Mitochondrial and Nuclear Mechanisms of Estrogen Receptor-Mediated Cardioprotecti

描述（由申请人提供）：绝经后妇女特别容易受到缺血性损伤，每年老年妇女因心血管疾病死亡的人数比老年男性多8万人。关于雌激素（E2）在调节缺血耐受性（IT）中的作用存在矛盾的数据，部分原因是E2替代改善IT的效果失败。已知受E2作用影响的心脏保护性信号通路包括(PI3K)-Akt、eNOS和GSK3-2。总的来说，这些信号影响对细胞存活至关重要的线粒体事件，以限制坏死和凋亡细胞死亡。在此，我们提出验证衰老心脏中雌激素受体（ER）表达失调导致氧化应激（ROS）增加和缺血/再灌注（I/R损伤）恶性循环的新范式，而选择性参与非基因组ER通路可以挽救这种表型。我们进一步提出，非基因组性内质网信号可导致PKC5蛋白激酶的激活，PKC5是一种众所周知的心脏保护介质。以下具体目标将被测试：1)确定哪些ER亚型（s)在衰老，性腺去角质的雌性大鼠中降低IT， 2)确定ER通过eNOS解耦，翻译后修饰（PTM）或PI3- Akt-GSK-32轴改变ROS （ONOO-和O-2）的分子机制，3）确定PKC5在快速ER信号传导中的作用，以及4)确定非基因组ER激活后新的线粒体PKC5结合靶点。使用全局缺血模型，我们将使用特异性ER和PKC5激动剂和EPR光谱来评估ROS。细胞凋亡将通过体内冠脉结扎、细胞内质网亚型靶向和下游信号的western blotting、内质网介导的基因表达的qRT- PCR和多种反应监测来评估内质网的PTMs。一种靶向的，最先进的蛋白质组学iTRAQ 8plex方法和LC MS/MS将用于鉴定新的心脏线粒体PKC5蛋白伴侣和磷酸化。我们提出，非基因组性的er激活可以弥补老年女性心脏中E2的缺乏，从而改善IT。我们假设的证实将有力地支持老年人群急性冠状动脉综合征治疗的新靶点，并解决ER/PKC5治疗作为改善绝经后妇女IT的可行方法的潜在应用。公共卫生相关性：75岁或75岁以上的人死亡的主要原因是心脏病，从1970年到2002年，80岁或80岁以上的人死于心脏病的人数实际上增加了60%。年龄也是心力衰竭（CHF）发生的主要危险因素，65岁及以上的美国人因CHF接受治疗和出院的人数多于其他任何诊断。有趣的是，老年女性因心力衰竭导致的死亡率明显高于老年男性。与老年男性相比，老年女性的心血管疾病发病率和相关死亡率也明显更高，大规模临床试验的数据表明，雌激素替代疗法对降低心血管疾病发病率无效，实际上可能会增加心血管疾病的发病率。总的来说，不断扩大的老年人口无疑将需要增加医疗资源的分配，增加所有美国人医疗费用的经济负担。然而，雌激素在衰老心脏中发挥作用的细胞机制尚不清楚。与这一建议相关的实验有望确定以前未研究过的雌激素缺乏的新蛋白靶点。如果我们关于雌激素受体的假设是正确的，将实现老年人群缺血性心血管疾病治疗的新靶点，即使用雌激素受体治疗剂作为提高老年女性缺血耐受性的可行途径。

DESCRIPTION (provided by applicant): Post-menopausal women are especially vulnerable to ischemic insult, and ~80,000 more cardiovascular deaths occur yearly in aged women than in aged men. Conflicting data exist on the role of estrogen (E2) in modulating ischemic tolerance (IT), due in part to the failed efficacy of E2 replacement to improve IT. Protective signaling pathways in the heart known to be influenced by E2 action include (PI3K)-Akt, eNOS and GSK3-2. Collectively, these signals impact mitochondrial events critical to cell survival to limit necrotic and apoptotic cell death. Here, we propose to test the novel paradigm that maladaptive estrogen receptor (ER) expression in the aged heart leads to a vicious cycle of increased oxidative stress (ROS) and ischemia/reperfusion (I/R injury), while selective engagement of non-genomic ER pathways can rescue this phenotype. We further propose that non-genomic ER signaling results in protein kinase C5 (PKC5) activation, a well-known mediator of cardioprotection. The following specific aims will be tested: 1) determine which ER subtype(s) reduce IT in aged, gonadectomized female rats, 2) determine the molecular mechanism(s) by which ERs alter ROS (ONOO- and O-2) through eNOS uncoupling, post-translational modification (PTM) or the PI3- Akt-GSK-32 axis, 3) determine the role of PKC5 in rapid ER signaling, and 4) identify novel mitochondrial PKC5 binding targets following non-genomic ER activation. Using a model of global ischemia, we will employ specific ER and PKC5 agonists and EPR spectroscopy to assess ROS. Apoptosis will be assessed through in vivo coronary ligation, western blotting for cellular ER subtype targeting and downstream signals, qRT- PCR for ER- mediated gene expression, and multiple reaction monitoring to assess PTMs of ERs. A targeted, state-of-the- art proteomic iTRAQ 8plex approach and LC MS/MS will be used to identify novel cardiac mitochondrial PKC5 protein partners and phosphorylation. We propose that non-genomic activation of ERs can compensate for E2 deficiency in aged female hearts, resulting in improved IT. The confirmation of our hypothesis will strongly support new targets for the treatment of acute coronary syndrome in the aging population and address the potential use of ER/PKC5 therapeutics as viable approaches to improve IT in post-menopausal women. PUBLIC HEALTH RELEVANCE: The leading cause of death among persons aged 75 years or older is heart disease, and deaths due to heart disease have actually increased by 60% from 1970 to 2002 among persons aged 80 years or older. Age is also the leading risk factor for the development of heart failure (CHF) and more Americans aged 65 and older are treated and discharged from hospitals with CHF than with any other diagnosis. Interestingly, mortality rates due to CHF are significantly greater in aged women vs aged men. CVD rates and associated mortality are also significantly greater in aged women when compared to aged men, and data from large scale clinical trials suggest that estrogen replacement therapy is ineffective in reducing, and may actually increase CVD outcomes. Collectively, this expanding aged demographic will undoubtedly require increased allocation of medical resources, raising the economic burden of health care costs for all Americans. The cellular mechanisms by which estrogen exerts effects in the aged heart, however, are poorly understood. The experiments associated with this proposal are expected to identify novel protein targets previously unstudied with estrogen deficiency. If our hypothesis on estrogen receptors is correct, new therapeutic targets for the treatment of ischemic cardiovascular disease in the aged population will be realized, i.e. use of estrogen receptor therapeutics as viable approaches to improve ischemic tolerance in aged women.