Theubiquitin-proteasomesystemincardiac proteinopathy: a quality control perspective

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1 Cardiovascular Research (2010) 85, doi: /cvr/cvp287 SPOTLIGHT REVIEW Theubiquitin-proteasomesystemincardiac proteinopathy: a quality control perspective Huabo Su and Xuejun Wang* Division of Basic Biomedical Sciences, Sanford School of Medicine of the University of South Dakota, Lee Medical Building, 414 E. Clark Street, Vermillion, SD 57069, USA Received 21 May 2009; revised 8 August 2009; accepted 14 August 2009; online publish-ahead-of-print 20 August 2009 Time for primary review: 44 days Protein quality control (PQC) depends on elegant collaboration between molecular chaperones and targeted proteolysis in the cell. The latter is primarily carried out by the ubiquitin-proteasome system, but recent advances in this area of research suggest a supplementary role for the autophagy-lysosomal pathway in PQC-related proteolysis. The (patho)physiological significance of PQC in the heart is best illustrated in cardiac proteinopathy, which belongs to a family of cardiac diseases caused by expression of aggregation-prone proteins in cardiomyocytes. Cardiac proteasome functional insufficiency (PFI) is best studied in desmin-related cardiomyopathy, a bona fide cardiac proteinopathy. Emerging evidence suggests that many common forms of cardiomyopathy may belong to proteinopathy. This review focuses on examining current evidence, as it relates to the hypothesis that PFI impairs PQC in cardiomyocytes and contributes to the progression of cardiac proteinopathies to heart failure Keywords Protein quality control Ubiquitin Proteasome Desmin-related cardiomyopathy Chaperone Autophagy This article is part of the Spotlight Issue on: The Role of the Ubiquitin-Proteasome Pathway in Cardiovascular Disease 1. Introduction Proteins not only are the building blocks of the cell body but also execute nearly all the cellular functions. The long-term wellbeing of the cell is thus inextricably associated with protein quality control (PQC), which functions to minimize the production of abnormal proteins in the cell, remove unsalvageable abnormal proteins, and prevent abnormal proteins from damaging the cell. Abnormal proteins can arise from genetic mutations, errors in transcription, translation and folding, or damages resulting from environmental insults. When a misfolded or damaged protein fails to be repaired by chaperones-mediated processes, it will be degraded by targeted proteolysis. 1 When PQC is impaired or overloaded, abnormal proteins accumulate and cause aberrant aggregation in the cell, thereby injuring the cell and ultimately leading to cell death. This can be quite detrimental to post-mitotic organs such as the heart and brain, due to their very limited, if any, self-renewal capacity. 2 PQC depends on sophisticated collaboration between molecular chaperones and targeted proteolysis. The latter was previously believed to be carried out solely by the ubiquitin-proteasome system (UPS), but recent advances in this area of research suggest an important role for autophagy in PQC-associated proteolysis (Figure 1). 1 Since the targeted proteolysis, especially that mediated by the UPS, is also responsible for the degradation of the majority of normal cellular proteins that are no longer needed, dysfunctional protein degradation will affect not only PQC but also many other cellular processes as reviewed by other articles in this Spotlight Issue. 3 6 This review article focuses on the PQC perspective. The importance of PQC in cell function and viability is demonstrated by overexpression of a molecular chaperone, and an ATPdependent protease essential to mitochondrial PQC increased the healthy lifespan of Podospora anserine. 7 Conversely, inhibitors of molecular chaperones and proteasomes have become promising new drug candidates for cancer chemotherapies. 8,9 The (patho)physiological significance of PQC in the heart is best illustrated in cardiac proteinopathy, which is a family of cardiac disease caused by expression of aggregation-prone proteins in cardiomyocytes. Here, we will examine the current evidence, as it relates to the hypothesis that proteasome functional insufficiency (PFI) impairs PQC in cardiomyocytes and contributes to the progression of cardiac proteinopathy to congestive heart failure (CHF). 2. PQC in cardiomyocytes The heart is arguably the most stressed organ in the body, and polypeptides face enormous challenge to attain and maintain a * Corresponding author. Tel: þ ; Fax: þ address: xuejun.wang@usd.edu Published on behalf of the European Society of Cardiology. All rights reserved. & The Author For permissions please journals.permissions@oxfordjournals.org.

