1

Myofilament Response to Ca2+ and Na+/H+ Exchanger Activity in Sex Hormone Related

Protection of Cardiac Myocytes from Deactivation in Hypercapnic Acidosis

Tepmanas Bupha-Intr*, Jonggonnee Wattanapermpool*, James R. Peña†, Beata M Wolska†,

and R John Solaro

Dept. of Physiology & Biophysics, Medicine†, and Center for Cardiovascular Research,

College of Medicine, University of Illinois at Chicago, IL, USA

and

*Department of Physiology, Faculty of Science, Mahidol University, Thailand

Running title: Sex hormones, acidosis, and cardiac myocyte function

Address for correspondence: R. John Solaro, PhD Department of Physiology and Biophysics College of Medicine University of Illinois at Chicago 835 S. Wolcott Ave. Chicago, IL 60612-7342 Tel: 312-996-7620 Fax: 312-996-1414 email: solarorj@uic.edu

Page 1 of 30Articles in PresS. Am J Physiol Regul Integr Comp Physiol (October 12, 2006). doi:10.1152/ajpregu.00376.2006

Copyright © 2006 by the American Physiological Society.

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Abstract Compared to sham operated controls, myofilaments from hearts of ovariectomized ( OVX) rats

demonstrate an increase in Ca2+ sensitivity with no change in maximum tension (Wattanapermpool and

Reiser, Am J Physiol 1999;277:H467-73). To test the significance of this modification in intact cells, we

compared intracellular Ca2+ transients and shortening of ventricular myocytes isolated from sham and 10

week OVX rats. There was a decrease in the peak Ca2+ transient with prolonged 50% decay time in

OVX cardiac myocytes without changes in the resting intra-cellular Ca2+ concentration. Per cent cell

shortening was also depressed and relaxation was prolonged in cardiac myocytes from OVX rats

compared to shams. Ovariectomy induced a sensitization of the myofilaments to Ca2+. Hypercapnic

acidosis suppressed the shortening of OVX myocytes to a lesser extent than that detected in shams.

Moreover, a larger compensatory increase in % cell shortening was obtained in OVX myocytes during

prolonged acidosis. The elevated compensation in cell shortening was related to a higher amount of

increase in the amplitude of the Ca2+ transient in OVX myocytes. However, these differences in Ca2+

transients and % cell shortening were no longer evident in the presence of 1µM cariporide, a specific

inhibitor of Na+/H+ exchanger type 1 (NHE1). Our results indicate that deprivation of female sex

hormones modulates the intracellular Ca2+ concentration in cardiac myocytes possibly via an increased

NHE1 activity, which may act in concert with Ca2+ hypersensitivity of myofilament activation as a

determinant of sex differences in cardiac function.

Keywords: heart, estrogen, acidosis, sarcomeric proteins, sodium, proton exchanger

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Introduction

Sex steroid hormones exert significant control over cardiac physiology, but the molecular and

cellular mechanisms for these effects remain unclear. Understanding these mechanisms is crucial for

translation to hormone therapy (30). Scheuer and co-workers (24,25) first demonstrated a suppression

of function in isolated heart preparation in both gonadectomized male and female rats. Moreover,

these investigators reported that the decreases in ejection fraction, peak systolic pressure, and cardiac

output in the hearts from gonadectomized rats could be normalized with hormone supplementation.

These results, which pointed to an important role of both male and female steroid hormones in

regulating cardiac function, fit with the hypothesis of a cardioprotective role of female sex hormones.

Epidemiological studies also supported this hypothesis by demonstrating that men have a higher

incidence of heart disease than pre-menopausal women (14). Echo-cardiographic assessment of

cardiac function in pre-menopausal women also demonstrated enhanced contractile function compared

to post-menopausal women (20). Evidence of improved prognosis in female heart failure patients

taking estrogen compared to those off estrogen also supports the hypothesis of the importance of sex

hormones in heart diseases (16,21).

Although, anti-apoptotic effects of estrogen have been reported (19), depression in Ca2+ fluxes

to and from the myofilaments appears to account in part for the depression of contractility in hearts of

ovariectomized (OVX) rats. Ren et al. (22) reported that myocytes from 6-week OVX rat hearts

demonstrated slowing of relaxation and contraction kinetics associated with a slower decay and

depressed peak amplitude of Ca2+ transients. Bupha-Intr and Wattanapermpool (5) reported a

depression in Ca2+ uptake rate and ATPase activity by vesicles of the cardiac sarcoplasmic reticulum

(SR) derived from hearts of rats 10 weeks following ovariectomy, compared to sham controls.

