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Ann Thorac Surg 1998;66:1658-1661
© 1998 The Society of Thoracic Surgeons

Protective action of 17ß-estradiol in cardiac cells: implications for hyperkalemic cardioplegia

^a Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota, USA

Accepted for publication May 22, 1998.

Address reprint requests to Dr Terzic, Division of Cardiovascular Diseases, Department of Medicine, Guggenheim-7F, Mayo Clinic and Foundation, Rochester, MN 55905

	Abstract

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Background. Hyperkalemic cardioplegic solutions effectively arrest the heart, but may also induce intracellular Ca²⁺ loading and cellular hypercontracture, which could contribute to ventricular dysfunction associated with global surgical ischemia. Recently, it has been proposed that 17ß-estradiol may possess protective properties in the ischemic myocardium. The purpose of the present study was to examine the action of 17ß-estradiol on cardiac cells exposed to hyperkalemic stress.

Methods. Single ventricular cardiomyocytes, a preparation devoid of vascular and neuronal elements, were isolated from guinea pig hearts, loaded with a Ca²⁺-sensitive fluorescent probe, and imaged by digital epifluorescent microscopy. The emitted fluorescence of the probe, a measure of intracellular Ca²⁺ concentration, and cell length were simultaneously recorded during hyperkalemic challenge, in the absence or presence of 17ß-estradiol.

Results. In control cardiomyocytes, the cytosolic concentration of Ca²⁺ was 138 ± 11 nmol/L and cell length 93 ± 11 µm. Exposure to high K⁺ (+16 mmol/L KCl) significantly increased cytosolic Ca²⁺ to 2,191 ± 187 nmol/L (p < 0.001), and produced cell shortening (length at 39 ± 5 µm; p < 0.001). 17ß-Estradiol (10 µmol/L) acutely prevented high K⁺ to induce either intracellular Ca²⁺ loading (144 ± 13 nmol/L, p < 0.001) or hypercontracture (91 ± 10 µm, p < 0.001). Tamoxifen (10 µmol/L), an antiestrogen, abolished the protective effect of 17ß-estradiol.

Conclusions. We conclude that 17ß-estradiol prevents hyperkalemia-induced Ca²⁺ loading and hypercontracture through a direct and tamoxifen-sensitive action in cardiomyocytes. This study raises the possibility that 17ß-estradiol should be considered as a cardioprotective adjunct toward a safer hyperkalemic cardioplegia.

	Introduction

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Hyperkalemic cardioplegia is a widely used method to arrest the heart during open heart operations [1]. Potassium, present at high concentration in cardioplegic solutions, induces depolarization of the cardiac cell membrane and thereby, electromechanical arrest of the heart. This is necessary for the surgeon to operate on a calm operating field. However, membrane depolarization can also lead to intracellular Ca²⁺ loading, which has been observed in myocardial cells exposed to hyperkalemic challenge [2–6]. K⁺-induced Ca²⁺ loading is undesirable, and could impair both contraction and relaxation of a cardiac cell, perturb proper membrane excitation, and induce abnormal gene expression [7–10]. If high levels of Ca²⁺ persist within the cytosol, this may further lead to irreversible cellular hypercontracture and ultimately cell death [7]. Although the pathophysiology underlying cardioplegia-related ventricular dysfunction is complex [1, 8, 9], impaired intracellular Ca²⁺ homeostasis has been identified as a major contributing factor [1, 10]. Therefore, the search for substances that could promote protection of the myocardium under hyperkalemic cardioplegia is warranted [10, 11]. Such cardioprotective agents should inhibit K⁺-induced Ca²⁺ loading in cardiac cells, thus reducing the risk of cellular damage during global surgical ischemia.

Estrogen supplementation is known to reduce the incidence of ischemic heart disease [12]. Although the major mechanism responsible for the protective effect of estrogens is believed to be attributable to an antiatherogenic action on the lipid profile [12], more recently 17ß-estradiol (E2) has also been found to acutely inhibit cardiac Ca²⁺ channels [13, 14], and to limit the size of myocardial infarction [15, 16]. However, whether E2 has a beneficial effect in the setting of hyperkalemic cardioplegia is, at present, unknown.

Therefore, the purpose of the present study was to examine the action of E2 on single cardiomyocytes under conditions of K⁺-induced Ca²⁺ loading and hypercontracture. We report a novel cardioprotective property of E2 that may be of benefit toward the development of safer hyperkalemic cardioplegia.

	Material and methods

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Isolation of single ventricular cardiomyocytes
Ventricular cardiomyocytes were enzymatically dissociated from pentobarbital-anesthetized, sexually mature, female guinea pigs, as previously described [5, 17]. In brief, hearts were perfused (at 37°C) with medium 199, Ca²⁺–ethylene glycol-B/S(ß-aminoethyl ether) N,N,N',N',tetraacetic acid-buffered low Ca²⁺ medium, and low Ca²⁺ medium containing pronase E (8 mg/100 mL), proteinase K (1.7 mg/100 mL), bovine albumin (0.1 g/100 mL), and 200 µmol/L CaCl₂. Ventricles were cut into fragments, and single cells were isolated by stirring the tissue in pronase E, proteinase K, and collagenase (5 mg/10 mL).