2 254 H. Su and X. Wang Figure 1 A schematic illustration of PQC in the cell. Chaperones facilitate the folding of nascent polypeptides and the unfolding/refolding of misfolded proteins, prevent the misfolded proteins from aggregating, and escort terminally misfolded proteins for degradation by the UPS. The UPS degrades both misfolded/damaged proteins and most unneeded native proteins in the cell. This process involves two steps: first, covalent attachment of ubiquitin to a target protein by a cascade of chemical reactions catalysed by the ubiquitin-activating enzyme (E1), ubiquitinconjugating enzymes (E2), and ubiquitin ligase (E3); and then the degradation of the target protein by the proteasome. The autophagy-lysosomal pathway participates in PQC by helping remove protein aggregates formed by the misfolded proteins that have escaped from the surveillance of chaperones and the UPS. Protein aggregates or defective organelles are first segregated by an isolated double membrane (phagophore) to form autophagosomes, which later fuse with lysosomes to form autophagolysosomes, where the segregated content is degraded by lysosomal hydrolases. p62/sqstm1 and NBR1 (neighbour of BRCA1 gene 1) mediate the activation of autophagy by aggregated ubiquitinated proteins. The legend for symbols used is shown in the box at the lower left. proper conformation in heart muscle cells. Hence, PQC is a vital cellular process to cardiomyocytes. The major PQC players and their functional relationship (Figure 1) are briefly reviewed in this section. 2.1 Chaperones in cardiac PQC As an essential player in PQC, chaperones participate in protein folding/unfolding, repairing misfolded proteins, and preventing the aggregation of unfolded/misfolded proteins. Chaperones also escort terminally misfolded proteins to the proteolytic pathway for degradation. 10,11 For instance, C-terminus of Hsc70-interacting protein (CHIP) can function as both a chaperone and a ubiquitin ligase, cooperating with UNC-45 to target unassembled myosin for proteasome degradation. 12 BAG1 (Bcl-2-associated athanogene 1), another co-chaperone that binds to heat shock protein 70 (HSP70) and HSP90, protects the heart against ischaemia/reperfusion (I/R) injury by facilitating both UPS-mediated and autophagy-lysosome-mediated protein degradation. 13,14 It seems that in the chaperone co-chaperone complex, the chaperones are responsible for capturing the misfolded proteins and preventing their aggregation, whereas the co-chaperones escort captured substrates to the degradation machinery by virtue of their respective ubiquitin ligase, ubiquitin-binding or proteasome-binding domains. 10 Notably, some chaperones actually promote the efficient and correct assembly of both 19S and 20S proteasome complexes. 15,16 Although these chaperones are not associated with mature 26S proteasomes, mutations in these genes display proteasome loss-of-function phenotypes. 16 Therefore, rapid upregulation of certain chaperones in response to stress may have a direct effect on proteasome function via regulation of proteasome assembly. In cardiomyocytes, molecular chaperones are either constitutively highly expressed, likely due to the extremely hostile environment for protein homeostasis, or rapidly upregulated in response to different physiological or pathological stress. Many gain- or

3 Proteasomes in cardiac proteinopathy 255 loss- of-function studies have examined the role of chaperones in cardiac hypertrophy, 17,18 I/R injury, 19 and doxorubicin-induced cardiomyopathy. 20 The consensus of the chaperone studies appears to be that loss-of-function compromises the ability of the heart to handle stress, whereas gain-of-function confers cardiac protection against cell injury. The latter actually suggests that PQC, at least the chaperone part, is inadequate in all these cardiac disorders. The findings that gain-of-function of small HSPs (e.g. ab-crystallin) suppresses pressure overload or adrenergic stimulation-induced cardiac hypertrophic responses also suggest that at least a portion of the cardiac hypertrophic responses is caused by PQC inadequacy The UPS in PQC PQC-associated protein degradation is primarily carried out by the UPS. The UPS degrades a target protein usually via two sequential steps: the covalent attachment of ubiquitin moieties to the protein (termed ubiquitination) and subsequent degradation of the ubiquitinated protein by proteasomes Ubiquitination of abnormal proteins With a few known exceptions, polyubiquitination is required for a protein to be degraded by the proteasome. Polyubiquitination is accomplished by forming an isopeptidyl bond between the carboxyl terminus of ubiquitin and a lysine residue from the substrate or from the proceeding ubiquitin. All seven lysine residues (K6, K11, K27, K29, K33, K48, and K63) within ubiquitin can be used for chain extension in vivo. 21 An ubiquitin chain linked via K63 usually does not elicit proteasomal degradation but rather signals for a non-proteolytic fate for the modified protein Ubiquitin chains linked via K48 (the dominant form), K6, K11, K27, K29, or K33 can all target the modified substrates for proteasomal degradation. 