© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Male and female mice overexpressing the β2-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide
aDepartment of Pathology, Box 3712, Duke University Medical Center, Durham, NC 27710, USA
bDepartment of Surgery, Duke University Medical Center, Durham, NC 27710, USA
cNIEHS, Research Triangle Park, Durham, NC 27709, USA
cross017{at}mc.duke.edu
* Corresponding author. Tel.: +1-919-541-5411; fax: +1-919-541-3385
Received 6 August 2001; accepted 26 October 2001
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Abstract |
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 Top Abstract 1. Introduction2. Methods3. Results4. DiscussionReferences |
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Objective: Cardiac overexpression of β2-adrenergic receptors (β2ARs) in male mice (MTG4) results in increased contractility and increased ischemic injury. Considering recent clinical data indicating that premenopausal women are protected from cardiovascular injury, we assessed the consequences of β2AR overexpression in females (FTG4). Since protection in females is mediated via estrogen, which activates endothelial and inducible nitric oxide synthases (eNOS and iNOS) we also examined the role of NOS in ischemia/reperfusion injury in male and female TG4 and wild-type (WT) mice. Methods: Hearts from MTG4, FTG4, MWT and FWT mice were isolated and perfused in the Langendorff mode. Hearts were pretreated with either 1 µmol/l of the nonspecific NOS inhibitor, L-NAME, or 100 nmol/l of the specific iNOS inhibitor, 1400W. Control hearts received no treatment. All hearts were subjected to 20 min ischemia and 40 min reperfusion while 31P-NMR spectra were acquired. Results: During ischemia, ATP and pH fell lower in MTG4 hearts than in FTG4 or WT hearts. Hearts from MTG4 mice exhibited increased ischemia/reperfusion injury as indicated by lower recoveries of postischemic contractile function, ATP and PCr than WT. Despite contractility being elevated in FTG4 hearts to the same level as MTG4 hearts, ischemia/reperfusion injury was not increased, as indicated by similar postischemic recoveries of contractile function, ATP and PCr in FTG4 hearts compared to WT. ATP and pH fell lower during ischemia in L-NAME-treated FTG4 hearts than in untreated FTG4 hearts, falling as low as untreated MTG4s. Recoveries of contractile function, ATP and PCr were as low in L-NAME-treated FTG4 hearts as in untreated MTG4 hearts and lower than untreated FTG4 hearts. In contrast, 1400W had no effect on FTG4 hearts. MTG4 hearts were unaffected by L-NAME or 1400W. Conclusions: β2AR overexpression increased ischemia/reperfusion injury in males but not females, thus females were protected from the detrimental effects of β2AR overexpression. Protection was abolished by treatment with L-NAME, but not 1400W, implying that protection was mediated by eNOS not iNOS.
KEYWORDS Energy metabolism; Gender; Ischemia; Nitric oxide; NMR
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1. Introduction |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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Cardiac β-adrenergic receptors (βARs) mediate the contractile response to the sympathetic transmitters, epinephrine and norepinephrine. Binding of agonist to the β2AR leads to activation of adenylate cyclase and protein kinase A (PKA). In the heart, PKA phosphorylates several proteins. Phosphorylation of the L-type Ca2+ channel by PKA leads to increased Ca2+ influx, phosphorylation of the sarcoplasmic reticulum (SR) Ca2+ ATPase inhibitor phospholamban leads to increased SR Ca2+ levels and increased SR Ca2+ release and phosphorylation of the myofibrillar protein troponin I leads to altered Ca2+ sensitivity [1]. These phosphorylation events result in increased cardiac contractility. Correspondingly, transgenic mice with a 200-fold cardiac-specific overexpression of the β2AR exhibit increased basal myocardial contractility [2]. There was no evidence of cardiac abnormalities nor increased mortality in this transgenic model [2], however, the male β2AR overexpressor mice exhibited greater myocardial energy utilization during ischemia and increased ischemia/reperfusion injury when compared to wild-type littermates [3].
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 [4] and this protection is lost after menopause [5]. Estrogen replacement therapy has been shown to decrease the occurrence and mortality of myocardial infarction [6] and increase survival from coronary artery bypass [7]. To determine whether female β2AR overexpressor mice were protected from the exacerbation of ischemia/reperfusion injury observed in the males, we subjected isolated hearts from male and female wild type and β2AR overexpressor mice to global ischemia and reperfusion. Ischemia/reperfusion injury was determined by the extent of recovery of postischemic contractile function and energy metabolites. Interestingly, female hearts were protected from the detrimental effects of β2AR overexpression, with respect to ischemia/reperfusion injury.
