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Am J Physiol Heart Circ Physiol 278: H1640-H1647, 2000;
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Vol. 278, Issue 5, H1640-H1647, May 2000

Myocardial ischemia-reperfusion injury in estrogen receptor-alpha knockout and wild-type mice

Peiyong Zhai1, Thomas E. Eurell1, Paul S. Cooke1, Dennis B. Lubahn2, and David R. Gross1

1 Department of Veterinary Biosciences, University of Illinois, Urbana-Champaign, Illinois 61802, and 2 Departments of Biochemistry and Child Health, University of Missouri, Columbia, Missouri 65211


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the function of estrogen receptor-alpha in global myocardial ischemia and reperfusion injury in male estrogen receptor-alpha knockout (ERKO) and wild-type mice. Mouse hearts were subjected to 45 min of global ischemia followed by 180 min of reperfusion. The hearts were excised, cannulated, and maintained in a chilled (4°C) cardioplegia solution until warm (37°C) oxygenated Krebs-Henseleit bicarbonate buffer was perfused through the coronary arteries. ERKO hearts started beating later and had a higher incidence of ventricular fibrillation and/or tachycardia than control hearts. Coronary flow rate was significantly lower in ERKO hearts during the 90- and 120-min periods of reperfusion. Ca2+ accumulation was significantly greater following 30, 90, 120, 150, and 180 min of reperfusion in ERKO hearts. Nitrite production was significantly less in ERKO hearts following 90, 120, and 150 min of reperfusion. Myocardial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was significantly lower in experimental ERKO hearts. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of ischemia-reperfused ERKO hearts but not in control tissues. Hematoxylin-basic fuchsin-picric acid-stained sections from experimental ERKO hearts had fewer viable myocytes compared with controls. Transmission electron microscopy revealed swollen and fragmented mitochondria with amorphous and granular bodies, loss of matrix, and rupture of cristae in experimental ERKO hearts. This is the first demonstration that estrogen receptor-alpha plays a cardioprotective role in ischemia-reperfusion injury in males.

calcium; nitric oxide; mitochondrial function; myocardial ultrastructure


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE RISK OF CORONARY HEART disease in women between puberty and menopause is much lower than that in age-matched men, but this significant gender difference diminishes when postmenopausal women and men of similar age are compared (35). Anecedotal evidence suggests that premenopausal women withstand elective ischemia-reperfusion injury, i.e., cardioplegic arrest, during open heart surgery better than males. A man with a disruptive mutation in estrogen-receptor gene was reported to have impaired flow-mediated, endothelium-dependent vasodilatation (51) and premature coronary artery disease (52) in the presence of circulating estrogen. Therefore, endogenous estrogen may have cardioprotective effects in both males and females when a functional estrogen receptor is present.

Estrogen replacement therapy, which provides exogenous estrogen to postmenopausal women, increases the circulating estrogen concentration and significantly decreases the morbidity and mortality of coronary heart disease in these patients (50). 17beta -Estradiol (E2) appears to preserve endothelium-dependent coronary artery dilation, reduce infarct size, and decrease the occurrence of ventricular arrhythmias in experimental models of regional ischemia-reperfusion (14, 21, 37).

The mechanisms by which E2 may exert cardioprotective effects during ischemia-reperfusion are unclear. Because E2 actions are mediated through its cognate receptors, studying the function of estrogen receptors may be helpful to the elucidation of the mechanisms of the cardiovascular effects of estrogen. The classic subtype of estrogen receptors (ER), ER-alpha , is known to be expressed in the male cardiovascular system (25). It is not known if ER-alpha has any function in myocardial ischemia-reperfusion. Although the cardioprotective effects of endogenous and exogenous estrogen have received extensive attention in females, much less work has been directed toward elucidating the possible role of estrogen in males. These experiments were designed to explore a possible role for ER-alpha in global ischemia-reperfusion injury in male mice.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental animals. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the University of Illinois and were conducted in strict accordance with the "Guiding Principles for the Care and Use of Research Animals." ER-alpha knockout (ERKO) mice were obtained by mating mice of a mixed C57BL6/129SV background that were heterozygous for the ER-alpha gene disruption as described previously (33). After genotyping, only homozygous ERKO males and homozygous wild-type C57BL6 males (control) were used in these experiments. Eight male ERKO mice (group 1) and 12 male control mice (group 2), 40 to 60 days old, were used for myocardial ischemia-reperfusion studies. An additional 6 male ERKO mice (group 3) and 6 male control mice (group 4), 40 to 60 days old, were used to study mitochondrial function, myocardial histology, and ultrastructure without ischemia-reperfusion.

