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Keywords:

  • cardiomyocyte;
  • growth;
  • heart;
  • hypertrophy;
  • ribosomal gene transcription;
  • ribosomes;
  • translation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Regulation of translation in eukaryotes
  5. Regulation of translation in cardiac myocytes during hypertrophic growth
  6. Concluding remarks
  7. References

1. Left ventricular hypertrophy (LVH) of the heart is an adaptive response to sustained increases in blood pressure and hormone imbalances. Left ventricular hypertrophy is associated with programmed responses at the molecular and biochemical level in different subsets of cardiac cells, including the cardiac muscle cells (cardiomyocytes), fibroblasts, conductive tissue and coronary vasculature.

2. Regardless of the initiating cause, the actual increase in chamber enlargement is, in each case, due to an increase in size of a pre-existing cardiomyocyte population, with little or no change in their number; a process referred to as cellular hypertrophy.

3. An accelerated rate of global protein synthesis is the primary mechanism by which protein accumulation increases during cardiomyocyte hypertrophy. In turn, increased rates of synthesis are a result of increased translational rates of existing ribosomes (translational efficiency) and/or synthesis and recruitment of additional ribosomes (translational capacity).

4. The present review examines the relative importance of translational capacity and translational efficiency in the response of myocytes to acute and chronic demands for increased protein synthesis and the role of these mechanisms in the development of LVH.


List of abbreviations
4E-BP1

eIF4E-binding protein-1

eEF

Eukaryotic elongation factor

eIF

Eukaryotic initiation factor

HMG

High mobility group

LVH

Left ventricular hypertrophy

m7G

7-Methylguanosine

MHC

Myosin heavy chain

Met-tRNAi

Initiator methionyl transfer RNA

mTOR

Mammalian target of rapamycin

PI3-K

Phosphoinositide 3-kinase

r-proteins

Ribosomal proteins

rDNA

Ribosomal genes

rRNA

Ribosomal RNA

S6K1

S6 kinase 1

SL-1

Selectivity factor-1

5′-TOP

5′-Terminal oligopyrimidine tract

tRNA

Transfer RNA

UBF

Upstream binding factor

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Regulation of translation in eukaryotes
  5. Regulation of translation in cardiac myocytes during hypertrophic growth
  6. Concluding remarks
  7. References

The size of the heart in mammals is modulated during the course of life. Left ventricular hypertrophy (LVH) of the heart is a response to sustained increases in blood pressure and hormonal imbalances, in order to increase pump function and adapt to new work conditions.1,2 Left ventricular hypertrophy is defined by increases in left ventricular mass with or without increases in wall thickness (i.e. concentric or eccentric LVH). Although such hypertrophic growth is generally considered as an adaptive response of the organism at the initial stage of onset, sustained and uncontrolled growth ultimately leads to diminished cardiac performance, resulting finally in the onset of heart failure.3 This is reflected by the observation that the latest stages of LVH highly correlate with various cardiopathological states (e.g. myocardial ischaemia, impaired contractility, impaired left ventricular filling and ventricular arrhythmias).4 Despite the fact that LVH is considered to be one of the strongest blood pressure-independent risk factors for sudden death, acute myocardial infarction, congestive heart failure and other events that contribute to cardiovascular related morbidity and mortality,5 the exact mechanisms by which LVH leads to heart dysfunction remain unresolved.

The major constituent cells of the heart, cardiac myocytes, stop dividing early in development (after birth) and irreversibly withdraw from the cell cycle (terminally differentiate).6 Thus, regardless of the initiating cause (e.g. exercise, hypertension, infarction and hormone imbalances), hypertrophic growth of the heart, by definition, results from an increase in cardiomyocyte size and in the absence of significant cell division. The majority of our information concerning the molecular mechanisms that regulate LVH has come from cell culture models, which use cultured cardiomyocytes derived from the left ventricle of neonatal or adult rat hearts. Like cardiac hypertrophy in vivo, hypertrophy of neonatal cardiomyocytes in culture is characterized by a series of phenotypic changes. These changes include increases in total protein content, changes in cell size and morphology, altered contractile activity and re-induction of genes normally expressed restrictively in the fetal heart or upregulation of genes constitutively expressed but subject to further regulation. Altered transcription of a subset of genes appears to be conserved to the hypertrophic response induced by a diverse range of hypertrophic stimuli both in vivo and in vitro. These genes include atrial natriuretic peptide (ANP), B-type natriuretic peptide, the cytoskeletal proteins α-actin and myosin heavy chain (MHC) and various early immediate response genes, such as c-myc and c-fos. Consequently, these are broadly monitored as markers for hypertrophic growth.7–9 Although the regulation of expression of these individual genes and, in some cases, their impact on cardiac muscle function has been studied extensively, the molecular mechanism(s) by which protein synthesis and cardiac growth is regulated has received far less attention. This is somewhat surprising given that the altered chamber geometry and contractile dysfunction that occurs during transition from adaptive compensatory cardiac growth to the onset of heart failure is likely to be the net result of both of changes in the expression of specific genes and inappropriate cardiomyocyte enlargement.1 Clearly, the development of new therapeutic regimens to prevent or regress pathological LVH requires an intimate understanding of both processes and their interrelationships.