25 Interestingly, ubiquitin chains linked via K11 were recently found to function in endoplasmic reticulum (ER)-associated degradation (ERAD). 25 ERAD degrades misfolded proteins that are retrograde-transported from the ER to the cytosol. It is unknown whether K11 ubiquitin linkage serves as a general signal for the UPS-mediated degradation of all misfolded proteins. The ubiquitination of a target protein is activated by the maturation of a degradation signal (degron) on the target protein. The maturation often requires post-translational modification, such as N-terminal residue processing, phosphorylation, prolyl hydroxylation, and glycosylation, and provides a specific binding surface for its specific E3 ubiquitin ligase. 26 The degradation signal borne by misfolded or abnormal proteins is not yet well defined. Unfolding and misfolding associated with abnormal proteins may expose normally cryptic degrons existing in many proteins and activate the UPS. 26 A helical surface-exposed hydrophobic structure is proposed as a recognition motif within ERAD substrates. 26 Indeed, a helical representation of the degron CL1 sequence isolated from yeast does bare a hydrophobic surface. 27 When fused with an otherwise long half-life protein, degron CL1, as well as its hydrophobic residue-conserved mutants, could destabilize the protein by targeting it for degradation via a sub-pathway of the UPS known to be used by ERAD. 27 This suggests that the surface exposure of a patch of hydrophobic amino acids may serve as a common signal for misfolded proteins to activate ubiquitination Proteasomal degradation of abnormal proteins In general, polyubiquitinated proteins are degraded by the 26S proteasome in an ATP-dependent manner and this is likely true for polyubiquitinated misfolded proteins, at least for the soluble ones. Elegant proteomics studies revealed that murine cardiac 26S proteasomes contain at least 34 subunits, some of which undergo cardiac-specific modification. 28,29 Variation in proteasome subunit composition alters the proteasome selectivity and specificity and thus may play an important role in regulating proteasome function. 29 In addition, the cardiac-specific associating partners of the proteasome with regulatory activities may increase the diversity of proteasome function in the heart. 29 A very interesting idea emerging from these recent proteomics studies is that proteasomes may be differentially composed and modified to degrade different families of substrates. Consistent with this idea, different subtypes of cardiac proteasomes were found to respond differently to pharmacological proteasome inhibitors. 30 It will be interesting to test whether proteasomes involved in PQC differ from those for the degradation of normal proteins. In addition to regulating cullin-based RING E3 ligases via deneddylation, the COP9 signalosome (CSN) may function as an alternative lid for the 19S proteasome. 31 In a recent study, we were able to show that the downregulation of the CSN by knocking down its subunit 8 facilitated the proteasome-mediated degradation of a surrogate misfolded protein in cultured cells, 32 suggesting that the homeostatic level of CSN8/CSN suppresses the proteasome-mediated degradation of misfolded proteins. The ubiquitinated proteins reach the proteasome by diffusion, factor-assisted shuttling, or proteasome translocation to the substrates. Rpn10 and Rpt5 of 19S proteasomes contain the ubiquitin-interacting motif and thereby may serve as a docking site for substrates. 33 However, only a small fraction of Rpn10 is associated with 19S proteasomes at a given time, hinting the possible role of Rpn10 as a shuttling factor for the substrates. 34 Additional shuttling factors include Rad23, Dsk2, Ddi1, the p97/ cdc48 complex, and p62. These factors are capable of binding to polyubiquitin and also to 19S proteasomes, thereby likely delivering polyubiquitinated substrates to the proteasome. 35,36 The poor accessibility of polyubiquitinated misfolded proteins to the proteasome may be an important factor causing PQC inadequacy. It should be noted that an increasing number of proteins, such as L-ornithine decarboxylase, p21, c-fos, p53, HIF1a, and Rb, can also be degraded by proteasomes in an ubiquitin-independent manner. 