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Endocrinology Vol. 147, No. 10 4569-4577
Copyright © 2006 by The Endocrine Society

Testosterone Is Required for Delayed Cardioprotection and Enhanced Heat Shock Protein 70 Expression Induced by Preconditioning

Jing Liu, Sharon Tsang and Tak Ming Wong

Department of Physiology and Institute of Cardiovascular Sciences and Medicine, The University of Hong Kong, Hong Kong SAR, China

Address all correspondence and requests for reprints to: Professor T. M. Wong, Ph.D., Department of Physiology, Faculty of Medicine, The University of Hong Kong, 4/F Laboratory Block, Faculty of Medicine Buildings, 21 Sassoon Road, Pokfulam, Hong Kong SAR, China. E-mail: wongtakm{at}hkucc.hku.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ischemic preconditioning fails to confer immediate cardioprotection in the absence of testosterone, indicating that the hormone is required for the process. Here we set out to determine whether testosterone is also necessary for delayed cardioprotection and, if so, how it acts. Male Sprague Dawley rats (7–8 wk) underwent sham operation or gonadectomy without (G) or with testosterone replacement (GT) for 8 wk. Isolated ventricular myocytes were preconditioned either by metabolic inhibition or with U50,488H, a {kappa}-opioid receptor agonist. In intact rats, U50,488H was administered systemically and 24 h later the hearts were removed. Ventricular myocytes were then subjected to metabolic inhibition and anoxia and isolated hearts to regional ischemia, followed by reperfusion to induce injury. Both types of preconditioning significantly increased the viability and decreased the lactate dehydrogenase release in ventricular myocytes from sham rats. They also activated heat shock transcription factor-1 and increased heat shock protein 70 expression. In contrast, all these effects were absent in myocytes from G rats and were restored by testosterone replacement. Parallel results were found in isolated hearts. In addition, preconditioning improved contractile functions impaired by ischemic insults in sham and rats gonadectomized with testosterone replacement but not G rats. The effects of testosterone replacement in ventricular myocytes were abolished by androgen receptor blockade. In conclusion, preconditioning requires testosterone to increase heat shock protein 70 synthesis, which mediates delayed cardioprotection in the male. These effects of testosterone are mediated by the androgen receptor.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ISCHEMIC PRECONDITIONING (IP) is the most potent mechanism known to protect against prolonged periods of ischemia (1). The effect of IP appears to be reduced or even eliminated with aging, as demonstrated in both clinical (2, 3) and experimental animal studies (4, 5). Testosterone also declines with advancing age. A recent study showed that in mice with testes removed, the immediate cardioprotection of IP is abolished (6). Although a testosterone replacement study was not performed, the observation indicated that testosterone is needed for acute cardioprotection of preconditioning. Whether testosterone is responsible for the delayed cardioprotection of IP has not been investigated. Neither has the underlying mechanism been studied.

As early as 1993, a correlation between enhanced heat shock protein (HSP) 70 content after IP and the ensuing reduction in postischemic infarction was first observed (7). In confirmation, protection against ischemia was observed in transfected rat cardiac myocytes or transgenic mice overexpressing HSP70 (8, 9). We previously showed that the expression of HSP70 is enhanced by preconditioning either by metabolic inhibition or with U50,488H, a {kappa}-opioid receptor agonist, which is accompanied by delayed cardioprotection in ventricular myocytes. Furthermore, blockade of HSP70 synthesis with a selective antisense oligonucleotides abolishes the protective effect of preconditioning (10). These observations provide evidence that HSP70 mediates delayed cardioprotection of preconditioning. The role of HSP70 in this process was confirmed in our subsequent studies (11, 12, 13).

Transcriptional regulation of HSP70 expression is mediated by the binding of heat shock transcription factor (HSF)-1 to the specific regulatory element, heat shock element, in the promoter region of the HSP gene. A recent demonstration that HSF1 DNA binding is uncoupled from its transcriptional activity in mammalian cells (14) led to the finding that phosphorylation of HSF1 is an important determinant of its transactivating potency (15, 16).

Expression of HSP70 in response to heat stress (17) or exercise (18) also decreases with age, when testosterone level declines. In support of a direct link between testosterone and HSP70, addition of testosterone to a fungal growth medium up-regulates HSP70 mRNA expression and induces a general stress response (19). We therefore hypothesized that testosterone at physiological concentrations is required for the enhanced expression of HSP70 in response to preconditioning, which mediates the delayed cardioprotection of preconditioning.