Depression in expression of SERCA at the protein and mRNA levels appeared to account for this

depression in myocyte function and Ca2+ fluxes (5). There is also evidence of sex related differences in

fluxes through the Na+,/Ca2+ exchanger (NCX). Sugishita et al. (29) reported that metabolic inhibition

induced a relatively large increase in intracellular Ca2+ in myocytes of transgenic male versus female

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mouse hearts over expressing (NCX). Treatment of the males with 17β-estradiol (E2) significantly

attenuated this difference. Whether these effects on intracellular Ca2+ are due directly to NCE activity

or indirectly through modification of the activity of the Na+,H+ exchanger (NHE1) remains unclear.

There is, however, evidence of acute effects of E2 on regulation of NHE1 activity. In demonstrating

that acute treatment with E2 improved recovery of hearts stressed by normoxic acidification, Anderson

et al. (2) proposed a mechanism involving in part promotion of NO synthesis that increased NHE1

activity.

Alterations in myofilament response to Ca2+ also appear to play an important role in effects of

estrogen on cardiac function. There is a reduction in ATPase rate in myofilaments isolated from hearts

of OVX rats, which is attributable to a shift in myosin isoform population from one predominant in the

alpha-myosin heavy chain to one containing significant amounts of beta-myosin heavy chain (33).

Moreover, compared to preparations from shams, ATPase activity (33) and tension (34) of

myofilaments from OVX rat hearts were more sensitive to Ca2+. These changes in myofilament

response to Ca2+ were reversed in preparations from OVX rats supplemented with E2 (36) or in animals

subjected to exercise training (4).

In experiments reported here, we have extended our studies of the relation between sex

hormones and myofilament response to Ca2+ with the objective of understanding the relevance of this

effect in isolated cardiac myocytes. We think this understanding is important in view of evidence that

an increase in myofilament response to Ca2+ is a common functional consequence of mutations in

sarcomeric proteins that are linked to familial hypertrophic myopathies (10). Moreover, the role of

myofilament response to Ca2+ has not been generally considered in the cardio-protective effects of E2

in acidosis (2) and metabolic inhibition (29) even though there is evidence that altered myofilament

response to Ca2+ is a dominant mechanism responsible for the fall in tension associated with acidosis

(27). We therefore compared the effects of hypercapnic acidosis on shortening and intra-cellular Ca2+

of myocytes isolated from sham, OVX, and OVX + E2 rat hearts. We also compared tension-pCa

relations in detergent extracted fiber bundles from these hearts. To probe for changes in myofilament

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response to Ca2+, we compared responses at physiological pH to responses at acidic pH Our results

confirm that cardiac myofilaments functioning in intact cardiac myocytes from OVX rat hearts are more

sensitive to Ca2+ than in control myocytes. Our data also indicate that chronic deprivation of female

sex hormones modulates the intracellular Ca2+ concentration in cardiac myocytes via an increase in

NHE1 activity, which may act in concert with the hypersensitivity of myofilament activation as a

significant determinant of sex differences in cardiac function.

Materials and Methods

Animals

Our studies employed female Sprague-Dawley rats that were OVX or sham operated at 8

weeks of age by the supplier (Harlan, Indianapolis, IN) and killed at 18 weeks of age. We randomly

divided the OVX rats into two groups, and injected them subcutaneously with 0.1 ml of corn oil with or

without 5 µg of 17β-estradiol. We did these injections three times per week for 10 weeks, as previously

described (5). We verified deficiency of ovarian sex hormones by the absence of ovarian tissues and

by the shrinkage of uterine mass (6).

Cardiac Myocyte Isolation.

At the 10 week time point, we heparinized (1,000 unit/kg body wt) the rats; and, after 30 min

anesthetized them with an intra-peritoneal injection of pentobarbital sodium (25 mg/kg body weight).

