Heart failure and protein quality control

This review is part of a thematic series on Ubiquitination, which includes the following articles:

Regulation of G Protein and Mitogen-Activated Protein Kinase Signaling by Ubiquitination: Insights From ModelOrganisms

Heart Failure and Protein Quality Control

Ubiquitin and Ubiquitin-Like Proteins in Protein Regulation

Seven-Transmembrane Receptors and Ubiquitination Sudha K. Shenoy, Guest Editor

Heart Failure and Protein Quality ControlXuejun Wang, Jeffrey Robbins

Abstract—The heart is constantly under mechanical, metabolic, and thermal stress, even at baseline physiologicalconditions, and cardiac stress may increase as a result of environmental or intrinsic pathological insults. Cardiomyocytesare continuously challenged to efficiently and properly fold nascent polypeptides, traffic them to their appropriatecellular locations, and keep them from denaturing in the face of normal and pathological stimuli. Because deploymentof misfolded or unfolded proteins can be disastrous, cells, in general, and cardiomyocytes, in particular, have developeda multilayered protein quality control system for maintaining proper protein conformation and for reorganizing andremoving misfolded or aggregated polypeptides. Here, we examine recent data pointing to the importance of proteinquality control in cardiac homeostasis and disease. (Circ Res. 2006;99:1315-1328.)

Key Words: cardiac disease � cardiac failure � cardiac muscle � cardiomyocytes � cardiovascular disease� cardiovascular physiology

Because of alternative splicing of primary transcripts,posttranslational modifications, and the ability to assume

multiple conformations that differ in activity, the proteome,in terms of its informational content, is considerably morecomplex than the genome and transcriptome. Thus, it is notsurprising that controlling the quality of this information isessential for cell survival and function. Multiple layers ofquality control for protein production and maintenance exist.After their initial synthesis, proteins targeted for the mem-brane and secretory pathways are modified, folded, andassembled in the endoplasmic reticulum (ER), whereas othercellular proteins may be synthesized and processed indepen-dently of the ER in the cytosol. Accordingly, there exist bothER-associated protein quality control (PQC) and ER-independent PQC. In both cases, molecular chaperones andthe ubiquitin/proteasome system (UPS) play essential roles.In general, chaperones are responsible for protecting unfolded

or partially folded nascent or mature proteins, with manychaperones participating in protein repair,1 whereas the UPSis largely responsible for removing terminally misfoldedproteins permanently, thereby preventing misfolded proteinsfrom accumulating in the cell.2

Although the first lines of defense rest in the “proofread-ing” of the primary DNA and RNA sequences, the cell hasevolved multiple layers of control at the posttranslationallevel as well, and nascent proteins are subject to rigoroussurveillance as they are synthesized on the polysomes.Although small- and medium-sized proteins often can assumetheir correct tertiary and quaternary conformations spontane-ously, the majority of proteins cannot and depend on the helpof and interaction with other proteins to fold correctly. Thecomplete sequence is often necessary for assuming thecorrect conformation, but the linear process of protein syn-thesis presents unfinished proteins to the cellular environ-

Original received September 11, 2006; revision received October 6, 2006; accepted October 30, 2006.From the Division of Basic Biomedical Sciences (X.W.), Sanford School of Medicine of the University of South Dakota, Vermillion, SD; and

Children’s Hospital Research Foundation (J.R.), Cincinnati, Ohio.Correspondence to Jeffrey Robbins, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail

[email protected]© 2006 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/01.RES.0000252342.61447.a2

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ment. The solution lies in the surveillance of the process by alarge and diverse family of proteins, termed chaperones,which mediate the correct folding of nascent or incompletepeptides, preventing their misfolding and the subsequentformation of insoluble aggregates. Misfolding is often initi-ated by the exposed hydrophobic surfaces of the nascentprotein, and the chaperones bind tightly to these, preventingtheir interaction and subsequent aggregation. The complexityof this process and the diversity of peptide sequence arereflected in the large number and types of chaperones andcochaperones,1,3 such as the chaperonin family, the nuclearchaperones that help assemble nucleosomes, themitochondrial-specific chaperones for the respiratory chainproteins, and a class of chaperones whose synthesis isinduced in response to stress, the small heat shock proteins(HSPs).4

