Cardiac Angiotensin: Hypertrophy and Failure

描述（申请人逐字描述）：血管紧张素II 2型 (AT2)受体在肥大和心脏病的左心室（LV）中占主导地位 失败，AT 2受体激活抑制生长并促进凋亡 体外该项目将测试AT 2受体 体内信号传导介导压力下的抗生长和促凋亡作用 超负荷肥大我们将使用转基因小鼠， 由MLC 2 V启动子驱动的AT 2受体的过表达， 患有升主动脉瓣狭窄将研究功能性后果 通过超声心动图和血流动力学测量，分析分离的心肌细胞 使用荧光视频的收缩和细胞内离子调节 显微镜和共聚焦显微镜分析原位细胞形态， 凋亡信号通路将通过免疫组织化学和 使用针对特定信号传导分子的抗体的免疫印迹， 鉴定磷酸化状态。具体目标是：目标1。到 确定转基因小鼠中AT 2受体过表达是否抑制体内 慢性收缩压超负荷引起肥大性生长 主动脉瓣狭窄我们预测AT 2受体过度表达会严重抑制 左室肥厚的发展，并促进心脏的快速发育 失败和过早死亡。目标2.为了验证AT 2受体 过表达促进压力超负荷小鼠心肌细胞凋亡。 将使用共聚焦显微镜通过原位Tunel和连接酶测定来鉴定细胞凋亡。 显微镜，并补充测量细胞色素c泄漏到 胞质液目标3.为了确定AT 2受体在体内的过表达是否改变了 心肌细胞收缩功能，并干扰血管紧张素Ⅱ介导的收缩力。 这些实验将采用收缩性的测量以及 分离的小鼠心肌细胞内pH和Ca 2+，并检验假设 AT 2受体激活抑制Ang II与前向转运体的偶联， Na+-H+交换。目标4。为了确定AT 2受体是否在体内过表达， 激活激肽-cGMP通路。体外研究表明， 有助于AT 2受体信号传导，但它的贡献，如果有的话，在 大人的心不懂。将测量cGMP水平， 在LV组织（和心房组织，其中 转基因最低限度地表达）。此外，功能的影响， 抑制该途径对心脏生长和血液动力学性能影响将 在存在和不存在以下物质的情况下，在具有AT 2过表达的小鼠中进行体内测试： 压力过载这些综合分子生理学研究， 在体内和细胞心脏生理学，将提供新的见解， AT 2受体激活对心脏的保护作用与有害作用， 肥大和心力衰竭。

DESCRIPTION (the applicant's description verbatim): The angiotensin II type 2 (AT2) receptor predominates in the left ventricle (LV) in hypertrophy and heart failure, and AT2 receptor activation suppresses growth and promotes apoptosis in vitro. This project will test the overall hypothesis that AT2 receptor signaling in vivo mediates anti-growth and pro-apoptotic effects in pressure overload hypertrophy. We will use transgenic mice with ventricular targeted overexpression of the AT2 receptor driven by the MLC2V promoter which are subjected to ascending aortic stenosis. Functional consequences will be studied by echocardiography and hemodynamic measurements, analysis of isolated myocyte contraction and intracellular ion regulation using fluorescence video microscopy, and confocal microscopy analysis of in situ cell morphology and apoptosis. Signaling pathways will be studied by immunohistochemistry and immunoblotting using antibodies to specific signaling molecules and identification of phosphorylation state. The Specific Aims are: Aim 1. To determine if AT2 receptor overexpression in transgenic mice suppresses in vivo hypertrophic growth in response to chronic systolic pressure overload from aortic stenosis. We predict that AT2 receptor overexpression severely depresses the development of LV hypertrophy, and promotes the rapid development of heart failure and premature death. Aim 2. To test the hypothesis that AT2 receptor overexpression promotes myocyte apoptosis in mice with pressure overload. Apoptosis will be identified by in situ Tunel and ligase assays using confocal microscopy, and complementary measurement of cytochrome c leakage to the cytosol. Aim 3. To determine if AT2 receptor overexpression in vivo modifies myocyte contractile function, and interferes with Ang II mediated inotropy. These experiments will employ measurements of contractility as well as intracellular pH and Ca2+ in isolated mouse myocytes, and test the hypothesis that AT2 receptor activation suppresses the coupling of Ang II with forward Na+-H+ exchange. Aim 4. To determine if AT2 receptor overexpression in vivo activates the kinin-cGMP pathway. In vitro studies suggest that this system contributes to AT2 receptor signaling, but its contribution, if any, in the adult heart is not understood. Measurements will be made of cGMP levels and kininogenase activation in LV tissues (and atrial tissues in which the transgene is minimally expressed). In addition, the functional effects of inhibition of this pathway on cardiac growth and hemodynamic performance will be tested in vivo in mice with AT2 overexpression, in presence and absence of pressure overload. These integrated molecular physiology studies, which examine in vivo and cellular cardiac physiology, will provide new insights regarding cardioprotective versus deleterious effects of AT2 receptor activation in hypertrophy and heart failure.