37 These results suggest that this pathway may have been largely underestimated. In particular, some aged/oxidized/misfolded proteins were also found to be ubiquitin-independent substrates for the proteasome. 37 It is still unclear how 26S or 20S proteasomes directly degrade oxidized/misfolded proteins. Exposure of hydrophobic patches in oxidized and misfolded proteins might trigger substrate recognition by the proteasome, in the presence or absence of chaperones. Accessory proteins or the substrate itself might also open the entrance of the 20S proteasome gated by a-ring subunits. 37 A better understanding on the ATP- and ubiquitin-independent proteasome-mediated

4 256 H. Su and X. Wang degradation of abnormal proteins may help the search for measures to treat disease with PQC inadequacy. 2.3 Autophagy in cardiac PQC The involvement of macroautophagy (herein referred to as autophagy) in PQC has not been recognized until very recently. Autophagy begins with the formation of isolation membrane (phagophore). The phagophore then elongates and engulfs a portion of the cytoplasm to form a mature autophagosome, which then fuses with lysosomes to form autophagolysosomes, where the enclosed cytoplasmic components or organelles are degraded by lysosomal hydrolases. 38 In cardiomyocytes, autophagy occurs at the basal level and can be further induced by different physiological or pathological conditions such as starvation, 39,40 haemodynamic stress, 41, I/R, proteotoxicity, 46,47 and toxins. 48 The dual role of autophagy as both cell survival and cell death mechanisms has been extensively reviewed. 44,49 The capability of autophagy to perform bulk degradation of cytoplasmic components can be cytoprotective by removing misfolded proteins and protein aggregates and by recycling cytoplasmic components; however, excessive and relatively non-selective self-digestion can also promote cell death. It was shown in both yeast and cultured mammalian cells that misfolded cytosolic proteins are partitioned into two distinct PQC compartments, depending seemingly on their ubiquitination and aggregation status. 50 Soluble misfolded proteins accumulate in a juxtanuclear compartment presumably for the degradation by the proteasomal pathway because (i) these proteins are often ubiquitinated; (ii) this compartment is enriched in proteasomes, and (iii) enhancing the ubiquitination of a misfolded protein promotes its delivery to this compartment. 50 On the other hand, terminally aggregated proteins are partitioned in a perivacuolar inclusion. Interestingly, disease-associated Huntingtin and prion proteins are preferentially targeted to the perivacuolar inclusion. 50 It appears that ubiquitinated proteins can be degraded not only by the proteasome but also by autophagy. However, the aggregated form is mainly degraded by autophagy because of its limited accessibility to the proteasome. 51 p62/squstm1 and NBR1 (neighbour of BRCA1 gene 1) are two better characterized autophagy receptors which can oligomerize, bind ubiquitin on the ubiquitinated proteins via the ubiquitin-binding domain, and directly interact with ATG8/LC3 through their LC3-interacting region. 52 The HSPB8 BAG3 complex was shown to suppress the aggregation of misfolded proteins through the activation of autophagy. 53 The autophagic activation requires the phosphorylation of eif2a, which in turn shuts down translation and activates autophagy. 54 In studying PQC in models of cell ageing, Gamerdinger et al. 55 showed that BAG1 and BAG3 regulate proteasomal and autophagic pathways, respectively, for the turnover of polyubiquitinated proteins. More importantly, the aged cells more commonly use autophagy to degrade polyubiquitinated proteins and this is associated with an increased BAG3/BAG1 ratio in the aged cells, compared with the young cells. BAG3 recruits the autophagic system by teaming up with p62/sqstm. 55 It seems that concentrating aggregated proteins to the cell centre by the microtubule system facilitates the removal of the aggregated proteins by autophagy. 56 In a fruit fly model, genetically induced proteasome impairment caused a degenerative phenotype, and the activation of autophagy by HDAC6 rescued the phenotype. 57 Indeed, proteasome malfunction appears to be able to activate autophagy, and autophagy might ameliorate the pathogenesis of proteasome impairment in a mouse model of cardiac proteinopathy PFI in cardiac proteinopathies PFI has been observed in animal models of desmin-related cardiomyopathy, a bona fide cardiac proteinopathy, and several other forms of cardiomyopathy PFI is also implicated in common forms of human cardiomyopathy and CHF (Table 1). 