The small HSPs are a class of chaperones that have beenstudied extensively and play important roles in normalcellular function and disease development.5,6 The “heat-shockresponse,” in which an entire group of genes is activated inresponse to environmental stress, was first described inDrosophila. The small HSPs play important chaperone andprotective roles in the heart and the nomenclature, andclassification of the members in the family has recently beenreviewed in detail.7 They are abundant in cardiomyocytes andare upregulated during cardiac stress and disease,8,9 with asingle member, �-B crystallin (CryAB), making up as muchas 3% of the total soluble protein mass. CryAB binds bothdesmin and cytoplasmic actin and possesses molecular chap-erone function in vitro.10 All of these considerations, coupledwith genetic evidence linking an R120G mutation in CryAB(CryABR120G) to human cardiac disease, prompted a series ofexperiments demonstrating that CryABR120G expression wassufficient to cause heart failure in vivo.11

The Ubiquitin/Proteasome SystemThe integrity of a protein is constantly questioned by thePQC. Damage control is particularly important in long-livedcells, such as cardiomyocytes because they normally do notproliferate and thus are relatively sensitive to increasingconcentrations of misfolded protein. In this respect, theyshare an important feature with the neuronal populations,which also cannot proliferate under normal conditions. Ubiq-uitin/proteasome system (UPS)-mediated proteolysis consti-tutes a second line of defense for the PQC of a cell byremoving misfolded, oxidized, mutant, or otherwise damagedproteins.12,13 The UPS also degrades normal proteins that areno longer needed, providing temporal regulation of proteinactivity.12,14 UPS-mediated proteolysis includes 2 majorsteps: attachment of a polyubiquitin chain via isopeptidebonds to the target protein molecule through a process knownas ubiquitination and the subsequent degradation of theubiquitinated proteins by the 26S proteasome. Both ubiquiti-nation and proteasome-mediated degradation are highly reg-ulated cellular processes.15 Although monoubiquitinationdoes occur, polyubiquitination is the posttranslational processthat targets specific proteins for degradation by the 26Sproteasome.13 Ubiquitination is performed by a cascade ofenzymatic reactions that are catalyzed by 3 classes of en-

zymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (Ubc, E2), and ubiquitin ligase (E3).2,12

Recent data indicate that polyubiquitin chain assembly factor(E4) may also play an important role,16 as UFD2a, an E4exclusively expressed in cardiac muscle during mouse em-bryonic development, is essential for normal heart develop-ment.17 There is 1 ubiquitin E1, approximately 50 E2s, andmore than 400 potential E3s in the human genome, dependingon how these proteins are defined.18,19 To date, 2 E4s havebeen identified in mammalian cells.17

The high efficiency and exquisite specificity of ubiquiti-nation relies mainly on the E3 ligases.12 Nearly all known E3suse 1 of 2 catalytic domains: a HECT (Homologous to E6APCarboxyl Terminus) domain or a RING (Really InterestingNew Gene 1) finger domain.20,21 The U-box domain can alsoexecute ubiquitin ligase activity. The U-box is a distantrelative of the RING finger domain because it has a RING-like conformation but lacks the canonical Zn-coordinatingresidues.22 HECT domain E3s are typified by human E6AP.There are approximately 40 HECT domain–encoding genes,more than 380 RING finger protein genes, and 9 U-box genesin the human genome.19,23 They can possess ubiquitin ligaseactivity by themselves or as part of multisubunit E3s. HECTE3s are monomeric enzymes that directly participate in thechemistry of substrate ubiquitination reactions by forming aubiquitin–thioester intermediate and transferring a ubiquitinmonomer to the target protein molecule during each round ofcatalysis in the ubiquitin–isopeptide bond formation. Incontrast, the RING finger domain proteins usually form amultisubunit complex with partner proteins and bring acti-vated E2s and substrates into close proximity, facilitatingubiquitin–isopeptide bond formation.22

