Effect of ovariectomy on cardiac gene expression: inflammation and changes in SOCS gene expression

Karyn L. Hamilton, Li Lin, Yin Wang, Anne A. Knowlton


Basic research on estrogen-related changes in cardiomyocyte gene expression is needed to provide a greater understanding of the effects of estrogen, so that hormone replacement trials and treatment can be based on a true comprehension of estrogen's pleiotropic effects. Therefore, we compared gene expression in models of estrogen depletion and estrogen replacement. Using gene expression array analysis, we examined differences in expression in cardiac tissue from ovariectomized (OVX), ovariectomized with 17β-estradiol replacement (OVX/E2), and intact rats undergoing sham procedures (Sham). We found that OVX results in at least twofold changes in expression of genes involved in inflammation, vascular tone, apoptosis, and proteolysis compared with OVX/E2. With confirmation via real-time PCR, we found an OVX-induced increase in genes mediating inflammation (inhibin βa, IL-6, TNF-α, SOCS2, SOCS3), an OVX-related decrease in the myocardial mRNA expression of genes involved in regulating vasodilation (endothelial NOS, soluble guanyl cyclase), an OVX-associated increase in extracellular matrix genes (collagen12alpha1, connexin 43), and an OVX-related increase in proapoptotic genes (caspase 3, calpain). Because details of cardiac signaling by SOCS genes are virtually unknown, we examined the protein expression for these genes via Western analyses. Although we observed OVX-related increases in SOCS2 and SOCS3 mRNA, SOCS2 and SOCS3 protein did not differ among groups. In light of these findings, investigation into the net effect of OVX on inflammation is warranted. These experiments add to existing evidence that estrogen can protect against negative changes associated with estrogen removal.

  • estrogen
  • menopause
  • cytokine
  • cardiovascular

estrogen has pleiotropic effects that have yet to be fully defined. 17β-Estradiol (E2) activates intracellular signaling pathways by binding to the membrane-associated estrogen receptor (ER), or it can alter gene expression by binding the cytoplasmic/nuclear ER, which, as a transcription factor, directly interacts with gene promoters (30).

Estrogen has a wide variety of effects on cardiovascular tissues. Although initial controlled trials of estrogen therapy to reduce cardiovascular disease have been disappointing, results likely reflect the complex estrogenic actions in cardiovascular tissues. Retrospective studies supported estrogen replacement to minimize cardiovascular disease in postmenopausal women. However, several prospective trials, including the Heart and Estrogen/Progestin Replacement Study (HERS) and the Women's Health Initiative (WHI), suggested increased morbidity and mortality with estrogen, including increased incidence of thromboembolism, both when estrogen was used for the primary prevention (WHI) and secondary prevention (HERS) of heart disease (3, 19). Interpretation of the data drawn from these studies has been criticized (55), and it is also important to note more recent ancillary studies of the WHI revealing lower mean coronary-artery calcium scores among women receiving estrogen compared with those receiving placebo (29). Coronary-artery calcification serves as a marker of calcified atheroma and reflects the progression from simple fatty streaks to complex atherosclerotic plaques. Furthermore, ongoing trials including the Kronos Early Estrogen Prevention Study should shed light on the role of timing of estrogen treatment in contributing to cardiovascular benefits (16). Nonetheless, the conclusions drawn from these studies have been widely accepted as demonstrating that replacement with conjugated estrogens in postmenopausal women increased the risk of coronary heart disease, stroke, and venous thromboembolism.

The results from WHI and HERS create a sense of urgency to better identify the molecular effects of estrogen replacement in postmenopausal tissues that appear to respond “unpredictably” to treatment with this hormone. Awareness of the increased risk of all cause mortality associated with surgical ovariectomy in women younger than 45 yr (40) also punctuates the need for basic research on estrogen-related changes in gene expression. Such information will facilitate a greater understanding of the effects of estrogen on cardiovascular and other tissues, such that better hormone replacement clinical trials and recommendations can be made. Therefore, in this study, we compared rat models of surgical ovariectomy (OVX) and postovariectomy estrogen replacement. Using gene expression array analysis and real-time PCR, we examined differences in gene expression between rats undergoing OVX and ovariectomy plus estrogen replacement (OVX/E2) compared with intact controls.


Animals and treatment.