One example that particularly emphasizes the direct mechanistic link between altered expression of individual genes and cardiac growth is the immediate early response gene c-myc. c-myc was one of the first genes to be identified as a ‘marker’ for the induction of hypertrophy induced by a variety of stimuli, including pressure overload and catecholamines.10,11 For a long time, the specific contribution of c-myc to LVH pathology was unknown. However, advances in tissue-specific, inducible transgenics have led to the discovery that overexpression of c-myc in either liver12 or cardiac13 cells leads to cellular hypertrophy and selective increases in expression of proteins associated with the protein translation apparatus. Thus, it would appear that one of the cellular functions of c-myc in the context of hypertrophy is to regulate cellular metabolism at the level of protein translation and synthesis.

Hypertrophic growth of myocytes is accompanied by increases in both protein synthesis and proteolysis rates; however, the more rapid rate of protein synthesis relative to protein breakdown14 results in a net increase in protein accumulation and myocyte mass. Increases in cellular protein synthetic rates can, theoretically, have three sources of origin that are not mutually exclusive: (i) a general augmentation of transcription; (ii) an increase in post-transcriptional rates (i.e. splicing); and (iii) accelerated translation rates. Numerous studies have shown that changes in the rate of transcription and post-transcriptional modification can account for the qualitative changes in expression of specific genes during hypertrophic growth.15,16 For example, a majority of proteins that comprise the ‘fetal gene programme’ are regulated at the level of transcription. However, it is likely that selective alterations in the transcription rates of individual genes or groups of genes are insufficient to account for the global increases in protein synthesis observed during hypertrophy. Moreover, the availability of mRNA is not generally limiting for increases in overall cell protein synthesis during growth, suggesting that global protein synthesis rates are regulated at the level of mRNA translation.14 There are two general mechanisms that lead to accelerated rates of translation: (i) an increased efficiency of translation; or (ii) an increase in the capacity for protein synthesis (Fig. 1). Efficiency of translation is defined as the utilization of mRNA pools by existing ribosomes and is determined by protein synthesis rates relative to the amount of ribosomes per hour. The capacity of synthesis is the amount of existing ribosomes, transfer RNAs (tRNAs) and translation factors, which is calculated on the basis of the amount of total cellular RNA per g tissue.

image

Figure 1. Hypertrophic growth of cardiomyocytes is accompanied by various phenotypic changes. However, the primary cause of hypertrophic growth results from increased rates of protein synthesis. The regulation of protein synthesis rates in eukaryotes is controlled at the level of translation efficiency (i.e. the efficiency of translation of existing ribosomes) and of translation capacity (i.e. the amount of ribosomes actively translating mRNAs). Increases in both translation efficiency and capacity appear to be necessary for the rise in protein synthesis rates during the hypertrophic growth of cardiomyocytes. An increase of the translation initiation is rate limiting for the rise in translation efficiency and is stimulated acutely during the first phase of hypertrophic growth. An increase of translation capacity is subsequent to increases of translation efficiency and is necessary to face the demand for a sustained high protein level. AUG, initiation codon; m7G, 7-methylguanosine; eIF4F, eukaryotic initiation factor 4F.

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The present review is designed to provide the reader with a brief overview of the contribution of protein translation to the regulation of cardiac myocyte growth during LVH. Because it is often overlooked, despite its significant and essential contribution to cardiac growth, we have specifically highlighted the role of regulation of the cellular synthetic capacity (i.e. ribosome content and ribosome biogenesis) in this process.

