SD COBRE: MYOCYTE POLARITY AND SIGNAL TRANSDUCTION IN MYOCARDIAL INFARCTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 心肌细胞的大小通过两个纵向 通过肌节的串联添加的生长或通过肌节的平行添加的横截面生长。 心肌细胞如何合并肌节取决于负荷条件和由此产生的细胞骨架变化。细胞定向的钙粘蛋白-β-连环蛋白信号通路的激活可以将信号分子募集到极性质膜，并导致肌节的连续增加、进行性细胞延长和最终泵衰竭。糖原合成酶激酶-3 β（GSK-3 β）磷酸化丝氨酸和苏氨酸上的β-连环蛋白，调节细胞质β-连环蛋白水平并控制基因表达。我们假设GSK-3 β抑制剂激活β-连环蛋白信号通路触发了肌细胞延长和离心性肥大。在第一年，我们侧重于具体目标1和3。 这两个目标对于研究具体目标2和4至关重要。 在具体目标1中，我们对心肌梗死期间的β-连环蛋白信号传导进行了进一步的研究。 我们的初步结果表明，细胞质游离β-连环蛋白水平增加，在急性心肌缺血。 为了进一步证明β-连环蛋白信号调节心肌细胞中的基因调控，我们使用了在c-Fos最小启动子和三个Tcf结合位点的控制下表达lacZ的报告小鼠。 在β-连环蛋白信号传导过程中，游离β-连环蛋白易位到细胞核并结合TCF/LEF转录因子，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极性的变化。 我们的数据表明，在肌细胞延长过程中，肌细胞极性发生变化。 一旦产生心肌梗死小鼠，已建立的用于肌细胞极性的方案和技术就可以在本提议中使用。在具体目标3中，我们获得了在TA反式激活因子控制下条件性过表达β-连环蛋白的转基因小鼠。 这些小鼠目前与在MHC或CMV启动子控制下表达TA反式激活因子的小鼠一起繁殖。 我们将用强力霉素诱导基因表达，并在小鼠可用后分析表型。 这些研究将进一步加深我们对心肌肥大和心力衰竭中肌细胞形状和极性调节的理解，并确定参与肌细胞形状和极性调节的关键分子，为未来的遗传操作和基因治疗提供参考。

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.