HORMONE REGULATION OF CARDIAC INJURY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这一建议的中心假设是，高密度脂蛋白相关的性激素(雌激素和睾酮)调节心肌细胞结构性一氧化氮合酶的活性，从而影响缺血损伤的程度。 流行病学研究表明，女性比男性更不容易患缺血性心脏病。尽管目前的研究重点是性激素，特别是雌激素和睾丸素，但导致男性和女性缺血性损伤差异的机制尚不清楚。大量研究表明，雌激素诱导的结构性一氧化氮合酶(CNOS)的刺激对心肌细胞的缺血性损伤具有保护作用。关于睾酮对缺血性损伤的保护作用和睾酮促进缺血性损伤的报道，其作用尚不清楚。大多数关于雌激素和睾酮的研究没有区分心脏内皮细胞和心肌细胞介导的作用。这项研究的一个新方面是，初步数据表明，雌激素和睾酮对心肌细胞有直接影响，不依赖于内皮。这项研究的另一个新方面是，与高密度脂蛋白相关的雌激素或睾酮负责在心肌细胞中产生一氧化氮，保护/促进缺血性损伤。雌激素限制缺血性损伤和睾酮增加缺血性损伤的分子机制将从两个方面进行测试。 目的1：探讨高密度脂蛋白相关雌激素刺激一氧化氮产生和限制缺血损伤的机制。初步研究表明，雌激素受体α和小窝蛋白-1形成一个复合体，通过AMP激酶刺激cNOS。从野生型小鼠、小窝蛋白-1缺失小鼠或雌激素受体α缺失小鼠分离的心肌细胞将与显性负性腺病毒构建体、激动剂/拮抗剂和酶分析一起用于剖析其机制(S)。此外，野生型小鼠、SR-BI缺失小鼠、小窝蛋白-1缺失小鼠、雌激素受体α缺失小鼠以及卵巢切除和卵巢切除/雌激素替代将用于确定其对缺血性损伤的影响。 目的2：探讨高密度脂蛋白相关睾酮减少一氧化氮生成、增加缺血损伤的机制。初步研究表明，与高密度脂蛋白相关的睾酮减少了一氧化氮的产生，而不影响cNOS的细胞水平。分离的心肌细胞将被用来研究几种可能的机制，如改变小窝与cNOS的结合，清除小窝胆固醇，重新定位cNOS，以及改变cNOS和/或小窝蛋白-1，来研究高密度脂蛋白-睾酮对cNOS的抑制作用。其他研究将确定雄激素受体和/或睾酮摄取是否参与这一过程。野生型小鼠、SR-BI缺失小鼠和小窝蛋白-1缺失小鼠将与睾丸切除和睾丸切除/睾酮替代一起用于确定对缺血性损伤的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The central hypothesis of this proposal is that HDL-associated sex hormones (estrogen and testosterone) regulate the activity of cardiac myocyte constitutive nitric oxide synthase which subsequently affects the extent of ischemic injury. Epidemiological studies demonstrate that women are less prone to ischemic heart disease than men. The mechanisms responsible the differences in ischemic injury observed between men and women are not know although the current focus is on sex hormones, in particular estrogen and testosterone. A large number of studies have implicated estrogen-induced stimulation of constitutive nitric oxide synthase (cNOS) as providing protection against ischemic injury of cardiac myocytes. The role of testosterone is unclear with reports of protection against ischemic injury and reports of testosterone promoting ischemic injury. The majority of the studies on estrogen and testosterone do not distinguish between effects mediated by the endothelium in hearts and cardiac myocytes. One of the novel aspects of the proposed studies is that the preliminary data indicate that estrogen and testosterone have direct effects on cardiac myocytes, independent of the endothelium. Another novel aspect of the proposed studies is that it is estrogen or testosterone associated with HDL that is responsible for the generation of nitric oxide in cardiac myocytes and protection/promotion of ischemic injury. The molecular mechanism whereby estrogen limits ischemic injury and testosterone increases ischemic injury will be tested in two Aims. Aim 1: To determine the mechanism whereby HDL-associated estrogen stimulates the production of nitric oxide and limits ischemic injury. The preliminary studies suggest that estrogen receptor alpha and caveolin-1 form a complex that stimulates cNOS via AMP kinase. Cardiac myocytes isolated from wild type mice, caveolin-1 null mice, or estrogen receptor alpha null mice will be used along with dominant negative adenoviral constructs, agonist/antagonists, and enzyme assays to dissect the mechanism(s). In addition, wild type mice, SR-BI null mice, caveolin-1 null mice, and estrogen receptor alpha null mice along with ovariectomies and ovariectomies/estrogen replacement will be used to determine the effect on ischemic injury. Aim 2: To determine the mechanism whereby HDL-associated testosterone decreases the generation of nitric oxide and increases ischemic injury. The preliminary studies demonstrate that HDL-associated testosterone decreases the generation of nitric oxide without affecting the cellular levels of cNOS. Isolated cardiac myocytes will be used to examine several possible mechanisms for the HDL-testosterone mediated inhibition of cNOS such as, altered caveolin binding to cNOS, depletion of caveolae cholesterol, re-localization of cNOS, and modification of cNOS and/or caveolin-1. Additional studies will determine if the androgen receptor and/or testosterone uptake are involved in this process. Wild type mice, SR-BI null mice, and caveolin-1 null mice along with orchidectomies and orchidectomies/testosterone replacement will be used to determine the effect on ischemic injury.