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Cardiovascular Research 2005 68(2):186-196; doi:10.1016/j.cardiores.2005.06.025
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Copyright © 2005, European Society of Cardiology

Protein turnover in cardiac cell growth and survival

Nadia Hedhli, Michel Pelat and Christophe Depre*

Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry New Jersey, New Jersey Medical School, 185 South Orange Avenue, MSB G-609, Newark, NJ 07103, United States.

* Corresponding author. Email address: deprech{at}umdnj.edu

Received 11 May 2005; revised 8 June 2005; accepted 24 June 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Regulation of protein...
 3. Protein turnover and...
 4. Protein turnover and...
 5. Future directions
 References
 
Protein turnover represents the balance between protein synthesis and degradation. It can be controlled quantitatively, for instance by an activation of protein synthesis during cardiac hypertrophy or by activating protein degradation during ventricular unloading. It can also be regulated qualitatively by changing the steady state concentration of specific proteins and enzymes. The recent literature points to an emerging role for the mammalian target of rapamycin (mTOR) and for the ubiquitin–proteasome system (UPS) in this process, and both pathways interact in the regulation of cell growth and survival. We highlight the critical role played by such interaction in different cellular functions, including insulin signaling, stress response to hypoxia, adaptation to variations in workload, regulation of protein phosphatase activity, apoptosis and post-ischemic recovery. A deregulation of these pathways participates in the mechanisms of cardiac ischemia, hypertrophy and failure, and controlling their activity represents an opportunity for novel therapeutic avenues.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Regulation of protein...
 3. Protein turnover and...
 4. Protein turnover and...
 5. Future directions
 References
 
Because fully differentiated cardiac myocytes are post-mitotic cells, the regulation of cell size is the main mechanism of adaptation to variations in workload in the heart. In addition, cardiac myocytes have developed remarkable mechanisms of cytoprotection and cell survival, also due to this absence of cell division. Cellular pathways participating in cell growth, survival and death are controlled by the steady state level of regulatory proteins and enzymes, which results from a balance between the rate of protein synthesis and degradation. The recent literature points to an emerging role for the mammalian target of rapamycin (mTOR) in the synthetic pathway and for the ubiquitin–proteasome system (UPS) in the proteolytic pathway. Both pathways are regulated by growth factors, substrate availability and energy depletion. They control the normal size of the cell as well as the adaptation of cell size to changes in workload. They affect the extent of irreversible damage following ischemic injury. The main goal of this review is to illustrate how both pathways are coordinated and interact in heart. First, we describe the mechanisms regulating mTOR and the UPS, and the links between both systems (Section 2). Second, we focus on the heart to illustrate the role of mTOR and the UPS in cardiac cell growth (Section 3) and survival (Section 4), with a special emphasis on the molecular interactions between both systems.


   2. Regulation of protein turnover
Top
 Abstract
 1. Introduction
 2. Regulation of protein...
3. Protein turnover and...
 4. Protein turnover and...
 5. Future directions
 References
 
By controlling the initiation of translation, which represents the limiting step of protein synthesis, mTOR can activate the global ribosomal machinery, but it can also increase the translational rate of specific transcripts depending on their structure and post-transcriptional modifications. Protein degradation by the UPS also can be a global process, such as the degradation of denatured proteins after ischemia/reperfusion or the degradation of myofilaments during unloading, or it can be a specific process for regulatory proteins involved in cell cycle, cell growth or apoptosis.

2.1 Regulation of the mTOR pathway
Cellular pathways adapting cardiac cell size to growth factors and nutrients converge on mTOR. Rapamycin is a natural bacterial macrolide that binds the intracellular protein FKBP12. The rapamycin/FKBP12 complex inhibits mTOR upon binding on a specific C-terminal site of the enzyme [1]. The serine/threonine kinase Akt is the major mediator of mTOR activation. Akt is activated downstream phosphatidylinositol 3-kinase (PI3K) in response to the activation of growth factor receptors or G protein-coupled receptors (Fig. 1). mTOR is also activated by the MAP kinase pathway upon stimulation of the protooncogene Ras [2].


