The ubiquitin proteasome system: Focus on the heart

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1 Cardiovascular Research 70 (2006) Review The ubiquitin proteasome system: Focus on the heart Oliver Zolk *, Carolus Schenke, Antonio Sarikas Abstract Institute of Experimental and Clinical Pharmacology and Toxicology, Friedrich-Alexander-University Erlangen-Nuremberg, Germany Received 29 September 2005; received in revised form 5 December 2005; accepted 28 December 2005 Available online 23 February 2006 Time for primary review 27 days Proteasomes are the main non-lysosomal multicatalytic protease complexes that are involved in the degradation of most intracellular proteins. The substrate proteins are marked by ubiquitin, which serves as a tag for their regulated proteasomal destruction. One major function of the ubiquitin proteasome system (UPS) is to prevent accumulation of non-functional, potentially toxic proteins. Moreover, it has become clear that the UPS is involved in most aspects of eukaryotic biology, such as intracellular signaling, transcriptional control, or regulation of cell death. Recent studies demonstrated that the UPS regulates receptor internalization, hypertrophic response, apoptosis, and tolerance to ischemia and reperfusion in cardiomyocytes. Since structural remodeling of the myocardium, ischemia reperfusion injury, and myocardial cell loss are important components in the genesis of progressive heart failure, these findings suggest a pathophysiological role of the UPS. This review briefly summarizes present knowledge about structure and function of the proteasome in the heart and discusses the relevance of the UPS for cardiac diseases. D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. Keywords: Cardiomyopathy; Heart failure; Ischemia reperfusion; Proteasome; Proteasome inhibitor; Ubiquitin 1. Introduction In view of the intimate involvement of different types of protease in maintaining cellular structure and function, the role of proteases in the pathogenesis and progression of various cardiac diseases has become a topic of recent research. This article focuses on an important pathway for protein degradation, namely the ubiquitin proteasome system (UPS), and reviews its physiological and pathophysiological relevance in cardiac biology. * Corresponding author. Institut für Experimentelle und Klinische Pharmakologie und Toxikologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Fahrstr. 17, Erlangen, Germany. Tel.: ; fax: address: Zolk@pharmakologie.uni-erlangen.de (O. Zolk). 2. Structure and function of the ubiquitin proteasome system (UPS) Cardiac proteins are in a dynamic state of continual degradation and resynthesis. This process is highly selective, precisely regulated and pivotal for normal cellular function. Proteases are located in a number of organelles. Among these, the lysosomes and the proteasomes play important roles in the degradation of cardiac proteins. While the lysosomes degrade the majority of endocytosed (membrane) proteins, the ubiquitin proteasome system (UPS) degrades most long- and short-lived normal and abnormal intracellular proteins [1]. In fact, the bulk of proteins in mammalian cells (up to 80 90% of all intracellular proteins) are degraded via the UPS, which is hence considered to be the major pathway of intracellular protein degradation. In this pathway, which is present in both the nucleus and the cytosol, most substrates are first marked for degradation by covalent linkage to multiple ubiquitin molecules. Ubiquitin, an evolutionary highly conserved 76 amino acid protein, is covalently linked to proteins in a /$ - see front matter D 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved. doi: /j.cardiores

2 O. Zolk et al. / Cardiovascular Research 70 (2006) multistep process (Fig. 1A) involving E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) enzymes (Tables 1 and 2). Polyubiquitin chains are assembled via an isopeptide linkage between the lysine residue of the previous ubiquitin and the C-terminal glycine residue of the subsequent Fig. 1. (A) The conjugation of ubiquitin (Ub) to substrates usually involves three steps: an initial activation step catalyzed by E1; an intermediate step in which the ubiquitin is covalently linked to a conjugating enzyme, E2; and a final step in which the ubiquitin reaches its ultimate destination of the substrate amino group. The last step is facilitated by the E3 ligase enzyme family. Some types of E3s facilitate ubiquitin conjugation to the substrate directly from E2 by acting as a bridging factor. Another type of E3 forms a ubiquitin thiol ester intermediate before the ubiquitin is transferred to the substrate. After the linkage of a polyubiquitin chain onto the protein that is to be degraded, its recognition occurs at the 19S cap of the 26S proteasome. Degradation is brought about by the proteolytic activity of a pair of three different h-subunits, h 1, h 2, and h 5, each located in the cylinder of the 20S core unit of the 26S proteasome. (B) Schematic representation of the ubiquitin-conjugation cascade. Eukaryotic cells contain one ubiquitin-activating enzyme, E1, but multiple E2s and E3s. Substrate specificity depends mainly on the identity of the E3 ligase.

