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1 Department of Pathology, Duke University Medical Center, Durham, 27710; and 2 National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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To
compare ischemia-reperfusion injury in males versus females
under hypercontractile conditions, perfused hearts from 129J mice
pretreated with 3 mmol/l Ca2+ or 10
8 mol/l
isoproterenol ± 106 mol/l
N
-nitro-L-arginine methyl ester
(L-NAME) were subjected to 20 min of ischemia and
40 min of reperfusion while 31P NMR spectra were acquired.
Basal contractility increased equivalently in female versus male hearts
with isoproterenol- or Ca2+ treatment. Injury was
equivalent in untreated male versus female hearts but was greater in
isoproterenol or Ca2+-treated male than female hearts, as
indicated by lower postischemic contractile function, ATP, and
PCr. Endothelial nitric oxide (NO) synthase (eNOS) expression was
higher in female than male hearts, neuronal NOS (nNOS) did not differ,
and inducible NOS (iNOS) was undetectable. Ischemic NO
production was higher in female than male hearts, and
L-NAME increased injury in female isoproterenol-treated hearts. In summary, isoproterenol or high Ca2+ pretreatment
increased ischemia-reperfusion injury in males more than
females. eNOS expression and NO production were higher in female than
male hearts, and L-NAME blocked female protection. Females
were therefore protected from the detrimental effects of adrenergic
stimulation and Ca2+ loading via a NOS-mediated mechanism.
energetics; gender; nitric oxide synthase; nuclear magnetic resonance spectroscopy
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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CLINICAL FINDINGS indicate that females are protected from cardiovascular injury and that this protection is mediated via estrogen. Premenopausal women possess a lower risk for ischemic heart disease than age-matched males (10), and this protection is lost after menopause (7). Estrogen replacement therapy has been reported to decrease the occurrence and mortality of myocardial infarction (8) and increase survival in patients following coronary artery bypass surgery (16). Despite numerous clinical observations, no experimental study has demonstrated male/female differences in the susceptibility to myocardial ischemia-reperfusion injury in untreated wild-type isolated hearts from any species, and few studies have demonstrated cardioprotection in isolated hearts treated with physiological levels of estrogens (11).
In previous studies, we likewise observed no male/female differences in the susceptibility to ischemia-reperfusion injury under basal conditions in hearts isolated from wild-type mice (3). However, in mice with cardiac overexpression of the Na+/Ca2+ exchanger, ischemia-reperfusion injury was exacerbated in males but not females (3). Because the Na+/Ca2+ exchange overexpressor mice exhibit increased contractility, in addition to increased cytosolic and sarcoplasmic reticular (SR) Ca2+ levels (3, 18), the possibility exists that cardioprotection in females may be due to a lesser susceptibility to the detrimental effects of contractile stimulation or Ca2+ overload. To test this hypothesis, hearts from male and female wild-type mice were pretreated with isoproterenol (Iso) or high Ca2+ before being subjected to global ischemia and reperfusion. Ischemia-reperfusion injury was determined by the extent of recovery of postischemic contractile function and energy metabolites.
Because male/female differences were observed previously under
conditions of altered Ca2+ homeostasis and because nitric
oxide (NO) affects calcium homeostasis (13, 19, 23), we
studied the estrogen effector NO synthase (NOS). Expression of both
inducible NOS (iNOS) and endothelial NOS (eNOS) has been reported to be
increased by estrogen in the rat myocardium (15), and NO
produced via iNOS, eNOS, or NO donors can be cardioprotective (9,
17, 20). To examine the role of NOS in
ischemia-reperfusion injury in males versus females, we studied
the energetic and functional response to ischemia of male and
female hearts pretreated with the nonisoform-specific NOS inhibitor
N-nitro-L-arginine methyl ester
(L-NAME). We also measured expression of iNOS, eNOS, and
neuronal NOS (nNOS) in male and female hearts and NO production under
basal and ischemic conditions in male and female untreated and
Iso-treated hearts.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals
Male wild-type 129J strain mice (n = 59) and female wild-type 129J mice (n = 60) were used. All animals were adult and reproductively viable, being between the age of 12 and 24 wk old at the time of experimentation. All animals were treated in accordance with National Institutes of Health guidelines and the "Guiding Principles for Research Involving Animals and Human Beings."Ischemia-Reperfusion Protocol
Hearts were isolated and perfused in the Langendorff mode as described previously (4). Hearts were perfused for 30 min before being subjected to 20 min no-flow ischemia and 40 min reperfusion. Left ventricular developed pressure (LVDP), maximum rate of contraction (+dP/dtmax), minimum rate of contraction (
dP/dtmin), and heart rate were
monitored via a water-filled latex balloon in the left ventricle.