Figure 1
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  		Fig. 1 Integrated view of the mTOR pathway and the UPS. Abbreviations: AMPK, 5'AMP-activated protein kinase; Ask-1, Apoptosis signal-regulating kinase 1; GFR, Growth factor receptor; GPCR, G protein-coupled receptor; GSK-3β, Glycogen synthase kinase 3β; HIF-1{alpha}, Hypoxia-inducible factor 1{alpha}; HSP, Heat shock protein; IRS-1, Insulin receptor substrate 1; mTOR, Mammalian target of rapamycin; PDK-1, 3' phosphatidylinositol-dependent kinase 1; PI3K, Phosphatidylinositol-3-kinase; PP, Protein phosphatase; U, Ubiquitin; 26Sp, 26S proteasome. The 26S proteasome includes a 20S central subunit made of {alpha} (yellow) and β proteins (green), and capped with two 19S subunits.

 
The two most widely described substrates of mTOR are the eukaryotic translation initiation factor 4E (eIF4E)-binding protein (4E-BP) and the p70S6 kinase (S6K). Interaction between mTOR and these two substrates is controlled by the adaptor protein raptor [3]. Rapamycin dissociates mTOR from raptor, which inhibits the association of mTOR with S6K and 4E-BP [3]. 4E-BP is a family of translational repressors that bind eIF4E, a translation initiation factor adapting the 5' cap of messenger RNAs on the ribosome. When eIF4E is bound to 4E-BP, this adaptor role is blocked and protein translation is interrupted. Upon phosphorylation by mTOR, 4E-BP releases eIF4E, which activates translation. The S6K proteins exist in two isoforms, S6K1 and S6K2, which are produced from different genes. Each isoform is expressed either as a cytosolic protein, or as an alternatively spliced variant with a nuclear localization signal. S6K phosphorylates the 40S ribosomal subunit S6, which promotes the binding and translation of messenger RNAs containing a 4–14 nucleotide polypyrimidine stretch adjacent to the 5' cap [4]. These 5' TOP (terminal oligopyrimidine tract) mRNAs encode mainly ribosomal proteins and elongation factors. S6K also activates translation by phosphorylation of the factor eIF4B, which participates in the unwinding of the 5' UTR of mRNAs upon binding to the ribosome.

Both targets of mTOR participate in a relatively similar function, which is to optimize the attachment of mRNAs to the ribosome. This activity is crucial because the binding of transcripts on ribosomes and their unwinding represents the rate-limiting step of translation initiation [5]. mTOR activates translation not only by phosphorylation of its substrates, but also by inhibiting their dephosphorylation through the control of the protein phosphatase (PP) 2A (PP2A) [6]. mTOR inactivates PP2A by "locking" the phosphatase with the adapter protein {alpha}4 [7]. Inhibition of this interaction is followed by dephosphorylation of S6K [8].

Direct phosphorylation of mTOR by Akt is not necessarily accompanied by a change in mTOR enzymatic activity [9], suggesting an alternative mechanism of regulation. Such mechanism was unraveled recently by studies on the tuberous sclerosis complex (TSC). Tuberous sclerosis is an autosomal dominant disease characterized by the presence of hamartomas, or benign tumors growing in multiple organs [10]. The disease is due to a mutation in one of two interacting tumor suppressors, TSC1 (or hamartin) and TSC2 (or tuberin) [11]. The TSC1/TSC2 complex inhibits mTOR and represents a sensitive mechanism controlling cell growth in response to nutrient availability and growth factors (Fig. 1). This regulation relies on the fact that TSC2 is a substrate for both Akt and the 5' AMP-activated protein kinase (AMPK). AMPK is a stress-responsive kinase activated by an increase in the AMP/ATP ratio, which typically occurs in the heart during myocardial ischemia. Activation of AMPK is followed by a stimulation of anaerobic utilization of glucose to provide more ATP, and by a genomic activation of glucose utilization through the stabilization of the hypoxia-inducible transcription factor 1-{alpha} (HIF-1{alpha}). Whereas the phosphorylation of TSC2 by Akt inhibits the TSC complex [12], the phosphorylation by AMPK on another site has a reciprocal effect [13]. Therefore, Akt, via TSC1/TSC2 inhibition, activates mTOR, whereas AMPK activates the TSC complex and thereby inhibits mTOR. When the PI3K pathway is activated, the Akt-mediated inhibition of TSC1/TSC2 stimulates mTOR, which induces cell growth (Fig. 1). When AMPK is activated by energy starvation (lack of nutrients and oxygen, such as ischemia), the mTOR pathway is inhibited.

The regulation of mTOR by TSC is mediated by the small GTPase protein Rheb (Fig. 1), which exists in equilibrium between two configurations, the GDP-bound inactive form and the GTP-bound active form [14]. TSC2 is a Rheb GTPase-activating protein that promotes the Rheb-GDP configuration. This occurs when TSC2 is activated by AMPK, which results in the inhibition of mTOR activity. When Akt inhibits TSC2, Rheb remains in the active, GTP-bound configuration and stimulates mTOR (Fig. 1).