The present study attempted to test this hypothesis by investigating the effects of preconditioning on cardiac injury, expression of HSP70 and HSF1 phosphorylation in both isolated ventricular myocytes and perfused hearts from sham operated rats and gonadectomized rats without (G) and with testosterone replacement (GT). Ventricular myocytes were preconditioned either by metabolic inhibition (MP) or with U50,488H (UP; Roche, Indianapolis, IN) in vitro, as previously described (10, 11, 12). Furthermore, the hearts of intact rats were preconditioned by the systemic administration of U50,488H (13, 20). The most important observation was that preconditioning, either in vitro or in vivo, conferred delayed cardioprotection and improved the contractile functions of isolated hearts, and these responses were accompanied by HSF1 activation and increased HSP70 expression. All effects were absent in the absence of testosterone but restored by testosterone replacement. The effects of testosterone replacement were abolished by blockade of the androgen receptor.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Experimental animals
Male Sprague Dawley rats (7–8 wk) were anesthetized with sodium pentobarbital (60 mg/kg, ip). One group underwent sham operation and the other group underwent gonadectomy with both testes removed (G). One week after gonadectomy, a subgroup was treated with a physiological dose of testosterone (200 µg/100 g body weight, sc) daily for 8 wk (GT) (21), whereas the rest received saline injection as control. At the end of the period, blood samples were collected for determination of total serum testosterone using a commercially available RIA kit (Diagnostic Products, Los Angeles, CA).

The protocol was approved by the Committee on the Use of Experimental Animals for Teaching and Research, The University of Hong Kong.

Isolated ventricular myocyte preparation
Nine weeks after surgery, ventricular myocytes were isolated from the hearts of sham and G rats using a collagenase method described previously (22). After stabilization, myocytes were subjected to 30 min pretreatment with either a selective {kappa}-opioid receptor agonist, 30 µmol/liter UP, or MP in a glucose-free Krebs buffer (pH 6.5) containing 20 mmol/liter lactate and 10 mmol/liter 2-deoxy-D-glucose (2-DOG), an inhibitor of glycolysis. In the control group, myocytes were subjected to pretreatment with normal Krebs solution only. After 24 h incubation with or without 10–8 mol/liter testosterone, myocytes were subjected to 10 min metabolic inhibition and anoxia with glucose-free Krebs solution containing 10 mmol/liter 2-DOG and 10 mmol/liter sodium dithionite, an oxygen scavenger (23). Finally, the myocytes were transferred back to normal Krebs solution for 10 min to simulate reperfusion (Fig. 1Go).


Figure 1
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  		FIG. 1. Upper panel, Experimental protocols. After isolation and stabilization, ventricular myocytes from sham and G rats were subjected to 30 min preconditioning with 30 µmol/liter U50,488H (UP) or glucose-free Krebs solution containing 20 mmol/liter lactate and 10 mmol/liter 2-DOG (MP). For testosterone replacement, myocytes from G rats were incubated with testosterone at 10–8 mol/liter for 24 h (GT); 10–6 mol/liter cyproterone acetate (cy) was used to block the androgen receptor. Myocytes were then subjected to 10 min metabolic inhibition and anoxia (MeI/A) with glucose-free Krebs solution containing 10 mmol/liter 2-DOG and 10 mmol/liter Na2S2O4 followed by 10 min reperfusion (RE). Lower panel, Effects of preconditioning with UP or MP on viability (A) and LDH release (B) in ventricular myocytes subjected to MeI/A followed by reperfusion. Both parameters were measured at the end of reperfusion. Values are mean ± SEM; n = 6–8 rats. *, P < 0.05, **, P < 0.01 vs. sham group; #, P < 0.05 vs. GT group; {dagger}, P < 0.05, {dagger}{dagger}, P < 0.01 vs. corresponding group with cy.