Recovery of contractile function was assessed by measurement of LVDP at
the end of reperfusion and expressed as a percentage of
preischemic LVDP.
Isoproterenol, L-NAME, and High Ca2+ Treatment
A group of hearts from male (male + Ca; n = 5) and female (female + Ca; n = 5) mice were treated with 3 mmol/l extracellular Ca2+ (high Ca
8
mol/l Iso 1 min before ischemia (male + Iso,
n = 11; female + Iso, n = 11),
106 mol/l of the nonisoform-specific NOS inhibitor
L-NAME 5 min before ischemia (male + L-NAME, n = 6; female + L-NAME, n = 7), or 106 mol/l
L-NAME 5 min before ischemia plus 108
mol/l Iso beginning 1 min before ischemia (male + Iso + L-NAME, n = 6; female + Iso + L-NAME, n = 6). All treatments were
continued until 30 min of reperfusion, followed by 10 min of washout.
Appropriate treatment concentrations were predetermined from
dose-response curves; 108 mol/l Iso and 3 mmol/l
Ca2+ were selected to provide similar hypercontractility to
that observed in the Na+/Ca2+ exchange
overexpressor mice studied previously, whereas 106 mol/l
L-NAME was selected as the highest effective dose having no
measurable effect on coronary flow.
Nuclear Magnetic Resonance Spectroscopy
Relative changes in concentrations of phosphorus metabolites were observed during the ischemia-reperfusion protocols by acquiring consecutive 31P NMR spectra as described previously (4). The areas of the spectral peaks were expressed as a percentage of the peak areas of an initial, preischemic control spectrum from each heart. Phosphocreatine (PCr) values were normalized by expressing PCr peak areas as a percentage of the ATP peak area in the initial preischemic control spectrum from each heart. Intracellular pH was estimated from the chemical shift of the Pi peak relative to PCr using previously obtained titration curves.NOS Expression and Activity
Male (n = 3) and female (n = 3) hearts were perfused for 30 min and frozen in liquid nitrogen. Lysates were prepared, 80 µg (for eNOS and nNOS) or 200 µg (for iNOS) protein was loaded onto 8% SDS gels, and proteins were separated by electrophoresis and transferred to nitrocellulose as described previously (3). Membranes were incubated with eNOS, iNOS, or nNOS rabbit polyclonal antibodies at 1:400 dilution (Santa Cruz Biotechnology; Santa Cruz, CA) followed by incubation with horseradish peroxidase-conjugated anti-rabbit IgG at 1:5,000 dilution before visualization by enhanced chemiluminescence. The optical density of immunoreactive bands was quantified using NIH Image 1.61.Total nitrate concentrations (NOx) were determined in
untreated male (n = 10) and female (n = 12) hearts using a nitrate/nitrite colorimetric assay kit (Cayman
Chemical). NOx concentrations were also determined in male
(male + Isc, n = 8) and female (female + Isc,
n = 8) untreated hearts subjected to 10 min
ischemia (Isc) and in male (male + Iso + Isc,
n = 6) and female (female + Iso + Isc,
n = 6) hearts pretreated for 1 min with
108 mol/l Iso before 10 min ischemia. Because of
the low myocardial NOx levels, two hearts were combined for
each measurement.