The SCF (Skp1-Cul1–F-box protein) complexes are theprototype of a superfamily of cullin (Cul)-based RINGfinger–type E3s that represent the largest family of ubiquitinligases and mediate the ubiquitination of a variety of regula-tory and signaling proteins in diverse cellular pathways.24 TheSCF consists of 3 invariant components, Skp1, Cul1, andRbx1 (a RING finger protein, also known as Roc1 or Hrt1)and an interchangeable F-box protein subunit. Cul1-Rbx1forms the catalytic core of the complex and is responsible forrecruiting E2, whereas Skp1 serves as an adaptor that is ableto bind different F-box proteins.25 The F-box proteins, carry-ing a Skp1-interacting F-box motif and a protein–proteininteraction domain, can dock different protein substrates thatare often phosphorylated to the SCF complex for ubiquitina-tion. In addition to Cul1, the cullin protein family has at least6 additional closely related paralogs in humans (Cul2, -3,-4A, -4B, -5, and -7).26 Cul-1 is required for cell cycle exit inCaenorhabditis elegans and is part of a novel gene family.26

All of these cullins can bind Rbx1 and form ubiquitin ligases.Thus, a large number of ubiquitin E3s are cullin dependent,and cullin appears to be a focal point for regulation ofubiquitin E3 assembly by neddylation, a posttranslationalmodification similar to ubiquitination (see below).27

The carboxyl terminus of the Hsc70-interacting protein(CHIP), originally identified as a cochaperone of Hsc70, isalso a ubiquitin E3 ligase.28 It has both a tetratricopeptiderepeat motif and a U-box domain. The tetratricopeptide repeat

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motif associates with Hsc70 and Hsp90, whereas the U-boxmediates ubiquitin ligation. Hence, CHIP is an ideal candi-date E3 for PQC, which selectively leads the abnormalproteins recognized by molecular chaperones to degradationby the proteasome,29 and evidence continues to accumulateindicating that this hypothesis is correct.30,31 Compared withwild-type controls, mouse hearts lacking CHIP were compro-mised in their ability to respond to and cope with myocardialinfarction.32

Only a few ubiquitin E3 ligases responsible for ubiquiti-nation of cardiac proteins have been described. Atrogin-1, anF-box protein that was identified in skeletal muscle and isinvolved in myofibrillar protein degradation, can inhibitcalcineurin-mediated cardiac hypertrophy by participating inSCF complex activity.14 Muscle-specific RING finger(MuRF) proteins are the other known major family of E3proteins involved in muscle protein degradation,33,34 andMuRF1 is a ubiquitin E3 for troponin I.35 Interestingly,MuRF1 can also inhibit protein kinase C� activation andprevent phenylephrine- and 4-phorbol myristate 13-acetate–induced, but not insulin-like growth factor-1–induced, car-diomyocyte hypertrophy.36 These findings have raised thepossibility of affecting cardiac hypertrophy by activatingUPS-mediated degradation of specific cardiac proteins.37 Insupport of this hypothesis, levels of connexin43, the mostabundant gap junction protein in ventricular myocardium, arepartially regulated by the proteasomal (as well as the lysoso-mal) pathway.38,39 The specific E3 mediating connexin43ubiquitination has not been identified, but ubiquitinationappears to be involved in both proteasome- and lysosome-mediated connexin43 degradation.40,41