Regulation of translation in eukaryotes

  1. Top of page
  2. Summary
  3. Introduction
  4. Regulation of translation in eukaryotes
  5. Regulation of translation in cardiac myocytes during hypertrophic growth
  6. Concluding remarks
  7. References

Ribosomes

Ribosomes form the core of the eukaryotic translation machinery (for a recent review, see Lafontaine and Tollervey17) and consist of one small (40S) and one large (60S) subunit. The small and large subunits are constituted of one and three ribosomal RNA molecules (rRNA), respectively, and more than 80 different ribosomal proteins (r-proteins). The rRNAs not only form the core of the ribosomal subunits, but also contain the catalytic activity necessary for the synthesis of proteins. The small ribosomal subunit catalyses the decoding of mRNA, whereas the large subunit is responsible for the formation of peptidyl bonds during peptide chain elongation.

Regulation of translation efficiency

Regulation of protein synthesis by modulating the efficiency of translation of an existing pool of mRNA on existing ribosomes enables cells to respond rapidly to growth stimuli. Although there are, theoretically, three steps in which translation efficiency can be regulated (i.e. initiation, elongation and termination), in mammals translation initiation is the key point of control in most instances.18 Translation begins after assembly of the initiator methionyl tRNA (Met-tRNAi) to several different eukaryotic initiator factors (eIFs) and to the 40S ribosomal subunit to form the 43S pre-initiation complex (Fig. 1). Then, 43S binds the eIF4F complex (composed of the initiation factors eIF4A, eIF4E and eIF4G), which catalyses the search for most of 5′ end mRNAs harbouring a 7-methylguanosine (m7G) cap. Once the m7G cap has been found, the ribosome complex moves along the 5′ non-translated region of the mRNA from its initial binding site until the initiation codon is paired to Met-tRNAi. Finally, the eIFs are displaced and the large ribosomal subunit (60S) associates with the pre-initiation complex to form the 80S ribosome. The ribosome complex then leaves the Met-tRNAi and starts peptide chain elongation. Although, in most cases, global translation efficiency is controlled at the level of initiation by eIFs, recently a class of proteins and non-coding RNA molecules have been shown to regulate elongation through their inhibitory effects on elongation.19

Regulation of translational capacity (synthesis of ribosomes)

Sustained cell growth is usually accompanied by an increased production of the ribosomal apparatus, which is needed to cope with increased demand of protein synthesis. The major steps of ribosomal biogenesis are as follows:17 (i) transcription of the 45S pre-rRNA and 5S rRNA; (ii) post-modifications of the 45S pre-rRNA precursor; (iii) processing of the 45S pre-rRNA into mature rRNA (i.e. 28S, 5.8S and 18S); and (iv) assembly of the rRNA subunits with the ribosomal proteins (Fig. 2). Synthesis of the 18S, 5.8S and 28S rRNA by RNA polymerase I and their assembly with r-proteins take place in the nucleolus, a subcompartment of the nucleus. Transcription of the 5S rRNA occurs in the nucleoplasm rather than nucleoli and is performed by RNA polymerase III. Compelling evidence demonstrates that the key step in the formation of ribosomes in eukaryotes during cell growth is the rate of transcription of the 45S ribosomal gene (rDNA) by RNA polymerase I.20–22 Indeed, almost invariably, sustained changes in cell growth rates are associated with concomitant changes in rDNA transcription. The rate of processing of the 45S pre-RNA in most cases does not appear to be rate limiting for the synthesis of mature rRNA and is either concomitantly raised during growth, keeping pace with transcription, or remains constant with regard to the stimulus. In either case, the increased rate of transcription of rDNA results in a net increase of ribosomal RNA content (18S, 28S and 5.8S).

image

Figure 2. Hypertrophic growth of cardiomyocytes requires an increase of ribosome biogenesis in the long term. The major steps of ribosome synthesis are summarized in this figure: (i) synthesis of ribosomal RNA; (ii) rRNA processing into mature 18S, 5.8S and 28S; and (iii) assembly of rRNA with ribosomal proteins to form mature ribosomes. The rate-limiting step of ribosome biosynthesis in eukaryotes is the transcription of rDNA by RNA polymerase I, which is highly regulated during hypertrophic growth in cardiac myocytes.