A novel mTOR-binding adapter named rictor was recently discovered [15], which is mutually exclusive with raptor (Fig. 1). Remarkably, when mTOR binds rictor instead of raptor, the complex becomes insensitive to rapamycin [15]. In addition, mTOR/rictor does not phosphorylate S6K but it phosphorylates Akt and indirectly activates PKC{alpha} [15]. Presently, it is not known whether rictor is expressed in the heart. If this is the case, it would represent a powerful tool by which mTOR can switch between a function in cell growth (phosphorylation of S6K by mTOR-raptor) and a role in cell survival (phosphorylation of Akt by mTOR-rictor).

2.2 Regulation of the ubiquitin–proteasome system
The UPS represents the most important mechanism of proteolysis in the cardiac cell, degrading about 80% of the intracellular proteins [16]. The UPS has two main functions that can be defined as quality control and biological control. The quality control includes the degradation of denatured and misfolded proteins. The biological control consists in modifying the steady state level of specific proteins acting as regulators of biological functions, which are usually expressed in low amount and have a short half-life. This second role is particularly important to adapt the expression of proteins controlling the progression of the cell cycle (cyclins and inhibitors of cyclin-dependent kinases), the metabolic adaptation to substrate supply (glucose transporters, acyl-carnitine shuttles) or the activity of signaling pathways (stabilization of NF{kappa}B or HIF-1{alpha}, for instance).

Ubiquitin binds the proteins to be degraded (ubiquitination) and transfers them to the proteasome for degradation (proteolysis). Ubiquitination results from the formation of a peptidyl bond between the C-terminal carboxy group of ubiquitin and the {varepsilon}-amino group of a lysine residue of the target protein, followed by the synthesis of a multi-ubiquitin chain in which the {varepsilon}-amino group of the lysine-48 residue of the conjugated ubiquitin is bound to the C-terminal carboxy group of the incoming ubiquitin moiety (Fig. 1). Three specific enzymes catalyze this mechanism of conjugation. The ubiquitin-activating enzyme (E1) binds ubiquitin by a thioester bond in a reaction that requires ATP hydrolysis. The ubiquitin-conjugating enzyme (E2) transfers the ubiquitin moiety from E1 to the target protein, which has been recognized by the ubiquitin–protein ligase (E3). E3 ubiquitin ligase catalyzes the formation of the peptidyl bond between ubiquitin and the target protein. Once the first ubiquitin is bound to its target, the elongation of the poly-ubiquitin chain is performed by E2 and E3.

Several mechanisms tightly regulate ubiquitination. The first mechanism, known as the N-end rule, consists in the recognition by E3 of a specific amino acid (especially arginine, lysine or aspartate) at the N-terminus of the target protein. In other cases, the participation of the heat-shock protein Hsc70 is essential for the conjugation reaction between E3 and the target protein [17]. Some proteins may be targeted by E3 when they are phosphorylated on specific residues, as described below for I{kappa}B. E3 also recognizes proteins that have been altered by other post-translational modifications, such as acetylation or methylation [18]. In addition, there are multiple isoforms of E2 and E3 enzymes. The pairing between E2 and E3 is isoform-specific, and each E2/E3 complex recognizes only a limited subset of target proteins.

After poly-ubiquitination, the target protein is directed to the proteasome for degradation (Fig. 1). The 26S proteasome is made of two subunits, the catalytic 20S unit and the regulatory 19S unit. The 20S unit is a "barrel-shaped" structure that is capped on each side by a 19S unit. The barrel is made of four stacked rings, composed of a 7-mer of {alpha} proteins in the outer rings and β proteins in the two inner rings, with the catalytic activity located inside the β rings [19]. The 19S cap contains regulatory proteins binding and denaturing the target, releasing ubiquitin, and leading the denatured target inside the central barrel. Inside the 20S subunit, the protein is degraded by five proteolytic activities: trypsin-like (cutting the petidyl bond involving basic amino acids), chimotrypsin-like (for hydrophobic amino acids), peptidylglutamyl hydrolase (acid amino acids), branched-chain acyl hydrolase and neutral acyl hydrolase [20]. These catalytic activities interact with each other to achieve a digestion that results in the production of peptides containing less than 20 amino acids, which will be hydrolyzed completely by cytosolic peptidases. Proteolytic activity can be modulated by phosphorylation of the 20S unit, although the full consequences of proteasome phosphorylation remain to be determined [21]. Also, the analysis of the structure, composition and regulation of the proteasome in the heart in particular is at its earliest stage. It is likely that precise characterization of the proteasome in human cells will unravel novel mechanisms of regulation.