ProteasomesThe 26S proteasome is the cellular machinery responsible fordegrading polyubiquitinated proteins. The 26S is composedof a 20S proteolytic core particle (the 20S proteasome) and aproteasome activation particle (the 19S proteasome), which islocated at 1 or both ends of the 20S proteasome. The 20S isa hollow cylindrical structure formed by a stack of 4heptameric rings: 2 central antiparallel identical � ringsflanked by 2 identical outer � rings (Figure 1). Each � or �ring consists of 7 unique protein molecules (�1 to �7, �1 to�7). The unfolded protein is degraded in the cavity of the 20Sby the chymotrypsin-like, trypsin-like, and caspase-like (alsoknown as peptidylglutamyl-peptide hydrolase) activities,

which reside in the �5, �2, and �1 subunits, respectively. Theouter � ring forms the gate, which regulates substrate entryinto the proteolytic chamber and also controls the exit of theproteolytic peptide products.12

The 19S (also known as PA700) binds the 20S through�-ring interactions. The 19S from Saccharomyces cerevisiaeconsists of at least 17 subunits: regulatory particle non-ATPase (Rpn) 1 to 12 and regulatory particle tripleA�-ATPase (Rpt) 1 to 6. The 6 tripleA�-ATPase subunits, alongwith Rpn1 and Rpn2, form the base of the 19S, whereasRpn3, Rpn5 to Rpn9, Rpn11, and Rpn12 form its lid, withRpn10 linking the lid and base. The base directly interactswith the � ring of the 20S. In murine hearts, an alternativelyspliced isoform of Rpn10 has been described.42 The 19Srecognizes ubiquitinated protein molecules, deubiquitinatesand unfolds them, and channels the unfolded protein to the20S where peptide cleavage takes place. The proteasome alsoplays an important role in antigen processing and antigenpresentation through the class I major histocompatibilitycomplex. Certain conditions, such as viral infection or induc-tion with interferon � or tumor necrosis factor, remodel the20S, resulting in the constitutive peptidase subunits �5, �2,and �1 being replaced with the inducible �5i, �2i, and �1ipeptidase subunits, respectively. Remodeling can alter pro-teasome peptidase activity and proteomic analyses show thatpurified murine cardiac 26S proteasomes contain both the 3constitutive and 3 inducible peptidase subunits.42

The 20S can also associate with the 11S proteasome.43 The11S is also known as proteasome activator 28 (PA28) or REGbecause its subunits, PA28�, PA28�, and PA28�, withmolecular masses of approximately 28 kDa, have the capacityto enhance the peptidase activities of the 20S.43 Of the 3PA28s, PA28� and PA28� assemble into heteroheptamers(�3�4 or �4�3) and PA28� forms homoheptamers. Bothtypes of 11S proteasomes have a molecular mass of �200kDa.43 All 3 PA28s are expressed in cardiomyocytes. PA28�and PA28� are found in the cytoplasm and the nucleus, butPA28� is located predominantly in the nucleus.43 The 20Scan be capped by 19S or 11S proteasomes at both ends(19S-20S-19S or 11S-20S-11S) or by the 19S at one end andthe 11S at the other (19S-20S-11S) (Figure 1). Because the11S does not contain ATPase activity and it can enhance thepeptidase activities of the 20S, it has been hypothesized thatit enhances 20S proteasome peptide cleavage but does notfacilitate substrate entry. However, a recent study showedthat PA28� directs degradation of the steroid receptor coac-tivator SRC-3 by the 20S in an ATP- and ubiquitin-independent manner, indicating that entry may be affect-ed.44,45 In contrast to data obtained from red blood cells andliver cell preparations, Gomes et al recently reported that 11Sproteasomes were not detected in murine cardiac 20S protea-some preparations and were only occasionally detected in the26S proteasome preparations, although PA28� is fairly abun-dant in the heart.42 It is possible that the purification proce-dures selectively leave out 11S-associated 20S proteasomes.Using gel filtration, a significant amount of 20S proteasomescoeluted with the 11S proteasome in native murine myocar-dial proteins. Consistent with the hypothesis that this pathwayplays an important role in cardiomyocyte function, a substan-

Figure 1. Proteasome complexes. Shown are the various parti-cles and their subunit compositions.