Further studies were performed to determine the mechanism of protection in the female β2AR overexpressor hearts. Because the detrimental effects of β2AR overexpression are likely to be mediated by calcium overload, and nitric oxide (NO) is known to affect calcium homeostasis [8–10], we studied the role of the estrogen effector, NO synthase (NOS). Expression of both inducible NOS (iNOS) and endothelial NOS (eNOS) is increased by estrogen in the myocardium [11] and a number of studies have found NO produced via iNOS, eNOS or NO donors to be cardioprotective [12–14]. By pretreating male and female β2AR overexpressor mouse hearts with either the nonspecific NOS inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME) or the specific iNOS inhibitor, N-[3-(aminomethyl)benzyl]acetamidine (1400W), and studying the ischemic response, we further aimed to determine the role of NOS in the protection from ischemia/reperfusion injury observed in female β2AR overexpressor hearts.
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2. Methods |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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2.1. Animals
The transgenic mice (TG4) exhibited a 200-fold, cardiac-specific overexpression of the β2AR; expression levels were determined as described previously [2]. Twenty-four male adult heterozygous transgenic mice of body weight 30±1 g and 26 female adult heterozygous transgenic mice of body weight 24±1 g were used. Twenty male and 12 female wild-type littermate mice of body weights 33±2 and 22±1 g, respectively, were employed as controls. All animals were adult and reproductively viable at the time of experimentation. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
2.2. Ischemia/reperfusion protocol
Hearts were isolated and perfused in the Langendorff mode as described previously [15]. Hearts were perfused for 30 min before being subjected to 20 min no-flow ischemia and 40 min reperfusion. Left-ventricular developed pressure (LVDP), ±dP/dt and heart rate were monitored via a water-filled latex balloon inserted into 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.
2.3. Nitric oxide synthase inhibitors
To inhibit both eNOS and iNOS, hearts from six of the male transgenic, six of the female transgenic, five of the male wild-type and seven of the female wild-type mice were perfused with 1 µmol/l L-NAME, a non-specific NOS inhibitor, 5 min prior to ischemia. The concentration of L-NAME used corresponded to the concentration required to inhibit eNOS and iNOS without inducing vasoconstriction [16]. To inhibit specifically iNOS, five of the male transgenic and seven of the female transgenic mice were perfused with 100 nmol/l 1400W, 5 min prior to ischemia. Specificity of 1400W was confirmed as described below.
2.4. Nuclear magnetic resonance spectroscopy
Relative changes in concentrations of phosphorus metabolites were observed during the ischemia/reperfusion protocol by acquiring consecutive 5 min 31P nuclear magnetic resonance (NMR) spectra as described previously [3]. The areas of the spectral peaks were expressed as a percentage of the peak areas of an initial, preischemic control spectrum from each heart. The ratios of the PCr/ATP peaks in the preischemic control spectra were lower in the male transgenic hearts, compared to male wild-type. The lower PCr/ATP ratio implies that preischemic PCr levels were lower in male transgenic mice. PCr values were therefore 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.
2.5. Specificity of 1400W and NO levels in male and female hearts
To confirm specificity of 1400W, 13 male wild-type mice were injected with 2.5 mg/kg i.p. monophosphoryl lipid A (MLA) to induce iNOS expression, 8 h prior to heart perfusion, 10 min ischemia and freeze-clamping [17]. Four of these hearts were perfused with 100 nmol/l 1400W, three hearts were perfused with 1 µmol/l L-NAME, three hearts were perfused with 1 µmol/l L-NAME plus 100 nmol/l 1400W, all for 5 min before ischemia; three hearts received no additional treatment. Total nitrate concentrations (NOx) were then determined using a nitrate/nitrite colorimetric assay kit (Cayman Chemical, MI, USA) and compared to NOx levels in non-MLA treated male wild-type hearts also subjected to 10 min ischemia before freeze-clamping.
To determine NOx levels in male vs. female hearts, five untreated male wild-type and five untreated female wild-type hearts were isolated, perfused and freeze-clamped. A further three male wild-type and three female wild-type untreated hearts were subjected to 10 min ischemia before freeze-clamping and determination of NOx levels as described above.