Experimental protocol. Mice were anesthetized with ketamine (20 µg/g ip) and xylazine (0.5 µg/g ip) and treated intraperitoneally with 50 units of heparin. The heart was quickly removed and immersed in 4°C cardioplegic solution [Plegisol (Abbott Labs) + 25 mmol/l NaHCO3 and 2 U/ml heparin], pH 7.4. Hearts from groups 1 and 2 had the aorta isolated and catheterized with a 22-gauge polypropylene tube. After 45 min of cold cardioplegia, these hearts were mounted on a Langendorf-type isolated heart perfusion system and subjected to 3 h of retrograde coronary artery reperfusion with 37°C oxygenated Krebs-Henseleit bicarbonate buffer (Sigma), pH 7.4, at a constant pressure of 120 cmH2O. Coronary flow, coronary effluent nitrite concentration, and calcium concentrations in both coronary influent and effluent were measured at various time points during reperfusion. The time the heart required to resume regular beating and the occurrence of ventricular arrhythmias were recorded during reperfusion. After 180 min of reperfusion, myocardial samples were prepared for measuring mitochondrial function, myocardial histology, and ultrastructure. Hearts from groups 3 and 4 were prepared, after being rinsed with the cold cardioplegia, for measuring mitochondrial function, myocardial histology, and ultrastructure.

Measurement of coronary flow rate. The coronary effluent volume was measured at 30-min intervals for a total of 180 min. Coronary flow rate (CFR , in ml · min-1 · g-1) was defined as the total volume collected during the reperfusion interval divided by the time, normalized by the heart wet weight, later determined at the end of the reperfusion period.

Measurement of nitrite concentration in coronary effluent. Nitrite concentration in coronary effluent was measured using the Griess reaction (38). One milliliter of coronary effluent was incubated with 200 µl of sulfanilamide (5 mM in 0.5 N HCl) and 20 µl of napthylenediamine dihydrochloride (20 mM in distilled water) at room temperature. Effluent nitrite concentration was obtained from a standard curve for known concentrations of sodium nitrite [optical density (OD) at a wavelength of 545 nm]. Myocardial nitrite production (nmol/g) was estimated as the product of nitrite concentration and coronary effluent volume normalized by heart wet weight.

Estimation of myocardial Ca2+ accumulation. The Ca2+ concentrations in coronary perfusates and effluents were measured by inductively coupled plasma atomic emission spectrometry at the end of each 30-min period of reperfusion. Myocardial Ca2+ accumulation (µmol/g) was estimated from the calcium concentration (µmol/ml) difference between perfusate and effluent times coronary flow (ml) per heart wet weight (g).

Myocardial 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. The conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to an insoluble formazan dye product provides an estimate of mitochondrial respiratory function (30, 49). A 1-mm-thick section of the right and left ventricle was cut parallel to the atrioventricular groove within 2 min after perfusion was stopped. The section was incubated with 1 ml of Dulbecco's modified Eagle's medium without phenol red and 1 ml of MTT solution (0.5 mg/ml) for 24 h at 37°C. The MTT medium solution was then gently aspirated and the formazan dye extracted from the tissue with 0.5 ml of isopropanol and DMSO (in equal volumes). The OD570 was measured and corrected for tissue wet weight (in g).

Light microscopy. The heart was fixed by immersion in 10% neutral buffered Formalin. Serial sections (6 µm) were made parallel to the atrioventricular groove. Standard hematoxylin and eosin (HE) and hematoxylin-basic fuchsin-picric acid (HBFP) stain (29) were used for morphological evaluation. Four digital images of each sample were randomly taken for morphometric analysis using NIH Image software. The contrast and magnification of all the images were identical. The percentage and the mean density (gray value) of myocardium with a positive HBFP stain were calculated.

Ultrastructure study. Small tissue blocks (~1 mm3) were cut from the left ventricular free wall, fixed in Karnovsky's fixative for 24 h at room temperature, and stored at 4°C until processed. The sample was postfixed in osmium tetroxide, dehydrated in a graded series of alcohol, treated with propylene oxide, and embedded in epoxy. After polymerization, 0.5-µm sections were examined under light microscopy, and representative areas of tissue samples were chosen for ultrathin sectioning (0.1 µm). The ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate and lead citrate, and examined with a Hitachi 600 transmission electron microscope. Four negative films per sample were randomly taken for quantitative analysis. The films were scanned to obtain digital images, which were then analyzed using NIH Image software. The mitochondrial cross-sectional area was measured. The number of fragmented mitochondria, the number of mitochondria with amorphous matrix densities or granular densities, and the total number of mitochondria studied in each group were counted.