Wang and Robbins Protein Quality Control 1317



tial subset of 20S proteasomes appears to associate with thesarcomere in striated muscle and display a characteristicstriated pattern when immunolabeled in striated muscle.42,46

The physiological significance of this association has notbeen established, but it is conceivable that the proteasomemay be involved in sarcomere genesis and maintenance. Inthe future it will be important to define the (patho)physiolog-ical role of 11S proteasomes in the heart.

Regulation of Proteasomal Proteolytic ActivityThe potential of therapeutic manipulation of protein degra-dation in a specific organ or tissue type has been recog-nized47,48 but is limited by an incomplete understanding ofhow proteasomal specificity and activity are regulated. Polyu-biquitination is almost always required for a specific proteinto be degraded by the 26S proteasome.12 Thus, the decisionfor a protein to be degraded by the UPS is made by exposureor maturation of its ubiquitination signal (degron) to itsspecific E3 ligase. Directly enhancing proteasomal activitywould unlikely increase degradation of normal proteins be-cause posttranslational modification within or nearby thedegron of a normal protein is usually required for the bindingof a specific E3 complex and ensuing degradation of thenormal protein.20 Taking the opposite approach, inhibition ofthe 20S proteasome would affect degradation of the majorityof cellular proteins, whereas targeting a specific E3 ligasewould likely affect a family of proteins. The most preciseapproach would be to manipulate the ubiquitination signal ofthe target protein. An example of the general approach is theubiquitin E3 inhibitor nutlin, which specifically binds to thep53 binding pocket of MDM2 (Murine Double Minute 2), anE3 ligase for p53, and prevents p53 from being degraded.47

As described earlier, the binding of 19S (PA700) or 11S(PA28) proteasomes to the 20S activates the proteasome.Although 11S binding does not require ATP, the associationof the 19S and 20S particles to form the 26S proteasome isATP dependent. Unfolding the substrate protein and channel-ing it into the 20S proteasome by the base of 19S proteasomesalso requires ATP hydrolysis.12 Thus, an overall negativeenergy balance, which can occur in some cardiac disease,might inhibit UPS proteolytic function directly.

The 20S proteasome appears to be able to selectivelyrecognize and degrade oxidized proteins in the heart in aubiquitin-independent manner.15,49 Protein oxidation oftenleads to denaturation and/or partial unfolding, exposinghydrophobic surfaces or domains that are normally buried inthe native polypeptide. Proteasomes have a relatively highaffinity for hydrophobic amino acids,50 and, thus, partiallydenatured proteins that have undergone mild oxidation maywell be preferentially bound by the 20S proteasome andtargeted in a ubiquitin-independent manner for degradation.51

It should be emphasized, however, that this activity isdependent on only mild oxidation, and if significant oxidativeinjury takes place, such as what occurs during a major cardiacischemic event, proteasomal proteins themselves are signifi-cantly damaged and proteasomal activity decreased.52,53 Pro-teasomal dysfunction during ischemia has been extensivelydiscussed.13 The exact mechanism of this inhibition remainsto be determined as the ischemic cell will also be ATP

deficient, and, thus, the ATP-dependent ubiquitination path-ways will also be affected.

Posttranslational modifications, including phosphorylation,N-terminal acetylation, and N-terminal myristoylation, areobserved in proteasome subunits,15,54 but their functionalsignificance is largely undefined. The �3 and �7 subunits in20S proteasomes isolated from Rat-1 fibroblasts and humanembryonic lung cells (L-132) are phosphorylated. Followingdephosphorylation by acidic phosphatase, immunoprecipi-tated proteasomes displayed a significantly lower activitycompared with the phosphorylated proteasomes.55 The �7subunit is phosphorylated by proteasome-associated caseinkinase II (CK2) at Ser243 and Ser250.56 Phosphorylation of�7 may play an important role in stabilizing the 26S protea-some.57 Several subunits of the 19S particle in animal cellsare also phosphorylated, and this may be important inmediating 26S proteasome assembly.58,59 Using a proteomicapproach, Ping and colleagues detected N-terminal acetyla-tion of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and20S subunits (�2, �5, �7, �3, and �4), N-terminal myristoyl-ation of a 19S subunit (Rpt2), and phosphorylation of 20Ssubunits (eg, �7).42 They also identified 2 previously unrec-ognized functional partners in the endogenous intact cardiac20S proteasomes: protein phosphatase 2A (PP2A) and proteinkinase A (PKA).54 Multiple individual subunits of 20S (eg,�1 and �2) appear to be the targets of PP2A and PKA;inhibition of PP2A or the addition of PKA significantlymodified both the serine- and threonine-phosphorylationprofile of proteasomes, and phosphorylation of the 20Scomplex enhances the peptidase activity of the individualsubunits in a substrate-specific fashion. Taken together, thesestudies show that the peptidase activities of cardiac 20Sproteasomes are modulated by associating partners and phos-phorylation may be a key mechanism for regulation.15