2.6. Statistics
Results are expressed as means±S.E.M. Pre- and post-infusion contractile function values were compared within each treated group by a paired t-test. Discrete variables of contractile function were analyzed by a two-way analysis of variance for sex, genotype and the interaction between the two factors or for sex, treatment and the interaction between the two factors. Where the two-way analysis revealed significant differences, pairwise comparisons were made using a Fisher's LSD test and significance was taken as p<0.05. For the energetic measurements, a two-way analysis of variance and Fisher's LSD test were performed as above, comparing the end-point value of ischemia between groups and the end-point value of reperfusion between groups. In all cases where the two-way analysis was used, the following groups were compared: (a) male transgenic, female transgenic, male wild-type, female wild-type; (b) male transgenic, female transgenic, L-NAME-treated male transgenic, L-NAME-treated female transgenic; (c) male transgenic, female transgenic, 1400W-treated male transgenic, 1400W-treated female transgenic.
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3. Results |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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3.1. β2AR density
β2AR density was the same in female transgenic hearts, at 11.2±1.5 pmol/mg protein (220-fold increase over wild-type; n=3), as in male transgenic hearts, at 9.0±0.9 pmol/mg protein (180-fold increase over wild-type; n=3).
3.2. Contractile function
3.2.1. Effects of overexpression of the β2AR
During the preischemic period, LVDP, +dP/dt and –dP/dt were higher in male transgenic than male wild-type hearts (p<0.05; Table 1) and higher in female transgenic than female wild-type hearts (p<0.05). Overexpression of the β2AR, therefore, increased basal contractility in both male and female hearts.
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Ischemic contracture, the sigmoidal increase in left-ventricular diastolic pressure during ischemia, began earlier in male transgenic than male wild-type hearts (p<0.01; Table 2). However, there was no difference in the onset of contracture in female transgenic, compared to female wild-type, hearts.
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By the end of reperfusion, recovery of contractile function was lower in male transgenic, at 8% initial LVDP, than male wild-type hearts, at 31% initial LVDP (p<0.0001); Fig. 1. However, there was no difference in functional recovery in female transgenic, at 31% initial LVDP, compared to female wild-type hearts, at 37% initial LVDP. Overexpression of the β2AR, therefore, resulted in increased susceptibility to ischemia/reperfusion injury in male, but not female, hearts.
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: significant difference from FTG4 (p<0.05).3.2.2. Effects of pretreatment with L-NAME
To inhibit both eNOS and iNOS, male and female wild-type and male and female transgenic hearts were perfused with 1 µmol/l L-NAME prior to ischemia. During normoxic perfusion, L-NAME had no effect on heart rate LVDP, +dP/dt or –dP/dt in male or female transgenic hearts (Table 1). Likewise, L-NAME had no effect on contractility in male and female wild-type hearts (data not shown). L-NAME also had no significant effect on coronary flow, 1.9±0.1 ml/min pre-infusion and 1.9±0.2 ml/min post-infusion in male transgenic hearts and 1.7±0.1 ml/min pre-infusion and 1.5±0.1 ml/min post-infusion in female transgenic hearts.
There was no effect of L-NAME pretreatment on the timing of contracture in male transgenic hearts (Table 2). However, ischemic contracture was earlier in L-NAME-treated female transgenic hearts than in untreated female transgenic hearts (p<0.05). There was no significant difference in timing of contracture between L-NAME-treated female transgenic and untreated male transgenic hearts.
There was no difference in post-ischemic functional recovery between L-NAME-treated male transgenic hearts, at 10% initial LVDP, and untreated male transgenic hearts, at 8% initial LVDP (Fig. 1). However, by the end of reperfusion, recovery of contractile function was lower in L-NAME-treated female transgenic hearts, at 11% initial LVDP than in untreated female transgenic hearts, at 31% initial LVDP (p<0.001) and as low as in male transgenic hearts. There was no effect of L-NAME pretreatment on functional recovery in male, at 35±6% initial LVDP, and female, at 31±4% initial LVDP, wild-type hearts. In summary, the protection from ischemia/reperfusion injury in female, compared to male, β2AR overexpressors was abolished by pretreatment with L-NAME. These results imply that the protection in female β2AR overexpressors is mediated via eNOS or iNOS.