2.3 Interactions between mTOR and the UPS
In addition to the interactions between mTOR and the UPS that have been described in cardiac cell growth and survival (Sections 3 and 4), important links between mTOR and the UPS include insulin sensitivity through the regulation of the insulin receptor substrate-1 (IRS-1), and the regulation of protein phosphatases. These links are described here because, although they are of potential importance for the myocardium, they have not been investigated fully in that specific tissue.

2.3.1 Insulin signaling
Binding of insulin to its receptor results in a tyrosine phosphorylation of IRS-1, which docks the regulatory subunit (p85) of PI3K. When associated to IRS-1, p85 binds the catalytic (p110) subunit, which activates PI3K, a lipid kinase that phosphorylates the membrane-bound phosphatidylinositol 4,5-bisphosphate on the 3' position. This phosphorylation creates a membrane-binding site for the serine/threonine kinase Akt (Fig. 1). Upon binding to the membrane, Akt is activated by the 3'-phosphoinositide-dependent protein kinase 1 (PDK 1), which also activates PKC{varepsilon}. As mentioned above, Akt in turn activates mTOR through the TSC complex. Akt also inhibits by phosphorylation the glycogen synthase kinase-3β (GSK-3β). This signaling pattern explains the pleiotropic effects of insulin on cell growth (mTOR), anti-apoptosis (Akt), cytoprotection (PKC{varepsilon}) and glucose metabolism (GSK-3β). Such powerful system needs to be controlled by a negative feedback mechanism. It has been known for some time that prolonged exposure to insulin decreases the content in IRS-1 protein [22]. The precise molecular mechanism was elucidated recently with the observation that IRS-1 is a substrate for S6K [23]. Upon chronic activation of the PI3K/Akt/mTOR/S6K pathway, S6K phosphorylates IRS-1 on specific serine residues. Although this phosphorylation pattern does not affect the function of IRS-1, it tags the protein for ubiquitination and subsequent degradation by the proteasome [24]. This mechanism of regulation explains why a deletion of S6K1 increases insulin sensitivity [25].

2.3.2 Ubiquitination of {alpha}4-PP2A
We mentioned above that mTOR restrains the activity of PP2A through {alpha}4, which controls the action of PP2A on S6K1 and other substrates. Several studies in non-cardiac systems have shown that an excessive activity of PP2A or its dissociation from {alpha}4 leads to apoptosis [26,27]. This mechanism of regulation could be particularly significant in the heart because overexpression of PP2A induces dilated cardiomyopathy with cell death and fibrosis [28]. In addition, {alpha}4 binds the Midline-1 (Mid1) protein, which is an E3 ubiquitin ligase targeting specifically PP2A for proteasomal degradation [29].

2.3.3 Regulation of PP5
The apoptosis signal-regulating kinase 1 (ASK-1) is a MAP kinase kinase kinase that is activated by hypoxia, amino acid deprivation or oxidative stress [30]. ASK-1 phosphorylates the MAP kinase JNK, which results in the phosphorylation of c-Jun and in subsequent cell death by apoptosis (Fig. 1). This system can be activated in the heart in response to oxidative stress [31]. The activity of ASK-1 can be inhibited by direct interaction with the protein phosphatase 5 (PP5), and this complex physically associates with PP2A [32]. Upon inhibition of mTOR by rapamycin, PP2A dissociates from ASK-1/PP5, which is followed by an inhibition of PP5 that results in the activation of the pro-apoptotic ASK-1 [32]. Reciprocally, expression of PP5 is increased by HIF-1{alpha} [33]. Therefore, through the regulation of both HIF-1{alpha} and PP5, the interaction between mTOR and the UPS controls a dual mechanism of cell survival (Fig. 1).


   3. Protein turnover and cardiac cell growth
Top
 Abstract
 1. Introduction
 2. Regulation of protein...
 3. Protein turnover and...
4. Protein turnover and...
 5. Future directions
 References
 
Both mTOR and the UPS control the adaptation of cardiac cell size in response to increased workload (hypertrophy), and in the reciprocal condition of ventricular unloading (atrophy). These systems also determine the normal size of a cardiac cell.

