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1Cardiology Research, Veterans Affairs Medical Center and Baylor College of Medicine, Houston 77030; 2Michael E. DeBakey Institute for Comparative Cardiovascular Science and 3Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843; 4Department of Physiology and Cardiovascular Research Institute, Maastricht University, 6200 MD Maastricht, the Netherlands; and 5Department of Anesthesiology and Intensive Care Medicine, University of Bonn, Bonn, D-53012 Germany
Submitted 25 November 2002 ; accepted in final form 17 April 2003
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ABSTRACT |
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-estradiol increases levels of HSPs in male
rat cardiac myocytes. We hypothesized that there were gender differences in
HSP72 expression in the heart secondary to estrogen. To test this hypothesis,
we examined cardiac levels of HSP72 by ELISA in male and female Sprague-Dawley
rats. In addition, three other HSPs were assessed by Western blot (HSP27,
HSP60, and HSP90). To determine whether estrogen status affected HSP72
expression in other muscles or tissues, two other muscle tissues, slow twitch
muscle (soleus muscle) and fast twitch muscle (gastrocnemius muscle), were
studied as well as two other organs, the kidney and liver. Because HSP72 is
cardioprotective, and females are known to have less cardiovascular disease
premenopause, the effects of ovariectomy were examined. We report that female
Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy
reduced the level of HSP72 in female hearts, and this could be prevented by
estrogen replacement therapy. These data show that the expression of cardiac
HSP72 is greater in female rats than in male rats, due to upregulation by
estrogen.
hormones; cardiovascular diseases; ischemia
-estradiol increases levels of HSP72 in male rat cardiac myocytes
but has no effect on HSP27, HSP60, or HSP90
(14). We hypothesized that
there were gender differences in HSP72 expression related to estrogen. To test
this hypothesis, we examined cardiac levels of HSP72 by ELISA. In addition,
three other HSPs were compared by Western blot (HSP27, HSP60, and HSP90). To
determine whether gender differences were found in other tissues, skeletal
muscle [slow twitch muscle (STM; soleus muscle) and fast twitch muscle (FTM;
gastrocnemius muscle)] as well as the kidney and liver were studied. Because HSP72 is cardioprotective, and females are known to have less cardiovascular disease premenopause, the effects of ovariectomy were compared with estrogen replacement on HSP72. We report that female Sprague-Dawley rat hearts have twice as much HSP72 as male hearts. Ovariectomy reduced the level of HSP72 in female hearts, and this could be prevented by estrogen replacement therapy.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-estradiol 60-day
slow-release pellets (Innovative Research; Sarasota, FL) implanted
subcutaneously. This estrogen preparation produces sustained plasma estrogen
levels in the physiological range
(17,
33). At selected time points,
the rats were euthanized, and tissues were collected and snap frozen in liquid
nitrogen. The soleus muscle was used for STM, and the gastrocnemius muscle was
used for FTM. Plasma samples were collected at the same time for measurement
of estrogen levels. Where needed, the phase of the estrous cycle in female rats was determined by daily vaginal smear. At least two or more complete estrous cycles (4–5 days in length) were followed before a rat was used in an experiment, and a vaginal smear was always obtained immediately before death for an experiment. Tissue samples from intact female rats were collected at metestrus and proestrus. Initial analysis showed no difference between proestrus and metestrus samples, and samples were pooled for analysis. It should be noted that the rat has a 4-day cycle. To ensure that all animals were treated uniformly, male rats were also handled daily before use in an experiment.
The animal protocol was approved by the Baylor College of Medicine Animal Research Committee and the Texas A&M University Laboratory Animal Care Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1985).
HSP72 levels were measured by a sandwich ELISA using a kit (StressGen). Samples were assayed at two different dilutions, and the results were averaged and expressed as picograms per microgram of soluble protein. As an additional control, samples with only secondary antibody were tested to exclude a high background signal, and these were found to be zero. Details of Western blotting and protein measurements have been previously described (13, 14, 22). Samples were homogenized using a Brinkmann Polytron in ice-cold RIPA buffer supplemented with protease inhibitors as previously detailed. Protein concentrations were determined using the bicinochonic assay (Pierce). Ponceau red staining of the membranes was used to verify equal protein loading. Antibodies were purchased from StressGen except for the antibody to HSP90 (Transduction Laboratories). For Western blots, all samples were run simultaneously, and densitometric values were normalized to internal controls (all male samples combined) on each blot to facilitate comparison between blots.
Plasma estrogen levels were measured by radioimmunoassay (RIA) of unextracted plasma collected at the time of tissue collection. The RIA is a clinically based kit that has been validated by one of the authors (J. N. Stallone) and others for measurement of estrogen in rat plasma (Coat-A-Count, Diagnostic Products; Los Angeles, CA). Duplicate determinations were made for all samples.