Adult female Sprague-Dawley rats (3–4 mo old) were ovariectomized (OVX) under sterile conditions by standard methods. In one-half of the OVX rats, hormone replacement was initiated via subcutaneous placement of 0.100 mg 17β-estradiol 60-day slow-release pellets (OVX/E2; Innovative Research; Sarasota, FL). We and others have previously shown that this estrogen preparation typically produces sustained plasma estrogen levels in the physiological range (25, 57). Hormone replacement was initiated 3 wk postovariectomy and continued for 6 wk (25). We chose this estrogen replacement protocol because we were interested in the effect of the return of estrogen after OVX to “pattern” the late start of estrogen replacement following menopause or surgical OVX, especially given recent evidence that suggests the timing of estrogen replacement may be significant with respect to coronary artery disease risk (16, 41). Following 6 wk of placebo or estrogen replacement, the rats were euthanized with a lethal dose of ketamine and xylazine, and left ventricular tissues were collected and snap frozen in liquid nitrogen. Our time period, which is longer than the period of estrogen washout used in some studies, was chosen based on our observation that changes in protein expression following OVX could take 9 wk to be manifest (57). Sham animals underwent all surgical manipulation but without removal of ovaries. The animal protocols were approved by the Baylor College of Medicine Animal Research Committee and the University of California Davis Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Expression arrays.

Isolation of total RNA from left ventricular tissue was accomplished via standard TRIzol (Life Technologies) extraction following manufacturer's instructions. Isolated RNA was further purified by a phenol-chloroform extraction, followed by ethanol precipitation. Biotin-labeled array probes were synthesized according to Clontech recommendations (Atlas SpotLight Labeling Kit; Clontech Laboratories, Mountain View, CA). This method generates biotin-labeled array probes from 50 mg of total RNA using PowerScript Reverse Transcriptase. The high affinity of biotin for streptavidin (kd = ∼10–15) permits sensitive detection of probe-target hybrids and rapid detection and analysis with AtlasImage software (Clontech). Hybridization of labeled samples was carried out according to manufacturer recommendations for Clontech Atlas Nylon Arrays using the SpotLight Chemiluminescent Hybridization & Detection Kit. Two rat arrays were screened, totaling over 2,300 carefully selected, well-characterized cDNAs. Expression array analyses were performed in OVX (n = 3) and OVX/E2 (n = 3) groups.

Real-time RT-PCR.

To better delineate changes in gene expression in OVX vs. OVX/E2 identified by expression arrays, we added a Sham group when confirming genes by real-time PCR. To confirm a selected set of genes in OVX, OVX/E2, and Sham hearts, RNA was extracted from left ventricular samples (n = 4 rats per group) and processed by a “two-step” RT-PCR. Real-time quantitative PCR was performed using an ABI Prism 7000 Sequence Detection System and SYBR Green as double-strand DNA binding dye (Applied Biosystems). Primers, listed in Table 1, were either purchased from SuperArray (http://www.superarray.com) or synthesized based on published references and RTPrimer database (http://medgen.ugent.be/rtprimerdb/index.php). Results were expressed as the ratio relative to an internal control (GAPDH).

View this table:
Table 1.

Primers used in PCR reactions for template synthesis

Western blotting.

Using standard techniques described previously (14, 15), we carried out Western blot analyses to verify protein expression of suppressor of cytokine signaling (SOCS)2 as well as SOCS3. Samples were separated on 10% polyacrylamide gels, transferred to nitrocellulose, and developed with appropriate antibodies (SOCS3, 1:250; Zymed, San Francisco, CA; SOCS2, 1:10,000, Abnova, Taiwan; GAPDH, 1:20,000, Fitzgerald Industries Intl., Concord, MA; anti-mouse horseradish peroxidase, 1:1,000, GE Healthcare, Piscataway, NJ) and developed using chemiluminescence. Nitrocellulose was blocked with Blotto (Bio-Rad), except for GAPDH antibody, where blocking was done with 3% BSA. Following densitometry, SOCS2 and SOCS3 were normalized to GAPDH.

Data analyses.

Changes in genes expression confirmed via real-time PCR, and Western blot analysis were statistically analyzed using an ANOVA on Ranks, followed by a Student Neuman Keuls test, where appropriate, with P ≤ 0.05 considered significant.