Statistics
Results are expressed as means ± SE. Significance (P
0.05) was determined for discrete variables by
analysis of variance (ANOVA) and for continuous variables by ANOVA for
repeated measures; both analyses were followed by a Fisher's post hoc test.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Body and Heart Weights
The male mice had a body weight of 26 ± 1 g and heart weight of 0.14 ± 0.01 g, whereas the female mice had a body weight of 21 ± 1 g and a heart weight of 0.12 ± 0.01 g. Heart weight-to-body weight ratios did not differ between male and female mice.Contractile Function
Untreated hearts.
During the preischemic period, LVDP did not differ
between male and female untreated hearts, both being ~110
cmH2O (Table 1). Heart rate
was also the same, at ~370 beats/min, in female untreated hearts
as in male untreated hearts, as was the
+dP/dtmax and dP/dtmin
at ~3.7 cmH2O/ms and approximately 2.8
cmH2O/ms, respectively, in both groups (Table 1). Thus
there were no apparent male/female differences in basal contractility
in untreated hearts.
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High Ca
dP/dtmin, at
approximately 6.2 cmH2O/ms, also increased significantly
in magnitude in male and female hearts upon high CadP/dtmin between high
Ca
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Iso treatment.
Treatment with 108 mol/l Iso resulted in a significant
increase in LVDP to ~190 cmH2O in both male and female
hearts (Table 1; P < 0.0001).
+dP/dtmax, at ~8.0 cmH2O/ms, and
dP/dtmin, at approximately 7.8
cmH2O/ms, also increased significantly in male and female
hearts upon Iso treatment (Table 1; P < 0.0001). In contrast to treatment with high CadP/dtmin between Iso-treated female hearts and
Iso-treated male hearts. Iso treatment, therefore, increased basal
contractility equivalently in both male and female hearts.
L-NAME treatment.
Infusion of 106 mol/l L-NAME had no effect on
preischemic LVDP, heart rate, +dP/dtmax,
or dP/dtmin in male or female hearts (Table
1). Coronary flow was also unaffected by infusion of 106
mol/l L-NAME, being 1.65 ± 0.11 ml/min preinfusion
and 1.82 ± 0.13 ml/min postinfusion in male hearts and 1.74 ± 0.15 ml/min preinfusion and 1.64 ± 0.09 ml/min postinfusion in
female hearts. Preinfusion of 106 mol/l
L-NAME had no significant effects on the contractile
response to 108 mol/l Iso in male or female hearts (Table
1). There were no male/female differences in LVDP, heart rate,
+dP/dtmax, or dP/dtmin in L-NAME-treated or Iso plus L-NAME-treated
hearts. Infusion of 106 mol/l L-NAME,
therefore, had no effect on basal contractility or coronary flow in
male or female hearts. Similarly, 106 mol/l
L-NAME had little effect on the contractile response to Iso
in male and female hearts.
8 mol/l Iso plus
106 mol/l L-NAME exhibited the same
postischemic recovery of function as male hearts treated with
108 mol/l Iso alone, both ~12% initial LVDP. However,
pretreatment with L-NAME lowered postischemic
functional recovery in Iso-treated female hearts from 33% initial LVDP
in female hearts with Iso alone to 10% initial LVDP in female hearts
treated with Iso plus L-NAME (P < 0.001;
Fig. 1). Treatment with L-NAME alone had no significant
effect on postischemic functional recovery in either male or
female hearts, both recovering ~40% initial LVDP, compared with
~45% initial LVDP in untreated male and female hearts (Table 2).
Therefore, despite having no effect alone, L-NAME treatment abolished the protection observed in female Iso-treated hearts versus
male Iso-treated hearts.
Phosphate Metabolites and Intracellular pH
Phosphate metabolites and intracellular pH were measured to determine whether there were male/female differences in energetics and pH in untreated hearts and whether the energetic response to Iso, L-NAME, or high CaUntreated hearts.
During ischemia, ATP fell to the same level, ~35% of initial
ATP, in both male and female untreated hearts (Fig.
