J Mol Cell Cardiol 31, 1539–1549 (1999)

Article No. jmcc.1999.0986, available online at http://www.idealibrary.com on

Enhancement of Post-ischemicMyocardial Function by Chronic 17b-Estradiol Treatment: Role of Alterationsin Glucose MetabolismHeather Fraser1,2, Sandra T. Davidge1,3 and Alexander S. Clanachan1,2

1Cardiovascular Research Group, 2Departments of Pharmacology, 3Physiology and Obstetrics andGynecology, University of Alberta, Edmonton, Alberta, Canada

(Received 24 November 1998, accepted in revised form 13 May 1999)

H. F, S. T. D A. S. C. Enhancement of Post-ischemic Myocardial Function by Chronic17b-estradiol Treatment: Role of Alterations in Glucose Metabolism. Journal of Molecular and Cellular Cardiology(1999) 31, 1539–1549. This study was designed to assess the effects of chronic estrogen replacement therapyon mechanical function and glucose utilization in aerobic and post-ischemic hearts. Ovariectomized female ratswere either untreated or were treated subcutaneously with 17b-estradiol (0.25 mg 21-day slow release pellets).After 14 days, when serum concentrations of 17b-estradiol were 3.8±0.8 and 148±15 pg/ml, respectively,hearts were isolated and perfused in working mode with Krebs–Henseleit solution containing 1.2 m palmitateand 11 m [5-3H/U-14C]glucose. Hearts were perfused aerobically (60 min) and then subjected to low-flowischemia (0.5 ml/min, 60 min) followed by reperfusion (30 min). During reperfusion, hearts from rats treatedchronically with 17b-estradiol had an improved (two-fold) recovery of mechanical function. 17b-estradiol (400 p,109 pg/ml), when present acutely in heart perfusate during ischemia and reperfusion, did not improve recovery.Chronic 17b-estradiol increased glucose oxidation during reperfusion as well as during aerobic perfusion but hadno effect on glycolysis. Chronic 17b-estradiol also altered post-ischemic glycogen metabolism and increasedglycogen content and glycogen synthase activity at the end of reperfusion. As stimulation of glucose oxidationhas been shown previously to be cardioprotective, and as the enhanced rate of glucose oxidation was not simplya consequence of enhanced recovery of mechanical function, alterations in glycogen and glucose utilization maycontribute to the direct cardioprotective effects of chronic estrogen treatment.

K W: Estrogen; Myocardial ischemia; Myocardial reperfusion; Glucose metabolism; Glycogen; Leftventricular function.

considered to reduce the risk of cardiovascularIntroductiondisease in post-menopausal women (Stampfer andColditz, 1991; Grady et al., 1992), recent evidenceCardiovascular disease is a major cause of death in

both men and women over the age of 60 years. from a randomized and blinded secondary pre-vention trial of a large (n=2763) number of post-However, in women at age 40 (prior to menopause)

there is a six-fold lower incidence of death due to menopausal women found that combined therapywith conjugated estrogens and medroxyprogest-ischemic heart disease than in men (Furman,

1968). These data indicate that pre-menopausal erone exerted no significant beneficial actions(Hulley et al., 1998). However, the cardioprotectivewomen possess an endogenous protective mech-

anism. Although estrogen replacement therapy is efficacy of estrogen per se was not assessed. A direct

Please address all correspondence to: Dr A. S. Clanachan, 9-70 Medical Sciences Building, Department of Pharmacology, Faculty ofMedicine, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada. Fax: 780-492-4325; E-mail: sandy.clanachan@ualberta.ca

0022–2828/99/081539+11 $30.00/0 1999 Academic Press

H. Fraser et al.1540

cardioprotective effect of 17b-estradiol is dem- glycogen deposition by estrogen has been observedin uterus, skeletal muscle, heart and liver (Car-onstrable under experimental conditions where

beneficial effects are manifest either as a reduction rington and Bailey, 1985). Moreover, estrogen in-hibits exercise-induced glycogenolysis in liver andin myocardial necrosis after ischemia and re-

perfusion in rabbits (Hale et al., 1996) or as an skeletal muscle (Rooney et al., 1993), improvesexercise tolerance and prevents depletion of myo-improvement in the recovery of mechanical func-

tion in isolated rat hearts reperfused following global cardial glycogen (Kendrick et al., 1987). Con-sequently, we hypothesized that chronic estrogenischemia (Kolodgie et al., 1997). The salutary mech-