Statistical analysis. All values are presented as means ± SE unless otherwise stated. Data were first analyzed using a two-way ANOVA for repeated measures or a single-factor ANOVA as appropriate. If significant differences were observed, the Bonferroni's t-test was applied to compare differences between ERKO and control groups and differences between 30-min and other time periods within groups subjected to ischemia-reperfusion. All proportions were compared using a chi-square test. The alpha  level was set at 0.05, and adjustment was made to control experimentwise type-I error rate where appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ERKO hearts (group 1) took 5.5 ± 0.5 min to resume regular beating, whereas control hearts (group 2) took only 1.8 ± 0.3 min, which was significantly shorter. Group 1 hearts also had a significantly higher incidence (3/8; 37.5%) of ventricular arrhythmias than group 2 hearts (0/12; 0%).

CFR. During the first 30 min of reperfusion, CFR was significantly higher in both ERKO (group 1) and control hearts (group 2) than during other collection periods (Fig. 1). The CFR of group 1 hearts tended to diminish faster than that of group 2 hearts. The CFR measured during 90 and 120 min of reperfusion was significantly lower in group 1 than in group 2. The heart wet weight that is used for normalized coronary flow is not significantly different between group 1 and group 2. The wet weight of the hearts that are not subjected to ischemia-reperfusion is not significantly different between ERKO (group 3) and control (group 4) hearts (Table 1). Table 1 also provides peak response values for coronary flow rate, nitrite levels, and Ca2+ accumulation. There were significant differences in coronary flow, nitrite production, and Ca2+ accumulation between group 1 and group 2 hearts.


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  		Fig. 1.   Coronary flow rate (ml · min-1 · g-1, means ± SE) of control (wild-type) hearts and estrogen receptor-alpha knock out (ERKO) hearts during 180 min of reperfusion after 45 min of ischemia. *Significant difference from control. #Significant difference with time, within groups, compared with 30-min values.


                              
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  		Table 1.   Heart wet weight, the weight of the tissue for MTT reduction, and values for measured parameters during 60-90 min of reperfusion

Nitrite production. Estimated nitrite production was the highest during the first 30 min of reperfusion. ERKO hearts in group 1 produced significantly less nitrite than control hearts in group 2 during 90, 120, and 150 min of reperfusion (Fig. 2).


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  		Fig. 2.   Nitrite production (nmol/g, means ± SE) in control (wild-type) hearts and ERKO hearts during 180 min of reperfusion after 45 of ischemia. *Significant difference from control. #Significant difference with time, within groups, compared with 30-min values.

Myocardial calcium accumulation. A significantly greater amount of calcium accumulated in group 1 hearts than in group 2 hearts during the 30-, 90-, 120-, 150-, and 180-min time periods (Fig. 3). In group 1 hearts calcium accumulation during the first 30 min was significantly greater than that during other reperfusion times. In group 2 hearts calcium accumulation tended to decrease after 60 min of reperfusion, but no significant difference was observed between the first 30 min and other times of reperfusion.


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  		Fig. 3.   Myocardial Ca2+ accumulation (µmol/g, means ± SE) during 180 min of reperfusion after 45 min of ischemia in both control (wild-type) hearts and ERKO hearts. *Significant difference from control. #Significant difference with time, within groups, compared with 30-min values.

MTT extraction. Before ischemia-reperfusion, there was no significant difference in MTT reduction between group 4 control and group 3 ERKO hearts. After ischemia-reperfusion MTT reduction of group 2 control hearts was significantly higher than that of group 1 ERKO hearts (Fig. 4). This indicates that ERKO hearts had more severe impairment of mitochondrial respiratory function than control hearts after ischemia-reperfusion. The weight of the tissues used for MTT reduction is not significantly different between group 1 ERKO and group 2 control hearts that are subjected to ischemia-reperfusion, and neither is it significantly different between group 3 ERKO and group 4 control hearts that are not subjected to ischemia-reperfusion (Table 1).


