Cardiac Angiotensin: Hypertrophy and Failure

描述(申请人逐字描述)：血管紧张素II 2型 (AT2)受体在肥厚和心脏左室起主导作用 失败，AT2受体激活抑制生长并促进细胞凋亡 在试管中。该项目将检验AT2受体的总体假设 体内信号转导在压力下的抗生长和促凋亡作用 超负荷肥大。我们将使用具有脑室靶向的转基因小鼠 由MLC2V启动子驱动的AT2受体的过表达 患有升主动脉狭窄。功能后果将被研究 通过超声心动图和血流动力学测定，分析分离的心肌细胞 利用荧光视频实现细胞收缩和细胞内离子调节 显微镜和共聚焦显微镜分析细胞的原位形态和 细胞凋亡。信号通路将通过免疫组织化学和 免疫印迹使用特定信号分子和抗体 磷酸化状态的鉴定。具体目标是：目标1.至 确定转基因小鼠体内AT2受体的过度表达是否受到抑制 肥厚性生长对慢性收缩压超负荷的反应 主动脉狭窄。我们预测AT2受体的过度表达会严重抑制 发展左室肥厚，促进心脏的快速发展 失败和过早死亡。目的2.验证AT2受体的假说 过度表达促进压力超负荷小鼠心肌细胞的凋亡。 用共聚焦原位末端标记法和连接酶分析鉴定细胞凋亡 显微镜和细胞色素c渗漏到细胞的补充测量 胞浆。目的3.确定AT2受体在体内的过度表达是否会改变 心肌细胞收缩功能，并干扰Ang II介导的变力作用。 这些实验将使用收缩能力的测量以及 分离的小鼠心肌细胞内pH和钙离子，并验证这一假说 AT2受体激活抑制Ang II与Forward的偶联 Na+-H+交换。目的4.确定AT2受体在体内是否过表达 激活激动素-cGMP途径。体外研究表明，这个系统 对AT2受体信号的贡献，但它的贡献，如果有的话，在 成年人的心脏是不被理解的。将测量cGMP水平和 激肽原酶在左心室组织(和心房组织，其中 转基因是最低限度的表达)。此外，它的功能效果 抑制这一途径对心脏生长和血流动力学性能的影响 在存在和不存在AT2过表达的小鼠身上进行体内测试 压力过载。这些综合的分子生理学研究，检查 活体和细胞心脏生理学，将提供关于 AT2受体激活对大鼠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.