3.1 PFI in mouse models of desmin-related cardiomyopathy Desmin-related myopathy is a family of heterogeneous myopathies with a pathological hallmark of the presence of aberrant desminpositive aggregates in myocytes. 62 Desmin-related myopathy often affects cardiac muscle, and the resultant cardiomyopathy is referred to as desmin-related cardiomyopathy, which can present as restrictive, hypertrophic, or dilated cardiomyopathies. Aberrant protein aggregation in desmin-related cardiomyopathy is caused by mutations in either desmin itself or other proteins that are essential to desmin filament formation and/or maintenance. 62 Several such mutations, for example, a 7-amino-acid (R173 through 179E) deletion of the desmin gene (D7-des) and a missense (R120G) mutation of the ab-crystallin gene (CryAB R120G ), have been investigated in transgenic mice Transgenic overexpression of D7-des or CryAB R120G in mouse hearts led to the formation of intrasarcoplasmic desmin-positive protein aggregates and cardiomyopathy, recapitulating aspects of human desminrelated cardiomyopathy Marked increases in polyubiquitinated proteins were detected in both the insoluble and the soluble fractions of myocardium from D7-des or CryAB R120G transgenic mice but not in mice overexpressing a comparable level of wild-type desmin or CryAB in the heart. 58,59 Given that polyubiquitinated proteins, more specifically, the soluble polyubiquitinated proteins, are designated substrates for the proteasome, this finding raises the possibility of PFI in desmin-related cardiomyopathy transgenic mouse hearts. 58,59 To evaluate UPS proteolytic function in vivo, a proteasome function reporter system was established, in which a green fluorescent protein (GFP) was fused with degron CL1, and the modified GFP is referred to as GFPdgn or GFPu. 66 GFPdgn/GFPu serves as a surrogate substrate for the UPS. In the absence of changes in synthesis, GFPdgn protein levels inversely reflect UPS proteolytic function Using this reporter system, we found that expression of aggregate-prone mutants but not wild-type proteins caused severe inadequacy in UPS function in mouse hearts, as reflected by GFPdgn accumulation in cardiomyocytes. Given that ubiquitinated proteins were significantly increased, the UPS inadequacy was likely caused by PFI. 58,59

5 Proteasomes in cardiac proteinopathy 257 Table 1 Alterations of the UPS in cardiac disease Disease UPS alterations References... Human DCM " Ubiquitinated proteins 60,73,74,105 " Ubiquitin-positive aggregates 106 " UCH 74 " Peptidase activities 105 " E3 (Mdm2) 105 Human ischaemic " Ubiquitinated proteins 74 heart disease Human diabetic " Peptidase activities 75 ischaemic cardiomyopathy Human sepsis-induced cardiomyopathy Experimental models Cardiac hypertrophy and failure Diabetic cardiomyopathy " Ubiquitinated proteins 107 " Ubiquitinated proteins 60 " Ubiquitin-positive aggregates 42 " E2 transcripts 108 " E3 (Cbl, E6AP, Mdm2, ciap) 109 " E3 (MuRF1; Atrogin-1) 108 # Peptidase activities 60 " Peptidase activities 94,95 " 19S subunits 95,110 " 20S subunits 95,110 " Ubiquitin 111 " Ubiquitinated proteins 75,76 # E3 (MuRF1; Atrogin-1) 112 " Peptidase activities 75,76,111 # Peptidase activities 76 " 11S subunits 76 " 20S subunits 76 Myocardial ischaemia " Ubiquitinated proteins 113 # Peptidase activities 113 Desmin-related " Ubiquitinated proteins 58,59 cardiomyopathy " Peptidase activities 58,59 " Proteasome surrogate 58,59 substrate " 20S subunits 59 # 19S subunits 59 Doxorubicin " E3 (Atrogin-1) 114 cardiomyopathy " Peptidase activities 66 # Proteasome surrogate 66 substrate TNFa-induced DCM " Ubiquitinated proteins 77 " Ubiquitin-positive aggregates 77 DCM, dilated cardiomyopathy; UCH, ubiquitin carboxyl terminal hydrolase. Modified and updated from Mearini et al, BBA 2008; 1782:754 with permission from Elsevier. 3.2 PFI can co-exist with increased proteasome peptidase activities in cardiac proteinopathy Interestingly, myocardial 20S proteasome peptidase activities (chymotrypsin-like, caspase-like, and trypsin-like) in D7-des and CryAB R120G transgenic mice as measured in vitro using conventional synthetic fluorogenic substrates were not decreased but rather significantly increased. 