3.2.3. Effects of pretreatment with 1400W
To confirm specificity of the iNOS inhibitor, 1400W, male wild-type mice were injected with MLA to induce iNOS expression and NOx was assayed in 1400W-treated, L-NAME-treated, 1400W+L-NAME-treated hearts and untreated hearts from these mice. NOx was 26±3 pmol/mg protein after 10 min ischemia in untreated hearts. NOx increased with MLA treatment, being 63±13 pmol/mg protein in MLA-only hearts, and was significantly lower in MLA+1400W-treated hearts, at 28±5 pmol/mg protein (p
0.05). NOx in MLA+1400W-treated hearts did not differ from that of untreated hearts, indicating that a 5 min perfusion with 100 nmol/l 1400W is sufficient to inhibit iNOS. It is possible that the full inhibition of MLA-induced NOx production by 1400W was not due to complete inhibition of iNOS, but to sub-maximal inhibition of iNOS plus partial inhibition of eNOS. However, based on the Ki values of Garvey et al. [18], 1400W is 7000 times more specific for iNOS than eNOS, therefore it is unlikely that inhibition of eNOS plays a significant role in our observations. The residual NOx in the MLA+1400W-treated hearts was presumably due to activity of other NOS isoforms. This was confirmed by treatment of hearts with MLA+L-NAME, NOx being negligible, at 2±5 pmol/mg protein, in MLA+L-NAME hearts. NOx was also negligible, at 3±8 pmol/mg protein, in MLA+L-NAME+1400W-treated hearts.
When male and female transgenic hearts were perfused with 100 nmol/l 1400W, 5 min prior to ischemia, 1400W had no effect on heart rate, LVDP, +dP/dt or –dP/dt in either group during normoxic perfusion (Table 1). There was no effect of pretreatment with 1400W on timing of ischemic contracture or postischemic recovery of contractile function in male or female transgenic hearts (Table 2; Fig. 1). Therefore, inhibition of iNOS alone had no effect on ischemia/reperfusion injury in female β2AR overexpressors, in contrast to inhibition of both iNOS and eNOS with L-NAME, implying that the protection in female β2AR overexpressors is mediated via eNOS.
3.3. Phosphate metabolite levels and intracellular pH
3.3.1. Effects of overexpression of the β2AR
During the preischemic period, the PCr/ATP ratio was lower in male transgenic, at 1.15±0.09, than in male wild-type hearts, at 1.50±0.05 (p<0.01). However, despite the increase in contractility in the female transgenic hearts, there was no difference in PCr/ATP ratio between female transgenic, at 1.40±0.11, and female wild-type hearts, at 1.39±0.09.
During ischemia, ATP levels fell lower in male transgenic hearts, reaching 16% initial ATP, than in either male wild-type, at 40% initial ATP (p<0.0001), or female transgenic hearts, at 32% initial ATP (p<0.05; Fig. 2a). There was no difference in ATP at the end of ischemia between female wild-type, at 36% initial ATP, and female transgenic hearts. During reperfusion, ATP levels remained lower in male transgenic hearts, reaching 29% initial ATP by the end of reperfusion, compared to 47% initial ATP in male wild-type (p<0.01) and 54% initial ATP in female transgenic hearts (p<0.01). There was no difference in end-reperfusion ATP levels between female wild-type, at 50% initial ATP, and female transgenic hearts.
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At the end of ischemia, there was no difference in PCr levels between any groups of hearts (Fig. 2b). At the end of reperfusion, PCr was lower in male transgenic hearts, at 54% initial ATP, than in either male wild-type, at 89% initial ATP (p<0.001) or female transgenic hearts, at 89% initial ATP (p<0.01). There was no difference in end-reperfusion PCr levels between female wild-type, at 95% initial ATP, and female transgenic hearts.
At the end of ischemia, pH was lower in male transgenic hearts, at pH 5.52, than in either male wild-type, at pH 5.85 (p<0.0001), or female transgenic hearts, at pH 5.87 (p<0.0001; Fig. 3). There was no difference in end-ischemic intracellular pH between female wild-type, at pH 5.85, and female transgenic hearts. During reperfusion, there were no differences in pH between any groups of hearts.