2A). Likewise, during
reperfusion, ATP increased to the same level, ~40% of initial ATP,
in untreated male hearts as in untreated female hearts (Fig. 2A).
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High Ca
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Iso treatment.
ATP fell lower during ischemia in the Iso-treated male hearts,
reaching 16% initial ATP, than in either untreated male hearts or
Iso-treated female hearts, at 35% initial ATP by the end of ischemia (P 0.05; Fig. 3A). There was
no significant difference in end-ischemic ATP levels between
the Iso-treated female hearts and untreated female hearts. ATP levels
were lower during reperfusion in the Iso-treated male hearts, reaching
22% initial ATP by the end of reperfusion, than in untreated male
hearts or Iso-treated female hearts, at 48% initial ATP (P 0.05; Fig. 3A). There was no significant difference in
end reperfusion ATP levels between Iso-treated female hearts and
untreated female hearts.
0.05; Fig.
3C). There was no significant difference in
end-ischemic pH between Iso-treated female hearts and untreated
female hearts. During reperfusion, there were no differences in pH
between any groups, all reaching ~pH 7.05 by the end of reperfusion.
In summary, treatment with Iso lowered ATP and intracellular pH levels
in the male hearts during ischemia but had no effect on ATP
levels or intracellular pH in female hearts. Likewise, during
reperfusion, Iso treatment decreased recovery of the energy metabolites
ATP and PCr in male but not female hearts. The lower postischemic recoveries of ATP and PCr in male Iso-treated
hearts correlated with the lower recovery of contractile function
observed, indicating greater myocardial injury. It appears, therefore,
that females are protected from the energetic, as well as functional, detrimental effects of Iso pretreatment.
L-NAME treatment.
ATP fell to the same level during ischemia in male Iso plus
L-NAME-treated hearts, reaching 21% initial ATP, as in
male hearts treated with Iso alone, at 16% initial ATP (Fig.
3A). However, ATP fell lower during ischemia in
female Iso plus L-NAME-treated hearts, reaching 20% ATP,
than in female hearts treated with Iso alone, at 37% ATP (P 0.05; Fig. 3A), falling as low as in male hearts treated
with Iso alone. During reperfusion, ATP levels were not significantly
different between male Iso plus L-NAME-treated hearts,
reaching 20% initial ATP, and male hearts treated with Iso alone, at
22% initial ATP (Fig. 3A). However, reperfusion ATP levels
remained lower in female Iso plus L-NAME-treated hearts, reaching 28% ATP, than in female hearts treated with Iso alone, at
48% ATP (P 0.05; Fig. 3A), being as low as in
male hearts treated with Iso alone. Treatment with L-NAME
alone had no effect on ischemic ATP levels, at 34 ± 5%
ATP in males and 28 ± 7% ATP in females by the end of
ischemia, compared with ~35% of the ATP in untreated male
and female hearts. Likewise, L-NAME alone had no effect on
reperfusion ATP levels at 36 ± 7% ATP in male and 42 ± 8%
ATP in females by the end of reperfusion, compared with ~40% ATP in
untreated male and female hearts.
0.05; Fig. 3B), being as low as in male
hearts treated with Iso alone. Treatment with L-NAME alone
had no effect on ischemic PCr levels, at 5 ± 1% initial ATP in males and 8 ± 2% initial ATP in females by the end of
ischemia, compared with ~7% initial ATP in untreated male
and female hearts. Likewise, L-NAME alone had no effect on
reperfusion PCr levels at 90 ± 4% initial ATP in males and
92 ± 17% initial ATP in females by the end of reperfusion
compared with ~89% initial ATP in untreated male and female hearts.
At the end of ischemia, pH had fallen to the same level in male
Iso plus L-NAME-treated hearts, reaching pH 5.70, as in
male hearts treated with Iso alone, at pH 5.61 (Fig. 3C).
However, pH fell lower during ischemia in female Iso plus
L-NAME-treated hearts, reaching pH 5.58, than in female
hearts treated with Iso alone, at pH 5.81 (P 0.05; Fig.
