Mechanisms involved in male-female differences in cardioprotection

绝经前妇女患心血管疾病的风险降低，而绝经后心血管疾病增加。动物模型研究也表明，雌性小鼠在缺血再灌注（I/R）后损伤减轻。然而，一项名为“妇女健康倡议”的大型临床试验发现，接受激素替代疗法的女性心血管事件增加。综上所述，这些数据表明我们需要更好地了解在动物研究中观察到的保护机制。在一些研究中，特别是在大鼠中，雌性表现出较少的I/R损伤；然而，在许多动物研究中，没有观察到I/R损伤的性别差异。在缺血前钙升高或收缩力增加的情况下，有报道称女性的I/R损伤比男性少。此外，雌激素管理已被证明可以减少I/R损伤。在收缩性增强的条件下观察到的保护作用已被证明涉及一氧化氮信号的增加，导致l型钙通道的s -亚硝基化，从而减少缺血和早期再灌注期间的钙负荷，从而减少I/R损伤。雌激素结合核雌激素受体导致一些心脏保护基因如一氧化氮合酶和热休克蛋白的表达改变。雌激素也会改变一些参与代谢的基因，如脂蛋白脂肪酶、前列腺素D2合成酶和过氧化物酶体增殖物激活受体γ辅助激活因子1 α (pgc -1- α)。这些基因表达变化的影响可能取决于其他激素刺激和基因表达以及生理刺激的背景。此外，添加雌激素具有急性非基因组反应，涉及磷脂酰肌醇3-激酶（PI 3-激酶）途径的激活，该途径已被证明具有保护作用，至少在短时间激活时是如此。本研究的目的是了解男女在缺血再灌注损伤和心脏保护方面差异的机制。为了实现这些目标，我们研究了Langendorff灌注心脏模型的缺血再灌注和心脏保护。我们使用核磁共振和荧光测量跟踪高能磷酸盐和离子的变化。我们还使用标准方法测量收缩力和梗死面积。我们还分离了心脏提取物和线粒体，并将缺血再灌注损伤和心脏保护的变化与蛋白质水平、蛋白质定位和蛋白质翻译后修饰的变化联系起来。

Pre-menopausal women have reduced risk for cardiovascular disease, and cardiovascular disease rises after menopause. Studies in animal models have also suggested that females have reduced injury following ischemia and reperfusion (I/R). However, a large clinical trial, the Women's Health Initiative, found an increase in cardiovascular incidents in women on hormone replacement therapy. Taken together, these data suggest that we need a better understanding regarding the mechanisms for the protection observed in the animal studies. In some studies, particularly in the rat, females show less I/R injury; however, in many animal studies no gender difference in I/R injury is observed. Under conditions where calcium is elevated or contractility is increased just prior to ischemia, females have been reported to have less I/R injury than males. Also, estrogen administration has been shown to reduce I/R injury. The protection observed under conditions of increased contractility has been shown to involve an increase in nitric oxide signaling leading to S-nitrosylation of the L-type calcium channel, which reduces calcium loading during ischemia and early reperfusion thereby reducing I/R injury. Estrogen binding to nuclear estrogen receptors results in altered expression of a number of cardioprotective genes such as nitric oxide synthase and heat shock proteins. Estrogen also alters a number of genes involved in metabolism such as lipoprotein lipase, prostaglandin D2 synthase, and peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1-alpha). The effects of these alterations in gene expression may depend on the context of other hormonal stimuli and gene expression as well as physiological stimuli. Furthermore, addition of estrogen has acute non-genomic responses that involve activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway, which has been shown to be protective, at least when activated for short durations. The goals of this study are to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. To accomplish these goals we study ischemia reperfusion and cardioprotection in a Langendorff perfused heart model. We follow changes in high energy phosphates and ions using nuclear magnetic resonance and fluorescent measurements. We also measure contractility and infarct size using standard methods. We also isolated heart extracts and mitochondria and correlate changes in ischemia-reperfusion injury and cardioprotection with changes in protein levels, protein location and protein post translational modifications. We were interested in determining whether treatment with an estrogen receptor-beta (ER-beta)-selective agonist (2,3-bis(4-hydroxyphenyl)-propionitrile, DPN) can provide cardioprotection in female mice lacking endogenous estrogen. To study the effect of ER-beta stimulation in ischemia-reperfusion injury, we treated ovariectomized (ovx) female mice with 0.1 mg/kg/day of 17beta-estradiol, 0.8 mg/kg/day of DPN, or vehicle for 2 weeks. Isolated hearts were Langendorff perfused for 25 min prior to a 1-min treatment with isoproterenol, followed by 20 min of normothermic global ischemia and 40 min of reperfusion. Left ventricular developed pressure (LVDP) and heart rate were