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In proliferating cells, r-protein synthesis and assembly is not considered rate limiting for ribosomal biogenesis and changes in r-protein expression rates are, under most circumstances, coordinately regulated with ribosomal DNA transcription.23,24 Expression of r-proteins in mammals is regulated at the level of translation and is due to the presence of a 5′-terminal oligopyrimidine tract in their mRNAs, referred to as 5′-TOP. In addition, several eIF translation factors are also encoded by 5′-TOP mRNAs. Translation rates of 5′-TOP mRNAs are increased rapidly in response to growth stimuli.24

The precise mechanisms underlying the control of translation of 5′-TOP mRNAs have not been fully elucidated, but the ribosomal protein S6 kinase 1 (p70/p85S6K; also termed S6K1) has been demonstrated to play a pivotal role in the regulation of expression of 5′-TOP mRNAs.25 The activation of S6K1 during growth results in the phosphorylation of the 40S ribosomal S6 protein, which leads to an increase of affinity of ribosomes for 5′-TOP mRNAs, resulting in an increased rate of initiation of their translation26 (Fig. 3). However, the phosphorylation of the S6 protein does not entirely account for the increased rates of translation of 5′-TOP mRNAs during growth, because conditional knockout of the S6 protein in murine hepatocytes did not alter the proportion of ribosomes in actively translating polysomes.27 Despite the absence of the 40S ribosomal subunit in S6-null cells, the total amount of ribosomes (which was uniquely constituted by the 60S complex) in active polysomes was unchanged. Surprisingly, the growth of S6-defficient hepatocytes was not affected.

image

Figure 3. Phosphorylation of the ribosomal protein S6 by S6 kinase 1 (S6K1) is determinant for the progression of hypertrophic growth in cardiomyocytes in culture. The activation of S6 results in the increase of translation of 5′-terminal oligopyrimidine tract (5′-TOP) mRNAs, including mRNAs coding for translation factors and most ribosomal proteins. Indeed, S6 protein appears to play an important role in the regulation of both translation efficiency and capacity. The activation of the inositol phospholipids metabolism pathways is a key step in the regulation of S6 kinase during hypertrophic growth. The treatment of myocytes with specific inhibitors of phosphatidylinositol 3-kinase (PI3-K) leads to the inhibition of both S6 protein phosphorylation and hypertrophic growth. In addition to its action on ribosomal S6 protein, S6K1 may also mediate the phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein-1 (4E-BP1) in myocytes during hypertrophy, resulting in an increase of activity of eIF4E and a rise in global translation efficiency.

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As outlined above, numerous steps are putative key targets for the control of translation during growth in eukaryotic cells. These steps include: (i) initiation and elongation of translation; (ii) increased rates of rDNA transcription and processing of rRNA; and, finally, (iii) ribosomal protein syntheses and their association with the ribosomes. Evidence for these processes in cardiomyocyte growth is presented in the following section.

Regulation of translation in cardiac myocytes during hypertrophic growth

  1. Top of page
  2. Summary
  3. Introduction
  4. Regulation of translation in eukaryotes
  5. Regulation of translation in cardiac myocytes during hypertrophic growth
  6. Concluding remarks
  7. References

Translation efficiency in cardiac myocytes

Initiation of translation

Increased rates of protein synthesis in cardiac myocytes have been shown to correlate with an increase of activity of translation initiation factors and with a concomitant rise in the rate of translation initiation28–30 (Fig. 1). For instance, the activity of the translation factor eIF4E is increased in models of pressure overload in cardiomyocytes subjected to hypertrophic stimuli.31,32 The regulation of eIF4E activity modulates the formation of the eIF4F complex, which alters the affinity of the ribosomal 43S pre-initiation subunit for the 5′ cap mRNA. Under most circumstances, this is the rate-limiting step of regulation of translation efficiency in eukaryotic cells. Both electrical pacing- and α1-adrenoceptor-induced hypertrophy of quiescent neonatal rat myocytes in culture is accompanied by an increase in expression of eIF4E protein after 24–48 h.31 However, increased expression of eIF4E by itself is probably insufficient alone to promote increases in translation rates because eIF4E specific activity and eIF4F complex formation are also regulated by eIF4E phosphorylation.33 In this respect, increased eIF4E phosphorylation was observed in adult feline cardiomyocytes in culture during electrical pacing and in canine cardiomyocytes in vivo after imposition of pressure overload.32 An increase of eIF4E phosphorylation in hypertrophied cardiomyocytes in vivo was found to be concomitant with an accelerated rate of translation efficiency.30 Indeed, an increase in eIF4F complex formation appears to be required for accelerated rates of protein synthesis during pressure-overload of myocytes, because overexpression of an inactive form of eIF4E, which decreases the affinity of the eIF4F complex for 5′ mRNA caps, resulted in an inhibition of cardiomyocyte hypertrophy in vitro.34 However, overexpression of eIF4E or an increase in its phosphorylation was not sufficient alone to increase protein accumulation in non-stimulated cardiac myocytes.34

