Recent clinical studies (1-4) have revealed the clinical features of 'Tako-tsubo (Ampulla)' cardiomyopathy mimicking acute myocardial infarction. This is characterized by: (i) acute onset, reversible left ventricular (LV) apical wall motion abnormalities (ballooning) with chest symptoms; (ii) electrocardiographic (ECG) changes (ST elevation); (iii) minimal myocardial enzymatic release; and (iv) no significant stenosis or spasm on coronary angiography. The backgrounds of these patients are unique, but have the following two characteristics: first, there is an emotional or physical stress event triggering the attack (33-86%); second, the patient is often an elderly (postmenopausal) female (75-93%). The precise etiology of this syndrome is yet to be determined, so finding a similar animal model would be extremely valuable for the study of the underlying pathological mechanisms of this condition. We have produced this syndrome by immobilization stress (IMO) in rats (5); this produces a reversible elevation of the ST segment on the ECG as well as the reversible LV apical ballooning in the left ventriculography (LVG), both of which were normalized by pretreatment with αβ-adrenoceptor blockers (6,7). In order to study whether these cardiac changes are especially predominant in elderly female subjects (8), we investigated LVG and ECG under IMO in ovariectomized (animal model of post-menopause) and estradiol- supplemented ovariectomized female rats.
Adult (24- to 32-week-old) female Wistar rats weighing 250-300 g, (n = 16) were purchased from Kiwa Laboratories (Wakayama, Japan). Estradiol 10 mg (β-estradiol; Sigma, St. Louis, MO, U.S.A.) was intro- duced into a silicone tube (Silastic Medical Grade Tubing, internal diameter 1.5 mm, outer diameter 2.0 mm; Dow Corning Corp., Midland, MI, U.S.A.) and was sealed with silicone paste (Silastic Medical Adhesive Silicone Type A; Dow Corning Corp.). Under anesthesia with sodium pentabarbital (40 mg/kg), a bilateral ovariectomy was performed by ligation and dissection of the ovaries (OVX, n = 8). Eight rats were also ovariectomized and subcutaneously implanted with silicone tube containing estradiol (OVX + E).
After 3 weeks, a polyethylene catheter (PE50; Becton Dickinson, Sparks, MD, U.S.A.) was inserted into the left ventricle via the common carotid artery. Under anesthesia with sodium pentobarbital (40 mg/kg), 1 ml of contrast medium (Iopamidol 370; Schering AG, Berlin, Germany) was injected and LVG was performed (control) using the digital subtraction system (DSA) (SERIES9600; OEC Medical Systems Inc., Salt Lake City, UT, USA). The LVG was recorded at 30 frames/s. The margins of the LV images were traced, and end-systolic or end-diastolic LV areas were also evaluated using NIH image 1.62. Heart rate was also recorded during LVG using ECG (FX-4100; Fukuda Denshi Co., Tokyo, Japan). The percentage contraction was determined as 100 × (end-diastolic area - end-systolic area)/end-diastolic area. Statistical analysis was performed by one-way analysis of variance followed by Fisher's protected least significant difference test, or Student's t-test using StatView software (Abacus Concepts, Berkeley, CA, U.S.A.).
The next day, the rats were exposed to IMO for up to 20 min by securing the rat on its back to a board using adhesive tapes, and LVG was performed without anesthesia (6,7). Three rats were excluded from our sample because of incomplete cannulation. At the end of IMO, the rats were decapitated and blood samples were collected. Serum estradiol levels were estimated with an estradiol radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA, U.S.A.) according to the manufacturer's protocol. Cross-reactivities to estrone and estriol were 1.1 and 0.32%, respectively. No cross-reactivities to other steroids were observed. The minimal limit of detection was 2 pg of estradiol in 1 ml of serum.
All animal manipulations were approved by Wakayama Medical University Animal Care and Use Committee.
