Selective translation of mRNAs in the left ventricular myocardium of the mouse in response to acute pressure overload

Abstract

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.

Figure 1

Time course of expression for marker genes of cardiac hypertrophy. Levels of ANF mRNA, Na⁺-Ca²⁺ Exchanger (NCX) mRNA and c-jun mRNA were quantified in total RNA samples derived from LV at the indicated time points after surgery. Values for TAC and Sham-operated mice are expressed as the fold increase relative to non-operated controls.

Figure 2

Initial screen for translationally regulated mRNAs. Equal volumes of mRNA were pooled to complete an initial screen of the candidate mRNAs. Shown are the 12 mRNA candidates with the greatest increase in percentage of polysome-bound mRNA. Values are expressed as the percentage of each mRNA pool that was polysome-bound after 24 h in sham-operated and TAC mice.

Figure 3

Secondary screen for translationally-regulated mRNAs. RNA samples derived from individual hearts were used to re-screen the 12 candidate mRNAs identified in Fig. 2. Values are expressed as the percentage of each mRNA pool that was polysome-bound after 24 h in sham-operated and TAC mice; mean ± SE, * p < 0.02 as determined by a Student's t-test.

Figure 4

Changes in the levels of candidate mRNAs in response to pressure overload. Total mRNA levels were quantified by real-time RT-PCR. Values in TAC mice were normalized to the corresponding values in Sham-operated controls; mean ± SE, * p < 0.05 as determined by a Student's t-test.