3 412 O. Zolk et al. / Cardiovascular Research 70 (2006) Table 1 E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzymes expressed in the heart and changes in cardiac disease Expression investigated References E1 (synonyms) Ubiquitin-activating enzyme E1 (UBE1) Protein (rat heart) [72] Protein (human left ventricular myocardium): unchanged in human heart failure [34] E2 (synonyms) Ubiquitin-conjugating enzyme E2N (UBE2N) mrna (human heart) [73] Ubiquitin-conjugating enzyme E2I (UBE2I, homolog of yeast UBC9) mrna (human heart) [74] Ubiquitin-conjugating enzyme E2D 2 (homolog of S. cerevisiae UBC4/5, ubiquitin-conjugating enzyme UBCH5B, UBCH5B, UBC4) mrna (human right atrial appendages): up-regulated in patients with atrial fibrillation [75] Ubiquitin-conjugating enzyme E2G 2 (UBE2G2, homolog of S. cerevisiae UBC7) Human Ubc2 (UBE2A/UBE2B, RAD6 homolog) ubiquitin. Due to the presence of seven lysine residues in the ubiquitin molecule, different multiubiquitin chains can be formed, depending on the lysine residues used for ubiquitin ubiquitin linkage. Monoubiquitins and structurally distinct polyubiquitin chains generally signal different fates for their target proteins. For example, chains of four or more ubiquitin moieties linked via Lys48 of ubiquitin usually signal proteasome proteolysis, whereas monoubiquitin usually acts as a signal for the internalization and subsequent endosomal sorting of many cell-surface proteins [2]. The process of ubiquitination is balanced by the process of de-ubiquitination, which is mediated by a number of enzymes (Table 3). Once marked by polyubiquitin chains, proteins are rapidly degraded by the 26S proteasome, which is a 2000-kDa ATP-dependent proteolytic complex (Fig. 1A). This large structure contains the central 20S proteasome, in which proteins are cleaved, and two 19S complexes, which provide substrate specificity and regulation. A detailed discussion of the structure of the proteasome is beyond the scope of this article, and the reader is referred to earlier reviews [3]. Notably, the catalytic activities of the proteasome most likely decline during senescence. Results of studies on the effects of aging on proteasome from rat cardiac tissue demonstrate age-dependent inhibition of the catalytic activities of the enzyme [4]. This was due, in part, mrna (human heart) [76] mrna (human left ventricular myocardium): up-regulated in end-stage heart failure [30] Protein (human left ventricular myocardium): [34] up-regulated in human heart failure to an age-dependent loss in the cardiac content and changes in the subunit composition of the 20S proteasome. Moreover, the presence of inhibitory proteins within the cellular milieu that may exist at greater concentrations in myocytes from senescent animals has been suggested [4]. One major function of the UPS is to protect the cell against misfolded or damaged proteins. The folding of newly synthesized proteins to their proper conformation might often be unsuccessful. As many as 30% of the newly synthesized proteins in eukaryotes might undergo degradation within minutes of their synthesis [5] and it seems likely that many newly synthesized polypeptides are destroyed because of the inherent inefficiency of protein folding [1]. Once synthesized and properly folded, proteins are constantly exposed to highly reactive molecules and to conditions that favor denaturation, in a process that is termed protein aging. The proteasome constitutes the cell s quality control system, which continually monitors proteins for signs of misfolding, postsynthetic denaturation or chemical damage [6]. Which of the E2s and E3s are involved in this cellular quality control system, and how they recognize misfolded or aged cardiac proteins are still open questions. Although the UPS first came to light in the context of protein destruction, it is now clear that it can also carry out various tasks in the heart, controlling activities as diverse as Table 2 E3 ubiquitin ligases expressed in the heart E3 (synonyms) Type Substrates (in the heart or References non-cardiac tissues and cells) STUB1 (CHIP) Single subunit U-box E3 ErbB2, p53 [77,78] MuRF1 Single subunit RING E3 Cardiac troponin I [79] Mdm2 (p53-binding protein, oncoprotein MDM2) Single subunit RING E3 p53, h-arrestin-2, h 2 -adrenoceptor [18,80,81] Neurl2 (Ozz) VCB multiple subunit RING E3 h-catenin [82] F-box only protein 32 (MAFbx, atrogin-1) SCF multiple subunit RING E3 Calcineurin A [11,27] UBE3A (human papilloma virus E6-associated protein, E6AP) HECT domain E3 [34]