3C), falling as low as in male hearts treated with Iso
alone. Treatment with L-NAME alone had no effect on
ischemic pH, at pH 5.77 ± 0.05 in males and pH 5.75 ± 0.07 in females by the end of ischemia, compared with ~pH
5.80 in untreated male and female hearts. During reperfusion there were
no differences in pH between any groups, all reaching ~pH 7.05 by the
end of reperfusion.
In summary, treatment with L-NAME lowered ischemic
intracellular ATP and pH in female Iso-treated hearts to the same level observed in male hearts treated with Iso alone. Likewise,
L-NAME treatment decreased recovery of the energy
metabolites ATP and PCr in female Iso-treated hearts during
reperfusion. In contrast, L-NAME had no measurable
energetic effect on male Iso-treated hearts. L-NAME had no
measurable energetic effect on non-Iso-treated male or female hearts.
It appears, therefore, that L-NAME abolished the protection
observed in Iso-treated females versus males, implying that the
protection was mediated by NOS.
NOS Expression and Activity
Protein expression levels of the three NOS isoforms (iNOS, eNOS, and nNOS) were measured in male and female untreated hearts under basal conditions. iNOS expression was not detected in any hearts, despite maximal protein loading and high iNOS antibody concentration. eNOS expression was 24 ± 10% greater in untreated female hearts than in untreated male hearts (P 0.05; Fig.
4A). We detected nNOS
expression in both male and female hearts, but there were no
differences between the two groups (Fig. 4B).
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We also assessed myocardial NO production via determination of tissue
total NOx levels in male and female untreated and
Iso-treated hearts. NOx levels were slightly but not
significantly greater in untreated female, at 53 pmol/mg protein, than
untreated male hearts, at 32 pmol/mg protein (Fig.
5). After 10 min of ischemia, NOx levels were significantly greater in untreated female
hearts, at 101 pmol/mg protein than in untreated male hearts, at 28 pmol/mg protein (P 0.05). NOx levels were
also significantly greater in Iso-treated female hearts after 10 min of
ischemia, at 89 pmol/mg protein, compared with Iso-treated male
hearts, at 31 pmol/mg protein (P 0.05). NO production
during ischemia, therefore, was greater in untreated and
Iso-treated female than male hearts.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effects of Isoproterenol or High Ca2+ on Contractility and Ischemia-Reperfusion Injury
In the present study, heart rate, peak contraction (LVDP), +dP/dtmax, anddP/dtmin
were the same in perfused hearts from untreated male and female mice.
Therefore, under unstimulated conditions, there were no gender
differences in basal myocardial contractility. Upon infusion of Iso,
heart rate, LVDP, +dP/dtmax, and
dP/dtmin increased to the same extent in the
male as in female hearts. In contrast to Iso treatment, high
CadP/dtmin to the same extent in the male hearts
as in the female hearts. Both Iso and high CaDuring ischemia and reperfusion there were no male/female
differences in energetics or contractile function in untreated hearts. However, despite having equivalent effects on contractility in male and
female hearts, Iso or high Ca
With Iso treatment, ATP depletion and H+ production
during ischemia were greater in the male Iso-treated hearts
compared with the male or female untreated hearts, indicating a greater
myocardial ischemic energy demand in the Iso-treated males. In
contrast, Iso treatment had no effect on ATP levels or intracellular pH in female hearts. Iso treatment increased ischemia-reperfusion injury in both male and female hearts; however, similar to high Ca
Role of NOS in Protection From Ischemia-Reperfusion Injury in Females
Stimulation of cardiac adrenergic receptors leads to phosphorylation of L-type Ca2+ channels and phosphorylation of the SR Ca2+-ATPase inhibitor phospholamban, resulting not only in increased contractility, but also in increased cytosolic and SR Ca2+ levels, respectively (5). As ischemia-reperfusion injury is exacerbated by Ca2+ overload (6, 12, 14), these increases in cytosolic and SR Ca2+ may be the mechanism by which infusion of the adrenergic agonist Iso leads to increased ischemia-reperfusion injury. Likewise, the other conditions in which we have observed male/female differences in ischemia-reperfusion injury, namely pretreatment with high Ca2+ in the present study or overexpression of the Na+/Ca2+ exchanger (3), both result in increased cytosolic and SR Ca2+ (18) without increased ischemic energy depletion (3). It appears, therefore, that females may be protected from the detrimental effects of cytosolic and SR Ca2+ overload.Clinical studies indicate that protection in females is mediated via estrogen (7, 8, 16). Consistent with a role for estrogen in female cardioprotection, we have shown previously that protection of hearts from female mice overexpressing the Na+/Ca2+ exchanger, which exhibit increased cytosolic and SR Ca2+ (18), is abolished by ovariectomy (3). The protective role of estrogen is also supported by other experimental studies in which chronic treatment of ovariectomized rats with physiological levels of estrogen results in protection from ischemia-reperfusion injury (11). In the present study, we aimed to elucidate the mechanism of estrogen-mediated cardioprotection by comparing activity and expression of a downstream target of estrogen, NOS, in male and female mouse hearts.