anisms proposed for the protective actions of es- treatment may elicit important alterations in myo-cardial glucose metabolism that may, in turn, affecttrogen include enhanced vascular smooth muscle

relaxation (Williams et al., 1990), improved plasma post-ischemic function.The present study was designed to measure dir-lipid profile (Stevenson et al., 1993), reduced ather-

oma formation (Williams et al., 1990), as well as ectly the effects of chronic treatment with 17b-estradiol on myocardial glycogen and glucose meta-an antioxidant activity (Delyani et al., 1996). In

addition, since an improvement in mechanical func- bolism and mechanical function in aerobic andpost-ischemic isolated working rat hearts.tion is demonstrable in hearts perfused ex vivo

(Kolodgie et al., 1997), chronic exposure to estrogenprobably causes changes in cardiac muscle whichelicit a direct cardioprotective effect and which are Materials and Methodsindependent of the aforementioned mechanisms.Identification of the mechanisms responsible for this

Animal treatmentsdirect beneficial alteration in cardiac function wouldhave important implications for the development

Female Sprague–Dawley rats (n=58, 275–300 g),of estrogen-like agents as cardioprotective drugs.treated according to standards of the CanadianEnergy substrate preference during reperfusionCouncil of Animal Care, were anesthetized withis an important determinant of post-ischemic myo-Brietal (50 mg/kg, i.p.) and ovariectomized. Im-cardial mechanical function (Lopaschuk, 1997; Lo-mediately following surgery, rats were randomlypaschuk and Stanley, 1997; Stanley et al., 1997).divided into two groups which were either untreatedIn hearts reperfused following ischemia, high rates(n=35) or treated with 17b-estradiol (n=23) sub-of fatty acid oxidation inhibit glucose oxidationcutaneously (17b-estradiol, 0.25 mg pellets, 21-and depress recovery of mechanical function (Lo-day release formulation; Innovative Research ofpaschuk et al., 1993). The fatty acid-induced in-America, Sarasota, FL, USA).hibition of glucose oxidation is greater than the

inhibition of glycolysis; consequently, this resultsin a marked uncoupling of glycolysis from glucoseoxidation (Lopaschuk et al., 1993), a condition Validation of 17b-estradiol therapywhich leads to proton production, which, in theabsence of adequate perfusion, causes acidosis, In order to validate the 17b-estradiol treatment

strategy as a means of achieving serum con-altered ionic homeostasis and impaired recoveryof mechanical function and myocardial efficiency centrations that approximated those associated

with estrogen replacement therapy in post-men-upon reperfusion (Liu et al., 1996). This is supportedby our previous studies showing that stimulation opausal women (Ginsburg et al., 1998), con-

centrations (pg/ml) of 17b-estradiol in serumof glucose oxidation improves the coupling betweenglycolysis and glucose oxidation (McVeigh and Lo- samples collected at the time of heart removal

were determined by radioimmunoassay (Diagnosticpaschuk, 1990; Lopaschuk et al., 1993), attenuatesproton production and improves recovery of mech- Products Corporation, Los Angeles, LCA, USA). As

an additional biological marker of 17b-estradiolanical function and cardiac efficiency in the post-ischemic heart (Liu et al., 1996). delivery, body, heart and uterine weights for animals

in each group were determined at the time ofInterestingly, estrogen-induced regulation ofglucose metabolism is demonstrable in a number sacrifice. One rat was excluded from the 17b-es-

tradiol-treated group due to a non-functioning pelletof tissues. Effects of estrogen include a stimulationof glucose uptake (rat uterus) (Smith, 1967) and as indicated by a low serum 17b-estradiol con-

centration and a low uterine weight. A second ratactivation of several gluconeogenic (rat liver)(Dahm et al., 1978) and glycolytic enzymes (rat from the 17b-estradiol-treated group was removed

due to a technical problem that resulted in unstablebrain) (Kostanyan and Nazaryan, 1992). Increased