The COP9 SignalosomeThe COP9 signalosome (CSN) can regulate UPS function. Inmammalian cells, the CSN is a multimeric protein complexconsisting of 8 unique protein subunits, referred to as CSN1through CSN8.27 The composition of the CSN complex andthe domain structure of its subunits resemble the 8 subunit–containing lid subcomplex of the 19S proteasome.27 Itsessential role in mammalian development is confirmed byembryonic lethality in csn2, csn3, and csn5 nulls.60 Thebiochemical activities and cellular functions of CSN inmammalian systems remain obscure, but studies in lowerorganisms point to its importance in controlling overallproteasomal activity. A major target of the CSN is thecullin-based ubiquitin E3 ligase complex. Cullin familymembers are hydrophilic proteins that were initially charac-terized as being involved in the control of yeast cell divisioncontrol and can be covalently modified by a ubiquitin-likeprotein, Nedd8/Rub1, via a process known as neddylation,which is similar to ubiquitination. The CSN is responsible forthe cleavage of the Nedd8 moiety (ie, deneddylation) fromcullins. CSN-associated deubiquitination activities have alsobeen described.61,62 These activities can be subdivided intothe ability to deconjugate ubiquitin from monoubiquitinatedsubstrates as well as depolarization of polyubiquitin.62 Col-




lectively, the CSN has both deneddylation and deubiquitina-tion activity, either by possessing the intrinsic catalyticactivity or by selectively recruiting different enzymes underdifferent circumstances.

The lid of the 19S proteasome consists of 8 subunits thatare paralogs of the 8 CSN subunits. Both CSN and the lid of19S proteasomes contain 6 subunits with a PCI (Proteasome,COP9, eIF3) domain and 2 subunits with a MPN (MOV34,PAD N-terminal) domain.60 CSN and the lid of the 19S alsohave a similar architecture. Recent studies indicate that CSNdirectly interacts with the 26S proteasome and may competewith the lid of the 19S proteasome, thereby influencingproteasomal activity.63 Both purified CSN and the lid of 19Sproteasomes can bind polyubiquitin chains in vitro.64 Al-though it is likely that CSN directly regulates the proteolyticfunction of the proteasome, this has not been demonstrateddirectly and it will be important to formally test CSN abilityto act cooperatively with the ubiquitination machinery toachieve ubiquitin-dependent degradation of specific regula-tors important for cardiac function.

Assessment of UPS Proteolytic FunctionSynthetic fluorogenic peptide substrates are widely used todetermine the chymotrypsin-like, trypsin-like, and caspase-like activities of the 20S proteasome in either tissue and celllysates or in purified proteasomes. However, there are anumber of technical concerns that limit this assay. Becausethese substrates can also be cleaved by nonproteasomalpeptidases, only the portion of peptidase activities inhibitedby a specific proteasome inhibitor can be attributed to theproteasome. An additional concern is that the syntheticpeptide substrates are usually very small and can diffuse intothe 20S proteolytic chamber, even in the absence of protea-some activators such as the 19S and the 11S proteasomes.65

Thus, although fluorogenic peptidase assays permit rapidassessment of the catalytic activity of the 20S proteasome,they may not accurately reflect UPS proteolytic function andneither the ubiquitination step nor the highly regulated entryof substrates into the 20S proteasome is effectively evaluatedby these assays.