Digital epifluorescent microscopy
Relaxed, rod-shaped cardiomyocytes, with clear striations and a smooth surface, were loaded with the esterified form of the fluorescent probe Fluo-3 (Fluo-3AM, Molecular Probes, Eugene, OR), excited at 488 nm and imaged as previously described [5, 18]. Fluorescence emitted at 520 nm was captured by an intensified charge coupled device camera, and digitized using an imaging system (Attoflor RatioVision, Atto Instruments, Rockville, MD) coupled to an inverted microscope (Zeiss Axiovert-135, Thornwood, NY). Cell length was simultaneously monitored [18]. An estimate of the cytosolic Ca²⁺ concentration, as a function of Fluo-3AM fluorescence, was calculated according to the equation: , where [Ca²⁺]r is resting cytosolic Ca²⁺, F_min and F_max minimal and maximal fluorescence intensity, K_d dissociation constant of the Fluo3-AM–Ca²⁺ complex and F intensity of fluorescence [2, 3, 5, 19, 20].

Experimental protocol
Single cardiomyocytes were bathed (37°C) in Tyrode solution containing (in mmol/L) NaCl 136.5, KCl 5.4, CaCl₂ 1.8, MgCl₂ 0.53, glucose 5.5, and HEPES–NaOH 5.5 (pH 7.4). A K⁺ challenge was induced by adding 16 mmol/L KCl [5]. To assess the effect of E2, myocytes were exposed first to Tyrode, then to Tyrode plus E2 (10 µmol/L), followed by Tyrode and E2 plus 16 mmol/L KCl (in the absence or presence of 10 µmol/L tamoxifen, an antiestrogen), and finally to Tyrode plus 16 mmol/L KCl.

Drugs
All chemicals were from Sigma Chemical Co (St. Louis, MO), with the exception of pronase E, and Fluo-3AM that were purchased from Serva (Heidelberg, Germany) and Molecular Probes, respectively. Estradiol and tamoxifen were dissolved in alcohol, and Fluo-3AM was dissolved in dimethyl sulfoxide plus pluronic acid. All substances were diluted in Tyrode solution immediately before the experiments. The final concentration of solvents in Tyrode solution was kept to less than 0.1%. At this concentration dimethyl sulfoxide did not affect Ca²⁺ levels nor cell length [5].

Statistical analysis
Results are expressed as mean ± standard error of the mean; n refers to the number of experiments. Significant differences between two means were determined with the Student’s t test, and with one-way analysis of variance when more than two groups were compared. A p value less than0.05 was considered significant.

	Results

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Effect of hyperkalemic challenge on cytosolic Ca²⁺ concentration and cell length in single ventricular cells
In control ventricular cardiomyocytes, the cytosolic concentration of Ca²⁺ was 138 ± 11 nmol/L and the cell length 93 ± 11 µm (n = 21). Exposure to K⁺-challenge (+16 mmol/L KCl) significantly increased cytosolic Ca²⁺ to 2,191 ± 187 nmol/L (p < 0.001), which in cardiac cells produced hypercontracture (cell length at 39 ± 5 µm; p < 0.001; Fig 1 ).

View larger version (20K): [in this window] [in a new window]   Fig 1. Effect of the addition of 16 mmol/L KCl (K+) on Ca2+ loading and cell length in single cardiac cells. Bars represent mean ± standard error of the mean (n = 21).

View larger version (59K): [in this window] [in a new window]   Fig 2. 17ß-estradiol (E2) prevents K+-induced Ca2+ loading and cell shortening. (A) Epifluorescent images from a cardiomyocyte under control conditions (frame 1), in E2 (frame 2), in E2 plus 16 mmol/L KCl (frame 3), and in 16 mmol/L KCl (frames 4 and 5). White horizontal bar indicates 30 µm. (A1 and A2) Fluo-3AM fluorescence and cell length as a function of time from myocyte in (A). Circles labeled 1 to 5 correspond to frames 1 to 5. (B and C) Fluorescence and cell length under indicated conditions. Bars represent mean ± standard error of the mean (n = 4).

View larger version (54K): [in this window] [in a new window]   Fig 3. Tamoxifen antagonizes the action of 17ß-estradiol (E2) against K+-induced Ca2+ loading and cell shortening. (A) Epifluorescent images from a cardiomyocyte under control conditions (frame 1), in E2 plus 16 mmol/L KCl (frame 2), and in tamoxifen and E2 plus 16 mmol/L KCl (frames 3 and 4). Corresponding graphs showing changes in intensity of Fluo-3AM fluorescence (A1) and cell shortening (A2) as a function of time. Circles labeled 1 to 4 correspond to frames 1 to 4. White horizontal bar indicates 30 µm. (B and C) Changes in maximal fluorescence and cell length under different experimental conditions. Bars represent mean ± standard error of the mean (n = 3).

