Investigation of Molecular Mechanism for Excitation-contraction Coupling in Heart

本研究旨在阐明心肌Ryanodine受体兴奋-收缩偶联的分子机制，并探讨Ryanodine受体的结构-功能关系，获得心脏特异性sorcine基因敲除小鼠，该基因敲除小鼠同时与Ryanodine受体和二氢吡啶受体相互作用。我们首先分离小鼠sorcine基因并对其进行分析，获得了靶向构建体，在第一和第二外显子插入了一段flox序列，并将其电穿孔到ES细胞中。经G418筛选后，用PCR和sourceBlot鉴定重组ES细胞。将ES细胞注射到小鼠子宫内，获得嵌合体小鼠，并将floxed ryanodine受体小鼠与MLC-2 v Cre小鼠杂交。这些缺少该基因一个等位基因的小鼠表现正常。Western印迹分析显示ryr 2蛋白水平降低约50%，但SEACA、NCX和CAQ无差异。超声心动图显示，与野生型小鼠相比，杂合子ryr 2基因敲除小鼠的心腔大小和射血分数没有显着差异。血流动力学研究也表明，基因的一个等位基因的消融导致心脏功能没有改变。

This study has been performed to elucidate a molecular mechanism for excitation-contraction coupling and examine the structure-function relationship of ryanodine receptor in heart.We attempted to obtain cardiac-specific sorcine knockout mice, which has been known to interact both with dihidropyridine receptor and with ryanodine receptor. We, first, isolated mouse sorcine gene and analyzed it to obtain targeting construct.We inserted a flox sequence in the first and second exon and electroporated it into ES cells. After selection with G418, we identified recombinant ES cells with PCR and sourthern blot analysis. We injected the ES cells into mouse uterus and obtained chimera mice.We have crossed floxed ryanodine receptor mice with MLC-2v Cre mice. The mice, which lack one allele of the gene, appeared normal. Western blot analysis revealed about 50 % decrease in ryr2 protein level but no differences in SEACA, NCX, and CAQ. Echocardiography indicated that heterozygous ryr2 knockout mice showed no significant differences in chamber size and ejection fraction compared with wild type mice. Hemodynamic study also indicated ablation of one allele of the gene led to no alteration in cardiac function.