3.1 Interaction between mTOR and the UPS in cardiac cell growth
IRS-1 is activated not only by insulin but also by other growth factors, including the insulin-like growth factor 1 (IGF-1). The release of IGF-1 by cardiac myocytes induces hypertrophy [34], through the IRS-1-mediated activation of PI3K/Akt/mTOR. There is a second mechanism, however, that also involves the UPS and the transcription factor Foxo (Fig. 2). Foxo represents a group of proteins that belong to the forkhead family of transcription factors and which regulates the expression of genes promoting apoptosis, cell cycle progression and differentiation. Phosphorylation by Akt promotes the transcriptional inactivation and nuclear exclusion of Foxo, followed by its ubiquitination and proteasomal degradation [35]. This inhibition is one of the mechanisms by which Akt sustains cell survival. Foxo also activates the transcription of two ubiquitin ligases, atrogin-1 and MurF1, which are expressed in skeletal and cardiac muscles [36]. These ubiquitin ligases target for degradation several components of the sarcomere, including titin and troponin I. Their activation is followed by atrophy in the skeletal muscle [37]. A similar mechanism was recently demonstrated in cardiac myocytes, where overexpression of Foxo3a blocks hypertrophy induced by IGF-1 or stretch, and induces the expression of atrogin-1 [38]. Therefore, activation of growth factor receptors concomitantly activates protein synthesis by mTOR and prevents protein degradation by the UPS (Fig. 2).


Figure 2
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  		Fig. 2 Role of mTOR and the UPS in cardiac cell growth. Abbreviations: AC, Adenylate cyclase; β-AR, Beta-adrenergic receptor; CREB, cAMP response element binding protein; ICER, Inducible cAMP early repressor; IGF-1, Insulin-like growth factor-1; PKA, cAMP-dependent protein kinase A. Other abbreviations as in Fig. 1.

 
3.2 Specific effects of mTOR on cardiac cell growth
Increased cardiac cell size is observed upon stimulation of PI3K and after inhibition of PTEN, the enzyme degrading inositol 3,4,5-trisphosphate [39]. This effect is reproduced by a constitutively active mutant (ca) of Akt [40]. The cardiac hypertrophy resulting from the expression of caAkt is accompanied by an increased phosphorylation of both 4E-BP1 and S6K1, and is attenuated after administration of rapamycin, which further demonstrates that mTOR is the mediating effector [40]. In skeletal muscles, a forced activation of mTOR can prevent muscle atrophy following denervation or unloading [41]. This latter aspect could be particularly relevant for the failing heart upon unloading with a left ventricular assist device.

The mTOR pathway also participates in the hypertrophic response to pressure overload. In vitro, rapamycin blocks myocyte hypertrophy induced by angiotensin II or catecholamines [42]. The impact of rapamycin treatment on cardiac cell size and contractile function during pressure overload induced by aortic banding has been reported in rodents [43,44]. In this condition, rapamycin blunts the increase in heart weight by about 70% and the increase in cell size by about 60% without inducing ventricular dysfunction [43]. However, this study considered only very short time points after banding (between a few hours and 1 week) and did not address the potential hemodynamic effects of the drug. Rapamycin can modify aortic blood pressure and thereby can affect the trans-stenotic gradient, which would have an impact on cardiac cell size. This potential effect is compatible with the observation that rapamycin can reduce established hypertrophy when administered 1 week after aortic banding [44]. This decrease in cardiac cell size is accompanied by an improvement in contractile function [44], which raises the possibility that rapamycin participates in the control of cardiac contractility, maybe through the control of the ryanodine receptor by FKBP12 [45].

3.3 Specific effects of the UPS on cardiac cell growth
Few studies addressed the role of UPS in the control of cardiac cell size, as opposed to the extensive work conducted in skeletal muscle. This is even more surprising when considering that both ubiquitin expression and proteasomal activity are higher in the heart than in skeletal muscles [46]. The increased stress that results from overload is accompanied by increased production of damaged and denatured proteins, which must be degraded to avoid the activation of pro-apoptotic signals. Also, a qualitative regulation of E3 ubiquitin ligases during overload will lead to the degradation of specific molecules interfering with an increase in cell size, such as tumor suppressors or inhibitors of cyclin-dependent kinases. The UPS is also activated during ventricular unloading [47]. In that case, the activation of the UPS results in increased degradation of contractile proteins, which leads to cardiac atrophy (Fig. 2). This condition also activates mTOR [47], as an attempt to balance proteolysis by a residual synthesis of proteins. Inhibition of mTOR in the unloaded heart accelerates atrophy [47].