Many studies that have examined the effect of pressure overload on cardiomyocyte translation rates have focused on the expression of the cardiac-specific MHC gene. These studies have shown that increases in expression of both the β-MHC isoform in adult feline cardiomyocytes35 and the α-MHC isoform in rat neonatal cardiomyocytes36 do not result from an increase of MHC gene transcription or from a change of the subcellular localization of MHC mRNA, respectively, during the first phase of hypertrophic growth. Instead, the increase of MHC protein expression resulted from an increase of active polysomes translating MHC mRNA.35 In these short-term experiments (4–6 h), the content of cell ribosomes was unchanged upon stimulation, showing that the capacity of translation was not affected and that efficiency of translation was the main mechanism by which MHC protein accumulation was elevated during the acute phase of stimulation. Further studies performed in vivo demonstrated that canine pressure overload mediated by aortic stenosis resulted in biphasic rises of MHC translation rates.30 The acute phase of increased protein accumulation was due to a rise of translation initiation, which was then followed by a combination of both rises in translation capacity (i.e. ribosome content) and high eIF4E activity after 5 and 10 days, respectively. Although the expression of a specific gene, such as MHC, does not necessarily reflect mechanisms of overall protein synthesis, this result suggests that hypertrophic stimuli activate intracellular pathways, leading to an increase of translation efficiency first, which is then ultimately followed by an increase of translation capacity.

Elongation

The level of expression and the phosphorylation status of the elongation factor eukaryotic elongation factor-2 (eEF-2) correlates with the growth rate of cardiomyocytes (Fig. 1). For example, hypertrophy of neonatal cardiac myocytes following treatment with angiotensin II is associated with mitogen-activated protein kinase-dependent decreases in eEF-2 phosphorylation.37 Dephosphorylation of eEF-2 generally results in the activation of translation elongation.38 Similarly, both a reduction of translation elongation and a decrease of eEF-2 expression39 accompany the decrease in general protein synthesis that occurs in hearts from diabetic rats relative to non-diabetic controls. While it is interesting to speculate that the modulation of eEF-2 activity observed during changes of protein synthesis rates in cardiomyocytes may directly affect elongation rates, it remains to be determined whether this event is sufficient and necessary for the regulation of global translation rates and hypertrophic growth.

Together, the above studies strongly suggest that an increase in the efficiency of translation initiation accounts for the initial rise in the rate of protein synthesis in cardiomyocytes. However, long-term stimulation of cardiomyocytes by hypertrophic growth agents results in a secondary increase in translation capacity (i.e. increased synthesis of ribosomes). This suggests that alterations at the level of translational efficiency are insufficient by themselves to supply adequate protein synthesis rates to meet the long-term requirement for hypertrophic growth of cardiomyocytes. For these reasons, the mechanism(s) by which hypertrophic agents upregulates ribosome biogenesis in cardiomyocytes has been the subject of investigation over the past few years.

Ribosome synthesis during cardiac myocyte hypertrophy

A number of independent observations indicate that increases in translation efficiency are not sufficient alone to account for the accumulation of protein associated with chronic hypertrophy and that alteration at the level of ribosome synthesis must occur.34,40 First, a majority of ribosomes (approximately 90%) are present in the form of polysomes in non-growing cardiomyocytes and the same proportion of active polysomes is engaged in translating mRNAs during growth, even though the total cell content of ribosomes is increased.14,41 In agreement with this, accelerated transcription of the 45S ribosomal gene (rDNA; Fig. 2) accompanies increased rates of protein synthesis and myocyte growth in vitro and in vivo.42–46 Given that ribosome biogenesis represents the most energy consuming process in eukaryotic cells,20,47 it would not seem likely, from an evolutionary perspective, that cells would unnecessarily waste a considerable proportion of available energy stores for synthesizing more ribosomes if there was a less calorific-consuming way to increase cell protein level in the long term. Direct evidence to support this hypothesis has come from recent studies, which have formally demonstrated that an increase in translational capacity (i.e. ribosome content) is required for accelerated growth of myocytes following stimulation by contraction or by phenylephrine.40