58,59 A logic explanation for these seemingly conflicting data is that proteasomal proteolytic capacity in DRC mouse hearts cannot meet the increased demand resulting from misfolded protein overload, even though the activities of 20S proteasomes are higher than normal. Apparently, in the presence of misfolded protein overload, PFI can be caused by either an insufficiency of overall proteasome abundance or the impairment of 20S and/or 19S proteasomes. Consistent with this interpretation, ubiquitinated proteins are significantly increased; the protein levels of some key 20S proteasome subunits were increased in CryAB R120G and D7-des transgenic hearts, whereas some key subunits of 19S proteasomes were significantly decreased. These data also suggest that the defects in the delivery of ubiquitinated proteins to the proteasome and/or impairment in the substrate uptake by 20S proteasomes likely contribute to PFI in desmin-related cardiomyopathy. 58,59 These potential defects were not, and cannot be, detected by the conventional proteasome peptidase activity assays, underscoring the importance of the use of a full-length protein (e.g. GFPdgn) as a surrogate substrate to assess the sufficiency of UPS proteolytic function in vivo. It has been reported that the cell responds to proteasome inhibition by increasing proteasome synthesis and assembly. 69 Apparently, a compensatory upregulation of proteasome abundance can lead to an increase in proteasome peptidase activities; however, this increase does not necessarily meet the cell s increased need on proteasome function. Therefore, increases in proteasome abundance and peptidase activities, when coupled with increases in polyubiquitinated proteins, as often reported for various forms of heart disease (Table 1), may very well reflect PFI rather than a proteasome function surplus in a diseased heart. 3.3 Protein aggregation causes PFI and marks PQC inadequacy in cardiomyocytes Protein aggregation is used here to describe the cellular process by which abnormal proteins form aggregates. Insoluble, microscopically visible aggregates (sometimes also known as inclusion bodies or aggresomes) may just be the tomb stone of aberrant protein aggregation and are no longer very toxic to the cell; however, the soluble, and often invisible, intermediate species of aggregates, such as the pre-amyloid oligomers during amyloid formation, are detrimental to the cell. 70,71 Impairment of UPS proteolytic function by aberrant protein aggregation has been demonstrated in both non-myocytes and cardiomyocytes. 58,59,67,68,72 Overexpression of human desminrelated cardiomyopathy-linked misfolded proteins (CryAB R120G or D7-des) caused aberrant protein aggregation and PFI in cultured cardiomyocytes. The PFI can be prevented and/or attenuated by diminishing aberrant protein aggregation via overexpression of chaperones or treatment of a pharmacological compound, 59,72 thus proving that aberrant protein aggregation is required for both CryAB R120G and D7-des to cause PFI in cardiomyocytes. The mechanism underlying proteasome impairment by protein aggregation remains unclear. Insoluble protein aggregates or inclusion bodies per se may not necessarily impair the proteasome, but the persistent presence of the aggregates in a cell marks PQC inadequacy. This is because the

6 258 H. Su and X. Wang inclusion bodies are not static but rather highly dynamic. They are the net result of a dynamic equilibrium between the deposit and the removal processes. The removal is mediated by intracellular proteolytic mechanisms including the UPS and autophagy, especially the latter; whereas, the new formation is fed by newly produced misfolded proteins. Therefore, the persistent presence of inclusion bodies indicates that the newly formed misfolded proteins are not cleared by the UPS in a timely fashion and keep depositing into the inclusion bodies. 3.4 Autophagy is activated by and compensates for PFI in CryAB R120G -based desmin-related cardiomyopathy Increases in autophagosomes were observed in CryAB R120G -expressing cultured cardiomyocytes as well as CryAB R120G transgenic mouse hearts. 46 Pharmacological inhibition of autophagy increased CryAB R120G protein aggregation in cultured cardiomyocytes. Suppression of autophagy by downregulating beclin1 significantly accumulated ubiquitinated proteins in CryAB R120G mouse hearts and exacerbated desmin-related cardiomyopathy progression, illustrating that autophagy is an adaptive response in desmin-related cardiomyopathy. 46 As described earlier, PFI was shown in a similar desmin-related cardiomyopathy mouse model. 