17b-Estradiol Alters Myocardial Glucose Metabolism 1541

myocardial mechanical function (left ventricle work untreated ovariectomized female rats were perfusedas described above either in the absence (n=6) ordecreased by more than 20% over the 60-min

period of aerobic perfusion). presence (n=6) of 17b-estradiol (final con-centration 400 p, c. 109 pg/ml) which was addedto the perfusate 30 min prior to the onset of low-flow ischemia. This concentration of 17b-estradiolHeart groups and perfusion protocolswas chosen to be similar to that predicted to be inthe serum of female rats following the 14-dayAfter 14 days of treatment, rats were anesthetized

with pentobarbital (60 mg/kg, i.p.) and hearts were treatment protocol with 17b-estradiol pellets. Atthe end of the perfusion protocol, hearts were rap-removed rapidly and immediately perfused at 37°C

via the aorta in Langendorff (non-working) mode idly frozen with Wollenberger clamps cooled to thetemperature of liquid N2 for subsequent de-(Liu et al., 1996) for a 10-min equilibration period.

Thereafter, Langendorff perfusion was stopped and termination of wet-to-dry weights, glycogen con-tents and enzyme activities. An additional series ofhearts were perfused in paced (5 Hz) working mode

(recirculating volume 100 ml) at a constant left hearts (untreated, n=7; 17b-estradiol, n=6) wasfrozen following 60 min of aerobic working per-atrial preload (11.5 mmHg) and aortic afterload

(80 mmHg). Working perfusate consisted of a modi- fusion in order to determine glycogen contents andenzyme activities prior to ischemia.fied Krebs–Henseleit solution containing 1.2 m

palmitate pre-bound to 3% bovine serum albumin,2.5 m Ca2+, 100 mU/l insulin, 11 m [5-3H/U-14C]glucose that was oxygenated with carbogen(5% CO2, 95% O2) (Liu et al., 1996). The inclusion Measurement of glycogen metabolismof insulin in the perfusate maintained glucose up-take and hence the relative rates of glycolysis and Myocardial glycogen content (lmol glucosyl units/

g dry weight) was determined by measuring theglucose oxidation were similar to those reportedpreviously for intact hearts in situ (Wisneski et al., glucose content in samples of frozen tissue which

were subjected to alkaline extraction (30% KOH)1990). Moreover, the presence of a high con-centration of fatty acid and glucose ensured an to separate glycogen from exogenous glucose. This

was followed by ethanol precipitation and acidadequate energy substrate supply for the workingheart and mimicked concentrations observed clin- hydrolysis (2N H2SO4) to release endogenous gluc-

ose from glycogen. The activities of glycogen syn-ically under conditions of post-ischemic stress (Lo-paschuk et al., 1994). thase and glycogen phosphorylase were also

determined in frozen tissue samples. Glycogen phos-Aortic pressures were determined using a GouldP21 pressure transducer and cardiac output, aortic phorylase activity, expressed as active (phos-

phorylase a) as a percentage of total, wasflow and coronary flow were measured using in-line ultrasonic flow probes connected to a Transonic determined as described previously (Dobson and

Fenton, 1993) from the formation of glucose-6-T206 ultrasonic flow meter. Left ventricular minutework (LV work, mmHg.l/min), calculated as cardiac phosphate in the presence of excess glycogen and

in the absence or presence of AMP (3 m). Glycogenoutput × LV developed pressure (aortic systolicpressure − preload pressure) was used as a con- synthase activity, expressed as active (synthase a) as

a percentage of total, was determined as previouslytinuous index of mechanical function. Coronaryvascular conductance was calculated as coronary described (Passonneau and Rottenberg, 1973) from

the consumption of UDP-glucose in the absenceflow divided by aortic diastolic pressure.One series of hearts was subjected to an ischemia- and presence of glucose-6-phosphate (5 m).

reperfusion protocol in which hearts (untreated,n=16; 17b-estradiol, n=15) were perfused underaerobic working conditions for 60 min and thensubjected to low-flow ischemia (0.5 ml/min cor- Measurement of glycolysis and glucose oxidationonary flow delivered by a calibrated infusion pumpvia the aorta) for 60 min followed by working mode Rates of glycolysis were measured directly as pre-

viously described (Finegan et al., 1995) from thereperfusion for 30 min.To determine the acute effect of 17b-estradiol on quantitative determination of 3H2O liberated from

labeled [5-3H]glucose at the enolase step of gly-post-ischemic mechanical function, an additionalgroup of hearts was subjected to the ischemia- colysis. Glucose oxidation was also determined dir-

ectly as previously described (Finegan et al., 1995;reperfusion protocol. In this series, hearts from