measured. Recovery of function at the end of 40 min of reperfusion was expressed as a percentage of pre-ischemic rate pressure product (RPP=LVDP x heart rate). Hearts from ovx female mice had a significantly lower recovery of LVDP than the hearts from intact female mice (12.4+/-1.6% vs. 19.6+/-1.6%, p<0.05, respectively). Furthermore, hearts from ovx female mice treated with DPN exhibited significantly better functional recovery than hearts from either vehicle-treated ovx female mice (20.1+/-2.2% vs. 12.4+/-1.6%, p<0.05, respectively) or wild type male mice (20.1+/-2.2% vs. 6.4+/-0.6%, p<0.05, respectively). DPN did not increase uterine weight in ovx females compared to vehicle treatment. Gene profiling showed that treatment with DPN resulted in upregulation of a number of protective genes such as heat shock protein 70, the antiapoptotic protein, growth arrest and DNA damage 45 beta, and cyclooxygenase 2. We also tested the hypothesis that estrogen protects by estrogen-receptor- (ER-) activation which leads to S-nitrosation of key cardioprotective proteins. To test this hypothesis, we treated bilaterally ovariectomized female mice with an ER- selective agonist, 2,2-bis(4-hydroxyphenyl)-proprionitile (DPN) (0.8 mg/kg/day), 17-estradiol (E2) (0.1 mg/kg/day) or vehicle for 2 weeks. Isolated hearts were Langendorff perfused for 20 minutes prior to 1 minute of isoproterenol treatment, followed by 20 minutes of global ischemia, and 120 minutes of reperfusion. Compared with vehicle, DPN and 17-estradiol treated hearts had significantly better post-ischemic recovery of left ventricular function as well as decreased infarct size. Using DyLight-maleimide fluors and a modified biotin switch method, we employed a 2D DyLight fluorescence difference gel electrophoresis (DIGE) proteomic method to quantify differences in protein S-nitrosation between our three treatment groups. We identified several cardiac proteins with a significant increase in S-nitrosation in the DPN and 17-estradiol groups, including F1-ATPase 1 subunit, malate dehydrogenase, aconitase, heat shock protein 60, cytochrome c oxidase subunit 5A and creatine kinase. In summary, chronic treatment with DPN or 17-estradiol increases S-nitrosation of many identical proteins in mouse hearts, consistent with our hypothesis that chronic estrogen exposure protects largely via ER- activation. We propose that S-nitrosation alters the activity of cardiac proteins such as mitochondrial F1-ATPase leading to protection during ischemia-reperfusion. Although estrogen has effects on the heart, little is known regarding which genes in the heart are directly responsive to estrogen. We have shown previously that lipoprotein lipase (LPL) expression was increased in female hearts compared with male hearts. To test whether LPL gene expression in heart is regulated by estrogen, we perfused mouse hearts from ovariectomized females with 100 nM 17beta-estradiol or vehicle for 2 h, after which hearts were frozen, and RNA was isolated. The SYBR green real-time PCR method was used to detect LPL gene expression. We found that addition of 17beta-estradiol to hearts from ovariectomized females resulted in a significant increase in LPL mRNA. This estrogen effect on LPL gene expression in mouse heart can be blocked by the estrogen receptor (ER) antagonist ICI 182,780 or by progesterone. We also identified a potential estrogen receptor element (ERE) enhancer sequence located in the first intron of the mouse LPL gene. The potential ERE sequence was linked to a TATA-luciferase (LUC) reporter plasmid in HeLa cells. Both ERalpha and ERbeta stimulated strong activity on the heterologous promoter reporter in Hela cells upon estrogen addition. Both ERalpha and ERbeta activities on the LPL ERE reporter were abrogated by the ER antagonist ICI 182,780. Progesterone also dose dependently inhibited the estrogen-mediated increase in LPL ERE reporter activity. These results show that heart LPL is an estrogen-responsive gene exhibiting an intronic regulatory sequence.