4 O. Zolk et al. / Cardiovascular Research 70 (2006) Table 3 Deubiquitinating enzymes expressed in the heart and changes in cardiac disease Enzyme (synonyms) Expression investigated References Ubiquitin C-terminal hydrolase UCHL1 (ubiquitin thiolesterase L1, mrna, protein (human left ventricular myocardium): [32] neuron cytoplasmic protein 9.5, PGP 9.5) up-regulated in human heart failure Ubiquitin-specific protease 5, USP5 (isopeptidase T) Protein (human left ventricular myocardium): up-regulated in human heart failure [34] Ubiquitin specific protease 20 (deubiquitinating enzyme 20, KIAA1003, ubiquitin thiolesterase 20, VDU2) mrna (human ventricular myocardium): up-regulated in human heart failure [31] receptor internalization, stress response and transcriptional regulation. Present knowledge about the function and role of the UPS system in the heart is summarized below. 3. Identification of UPS substrates in the heart 3.1. Structural proteins: sarcomeres and connexins Cardiac myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The balance between these opposing metabolic processes ultimately determines the number of functional contractile units within each cardiac muscle cell. Myosin heavy chains (MHCs), actin and other myofibrillar components are relatively long-lived compared with soluble cytosolic components. Nevertheless, subtle alterations in the rate of either the anabolic or catabolic process can markedly affect the myocardial mass [7]. Although alterations in myofibrillar protein degradation have been shown to contribute to cardiac growth and remodeling, surprisingly little information is available regarding the intracellular proteolytic systems that are responsible for degrading the cardiac myofibrillar proteins. Lactacystin, which is a highly specific proteasome inhibitor, was found to suppress MHC degradation effectively in cultured neonatal rat ventricular myocytes [8]. In this study, the MHC half-life markedly increased from 22 h in control cells to 43 h in cells treated with lactacystin. For other cardiac myofibrillar proteins, the role of proteasomal degradation is still unknown. Our best knowledge about the importance of the UPS in the degradation of sarcomeric (i.e. contractile) proteins comes from studies investigating the relevance of proteolytic systems for skeletal muscle wasting and atrophy (e.g., cachexia, AIDS-related wasting and disuse). Myofibrillar proteins that are predominantly degraded by the proteasome in skeletal muscle cells include troponin C, myosin lightchain-2 and myosin light-chain-3, a-actinin and tropomyosin [9,10]. The ubiquitination system, which marks substrates for degradation by the 26S proteasome, was found to be up-regulated in several forms of muscle wasting and atrophy. Increased expression of muscle-specific ubiquitin ligases (MuRF-1 and MAFbx/atrogin-1, both also expressed in the heart, see Table 2) is now known to precede the process of atrophy [11]. Although purified myosin, actin, troponin and tropomyosin were hydrolyzed rapidly by the ubiquitin proteasome pathway, these proteins were much more stable when present in myofibrils or as soluble actomyosin complexes [10]. These findings provide evidence that the specific associations between these proteins in the contractile apparatus protect them from degradation. Thus, the ratelimiting step in their degradation seems to be the dissociation from the contractile filaments. Experimental studies suggest that at least actin and myosin are released from the sarcomere by a calcium calpain-dependent mechanism before ubiquitination [12]. Other cardiac proteins thought to be degraded by the UPS include connexins. Connexins are constituents of cardiac gap junctions, which are important for electrical activation of the heart by current transfer from one cell to another. One of the most unusual aspects of gap-junction assembly is the exceptional metabolic lability of connexins. As assessed from the degradation rate of total cell-surface biotinylated proteins, the half-life of the great majority of plasma-membrane proteins exceeds 24 h [13]. By contrast, pulse-chase analysis has demonstrated that connexin family members, including those expressed in the heart e.g., connexin 43 (Cx43), Cx45 and Cx37 turn over with a half-life of only h, even after incorporation into gapjunctional plaques [14]. To date, two proteolytic pathways, the lysosomes and the proteasomes, have been implicated in connexin turnover in intact cells (Fig. 2). Chemical inhibitors of either pathway decreased the rate of turnover of Cx43, which is the principal gap-junction protein found in the ventricular myocardium [14,15]. However, our knowledge of the molecular mechanisms underlying the proteasome-dependent degradation of gap junctions is fragmentary. It has been reported that Cx43 is ubiquitinated at the plasma membrane following mitogen-activated protein kinase (MAPK)- and protein kinase C (PKC)- dependent hyperphosphorylation [16,17]. Interestingly, Cx43 is modified by multiple monoubiquitin residues rather than a polyubiquitin chain [16]. Moreover, recent studies indicate that the ubiquitination of Cx43 occurs prior to its internalization via a clathrin-dependent mechanism [17]. Thus, the monoubiquitination of Cx43 might be a signal for recruiting clathrin to the gap-junction plaque. It has been speculated that the monoubiquitination of Cx43 might also play a role in targeting Cx43 to lysosomal compartments, as demonstrated for other membrane proteins [2].