H. Fraser et al.1542

Liu et al., 1996) by measuring 14CO2 from [14C]glu- Resultscose liberated at the level of pyruvate de-hydrogenase (PDH) and in the TCA cycle. These The serum concentration of 17b-estradiol and uter-

ine weight were higher in rats from the 17b-techniques directly measure rates of glycolysis andglucose oxidation and do not require analysis of estradiol-treated group compared with the un-

treated group (Table 1). The concentration of 17b-lactate or pyruvate accumulation, which wouldonly provide indirect assessment of each component estradiol in the treated group (148 pg/ml) ap-

proximates that reported in post-menopausalof glucose metabolism. Perfusate samples were col-lected for analysis of 3H2O and 14CO2 at pre-de- women receiving estrogen replacement therapy

(112 pg/ml) (Ginsburg et al., 1998). Treatment withtermined intervals throughout each perfusionprotocol. Average rates of glycolysis and glucose 17b-estradiol also significantly increased the heart

weight/body weight ratio. This was due to an in-oxidation were calculated from linear time coursesof product accumulation and rates are expressed hibition by 17b-estradiol of the time-dependent gain

in body weight that normally occurs in responseas lmol glucose metabolized/min/g dry weight.Proton production attributable to the hydrolysis to estrogen deficiency, rather than to any changes

in either heart wet or dry weight.of ATP arising from glucose metabolism was cal-culated as 2×(rate of glycolysis−rate of glucose LV work, which was used as a continuous index

of mechanical function, was stable in both theoxidation). This accounts for the net production oftwo protons per molecule of glucose that passes untreated and 17b-estradiol-treated groups during

aerobic perfusion. LV work, based on pooled datathrough glycolysis that is not subsequently oxidized(Dennis et al., 1991). from hearts in both ischemia-reperfusion and aer-

obic protocols, was slightly higher (11%) in the17b-estradiol-treated group (7.3±0.2 mmHg.l/min,P=0.018) compared with the untreated group(6.6±0.2 mmHg.l/min). The increase in LV workPyruvate dehydrogenase activitywas due to a combination of small differences incardiac output and aortic pressures. The differenceTotal PDH activity (PDHt) and that in the active

dephosphorylated form (PDHa) (lmol/min/g dry was not due to changes in coronary flow, aorticcompliance or coronary vascular conductance asweight) were determined using a radioisotopic

coupled enzyme assay (Constantin-Teodosiu et al., these values were similar in hearts from the un-treated and 17b-estradiol-treated groups. Pre-isch-1991) as modified by Collins-Nakai et al. (1994).

PDH activity was determined as a function of acetyl emic baseline values are reported in Table 2.During the 60-min period of low-flow ischemia,coenzyme A (CoA) production, where acetyl CoA

formed from pyruvate via the PDH reaction was all external work ceased and only partial recoveryof mechanical function (14% compared with pre-condensed with excess [14C]oxaloacetate in the pres-

ence of citrate synthase to form [14C]citrate, which ischemic values) was evident during reperfusionafter ischemia. However, in hearts from the 17b-was then separated from the reaction mixture by

Dowex 50W-X8 (100–200 mesh) chromatography. estradiol-treated group, recovery of mechanicalfunction was two-fold greater (29% compared withpre-ischemic values) than in hearts from the un-treated group (P=0.037, Fig. 1). The enhancedpost-ischemic mechanical function was due mainlyStatistical analysisto an increase in cardiac output and aortic flow asheart rate and coronary vascular conductance wereData are expressed as the mean±standard error of

the mean (SEM). unaffected. Acute administration of 17b-estradiol(109 pg/ml, 400 p) to the perfusate of untreatedComparisons between untreated and 17b-es-

tradiol-treated groups were performed using the ovariectomized rat hearts 30 min prior to low-flowischemia had no effect on mechanical functionunpaired Student’s t-test (two-tailed). When sample

variances were significantly different, a non-para- either during aerobic perfusion prior to ischemia orduring post-ischemic reperfusion (data not shown).metric test was used (Mann–Whitney U-statistic,

unpaired, two-tailed test). Statistical analysis of During aerobic perfusion, rates of glucose ox-idation were 6.5-fold less (P<0.001) than rates ofglucose metabolism was performed using two-way

repeated measures analysis of variance followed by glycolysis (Fig. 2). This uncoupling of glycolysisfrom glucose oxidation resulted in a calculatedthe Tukey post-hoc test. Differences were judged to

be significant when P<0.05. rate of proton production from the hydrolysis of

17b-Estradiol Alters Myocardial Glucose Metabolism 1543

Table 1 Physiological parameters of untreated and 17b-estradiol-treated rats

Untreated 17b-estradiol P value∗(n=23) (n=21)

