SD COBRE: MYOCYTE POLARITY AND SIGNAL TRANSDUCTION IN MYOCARDIAL INFARCTION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。心肌细胞的大小可以通过连续添加肌节的纵向生长或通过平行添加肌节的横截面生长来增加。心肌细胞如何结合肌节取决于负荷条件和由此引起的细胞骨架变化。纵向定位的钙粘素-β-连环素信号通路的激活可能会将信号分子聚集到极质膜上，导致肌节的一系列增加，细胞的进行性延长，最终导致泵功能衰竭。糖原合成酶-3β(GSK-3β)使丝氨酸和苏氨酸上的β-连环蛋白磷酸化，调节细胞质中的β-连环蛋白水平，控制基因表达。我们假设GSK-3β抑制激活了β-连环蛋白信号通路，触发了心肌细胞的延长和离心性肥大。在第一年中，我们将重点放在特定的目标1和3上。这两个目标是研究特定的目标2和4的关键。在特定的目标1中，我们对心肌梗死过程中的β-连环蛋白信号进行了进一步的研究。我们的初步结果表明，在急性心肌缺血时，胞浆内游离β-连环素水平升高。为了进一步证明β-连环蛋白信号调节心肌细胞的基因调控，我们使用了一只在c-Fos最小启动子和三个Tcf结合位点控制下表达LacZ的报告小鼠。在β-连环蛋白信号转导过程中，游离的β-连环蛋白转位到细胞核，并与TCF/LEF转录因子结合，导致LacZ表达增加。我们继续研究高血压大鼠模型中心肌细胞的极性变化。我们的数据显示，在肌细胞延长的过程中，肌细胞的极性发生了变化。一旦建立了心肌梗死小鼠，已经建立的心肌细胞极性的方案和技术就可以在这个提案中使用了。在特定目标3中，我们获得了在TA反式激活剂控制下有条件地高表达β-连环蛋白的转基因小鼠。这些小鼠目前是在MHC或CMV启动子控制下表达TA反式激活因子的小鼠。我们将用多西环素诱导基因表达，并在小鼠出现后分析表型。这些研究将进一步加深我们对心肌肥厚和衰竭中心肌细胞形态和极性调节的理解，并为未来的基因操作和基因治疗寻找参与心肌细胞形态和极性调控的关键分子。

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. Cardiac myocytes size increases through either longitudinal growth by series addition of sarcomeres or cross-sectional growth by parallel addition of sarcomeres. How cardiac myocytes incorporate sarcomeres is dependent on loading condition and resultant cytoskeletal changes. The activation of longitudinally-oriented cadherin-beta-catenin signaling pathways may recruit signal molecules to the polar plasma membrane and lead to series addition of sarcomeres, progressive cell lengthening and eventually pump failure. Glycogen synthase kinase-3beta (GSK-3beta)phosphorylates beta-catenin on serine and threonine, regulating cytoplasmic beta-catenin level and controlling gene expression. We hypothesize that activation of the beta-catenin signaling pathway by GSK-3beta inhibition triggers myocyte lengthening and eccentric hypertrophy. During the first year, we have focused on specific aims 1 and 3. These two aims are critical to the study of specific aim 2 and 4. In specific aim 1, we have conducted further studies on beta-catenin signaling during myocardial infarction. Our preliminary results have demonstrated that cytoplasmic free beta-catenin levels increase during acute myocardial ischemia. To further prove that beta-catenin signaling regulates gene regulation in cardiac myocytes, we have used a reporter mouse expressing lacZ under the control of a c-Fos minimal promoter and three Tcf binding sites. During beta-catenin signaling, free beta-catenin translocates to the nucleus and binds to TCF/LEF transcription factors resulting in increased LacZ expression. We continue to investigate myocyte polarity changes in a hypertensive rat model. Our data have showed that myocyte polarity changes during myocyte lengthening. The established protocols and techniques for myocyte polarity are ready to use in this proposal once mice with myocardial infarction are created. In specific aim 3, we have acquired transgenic mice conditionally overexpressing beta-catenin under the control of TA transactivator. These mice are currently bred with mice expressing TA transactivator under the control of either MHC or CMV promoter. We will induce gene expression with doxycycline and analyze phenotype once mice are available. These studies will further our understanding of myocyte shape and polarity regulation in cardiac hypertrophy and failure and identify key molecules involved in myocyte shape and polarity regulation for future genetic manipulation and gene therapy.