5 414 O. Zolk et al. / Cardiovascular Research 70 (2006) Fig. 2. Role of the UPS in the degradation of connexins. Connexins are synthesized in the endoplasmic reticulum and are then transported through the Golgi apparatus towards the plasma membrane. They are oligomerized into connexons in the ER or in a post-er/golgi compartment. Misfolded (or inappropriately oligomerized) connexins are targeted to the proteasome by polyubiquitination. Connexons at the plasma membrane dock with connexons from the adjacent cell to form gap-junction channels, which cluster to form gap-junctional plaques. The ubiquitination of connexins at gap-junctional plaques has been proposed to induce internalization of a portion of the gap-junction plaque. Internalized gap junctions fuse with lysosomes and degradation of the connexins takes place. The exact role of the proteasomal pathway in the degradation of gap-junction plaques is not yet clear. The following alternative theories have been postulated: the proteasome pathway might act together with the lysosome in a sequential or parallel manner; an unknown short-lived protein might target the conformationally mature protein for proteasomal degradation; or the proteasome might be involved in the degradation of a protein that participates in the internalization of gap junctions Signaling molecules and transcription factors Cell-surface proteins, including receptor proteins, are generally thought to be degraded by lysosomal pathways. Nevertheless, the ubiquitination of several membrane receptors catalyzed by E3 ubiquitin ligases was observed [18]. In the case of membrane proteins, however, ubiquitination serves as a signal for the sorting and targeting of the internalized proteins to the lysosome, rather than to the proteasome. The best studied example is the h 2 -adrenergic receptor. In general, agonist-dependent phosphorylation of the h 2 -adrenoceptor leads to recruitment of the regulator protein, h-arrestin, which physically prevents the coupling of the receptor to G proteins in a process referred to as desensitization. Subsequent to desensitization, receptors are removed from the cell surface by a process of internalization via h-arrestin-dependent mechanisms. This paradigm of receptor sequestration has been extended by the observation that the h 2 -adrenoceptor protein and h-arrestin are both ubiquitinated in an agonist-dependent manner (Fig. 3) [18,19]. When further dissecting the role of h 2 - adrenoceptor ubiquitination, it became clear that it was required for proper sorting and degradation in lysosomes [18]. Mdm2 was identified to be one E3 ubiquitin ligase capable of catalyzing h 2 -adrenoceptor ubiquitination. Notably, Mdm2 physically interacts with and ubiquitinates also h-arrestin, and ubiquitination of h-arrestin turned out to be essential for receptor internalization [18]. Thus, the ubiquitination of h-arrestin and the receptor appears to serve important functions in regulating the life cycle of the h 2 -adrenoceptor. In addition to the role of the UPS for receptor regulation, there is growing evidence to indicate that ubiquitin and the proteasome are intimately involved in gene control. The two fundamental mechanisms involved are ubiquitin proteasome-mediated regulation of (a) the location and (b) the activity/abundance of the transcriptional activator. It is clear that if a transcription factor is not located in the nucleus, it cannot activate transcription. This simple mechanism of regulation is used extensively to control gene expression, and is achieved by phosphorylation, site-specific proteases and the UPS. The most straightforward example from the third category is nuclear factor (NF)-nB, which is held in the cytoplasm by interaction with the inhibitor protein InB (Fig. 4). Upon stimulation (e.g. by cytokines involved in inflammatory processes after myocardial infarction) an InB kinase (IKK) complex is activated and, in turn, phosphorylates InB proteins on specific serine residues [20]. The phosphorylation triggers the ubiquitination-dependent degradation of InB proteins by the 26S proteasome, resulting in the release of NF-nB [21]. Subsequently, NF-nB translocates into the nucleus, where it stimulates the transcription of specific target genes. NF-nB is known to be involved in inflammation. Additionally, recent evidence suggests that the activation of NF-nB plays a key role in myocyte hypertrophy [22,23]. At the same time, NF-nB

