EXCITATION/CONTRACTION COUPLING IN EMBRYONIC STEM CELL DERIVED CARDIAC MYOCYTES

本研究课题涉及胚胎干细胞（R1）和胚胎癌细胞（P19）来源的心肌细胞的体外分化模型。本研究旨在了解肌浆网钙释放通道的结构-功能关系及其与发育中心肌细胞火花形成和钙运动的关系。我们已经能够成功地将多能ES和EC细胞分化为含有收缩心脏样细胞的胚状体。已成功地进行了肌节肌动蛋白，肌钙蛋白T的单克隆和多克隆抗体的分化ES衍生的心肌细胞的免疫荧光分析，具体的工作，确定最早阶段的兰尼碱受体，SR钙ATP酶，受磷蛋白和二氢吡啶受体的表达正在进行中。我们还建立了几种技术来测量这些蛋白质的mRNA含量，所有这些都被用来开发一个分子模型的EC耦合和松弛的早期和晚期分化的ES细胞的发展。我们已经开发了一些克隆小鼠兰尼碱受体2基因，已被修改为引入R1 ES细胞。在正负选择之后，我们已经鉴定了一些在适当基因位点经历同源重组的克隆。这些细胞现在已经被表征用于心肌细胞中EC偶联的发展的体外分析。这项工作正在与心血管科学实验室的兴奋收缩耦合单元合作进行，他们已经使用电压钳位和共聚焦显微镜技术进行了许多非常初步的研究，以分析钙火花形成，钙瞬变和钙处理蛋白质的功能。该项目的主要目的是使用关键RyR 2外显子周围的loxP侧翼位点来制备嵌合分子。为此，我们已经成功地表达了Cre蛋白酶与loxP侧翼嘌呤霉素抗性基因在修饰的ES细胞。我们已经证明，floxed RyR 2外显子可以成功删除，嘌呤霉素耐药盒可以插入到同一个位点。现在正在制备新的构建体，以在内源性小鼠基因和人cDNA构建体之间形成嵌合体。- R1 ES细胞，体外分化，兰尼碱受体，P19 EC细胞

SUMMARY OF WORK This research area involves a model of in vitro differentiation of cardiomyocytes originating from embryonic stem cells (R1) and embryonic carcinoma cells (P19). The research is aimed at understanding the structure-function relationships of the sarcoplasmic reticulum calcium release channel and its relationship to spark formation and calcium movements in developing myocardial cells. We have been able to successfully differentiate pluriopotent ES and EC cells into embryoid bodies containing contracting cardiac-like cells. Immunofluorescence analyses of the differentiating ES-derived cardiac cells using monoclonal and polyclonal antibodies have been successfully performed for sarcomeric actins, troponin T; specific work on identifying the earliest stages of ryanodine receptor, SR CaATPase, phospholamban and dihydropyridine receptor expression are well under way. We have also established several techniques to measure the mRNA contents of these proteins, all of which are being used to develop a molecular model for the development of EC coupling and relaxation in early and late differentiating ES cells. We have developed a number of clones to the mouse ryanodine receptor 2 gene which have been modified for introduction into the R1 ES cells. Following positive-negative selection we have identified some clones which have undergone homologous recombination at the appropriate gene locus. These cells have now been characterized for in vitro analysis of the development of EC coupling in cardiomyocytes. This work is being performed in collaboration with the Excitation Contraction Coupling Unit of the Laboratory of Cardiovascular Science who have performed a number of very preliminary studies using the techniques of voltage clamping and confocal microscopy to analyze calcium spark formation, calcium transients and function of calcium handling proteins. A major aim of the project has been to use the loxP flanking sites around a critical RyR2 exon to make chimeric molecules. To this end, we have successfully expressed Cre Recombinase with a loxP flanking puromycin resistance gene in the modified ES cells. We have demonstrated that the floxed RyR2 exon can be successfully deleted and that a puromycin resistance cassette can be inserted into the same locus. New constructs are now being prepared to form chimeras between the endogenous mouse gene and a human cDNA construct. - R1 ES Cells, in vitro differentiation, ryanodine receptor,P19 EC Cells