Serum 17b-estradiol (pg/ml) 3.8±0.8 148±15 <0.0001Body weight (g) 352±4 302±3 <0.0001Heart weight (g) 1.52±0.05 1.58±0.05 NSHeart weight/body weight ratio (×103) 4.73±0.14 5.75±0.18 <0.001Uterine weight (g) 0.24±0.01 0.65±0.04 <0.0001

Data represent mean±...∗ P-value is for the comparison between untreated and 17b-estradiol-treated groups (unpaired t-test).

Table 2 Pre-ischemic baseline values for hemodynamic indices of hearts from untreated and 17b-estradiol-treated rats

Untreated (n=16) 17b-estradiol (n=15)

LV work (mm Hg.l/min) 6.6±0.2 7.3±0.3Heart rate (beats/min) 227±8 274±8Cardiac output (ml/min) 57.0±1.9 62.3±2.2Aortic flow (ml/min) 41.4±2.1 44.9±2.4Coronary flow (ml/min) 15.8±0.8 17.4±0.9Coronary vascular conductance (ml/min/mm Hg) 0.18±0.01 0.20±0.01

Data represent mean±... for aerobic baseline values prior to the onset of low-flow ischemia.

glycolytically derived ATP of 4.7±0.7 lmol/min/ was similar in both groups during low-flow isch-emia, reperfusion as well as during aerobic perfusiong dry weight. During aerobic perfusion, glucose

oxidation was substantially greater in hearts from (Fig. 2).The activity of PDH, the rate-limiting enzyme inthe 17b-estradiol-treated group (by 35%, P=

0.011) relative to the untreated group while rates glucose oxidation, was measured in hearts frozenat the end of reperfusion and after aerobic perfusionof glycolysis were similar in both groups. These

changes improved the coupling of glycolysis to (Table 3). As expected, total activity (PDHt) of themaximally dephosphorylated form of the enzymeglucose oxidation so that glycolysis was only 4.3-

fold higher than glucose oxidation. complex exceeded the activity of the normally activeform (PDHa), both at the end of reperfusion andAs expected, during low-flow ischemia, rates of

glucose oxidation decreased (P<0.001) in both after aerobic perfusion. However, the percentagesof PDH in the active form were not different betweengroups (Fig. 2). Rates were similar in hearts from

the untreated and 17b-estradiol-treated groups. hearts from the untreated and 17b-estradiol-treatedgroups, either after reperfusion or after aerobicSimilarly, glycolysis was not different between the

groups during low-flow ischemia. perfusion (Table 3).Glycogen content (lmol/g dry weight) at theDuring reperfusion, the rates of glucose oxidation

recovered to aerobic values. Moreover, as observed end of reperfusion was significantly greater (35%,P=0.026) in hearts from the 17b-estradiol-treatedduring aerobic conditions, the rate of glucose ox-

idation during reperfusion of hearts from the 17b- group relative to the untreated group (Fig. 3).However, after aerobic perfusion prior to the onsetestradiol-treated group was higher (by 28%, P=

0.042) compared with the untreated group. How- of low-flow ischemia, glycogen content was similarin both groups. Glycogen synthase activity wasever, during reperfusion, glycolysis was similar in

both the untreated and 17b-estradiol-treated significantly higher in the 17b-estradiol-treatedgroup both at the end of reperfusion (25%, P=groups.

Although a significant rate of proton production 0.047) and after aerobic perfusion (25%, P=0.047) (Fig. 3). In contrast, glycogen phos-occurs in response to the uncoupling of glycolysis

from glucose oxidation under aerobic conditions, phorylase activity was similar in each group bothat the end of reperfusion and after aerobicand despite a higher rate of glucose oxidation in

the 17b-estradiol-treated group, proton production perfusion.