Serum estradiol levels were significantly increased in OVX + E (mean ± SEM: OVX, 6.0 ± 2.4 pg/ml, n = 6, versus OVX + E, 515.9 ± 107.5 pg/ml, n = 7, p < 0.001). In response to IMO, the percentage contraction in LVG was significantly reduced in OVX (control: 50.3 ± 2.2%→ stress: 34.0 ± 4.5%, n = 6, p < 0.01), while it was not changed in OVX + E (control: 50.4 ± 1.2%→; stress: 46.0 ± 3.3%, n = 7, NS) (Fig. 1A). In response to stress, heart rate was significantly increased in OVX (control: 383 ± 18 bpm→; stress: 483 ± 15 bpm, n = 6, p < 0.01) and OVX + E (control: 347 ± 13 bpm→; stress: 402 ± 23 bpm, n = 7, p < 0.05). Heart rate in the control group was not significantly different between OVX and OVX + E but the heart rate in the stressed group was significantly higher in OVX than in OVX + E (p < 0.01) (Fig. 1B).
The study presented here demonstrates that reduction of LV contractility and the increase of heart rate in response to emotional stress were attenuated by supplement of estradiol in the ovariectomized rats.
Because there is a high incidence of Tako-tsubo cardiomyopathy in elderly post-menopausal female humans, it is likely that a reduction of estradiol may be involved in this myocardial disease. As shown here, changes in estradiol levels appear to be an important factor in this condition. The etiology of Tako-tsubo cardiomyopathy is unknown, but several suggestions have been proposed, including microvascular spasm (4,9), sympathetic nervous dysfunction, and catecholamine cardiotoxicity (3,10,11). Because estrogen has a vasodilatory effect by induction and activation of endothelial nitric oxide synthase (12), a decrease of estrogen may disturb the coronary microcirculation. In addition, several clinical investigations (13-16) as well as animal experiments (17,18) demonstrated that estrogen modulates cardiovascular reactivity by increasing vagal tone and decreasing sympathetic nervous activity. In fact, heart rate response was attenuated in OVX + E, suggesting that high levels of estradiol can affect the autonomic nervous activity. As previously demonstrated, pretreatment with αβ-adrenoceptor blockers completely normalized the stress- induced ECG change (6) and LV wall motion (7), and inhibited both the activation of mitogen-activated protein (MAP) kinase (19) and induction of immediate early genes (IEG) (20) in the heart as well.
These data strongly suggest that activation of the sympathetic nervous system in response to IMO stress is the major trigger for these physiological and molecular changes in the heart (21). Data from the present study suggest that estrogen may be partly involved in this process through the inhibition of sympathetic nervous activation. Immobilization stress activates multiple central nervous system sites, which are visualized by detection of IEG, such as c-fos mRNA (22). In preliminary experiments we have found that supplements of estrogen reduce IEG expression in several brain regions, such as the paraventricular hypothalamic and lateral septal nuclei, two central nervous system sites known to be involved in regulation of sympathetic functions (23,24). Previous work has shown that estrogen decreases uterine sympathetic innervation via an estrogen receptor α-medicated mechanism (25). Estrogen may have this effect on cardiac tissues as well. In a premenopausal non-pregnant female rat, serum estradiol levels vary during the estrous cycle. Basal estradiol levels are 7 pg/ml on estrus and reach peak levels of 50 pg/ml on pro-estrus (26). In our study, serum estradiol levels in OVX + E were much higher than these normal variations. Though high levels of estrogen attenuated the stress-induced cardiac responses, it is not clear that physiological levels of estrogen can also show similar effects. Therefore, it should be careful to conclude that the reduction of estradiol level is the main etiology in high incidence of Takotsubo cardiomyopathy in postmenopausal females. More detailed studies will be needed to obtain a better understanding of Tako-tsubo cardiomyopathy and the role of estrogen in this disease.
We thank Dr Arthur D. Loewy (St. Louis, MO, U.S.A.) for helpful comments and careful reading of the manuscript.
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