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  		Fig. 4.   Myocardial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (OD/g, means ± SE) after 45 min of ischemia followed by 180 min of reperfusion. Myocardial MTT reduction of control (wild-type) is significantly higher than that of ERKO. *Significant difference from control.

Myocardial histology. No significant differences in histological structure were found in either HE-stained or HBFP-stained sections between the group 4 control and the group 3 ERKO hearts without ischemia-reperfusion. After ischemia-reperfusion marked myocardial damage was found in group 1 ERKO hearts. Marked interstitial edema and contraction bands were evident in ERKO samples. Damaged myocytes, detected by a positive HBFP stain, were found in groups 1 and 2, but the extent and intensity of damaged cells (those that did not exclude the stain) were more prominent in group 1 ERKO hearts. The percentage of myocardium with a positive HBFP stain in the group 1 ERKO hearts was significantly higher than that of group 2 control. In addition, the mean gray value of the myocardium with a positive HBFP stain was also significantly higher in group 1 ERKO than in group 2 control hearts (Fig. 5).


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  		Fig. 5.   Hematoxylin-basic fuchsin-picric acid (HBFP) stain of myocardium after 45 min of ischemia followed by 180 min of reperfusion. A: control (wild-type) mouse. Stain was not taken up by a majority of cells, although some uptake (intensive dark areas) indicates myocardial damage. B: ERKO mouse. Stain was taken up by a large number of cells, indicating extensive myocardial damage. C: percentage of myocardium with positive stain (means ± SE). D: mean gray value of myocardium with positive stain (means ± SE). *Significant difference from control.

Myocardial ultrastructure. Before ischemia-reperfusion, no significant differences in myocardial ultrastructure were found between group 4 control and group 3 ERKO hearts. After ischemia-reperfusion group 1 ERKO hearts showed marked mitochondrial damage. In group 1 ERKO hearts, the mitochondria were swollen, with the average size of mitochondria significantly greater than in group 2 control hearts (Fig. 6A). The percentage of mitochondria with granular densities and amorphous matrix densities was significantly greater in group 1 ERKO hearts than in group 2 control hearts (Fig. 6B). Many more fragmented mitochondria were found in group 1 ERKO hearts (Fig. 6C). These severe mitochondrial changes of group 1 ERKO hearts compared with mild mitochondrial changes of group 2 control hearts are demonstrated in Fig. 7. The group 1 ERKO heart samples (Fig. 7, B and D) had a marked loss of characteristic myofibrilar structure, clear areas of sarcoplasmic space resulting from intracellular edema and loss of normal structures, and severely damaged mitochondria with prominent granular densities and amorphous matrix densities compared with group 2 control samples (Fig. 7, A and C). These mitochondrial densities could represent aggregation of proteins (such as denatured enzymes) and/or deposition of calcium and phosphate (17, 13, 48).


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  		Fig. 6.   Quantitative ultrastructural changes of myocardial mitochondria after 45 min of ischemia followed by 180 min of reperfusion. A: area of mitochondrial cross-section (µm2, means ± SE). B: percentage of mitochondria with granular densities and amorphous matrix densities from all mitochondria counted in each group. C: percentage of fragmented mitochondria from all mitochondria counted in each group. *Significant difference from control (wild-type).



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  		Fig. 7.   Transmission electron microscopy of myocardium after 45 min of ischemia followed by 180 min of reperfusion, original magnification is ×12,000 for A or B (scale bar = 1 µm) and ×25,000 for C or D (scale bar = 0.5 µm). A: control (wild-type) mouse; myofibrils are intact. Mitochondria are slightly abnormal in shape but most have sharply defined cristae. B: ERKO mouse; there is discontinuation and lysis of some myofibrils. Clarity of sarcoplasmic space indicates intracellular edema and loss of normal structures. Most of mitochondria are markedly abnormal in shape, with abnormal cristae, areas of loss of matrix, and localized high densities. C: control (wild-type) mouse; mitochondria have generally distinct cristae and normal matrix. D: ERKO mouse; mitochondria are markedly abnormal and greater in size compared with control tissue. Mitochondrial membranes are occasionally disrupted. Dense granular, amorphous matrix densities are visible within some mitochondria, suggesting mineral deposition.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In studies of regional ischemia reperfusion injury, administration of E2 was reported to markedly reduce myocardial necrosis (8), decrease the incidence of ventricular arrhythmias, and preserve ventricular function (21). Estrogen replacement in ovariectomized rats was shown to improve left ventricular contractile function in isolated hearts subjected to 15 min of global ischemia followed by 20 min of reperfusion (24). All of these studies indicate that estrogen plays a protective role in cardiac ischemia-reperfusion.

