Mechanisms involved in male-female differences in cardioprotection

尽管人们认识到心脏病的性别差异，但保护绝经前女性的机制尚未完全阐明。女性的心脏病发病率在绝经后增加，这表明雌激素在绝经前的心脏保护中发挥了作用。然而，临床试验发现激素替代疗法对心血管没有好处，这表明需要更好地了解机制。因此，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存在差异的机制。经典的雌激素诱导转录调控是由核雌激素受体(ER)ER-α和ER-β介导的。然而，ER-α和ER-β也定位在质膜上，并可通过激酶信号引起作用，导致翻译后修饰的S亚硝化(SNO)增加，与心脏保护相关。我们假设雌激素相关的心脏保护至少部分是由导致SNO增加的非核ER信号介导的。我们使用雌激素-树突状分子结合物(EDC)进行了测试，它已经在小鼠身上被证明是一种非核选择性ER调节器(SERM)，不会促进子宫癌或乳腺癌的生长。我们用EDC、树突状分子对照、17-β-雌二醇或赋形剂处理去卵巢的C57BL/6J小鼠两周。采用Langendorff模型灌流离体心，缺血30min，再灌流90min。正如先前报道的那样，与赋形剂治疗的心脏相比，雌激素治疗的心脏缩小了梗死面积(40.4-2.5%比62.9%)，提高了功能恢复(44.7-4.0%比27.0-2.7%)。与雌二醇相似，与树枝状大分子对照组相比，EDC缩小了梗塞面积(40.9±3.6%比63.8±4.7%)，改善了功能恢复(48.8±3.0%比28.6±2.5%)。给予EDC 5天的小鼠也有类似的保护作用(42.1%比63.8%梗死灶和38.9%比22.8%功能恢复比树突状分子)。双向差异凝胶电泳法显示，与树枝状大分子相比，EDC处理5天和2周的心脏SNO蛋白表达增加。许多已鉴定的蛋白质在其他心脏保护模型中增加了SNO。这些结果表明，EDC在小鼠缺血再灌注损伤中具有与雌二醇相同的心脏保护作用，其机制可能与增加SNO蛋白有关。他们进一步表明，非核ER行为在雌激素提供的保护中发挥着重要作用。因此，EDC和非核ER信号通路可用于临床，在不促进癌细胞生长的情况下提供心血管益处。 人们认为基因表达的改变有助于女性的心脏保护。MicroRNAs(MiRNAs)是一种小的、非编码的RNA，通过与mRNA结合来抑制基因的表达。我们假设miRNA的性别特异性表达模式有助于心肌保护蛋白表达的差异。用Affymetrix基因芯片miRNA阵列使用来自雄性或雌性小鼠心脏的miRNA来测定miRNA水平。5种miRNAs的表达在男性和女性之间存在显著差异。由于miR-222在eNOS表达的间接调控中的作用，我们选择进一步研究miR-222。定量逆转录聚合酶链式反应证实女性miR-222基因表达降低，这与enos基因表达增加相对应。转录因子Ets-1被TargetScan鉴定为miR-222的一个可能的直接靶标，并且在雌性中证实了Ets-1mRNA和蛋白的增加。在用miR-222抑制剂或模拟物处理的HEK293细胞中，通过转染含有可能的Ets-1 3UTR的荧光素酶报告基因，建立了miR-222对Ets-1表达的抑制作用。此外，我们证实了miR-222抑制剂或模拟物对成年大鼠心肌细胞eNOS水平的调节。因此，miRNA表达的固有性别差异，以及随后心脏保护蛋白表达的变化，可能有助于女性心脏保护。

Although gender disparities in cardiac disease are recognized, the mechanisms through which pre-menopausal females are protected have not been fully elucidated. Cardiac disease incidence in females increases post-menopause, suggesting a role for estrogen in pre-menopausal cardioprotection. However, clinical trials found no beneficial cardiovascular outcomes from hormone replacement therapy, indicating a better mechanistic understanding is needed. Thus the goal of this study is to understand the mechanism responsible for the male-female differences in ischemia-reperfusion injury and cardioprotection. Classical estrogen-induced transcription regulation is mediated by nuclear estrogen receptors (ER) ER-alpha and ER-beta. However, ER-alpha and ER-beta are also localized to the plasma membrane and can elicit effects through kinase signaling, leading to an increase in S-nitrosylation (SNO), a post-translational modification associated with cardioprotection. We hypothesized that estrogen-related cardioprotection is at least partially mediated by non-nuclear ER signaling leading to an increase in SNO. We tested this using an estrogen-dendrimer conjugate (EDC), which has been demonstrated in mice to be a non-nuclear selective ER modulator (SERM) that does not promote uterine or breast cancer growth. We treated ovariectomized C57BL/6J mice with EDC, dendrimer control, 17-beta-estradiol, or vehicle for two weeks. Isolated hearts were perfused in the Langendorff model and subjected to 30 minutes ischemia and 90 minutes reperfusion. As previously reported, estradiol-treated hearts had decreased infarct size (40.4 2.5% vs. 62.9 5.8%) and increased functional recovery (44.7 4.0% vs. 27.0 2.7%) compared to vehicle-treated hearts. Similar to estradiol, EDC decreased infarct size (40.9 3.6% vs. 63.8 4.7% total ventricle) and improved functional recovery (48.8 3.0% vs. 28.6 2.5%) compared to dendrimer control. Similar protection was seen when mice were treated with EDC for five days (42.1 4.7% vs. 63.8 6.4% infarct and 38.9 2.9% vs. 22.8 2.4% functional recovery for EDC vs. dendrimer). 2-D Difference Gel Electrophoresis showed an increase in protein SNO from hearts treated with EDC for 5 days and 2 weeks compared to dendrimer treatment. Many of the identified proteins have increased SNO in other models of cardioprotection. These results indicate that EDC is as effective as estradiol in providing cardioprotection during ischemia-reperfusion injury in mice, possibly due to increased protein SNO. They further suggest that non-nuclear ER actions play a major role in the protection afforded by estrogen. Thus, EDC and non-nuclear ER signaling pathways could be utilized clinically to provide cardiovascular benefit without promoting cancer cell growth. It is thought that altered gene expression contributes to female cardioprotection. MicroRNAs (miRNAs) are small, non-coding RNA that inhibit gene expression through binding to mRNA. We hypothesize that sex-specific expression patterns of miRNA contribute to differences in expression of cardioprotective proteins. miRNA levels were determined with the Affymetrix GeneChip miRNA array using miRNA from the hearts of male or female mice. Expression of five miRNAs was significantly different between males and females. Because of its role in indirect regulation of eNOS expression, we chose to further study miR-222. Decreased miR-222 in females was confirmed by qRT-PCR and this corresponded with increased eNOS mRNA. The transcription factor, ets-1, was identified as a putative direct target of miR-222 using TargetScan, and increased ets-1 mRNA and protein were confirmed in females. Inhibition of ets-1 expression by miR-222 was established by transfecting a luciferase reporter containing the putative ets-1 3UTR in HEK293 cells treated with either a miR-222 inhibitor or mimic. Furthermore, we confirmed modulation of eNOS levels by a miR-222 inhibitor or mimic in adult rat cardiomyocytes. Thus, inherent sex-specific differences in miRNA expression, and subsequent changes in the expression of cardioprotective proteins, may contribute to female cardioprotection.