H. Fraser et al.1544

Figure 1 Cardiac mechanical function measured during reperfusion of isolated rat hearts. Values (±SEM) are shownfor (a) left ventricular (LV) minute work, (b) coronary vascular conductance (CVC), (c) heart rate and (d) cardiac outputfor hearts from rats which were either untreated (Β, n=16) or were treated chronically with 17b-estradiol (Χ, n=15). ∗ P<0.05 for comparison of average reperfusion values for untreated and 17b-estradiol-treated groups.

determined whether alterations in myocardial gly-Discussioncogen and glucose metabolism might contribute toestrogen-induced cardioprotection. The data in-Although numerous mechanisms have been re-

ported to contribute to the cardioprotective efficacy dicate that chronic treatment of rats with 17b-estradiol elicits two important alterations in myo-of estrogen, little attention has been aimed at po-

tential alterations directly within cardiac muscle. cardial glucose metabolism. These comprise astimulation of glucose oxidation, as well as anThis study confirms the ability of chronic treatment

with 17b-estradiol, which achieved serum con- enhanced glycogen synthase activity. As these ef-fects were observed both during aerobic perfusioncentrations similar to those reported for re-

placement therapy in post-menopausal women as well as during reperfusion, it appears that theyare not simply a consequence of altered post-isch-(Ginsburg et al., 1998), to elicit a protective effect

directly on the myocardium. Hearts removed from emic mechanical function. Rather, these data, incombination with the well-documented benefits eli-17b-estradiol-treated rats retained a capacity to

resist ischemia-reperfusion damage as dem- cited by the optimization of myocardial glucosemetabolism (McCormack et al., 1996; Lopaschuk,onstrated by their enhanced recovery of post-isch-

emic mechanical function. This study also 1997; Lopaschuk and Stanley, 1997), suggest that

17b-Estradiol Alters Myocardial Glucose Metabolism 1545

Figure 2 Myocardial glucose metabolism measured dur-ing aerobic working perfusion, low-flow ischemia and Figure 3 Glycogen metabolism measured after aerobicworking reperfusion. Data are means±SEM for hearts perfusion and after reperfusion: (a) glycogen content; (b)from rats which were either untreated (Φ) or were glycogen synthase; (c) glycogen phosphorylase. Data aretreated chronically with 17b-estradiol (Ε). ∗ P<0.05 means±SEM for hearts from rats which were eitherwhen compared with the untreated group. (a), glucose untreated (Φ) or were treated chronically with 17b-oxidation; (b), glycolysis; (c), proton production. estradiol (Ε). ∗ P<0.05 when compared with the un-

treated group.

Table 3 Myocardial PDH activity in hearts from untreated and 17b-estradiol-treated rats at the endof aerobic perfusion and at the end of reperfusion

Aerobic Reperfusion

Untreated 17b-Estradiol Untreated 17b-Estradiol(n=7) (n=6) (n=15) (n=14)

PDHa (lmol/min/g dry weight) 1.20±0.11 1.24±0.08 2.67±0.58 1.61±0.35PDHt (lmol/min/g dry weight) 8.85±0.62 10.08±0.74 7.72±0.54 7.66±0.35Active (%) 13.7±0.8 12.3±0.2 31.6±5.5 20.7±3.7

Data represent means±... Values in hearts from the untreated and 17b-estradiol-treated groups were notsignificantly different (unpaired t-test).

changes in glucose metabolism arising from chronic action arising from 17b-estradiol replacement ther-apy, as well as measure directly glycogen and gluc-17b-estradiol treatment may indeed contribute to

the direct cardioprotective efficacy of estrogen. ose metabolism, studies were performed on isolatedworking hearts. With this approach, hearts wereIn order to investigate any direct cardioprotective

H. Fraser et al.1546

perfused under controlled and appropriate con- Kolodgie et al., 1997). Together, they suggest that17b-estradiol can reduce ischemia-reperfusion in-ditions of myocardial workload. An additional fea-

ture of the isolated working heart model is the jury in addition to reducing the incidence of cor-onary artery disease, as suggested by numerousinclusion in the heart perfusate of insulin and

both glucose and fatty acid as energy substrates observational studies of women taking estrogenreplacement therapy (Stampfer and Colditz, 1991;at concentrations that approximate those present

clinically during ischemia or stress (Lopaschuk et Grady et al., 1992).This study also examined whether chronic treat-al., 1994). This is an important consideration as

fatty acid exerts important alterations in glycogen ment with 17b-estradiol elicited alterations in myo-cardial glycogen and glucose oxidation. Beneficialand glucose metabolism such that relevant rates of

glycolysis and glucose oxidation cannot normally actions arising from the drug-induced optimizationof myocardial glycolysis and glucose oxidation havebe obtained unless fatty acid is available as an

energy substrate. Thus, in the current study, both been reviewed elsewhere (Lopaschuk and Stanley,1997; Stanley et al., 1997). While estrogen-inducedLV mechanical functon and rates of glycogen and

glucose metabolism were measured under ap- alterations of glycogen and glucose metabolismhave been observed in a number of tissues (Smith,propriate conditions of energy demand and supply.

