Changes in the phenotype of cells (e.g. proliferation, differentiation, hypertrophic growth) result from changes in gene expression. Emphasis is often placed on RNA expression, and the availability of microarray technology has enabled studies of the global transcriptome. However, gene expression is also influenced by the rate of translation into protein. The global rate of protein synthesis relates to the capacity for and efficiency of translation [1, 2]. Capacity is increased by synthesis of ribosomal subunits and other translational components, whereas efficiency is regulated by the rate of translational initiation (assembly of initiation factors, "unwinding" of RNA secondary structures, scanning and recognition of the initiation codon), and the rate of peptide chain elongation. Individual mRNAs are subject to additional levels of translational regulation, and elements in their 5' and 3' untranslated regions (UTRs) may interact with regulatory RNAs (e.g. antisense sequences, microRNAs) or RNA binding proteins to modulate ribosomal association . The 5' UTR particularly influences the rate of initiation via 5' terminal oligopyrimidine tracts (TOPs), inclusion of short upstream open reading frames (uORFs), GC content and UTR length [2, 3].
TOP mRNAs possess 5-15 pyrimidines at the 5' end, usually starting with C . They are subject to growth-associated translational regulation, and stimulation with serum increases their polysomal association. mRNAs encoding ribosomal proteins, elongation factors and some subunits of Eif3e initiation factor are all TOP mRNAs [4, 5]. Recruitment to polysomes increases their rate of translation, thus increasing translational capacity. Several studies have used microarrays to analyse RNA recruitment to polysomes [6–10], and bioinformatics approaches have been used to identify potential TOP mRNAs . However, the full panoply of TOP mRNAs is not known and the extent to which translational regulation is mediated through TOP mRNAs relative to other mechanisms (e.g. uORFs) remains to be established. Phosphoinositide 3' kinase (PI3K), signaling through protein kinase B (PKB, also known as Akt) and mammalian target of rapamycin (mTOR), is particularly implicated in translational regulation [1, 12]. mTOR complex 1 (mTORC1) promotes phosphorylation (activation) of p70 S6 kinases (p70S6Ks) that phosphorylate the small ribosomal subunit protein S6 (Rps6), and this was proposed to promote translation of TOP mRNAs. However, protein synthesis and recruitment of TOP mRNAs to polysomes in the presence of serum is not inhibited in p70S6K-null cells , and alternative mechanisms and signaling pathways may operate. For example, p90 ribosomal S6 kinases (p90RSKs), activated by extracellular signal-regulated kinases 1/2 (ERK1/2), also phosphorylate Eif4b and Eef2k . Additionally, the pathways are integrated and ERK1/2 can activate mTORC1 independently of PI3K [1, 12]. In a global context, PI3K signaling also influences the global rate of translation by promoting phosphorylation of 4E-BP1 [1, 12]. This promotes dissociation of 4E-BP1 from Eif4e, allowing Eif4e to bind to the 7-methylGTP cap of mRNAs and increase the rate of initiation.
Cardiomyocytes, the contractile cells of the heart, withdraw from the cell cycle perinatally. Maturational growth of the heart results from an increase in cell size, but cardiomyocytes also hypertrophy in response to physiological stresses (e.g. hypertension) . Cardiomyocyte hypertrophy is manifested in increased cell size and sarcomeric content. This reflects underlying changes in gene/protein expression, resulting from alterations in the transcriptome coupled with an increase in the rate of protein synthesis. Some would argue that the increased rate of protein synthesis is a crucial factor in facilitating hypertrophy . Various neurohumoral factors promote cardiomyocyte hypertrophy including endothelin-1 (ET-1) and other agonists that potently activate ERK1/2, and ERK1/2 signaling is particularly implicated in promoting hypertrophy . Insulin is associated with cardiomyocyte growth since it increases the rate of cardiac protein synthesis  and, as in other cells, insulin potently activates PKB/Akt via PI3K in cardiomyocytes . Insulin activates ERK1/2 to a degree, but this is substantially less than that induced by ET-1 and, although ET-1 activates PKB/Akt to a minor degree, this is substantially less than insulin . Notably, insulin does not induce the same hypertrophic phenotype as, for example, ET-1, and inhibition of ERK1/2 signaling, but not PI3K, attenuates cardiomyocyte hypertrophy induced by hypertrophic agonists . In hearts in vivo, pressure overload increases recruitment of Rpl32 mRNA (an example of a TOP mRNA) rather than non-TOP mRNAs (β-myosin heavy chain) suggesting that the TOP mRNA mechanism is an integral part of cardiac hypertrophy . Similar effects are seen in feline cardiomyocytes with ET-1 and insulin, both of which activate mTOR. However, only a single TOP mRNA was examined and whether this extends to other established TOP mRNAs, or if there are additional TOP mRNAs in the cardiac transcriptome is unknown.
Previously, we reported the acute effects of ET-1 on the cardiomyocyte transcriptome , identifying 1306 RNAs as temporally-regulated over the first 4 h of stimulation. Most of the protein-coding RNAs are approximately equally changed in total and polysomal RNA pools, suggesting that there is little translational regulation of these transcripts. Here, we compare the changes induced by ET-1 and insulin in the global cardiomyocyte transcriptome and in transcript recruitment to polysomes. Unlike ET-1, insulin did not have a substantial effect on global transcript expression, but both agonists differentially affected RNA recruitment to polysomes. Furthermore, whilst insulin did promote recruitment of TOP RNAs to the polysomes, not all recruited mRNAs possessed a TOP sequence.