Left ventricular myocytes were isolated as previously described (31, 39, 40). The heart was rapidly

removed, the aorta was cannulated and perfused with oxygenated nominally Ca2+-free Tyrode solution

until complete cessation of beating. The perfusion solution was then switched to 25 µM CaCl2 Tyrode

solution containing 1 mg/ml BSA, 160 unit/ml collagenase type II (Boehringer Mannheim), and 0.1

mg/ml protease type XIV (Sigma, St. Louis, MO). The hearts were then perfused for 16.5 min/g wet wt

with the above solution. At the end of the perfusion period, the hearts were removed from cannula and

placed into a Petri dish containing 50 µM CaCl2 Tyrode-BSA buffer. Ventricles were cut into small

pieces and gently triturated with a pipette and filtered through a cell collector. The cell suspension was

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placed into centrifuge tubes and gradually increased the extra-cellular CaCl2 concentration to a final

level of 0.5 mM CaCl2. Myocytes were studied within six hours after isolation.

Intracellular Ca2+ transients, pH, and Cell Shortening.

Myocytes were loaded with 5 µM Fura 2-AM for 15 min in 0.5 mM CaCl2 Tyrode buffer with 1

mg/ml BSA and 5% fetal bovine serum for 15 minutes. After washing off the excess Fura 2-AM, the

myocytes were electrically stimulated at a frequency of 0.5 Hz for 10 to 15 min or until the shortening

was stable with perfusion of 2 mM CaCl2 Tyrode buffer at room temperature. Fura 2 fluorescence

(340:380 ratio) and cell shortening were monitored simultaneously as described in detail by Wolska et

al. (38).

To test the effect of acidic pH on intracellular Ca2+ transients and cell shortening, we perfused

the myocytes with control Krebs-Henseleit buffer (KH) saturated with 95% O2 and 5% CO2 for 15 min,

with KH saturated with 85% O2 and 15% CO2 for 20 min, and then with control KH. In these

experiments, we also determined intracellular pH with the aid of BCECF as previously described (42),

and made the measurements in the presence and absence of 1.0 µM cariporide, an inhibitor of the

NHE1 (12).

Force measurements on skinned fiber bundles

We determined the relationship between pCa ( log of the molar free Ca2+) and tension

(force/cross-sectional area) in detergent (Triton X-100) extracted fiber bundles prepared from left

ventricular papillary muscles as previously described (39,41). The fiber bundles (~150-200 µm in

diameter and 4-5 mm long) were cut in ice cold high relaxing (HR) buffer: 10 mM EGTA, 2 mM free

Mg2+, 5 mM MgATP2-, 79.2 mM KCl, 12 mM creatine phosphate, 20 mM MOPS, pH 7.0, ionic strength

0.15 M, 2.5 µg/ml pepstatin A, 1 µg/ml leupeptin and 50 µM PMSF. After mounting the fiber bundles in

an apparatus for determination of force, sarcomere length was set at 2.2 µm as determined from a laser

diffraction pattern in HR solution and cross-sectional area determined. Isometric force was recorded on

a chart recorder at different pCa at room temperature. CaCl2 concentrations in HR were varied to

achieve a range of Ca2+ concentrations as computed using a computer program and binding constants

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summarized by Godt and Lindley (9). Only fiber bundles that developed 85-90% of their initial maximum

tension at the end of experiment were retained for analysis. We plotted isometric tension as a

function of pCa, fitted the curves to the A. V. Hill equation as previously described (41), and expressed

the relations as a per cent of maximum tension. pCa50 (half-maximally activating pCa values) was

computed from individual Hill fits of each pCa-tension relation and then averaged.

Expression of NHE1

To determine the level of expression of the NHE-1 gene, real-time one-step quantitative RT-

PCR with SYBR Green detection (Qiagen,Valencia, CA) was performed using a LightCycler

thermocycler (Roche Diagnostics) as previously described (1). Briefly, total RNA was isolated from

ventricular tissue with the RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA). The RNA was

reverse transcribed and the cDNA used to derive a PCR product for NHE-1 and ribosomal protein L7.

The PCR product was cloned into the vector, pBlue-Topo (Invitrogen, Carlsbad, CA). The plasmid

underwent amplification, DNA purification, and in vitro transcription. The resulting RNA was recovered,

quantified and used as standards in the RT-PCR reaction. Gene expression was normalized to

ribosomal protein L7. The following primers were used: NHE-1(forward) 5’-

TTTCCCCgATTTCCTTCTCT-3’; NHE-1(reverse) 5’-gCgTgTAAgACCTgggACAT-3’; Ribosomal protein

L7 (forward) 5’-gAAgCTCATCTATgAgAAggC-3’; Ribosomal protein L7 (reverse) 5’-

CAgACggAgCAgCTgCAgCAC-3’. We also determined the levels of NHE1 by Western blot analysis of

left ventricular proteins separated by 8% SDS-PAGE with anti-NHE1 antibody (Chemicon, 1:1000) using

an HRP labeled secondary antibody and visualization using ECL (Amersham Pharmacia).