Parameters were compared directly between 1967; Dahm et al., 1978; Carrington and Bailey,1985; Kendrick et al., 1987; Kostanyan andgroups of hearts removed from animals that had

either low (ovariectomy alone) or elevated serum Nazaryan, 1992; Rooney et al., 1993; Finegan etal., 1996), its effects in aerobic and post-ischemicconcentrations of estrogen (ovariectomy plus 17b-

estradiol replacement). This study design facilitated myocardium have hitherto not been addressed.However, it is interesting to note that many of thethe direct comparison of the mechanical and meta-

bolic consequences of chronic estrogen replacement effects of estrogen described in other tissues, if theywere to occur in heart, would elicit a cardio-therapy on the heart. Assay of serum estradiol

concentrations and the auxiliary biological markers protective action.Our results indicate that hearts from rats treatedof estrogen action (body and uterine weights) con-

firmed the efficacy of estradiol administration. Ad- chronically with 17b-estradiol had higher rates ofglucose oxidation during post-ischemic conditionsditional experiments will be required to investigate

the cardioprotective potential of 17b-estradiol in as well as during aerobic perfusion whereas ratesof glycolysis were not altered during any of themale animals.

The cardioprotective efficacy of estrogen has been phases of the perfusion protocol. Clearly the higherrate of glucose oxidation in hearts from 17b-es-observed previously both in animal studies (Hale et

al., 1996; Kolodgie et al., 1997) and in clinical tradiol-treated rats was not simply a consequenceof enhanced post-ischemic function as enhancedtrials (Stampfer and Colditz, 1991; Grady et al.,

1992). As anticipated, chronic treatment of rats for rates of glucose oxidation were also observed inhearts perfused under aerobic conditions. Thus,14 days with 17b-estradiol elicited changes within

the heart which conferred a direct cardioprotective chronic therapy with 17b-estradiol elicited an ac-tion similar to that of the cardioprotective agenteffect, manifested as an enhanced (two-fold) re-

covery of post-ischemic LV minute work. That this dichloroacetate (McVeigh and Lopaschuk, 1990).Although an increased rate of glucose oxidationeffect was observed only with chronic treatment,

and not with the acute addition of 17b-estradiol normally improves the metabolic coupling of gly-colysis to glucose oxidation (Lopaschuk et al., 1993;to heart perfusates, confirms that a change had

occurred within the heart which persisted during Finegan et al., 1996; Liu et al., 1996), this was notobserved in hearts from 17b-estradiol-treated rats.ex vivo perfusion, possibly as a consequence of

altered gene regulation. Further, the requirement Consequently, the calculated rate of proton pro-duction arising from glucose metabolism was notof chronic therapy indicates that, unlike many

cardioprotective drugs (McVeigh and Lopaschuk, reduced. Had proton production been reduced dur-ing reperfusion, the cardioprotective mechanism of1990; Finegan et al., 1993, 1996; Liu et al., 1996;

McCormack et al., 1996), no therapeutic benefit is chronic treatment with 17b-estradiol would havebeen consistent with that described for other meta-likely following acute administration of estrogen

immediately prior to ischemia or reperfusion. This bolic modulators such as N6-cyclohexyladenosine(Finegan et al., 1993, 1996). Proton productiondirect cardioprotective action resulting from chronic

treatment with 17b-estradiol is similar to that re- from glucose metabolism is calculated from thedifference in glycolysis and glucose oxidation, butported recently in other experimental models of

ischemia and reperfusion (Hale et al., 1996; as the rate of glycolysis far exceeds the rate of