6 O. Zolk et al. / Cardiovascular Research 70 (2006) Fig. 3. Agonist-dependent phosphorylation of the h 2 -adrenoceptor leads to the recruitment of h-arrestin (h-arr), whereupon h-arrestin and the receptor are both ubiquitinated (U; ubiquitin). The receptor-h-arrestin complex is targeted to clathrin-coated pits. h-arrestin then becomes deubiquitinated, leading to its dissociation from the receptor. The ubiquitinated receptor is finally transported into the late endosomes and lysosomes to be degraded. activation in the heart has more ambivalent effects on cellular survival, as it is involved in the direct regulation of both pro- and anti-apoptotic genes [24,25]. As well as its effects on transcription-factor location, the UPS can directly influence transcription by controlling the cellular abundance of transcription factors. Indeed, the 26S proteasome has been found to be involved in the degradation of several transcriptional regulators, such as c-jun, c- Fos, p53 and Ying Yang 1 (YY1), which are involved in the hypertrophic response of the myocardium or the temporal regulation of muscle development and differentiation [3,26]. A recent study by Li et al. established a link between the UPS and cardiac hypertrophy more directly [27]. They reported that overexpression of atrogin-1 attenuates calcineurin A signaling in cardiomyocytes [27]. Atrogin-1 is a skeletal muscle- and cardiac muscle-specific component of a so-called SCF E3 ubiquitin ligase complex. Within this SCF atrogin-1 E3 ligase complex, atrogin-1 is responsible for Fig. 4. A schematic presentation of the activation of the transcription nuclear factor-nb (NF-nB). In the cytoplasm of the resting cell, the NF-nB dimer, which often consists of the subunits p50/p65, is bound to inhibitory proteins known as InB. Stimuli activating the InB kinases cause nuclear translocation of the NFnB dimer, while InB is degraded by the proteasome. In the nucleus, NF-nB binds to consensus sites in promoter/enhancer regions of the genes that it regulates and the transcription of these substances commences. Proteasome inhibitors block NF-nB transcriptional activity by preventing InB degradation (red). ICAM- 2, intercellular adhesion molecule 2; IL, interleukin; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule 1.

7 416 O. Zolk et al. / Cardiovascular Research 70 (2006) substrate recognition. Atrogin-1 is capable of interacting with calcineurin A, which is ubiquitinated through the SCF atrogin-1 E3 ligase complex and thus targeted for proteasomal degradation [27]. Calcineurin A is a calciumactivated serine/threonine phosphatase that dephosphorylates and activates nuclear factor of activated T cells (NFAT) family members. First described as a regulator of immune function in T cells, calcineurin A lies downstream of G protein-coupled receptor activation in a signaling cascade that leads to cardiac hypertrophy [28]. Ubiquitination via the SCF atrogin-1 complex, therefore, represents a mechanism for regulating calcineurin A protein levels and activity in isolated cardiomyocytes, thereby attenuating hypertrophy and fetal gene expression [27]. Consistent with these in vitro observations, overexpression of atrogin-1 in transgenic mice blunted cardiac hypertrophy in response to pressure overload due to banding of the thoracic aorta [27]. Remarkably, however, transgenic overexpression of atrogin-1 resulted in ventricular dilatation and left ventricular dysfunction after 2-week aortic banding [27]. Thus, the impact of atrogin-1 as a calcineurin inhibitor on cardiac pathophysiology remains obscure. It has been hypothesized that the phenotypes of atrogin-1 transgenic mice are further modified by other substrates for the SCF atrogin-1 complex, or through other effects of atrogin-1 that are independent of its ubiquitin ligase activity [27]. Interestingly, atrogin-1 was shown to be regulated by the growth factor/akt signaling axis through direct transcriptional regulation by members of the FOXO subfamily of forkhead transcription factors [29]. These findings suggest that Akt signaling might promote cardiac hypertrophy in part by direct phosphorylation and inhibition of FOXO transcription factors, thereby reducing atrogin-1 gene expression and enhancing calcineurin signaling by inefficient SCF atrogin-1 -dependent calcineurin A ubiquitination and degradation. Atrogin-1, therefore, may constitute a link between two distinct signaling pathways of cardiac hypertrophy, namely the Akt and the calcineurin pathway. 4. Role of the UPS in cardiac diseases 4.1. Heart failure In heart failure, multiple alterations of the UPS have been described (Tables 1 and 3). Comparison of mrna expression in failing and non-failing human hearts revealed a significant down-regulation of the transcripts for some of the a and h subunits of the 20S proteasome core complex [30,31]. This was associated with an almost two-fold increase in the level of mrna expression of the ubiquitin-conjugating enzyme UBE2G2 [30]. Moreover, in failing ventricles, an up-regulation of the transcript for the deubiquitinating enzyme ubiquitin-specific protease 20 was observed [31]. Several findings support the notion that the changes described at the mrna level do indeed translate into changes at the functional level. Weekes et al. demonstrated that total ubiquitin conjugation was markedly increased in failing hearts from patients with hypertrophic and dilated