Ribosomal RNA synthesis and processing

Ribosomal RNA constitutes over 90% of total RNA in cardiomyocytes. Therefore, even small changes in the rate of rRNA synthesis result in a significant increase of total RNA content.48 In this regard, McDermott et al. demonstrated that the rate of synthesis of total RNA, as measured by [3H]-uridine incorporation, was increased when neonatal cardiomyocytes were stimulated by contraction compared with arrested cells, depolarized with 50 mmol/L KCl.44,46 The rate of RNA degradation also increased during stimulation, but the difference between synthesis and degradation resulted in a net RNA accumulation of 49% after 3 days of stimulation. Further studies have shown that the rate of rRNA synthesis in cultured neonatal cardiac myocytes is accelerated by twofold during contraction or during stimulation by phorbol ester.45,49 However, there is a slight difference between both stimuli in the way pre-rRNA was processed. Stimulation of myocytes with phorbol ester resulted in an increase of processing of the 45S pre-rRNA, leading to no change of the 45S pre-rRNA pool.45 In contrast, contraction did not stimulate the rate of processing, resulting in a rise of both the pre-rRNA and the 28S rRNA pools.49 During both stimuli, however, the accelerated rate of pre-rRNA synthesis resulted in a net increased accumulation of processed 28S rRNA. These results strongly suggest that the rate-limiting step of rRNA formation is the synthesis of rRNA by RNA polymerase I and that processing mechanisms of pre-rRNA are not limiting for the accumulation of rRNA during stimulation of cardiomyocytes (Fig. 2). Indeed, a number of studies have now confirmed that cardiac hypertrophy induced by diverse hypertrophic agents, including phenylephrine,50 endothelin-1,51 phorbol myristate acetate,51 contraction52 and angiotensin II (Y Brandenburger, unpubl. data, 2002), are characterized by a twofold increase in the rate of transcription of the 45S rDNA.

Regulation of RNA polymerase I activity during hypertrophic growth

Increased rates of rDNA transcription can be due to alterations in chromatin structure53–55 or changes in the amounts and/or activities of RNA polymerase I and/or of the rDNA transcription factors.56–58 However, in the context of LVH, one factor that has been strongly implicated in the regulation of ribosome biogenesis is the rDNA transcription factor termed upstream binding factor, or UBF59,60 (Fig. 4). In addition to the core rDNA-specific transcription factor selectivity factor-1 (SL-1), UBF is also required for the accurate and efficient transcription of vertebrate rRNA genes. Purified mammalian UBF contains two polypeptides (UBF1, 97 kDa; UBF2, 94 kDa),61 which result from alternative splicing of the same primary transcript.62–64 These proteins form hetero- and homodimers and interact with the rDNA promoter via DNA binding domains (high mobility group (HMG) boxes) homologous to those found in HMG1 proteins.65 Phosphorylated UBF is more active than dephosphorylated UBF1 in vitro and in vivo;66,67 hyperphosphorylated UBF is often associated with high rDNA transcription rates, whereas underphosphorylated forms are present when rDNA transcription is shut off.68–70 The cellular activity of UBF may also be regulated through its interaction with other proteins. For example, it has been shown that the hypophosphorylated form of the retinoblastoma gene product interacts directly with UBF and acts to block UBF-mediated rDNA transcription in vitro and in vivo by disrupting UBF/SL-1 complexes.71,72

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Figure 4. The nucleolar transcription factor upstream binding factor (UBF) is a key target in the regulation of hypertrophic growth in cardiomyocytes. The UBF stimulates ribosomal DNA transcription by RNA polymerase I (Pol I) in myocytes. Endothelin-1-mediated hypertrophic growth is accompanied by increases in UBF phosphorylation. Moreover, α1-adrenoceptor agents and contraction, which are both potent hypertrophic stimuli, stimulate UBF protein expression. We have recently shown that increased expression of UBF during contraction and α1-adrenoceptor stimulation is required for increases in rDNA transcription and hypertrophic growth in neonatal rat cardiac myocytes.40