As illustrated in Fig. 1A, OVX resulted in a model with significantly lower circulating estradiol concentrations compared with Sham and OXV/E2. The higher estradiol concentration observed in the Sham animals compared with the OVX/E2 group is, in part, due to timing of death, with the Sham animals being killed at peak estrus. At death, body weight and heart weight were greatest in the OVX group, with significantly lower uterus and uterus to body weight ratios in the OVX group compared with both of the other groups (Fig. 1, B and C).

Fig. 1.

Graphs summarize changes in serum estradiol concentrations and body/tissue weights in each experimental group. A: estradiol concentrations were measured in all 3 groups from plasma. Plasma was taken at peak estrus in the Sham group. B: body weights (BW) were measured on the day of death. C: organ weights were obtained postmortem. Sham = intact, sham operated; OVX = ovariectomized; OVX/E2 = ovariectomized with supplemental estrogen; Lv = Left ventricle; *different from all other groups for that measurement (P < 0.05).

In compliance with Microarray Gene Expression Data society, data have been submitted to the National Center for Biotechnology Information Gene Expression Omnibus. Of the 2,352 genes screened, expression of 67 genes differed twofold or greater between the treatment groups. These genes are summarized in Table 2. Of the genes with a twofold difference, the greatest number code for proteins playing a role in inflammation. Other functional categories of genes expressed differently in OVX, OVX/E2, and Sham groups were those involved in vascular tone; regulation of apoptosis, proteolysis, and calcium handling; cellular ion flux; and those comprising the extracellular matrix.

View this table:
Table 2.

Summary of mRNA showing twofold or greater change between ovariectomized and estrogen-replaced rats

Following expression array analysis, we selected 33 transcripts to verify via real-time PCR analyses. We selected the genes to establish a profile related to estrogenic impact on expression of genes involved in vascular tone and inflammation. Hence, we verified expression of inducible nitric oxide synthase (iNOS), endothelial NOS (eNOS), guanyl cyclase isoforms, epoxide hydrolase, and L-selectin to provide a profile of estrogenic regulation of genes involved in regulation of vascular tone and vascular adhesion. For a profile of inflammatory gene expression, we PCR verified IL receptors, TNF-α, inhibin βA, SOCS2 and SOCS3, complement proteins, and the advanced glycosylation end-product receptor (AGER). We also performed real-time PCR analysis on extracellular matrix proteins fibromodulin, fibronectin, collagen gene COL12A1, and connexin 43. Finally, to profile estrogenic effects on genes involved in proteolysis and apoptosis, we conducted real-time PCR analyses on calpains 1 and 2, caspases 3 and 9, and Bid3. Several of these genes were not in the original array screen or had very low levels of expression; however, as we had observed changes in these genes in response to estrogen in other models (Lin L and Knowlton AA, unpublished observations), we investigated these genes as well. In addition, to further validate our array findings, we used real-time PCR to verify eight additional genes exhibiting a fivefold or greater change by expression array analyses.

As shown in Fig. 2, real-time PCR analyses, overall, confirmed our expression array findings, revealing an OVX-induced increase in genes influencing the extracellular matrix (Fig. 2A), an OVX-related increase in epoxide hydroxylase and iNOS with a concomitant decrease in eNOS (Fig. 2B), and an OVX-associated increase in most of the genes involved in inflammation (Fig. 2C) compared with OVX/E2 and/or Sham. With regard to estrogenic influence on expression of the proapoptotic genes, caspases 3 and 9 (Fig. 2D), our results are mixed but accompanied by increases in the calcium-activated protease calpain, which has been shown to be capable of mediating caspase-independent apoptosis (9, 28, 43).

Fig. 2.

Graphs summarize key gene changes examined by real-time PCR. Results were normalized to GAPDH expression as an internal control. Data include mRNA for genes involved in extracellular matrix (A), vascular tone/adhesion (B), inflammation (C), apoptosis/proteolysis (D), and miscellaneous (E) genes exhibiting a 5-fold or greater difference via array analyses. *Different from all other groups for the same transcript; §different from OVX/E2; ‡different from sham (P < 0.05). COL12A1 = COL12A1 collagen gene; Cx43 = connexin 43; FMod = fibromodulin; Fnec = fibronectin; EH = epoxide hydroxylase; sGC-a and -b = soluble guanyl cyclase alpha and beta; IL-2ra = interleukin 2 receptor alpha; IL-6r = interleukin 6 receptor; TNF-a = tumor necrosis factor alpha; SOCS2 and 3 = suppressor of cytokine signaling 2 and 3; RAGE = advanced glycosylation end products receptor; C4BPa = complement 4 binding protein alpha; C8b = complement 8 beta; inhibin = inhibin βa; Bid3 = BH3 interacting (with BCL2 family) domain; Calpain 1 = calpain small subunit 1; Calpain 2 = calpain II, 80 kDa subunit; Plcb2 = phospholipase C, beta 2; Mylk2 = myosin light chain kinase 2; CSFr = colony stimulating factor receptor; JAG2 = Jagged 2 homolog; CLR = calcitonin-like receptor; GPX1 = epididymal secretory glutathione peroxidase; Scn2a1 = sodium channel, voltage-gated type2, alpha1.