 
Isolated heart preparation
Nine weeks after surgery, each of three groups of rats were divided into two subgroups: the preconditioned group was administered U50,488H by iv injection, and the other group was administered saline as control. A bolus dose of 10 mg/kg U50,488H was given as described previously (13, 20). After an interval of 24 h, when the cardioprotection of UP peaked (20), hearts were removed, mounted to the Langendorff apparatus, and perfused retrogradely at 100 cm H2O with Krebs-Henseleit solution oxygenated with 95% O2-5% CO2. Hearts were allowed to stabilize for 30 min and then subjected to 30 min regional ischemia induced by left anterior descending coronary artery ligation and 120 min reperfusion as previously described (24) (Fig. 2Go). Any hearts exhibiting arrhythmias during stabilization were discarded. Ischemia was confirmed by regional cyanosis and a substantial decrease in coronary flow.


Figure 2
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  		FIG. 2. Upper panel, Experimental protocol. Rats were treated iv either with saline or U50,488H (10 mg/kg). Twenty-four hours later, hearts were removed, perfused with Krebs-Henseleit solution, and then subjected to 30 min coronary occlusion followed by 120 min reperfusion. Lower panel, Effects of preconditioning with U50,488H on myocardial infarct (A) and LDH release (B) in isolated perfused hearts subjected to ischemia and reperfusion. IS determined at the end of reperfusion was expressed as a percentage of the area at risk (AAR). The coronary effluent was collected at 5 min into reperfusion for measurement of LDH release. Values are mean ± SEM; n = 8–12 rats. *, P < 0.05, **, P < 0.01 vs. sham group; #, P < 0.05, ##, P < 0.01 vs. GT group.

 
Cardiac variables
A latex balloon was inserted into the left ventricle and the end-diastolic pressure (LVEDP) was adjusted to 4–8 mm Hg. The cardiac parameters of heart rate, left ventricular developed pressure (LVDP), and + dp/dtmax and – dp/dtmax representing, respectively, velocities of contraction and relaxation (± dp/dtmax) were monitored continuously by a PowerLab/4SD system (AD Instruments, Castle Hill, Australia). Coronary arterial flow was measured by collecting the coronary effluent.

Viability assay
Viability was determined using CellTiter-Blue reagent (Promega, Madison, WI), which contains highly purified resazurin (dark blue). Viable cells can reduce resazurin into resorufin, which is pink and highly fluorescent, whereas nonviable cells cannot reduce this indicator dye. The absorbance of samples was measured at 570 nm with 600 nm as a reference wavelength. Each sample was tested in triplicate.

Measurement of infarct size
This procedure was carried out as described previously (13). Infarct size (IS) was expressed as a percentage of the area at risk.

Lactate dehydrogenase (LDH) assay
LDH release was measured with a cytotoxicity detection kit. The amount of color formed in the assay is proportional to the release of the enzyme, which indicates cytotoxicity. The absorbance of samples was measured at 490 nm. Data were expressed as a percentage of the total LDH release, which was obtained by adding 2% Triton X-100 to untreated cells.

The coronary effluent of isolated hearts was collected for LDH release measurement using a UV-rate assay kit as previously described (11). LDH activity was expressed as units per liter.

Western blotting
Proteins from left ventricular tissue were extracted as previously described (13). Sixty micrograms per lane of protein were loaded and membranes were probed with either a mouse anti-HSP70 antibody (SPA-810) or rat anti-HSC70 antibody (SPA-815), both at 1:2000, or rat anti-HSF1 antibody (MAB88078) at 1:500. According to the instructions of the manufacturer, MAB88078 recognizes a protein of 70 to 85 kDa, identified as HSF1, depending on the state of the cell. Therefore, it can detect different forms of HSF1, and this had also been confirmed by a previous study (16). To ensure that equal amounts of protein were loaded on each lane, the membrane was reblotted with a mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (ab9482) at 1:6000. Data are expressed as a ratio of the target protein to GAPDH.

Drugs and chemicals
U50,488H, 2-DOG, sodium dithionite, and testosterone were purchased from Sigma (St. Louis, MO); mouse anti-HSP70 (SPA-810), and rat anti-HSC70 antibody (SPA-815) from StressGen (Victoria, British Columbia, Canada); rat anti-HSF1 antibody (MAB88078) from Chemicon (Temecula, CA); mouse anti-GAPDH antibody (ab9482) from Abcam (Cambridge, MA); RIA kit (Coat A Count total testosterone kit) from Diagnostic Products; CellTiter-Blue cell viability assay (G8080/1/2) from Promega; cytotoxicity detection kit (1 644 793) from Roche and UV-rate assay kit from Stanbio Laboratory (Boerne, TX).