	Comment

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K⁺-induced Ca²⁺ loading and hypercontracture
In the present study, we exposed cardiomyocytes to an elevated extracellular concentration of K⁺, a challenge that occurs during cardioplegic cardiac arrest. We observed that addition of 16 mmol/L extracellular K⁺ induced significant intracellular Ca²⁺ loading. This is in accord with our previous studies, in which we demonstrated that elevation of extracellular K⁺ leads to intracellular Ca²⁺ overload [2–6]. The observed increase in cytosolic Ca²⁺ concentration was accompanied with cell hypercontracture. This finding confirms previous studies in which it was shown that when cytosolic Ca²⁺ occurs in a sustained manner, this could induce irreversible cell contracture due to the inability of contractile proteins to relax [7, 10]. Therefore, the present findings are in agreement with the notion that a hyperkalemic challenge may induce cellular injury [1].

17ß-estradiol prevents K⁺-induced Ca²⁺ loading and hypercontracture
The present study also demonstrates that E2 prevents K⁺-induced Ca²⁺ loading and cellular hypercontracture in single cardiomyocytes, a pure myocardial preparation with no neuronal and vascular elements. These data provide the first direct evidence for a cardioprotective action of estrogens in isolated cardiac cells.

Previously, it has been shown that chronic treatment with E2 may prevent the development of cardiac hypertrophy and lethal arrhythmia by promoting expression of genes encoding for myosin heavy chain and connexin proteins [21]. In addition to regulating gene expression [22], evidence for acute, nongenomic actions of estrogens that do not require de novo protein synthesis has also been obtained in the myocardium [16]. The rapid onset of E2 action reported in the present study is in accord with the notion that E2 may exert nongenomic effects in the myocardium [15, 16]. Herein, the effect of E2 was observed at a high pharmacologic concentration at which previously a "calcium antagonistic" action of E2 was observed [13, 14]. It has been proposed that this calcium antagonistic effect in the myocardium may be associated with activation of K⁺ channels, inhibition of Ca²⁺ currents, and suppression of Ca²⁺ influx [13–15, 23]. Known cardioprotective agents do activate cardiac K⁺ channels and prevent Ca²⁺ loading [2, 3, 10]. In this regard, E2 may share protective properties with established cardioprotective agents, including the neurohormone adenosine as well as potassium channel opening drugs [2–6, 10, 11].

Effects of 17ß-estradiol can be blocked by tamoxifen
In the present study, the antiestrogen tamoxifen blocked the E2-induced cardioprotection in the setting of a hyperkalemic challenge. The rapid onset of the observed effect of E2 suggests that cytosolic receptor binding, nuclear translocation, and transcription is not essential to mediate protection of cardiomyocytes against K⁺-induced intracellular Ca²⁺ loading and associated hypercontracture. Besides well-characterized cytosolic estrogen receptors, it has been recently demonstrated that estrogens can also bind to membrane receptors [24]. Therefore, the time course of the E2 action and the sensitivity of the E2 effects toward tamoxifen, a known ligand of estrogen receptors [22], suggests that the effect of E2 may be mediated through estrogen receptors that are independent from a genomic action. This is in accord with observations of a nongenomic, but receptor-mediated, cardioprotective effect of E2 at the whole heart level in the setting of ischemia–reperfusion injury [16].

Study limitations
To determine the direct effect of E2 on intracellular Ca²⁺ concentration and cell length after a challenge with high external concentration of K⁺, it was necessary to image isolated cardiomyocytes. Such an approach provided a direct visualization of the protective effect of E2 at the single cell level. However, it should be considered that in intact myocardium, additional cardiac, as well as extracardiac, mechanisms could further modulate the observed effect of E2. Moreover, all conditions associated with hyperkalemic cardioplegia in the setting of global surgical ischemia were not simulated in the present study. This includes the temperature at which cardiomyocytes were maintained. Therefore, further evaluation of the cardioprotective properties of E2 is warranted in the setting of open heart operations.

In conclusion, the present finding that E2 prevents cell injury in ventricular cardiomyocytes exposed to elevated extracellular K⁺ may have important implications toward a safer hyperkalemic cardioplegia. In view of the observed efficacy of E2, the possibility that this natural hormone may serve as a valuable supplement to hyperkalemic cardioplegia deserves to be considered, and the therapeutic profile of E2 further tested. For E2 to be considered as an adjunct to hyperkalemic cardioplegia, rigorous comparative studies with other cardioprotective agents, including adenosine and potassium channel openers, would be required.

	Acknowledgments

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This research was supported by a Merck Sharp & Dohme International Award in Clinical Pharmacology to Aleksandar Jovanovic and grants from the American Heart Association, the Miami Heart Research Institute, the Bruce and Ruth Rappaport Program in Vascular Biology and Gene Delivery, and the John Tainsh Heart Research Fund to Andre Terzic.

	References

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