While overall our real-time PCR analyses confirmed the expression array results, four genes had discrepant results. Expression array analyses showed that OVX resulted in an increase in both fibromodulin and fibronectin. While the trend persisted for fibronectin, significance was not reached. Curiously, PCR results suggested that fibromodulin was the greatest in OVX/E2 (Fig. 2A). Soluble guanyl cyclase-α (Fig. 2B), greater in OVX on the array, was not significantly different among groups by real-time PCR analysis. Guanyl cyclase-β (Fig. 2B) was higher in OVX on the expression array but was higher in OVX/E2 on analysis by real-time PCR. Inhibin βA (Fig. 2C) was lower in OVX on expression array analysis but was lower in OVX/E2 via real-time analysis. Finally, we selected eight additional genes, with various cellular functions, that exhibited a fivefold or greater increase by expression array analyses (Fig. 2E). With the exception of one gene, Jagged 2 homolog, all of the results for these PCR analyses paralleled the expression array results, though not all reached a level of significance of P < 0.05.

The SOCS genes have an important and poorly understood role in regulating the inflammatory response in cells. Details of cardiac signaling by SOCS genes are virtually unknown. Upon confirming the OVX-related increase in SOCS2 and SOCS3 mRNA by expression array and PCR analyses (Fig. 2C), we examined the protein expression pattern for these genes. Using Western blot analyses, we observed that although both SOCS transcripts were increased with OVX, protein expression was not significantly different among the groups (Fig. 3).

Fig. 3.

Protein levels of SOCS2 and SOCS3. A: graph summarizes expression of the 2 proteins normalized to GAPDH, which was used as a loading control. B: Western blots comparing expression of SOCS2 and SOCS3 in Sham, OVX, and OVX/E2.


The results of these experiments demonstrate that removal of estrogen has a profound impact on myocardial gene expression. A key aspect of our model was the sustained washout period after OVX allowing the full effect of estrogen withdrawal to be manifest. Our findings suggest that, in general, OVX results in a decrease in gene expression mediating vascular relaxation and an increase in gene expression mediating inflammation, apoptosis, and proteolysis compared with OVX followed by estrogen replacement or with continued endogenous estrogen production. The potential impact of changes in the expression of these genes following removal of estrogen is profound and could contribute to the mechanism(s) underlying the dramatic increase in cardiovascular diseases following menopause as well as the increased all cause mortality following surgical OVX (40). Surprisingly, previous work has not addressed the effect of changes in estrogen on cardiac gene expression.


Sex hormones are known modifiers of the inflammatory response to injury, an important aspect of myocardial dysfunction and cardiomyocyte death following ischemia and other challenges, such as endotoxin treatment. In the current experiments, the overall effect of OVX on myocardial gene expression was increased expression of genes involved in the inflammatory response. OVX increased IL-6 receptor, TNF-α, complement 8, and SOCS2 and 3 expression. These findings are consistent with reports of sexual dimorphism in myocardial TNF-α and IL-6 response following endotoxin, as well as endogenous estrogen mediating a higher threshold for endotoxin tolerance in female myocardium (37, 38). Estrogen may also exert anti-inflammatory effects involving TNF-α without altering expression, as demonstrated in a study of mice with congestive heart failure secondary to TNF-α overexpression. Estrogen treatment limited the severity of cardiomyopathy without altering TNF-α concentrations (21). IL-6 has been implicated in a broad range of cardiac pathologies and is thought to contribute to cardiac dysfunction (63). Activated by stresses including ischemia and endotoxin, IL-6 has been reported to decrease following estrogen administration (64) and, therefore, is thought to contribute to the sex-specific responses to cardiac injury. Consistent with these findings is our observation that expression of the IL-6 receptor increases following OVX compared with OVX with estrogen replacement.