cardiomyopathy, suggesting that the UPS is functionally impaired and net protein degradation is actually reduced in heart failure [32]. It has also been suggested that a disturbance in the homeostasis between protein synthesis and protein degradation results in the accumulation of modified proteins, which finally tend to form high-molecular-weight aggregates. Correspondingly, in an animal model of cardiac hypertrophy, augmented ubiquitin-positive lipofuscin deposits have been reported [33]. More recently, the hyperubiquitination of proteins and the deposition of nuclear or cytosolic ubiquitin-positive aggregates have also been observed in human dilated cardiomyopathy [34]. It might well be the case that protein aggregates in failing hearts become increasingly cross-linked and are therefore excellent substrates for the ubiquitination system, but are increasingly resistant to degradation of the proteasome. Moreover, these aggregates might be able to inhibit the proteasome, as demonstrated in several experimental studies [35,36]. Interestingly, the accumulation of protein aggregates and the inhibition of the proteasome seem to be much more dramatic in post-mitotic cells [37], suggesting that cardiac myocytes might be highly vulnerable to disturbances in the UPS. These observations support the hypothesis that the UPS might be involved in the disease etiology of heart failure Familial cardiomyopathies Recent studies suggest that alterations in the UPS might play a role in familial cardiomyopathies, namely in some cases of familial hypertrophic cardiomyopathy (FHC) and desmin-related myopathy (DRM). Despite the well-established genetic etiologies of FHC, the pathogenetic processes of these disorders are still unclear, especially in the case of cardiac myosin-binding protein C (cmybp-c)-related FHC, which accounts for a large number of the disease-causing mutations [38]. cmybp-c is an integral part of the heart muscle sarcomere. Most of the cmybp-c gene mutations result in a frameshift and are expected to lead to C-terminal truncated proteins lacking the major titin and/or myosinbinding sites [39]. At present, the most likely disease mechanism is that frameshift cmybp-cs act as null alleles leading to haploinsufficiency of cmybp-c in the sarcomere. The haploinsufficiency hypothesis was based on the observation that in cardiac tissue of patients known to be carriers of cmybp-c frameshift mutations, the expected truncated proteins were not seen in Western blots in a number of independent studies [40 42]. Using an adenovirus-based approach, a recent study analyzed the expression and localization of two different truncated cmybp-cs (M6t 3% and M7t 80% truncation, both of which mutations have been identified in FHC patients) in

8 O. Zolk et al. / Cardiovascular Research 70 (2006) neonatal rat cardiomyocytes [43]. Despite similar mrna levels, the protein expression of M6t and M7t was markedly lower than that of the wild-type cmybp-c [43]. Treatment of cardiomyocytes with the proteasome inhibitors MG132 or lactacystin markedly raised the protein concentrations of truncated cmybp-c to the level of the wild-type controls, suggesting that truncated cmybp-cs are rapidly degraded mainly by the UPS [43]. These results can be taken to explain the absence of mutated cmybp-c in cardiac tissue of patients, supporting the notion of haploinsufficiency being a trigger of disease. Of particular importance, however, was the demonstration that the truncated cmybp-cs were not only substrates of the UPS but also impaired the degrading system, as shown with a ubiquitin-ds-red (Ub G76V -DsRed) fusion protein reporter assay for proteasome activity in living cells. The mechanisms by which proteasome function is inhibited remain unclear. However, it is likely that mutant cmybp-cs provide an unusually strong degradation signal and effectively compete with other degradation-prone proteins for the proteasome (Fig. 5). Although these in vitro data have to be verified in vivo, it is tempting to speculate that the mutant cmybp-c protein imposes a continuous, lifelong additional workload on the protein-degradation machinery in the heart. The consequence might be the impaired degradation of abnormal proteins resulting from aging or oxidative stress (the accumulation of which could be toxic) and of many regulatory proteins (e.g., regulators of apoptosis) with short half-lives that determine their activities. The experimental data open the possibility that the UPS might be involved in the complex pathogenic process leading to the late-onset cardiomyopathy, which is characteristic for FHC secondary to mutations of the MYBPC3 gene [44]. Another example which demonstrates a possible role of the UPS for cardiac pathology is the familial desmin-related myopathy (DRM) caused by an R120G missense mutation of the chaperonic protein ab-crystallin. Initially discovered as a lens protein a century ago, ab-crystallin is the most