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Evidence to suggest that UBF is a target for regulation during cellular hypertrophy stems from a strong correlation between the expression and/or phosphorylation of this factor and the growth rate of cultured neonatal cardiomyocytes (Fig. 4). For example, hyperphosphorylation of a constant amount of UBF is associated with increased rDNA transcription during endothelin-1-induced hypertrophy of neonatal cardiomyocytes.51 In contrast, the elevated rDNA transcription associated with either α1-adrenoceptor- or contraction-mediated hypertrophy correlated with increases in UBF protein levels and UBF gene transcription in the absence of quantitative changes in phosphorylation.50,52 Subsequent studies demonstrated that overexpression of UBF1 in non-hypertrophied neonatal cardiac myocytes was sufficient alone to mediate an increase of rDNA transcription,73 suggesting that increased UBF expression/activity is at least part of the mechanism by which ribosome biogenesis is upregulated in response to hypertrophic stimuli. Subsequent studies have now formally demonstrated that increased levels of UBF are required for α1-adrenoceptor- and contraction-mediated hypertrophic growth of cardiomyocytes.40 In those studies, adenovirus vectors were used to efficiently deliver UBF antisense RNA into 100% of cardiomyocytes. This resulted in the findings that inhibition of UBF accumulation following stimulation with the α1-adrenoceptor agent phenylephrine or contraction led to a significant reduction in rRNA synthesis, RNA and protein accumulation and a decrease of cell size, which are the hallmarks of hypertrophic growth. Cardiac-specific genes, such as ANP and myosin light chain, the expression of which is regulated at the level of transcription by RNA polymerase II, were not effected by the UBF antisense RNA, indicating that activation of markers of the hypertrophic phenotype are not unconditionally linked to the growth process itself.

Together, these studies demonstrate that increases in the cellular translation capacity are required for sustained hypertrophic growth in cardiomyocytes and that activation of the rDNA transcription factor UBF plays an integral role in this process.

Synthesis of r-proteins and assembly of ribosomes

The regulation of ribosome assembly has been poorly investigated in eukaryotes and whether or not this step is regulated during hypertrophic growth in cardiomyocytes is not known. In contrast, the synthesis of r-proteins appears to be actively regulated during hypertrophy, which is not unexpected given that, for the efficient formation of functional ribosomes, r-proteins need to be produced in equimolar amounts as the ribosomal subunits.17 As outlined above, the expression of most mammalian r-protein species is controlled at the level of translation and there is compelling evidence that the 40S ribosomal protein S6 plays an important role in the regulation of translation of mRNAs that encode r-proteins harbouring a polypyrimidine 5′ tract (Fig. 3). The increase of translation rate of 5′-TOP mRNAs by the ribosomal S6 protein requires its phosphorylation in the carboxy-terminus by the mitogen-stimulated protein kinase S6K1.74 The activity of S6K1 is increased in cultured neonatal cardiomyocytes following treatment with various stimuli, including angiotensin II,75,76 insulin,77α-adrenoceptor agents,78 arginine vasopressin,79 leukaemia inhibitory factor80 and bradykinin.81In vivo studies have also shown that S6K1 is activated in left ventricles subjected to stretch-induced pressure overload.82,83 Furthermore, S6K1 can be inhibited by nanomolar amounts of rapamycin, which, at this concentration, blocks the activity of mammalian target of rapamycin (mTOR), an upstream activator of S6K1.86 Rapamycin treatment leads to inhibition of hypertrophic growth of neonatal cardiomyocytes stimulated with angiotensin II,75,76α-adrenoceptor agents,78 bradykinin81 or prostaglandin F84 and adult cardiomyocytes treated with β-adrenoceptor agents.85 However, in addition to S6K1, mTOR also activates Phas-1/eIF4E-binding protein-1 (4E-BP1), leading to stimulation of eIF4E activity and activation of more general protein translation initiation.86 Thus, it is unclear at this stage whether the effect of rapamycin to block cardiac growth is due to effects on Phas-1/4E-BP1, S6K1 or both (Fig. 3).

The exact contribution of second messenger(s) systems upstream or parallel to mTOR responsible for S6K1 activation during hypertrophic growth remains to be clearly defined. Recent studies suggest that phosphoinositide 3-kinase (PI3-K) is involved (for a recent review on PI3-K, see Cantrell;87Fig. 3). Indeed, two inhibitors of PI3-K, wortmannin and LY294002, inhibited α1-adrenoceptor-mediated activation of p70S6K and the concomitant growth of myocytes.78 The involvement of PI3-K in the hypertrophic growth of cardiac myocytes is also supported by the fact that cardiac-specific overexpression of PI3-K in transgenic mice results in the enlargement of the heart and, in contrast, the expression of a dominant negative form of PI3-K leads to an atrophied heart.88

