Mechanisms involved in male-female differences in cardioprotection

虽然心脏病的性别差异是公认的，通过保护绝经前女性的机制尚未完全阐明。绝经后女性心脏病发病率增加，提示雌激素在绝经前心脏保护中的作用。然而，临床试验发现激素替代疗法没有有益的心血管结局，这表明需要更好的机制理解。因此，本研究的目的是了解在缺血再灌注损伤和心脏保护方面男女差异的机制。 经典的雌激素诱导的转录调节由核雌激素受体（ER）ER-α和ER-β介导。然而，ER-α和ER-β也定位于质膜，并可通过激酶信号传导引起效应，导致S-亚硝基化（SNO）增加，这是一种与心脏保护相关的翻译后修饰。我们假设雌激素相关的心脏保护作用至少部分是由非核ER信号传导介导的，导致SNO增加。我们使用雌激素-树枝状聚合物缀合物（EDC）测试了这一点，该缀合物已在小鼠中被证明是不促进子宫癌或乳腺癌生长的非核选择性ER调节剂（SERM）。我们用EDC、树枝状聚合物对照、17-β-雌二醇或载体处理卵巢切除的C57 BL/6 J小鼠两周。在Langendorff模型中灌注离体心脏，并进行30分钟缺血和90分钟再灌注。如前所述，雌二醇治疗的心脏梗死面积减少（40.4 - 2.5%对62.9 - 5.8%）和增加功能恢复（44.7 - 4.0%对27.0 - 2.7%）相比，车辆治疗的心脏。与雌二醇相似，EDC与树枝状聚合物对照组相比，可减少梗死面积（40.9 ± 3.6% vs. 63.8 ± 4.7%）并改善功能恢复（48.8 ± 3.0% vs. 28.6 ± 2.5%）。当用EDC处理小鼠5天时，观察到类似的保护作用（对于EDC与树枝状聚合物，42.1 ± 4.7%对63.8 ± 6.4%的梗塞和38.9 ± 2.9%对22.8 ± 2.4%的功能恢复）。2-D差异凝胶电泳显示，与树枝状聚合物处理相比，来自用EDC处理5天和2周的心脏的蛋白质SNO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是有效的雌二醇在提供心肌保护在缺血再灌注损伤小鼠，可能是由于增加蛋白SNO。他们进一步表明，非核ER作用在雌激素提供的保护中起着重要作用。因此，EDC和非核ER信号传导途径可以在临床上用于提供心血管益处而不促进癌细胞生长。 扩张型特发性心肌病（DCM）是最常见的心肌病类型之一。已经提出心力衰竭中氧化应激的增加导致一氧化氮信号传导的减少，从而导致亚硝基氧化还原信号传导受损。为了验证这一假设，我们研究了发生蛋白质S-亚硝基化（SNO）和蛋白质氧化的活检组织从复苏的DCM和非失败的供体男性和女性的人类心脏。采用改良的艾德生物素转换法（DyLight-maleimide sulfhydryl-reactive uors）鉴定SNO蛋白，采用树脂辅助捕获Ox-RAC检测蛋白氧化。我们发现，与健康供体相比，DCM人类活检组织中的氧化应激水平升高。DyLight-马来酰亚胺凝胶电泳显示，与基线时的男性相比，女性的蛋白质SNO增加。有趣的是，许多在女性中显示SNO增加的蛋白质是线粒体蛋白质。 由于SNO在基线时存在性别差异，我们检查了心力衰竭中SNO的变化作为性别的函数，并发现这种反应存在性别差异。

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. Dilated idiopathic cardiomyopathy (DCM) is one of the most common types of cardiomyopathy. It has been proposed that an increase in oxidative stress in heart failure leads to a decrease in nitric oxide signaling, leading to impaired nitroso-redox signaling. To test this hypothesis we investigated the occurrence of protein S-nitrosylation (SNO) and protein oxidation in biopsies from explanted DCM and non-failing donor male and female human hearts. A modified biotin switch method using DyLight-maleimide sulfhydryl-reactive fluors was used to identify the SNO proteins and a resin-assisted capture Ox-RAC was used to measure protein oxidation. We found that oxidative stress rises in DCM human biopsies in comparison with healthy donors. DyLight-maleimide gel electrophoresis showed that females have an increase in protein SNO compared with males at baseline. Interestingly many of the proteins that showed an increase in SNO in females are mitochondrial proteins. Because there are sex differences in SNO at baseline, we examined changes in SNO in heart failure as a function of sex and found sex differences in this response.