Activation of the complement system in cardiac injury, including myocardial infarction, has been demonstrated in several studies, and complement activation is thought to be decreased by cardiac ischemic preconditioning (48, 49, 56). The current experiments suggest that estrogen removal results in a very large increase in expression of complement 8, but, interestingly, estrogen replacement is associated with an increase in complement 4 binding protein, a complement regulatory protein functioning as a cofactor in the degradation of complement 4b. The potential impact of altered myocardial complement gene expression due to estrogen removal has yet to be thoroughly investigated.

Interestingly, the current experiments revealed an OVX-induced decrease in expression of the AGER. Advanced glycosylation end products (AGE) result from sugars reacting with primary amino groups of proteins, are known to induce protein cross-linking and oxidant stress within various tissues, and are thought to play a role in the pathogenesis of normal aging and diabetes, as well as other chronic diseases associated with aging (44). Binding of AGE to receptors is thought to induce release of proinflammatory cytokines including IL-6 and TNF-α; however, available information on the impact of altered expression of AGERs remains quite puzzling (44). For example, as thoroughly reviewed by Simm et al. (2004), increased expression of AGERs are evident in diabetic rodent models and evidence predominantly suggests a pathological role for these receptors during aging and several models of chronic disease. Aging may alter cardiac AGERs in a receptor-specific manner (6). Even with respect to tumor growth, the apparent role of AGERs is contradictory, with reports of both increased and decreased receptor expression in various tumor-containing tissues (44). Therefore, future investigations should aim to identify the impact of estrogen and estrogen withdrawal on cardiac expression of specific isoforms of AGERs.

The inflammatory gene expression profile following OVX also included an increase in both SOCS2 and SOCS3, and this was confirmed by PCR analyses. Little is known about the SOCS genes and their function in the cardiovascular system. Their name, suppressor of cytokine synthesis, implies an anti-inflammatory action, but research has shown the situation to be more complex (10, 50). As little is known in the heart about these interesting proteins, we further analyzed expression by looking at protein levels by Western blot. Despite the increase in RNA, SOCS2 protein levels were lower in OVX vs. OVX/E2, indicating posttranscriptional regulation. Similarly, SOCS3 protein levels did not differ between the two groups, even though OVX had higher RNA levels. Transcription of SOCS genes is typically activated by cytokines, often through the JAK/STAT pathway (51). When induced, SOCS then inhibit JAK-mediated phosphorylation of the cytokine receptor, inhibiting activation of STAT signaling, thus providing negative feedback of JAK/STAT signaling, which is activated by the IL-6 family of cytokines (35).

More is known about cardiac SOCS3 than SOCS2. SOCS3 inhibits gp130 signaling by binding the cytokine receptor and inhibiting activation of it by JAK (22). The gp130 receptor is a mediator of IL-6 signaling and plays a substantial role in cardiomyocyte physiology (23). Recently, using a TNF-α receptor knockout, a study showed that TNF-α receptor deficiency protects the heart against TNF-mediated injury via the STAT3/SOCS3 pathway, resulting in decreased STAT3 activation, increased SOCS3 expression, and decreased IL-6 (58). Thus, SOCS3 seems to have an overall anti-inflammatory effect.

Interestingly, SOCS2 may have a dual suppressive and stimulating role depending on cellular concentration (50). While, in general it is thought that SOCS proteins target cytokine signaling intermediates for degradation, SOCS2 may antagonize the inhibitory effects of other SOCS proteins or increase their degradation, which has been found to enhance the cytokine response (10, 50). Therefore, after observing that OVX increased SOCS2, we were interested in whether increased SOCS2 expression would be associated with a decrease in SOCS3. However, Western blot analyses showed no clear differences among the groups in SOCS2 and SOCS3 protein. The dissociation between SOCS mRNA and protein suggests posttranscriptional regulation of SOCS genes, at least in face of the stress of estrogen removal. The role of the SOCS genes in the myocardium following estrogen removal is clearly in need of further investigation.

