins) acrylamide minigels (Bio-Rad) overlaid with a 4% stacking gel. A standard sample (pooled soleus obtained from 8- to 11-wk-old male rats) was loaded on each gel to use as a standard for Hsp70. After electrophoresis, the proteins were transferred at a constant voltage in cold transfer buffer (10% running buffer, 20% methanol in double-distilled H2O) to nitrocellulose membranes. The membranes were blocked in a 5% milk Blotto powder solution in Tris-buffered saline (TBS) + 0.05% Tween 20 (TTBS) (80 mM Tris base, 0.5 M NaCl) overnight. Membranes were then incubated in primary antibody specific to Hsp70 (anti-Hsp70 polyclonal antibody, Stressgen SPA-812), HSF1 (anti-HSF1 polyclonal antibody, Affinity Bioreagents PA3–017), or 1-adrenergic receptor (anti-1-adrenergic receptor polyclonal antibody, sc-568, Santa Cruz Biotechnology) diluted 1:4,000, 1:10,000, and 1:500, respectively, in TTBS with 2% milk Blotto powder for 2 h. Membranes were washed again in TTBS and then incubated in secondary antibody (goat anti-rabbit antibody conjugated to alkaline phosphatase for detection of Hsp70 or to horseradish peroxidase for detection of 1-adrenergic receptor and HSF1; Bio-Rad) in TTBS with 2% milk for 1 h. For detection of Hsp70, after washes in TTBS and TBS, the blots were developed with 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium color developing reagents (Bio-Rad 170-6532) in 100 ml of developing buffer. 1-Adrenergic receptor and HSF1 blots were developed with chemiluminescent detection according to manufacturers' directions and exposed to Kodak BioMax Light film. The blots were scanned, and then Scion Image blot analysis software was used for densitometric quantification.
Statistics.
Statistical analysis was performed with Sigma Stat for Windows, version 2.03. To compare treatment groups, a two-way ANOVA was used. When a significant F-ratio was found, a Tukey's post hoc test was used to determine significant (P < 0.05) differences between groups. All data are expressed as means ± SE.
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RESULTS |
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Body weights of intact, castrated, and castrated + DHT animals were similar upon arrival (8 wk of age). Intact and castrated + DHT rats gained more weight over the duration of the experiment, such that by 10 and 11 wk, respectively, significantly greater body weights were observed compared with castrated rats (P < 0.05; Fig. 1).
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Hormone levels.
Circulating hormone levels are presented in Table 1. Serum testosterone immediately before death was decreased to undetectable levels in castrated animals (P < 0.05). Serum DHT was greater in intact and castrated rats supplemented with DHT than in castrated animals (P < 0.05), indicating the effectiveness of both castration and the implanted hormone pellets in decreasing and returning hormone levels to normal, respectively. Intact males showed greater variability in circulating levels of DHT compared with supplemented and castrated animals. This is likely because of a continuous release from the implanted hormone pellets as opposed to a cyclic release in intact animals.
hsp70 mRNA and Hsp70 protein expression after exercise.
There was a significant main effect for exercise on the 30-min postexercise expression of hsp70 mRNA (P < 0.05); however, post hoc analysis only revealed a significant difference between sedentary and exercised animals in the intact (P < 0.05) and DHT-supplemented (P < 0.05) groups (Fig. 2A). Constitutive levels of cardiac Hsp70 protein were similar among sedentary intact, castrated, and castrated DHT-supplemented animals. Hsp70 accumulated in the hearts of intact male rats after exercise similarly to data we reported previously (18). Castrated rats did not show an increase of cardiac Hsp70 in response to exercise, whereas castrated animals supplemented with DHT exhibited an exercise-induced accumulation of Hsp70 similar to that in intact males (Fig. 2B). Moreover, when a separate group of castrated animals was allowed to age until their body weights approximated those of the intact and DHT-supplemented animals, no accumulation of cardiac Hsp70 was observed, suggesting that the differences observed in the present study were the result of effects of castration other than decreased work output due to smaller body mass.
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To determine whether castrated animals completely lost the ability to elevate Hsp70, intact and castrated animals were subjected to a 15-min 42°C heat shock and cardiac Hsp70 expression was determined 24 h later. In contrast to exercise, Hsp70 levels were significantly elevated in the hearts of both castrated and intact animals after heat shock to approximately seven times those of control animals (Fig. 3).
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Neither castration alone nor DHT supplementation altered HSF1 expression relative to intact animals (Fig. 4). Consistent with previous findings (17), exercise also did not affect the total cardiac content of this protein.
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1-adrenergic receptor expression.
1-Adrenergic receptor protein expression was increased in DHT-supplemented castrated rats relative to intact and castrated animals (Fig. 5A; P < 0.05). -Adrenergic receptor density was not different between any of the control groups (Fig. 5B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Sex differences in the constitutive and exercise-induced accumulation of Hsp70 are influenced by circulating levels of estrogen (20, 28). Here we show that the androgens testosterone and its 5-reduced metabolite DHT affect this response as well. Reduction of endogenous testosterone production by castration results in attenuation of the exercise-induced accumulation of Hsp70, whereas castrated animals supplemented with DHT exhibit a response similar to that of intact males. Moreover, these differences are observed even though the ability of castrated animals to initiate or manifest a stress response is still present, as castrated animals accumulate Hsp70 24 h after heat shock.
