The S1P1 receptor regulates sarcomere assembly and cardiac development

描述（由申请人提供）：对心脏发育的理解影响了儿童和成人心血管疾病的研究。心脏发育缺陷导致各种各样的病理，包括先天性心脏缺陷、心肌病和瓣膜疾病。鞘氨醇-1-磷酸（S1P）在脊椎动物的发育中起着重要的作用。S1P是细胞膜代谢的产物，在人体中无处不在。S1P曾经被认为是细胞膜破裂的最终产物，现在被认为是细胞增殖、迁移和死亡的重要调节因子。S1P的作用是由一个5个G蛋白偶联受体家族介导的，鞘氨醇-1-磷酸受体1-5 （S1P1-5）。它们以组织特异性的方式表达，并赋予对S1P的多样性反应。本项目拟研究S1P受体S1P1在心脏发育中的作用。合成寡核苷酸抑制斑马鱼S1P1表达导致心肌肌节发育受损。这些异常似乎是心肌细胞特有的。在Aim 1中，将检查S1P1功能在斑马鱼心脏发育中的时间。将光激活寡核苷酸注射到斑马鱼胚胎中，并使用紫外光在特定时间点启动对S1P表达的抑制。这将允许控制S1P1抑制的位置和时间。S1P1表达抑制的影响将通过评估心脏收缩力和肌节成分的免疫组织化学来检测。在Aim 2中，将在合成寡核苷酸抑制S1P1表达的斑马鱼中评估与肌节组装失败相关的细胞变化。肌动蛋白将使用荧光标记的方法可视化。这种方法不会干扰肌动蛋白的功能，并且可以实时评估体内的肌节。对其他重要的肌节成分进行免疫组化。该模型中导致肌节发育受损的信号通路也将被研究。在Aim 3中，将通过抑制小鼠中S1P1的表达来研究这种表型的保护。为了实现这一目标，将产生心房心肌、心室心肌或心肌和心内膜中S1P1基因被破坏的小鼠。然后通过超声心动图、免疫组织化学和电子显微镜分析这种破坏的影响。

DESCRIPTION (provided by applicant): An understanding of cardiac development has influenced the study of cardiovascular disease in children and adults. Defects in cardiac development lead to a wide variety of pathology, including congenital heart defects, cardiomyopathies, and valvular diseases. Sphingosine-1-phosphate (S1P) has been found to play an important role in vertebrate development. S1P is a product of cell membrane metabolism and found ubiquitously throughout the body. Once believed to simply be an end product of cel membrane breakdown, S1P is now recognized as an important regulator of cell proliferation, migration, and death. The effects of S1P are mediated by a family of five G protein-coupled receptors, sphingosine-1-phosphate receptors 1-5 (S1P1-5). They are expressed in a tissue specific manner and confer the diversity of responses to S1P. This project proposes to study the role of the S1P receptor S1P1 in cardiac development. Synthetic oligonucleotide inhibition of S1P1 expression in zebrafish leads to impaired cardiac sarcomere development. These abnormalities appear to be specific to cardiac myocytes. In Aim 1, the timing of S1P1 function in zebrafish cardiac development will be examined. Light-activated oligonucleotides will be injected into zebrafish embryos and ultraviolet light will be used to initiate inhibition of S1P expression at specific time points. This will allow control over the location and timing of S1P1 inhibition. The effects of S1P1 expression inhibition will be assayed by evaluating cardiac contractility and immunohistochemistry of sarcomere components. In Aim 2, the cellular changes associated with failed sarcomere assembly will be evaluated in zebrafish subjected to synthetic oligonucleotide inhibition of S1P1 expression. Actin will be visualized using a method of fluorescence tagging. This method does not interfere with actin function and will allow real-time assessment of the sarcomere in vivo. Immunohistochemistry of other important sarcomere components will be performed. The signaling pathways that lead to impaired sarcomere development in this model will also be investigated. In Aim 3, conservation of this phenotype will be investigated by inhibiting S1P1 expression in mice. To accomplish this, mice that have the S1P1 gene disrupted in the atrial myocardium, the ventricular myocardium, or both the myocardium and endocardium will be generated. The effects of this disruption will then be assayed by echocardiography, immunohistochemistry, and electron microscopy.