Statistical analysis.

Data are presented as means ± SE. All curve fittings were achieved using GraphPad Prism.

One-way ANOVA was used for statistically analysis and Student-Newman-Kuels test for multiple

comparisons . P < 0.05 was considered the significant difference among groups.

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Results

Data in Table 1 summarize heart, body, and uterine weights of sham, OVX, and OVX +E2

animals. Compared to the shams, OVX rats demonstrated a significant increase in both heart and

body weight, but no change in heart weight/ body weight ratio. As expected uterine weight was

significantly lower in the OVX rats compared to shams, and increased significantly upon E2

supplementation. However, uterine weight did return fully to control levels with E2 supplementation.

We think this is due likely to a requirement for other hormones in the re-growth of the uterus.

Cell Shortening and Intracellular Calcium Transients in myocytes.

Table 2 summarizes data comparing mechanics of ventricular myocytes from sham, OVX, and

OVX + E2 rat hearts. OVX induced a change in all parameters. The per cent cell shortening was

significantly depressed by ~22%, as was +dL/dt, the maximum velocity of shortening by ~30% and –

dL/dt, the maximum velocity of re-lengthening by ~50%. The prolongation of the time to peak

amplitude of shortening and the time to 80% full relaxation (T80) also reflected a significant alteration in

myocytes shortening and re-lengthening kinetics. These changes in mechanical properties were only

partially restored by E2 supplementation. Per cent cell shortening and T80 were both significantly

different from OVX myocytes, but other parameters were not. As indicated by the data in Table 3,

which summarizes our measurements of intracellular Ca2+ transients, the depression in per cent cell

shortening induced by OVX was associated with a significant fall in the peak amplitude of the Ca2+

transients with no change in diastolic levels of Ca2+ or the time to peak amplitude. The time to 50%

decay of the Ca2+ transient (T50) was significantly prolonged by OVX and restored to control levels by

E2 supplementation. Moreover, in contrast to the findings from measurements of per cent cell

shortening, the reduction in amplitude of Ca2+ transients and prolonged time of Ca2+ decay in OVX

myocytes could be completely restored with E2 supplementation.

Previous studies demonstrated that compared to controls, ATPase rate (33) and force (34) of

myofilaments isolated from hearts of OVX rats are more sensitive to Ca2+. To determine whether the

increase in myofilament sensitivity occurred in the myocytes from OVX hearts, we performed a set of

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experiments in which we varied extra-cellular Ca2+ to generate a range of increasing levels of the Ca2+

transient. We determined the relation between the peak amplitude of these Ca2+ transients (expressed

as 340:380 ratio), and the extent of cell shortening. Figure 1 summarizes the data for the level of Ca2+

inducing half-maximal activation of shortening in myocytes from shams, OVX and OVX + E2 hearts.

The value of the peak amplitude of Ca2+ transient that gave rise to 50% of the maximum cell shortening

was significantly diminished in OVX myocytes (Fura-2 ratio = 2.52 ± 0.05 in sham controls, and Fura-2

ratio = 2.11 ± 0.04 in OVX myocytes), and was partially restored in the myocytes from OVX + E2 (Fura-

2 ratio = 2.30 ± 0.04). These results indicated that the previous in vitro data indicating an enhanced

myofilament response to Ca2+ associated with OVX also occurs in the intact cardiac myocytes.

Acidosis, Myofilament Tension, and Myocyte Function.

In view of the cardio-protective effects of E2 in acidosis (2) and metabolic inhibition (29); and,

inasmuch as altered myofilament response to Ca2+ is a dominant mechanism responsible for the fall in

tension associated with acidosis (27), we compared the effects of pH on pCa-tension relations of

skinned fiber bundles isolated from sham, OVX, and OVX + E2 rat hearts. We also determined the

relative effects of hypercapnic acidosis on shortening and intra-cellular Ca2+ of myocytes isolated from

sham, OVX, and OVX + E2 rat hearts.