Data were compared by either ANOVA or ANOVA on ranks, where appropriate. Data were then analyzed by a Student-Newman-Keuls or Dunnett's test. The correlation between ELISA and Western blot results was determined by linear regression. P < 0.05 was considered significant. All data are expressed as means ± SE.
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RESULTS |
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Skeletal muscle. For comparison, two different skeletal muscle types were studied. In STM, male rats had 65% more HSP72 (P < 0.05; Table 1). Males had moderately more HSP27 (20%) and HSP90 (35%) than female rats (P < 0.05; Fig. 2A). HSP60 levels were the same in both sexes. In FTM, HSP60 levels were significantly higher in males than in females by a small but reproducible amount. HSP72 was slightly higher in males, but this difference was not significant. The higher HSP27 levels in the heart and STM might be related to the cytoskeletal functions of this protein and the larger muscles found in males, but FTM HSP27 levels did not differ between males and females (Fig. 2B). Similarly, there was no difference in HSP90 levels in FTM from males versus females (Fig. 2, A and B).
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Kidney and liver. Studies of the kidney and liver showed that
40% more HSP72 was present in female kidneys compared with male
(P < 0.05), whereas there were no differences in the liver. HSP60
levels were 80% higher in female than male kidneys (P < 0.05; data
not shown). There was no difference between males and females in renal levels
of HSP90 (Fig. 3). HSP27 was
not detected in the kidney. In the liver, there were no differences in any of
the HSPs (data not shown). The liver had the lowest level of HSP72 of all
tissues studied (Table 1). HSP27 was not detected in the liver (Fig.
4).
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Relative tissue expression. Tissue distribution comparisons demonstrated that high levels of HSP27 were present in muscle, whereas HSP90 was present in the greatest amounts in the liver and kidney (Fig. 4). The highest concentrations of HSP60 were in the heart and kidney, with much less in STM and FTM. Levels of HSP60 in the liver were intermediate to those of the kidney and FTM. HSP72 was most abundant in STM and least in the liver. The kidney, heart, and FTM had similar levels of HSP72, although male heart levels were lower. Males and females had similar tissue distribution patterns.
Estrogen withdrawal and its effect on HSP72. Female rats had higher levels of protective HSP72 in the heart. To determine whether this was estrogen sensitive, an ovariectomy was performed to create a rat model of menopause. Rats were divided randomly between estrogen replacement and no replacement groups. As shown in Fig. 5A, 6 wk postovariectomy, HSP72 levels in the hearts were reduced, but this difference was not significant. If rats were followed to 9 wk postovariectomy, HSP72 levels dropped significantly to 11.4 ± 0.9 pg/µg total protein, similar to levels in male rats. In contrast, replacement therapy maintained HSP72 at the level of intact females. These same results are illustrated in Fig. 5B, where, by Western blot, cardiac HSP72 levels are reduced postovariectomy to levels similar to those found in male hearts. There was good correlation by linear regression between the density by Western blot and the levels measured by ELISA (R = 0.906).
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Estrogen levels. Plasma samples were collected at the same time as tissues, and estrogen levels were measured on unextracted plasma in duplicate using a double-antibody RIA (Diagnostic Products). Estrogen levels in females varied significantly (P < 0.001) during the estrous cycle, with proestrus females averaging 24.30 ± 3.82 pg/ml and metestrus females averaging 5.62 ± 0.47 pg/ml, still significantly higher than males (1.62 ± 0.50 pg/ml). After ovariectomy, plasma estrogen dropped to 0.53 ± 0.28 pg/ml, whereas estrogen replacement resulted in levels of 21.43 ± 4.27 pg/ml.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Several other tissues were examined to determine whether there was a gender difference in HSP72 levels or if this increase in HSP72 was only seen in the heart. Only the kidney showed a significant increase in HSP72 in females versus males. There was no difference in FTM, and in STM males had 65% more HSP72 than females. Levels in the liver were slightly higher for females, but this did not reach significance.
There were smaller sex differences in the other HSPs. In the heart and STM, HSP27 was higher in males, possibly related to the cytoskeletal functions of this protein and the larger muscles found in males, although in FTM, HSP27 did not differ between males and females. HSP60 levels were similar in males and females except in the kidney, where females had twice as much HSP60 as males, and in FTM, where males had slightly higher levels than females. HSP90 was present in lower levels in female hearts than in male hearts. Statistical analysis showed no correlation between either HSP27 or HSP90 and HSP72 levels in the heart. Tissue distribution comparisons demonstrated that high levels of HSP27 were present in the muscle tissues, whereas HSP90 was present at higher amounts in the liver and kidney than in muscle. HSP60 was present in the greatest amount in the heart and kidney, with much less present in STM and FTM.