abundant small heat shock protein (HSP) in the heart, where its expression is limited to the cardiomyocytes [45]. Chaperonic proteins, such as ab-crystallin, facilitate the assembly, disassembly and folding/refolding of proteins, and, therefore, play important roles in the UPS system as they triage misfolded proteins for proteasomal degradation or repair. In vitro, ab-crystallin assists the assembly of desmin filaments and modulates interaction among desmin filaments. The R120G mutation significantly reduces the chaperonic function of ab-crystallin [46]. In the presence of ab-crystallin R120G, filaments formed by desmin protein appear to be less uniform in diameter and they tend to aggregate. Indeed, in patients carrying the ab-crystallin R120G mutation, desmin-positive aggregates were found Fig. 5. Effects of mutant myosin binding protein-c (MyBP-C) on the ubiquitin proteasome system (UPS) in cardiomyocytes. Unlike wild-type (WT) MyBP-C (A), truncated MyBP-C is preferentially degraded by the UPS (B). Truncated MyBP-C has been recognized not only as a substrate of the UPS but also as an inhibitor of the breakdown of other UPS substrates [43], such as damaged or misfolded proteins. It is likely that mutant MyBP-C provides an unusually strong degradation signal and effectively competes with other degradation-prone proteins for the proteasome. Moreover, mutant MyBP-C induces aggregate formation. In these aggregates, ubiquitinated proteins (including the mutant MyBP-C) were found. These protein aggregates may directly impair the function of the proteasome.

9 418 O. Zolk et al. / Cardiovascular Research 70 (2006) [47]. These aggregates were also found to contain large amounts of the mutant ab-crystallin protein [47]. ab-crystallin was shown to interact with the F-box protein FBX4 [48], which is an important component of a SCF-type E3 ubiquitin ligase and confers the substrate specificity to the SCF complex [49]. Interaction of abcrystallin with FBX4 promotes FBX4-dependent ubiquitination of substrates. Importantly, the R120G mutation of ab-crystallin results in increased interaction with FBX4, a specific translocation of FBX4 to the detergent-insoluble fraction, ubiquitination of yet unknown proteins, and accumulation of ubiquitinated products [48]. This mechanism might contribute to ab-crystallin R120G -induced aggregate formation. The transgenic expression of ab-crystallin R120G in the mouse heart caused aberrant desmin and ab-crystallin aggregation, as well as cardiomyopathy, in a dominantnegative manner, thereby reproducing the phenotype found in patients with DRMs [50]. Further analysis of this animal model of DRM revealed that aberrant protein aggregation induced by ab-crystallin R120G impaired the proteolytic function of proteasomes in the heart [51]. The UPS impairment was detected before cardiac hypertrophy and failure became discernible [51]. This observation suggests that defective protein turnover might contribute to cardiac remodeling and failure in this model and establishes an additional pathogenic mechanism of ab-crystallin R120G - related DRM. Similar to ab-crystallin R120G, other mutant proteins known to induce aggregate formation, such as mutant huntingtin or mutant cystic fibrosis membrane conductor protein, impair the UPS [36], suggesting that the formation of intracellular protein aggregates rather than loss of function of the respective gene causes UPS malfunction. This suggestion was supported by the observation that ab-crystallin R120G - induced UPS malfunction in cultured cardiomyocytes was significantly attenuated when aberrant protein aggregation was reduced by Congo red treatment [51] Ischemia reperfusion injury of the heart Early reperfusion of the ischemic myocardium plays an important role in minimizing myocardial tissue injury associated with acute myocardial infarction. However, the effects of reperfusion are complex and include some deleterious effects collectively referred to as reperfusion injury [52]. This reperfusion injury involves the activation of an inflammatory cascade and is manifest as functional impairment, arrhythmia, and accelerated progression of cell death in certain critically injured myocytes. Experimental studies with proteasome inhibitors strongly suggest a role of proteasomes in the process of ischemia reperfusion injury. In animal models of ischemia and reperfusion, proteasome inhibition significantly reduced the infarct size, in some studies by more than 50%, and improved functional parameters such as the left ventricular developed pressure (LVDP) and the + dp/dt max [53 56]. Pharmacological blockade of the UPS seems to confer cardioprotection due to the anti-inflammatory effects of proteasome inhibitors by a mechanism involving the inhibition of NF-nB [54]. Moreover, inhibition of proteasome activity by pharmacologic treatment leads to the induction of molecular chaperones, many of which are heat HSPs, such