17b-Estradiol Alters Myocardial Glucose Metabolism 1547

glucose oxidation, glycolysis dominates the equa- been excluded, the precise mechanism responsiblefor the 17b-estradiol-induced acceleration of myo-tion for proton production. The lack of any al-

teration of proton production when there was a cardial glucose oxidation was not identified in thepresent study.35% increase in glucose oxidation may have been

due to the small, but insignificant, increase in Data from this study also indicate that chronictreatment with 17b-estradiol elicits alterations inglycolysis which would tend to offset any potential

benefit arising from the higher rate of glucose myocardial glycogen metabolism. These includeimproved recovery of glycogen content duringoxidation. Thus, the increase in glucose oxidation,

in face of a small increase in glycolysis, was not reperfusion and increased glycogen synthase ac-tivity. Again, it is unlikely that the higher activitysufficiently great to cause a significant decrease in

proton production. of glycogen synthase in reperfused hearts wassimply a consequence of improved post-ischemicWhile inhibition of proton production from the

hydrolysis of ATP derived from glucose metabolism mechanical function since a similar increase inactivity was measured in aerobically perfusedcontributes to drug-induced cardioprotection in

fatty acid perfused working hearts subjected to hearts. The increase in glycogen synthase activityand the enhanced capacity to re-synthesize gly-severe ischemia (Lopaschuk, 1997; Lopaschuk and

Stanley, 1997), improved recovery of mechanical cogen may ultimately be beneficial for the post-ischemic heart. Recent studies indicate that endo-function may arise from improved utilization of

exogenous glucose (King and Opie, 1998) such genous glucose, namely that arising from gly-cogenolysis, is oxidized preferentially and thusas that associated with glucose-insulin-potassium

(Gradinac et al., 1989). Thus, improved recovery generates a greater amount of ATP relative toexogenous glucose (Henning et al., 1996; Fraserof mechanical function may have occurred simply

in response to an increase in the rate of ATP et al., 1998). Thus it is possible that a preferentialconsumption of glycogen during reperfusion mayproduction arising from the higher rates of glucose

metabolism, thereby maintaining better ionic have resulted in the increased rate of glucoseoxidation. However, in our other work (Fraser ethomeostasis and recovery of contractility.

Elucidation of the pharmacological mechanism of al., 1998), we have observed that during re-perfusion there is very little change in glycogenaction of 17b-estradiol in the regulation of glucose

oxidation is important as it may help identify novel content, all of the decrease in glycogen contentoccurring during low-flow ischemia. Moreover,targets for future therapeutic exploitation. Glucose

oxidation is controlled by the rate-limiting enzyme the higher glycogen content at the end of re-perfusion in the estrogen group appears due toPDH, an enzyme complex that is susceptible to

drug-induced modulation (Denton et al., 1996). an increase in glycogen synthesis rather than toa change in glycogen utilization (glycogenolysis).Surprisingly, the activities of neither the endo-

genously active nor the total component of PDH Thus, it is very unlikely that oxidation of glucosederived from glycogen during the 30-min periodwere different between the two groups of hearts,

suggesting a lack of 17b-estradiol-induced al- of reperfusion could explain the apparent increasein the oxidation of exogenous glucose.teration of this enzyme complex. However, the pos-

sibility remains that PDH activity was stimulated In conclusion, this study has confirmed that acomponent of the cardioprotective effect associatedin hearts from 17b-estradiol-treated rats, but ac-

tivation was not detectable as the appropriate in- with the chronic treatment of therapeutically rel-evant concentrations of 17b-estradiol is elicitedtracellular conditions were not mimicked by the in

vitro assay system. Alteration of the rate of b- directly on the myocardium. This is manifest by animprovement in post-ischemic mechanical functionoxidation of free fatty acids may also have affected

rates of glucose oxidation (Randle et al., 1963), in hearts isolated from 17b-estradiol-treated ratsand perfused ex vivo. Moreover, these experimentsalthough this is unlikely as PDH activities were

similar in both groups. show that chronic treatment with 17b-estradiolalso elicited important alterations in myocardialFacilitation of anaplerosis is an alternate mech-

anism that could account for a PDH-independent glycogen and glucose metabolism which may con-tribute to its cardioprotective efficacy. Althoughstimulation of glucose oxidation (Comte et al.,

1997), particularly in the presence of a marked these changes did not reduce proton productionfrom glucose metabolism, the enhanced rate offatty acid-induced inhibition of PDH activity. Un-

fortunately anaplerosis could not be measured in glucose oxidation may have contributed to theimproved recovery of mechanical function by en-the intact working hearts used in this study. Thus,

although a number of possible mechanisms have hancing ATP production.

H. Fraser et al.1548

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