Spruill Laura S, Baicu Catalin F, Zile Michael R, McDermott Paul J
Gazes Cardiac Research Institute, Department of Medicine, Medical University of South Carolina, Charleston, SC 29403, USA.
J Mol Cell Cardiol. 2008 Jan;44(1):69-75. doi: 10.1016/j.yjmcc.2007.10.011. Epub 2007 Oct 25.
During pressure overload hypertrophy, selective changes in cardiac gene expression occur that regulate growth and modify the structural and functional properties of the myocardium. To determine the role of translational mechanisms, a murine model of transverse aortic constriction was used to screen a set of specified mRNAs for changes in translational activity by measuring incorporation into polysomes in response to acute pressure overload. Candidate mRNAs were selected on the basis of two main criteria: (1) the 5'-untranslated region of the mRNA contains an excessive amount of secondary structure (DeltaG<-50 kCal/mol), which is postulated to regulate efficiency of translation, and (2) the protein product has been implicated in the regulation of cardiac hypertrophy. After 24 h of transverse aortic constriction, homogenates derived from the left ventricle were layered onto 15-50% linear sucrose gradients and resolved into monosome fractions (messenger ribonucleoprotein particles) and polysome fractions by density gradient ultracentrifugation. The levels of mRNA in each fraction were quantified by real-time RT-PCR. The screen revealed that pressure overload increased translational activity of 6 candidate mRNAs as determined by a significant increase in the percentage of total mRNA incorporated into the polysome fractions. The mRNAs code for several functional classes of proteins linked to cardiac hypertrophy: the transcription factors c-myc, c-jun and MEF2D, growth factors VEGF and FGF-2 and the E3 ubiquitin ligase MDM2. These studies demonstrate that acute pressure overload alters cardiac gene expression by mechanisms that selectively regulate translational activity of specific mRNAs.
在压力超负荷肥大过程中，心脏基因表达会发生选择性变化，这些变化可调节心肌的生长并改变其结构和功能特性。为了确定翻译机制的作用，我们使用了一种小鼠主动脉缩窄模型，通过测量急性压力超负荷后多聚核糖体中的掺入情况，来筛选一组特定mRNA的翻译活性变化。候选mRNA是根据两个主要标准选择的：（1）mRNA的5'非翻译区含有过量的二级结构（ΔG < -50 kCal/mol），据推测这可调节翻译效率；（2）蛋白质产物与心脏肥大的调节有关。在主动脉缩窄24小时后，将来自左心室的匀浆铺在15 - 50%的线性蔗糖梯度上，通过密度梯度超速离心将其分离为单核糖体组分（信使核糖核蛋白颗粒）和多核糖体组分。通过实时RT-PCR对每个组分中的mRNA水平进行定量。筛选结果显示，压力超负荷增加了6种候选mRNA的翻译活性，这可通过掺入多核糖体组分的总mRNA百分比显著增加来确定。这些mRNA编码与心脏肥大相关的几类功能蛋白：转录因子c-myc、c-jun和MEF2D、生长因子VEGF和FGF-2以及E3泛素连接酶MDM2。这些研究表明，急性压力超负荷通过选择性调节特定mRNA翻译活性的机制改变心脏基因表达。