Statistical analysis
All data were expressed as mean ± SE. One-way ANOVA followed by Newman-Keuls multiple comparison tests were carried out to test for differences. A difference of P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
General features of experimental animals
Nine weeks after surgery, the total serum testosterone level in G rats fell below the detection limit of 0.4 ng/ml. Body weight, heart weight, and the heart to body weight ratio also significantly declined. Daily injection of testosterone for 8 wk restored the testosterone level and body weight in GT rats to those of the sham-operated rats (Table 1Go). Heart weight was significantly but not completely restored, whereas the heart to body weight ratio was partially restored to the level of the sham control.


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  		TABLE 1. General feature of experimental animals

 
The changes in testosterone levels did not result in any significant differences in the contractile functions, heart rate, and coronary flow/heart weight among the three groups of rats (Fig. 3Go).


Figure 3
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  		FIG. 3. Effects of preconditioning with UP on cardiac variables in isolated perfused hearts subjected to ischemia and reperfusion. The experimental protocol shown in Fig. 2Go was used. Analyses were made during stabilization (baseline) and at the end of reperfusion (RE). A, LVDP. B, LVEDP. C and D, ± dp/dtmax. E, Heart rate. F, Coronary flow. Values are mean ± SEM; n = 8–10 rats. *, P < 0.05, **, P < 0.01 vs. sham group; #, P < 0.05, ##, P < 0.01 vs. GT group.

 
Effects of UP or MP on injury/viability after ischemic insult in ventricular myocytes
In ventricular myocytes from sham rats, both types of preconditioning, UP and MP, significantly increased the viability and decreased the LDH release after ischemic insult (Fig. 1Go, A and B), confirming the cardioprotection of preconditioning. Interestingly, neither parameter was changed after either UP or MP in myocytes from G rats. Incubation with testosterone for 24 h restored the ability of myocytes from G rats to respond to UP or MP. This cardioprotective effect of preconditioning was abolished by a testosterone antagonist, cyproterone acetate. These observations indicate that testosterone is required for the delayed cardioprotection of preconditioning and that the effect is mediated by the androgen receptor.

Without any pretreatment, the viability and LDH release were the same in myocytes from sham and G rats, indicating their similar susceptibility to ischemic insult. This was also unaffected by incubation with testosterone for 24 h (Fig. 1Go).

Effects of UP on injury after ischemic insult in isolated perfused hearts
Isolated perfused hearts (Fig. 2Go) were used to confirm the observations on myocytes. U50,488H was injected iv 24 h before the heart was removed (UP); this procedure is known to confer delayed cardioprotection (20). Then the isolated hearts were subjected to perfusion and ischemic insult. UP significantly reduced the infarct size (Fig. 2AGo) and LDH release (Fig. 2BGo) in hearts from sham rats. In contrast, this cardioprotection was absent in hearts from G rats. Testosterone replacement for 8 wk in G rats (GT) restored the effects of UP. These changes were consistent with those observed in myocytes (Fig. 1Go).

In contrast to the observations in myocytes, without UP, infarct size and LDH release were significantly greater in hearts from G rats than from either sham or GT rats.

Effects of UP on cardiac functions after ischemic insult in isolated perfused hearts
In all groups, ischemic insult caused marked decreases in LVDP (Fig. 3AGo), ± dp/dtmax (Fig. 3Go, C and D) and coronary flow (Fig. 3FGo) and a marked elevation in LVEDP (Fig. 3BGo) at the end of reperfusion. In hearts from sham rats, UP significantly attenuated the reductions in LVDP, ± dp/dtmax and the elevation in LVEDP, indicating improved left ventricular contractile functions. However, no improvements in these parameters were observed after UP in hearts from G rats. Similar to the effects on injury/viability, the beneficial effects of UP reappeared in GT rats, as reflected by higher LVDP and ± dp/dtmax, and lower LVEDP in the GTUP group, compared with the GT group.

In agreement with the observation that hearts from G rats suffered the most severe injury induced by ischemic insults (Fig. 2Go), the LVDP and ± dp/dtmax were the smallest and LVEDP was the greatest in hearts from G rats among the three groups of rats without UP (Fig. 3Go).

No differences were found in the heart rate and coronary flow among all groups (Fig. 3Go, E and F).

