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。心脏发育缺陷导致多种病理，包括先天性心脏缺陷、心肌病和瓣膜疾病。鞘氨醇-1-磷酸（S1 P）在脊椎动物的发育过程中起着重要的作用。S1 P是细胞膜代谢的产物，在整个身体中无处不在。S1 P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。S1 P的作用是由一个由五个G蛋白偶联受体组成的家族介导的，即鞘氨醇-1-磷酸受体1-5（S1 P1 -5）。它们以组织特异性方式表达，并赋予对S1 P的反应的多样性。本项目旨在研究S1 P受体S1 P1在心脏发育中的作用。合成寡核苷酸抑制斑马鱼S1 P1表达导致心肌肌节发育受损这些异常似乎是特定于心肌细胞。在目标1中，将检查斑马鱼心脏发育中S1 P1功能的时间。将光激活的寡核苷酸注射到斑马鱼胚胎中，并使用紫外线在特定时间点开始抑制S1 P表达。这将允许控制S1 P1抑制的位置和时间。将通过评价心肌收缩力和肌节组分的免疫组织化学来测定S1 P1表达抑制的作用。在目的2中，将在斑马鱼中评价与失败的肌节组装相关的细胞变化，所述斑马鱼经受合成的寡核苷酸抑制S1 P1表达。肌动蛋白将使用荧光标记方法可视化。这种方法不干扰肌动蛋白功能，并将允许实时评估的肌节在体内。将进行其他重要肌节组分的免疫组化。在这个模型中，导致肌节发育受损的信号通路也将被研究。在目标3中，将通过抑制小鼠中的S1 P1表达来研究该表型的保守性。为了实现这一点，将产生在心房心肌、心室心肌或心肌和内膜两者中具有破坏的S1 P1基因的小鼠。然后通过超声心动图、免疫组织化学和电子显微镜检查来测定这种破坏的影响。

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.