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

钙网蛋白（Calreticulin， CRT）是一种内质网Ca^<2+>结合蛋白，对心脏发育至关重要。a-肌球蛋白重链启动子在心脏特异性过表达钙调蛋白可导致转基因小鼠完全心脏传导阻滞和猝死。为了进一步研究钙网蛋白在心脏发育中的作用机制及其功能，我们利用cre-loxP系统构建钙网蛋白时空过表达转基因小鼠。将含有CAG启动子、loxp侧氯霉素乙酰转移（CAT）基因和CRT cDNA的转基因基因微注射到C57BL/6小鼠的原核细胞中，制备了loxP-CRT转基因小鼠。这些小鼠与Nkx2.5- cre小鼠杂交，后者表达受Nkx2.5启动子控制的cre重组酶，Nkx2.5启动子是早期心脏发育所必需的转录因子。培育出心脏特异性过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调蛋白水平比对照小鼠提高了44倍。钙调钙蛋白的过表达与6 ~ 10周龄的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分析显示，Nkx2.5-CRT转基因小鼠心脏中超极化激活环核苷酸门控通道（HCN1）的表达减少，HCN1是心脏起搏器活性的重要组成部分。这些结果表明，钙网蛋白通过破坏心脏信号来影响心律失常，如HCN家族。总之，这些转基因小鼠是一种新的致死性心律失常模型，并为分析钙网蛋白等内质网蛋白在心脏发生中的作用提供了有用的模型。

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.