Effects of UP or MP on the expression of HSP70 after ischemic insult in ventricular myocytes and isolated perfused hearts
In both myocytes (Fig. 4Go, A and B) and isolated hearts (Fig. 4DGo) from sham rats, UP or MP induced a significant elevation in the expression of HSP70. Similar to the inability to induce cardioprotection, preconditioning in G rats failed to elevate HSP70 expression. Once again, testosterone replacement either in vitro (Fig. 4Go, A and B) or in vivo (Fig. 4DGo) restored the enhanced expression of HSP70 after preconditioning.


Figure 4
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  		FIG. 4. Effects of preconditioning on the expression of HSP70 after ischemic insult in ventricular myocytes (A–C) and isolated perfused hearts (D). Experimental protocol shown in Fig. 1Go was used for A and B and Fig. 2Go for C and D. Upper panels, Representative protein bands. Lower panels, Relative levels of protein expression assessed by densitometry. Measurements were normalized to the value obtained for the sham group in A, B, and D and the GT group in C. Values are mean ± SEM; n = 5–6 rats. **, P < 0.01 vs. sham group; ##, P < 0.01 vs. GT group; {dagger}{dagger}, P < 0.01 vs. corresponding group with cyproterone acetate (cy).

 
Cyproterone acetate also abolished the restorative effects of testosterone replacement on the activation of HSP70 on preconditioning (Fig. 4CGo), similar to the observations on viability/injury (Fig. 1Go).

In both preparations, there was no difference in the expression of HSP70 among sham-operated, G, and GT groups without preconditioning (Fig. 4Go, A, B, and D).

Effects of UP or MP on the expression of HSF1 after ischemic insult in ventricular myocytes and isolated perfused hearts
In control male rats without any treatment, HSF1 was recognized on the gel as an approximately 70-kDa protein (Fig. 5Go). After ischemic insult, not only was the expression markedly augmented, but also there was a shift to the higher molecular weight form due to phosphorylation in HSF1 (16, 25). Paralleling the changes in HSP70 expression, HSF1 expression was further increased significantly after UP or MP in both myocytes (Fig. 5Go, A and B) and hearts (Fig. 5DGo) from sham rats. This effect of preconditioning disappeared after gonadectomy and was restored by testosterone replacement.


Figure 5
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  		FIG. 5. Effects of preconditioning on the expression of HSF1 after ischemic insult in ventricular myocytes (A, B, and C) and isolated perfused hearts (D). Experimental protocol shown in Fig. 1Go was used for A and B and Fig. 2Go for C and D. Upper panels, Representative protein bands. Lower panels, Relative levels of protein expression assessed by densitometry. Measurements were normalized to the value obtained for the sham group in A, B, and D and the GT group in C. Values are mean ± SEM; n = 5–6 rats. **, P < 0.01 vs. sham group; ##, P < 0.01 vs. GT group; {dagger}{dagger}, P < 0.01 vs. corresponding group with cyproterone acetate (cy).

 
Similar to the change in HSP70, cytoperone acetate abolished the restorative effects of testosterone replacement on the activation of HSF1 upon preconditioning (Fig. 5CGo).

Similarly, in both preparations, neither gonadectomy nor testosterone replacement after gonadectomy changed the HSF1 expression after ischemic insults without preconditioning (Fig. 5Go, A, B, and D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that in the absence of testosterone, preconditioning with metabolic inhibition or U50,488H in vitro or U50,488H in vivo failed to confer delayed cardioprotection against ischemic insult in ventricular myocytes or isolated perfused hearts from male rats, respectively. This finding is in agreement with the previous observation that the acute cardioprotection of IP disappears after gonadectomy in hearts from male mice (6). We further showed that testosterone replacement, which restored the testosterone level to the physiological range, restored the delayed cardioprotection. This is the first evidence that testosterone at physiological concentrations is needed for the delayed cardioprotection of preconditioning. That an androgen receptor blocker, cyproterone acetate, abolished the effect of testosterone replacement indicates that the effect of testosterone is androgen receptor mediated and excludes the possibility that the effects were due to estrogen converted from testosterone by the enzyme aromatase.