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In summary, male β2AR overexpressor hearts had a lower PCr/ATP ratio during basal perfusion than wild-type hearts. During ischemia, these hearts exhibited a greater loss of ATP and a lower intracellular pH than wild-type hearts. During reperfusion, recoveries of ATP and PCr were lower in male β2AR overexpressor hearts than in wild-type hearts, therefore correlating with the lower recovery of contractile function observed and indicating greater injury. In contrast, despite contractility being elevated in the female β2AR overexpressor hearts to the same extent as in males, PCr/ATP ratios during basal perfusion were the same as in wild-type hearts and there were no differences in energy metabolites or intracellular pH during ischemia and reperfusion compared with wild-type hearts. Overexpression of the β2AR, therefore, resulted in increased energy demand and increased susceptibility to ischemia/reperfusion injury in male, but not female, hearts.
3.3.2. Effects of pretreatment with L-NAME
There was no effect of L-NAME treatment on energetics or intracellular pH in either male wild-type or female wild-type hearts during ischemia or reperfusion (data not shown).
During ischemia, ATP levels fell lower in L-NAME-treated female transgenic hearts, reaching 18% initial ATP, than in untreated female transgenic hearts, at 32% initial ATP (Fig. 4a; p=0.05). ATP fell as low in L-NAME-treated female transgenic hearts during ischemia as in untreated male transgenic hearts, at 16% initial ATP. During reperfusion, ATP remained lower in L-NAME-treated female transgenic hearts, reaching 32% initial ATP, compared to 54% initial ATP in untreated female transgenic hearts (p<0.05). There was no difference in end-reperfusion ATP levels between L-NAME-treated female transgenic hearts and untreated male transgenic hearts, at 29% initial ATP. At no time during the protocol were there differences in ATP levels between L-NAME-treated male transgenic and untreated male transgenic hearts (Fig. 4a).
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At the end of ischemia, there was no difference in PCr levels between any groups of hearts (Fig. 4b). At the end of reperfusion, PCr was lower in L-NAME-treated female transgenic hearts, reaching 52% initial ATP, compared to 89% initial ATP in untreated female transgenic hearts (p<0.05) and as low as in untreated male transgenic hearts, at 54% initial ATP. At no time during the protocol were there differences in PCr levels between L-NAME-treated male transgenic and untreated male transgenic hearts (Fig. 4b).
At the end of ischemia, intracellular pH was lower in L-NAME-treated female transgenic hearts, at pH 5.64, than in untreated female transgenic hearts, at pH 5.87 (p<0.05; Fig. 5). There was no difference in end-ischemic pH between L-NAME-treated female transgenic and untreated male transgenic hearts, at pH 5.52. During ischemia there was no significant difference in pH between L-NAME-treated male transgenic and untreated male transgenic hearts (Fig. 5). During reperfusion, there were no significant differences in pH between any groups of hearts.
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To summarize, L-NAME-treated female β2AR overexpressor hearts exhibited a greater loss of ATP during ischemia than untreated female β2AR overexpressor hearts and were not different from male β2AR overexpressor hearts. During reperfusion, recoveries of ATP and PCr were also lower in L-NAME-treated female β2AR overexpressor hearts than in untreated female β2AR overexpressor hearts, therefore correlating with the lower recovery of contractile function observed and indicating greater injury. There was no effect of L-NAME pretreatment, with respect to ischemic energetics or pH, on male β2AR overexpressor hearts, consistent with the lack of functional effects. L-NAME pretreatment, therefore, abolished the protection of female hearts from the increased energy demand and ischemia/reperfusion injury resulting from β2AR overexpression, implying that protection was mediated by iNOS or eNOS.
3.3.3. Effects of pretreatment with 1400W
Consistent with the lack of functional effects, there was no effect of pretreatment with 1400W on intracellular pH or levels of ATP or PCr during ischemia or reperfusion in male transgenic or female transgenic hearts (Figs. 6a, b and 7
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In summary, inhibition of iNOS alone had no effect on myocardial energetics during ischemia and reperfusion in female β2AR overexpressors, in contrast to the effects of inhibition of both iNOS and eNOS with L-NAME. These results are consistent with the lack of an effect of 1400W on post-ischemic functional recovery in female β2AR overexpressors and supports the hypothesis that protection in female β2AR overexpressors is mediated via eNOS.
3.4. NO levels in male and female hearts
Since estrogen increases expression of eNOS and iNOS, NO levels would be expected to be elevated in female hearts. We found that total nitrate levels were slightly but not significantly greater under basal conditions in female wild-type, at 53±21 pmol/mg protein, than male wild-type hearts, at 32±4 pmol/mg protein. After ischemia, total nitrate levels were significantly greater in female wild-type hearts, at 101±26 pmol/mg protein than in male wild-type hearts, at 28±4 pmol/mg protein (p0.05). NO production was therefore slightly greater under basal conditions and significantly greater during ischemia in female than male hearts, consistent with reports that expression of eNOS and iNOS is increased by estrogen.