Although the consensus for the above studies is that S6K-dependent activation of r-protein synthesis is important for protein synthesis in cardiomyocytes, few experiments have specifically addressed whether r-protein synthesis is limiting for hypertrophy induced in response to hypertrophic agents. For example, in our hands, rapamycin inhibits basal levels of protein synthesis in unstimulated cardiomyocytes, as well as in stimulated cells (RD Hannan et al., unpubl. data, 2000). Further studies in which dominant negative acting versions of S6K189 are used to quantitatively titrate out S6K1 activity are required before conclusions can be made on whether r-protein synthesis is limiting for cardiomyocyte hypertrophy. Finally, it is also formally possible that S6K1 has targets in addition to S6 or, alternatively, that rapamycin inhibits regulatory pathways in addition to mTOR/Phase1/S6K1 signalling.77 With respect to the former, we have preliminary data to demonstrate that S6K1, in addition to effects on r-proteins, also directly activates rDNA transcription in cardiomyocytes (RD Hannan et al., unpubl. data, 2000). This observation raises the intriguing possibility that S6K1 regulates, in a coordinate manner, both r-protein synthesis and rRNA synthesis and, thus, represents a critical step in the regulation of ribosome biogenesis and the growth of cardiomyocytes.

Concluding remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Regulation of translation in eukaryotes
  5. Regulation of translation in cardiac myocytes during hypertrophic growth
  6. Concluding remarks
  7. References

Current studies suggest that cardiomyocyte growth is not merely an unrelated bystander in the progression of LVH to heart failure. Comprehension of the signalling pathways and cellular mechanism(s) that regulate this anabolic process are therefore likely to be an important part of understanding the pathophysiology associated with this disease syndrome. Regardless of the stimuli and the subsequent phenotypic changes that result from those stimuli, the regulation of overall rates of protein synthesis in cardiomyocytes is the primary determinant for the growth associated with LVH. A sustained increase in protein accumulation during hypertrophy is dependent on both an increase in efficiency of translation and an elevated capacity to synthesize protein (i.e. an increase in functional ribosomes). An increase of translation efficiency during stimulation by hypertrophic agents is rapid and can be measured both in vitro and in vivo in a matter of hours. Under most circumstances, the increase of translation efficiency during hypertrophic growth is sustained and is followed by a secondary rise in ribosome biogenesis after 12–24 h.

The reason why the growth response is biphasic and its relevance to the development of hypertrophy is not completely understood. One possibility is that the increase in translational efficiency accounts for the majority of the hypertrophic growth of the heart and that the secondary increase in ribosome synthesis merely serves as a mechanism to restore the ‘hypertrophic reserve’ or capacity of the heart to mount a subsequent rapid response. However, the fact that a majority of ribosomes are present in the form of polysomes in both hypertrophic and non-hypertrophic myocytes14,41 suggests that there is limited capacity for a latent pool of ribosomes to be recruited rapidly on demand. Moreover, the demonstration that inhibiting the increase in rRNA synthesis blocks the hypertrophic response in neonatal cardiomyocytes40 suggests that the steady state level of ribosomes in non-hypertrophic cells eventually becomes functionally limiting for optimal cardiac growth.

Interestingly, the lag phase in the activation of rRNA synthesis in response to growth stimuli observed for differentiated cardiomyocytes differs from proliferating cells, where changes in rDNA transcription rates are a primary and sometimes immediate response to growth/proliferation signals.90 Thus, an alternative explanation for the biphasic response may lie in the intrinsic differences between hypertrophic and proliferative growth. For dividing cells, growth is tightly coupled to the rate of proliferation and, in response to mitogenic signals, they rapidly and concomitantly increase translational efficiency and capacity.90 This both facilitates growth and provides sufficient ribosomes to ensure ‘daughter’ cell survival. In contrast, by definition, the growth of terminally differentiated cells is uncoupled from the cell cycle and can be up to an order of magnitude lower than proliferating cells. Thus, for differentiated cells, such as cardiomyocytes, the signal for growth does not unconditionally result in the need for additional ribosomes. Instead, these cells need to distinguish between temporary increases in demand for protein synthesis, which may be achieved at the level of translational efficiency, and long-term changes in the requirements for growth, as observed during chronic pressure overload, which may require an additional increase in steady state levels of ribosomes. Thus, by delaying the response to activate rDNA transcription until absolutely necessary, cardiomyocytes do not needlessly waste energy on the synthesis of new ribosomes, one of the most energetically costly processes in the cell.

Understanding the exact origin of the molecular trigger that switches on the secondary response to increase rDNA transcription rates and ribosome biogenesis may be a key step in unravelling the complexities of cardiac growth during normal development and disease states. It is likely that new approaches to tissue-specific gene delivery and knockout applied for factors affecting translational efficiency and ribosome biogenesis in living animals will soon provide answers to these important questions.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Regulation of translation in eukaryotes
  5. Regulation of translation in cardiac myocytes during hypertrophic growth
  6. Concluding remarks
  7. References
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