Another important observation is that, in addition to the loss of delayed cardioprotection, preconditioning also failed to enhance HSP70 expression in G rats, and this was also restored by testosterone replacement, an effect abolished by cyproterone acetate. Together with previous findings by others (7, 26) and in our laboratory (10, 11, 12, 13) that HSP70 mediates the delayed cardioprotection of preconditioning, this finding indicates that testosterone deficiency impairs HSP70 synthesis on preconditioning, and this is responsible at least partly for the lack of delayed cardioprotection of preconditioning. Another study reported that in aged rats, loss of protection against ischemia by preconditioning with heat stress is accompanied by a blunted elevation in HSP70 (17). This may also be due to testosterone deficiency, although its level was not measured. In contrast, no differences were found in the expression of HSP70 among both isolated myocytes and perfused hearts from sham, G, and GT rats without preconditioning. These observations further indicate that testosterone is required for the HSP70 response to preconditioning.

We also showed that the failure in HSP70 up-regulation after MP or UP in the absence of testosterone was accompanied by impaired HSF1 phosphorylation, and testosterone replacement reversed this impairment. This is in agreement with previous observations that the time course of HSF1 phosphorylation parallels that of the altered HSP70 expression induced by heat stress in spontaneously hypertensive rats (16) and that induction of HSP70 mRNA and protein after nitric oxide treatment in vascular smooth muscle cells is associated with the phosphorylation and translocation of HSF1 (25). So it is reasonable to suggest that testosterone may be involved in the stimulation of HSF1 phosphorylation, thus leading to increased HSP70 expression on preconditioning. It would be of interest to know the mechanism by which testosterone leads to activation of HSF1 on preconditioning. Unfortunately, there is no evidence for the interaction between testosterone and HSF1 activation. There is, however, evidence that nuclear factor-{kappa}B (NF{kappa}B) activation is necessary for estrogen-induced activation of HSF1 and HSP70 in female cardiomyocytes (27) and that NF{kappa}B induced HSPs (28). Moreover, NF{kappa}B is activated by IP, and inhibition of the activation abolishes the cardioprotection 24 h later, indicating that NF{kappa}B plays a critical role in the genesis of late IP (29). Interestingly, both estrogen and testosterone reduce intracellular iAbeta1–42 toxicity in human neurons by increasing HSP70 expression (30). It is therefore not unreasonable to suggest that testosterone also has a similar relationship with NF{kappa}B and HSF1 as does estrogen. Further studies are warranted.

In the present study, preconditioning was performed both in vitro and in vivo. In the former, isolated ventricular myocytes were subjected to either metabolic inhibition or U50,488H. In the latter, U50,488H was administered iv before hearts were removed for study. The effects of testosterone replacement were also determined in both models. In the ventricular myocyte preparation, testosterone replacement was achieved by incubation with testosterone for 24 h. In the isolated perfused heart preparation, hearts were removed from sham, G, and GT (8 wk replacement) rats. Findings from the in vitro preparation indicated that the myocardium is the target of action of preconditioning and testosterone, whereas those from in vivo preparation provided information useful for future consideration in clinical application.

In the present study, testosterone replacement was achieved by daily injection for 8 wk or incubation of myocytes with testosterone for 24 h. That prolonged testosterone replacement was needed suggests that its action may be genomic. More importantly, the finding that the effects of testosterone replacement in myocytes were abolished by cyproterone acetate indicates that the actions of testosterone were mediated by the classical androgen receptor, known to act via a genomic pathway.

In addition to the finding that testosterone is needed for the cardioprotection of preconditioning, we also found that both cardiac injury and functional impairment induced by ischemia and reperfusion were significantly greater in hearts from G than from sham rats, and this was reversed by testosterone replacement. This indicates that testosterone has a cardioprotective effect and is consistent with the findings from cross-sectional studies that patients with coronary artery disease have lower levels of testosterone (31) and that testosterone supplements cause short-term improvements in exercise electrocardiogram (32, 33, 34). Similarly, in normal male rats, administration of testosterone improved postischemic recovery of contractile function, whereas removal of testes led to an impaired recovery (35). There is also a study demonstrating an acute protective effect of testosterone in rat ventricular myocytes (36). However, testosterone has also been shown to exert detrimental effects. Isolated rat hearts from castrated males and males treated with flutamide, an androgen receptor blocker, show better myocardial functional recovery after global ischemia and reperfusion than untreated controls (37). Moreover, cardiac rupture rate and infarct expansion index after myocardial infarction were found to be higher in normal male mice than in castrated ones (38). These discrepancies may be due to different experimental protocols, parameters measured, and preparations used.