59 Hence, autophagy activation in the desmin-related cardiomyopathy mouse hearts may be caused by PFI, which was later supported by a report that illustrated proteasome inhibition activates autophagy and vice versa in cultured cells UPS dysfunction in animal models of other forms of cardiomyopathy As reviewed by many others in this Spotlight Issue, 3,4 altered UPS function has also been observed in animal models of many other cardiomyopathies. Notably, nearly all the reports so far have observed increased ubiquitinated proteins in diseased hearts regardless of the form of primary cardiac disease. Also, the reported changes in proteasome peptidase activities vary among models of different cardiac disorders, and the majority of these studies observed increased proteasome peptidase activities 60,73 77 (Table 1). Intriguingly, these resemble what we have observed in the desmin-related cardiomyopathy mouse models. 58,59 Because polyubiquitinated proteins are normally very efficiently degraded by the proteasome, we believe that the increases in polyubiquitinated proteins implicate PFI in these diseased hearts as well. Of course, this remains to be tested because alternative interpretations are possible. For instance, one possibility is that some of the accumulated ubiquitin conjugates might be formed by abnormal ubiquitin linkage that cannot be recognized and degraded by the proteasome. A global change in the ubiquitin system was recently revealed by careful proteomics analyses of brain tissue samples from Huntington disease humans and animal models. 78 Accumulation of K48-linked polyubiquitins which signal for proteasomal degradation was consistently detected in the early stage of pathogenesis of Huntington disease, consistent with a pathogenic role of PFI, whereas K63-linked and K11-linked polyubiquitin chains were also increased. 78 It will be interesting and important to determine whether a similar change also occurs in cardiomyopathy, especially cardiac proteinopathy. 3.6 Inadequate PQC in human cardiomyopathy and CHF Many common factors during the progression of various heart diseases to CHF can conceivably increase the load of misfolded proteins while impairing UPS proteolytic function. Consequently, UPS function may become inadequate. 2 The most common and powerful cardiac response to a variety of stresses is hypertrophy, cardiomyocyte growing larger, which requires not only increases in protein synthesis but also counter-intuitively increased protein degradation. This is because: (i) during protein synthesis, 30% of newly synthesized polypeptides need to be co-translationally degraded by the UPS presumably for PQC 79 ; (ii) the addition of new sarcomeres to the myofibrils may require tearing down some of the existing sarcomeres, and most, if not all, sarcomeric proteins are degraded by the UPS 2 ; and (iii) a hypertrophied cardiomyocyte has more sarcomeres needing to be periodically renewed. In addition, UPS-mediated proteolysis requires ATP, but energy expenditure is usually challenged in diseased hearts. Using antibodies that recognize the conformation of preamyloid oligomers, Sanbe et al. 71 detected pre-amyloid oligomers in failing human hearts with dilated or hypertrophic cardiomyopathies but not normal hearts. Increased ubiquitinated proteins were also detected in explanted human hearts with dilated cardiomyopathy or ischaemic heart disease. 74 These findings arguably situate a large subset of human cardiomyopathy and CHF into the category of proteinopathy, suggesting that PFI and resultant PQC inadequacy are common phenomena in these disorders. In support of this notion, both the accumulation of polyubiquitinated proteins and the formation of the aggresome-like structure were recently reported in a severe transverse aortic constrictioninduced mouse model of cardiac hypertrophy and failure Does PFI contribute to the progression of proteinopathies to CHF? Since PFI and the activation of autophagy have been observed or implicated in many forms of cardiomyopathy, especially proteinopathies, we have submitted that inadequacy in cardiac PQC contributes to cardiac remodelling and failure (Figure 2). 1,2 Obviously, it will take a tremendous amount of work to test this hypothesis, but some published studies have shed light on the pathophysiological significance of PQC inadequacy in the heart. For instance, a protective role of chaperones and autophagy has been well established in desmin-related cardiomyopathy and some other cardiac disorders. 17,46,70,80 The pathogenic significance of PFI in the heart, on the other hand, remains uncertain. Because a reliable measure to normalize PFI in cardiomyocytes is currently lacking, no study has been reported to determine the necessity of PFI in the pathogenesis of any cardiac diseases. As summarized in the following, the current evidence on the sufficiency of PFI to cause cardiac dysfunction is quite conflicting.