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4. Discussion |
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TopAbstract1. Introduction2. Methods3. Results4. DiscussionReferences |
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4.1. Effects of β2AR overexpression on contractility and ischemia/reperfusion injury in male and female hearts
In the present study, perfused hearts from both male and female β2AR overexpressor mice exhibited increased peak contraction (LVDP), rate of contraction (+dP/dt) and rate of relaxation (–dP/dt), compared to wild-type hearts. During ischemia, ATP levels fell lower in the male β2AR overexpressor hearts than male wild-type, reflecting greater ischemic energy utilization. The faster rate of ATP utilization in the male β2AR overexpressor hearts was consistent with the earlier onset of contracture observed and, as H+ are produced by ATP degradation, the faster rate of ATP utilization was also consistent with the lower pH observed in these hearts. However, ischemic ATP depletion, timing of contracture onset and ischemic intracellular pH in female β2AR overexpressor hearts were not different from that in male or female wild-type. Ischemia/reperfusion injury was increased in hearts from male β2AR overexpressor mice, as indicated by lower postischemic recoveries of contractile function, ATP and PCr than wild-type hearts. Despite contractility being elevated in female β2AR overexpressor hearts to the same level as males, ischemia/reperfusion injury was not increased, as indicated by higher postischemic recoveries of contractile function, ATP and PCr in female vs. male β2AR overexpressors. Notably, recoveries of contractile function, ATP and PCr in female β2AR overexpressors were no different from male or female wild-type. In summary, in contrast to the observation in males, β2AR overexpression in female mouse hearts did not result in greater ischemic energy utilization and increased ischemia/reperfusion injury.
4.2. Role of NOS in protection from ischemia/reperfusion injury in female β2AR overexpressor mouse hearts
Adrenergic stimulation leads to phosphorylation of the L-type Ca2+ channel and the SR Ca2+ ATPase inhibitor phospholamban, resulting not only in increased contractility, but also in increased cytosolic and SR Ca2+ levels, respectively [1]. Since Ca2+ overload is a major mediator of ischemia/reperfusion injury [19–21], these increases in cytosolic and SR Ca2+ may be the mechanism by which overexpression of the β2AR leads to increased ischemia/reperfusion injury. Interestingly, under our experimental conditions, we found no male/female difference in susceptibility to ischemia/reperfusion injury in wild-type mouse hearts. However, male/female differences in susceptibility to ischemia/reperfusion injury have been observed under our experimental conditions in mice which overexpress the Na+/Ca2+ exchanger [15], another transgenic model which exhibits increased cytosolic and SR Ca2+ [22]. It appears, therefore, that females may be protected from the consequences of cytosolic and SR Ca2+ overload. Clinical studies indicate that protection in females is mediated via estrogen [4–7]. Consistent with a role for estrogen, we have shown previously that protection of female Na+/Ca2+ exchange overexpressor hearts is abolished by ovariectomy [15] and chronic treatment of ovariectomized rats with physiological levels of estrogen has been shown to result in protection from ischemia-reperfusion injury [23]. Estrogen increases expression of both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in the myocardium [11]. Consistent with an estrogen-mediated increase in NOS expression, we found that NO levels were higher in female than male hearts. Since NO is known to affect Ca2+ homeostasis, modulating sarcolemmal Ca2+ channels [10] and SR Ca2+ transport proteins [8,9], and since, although contradictory findings exist [16,24,25], a number of studies have found NO to be cardioprotective [12–14], we studied the role of NOS in the protection of female β2AR overexpressor mouse hearts.