There are three isoforms of NOS: iNOS, the activity and expression of which is increased by stress stimuli such as ischemia or toxins, and eNOS and nNOS, both of which exhibit constitutive activity and expression. iNOS has been detected in rat myocardium at low levels in the unstressed state (2, 15), whereas eNOS is primarily found in endothelial cells but has been shown to be present in myocytes (1, 15). It was assumed previously that nNOS was not present in the heart; however, with the advent of new NOS isoform-specific antibodies, Xu et al. (21) recently demonstrated cardiac nNOS expression. Expression of both iNOS and eNOS has been reported to be increased by estrogen in the rat myocardium (15).
NOS catalyzes the conversion of L-arginine to L-citrulline, resulting in the production of NO. NO is known to affect Ca2+ homeostasis, modulating sarcolemmal Ca2+ channels (13) and SR Ca2+ transport proteins such as the ryanodine receptor (23) and SR Ca2+-ATPase (19). In addition, a number of studies have found that NO produced via iNOS, eNOS, or NO donors to be cardioprotective (9, 17, 20), although iNOS-mediated protection is usually demonstrated over a delayed time period and is therefore unlikely to explain the short-term protection observed in the present study. Because NOS is a downstream target of estrogen, since NO is involved in Ca2+ homeostasis and since NO is cardioprotective, we investigated whether NOS activity or expression was altered in the protected female hearts and whether protection was affected by pretreatment with the NOS inhibitor L-NAME. Because Iso treatment is more physiologically relevant than high extracellular Ca2+ treatment, the mechanism of protection was pursued in the Iso-treated hearts.
We found that eNOS expression was higher in female than male mouse hearts, similar to previous findings in rat hearts (15) and consistent with previous studies showing that myocardial eNOS expression can be induced by estrogen (15). nNOS expression levels were the same in male and female hearts. As far as we are aware, this is the first study to assess whether gender differences in nNOS expression exist in the myocardium. We failed to detect iNOS protein in any hearts, despite maximal protein loading and high antibody concentration. iNOS expression has been detected at low levels in the rat myocardium (2, 15). It is possible that basal iNOS expression is greater and therefore more easily detected in the rat heart than in the mouse hearts analyzed in the present study.
NO production was slightly greater in female than male hearts under
basal conditions and significantly greater in female than male
Iso-treated and untreated hearts during ischemia. Pretreatment with 106 mol/l L-NAME had no effect on the
energetics or function during either ischemia or reperfusion in
male or female hearts without Iso or in Iso-treated male hearts.
