Horsthuis Thomas, Houweling Arjan C, Habets Petra E M H, de Lange Frederik J, el Azzouzi Hamid, Clout Danielle E W, Moorman Antoon F M, Christoffels Vincent M
Heart Failure Research Center, AMC, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
Circ Res. 2008 Apr 11;102(7):849-59. doi: 10.1161/CIRCRESAHA.107.170571. Epub 2008 Feb 14.
Nppa, encoding atrial natriuretic factor, is expressed in fetal atrial and ventricular myocardium and is downregulated in the ventricles after birth. During hypertrophy and heart failure, Nppa expression is reactivated in the ventricles and serves as a highly conserved marker of heart disease. The Nppa promoter has become a frequently used model to study mechanisms of cardiac gene regulation. Nevertheless, the regulatory sequences that provide the correct developmental pattern and ventricular reactivation during cardiac disease remain to be defined. We found that proximal Nppa fragments ranging from 250 bp to 16 kbp provide robust reporter gene activity in the atria and correct repression in the atrioventricular canal and the nodes of the conduction system in vivo. However, depending on fragment size and site of integration into the genome of mice, the fetal ventricular activity was either absent or present in an incorrect pattern. Furthermore, these fragments did not provide ventricular reactivation in heart disease models. These results indicate that the proximal promoter does not provide a physiologically relevant model for ventricular gene activity. In contrast, 2 modified bacterial artificial chromosome clones with partially overlapping genomic Nppa sequences provided appropriate reactivation of the green fluorescent protein reporter during pressure overload-induced hypertrophy and heart failure in vivo. However, only 1 of these bacterial artificial chromosomes provided correct fetal ventricular green fluorescent protein activity. These results show that distinct distal regulatory sequences and divergent regulatory pathways control fetal ventricular activity and reactivation of Nppa during cardiac disease, respectively.
Nppa基因编码心钠素，在胎儿心房和心室心肌中表达，出生后在心室中表达下调。在心肌肥大和心力衰竭期间，Nppa基因在心室中的表达会重新激活，并作为心脏病的一个高度保守的标志物。Nppa基因启动子已成为研究心脏基因调控机制的常用模型。然而，在心脏病期间提供正确发育模式和心室重新激活的调控序列仍有待确定。我们发现，长度从250 bp到16 kbp的近端Nppa片段在心房中能提供强大的报告基因活性，并在体内房室管和传导系统节段中实现正确的抑制。然而，根据片段大小和整合到小鼠基因组中的位点不同，胎儿心室活性要么缺失，要么呈现错误的模式。此外，这些片段在心脏病模型中并未实现心室重新激活。这些结果表明，近端启动子不能为心室基因活性提供一个生理相关的模型。相比之下，2个具有部分重叠基因组Nppa序列的修饰细菌人工染色体克隆，在体内压力超负荷诱导的肥大和心力衰竭期间，能使绿色荧光蛋白报告基因实现适当的重新激活。然而，这些细菌人工染色体中只有1个能提供正确的胎儿心室绿色荧光蛋白活性。这些结果表明，不同的远端调控序列和不同的调控途径分别控制胎儿心室活性和心脏病期间Nppa基因的重新激活。