More specifically, a mechanism by which the UPS can control cardiac hypertrophy involves the regulation of the inducible cyclic AMP early repressor (ICER). A major pathway leading to cardiac hypertrophy starts with the activation of the adrenergic system (Fig. 2). Upon stimulation of β-adrenergic receptors, increased cAMP production followed by activation of the cAMP-dependent protein kinase leads to an activation of the cAMP response element binding protein (CREB), a transcription factor activating the expression of genes involved in cardiac cell growth, contractility and protection against apoptosis [48]. By binding the cAMP response element, ICER prevents the transactivation of these genes by CREB, which is followed by an inhibition of hypertrophy and increased apoptosis [49]. As its name indicates, ICER is an inducible molecule, which means that its activity results more from its expression level than from post-translational modifications or protein–protein interactions. It was shown in cardiac myocytes that the UPS controls the degradation rate of ICER, and thereby its activity [50], by the binding of specific E3 ligases that are themselves transcriptionally regulated [51].

Data collected from both patients and animal models offer concrete evidence for a regulation of the UPS in chronic conditions of prolonged left ventricular hypertrophy and heart failure. In patients with aortic valve stenosis, for example, UPS activity (as measured by autophagy) was increased by 2-fold versus control during the stage of compensated hypertrophy, and was further increased by 12-fold versus control after the transition into heart failure [52]. Increased expression of regulatory proteins of the UPS was further confirmed by functional proteomics in myocardial samples from patients with idiopathic, dilated cardiomyopathy [53]. It is likely that the increased activity of the UPS during myocardial hypertrophy represents an adaptive mechanism. An increased rate of peptide synthesis inevitably leads to accumulation of misfolded proteins that need to be degraded to keep available the much needed pool of amino acids [54]. It is possible that a dysfunction in proper ubiquitination and proteasomal degradation of proteins during chronic hypertrophy might be interpreted as a signal of decompensation that would precipitate heart failure [55].


   4. Protein turnover and cardiac cell survival
Top
 Abstract
 1. Introduction
 2. Regulation of protein...
 3. Protein turnover and...
 4. Protein turnover and...
5. Future directions
 References
 
Although the role of mTOR and the UPS in the control of cell size mainly involves the quantitative aspect of protein turnover, their role in cell survival represents an illustration of the qualitative regulation of these pathways. By changing the translation, stability or degradation of specific effectors and adaptor proteins, both mTOR and the UPS are strongly involved in the resistance against cell death.

4.1 Interaction between mTOR and the UPS in cell survival
A major mediator of the cardiac stress response is HIF-1{alpha}, a transcription factor that transactivates genes encoding proteins involved in cell survival during hypoxia, such as glucose transporters, glycolytic enzymes, growth factors, heme oxygenase and the inducible isoform of nitric oxide synthase (Fig. 3). Expression of these genes is activated when HIF-1{alpha} forms a heterodimer with HIF-1β and binds to the promoter region of the target genes. Both HIF subunits are expressed constitutively. However, in presence of oxygen, HIF-1{alpha} is hydroxylated on two specific proline residues present in an oxygen-dependent degradation domain (ODD). The hydroxylation is catalyzed by prolyl 4-hydroxylases using O2 as substrate [56]. These hydroxyprolines are recognized by the Von Hippel–Lindau (VHL) protein, a tumor suppressor which, together with cofactors, acts as an E3 ubiquitin ligase for HIF-1{alpha} [57]. Because O2 is the limiting substrate, this mechanism of degradation is interrupted in hypoxia. Inhibition of the targeting of the ODD by VHL in heart leads to increased glucose utilization and cardioprotection during metabolic inhibition [58]. In addition, O2 is the substrate of the asparagine hydroxylase FIH-1 (Factor Inhibiting HIF-1), which catalyzes the hydroxylation of an asparagine residue in the C-terminal activation domain of HIF-1{alpha} and thereby prevents its interaction with transcriptional co-activators [59].


Figure 3
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  		Fig. 3 Role of mTOR and the UPS in cardiac cell survival. Abbreviations: HIF-1β, Hypoxia-inducible factor 1β; VHL, Von Hippel–Lindau protein. Other abbreviations as in Fig. 1.