The upper panel of Figure 2 shows relations between pCa and relative tension at pH 7.0, pH

6.8, and pH 6.5 for skinned fiber bundles from the three groups of rat hearts. These results

demonstrate the well-known effect of acidic pH to desensitize cardiac myofilaments to Ca2+. Data in the

lower panel of Figure 5 summarize the half-maximal activating pCa values (pCa50). In agreement with

previous results (34), the skinned fibers from OVX rat hearts demonstrated a significant increase in

sensitivity to Ca2+ at pH 7.0 (pCa50 = 5.84 ± 0.02) compared to fibers from shams (pCa50 = 5.77 ± 0.02).

Skinned fibers from hearts of OVX rats supplemented with E2 had a sensitivity to Ca2+ essentially the

same as controls (pCa50 = 5.75 ± 0.03). However, at pH 6.8 and 6.5 there were no significant

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differences in the pCa50 among the fiber bundles from the three groups. In all cases there were no

significant differences in the Hill coefficient among the fiber groups.

We also determined the relative effects of hypercapnic acidosis on shortening and intra-cellular

Ca2+ of myocytes isolated from sham, OVX, and OVX + E2 rat hearts. Intra-cellular pH fell from a value

of pH 7.25 ± 0.08 in control conditions to pH 6.65 ± 0.07 during perfusion with KH buffer saturated with

15% CO2, 85% O2. The top 3 records in Figure 3A show representative traces of myocytes shortening

before, during, and after these changes in pH. In myocytes from sham rat hearts, about 3 min after

introduction of the hypercapnic KH buffer, the extent of cell shortening fell significantly before rising to a

steady-state value after about 5 min. This steady-state level of extent of shortening remained

significantly lower (56.4 ± 3.9 % reduction) than the value before acidosis and abruptly returned to this

control level with an over-shoot after re-introduction of the control KH buffer. However, although

myocytes from OVX hearts demonstrated the initial fall in extent of shortening similar to that of the

sham control myocytes, extent of shortening rose to a steady-state level 99.8 ± 5.4 % of the value in

the control KH buffer. This resistance to hypercapnic acidosis was partially reversed (72.0 ± 2.8 %

reduction in cell shortening), in myocytes from hearts of the OVX + E2 rats but remained significantly

higher (P < 0.01) than during the control period. The lower 3 traces in Figure 3A show typical records

of myocyte shortening obtained in the presence of cariporide, an inhibitor the NHE1 exchanger.

Regardless of the source, the myocytes demonstrated a lack of biphasic response and essentially the

same extent of severe reduction in steady-state shortening with hypercapnic acidosis. Figure 3B

summarizes the change in extent of shortening at 5 min, 10 min, and with recovery from acidosis.

These data show that the significant protection of OVX myocytes from acidosis was not present in the

myocytes treated with cariporide. To determine whether cariporide might affect myofilament response

to Ca2+, we compared pCa-tension relations in the presence and absence of 1 µM cariporide. These

experiments (data not shown) demonstrated that cariporide does not exert any direct effects on

myofilament response to Ca2+.

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Data in Figure 4 summarize the per cent change in peak amplitude of intra-cellular Ca2+ transients in

the cardiac myocytes from the three groups in the absence and presence of cariporide before, at 5 min

and at 10 min of acidosis, and during recovery after hypercapnic acidosis. In the absence of

cariporide, the Ca2+ transient increased significantly at 10 min in myocytes from the OVX and OVX +E2

rat hearts. However, there were no changes in the peak amplitudes of the Ca2+ transients at any time

in the presence of cariporide. The data in Figures 3 and 4 indicate that the contribution of the activity

of the NHE1 and NCX in regulation of intra-cellular Ca2+ was relatively high in myocytes from OVX rat

hearts compared to shams. We therefore determined the level of expression of NHE1 message and

protein in the hearts from the three groups of rats. Figure 5A shows results of experiments, in which we

quantified the level of expression of the NHE-1 gene employing real-time quantitative RT-PCR with

SYBR Green detection. The data showed no significant differences. This was also true at the protein

level as illustrated in Figure 5B, which shows a representative Western blot indicating that there was no

significant difference in expression of NHE1 among the sham, OVX, and OVX + E2 hearts. This

determination was made three times with no difference in protein level detected.