7 Proteasomes in cardiac proteinopathy 259 Figure 2 A diagram to depict the hypothesis that PFI contributes to cardiac dysfunction. Under stress conditions, the increased production of abnormal proteins overwhelms the UPS, and the resultant aberrant protein aggregation impairs proteasome proteolytic function, leading to PFI. PFI in turn accumulates misfolded proteins, thereby forming a vicious cycle between accumulation of misfolded proteins and PFI. PFI can induce cardiomyocyte dysfunction and/or cardiomyocyte death, and the dysfunctional cardiomyocytes produce more misfolded proteins, thereby forming a vicious cycle between cardiac dysfunction and PFI. Aberrant protein aggregation and/or PFI activate the autophagy-lysosome pathway, which may help relieve proteotoxic stress and attenuate PFI-induced cardiomyocyte dysfunction. The legend for the symbols is the same as in Figure Genetic perturbations of specific UPS components compromise cardiac function Multiple lines of genetic evidence indicate that perturbation of the UPS is sufficient to compromise cardiac function. Deficiency of CHIP, a co-chaperone and a likely important ubiquitin ligase for PQC, was found to accumulate damaged proteins in the heart, cause cardiac hypertrophy, decrease mouse longevity, and make the heart more susceptible to myocardial infarction (MI)-induced dysfunction. 19,81 Loss of MuRF1 and MuRF3, two muscle-specific ubiquitin ligases, caused cardiac hypertrophy and sensitized the mouse heart to MI injury. 82,83 The von Hippel Lindau protein (VHL) is an F-box protein that constitutes an important E3 ligase with cullin2 and mediates the proteasomal degradation of HIF1a under normoxia. 84,85 Loss of VHL in mouse hearts accumulated HIF1a and caused severe foetal heart failure, accompanied by lipid accumulation, aberrant nuclear and myofibril morphology, myocyte loss, and fibrosis. 86 Mutations in VCP (CDC48 or p97), which is involved in coordinating substrates recruitment, ubiquitin chain assembly, and proteasomal targeting, 87,88 caused the formation of inclusion bodies and cardiomyopathy in humans. 89,90 Finally, deficiency of an inducible proteasome subunit low molecular mass polypeptide-2 in mice led to the loss of ischaemic preconditioning-induced cardioprotection against I/R injury, likely through stabilizing PTEN in the heart. 91 To date, no studies on genetic perturbation of the 26S proteasome in the heart have been reported. Interestingly, conditional deletion of a 19S proteasome base subunit Psmc1 in mice resulted in 26S proteasome depletion in targeted neurons but left 20S proteasomes intact, leading to the development of neural proteinopathy in mice Pharmacological proteasome inhibition produced mixed results Both detrimental and beneficial effects were reported for pharmacologically induced proteasome inhibition on I/R injury. 93 Differences in the timing, degree, and duration of proteasome inhibition may account for the conflicting results. Interestingly, nearly all reported studies on pressure overload cardiac hypertrophy have shown that acute or chronic pharmacological proteasome inhibition of the 20S proteasome prevented or reversed cardiac hypertrophy without discernibly affecting heart function in rodents These studies provided compelling evidence that normal proteasome function is required for hypertrophic

8 260 H. Su and X. Wang growth. 97 Meanwhile, the experiments also showed that cardiac function remained intact even during chronic proteasome inhibition, and proteasome inhibition after the initiation of pressure overload suppressed cardiomyocyte apoptosis. 94,95 This evidence appears to go against the hypothesis that PFI may play an important role in cardiac proteinopathies. However, the relatively mild systemic proteasome inhibition, which affects both the cardiomyocyte and non-cardiomyocyte compartments in the heart in addition to other organs, may not necessarily recapitulate cardiomyocyterestricted severe PFI in cardiac proteinopathies. Notably, clinical reports seem to support the PFI hypothesis quite well. Chronic use of proteasome inhibitor bortezomib in cancer chemotherapies was found in some cases to cause cardiac complications including CHF, angina, atrial fibrillation, and bradycardia Interestingly, most of these patients are the elderly. Given the decreasing proteasome function during ageing, 102,103 it is possible that aged hearts are more vulnerable to the cardiotoxicity of proteasome inhibition. In situations where PFI is likely responsible for cardiac dysfunction, the underlying mechanism remains to be investigated. Nonetheless, we found that proteasome inhibition was sufficient to activate a signalling transduction pathway that is known to mediate cardiac pathological remodelling (X.W., unpublished data) and cause cardiomyocyte death in tissue culture settings Conclusion and future directions PFI and resultant PQC inadequacy have been observed or implicated in animal models of bona fide cardiac proteinopathies and other common forms of cardiac disease; however, their pathophysiological significance in these diseases remains to be established. Evidence that either supports or contradicts a significant role of PFI in cardiac remodelling and failure has been reported. Additional, more definitive studies are warranted. Cardiomyocyte-restricted proteasome perturbation and measures to enhance cardiac proteasome function should be very helpful in defining the role of proteasome dysfunction in the heart. Acknowledgements We thank Ms Emily McDowell for critically reading this manuscript. X.W. is an established investigator of the American Heart Association. Conflict of interest: None. Funding Research in H.S. and X.W. s laboratory is supported in part by grants R01HL072166, R01HL085269, and R01HL from NIH and grant N from the American Heart Association (to X.W.) and by the MD/PhD Program of the University of South Dakota. References 1. Wang X, Su H, Ranek MJ. Protein quality control and degradation in cardiomyocytes. J Mol Cell Cardiol 2008;45: Wang X, Robbins J. Heart failure and protein quality control. Circ Res 2006;99: Luo H, Wong J, Wong B. Protein degradation systems in viral myocarditis leading to dilated cardiomyopathy. 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