Nearly everything that is known about activins and inhibins, members of the transforming growth factor (TGF)-β superfamily, is known in the context of activity in the reproductive axis (52); thus, their roles in cardiovascular diseases are largely unknown. Activins are thought to play a role in cardiac remodeling associated with heart failure and in progression of fibroproliferative vascular diseases (20, 31, 36, 65). Inhibin, on the other hand, is an activin antagonist (52, 61). In the current work, cardiac expression of inhibin A was increased in Sham and OVX compared with OVX/E2. While one might be tempted to hypothesize that high inhibin levels should antagonize the potential vascular proliferative effects of activins, differential binding properties of the inhibins to activin receptors have been described. For example, neither inhibin A nor inhibin B appears capable of binding to one activin receptor type (ActRIIA) in the absence of the co-receptor betaglycan. Conversely, both inhibin A and inhibin B bind a different activin receptor type (ActRIIB2) even in the absence of betaglycan, though inhibin B bound this receptor with 1.3- to 4-fold higher affinity than did inhibin A (52). Thus while it is interesting to hypothesize that these patterns suggest an inhibin isotype-specific control on binding of target tissue, this is purely speculative at this time.

Vascular tone.

It is commonly accepted that estrogen can regulate expression of neuronal NOS and eNOS (11). Estrogen has also been shown to play a role in regulation of iNOS activation in a variety of cell types including cardiac myocytes (34). In the current experiments, OVX resulted in a decrease in eNOS and compared with OVX/E2 and sham. This finding is in agreement with numerous other published reports of OVX causing significant decreases in NO formation in rat and rabbit aorta and rat mesenteric artery (8, 17, 18, 26, 45) with estrogen replacement restoring the basal endothelial formation of NO (2, 26, 39, 45). In contrast, iNOS expression was increased with OVX compared with OVX/E2 and sham. Although iNOS has recently been shown to have protective functions, it is more frequently associated with overproduction of NO leading potentially to the synthesis of the highly reactive peroxynitrite.

The current literature is not without discrepancy with regard to estrogen and regulation of vasodilatory gene expression. Acetylcholine-induced NO mediated relaxation of rat mesenteric arteries was enhanced following OVX, with no change in eNOS protein levels (7). However, others report that acetylcholine-induced NO-mediated relaxation is unaffected by OVX plus E2 treatment (26, 42). Similarly, we have observed that OVX in aged, but not adult, rats affects vascular relaxation in response to acetylcholine (Stice JP, Eiserich JP, and Knowlton AA, unpublished observations).

Interestingly, soluble guanyl cyclase-β was highest in the OVX/E2 group. This finding is consistent with previous work from our laboratory (Stice JP, Eiserich JP, and Knowlton AA, unpublished observations). These experiments showed that in aged compared with adult OVX Brown Norway rats with and without immediate estrogen replacement, aging plus OVX decreases both α- and β-isoforms of soluble guanyl cyclase. Estrogen replacement prevented this decrease in soluble guanyl cyclase expression. However, in adult, OVX resulted in no differences in soluble guanyl cyclase isoforms.

Epoxide hydrolase, which hydrolyzes epoxyeicosatrienoic acids (EETs), was increased in OVX vs. OVX /E2. EETs play important roles in intracellular signaling in cardiac tissues, including activation of calcium-sensitive potassium channels in vascular smooth muscle cells, resulting in vasodilation of the coronary circulation (5). Others have reported that EETs possess anti-inflammatory and thrombolytic properties within the vasculature (32, 33, 59). In endothelial cells, EETs increase intracellular cAMP levels, upregulate expression of NOS, and protect against hypoxia-reoxygenation injury (62). Thus, increased hydrolysis of EETs via increased epoxide hydrolase would have a proinflammatory effect.

Extracellular matrix and cell-cell interactions.

Expression array analyses showed an OVX-related increase in fibromodulin, a small proteoglycan that associates with collagen in the extracellular matrix. In vascular tissue, proteoglycans are upregulated at sites of intimal hyperplasia and reportedly play a role in the development of atherosclerosis by binding apolipoprotein-B containing lipoproteins and regulating vascular cell growth (4, 47). Interestingly, PCR analyses showed OVX/E2 to have the greatest fibromodulin expression. Consistent with this finding of increased extracellular matrix components was an OVX-associated increase in the expression of the collagen gene COL12A1. Fibronectin, another component of the extracellular matrix, although increased with OVX by array and frequently increased in aging models, was not found to be significantly increased by real-time PCR analyses in the current study.