Data presented in this study indicated more severe myocardial damage in male ERKO hearts subjected to 45 min of global hypothermic ischemia, followed by 180 min of reperfusion, than in male control hearts. Male ERKO hearts also had a higher incidence of ventricular arrhythmias or required more time to resume regular sinus rhythm. Our study suggests that ER-alpha plays an important protective role in male global myocardial ischemia-reperfusion. Interestingly, there were no significant differences in the parameters we studied between control and ERKO without ischemia-reperfusion. Therefore, without pathological challenges the absence of ER-alpha does not appear to result in physiological or histological myocardial abnormalities.

To our knowledge, this report is the first study of the cardioprotective function of ER-alpha in global ischemia-reperfusion in males. Men have measurable circulating concentrations of E2 (39, 44), though those concentrations are clearly less than those observed in premenopausal women. Similarly, male mice also have measurable circulating concentrations of E2 (15). In addition, the myocyte itself may produce estrogen that could have local effects (12). Regardless of its sources, estrogen exerts physiological effects through activating estrogen receptors (3, 26, 41). The demonstration of both subtypes of estrogen receptors, ER-alpha and ER-beta , in cardiovascular tissue provides the major basis for speculation about the cardiovascular effects of estrogen. Both ER-alpha and ER-beta have been demonstrated in myocytes (12) and coronary arteries (22, 45). ER-alpha mRNA occurs predominantly in wild-type mouse hearts, whereas only minimally detectable levels of ER-beta mRNA have been reported in the nuclei of ventricular muscle cells of both ERKO and wild-type mice (25). A relationship between estrogen receptor expression and the absence of atherosclerotic lesions in coronary arteries was observed in premenopausal women (32). Similarly, coronary artery disease was associated with the absence of a functional estrogen receptor caused by a disruptive mutation in the estrogen receptor gene in a human (52). Therefore, both ER-alpha and ER-beta may be involved in the cardioprotective effects of estrogen, and it is critical to understand the relative roles of each receptor. Our present results obtained from isolated perfused mouse hearts indicate that ER-alpha may be essential for the protective function of estrogen in global ischemia-reperfusion. The involvement of ER-alpha in this process does not, however, completely rule out a role for ER-beta in mediating the protective effects of estrogen in ischemia-reperfusion injury.

In our study, ERKO hearts had decreased nitrite production [i.e., nitric oxide (NO) release] compared with control hearts during reperfusion. E2 was shown to enhance the activity of NO synthase (NOS-3) and thereby the release of NO from human umbilical vein endothelial cells. This effect was inhibited by ICI-182,780, a specific anti-estrogen that appears to block both ER-alpha and ER-beta (13). Aortic endothelial NO production was reportedly correlated with the amount of estrogen receptor expressed in aortas from wild-type mice (46). Furthermore, aortas from male ERKO mice had less basal NO release than those from wild-type controls (46). Collectively, the results of our study and others suggest that the function of ER-alpha may be critical in maintaining NO production. Via the function of estrogen receptor (mainly ER-alpha ), estrogen probably increases NO production through one or more of the following mechanisms: 1) inhibiting the downregulation of NOS-3 gene expression, 2) regulating NOS-3 protein, 3) activating second messenger systems and tyrosine kinase, and 4) inhibiting the function of NO-degrading systems (20). With regard to the last mechanism, estrogen was reported to decrease superoxide generation in bovine endothelial cells by an estrogen receptor-mediated action (1). During ischemia-reperfusion, massive oxygen free radicals, including superoxide, are reported to be produced (2, 6, 10, 36, 56) and believed to be responsible for coronary endothelial dysfunction (27). Because the reaction of superoxide with NO to form peroxonitrite is one of the NO degradation pathways (5), decreasing superoxide by estrogen through estrogen receptor may contribute to the maintained NO release in the wild-type control hearts subjected to ischemia-reperfusion. Early experimental investigations have demonstrated that NO has protective effects against ischemia-reperfusion injury (9, 16, 40, 47, 54). NO could improve myocardial perfusion by ameliorating coronary dysfunction (16, 40, 47) and reduce tissue edema by decreasing microvascular permeability (28). Our experimental findings also demonstrated that, in association with the impaired NO production, decreased coronary flow rate and marked myocardial edema were present in ERKO hearts subjected to ischemia-reperfusion. The function of ER-alpha during ischemia-reperfusion seems to be coupled with the improvement of NO release.