Male and female β2AR overexpressor mouse hearts were pretreated with 1 µmol/l of the nonspecific NOS inhibitor, L-NAME, prior to ischemia. The concentration of L-NAME used corresponded to the concentration required to inhibit eNOS and iNOS without inducing vasoconstriction [16]. Correspondingly, we observed no effect of 1 µmol/l L-NAME on contractile function and coronary flow in any hearts during normoxic perfusion. During ischemia, ATP levels and intracellular pH fell lower and the onset of contracture was earlier in the L-NAME-treated female β2AR overexpressor hearts than in the untreated female β2AR overexpressor hearts, reflecting greater ischemic energy utilization. Notably, ischemic ATP and pH and postischemic recoveries of contractile function and energy metabolites were as low in the L-NAME-treated female β2AR overexpressor hearts as in untreated male β2AR overexpressor hearts. The post-ischemic recoveries of both contractile function and ATP were less in the L-NAME-treated female β2AR overexpressor hearts compared to untreated female hearts, indicating greater injury. Therefore, the protection from ischemia/reperfusion injury observed in the female β2AR overexpressors, compared to male β2AR overexpressors, was abolished by pretreatment with L-NAME. These results imply that the protection in female β2AR overexpressors is mediated via eNOS or iNOS.
To determine the relative roles of eNOS and iNOS in the observed female cardioprotection, we studied the ischemic response of β2AR overexpressor hearts pretreated with 100 nmol/l of the potent and specific iNOS inhibitor, 1400W. The Ki of 1400W is 7 nmol/l for iNOS, 2 µmol/l for nNOS and 50 µmol/l for eNOS [18], therefore 100 nmol/l 1400W should be sufficient to inhibit iNOS without affecting other NOS isoforms, as shown by Cavicchi and Whittle [26]. Inhibition of iNOS by 100 nmol/l 1400W was also confirmed independently in the present study. There was no effect of pretreatment with 1400W on timing of ischemic contracture, ischemic ATP levels and intracellular pH or post-ischemic recovery of contractile function and energy metabolites in female β2AR overexpressor hearts. Therefore, inhibition of iNOS alone had no effect on ischemia/reperfusion injury in female β2AR overexpressors, in contrast to inhibition of both iNOS and eNOS with L-NAME, implying that the protection in female β2AR overexpressors is mediated via eNOS.
From our findings, we can speculate on a model of female cardioprotection. When cytosolic and SR Ca2+ overload are increased, such as by β2AR overexpression, secondary Ca2+-mediated mechanisms of injury may be invoked during ischemia/reperfusion resulting in injury being exacerbated in male transgenic hearts. Female hearts, however, exhibit increased NO production which appears to oppose the Ca2+-mediated injury induced by β2AR overexpression, leading to cardioprotection in the female transgenics. In the wild-type hearts, the secondary mechanisms of injury may not be active thus the increased NO production in female wild-type hearts has no protective role and, as observed in the present study, susceptibility to ischemia/reperfusion injury remains equivalent in male and female wild-type hearts. Interestingly, our observation that NO has no protective role under basal conditions, but does have a role under stimulated conditions is similar to reports that NO does not modulate contractility under basal conditions but does modulate contractility in stimulated hearts [27].
The findings of this study have clinical relevance. Overexpression of the β2AR has been proposed as a contractile therapy for heart failure [2], therefore our observation that basal contractility can be increased by expression of the β2AR in females without a concomitant increase in ischemia/reperfusion injury has important implications for sex-specific therapies for heart failure. In addition, our finding that females are protected from the detrimental effects of adrenergic stimulation and Ca2+ overload suggest that under conditions of adrenergic stimulation or enhanced cytosolic or SR Ca2+ overload, such as occur clinically during cardioplegia, hypercalcemia or treatment with adrenergic agonists, females may be protected from cardiovascular injury via an eNOS-mediated mechanism.
In summary, by comparing the ischemic response of hearts from male and female β2AR overexpressor mice, we demonstrated that females were protected from the detrimental effects of β2AR overexpression observed in males, namely a greater ischemic energy utilization and increased ischemia/reperfusion injury. By pretreating male and female β2AR overexpressor mouse hearts with the nonspecific NOS inhibitor, L-NAME, and studying the ischemic response, we demonstrated that the protection in female β2AR overexpressors was mediated via either eNOS or iNOS. By also studying the ischemic response of β2AR overexpressor hearts pretreated with the specific iNOS inhibitor, 1400W, we found evidence that it was eNOS, not iNOS, which mediated protection in the female β2AR overexpressors. These results highlight the role of eNOS in female cardioprotection and may be relevant to the protection from cardiovascular injury observed clinically in premenopausal females.
Time for primary review 29 days.
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Acknowledgements |
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W.J.K. and C.S. thank the NIH for grant support. The authors thank Sandy J. Duncan and Rachel McAdam for mouse phenotyping, Robert E. London for use of NMR facilities and Joseph Haseman for assistance with statistical analyses.
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