Discussion

Data reported here are the first to report an effect of ovariectomy to enhance Ca2+-sensitivity of

myofilaments functioning in the intact cardiac myocyte. An important new finding with regard to this

altered response to Ca2+ is that although it appears to occur at pH 7.0, the sensitization did not occur at

pH 6.8 or pH 6.5. This result adds new insights with regard to the interpretation of the significance of

the altered myofilament response to Ca2+ in the physiology and pathology of the myocardium. Our data

are also the first to demonstrate a significant role of enhanced NHE1 activity in altering the response of

heart cells from OVX rats to the stress of acidosis.

There is now general agreement that altered myofilament response to Ca2+ is a significant

aspect of the integrated biology of cardiac function (28). A mechanism involving sarcomere length

dependent alterations in myofilament response to Ca2+ remains the dominant theory for the cellular

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basis of Starling’s Law of the heart (23). Moreover, there is substantial evidence for the functional

significance of myofilament protein phosphorylation as a significant factor controlling cardiac dynamics

(29). As illustrated by the data presented here, depressed cardiac function in acidosis also reflects an

influence of intra-cellular environment to depress myofilament response to Ca2+, a mechanism which is

dominant in determining cellular function. For example, Figures 4 and 5 summarized data showing a

severe depression in cell shortening with acidosis in the presence or absence of cariporide with little or

no change in the peak amplitude of the Ca2+ transient. Chronic increases in cardiac myofilament

response to Ca2+, which our previous (33,34) and present data indicate occur with ovariectomy, are

now also recognized to be of major significance as a cause of hypertrophy associated with mutations in

sarcomeric proteins (1012). The increased Ca2+ sensitivity of OVX cardiac myofilaments is also similar

to that reported in cardio-myopathic and heart failure models (11,37). The elevated myofilament

response to Ca2+ may induce diastolic defects leading to cell stretch and elaboration of signals

promoting hypertrophic growth (8).

The effects of chronic increases in myofilament response to Ca2+ are, however, complex

inasmuch as there is evidence of the beneficial effects of therapeutic agents, which induce myofilament

Ca2+ -sensitization (13). Moreover, hearts of transgenic mouse models with constitutive myofilament

Ca2+-sensitization associated with modification of troponin I demonstrate remarkable resistance to

acidosis (32,35), sepsis (15), and ischemia/reperfusion (3,7). An important distinction is that compared

to controls the myofilaments containing modified troponin I or treated with Ca2+-sensitizing agents are

not only more sensitive to Ca2+ they are resistant to deactivation by acidic pH (30,45), whereas the

myofilaments from OVX hearts are not.

To more clearly understand whether the increase in myofilament response to Ca2+ associated

with ovariectomy is an adaptive or a maladaptive response, we stressed myocytes with acidosis. We

chose this approach because of the dominance of depressed myofilament Ca2+ responsiveness in

acidosis, and because of its relevance to ischemia and ischemia/reperfusion injury (3). We found that

the acidosis induced depression of shortening of myocytes isolated from hearts of OVX rats was

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blunted compared to the controls. Although this blunting may have been due in part to the enhanced

sensitivity of the myofilaments to Ca2+, data shown in Figure 2 indicate that the sensitization is apparent

only at pH 7.0.

It is likely that this increase in Ca2+ during acidosis in myocytes from OVX hearts and not in

controls occurred as a result of increased Na exchange associated with stimulation of the NHE1 and a

resultant decrease in the efflux of Ca2+ through NCX. This could be due either to a reduced Ca2+ -efflux

or increased influx via reverse mode activity of the NCX. It is also possible that the elevation of

intracellular Ca2+ is due in part to a concerted depression in Ca2+ uptake by the SR, which was

demonstrated in earlier studies to occur in OVX rat hearts (5). Yet, inhibition of the differential effects of

acidosis on myocytes from OVX and sham hearts by cariporide demonstrated the critical role of NHE1

in this response. We did not find a difference in expression of NHE1 among the three groups studied.