Plasma estrogen levels demonstrated both variation with the stage of estrus (data not shown) and with withdrawal and supplementation with estrogen. Estrogen levels are typically higher in primates. Primates have a menstrual cycle, whereas rodents have an estrous cycle. These estrogen levels are lower than levels reported for humans; however, the values measured in rat serum are similar to those reported elsewhere (32).
Few studies have addressed the effects of estrogen on HSP expression, and
gender differences have not previously been reported except for higher levels
of HSP72 in serum of normal adult females versus males
(27). Previously, we observed
that 10 h of 17-estradiol treatment (concentration tested: 0.1–10
µM) doubled the level of HSP72 in adult cardiac myocytes from male rats
(14). Although estrogen
treatment resulted in an increase in HSP72 similar to the differences observed
between male and female hearts in the present study, further work will be
needed to fully identify the underlying mechanisms. Others
(23) have reported that
estrogen induces HSP90 expression in the murine uterus. In the ventromedial
hypothalamus, estrogen increases heat shock cognate 70 and HSP90 expression
(15,
23). Similarly, estrogen
increases transcription and translation of heat shock factor (HSF)-1 and HSF-2
in the uterus (34). Gender
differences in the response of skeletal muscle HSPs to exercise have been
reported, but these levels were not compared with control tissue
(25). A more recent study
(24) reported that HSP72
levels were increased in the hearts of male rats after exercise but not
females. After ovariectomy, females also had an increase in cardiac HSP72 with
exercise, although it was less marked. The rats studied were only 8 wk of age,
in contrast to the 12- to 16-wk-old rats used in the present study. Some of
the discrepancy in findings may be related to the fact that these rats were
juveniles rather than adults. In addition, some of the differences between the
present study and these observations may be isoform related. The prior study
used a polyclonal antibody, which may bind more than one HSP70 protein,
whereas in the present study we measured specifically the major inducible
protein HSP72. The differences in these antibodies are notable in that for the
Western blots of the present study, 10 µg of protein were used compared
with 50 µg of protein in the study of Paroo et al
(24). The present results are
consistent with the observations of Pockley et al.
(27). There are likely other
variables in experimental conditions that affect HSP levels, and further
studies will be needed.
The different patterns of HSP72 expression among cardiac tissue, STM, and FTM for males and females most likely reflects that the regulation of expression of this protein is complex. Although a gender/estrogen-related difference in expression was observed for the heart, in STM the reverse effect was seen and in FTM there was no gender difference. Further work will be needed to delineate the regulation of expression of HSP72 in these different muscles.
Only many weeks after ovariectomy was a significant decrease in cardiac HSP72 levels seen. Estrogen levels after ovariectomy fall quickly. This gradual decline in HSP72 suggests that the effect of estrogen on HSP72 expression may be indirect and that secondary changes resulting from ovariectomy lead to the drop in HSP72. This in turn suggests that the effects of estrogen withdrawal are complex and that the effects of withdrawal evolve over time. This could have important implications for clinical studies of estrogen replacement.
At the transcriptional level, HSP27 has been found to have an estrogen response element in its promoter and has been shown to be estrogen responsive (4, 28); however, we found no increase in HSP27 levels in females. Estrogen response elements have not been identified in the promoters of other HSPs, nor has a pattern of a selective increase by estrogen been found in the studies of these other proteins to date.
It is well established that premenopausal women have a lower incidence of cardiovascular disease than men, but postmenopausal women have a greatly increased incidence of heart disease, presumably resulting from the loss of ovarian steroid hormone effects. Although about one-third of the protective effects of estrogen can be explained by the changes in lipoprotein profiles, other unknown, but important, factors are involved (20). Likewise, women with heart failure survive longer than men, and estrogen replacement has been found to be associated with improved survival (1, 8, 9, 30). Higher levels of HSP72 in female hearts may contribute to some of the benefits of estrogen; however, further studies are needed to investigate this possible mechanism. Results of clinical estrogen replacement studies have not demonstrated the expected benefit from estrogen treatment (7, 10). Estrogen has a myriad of effects, the complexity of which is just being realized (2). Important functions such as the activation of endothelial nitric oxide synthase have only recently been identified (2, 31). More recent work suggests an adverse effect of estrogen on endothelium and vascular smooth muscle, leading to enhanced vascular tone (6). Further basic research is needed to comprehend the many effects, both beneficial and disadvantageous, that estrogen has on the cardiovascular system (5).
In conclusion, we report an increase in HSP72 expression in the adult female rat heart. Ovariectomy in females reduces the level of HSP72 to that of males, and this can be prevented by estrogen replacement therapy. These differences may account for some of the differences in cardiovascular mortality between males and females.
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DISCLOSURES |
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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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-skeletal actin on the same blot
for comparison.