However, pretreatment with L-NAME lowered ATP and pH during
ischemia and lowered reperfusion ATP, PCr, and contractile function in
female Iso-treated hearts to the same level observed in male hearts
treated with Iso alone. Thus L-NAME blocked the
protection from the energetic and functional effects of Iso
pretreatment observed in female compared with male Iso-treated hearts,
implying that the protection was mediated by NOS. Our findings that the
protection in the female compared with the male Iso-treated hearts
could be blocked by L-NAME, coupled with the demonstration
of greater ischemic NO production in female versus male hearts,
provides strong evidence for the role of NOS in mediating the female
cardioprotection. Although not conclusive, our finding that eNOS is the
only NOS isoform with elevated expression in female hearts provides
some indication that eNOS may be the NOS isoform responsible for the
increased ischemic NO production and for mediating the
protection. Such a role for eNOS would be consistent with reports that
eNOS is activated rapidly in response to ischemia, in contrast
to iNOS, which is not activated until 24 h after ischemia
(22).
Taken together, the findings of the present study indicate that adrenergic stimulation or Ca2+ overload leads to increased injury, but estrogen-mediated production of NO can counteract the increased injury. In the unstimulated hearts, NOS activity is higher in females than males, but the beneficial effects of NO do not appear to result in a significant level of protection. However, with adrenergic stimulation or high Ca2+ perfusion, injurious mechanisms appear to be exacerbated, the beneficial effects of NO reach significance, and male hearts exhibit increased injury, whereas female hearts are protected. We have no direct evidence for the mechanism of NOS-mediated protection. However, given that adrenergic stimulation, high Ca2+ perfusion, and NO all modulate cytosolic and SR Ca2+ homeostasis (13, 18, 19, 23), some possibilities include an NO-mediated decrease in ischemic SR Ca2+ release, SR Ca2+ cycling, or Ca2+ influx across the sarcolemma. Our observations would be consistent with ischemic SR Ca2+ release, SR Ca2+ cycling, or Ca2+ influx being low in unstimulated hearts and thus not contributing to injury, but increasing with Iso or high Ca2+ treatment to a level that exacerbates injury, and decreasing below the injury threshold with the NO produced in female hearts. NO could act directly on SR Ca2+ release, SR Ca2+ cycling, or Ca2+ influx or may act downstream by preventing Iso or high Ca2+-induced increases in these Ca2+-handling processes from exacerbating injury. These mechanisms of NO-mediated protection are highly speculative and will be the subject of future investigation.
In summary, we have demonstrated that females are protected from the detrimental effects, namely increased susceptibility to myocardial ischemia-reperfusion injury, of Iso or high Ca2+ treatment. We also measured expression of iNOS, eNOS, and nNOS and found that expression of eNOS was higher in female than male hearts. NO production was also higher in both Iso-treated and untreated female than male hearts during ischemia, and protection in the Iso-treated female hearts was abolished by the NOS inhibitior L-NAME, consistent with a role for NOS in mediating the protection observed in female hearts.
Taken together, the results of this study indicate that cardioprotection in females may be due to a lesser susceptibility to the detrimental effects of Ca2+ overload and that this cardioprotection is likely to be via a NOS-mediated mechanism. The finding that male/female differences are only revealed in stimulated hearts may explain the lack of observation of male/female differences in ischemia-reperfusion injury in isolated heart studies, in which systemic catecholamine stimulation is absent, in comparison to clinical observations. One could further speculate, based on the findings of the present study, that under conditions of enhanced cytosolic or SR Ca2+ overload, such as occur clinically during cardioplegia, hypercalcemia, or treatment with adrenergic agonists, that females may be protected via a NOS-mediated mechanism.
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ACKNOWLEDGEMENTS |
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Charles Steenbergen thanks the National Institutes of Health for grant support. The authors thank Robert E. London for use of NMR facilities and Diane M. Magnuson for technical assistance with the protein expression studies.
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FOOTNOTES |
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Address for reprint requests and other correspondence: H. R. Cross, Dept. of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710 (E-mail: cross017{at}mc.duke.edu).
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. Section 1734 solely to indicate this fact.
April 4, 2002;10.1152/ajpheart.00790.2001
Received 5 September 2001; accepted in final form 4 April 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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female vs.
male; *female + Ca vs. male + Ca; P 
female + Iso + L-NAME
vs. male + Iso + L-NAME; P 