The constitutive expression of Hsp70 in intact female rat hearts has been reported to be twice that of males (28), and similarly, exogenous estrogen, but not testosterone administration, elevates Hsp70 in male cardiomyocytes (10). In support of the latter, we did not observe any significant differences in the constitutive levels of cardiac Hsp70 between intact and castrated males, suggesting that the expression of Hsp70 in the unstressed male heart is unrelated to androgens. Although the authors of the previous study reasoned that testosterone had no effect in cardiomyocytes because of the absence of androgen receptors (10), others have shown that these receptors are present in male rodent whole hearts and that androgens do affect this tissue (16). The present data suggest that androgens per se do not induce the stress response, as evidenced by the lack of a difference in Hsp70 expression between sedentary groups, but rather they somehow influence the heart such that exercise is able to induce an increase in myocardial Hsp70. Additional studies will be required to elucidate how androgens are permissive in this regard.
Castration does not alter ability of heart to initiate stress response.
Similar to Hsp70 protein levels, basal expression of the transcription factor for the hsp70 gene, HSF1, and hsp70 mRNA were unaltered in male cardiac tissue regardless of androgen hormone status. Moreover, after exercise, mRNA was significantly increased only in the intact and DHT-supplemented groups. However, when a different physiological stress was applied (i.e., heat shock), castrated animals exhibited an accumulation of Hsp70 equal to that of heat-shocked intact male rats. As we reported previously (18), exercise intensity and temperature are likely important factors in the exercise-induced increase of cardiac Hsp70. Because castrated animals were lighter (i.e., did not work as hard as the other groups; Ref. 19) and exhibited the lowest (although not statistically different) peak exercise temperatures, it could be argued that this was at least partially responsible for their lack of a response. However, when we examined Hsp70 accumulation in a separate group of castrated animals that were matched for weight, they still did not show an accumulation of Hsp70 after exercise (Fig. 2B). This suggests that the effect of castration was independent of potential body mass or temperature confounds.
Castration and 1-adrenergic receptor expression.
In an effort to determine a mechanism that could explain the effect of altered androgen status on the Hsp70 response to exercise, 1-adrenergic receptor expression and density were examined. Our lab has recently published (17) data suggesting that blockage of a downstream member of the -receptor signaling cascade, PKA, inhibits the accumulation of Hsp70 after exercise. Moreover, others have found that the mRNA coding for the 1-adrenergic receptor is reduced in the hearts of castrated rats (7). Hence, we expected to see a reduction in the expression of cardiac 1-adrenergic receptor in the hearts of castrated animals that would be returned to intact levels with DHT supplement and hypothesized that this was a potential method by which the androgens were regulating the exercise stress response. However, we were unable to show significant differences in 1-adrenergic receptor expression between intact and castrated animals by Western blotting, although DHT-treated castrated animals exhibited above-normal levels (Fig. 5A). Moreover, when we examined 1-adrenergic receptor density at the membrane of these hearts, there were no significant differences between any of the groups (Fig. 5B). Therefore, the present data do not provide evidence for a link between androgen level, 1-adrenergic receptor density, and exercise-related Hsp70 expression. It is clear from a number of studies that the heat shock response is important to cell survival and therefore able to be activated by a number of signaling cascades (8, 27, 31), of which those emanating from the 1-adrenergic receptor are only one. Moreover, Paroo and Noble (21) showed that nonspecific blockage of the -adrenergic receptor with nadolol does not inhibit the exercise-induced accumulation of Hsp70 in the male rodent heart, although the -adrenergic agonist isoproterenol augments it. As such, although it seems that the -adrenergic receptor may be important to the induction of Hsp70 after exercise, it is not vital.
In conclusion, the present findings indicate that sex differences in the expression of Hsp70 after exercise are not simply due to estrogen. Androgens play a significant role in this sexual dimorphism in vivo and need to be investigated further. Also, there is a growing body of work suggesting that testosterone may exert beneficial effects on a number of coronary risk factors in men and is associated with reduced susceptibility and severity of ischemic injury (3, 14, 30), even without prior exercise. In the present study, we showed that Hsp70 expression was similar among sedentary animals regardless of hormonal status, but with exercise, androgen was necessary to elicit an increase in cardiac Hsp70. With the cardioprotective role of Hsp70 already established, androgens may be just as vital to cardiac function as estrogen is thought to be.
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GRANTS |
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FOOTNOTES |
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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.
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REFERENCES |
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B signaling. J Mol Cell Cardiol 36: 577–584, 2004.[CrossRef][Web of Science][Medline]-adrenergic receptors in heart failure due to volume overload. Am J Physiol Heart Circ Physiol 289: H151–H159, 2005.This article has been cited by other articles:
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  E. Bombardier, C. Vigna, S. Iqbal, P. M. Tiidus, and A. R. Tupling Effects of ovarian sex hormones and downhill running on fiber-type-specific HSP70 expression in rat soleus J Appl Physiol, June 1, 2009; 106(6): 2009 - 2015. [Abstract] [Full Text] [PDF]
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D. A. Brown, M. S. Johnson, C. J. Armstrong, J. M. Lynch, N. M. Caruso, L. B. Ehlers, M. Fleshner, R. L. Spencer, and R. L. Moore Short-term treadmill running in the rat: what kind of stressor is it? J Appl Physiol, December 1, 2007; 103(6): 1979 - 1985. [Abstract] [Full Text] [PDF]
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 D. S. Hydock, C.-Y. Lien, C. M. Schneider, and R. Hayward Effects of voluntary wheel running on cardiac function and myosin heavy chain in chemically gonadectomized rats Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3254 - H3264. [Abstract] [Full Text] [PDF]
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