The Role of Calcium Signaling in the Metabolic Syndrome during Childhood Onset

钙网蛋白(CRT)是一种内质网钙结合蛋白，对心脏发育至关重要。由α-肌球蛋白重链启动子介导的心脏特异性钙网蛋白过表达可导致转基因小鼠心脏完全阻断和猝死。为了进一步研究钙网织蛋白在心脏发育中的机制及其功能，我们利用cre-loxP系统建立了导致钙网蛋白在时间和空间上过表达的转基因小鼠。将含有CAG启动子、loxP侧翼氯霉素乙酰转移酶(CAT)基因和CRT基因的转基因小鼠显微注射到C57BL/6小鼠原核细胞中，获得loxP-CRT转基因小鼠。这些小鼠在NKX2.5启动子的控制下与表达cre重组酶的NKX2.5-Cre小鼠杂交，NKX2.5启动子是心脏早期发育所必需的转录因子。选育出心脏特异高表达钙网蛋白的转基因小鼠(NKX2.5-CRT转基因小鼠)。NKX2.5-CRT转基因小鼠的心肌钙网织蛋白水平是对照小鼠的44倍。在6~10周龄，钙网织蛋白的过度表达与PR间期、QRS间期延长、心动过缓和猝死有关。此外，一些NKX2.5-CRT转基因小鼠在7周龄时出现明显的水肿。RT-PCR分析表明，超极化激活的环核苷酸门控通道(HCN1)在NKX2.5-CRT转基因小鼠心脏中的表达减弱，这是心脏起搏活动的重要组成部分。结论：这些转基因小鼠是一种新的致死性心律失常模型，可用于分析钙网蛋白等内质网蛋白在心脏发生中的作用。

Calreticulin(CRT) is a Ca^<2+>-binding protein of the endoplasmic reticulum and essential for cardiac development. Cardiac specific overexpression of calreticulin by a-myosin heavy chain promoter results in complete heart block and sudden death in transgenic mice. For further investigating mechanism of calreticulin in cardiac development and its function, we generate transgenic mice that cause temporal and spatial overexpression of calreticulin using cre-loxP system. By microinjection of the transgene which contains a CAG promotor, a loxP-flanked chloramphenicol acetyltransferas(CAT) gene, and CRT cDNA into pronuclear cells of C57BL/6 mice, we made loxP-CRT transgenic mice. The mice were cross-bred with Nkx2.5-Cre mice which express cre recombinase under control of Nkx2.5 promoter, a transcription factor essential for early cardiac development. The breeding were resulted in transgenic mice with cardiac specific overexpression of calreticulin(Nkx2.5-CRT transgenic mice). Cardiac calreticulin protein levels of Nkx2.5-CRT transgenic mice were increased by 44-fold compared with control mice. Overexpression of calreticulin was associated with PR interval, QRS interval prolongation, bradycardia, and sudden death observed from 6- to 10-week-old. Furthermore, some NKx2.5-CRT transgenic mice revealed marked edema in 7-week-old. RT-PCR analysis revealed that the expression of hyperpolerization-activated cyclic nucleotide-gated channell(HCN1), essential component for cardiac pace maker activity, was receded in the heart of Nkx2.5-CRT transgenic mice. These suggested that carleticulin affect cardiac arrhythmia with disruption of cardiac signaling, such as HCN families In conclusion, these transgenic mice are a novel model of fatal arrhythmia, and are useful model for analysis of endoplasmic reticulum protein such as calreticulin in cardiogenesis.