Our data showed that calcium accumulation in ERKO hearts was significantly higher than that in control hearts during reperfusion. This finding is supported by a previous report that the cardiac L-type calcium channel is overexpressed in ERKO mice (19). E2 was reported to transiently decrease the inward calcium current and intracellular free calcium in ventricular myocytes (18) and to specifically inhibit L-type Ca2+ channel currents (4). Estrogen was also shown, during ischemia-reperfusion, to modify the function of a genetically overexpressed Na+/Ca2+ exchanger (7). Taken together, experimental findings from our study and others suggest that ER-alpha may play a key role in modulating these Ca2+ channels and/or exchangers. This may be important because calcium channels and exchangers are probably involved in calcium overload during ischemia-reperfusion (42, 53). Our results showed that Ca2+ was deposited in many myocardial mitochondria of ERKO during ischemia-reperfusion. Accumulated calcium in the cytosol and mitochondria of myocytes is believed to have several harmful effects. It depletes ATP by activating Ca2+-activated ATPases and inhibiting high-energy phosphate production in mitochondria, degrades cellular membrane systems by activating phospholipases and lipases, and accelerates oxygen free radical production via the endothelial xanthine oxidase system (55). In agreement with these findings, our study demonstrated that ERKO hearts subjected to ischemia-reperfusion had myocardial contraction bands, more severe myofibrilar destruction, and more prominent mitochondria damage than control hearts going through identical experimental procedures. Therefore, through the function of ER-alpha , E2 appears to inhibit calcium influx, thereby preventing the harmful effects caused by calcium overload during myocardial ischemia-reperfusion.

In our study myocardial MTT reduction, an indirect indicator of mitochondria respiratory function, was significantly lower in ERKO hearts than in controls after ischemia-reperfusion. MTT is a tetrazolium salt that can be reduced by active mitochondria enzymes (49). Two sites on the mitochondria electron transport chain, coenzyme Q and cytochrome c, are thought to catalyze the reduction of MTT to formazan (49), which accumulates in the endosomes and lysosomes or is exported by exocytosis (31). MTT formazan can be extracted by permeablizing the cell with agents such as DMSO and isopropanol. In our study, the significantly impaired mitochondrial function observed in ERKO was correlated with granular densities, which are thought to be calcium deposits (48), and with the amorphous matrix densities, which presumably are aggregation of denatured proteins (such as enzymes) (17) or calcium deposits containing lipids (23). These densities could substantially impair cellular respiratory function because mitochondrial calcium overload has been reported to decrease ATP synthesis (43). In addition to denaturation of enzymes, the substantial loss of mitochondrial enzymes because of the loss of cristae (Fig. 7, B and D), which provide most of the capacity for oxidation and phosphorylation, may also contribute to the impairment of mitochondrial function in ERKO subjected to ischemia-reperfusion. The impaired mitochondrial function, calcium accumulation, and other changes probably form a vicious circle that leads to progressive myocardial damage. Progressive decrease in mitochondrial MTT reduction occurred in enterocytes subjected to ischemia and reperfusion (34) and occurred in cardiac myoblasts in response to lipopolysaccharide challenge (11). In a series of preliminary experiments we found that myocardial MTT reduction was significantly lower after 45 min of ischemia than that from hearts harvested without ischemia and decreased further after 180 min of reperfusion. The data in this study suggest that ER-alpha may be necessary for estrogen to protect the myocardium against reperfusion injury by preserving mitochondrial structure and respiratory function.

In conclusion, this study is the first indication that ER-alpha may play a significant protective role in myocardial ischemia-reperfusion in males. The results suggest that the absence of ER-alpha is associated with more severe damage following ischemia-reperfusion injury. The functions of ER-alpha in myocardial ischemia and reperfusion appear to be 1) improving NO release, 2) attenuating myocardial calcium accumulation, and 3) preserving mitochondria structure and function.


    ACKNOWLEDGEMENTS

This work was supported by National Institute on Aging Grant AG-15500 and Illinois Council for Food and Agricultural Research Grant 99I-066-4.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. R. Gross, Dept. of Veterinary Biosciences, 3516 VMBS Bldg., 2001 S. Lincoln Ave., Urbana, IL 61802 (E-mail: dgross{at}cvm.uiuc.edu).

Received 30 July 1999; accepted in final form 16 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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