In addition to activation of HSF1 and the subsequent up-regulation of HSP70, which mediates delayed cardioprotection, other mechanisms have been reported to account for the cardioprotective effects of testosterone. A previous study from our laboratory showed that testosterone at physiological concentrations confers cardioprotection against ischemic insult by enhancing the cardiac response to {alpha}1-adrenoceptor stimulation, an effect mediated by the androgen receptor (39). Superphysiological levels of testosterone also confer acute protection against ischemic insult by opening the mito-ATP-sensitive potassium channel, which is independent of the androgen receptor (36).

Here the protective effects of testosterone were observed in isolated hearts. However, no differences were observed in viability and LDH release in isolated myocytes from sham and G rats without or with testosterone replacement. This discrepancy may have resulted from the fact that the viability of isolated myocytes was lower than that of perfused heart due to the isolation procedure. Therefore, small to moderate changes in viability/injury induced by ischemic insult may not have been enough to reach statistical difference. This explanation is supported by another study from our laboratory showing that metabolic inhibition and anoxia can only lead to significantly higher viability and lower LDH release in myocytes from sham than G rats when coupled with adrenergic stimulation (39).

There is now evidence that gender differences exist in the protection of IP. A previous study (6) reported that normal female mice cannot be preconditioned as reflected by no reduction in infarct size in isolated perfused hearts after preconditioning. In contrast, a significant antiarrhythmic effect of IP occurs in both isolated perfused hearts and intact hearts from female rats (40), an observation supported by the report that female dogs benefit from IP in myocardial infarction (41). A possible explanation for this discrepancy is that a higher threshold, required to initiate preconditioning (42, 43), exists in the female. In support of this notion, a recent study found that the cardiac function in male rat hearts improves after preconditioning with both high and low doses of endotoxin, whereas female hearts can be protected only with the high dose (44). In the present study, we showed that testosterone was required for the enhanced expression of HSP70, which is responsible for the delayed cardioprotection of preconditioning. So is estrogen required for the delayed cardioprotection of preconditioning, and if so, is it also required for enhanced expression of HSP70 in the female? A previous study reported that the expression of cardiac HSP70 is greater in female than male rats due to the presence of estrogen (45). On the other hand, after exercise female rats exhibit much lower levels of cardiac HSP70 than male rats (46). In addition, ovariectomized rats show an exercise-mediated induction of HSP70 similar to that in males and this is reversed by estrogen replacement. These results, although not from preconditioning models, suggest that the signals involved in the preconditioning pathways may be affected by gender-specific mechanisms. Further studies are warranted to determine the relationship between estrogen and HSP70 and whether estrogen and testosterone affect HSP70 differently on preconditioning.

In conclusion, the present study has provided novel evidence that testosterone at physiological concentrations is required for the delayed cardioprotection of preconditioning, concomitant increased HSF1 phosphorylation, and HSP70 expression in the male. This action of testosterone is mediated by the androgen receptor in the heart. Because HSP70 is known to mediate the delayed cardioprotection of preconditioning, our observations indicate that testosterone at physiological concentration is needed for preconditioning to activate HSP70 synthesis, which then mediates the delayed cardioprotection.


   Acknowledgments
 
We thank Dr. I. C. Bruce for his comments on the manuscript and Mr. C. P. Mok for technical assistance.


   Footnotes
 
This work was supported by The Research Grants Council (Hong Kong), The Strategic Theme on Healthy Aging (The University of Hong Kong), and the Cardiovascular Research Fund donated by L.C.S.T. (Holdings) Ltd., Hong Kong.

Disclosure summary: all authors have nothing to declare.

First Published Online June 22, 2006

Abbreviations: 2-DOG, 2-Deoxy-D-glucose; G, gonadectomized male rats; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GT, gonadectomized male rats with testosterone replacement; HSF1, heat shock transcription factor-1; HSP, heat shock protein; IP, ischemic preconditioning; IS, infarct size; LDH, lactate dehydrogenase; LVDP, left ventricular developed pressure; LVEDP, left ventricle and end-diastolic pressure; MP, preconditioned with metabolic inhibition; NF{kappa}B, nuclear factor-{kappa}B; UP, preconditioned with U50,488H.

Received March 7, 2006.

Accepted for publication June 13, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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