We therefore conclude that compared to controls, the loss of ovarian sex hormones may induce a

sensitization of NHE1 activity to pH. This conclusion fits with studies investigating acute effects of E2,

which reported administration of E2 to hearts during ischemia and reperfusion limited the rise in Ca2+

occurring with ischemia and reperfusion (2). By use of inhibitors, these studies provided evidence that

acute administration of E2 increased NOS activity and NO release, and thereby depressed the activity

of the NHE1 resulting in a decreased intra-cellular Na and a limited rise in Ca2+ during

ischemia/reperfusion. A role for NOS has been documented as a basis for the difference in control of

NCX activity between hearts of male and female rats (6,18). Our data provide evidence that the

protective effect of E2 in ischemia and reperfusion occurs during chronic depressions in levels of

ovarian sex hormones. In this case the withdrawal of E2 would release NHE1 from inhibition by NO

resulting in an enhanced activity, which in concert with altered NCX activity led to elevated intracellular

Ca2+ Morimoto et al. (17) reported an increase in mesenteric eNOS expression associated with E2

treatment of OVX rats. An effect of chronic deprivation of ovarian sex hormones on NOS activity is an

attractive mechanism, but whether this occurs with ovariectomy awaits further investigation.

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Grant Support

This work was supported by NIH Grants RO1 HL 64035 (RJS), PO1 HL 62426 (RJS), and

RO1 HL 64209 and 79032 (BMW). Dr. Bupha-Intr also received support as a Golden Jubilee pre-

doctoral fellow of Mahidol University, Bangkok, Thailand.

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Figure Legends

Figure 1. Effects of ovariectomy on relationship between the per cent maximum shortening

and amplitude of calcium transient (reported as the Fura-2 ratio) of cardiac myocytes isolated

from hearts of shams, OVX rats, and OVX rats supplemented with E2. Data are means ± SEM

from 10 myocytes. * Significantly different from shams, P < 0.05; # significantly different from

OVX, P < 0.05.

Figure 2. A. pCa-tension relations of skinned fiber bundles from sham, OVX and OVX + E2

rats at three different pH values. B. Comparison of pC50 values. Data are means ± SEM

from 10 preparations. * Significantly different from shams, P < 0.05; # significantly different

from OVX, P < 0.05.

Figure 3. A. Representative tracings showing effects of acidosis on shortening during twitch

contractions with and without the NHE1 inhibitor cariporide of myocytes isolated from hearts of

shams, OVX rats, and OVX rats supplemented with E2. B. Data summarizing extent of

shortening in the absence (top panel) and presence (bottom panel) of cariporide at 5 and 10

min of acidosis and following recovery in myocytes from hearts of shams, OVX rats, and OVX

rats supplemented with E2. Percent shortening under control conditions was taken as 100%.

Data are means ± SEM from 10 myocytes. * P < 0.05 = significantly different from sham.

Figure 4. Data summarizing of Ca2+ transient amplitude in the absence (top panel) and

presence (bottom panel) of cariporide at 5 and 10 min of acidosis and following recovery in

Page 20 of 30

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myocytes from hearts of shams, OVX rats, and OVX rats supplemented with E2. Percent

shortening under control conditions was taken as 100%. Data are means ± SEM from 10

myocytes. * P < 0.05 = significantly different from sham.

Figure 5. Data demonstrating no change in expression of Na+-H+ exchanger 1 (NHE1). A.

NHE1 expression of studied in 7 separate hearts from sham-operated (SHAM), 7 from

ovariectomized (OVX) and 9 from OVX with estrogen supplementation (OVX+E2). Expression

was normalized to ribosomal protein L7 and the data are means ± SEM. B. Representative

immunoblot image of proteins and calsequestrin (CQ) of SHAM, OVX, and OVX+E2.

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TABLE 1. General characteristics of sham controls, ovariectomized, and ovarectomized With

estrogen supplementation rats

Parameters SHAM OVX OVX+E2

n 18 19 19

Body Weight, g 267 ± 3.3 323 ± 3.4* 251 ± 3.2

Heart Weight, g 1.11 ± 0.01 1.26 ± 0.02* 1.10 ± 0.02

HW/BW Ratio 0.41 ± 0.01 0.39 ± 0.01 0.438 ± 0.01

Uterine Weight, g 0.56 ± 0.02 0.12 ± 0.01* 0.34 ± 0.02 *#

Results are means ± SEM from each group. SHAM represents sham-operated control;

OVX, ovariectomized rats; OVX+E2, ovariectomized plus estrogen supplementation. *P< 0.05

vs SHAM, #P<0.05 vs OVX.