as ab-crystallin or HSP70. HSPs protect cardiomyocytes against hypoxia, and have direct antiapoptotic activities [57,58]. Specifically, ab-crystallin inhibits apoptosis during myocardial ischemia and reperfusion [59,60] by preventing the activation of procaspase-3 [61], and its association with cytoskeletal components during stress [59,62]. A recent study focussing on the cochaperone/ubiquitin ligase CHIP (for carboxyl terminus of Hsp70 interacting protein) provides additional evidence that the molecular chaperone machinery plays an important role in protection against ischemia reperfusion injury. Mice deficient for CHIP were less tolerant against ischemia reperfusion injury with more frequent reperfusion arrhythmias and increased infarct size compared to wild-type mice [63]. CHIP, which is abundantly expressed in the heart, ubiquitinates damaged proteins and triggers their proteasome-dependent degradation in a process requiring the molecular chaperones HSP70 or HSP90, which bind to unfolded domains thereby facilitating substrate recognition by CHIP [64,65]. In addition, CHIP regulates activation of the stress-chaperone response through induced trimerization and transcriptional activation of heat shock factor 1 (HSF1) [66]. These diverse functions of CHIP may explain its protective properties against cardiac ischemia reperfusion injury. 5. Proteasome inhibition: a future therapy for cardiac diseases? During recent years, several types of low-molecular weight inhibitors of the proteasome have been identified that can readily enter cells and selectively inhibit the proteolytic function of the proteasome complex [3]. Bortezomib (Velcadei) is a novel dipeptide boronic acid that is the first proteasome inhibitor to have progressed to clinical trials. Bortezomib has now been approved by the US Food and Drug Administration (FDA) and the European Agency for the Evaluation of Medicinal Products (EMEA) for the treatment of multiple myeloma patients who have received at least two prior therapies and have demonstrated disease progression during the last treatment. Because the first proteasome inhibitors for clinical use were developed as novel antineoplastic agents, the majority of data relating to the clinical effects and side effects of proteasome inhibitors have been obtained from cancer studies. The risks that are associated with bortezomib therapy include new or worsening peripheral neuropathy,

10 O. Zolk et al. / Cardiovascular Research 70 (2006) orthostatic hypotension, gastrointestinal adverse events and thrombocytopenia. Moreover, the acute development or exacerbation of congestive heart failure has been seen in patients with risk factors for, or pre-existing, heart disease [67]. Although there seems to be some risk of cardiovascular side effects when proteasome inhibitors are applied as cancer treatment intermittently for several weeks, it has been proposed that proteasome inhibitors might have beneficial effects due to their potential anti-inflammatory properties when administered for a short time immediately after acute ischemic events, such as myocardial infarction or stroke. Proteasome inhibitors are known to act on a key intracellular mechanism, the NF-nB pathway, which controls the activation of inflammatory molecules. Based on animal studies, it has been proposed that proteasome inhibitors might have the potential to attenuate reperfusion injury and, thus, might work synergistically with current myocardial infarction reperfusion therapies [56]. Existing thrombolytic agents have a narrow window of time for therapeutic application. The hope for the future is to increase this period using proteasome inhibitors, which would expand the patient population that could receive thrombolytic therapy [68,69]. The anti-inflammatory action of proteasome inhibitors might also offer an opportunity for tolerance induction in transplant recipients. It has been demonstrated in vitro that proteasome inhibitors could suppress the proliferation and induce the apoptosis of activated T cells. This finding suggests that such inhibitors could be used as a novel category of immunosuppressants in blocking allograft rejection. Indeed, in a mouse heterotopic heart allograftrejection model, the proteasome inhibitor dipeptide boronic acid prolonged heart allograft survival from 7 to 35 days [70]. Initial reports on the effects of proteasome inhibitors indicate that proteasome inhibition might also be an effective therapeutic strategy for the reduction of restenosis after balloon angioplasty of coronary arteries and stent implantation. In a balloon-injury model of the rat carotid artery, local administration of the proteasome inhibitor MG132 effectively reduced neointima formation, which was associated with strong antiproliferative and proapoptotic effects on vascular smooth muscle cells and reduced infiltration of macrophages [71]. 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