In the current experiments, OVX resulted in a significant increase in connexin 43, a gap junction protein with a critical role in intercellular communication. Decreased vascular expression of connexin 43 after OVX has been reported in mesenteric artery of rats (27). This study differed from the current work in the use of immature rats (only 8 wk at time of OVX) and only 4 wk from OVX to study. It is now fairly well accepted that gap junctions play a crucial role in endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation and hyperpolarization of vascular smooth muscle (12). These findings suggest that connexins play key roles in EDHF sensitivity to estrogens. It is possible, therefore, that changes in their expression level might explain some of the variability in EDHF-mediated responses during the estrus or after OVX of rats (7). While our finding of an OVX-associated increase in connexin 43 is in contrast to findings in vascular tissue, it is consistent with literature specific to the myocardium. Myocardial connexin 43 mediates cell-to-cell movement of calcium ions; this connexin-mediated calcium movement may induce calcium overload and contractile dysfunction postischemia (53, 54). Dephosphorylation of connexins has been related to cardioprotection and electrical uncoupling during ischemia, and estrogen has been found to decrease phosphorylation of this protein (13, 24).

Apoptosis and proteolysis.

Apoptotic death of cardiomyocytes contributes to morbidity and mortality associated with heart disease (1). Caspase 3 has a central role in the apoptotic cascade. The current experiments demonstrate an increase expression of caspase 3. Whether this increase in caspase 3, which is synthesized as an inactive procaspase, translates into more cell death in response to apoptotic signals remains to be determined. However, consistent with this finding is an OVX-associated increase in Bid3, a BH3 interacting domain death agonist. Bid regulates in apoptotic cascades in that when it is cleaved by death receptor-activated caspases generating truncated Bid (tBid), it then functions as an operational BH3 domain-only protein causing cell death (60). Collectively, the OVX-associated increases in caspase 3 and bid suggest an overall increase in the proapoptotic cardiac profile.

Two subunits of calpain, a calcium-dependent cysteine protease, were also increased in OVX by both array and real-time PCR. Calpain, once activated, can induce apoptosis in heart cells and may do so in a caspase-independent manner (9). Collectively the results of these experiments suggest that OVX increases expression of proapoptotic genes.

Concluding Remarks

Estrogen has a plethora of effects on cardiovascular tissues. Using rat models of estrogen depletion and replacement, these experiments, at this stage still correlative, demonstrated OVX-related changes in myocardial gene expression that could impose increased inflammation with less suppression of cytokine signaling, impaired regulation of vascular tone, and accelerated apoptosis and proteolysis compared with heart tissues of estrogen-replaced animals. We acknowledge that since human aging/onset of menopause imposes more of a continuum of estrogen changes, our model may result in changes in gene expression that may differ from those imposed by menopause in humans. However, in light of increasing awareness that the risk of all cause mortality associated with surgical ovariectomy in women younger than 45 yr is increased (40), we believe that this model is still provides information quite relevant to basic research on estrogen related changes in gene expression.

While basic science, including the current experiments, provides evidence that estrogen should protect against negative changes associated with estrogen removal and development of cardiovascular disease, results of randomized clinical trials assessing hormone replacement in the prevention of cardiovascular disease challenge this existing dogma. Indeed, investigation of the effects of estrogen on the cardiovascular system have demonstrated benefits with respect to metabolic coronary artery disease risk factors, arterial function, and surrogate clinical markers of cardiovascular disease risk (46). Nonetheless, while it seems implausible that estrogen should not have beneficial cardiovascular effects, the risks of adverse health effects must be balanced against the benefits associated with estrogen replacement following either surgical OVX or menopause. To guide future research aimed at improving hormone formulations, optimizing timing of treatments, and identifying patients who are most likely to benefit from treatment with respect to limiting risk and progression of cardiovascular disease, improved understanding of the widely varied estrogenic effects in cardiovascular tissues is essential.


Extramural funding for this project was provided by Medtronic, National Institutes of Health Grants AG-19327, HL-077281, and HL-079071, and a Department of Veterans Affairs Merit Award, all to A. A. Knowlton.


  • Address for reprint requests and other correspondence: K. L. Hamilton, 220 Moby B. Complex, Colorado State Univ., Fort Collins, CO 80523-1582 (e-mail: karynh{at}cahs.colostate.edu)

    Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


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