 
Reciprocally, the PI3K/Akt/mTOR pathway increases HIF-1{alpha} stability (Fig. 3). First, phosphorylation of 4E-BP1 and S6K1 by mTOR stimulates the translation of the HIF-1{alpha} transcript by improving the docking of the mRNA on the ribosome. Second, mTOR stabilizes HIF-1{alpha} by targeting the ODD. It is known that HIF-1{alpha} can be phosphorylated, and the ODD contains putative target sites for mTOR. However, it remains undetermined whether the ODD is a direct substrate for mTOR. Third, Akt inhibits pro-apoptotic effectors, including GSK-3β and Foxo, which leads to a transcriptional activation of genes encoding the cytoprotective heat-shock proteins HSP70 and HSP90 [60]. As chaperones, HSPs bind multiple signaling molecules that promote cell survival, including HIF-1{alpha}. The half-life of HIF-1{alpha} is markedly increased upon binding to HSP70 and HSP90, which mask the ODD and thereby prevent the binding of VHL [61].

4.2 Specific role of mTOR in cardioprotection
The mechanism of stabilization of HIF-1{alpha} described above represents an important survival mechanism by mTOR, but other potential mechanisms are suggested from the literature. For instance, the regulation of PP2A and PP5 by mTOR (see above) is potentially important for cardiac cell survival, although it has not been fully investigated. mTOR can localize in the nucleus [62], where it could potentially regulate the activity of transcription factors. As mentioned above, both S6K isoforms have a nuclear splice variant, which suggests another role for this enzyme beside the phosphorylation of the ribosomal subunit S6. These potential pathways remain to be tested in the heart.

Several reports indicate that mTOR is involved in preconditioning, i.e., the protection conferred against irreversible damage by short episodes of ischemia/reperfusion preceding a potentially lethal ischemia. The time course of cardioprotection by preconditioning includes an early phase (starting immediately after ischemia and lasting for 12 h), due to the activation of pro-survival signaling pathways, and a second window (1 to 3 days after ischemic injury) that results from changes in gene expression. Activation of the PI3K pathway participates in the mechanisms of early preconditioning [63]. A role for mTOR has been addressed recently by pharmacological inhibition with rapamycin, which blocks the second window of preconditioning [64]. Inhibition of mTOR also prevents the cardioprotective effect of insulin at reperfusion [65]. In addition, rapamycin abrogates the cardioprotection conferred by opioids [66], which represents a pharmacological form of preconditioning. More work is needed to decipher the molecular mechanisms of cardioprotection by mTOR during preconditioning, and especially to determine whether it is mediated by HIF-1{alpha}.

4.3 Inhibition of the UPS and cardiac cell survival
Inhibition of the UPS promotes cardiac cell survival during ischemia/reperfusion [67,68]. Accordingly, expression of the proteasome is decreased in hibernating myocardium [69], which represents an adaptive condition to prolonged ischemia in which contractile function is decreased and survival mechanisms are activated [70]. Inhibition of the UPS in the ischemic heart limits its role in protein quality control. Accumulation of denatured proteins triggers a heat shock response that leads to the induction or increased expression of multiple HSPs (Fig. 3). As chaperones, HSPs bind to the denatured proteins in order to restore their proper configuration. In addition to this chaperone function, most HSPs are also involved in cell survival mechanisms by blocking the activation of pro-apoptotic pathways. As a consequence, the upregulation of HSPs by proteasome inhibitors before ischemia confers protection.

Another mechanism relates to the regulation of the transcription factor NF{kappa}B, which participates in the inflammatory reaction triggered by necrotic cells following ischemia. NF{kappa}B is a heterodimer bound in the cytoplasm to the inhibitory protein I{kappa}B. Upon stimulation by stress factors (hypoxia, reactive oxygen species, lipopolysaccharide, TNF{alpha}...), I{kappa}B is phosphorylated by specific I{kappa}B kinases, which targets I{kappa}B for ubiquitination and proteolysis. As a result, NF{kappa}B moves to the nucleus and activates the transcription of genes encoding cytokines, adhesion molecules and interleukins, which activate the inflammatory reaction characterizing the acute phase of myocardial infarction. Blocking NF{kappa}B activity with proteasome inhibitors limits the infiltration of ischemic myocardium by leukocytes and decreases infarct size by about 50% in vivo [68,71].

However, NF{kappa}B also triggers the expression of the inducible isoform of nitric oxide synthase, the main effector of the late phase of ischemic preconditioning. Accordingly, inhibition of NF{kappa}B activity prevents ischemic preconditioning [72]. In addition to inflammatory mediators, NF{kappa}B transactivates genes encoding anti-apoptotic proteins, and promotes the activity of survival pathways [73]. Whether proteasome inhibitors block the cardioprotection conferred by ischemic preconditioning has not been investigated. It is well possible that an inhibition of NF{kappa}B would be beneficial when performed in the inflammatory cells infiltrating the ischemic myocardium, but deleterious when performed in cardiac myocytes.