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TABLE 2. Contraction/relaxation kinetics of myocytes isolated from hearts of sham controls,

ovariectomized, and ovariectomized with estrogen supplementation rats.

Parameters SHAM OVX OVX+E2

Hearts 4 5 5

Cells, n 14 19 18

% Shortening 8.35 ± 0.39 6.50 ± 0.27* 8.23 ± 0.37#

Time to Peak Ampitude of

shortening, msec

173 ± 11 228 ± 13* 233 ± 8.9*

T80, msec 170 ± 13 334 ± 22* 236 ± 18* #

+dL/dt, µm/sec 104.3 ± 6.8 71.0 ± 4.9* 74.2 ± 5.5*

-dL/dt, µm/sec 92.0 ± 7.9 48.6 ± 4.7* 57.6 ± 5.2*

Results are mean ± SE; n = number of cells; T80, time required for cell to 80% of full

relaxation from the peak of shortening; +dL/dt, rate of cell shortening; -dL/dt, rate of cell re-

lengthening. * P< 0.05 vs SHAM; #P < 0.05 vs OVX.

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TABLE 3. Ca2+ transient kinetics of myocytes isolated from hearts of sham controls,

ovariectomized, and ovariectomized with estrogen supplementation rats.

Parameters SHAM OVX OVX+E2

Hearts 7 6 6

Cells, n 20 20 20

Baseline, ratio unit 1.36 ± 0.04 1.33 ± 0.04 1.37 ± 0.03

Amplitude, ratio unit 2.12 ± 0.13 1.74 ± 0.10* 2.26 ± .14#

Time to Peak, msec 95.3 ± 4.3 95.5 ± 1.9 97.7 ± 2.2

T50, msec 180 ± 10.7 232 ± 9.3* 196 ± 9.3

Results are means ± SEM; n = number of cells; baseline, 340/380 ratio at rest;

amplitude, peak of 340/380 ratio minus baseline ratio; T50, times required for cell to achieve

50% of full relaxation of Ca2+ signal to baseline ratio from peak ratio. * P< 0.05 vs SHAM.

Page 24 of 30

Figure 1. Effects of ovariectomy on relationship between the per cent maximum shortening and amplitude of calcium transient (reported as the Fura-2 ratio) of cardiac myocytes isolated from hearts of shams, OVX rats, and OVX rats supplemented with E2. Data are means ± SEM from 10 myocytes. * Significantly different from shams, P < 0.05;

# significantly different from OVX, P < 0.05.

Page 25 of 30

Figure 2. A. pCa-tension relations of skinned fiber bundles from sham, OVX and OVX + E2 rats at three different pH values. B. Comparison of pC50 values. Data are means ± SEM

from 10 preparations. * Significantly different from shams, P < 0.05; # significantly different from OVX, P < 0.05.

Page 26 of 30

Figure 3. A. Representative tracings showing effects of acidosis on shortening during twitch contractions with and without the NHE1 inhibitor cariporide of myocytes isolated

from hearts of shams, OVX rats, and OVX rats supplemented with E2.

Page 27 of 30

B. Data summarizing extent of shortening in the absence (top panel) and presence (bottom panel) of cariporide at 5 and 10 min of acidosis and following recovery in

myocytes from hearts of shams, OVX rats, and OVX rats supplemented with E2. Percent shortening under control conditions was taken as 100%. Data are means ± SEM from 10

myocytes. * P < 0.05 = significantly different from sham.

Page 28 of 30

Figure 4. Data summarizing of Ca2+ transient amplitude in the absence (top panel) and presence (bottom panel) of cariporide at 5 and 10 min of acidosis and following recovery in myocytes from hearts of shams, OVX rats, and OVX rats supplemented with E2. Percent shortening under control conditions was taken as 100%. Data are means ± SEM from 10

myocytes. * P < 0.05 = significantly different from sham.

Page 29 of 30

Figure 5. Data demonstrating no change in expression of Na+-H+ exchanger 1 (NHE1). A. NHE1 expression of studied in 7 separate hearts from sham-operated (SHAM), 7 from

ovariectomized (OVX) and 9 from OVX with estrogen supplementation (OVX+E2). Expression was normalized to ribosomal protein L7 and the data are means ± SEM. B.

Representative immunoblot image of proteins and calsequestrin (CQ) of SHAM, OVX, and OVX+E2.

Page 30 of 30