EXCITATION/CONTRACTION COUPLING IN EMBRYONIC STEM CELL DERIVED CARDIAC MYOCYTES

本研究领域包括： 心肌细胞的体外分化模型 胚胎干细胞（R1）和胚胎癌细胞 （P19）。该研究旨在了解 肌浆网的结构与功能关系 钙释放通道及其与火花形成的关系 发育中的心肌细胞中的钙运动。我们一直 能够成功地将多能ES和EC细胞分化为 含有收缩的心脏样细胞的胚状体。到 鉴定，心房与心室样细胞，表达载体 已经构建了将心室标记物 绿色荧光蛋白（GFP），包括2.1kb的 MLC 2 v-GFP构建体。这种结构已经被证明是有效的 在瞬时转染后， ES细胞和新霉素抗性克隆的筛选。 分化的ES-衍生细胞的免疫荧光分析 使用单克隆和多克隆抗体的心肌细胞也 成功地进行了肌节肌动蛋白，肌钙蛋白T; 确定ryanodine最早阶段的具体工作 受体，SR CaATP酶，受磷蛋白和二氢吡啶 受体表达正在顺利进行。我们还建立 几种技术来测量这些细胞的mRNA含量， 蛋白质，所有这些都被用来开发一个分子模型， EC偶联和松弛的发展在早期和晚期 分化ES细胞。随后，我们开发了一些 小鼠兰尼碱受体2基因的克隆， 修饰以引入R1 ES细胞。以下 正-负选择，我们已经鉴定了一些克隆， 在适当的基因上进行同源重组 基因座这些细胞目前正在进行体外分析 EC偶联的发展。这项工作是 与兴奋收缩 心血管科学实验室的耦合单元， 进行了一些非常初步的研究， 电压钳位和共聚焦显微镜技术分析 钙火花形成、钙瞬变和钙的作用 处理蛋白质

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. To identify, atrial versus ventricular like cells, expression vector constructs have been constructed that link ventricular markers to the green florescent protein (GFP), including the 2.1kb MLC2v-GFP construct. This construct has been shown to work very well after transient transfections, and thus were introduced into ES cells and neomycin resistance clones selected. Immunofluorescence analyses of the differentiating ES-derived cardiac cells using monoclonal and polyclonal antibodies have also 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. Subsequently, 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 are now being 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.