   5. Future directions
Top
 Abstract
 1. Introduction
 2. Regulation of protein...
 3. Protein turnover and...
 4. Protein turnover and...
 5. Future directions
References
 
Coordinated regulation of mTOR and the UPS represents a powerful mechanism controlling cell growth and survival. Recent findings highlight both the biological and therapeutic impact of these pathways.

5.1 Biological impact
Although the function of mTOR and the UPS in cardiac cell growth and survival is rapidly emerging, multiple molecular mechanisms remain to be explored. It has been shown only very recently that mTOR/rictor can activate Akt, and the potential consequence of such activation on cardiac survival mechanisms is unexplored. The transcription factors potentially regulated by mTOR remain largely unknown. How mTOR stabilizes HIF-1{alpha} also remains to be elucidated. Other kinases have a structure similar to mTOR but their function remains largely unknown. This includes SMG-1, a member of the phosphatidylinositol kinase-related kinases [74], which phosphorylates 4E-BP1 and which is under the control of PI3K. SMG-1 has a rapamycin-FKBP12 binding domain, but a single amino acid substitution in this domain renders SMG-1 insensitive to the inhibitor [74]. SMG-1 is expressed in the heart, where it has not been characterized.

Both the composition and the regulation of expression of the proteasome subunits remain to be investigated in the heart. Multiple subunits can be phosphorylated, although the impact on the catalytic activity of the proteasome remains to be determined. The fact that IGF-1 controls the expression of atrogin-1 suggests that other E3 ubiquitin ligases can be regulated. A proteomic profile of E3 ligases during cardiac hypertrophy or ischemia would improve our understanding of the mechanisms regulating UPS activity.

5.2 Therapeutic impact
The same mechanisms described above for the heart participate in oncogenesis. Inhibitors of mTOR and the UPS are currently tested in oncology trials. Heart disease and cancer represent the two main forms of morbidity and mortality in western countries, counting to up to 60% of the total causes of mortality. Although these two forms of disease appear very different, they rely on identical biological processes that relate to the death or survival of the cell. Whereas the purpose of cancer research is to elucidate new ways to block cell proliferation, to provoke cell death and to prevent angiogenesis, a major goal of cardiovascular research is to improve cell survival mechanisms, to reenter the cardiac myocyte in the cell cycle, and to promote myocardial revascularization. Due to this biological similarity, the heart may represent a model to investigate the survival mechanisms that relate to cancer development and cancer research may open novel avenues for cardiac salvage.

Novel transgenic models not only illustrate the specific role of mTOR in oncogenesis, but also help to better define the therapeutic rationale to manipulate the activity of this pathway in cancer cells. For example, in a transgenic form of Akt-dependent prostate cancer, inhibition of mTOR by the rapamycin derivative RAD001 blunts the expression of HIF-1{alpha}, promotes apoptosis [75] and decreases metastatic spreading by limiting the production of vascular endothelial growth factor [76]. The interest of such approach includes the generation of a transgenic form of cancer in a suitable animal model (as opposed to implanting human tumors in nude mice), and the effect thereupon of a drug that is already used in clinical trials. This model tracks the specific effects of mTOR inhibition in this particular form of cancer and thereby exposes the molecular markers that demonstrate a therapeutic response of the tumor. It also unravels the potential activation of inducible mechanisms of drug resistance, such as an increased expression of the anti-apoptotic effector Bcl-2 [75]. Based on such studies, inhibitors of Bcl-2 have been developed that block the progression and induce some regression of tumors [77]. Another study in the same line of research showed that 4E-BP1 acts as a tumor suppressor by limiting the activity of eIF4E [78], whereas transgenic overexpression of eIF4E promotes malignancies [79]. The UPS also participates in the development and progression of cancer. For instance, the ubiquitination and degradation of the tumor suppressors p53 and β-catenin is altered in some forms of neoplasia. Proteasome inhibitors are used in clinical practice to reverse the drug resistance during chemotherapy for myeloma, lymphoma, renal cancer or lung cancer [80].

In conclusion, mTOR and the UPS represent two pathways that control the life and death of the cell, and which are potentially involved in multiple forms of disease. The intense interest in these pathways is explained both by the discoveries of novel cellular functions, and by the possibility of designing new families of drugs. Therefore, it is likely that the potential therapeutic aspect of controlling protein turnover is just beginning to unravel.


   Notes
 
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   References
Top
 Abstract
 1. Introduction
 2. Regulation of protein...
 3. Protein turnover and...
 4. Protein turnover and...
 5. Future directions
 References
 

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