To better assess UPS proteolytic function in cells, tissues,and the whole animal, a series of fluorescence proteinreporters were developed.66–68 Biological reporters such asgreen fluorescence protein (GFP) or luciferase were modifiedsuch that they were targeted for ubiquitination and degrada-tion by the UPS. These reporters use different ubiquitinationsignals, such as a noncleavable ubiquitin (Ub) fusion con-struct (eg, UbG76V-GFP),67 a cleavable Ub fusion peptide thatpermits the creation of an N-end rule substrate (eg, Ub-Arg-GFP), and the CL1 degron (GFPu) (Figure 2).69,70 Reporterscarrying different ubiquitination signal sequences conceiv-ably could detect different ubiquitin conjugation pathwaystraversed en route to the proteasome. An example of thisgeneral strategy is illustrated by the degron CL1, a peptidesequence consisting of 16 amino acids (ACKNWFSSLSH-FVIHL). Structural predictions indicate that the CL1 peptidesequence can form an amphipathic helix with surface expo-sure of a patch of hydrophobic amino acids, which may be astructural feature that is shared by misfolded proteins and

signals for ubiquitination. The fusion of CL1 to the carboxylterminus of GFP creates GFPu (or GFPu). GFPu is a specificUPS substrate in cultured human embryonic kidney (HEK)cells and neonatal rat ventricular myocytes.69 In HEK cells,GFPu has a half-life of �30 minutes, which is much shorterthan unmodified GFP. Cell lines stably expressing GFPu andrecombinant adenoviruses capable of delivering the GFPu

transgene have been successfully used.71,72

Transgenic (Tg) mouse lines that ubiquitously express asimilarly engineered, but a slightly different GFP referred toas GFPdgn, have been made.73 The GFPdgn mice were usedfor monitoring dynamic changes in UPS proteolytic functionin vivo. Systemic inhibition of the proteasome by pharmaco-logical agents such as MG-262 and lactacystin resulted insignificant accumulation of GFPdgn in all organs examined,including the heart.73 Interestingly, a similar approach di-rected at inhibiting the proteasome did not lead to accumu-lation of a different UPS reporter (UbG76V-GFP) in UbG76V-GFP Tg mouse hearts, skeletal muscle, and brain, suggestingthat GFPdgn mice are better suited for monitoring UPSproteolytic function in these organs.68 GFPu and GFPdgn aredistributed in both the cytoplasm and the nucleus. Recently,nuclear and cytoplasmic variants of GFPu reporters werecreated by inserting either a nuclear localization sequence(NLS) or a nuclear export sequence (NES) at the N-terminusof a tandem GFP construct. The nuclear and cytoplasmicGFPu reporters (NLSGFPu and NESGFPu) can be used to

Figure 2. Schematic diagrams of the GFP reporters for UPSproteolytic function. A, C-terminal fusion of the degron CL1sequence renders an enhanced GFP (EGFP) as a specific sub-strate for the UPS. The EGFP signal is inversely correlated toUPS function. GFPdgn and GFPu distribute to the cytoplasmand the nucleus, serving as reporters for both compartments.NES-GFPu, a reporter for cytoplasmic UPS, was engineered byfusion of 2 EGFPs in tandem, insertion of a nuclear exclusionsequence (NES) at the N-terminal of each EGFP, and C-terminalfusion of the degron CL1. NLS-GFPu, a reporter for nuclearUPS, was made similarly, but a nuclear localization sequence(NLS) rather than NES was used. B, N-terminal fusion of a wild-type ubiquitin (Ub) to an unmodified GFP does not change GFPprotein stability but creates an N-end rule degradation signal forthe UPS if the first amino acid of GFP is mutated from Met toArg, rendering Ub-R-GFP a reporter for the N-end rule degrada-tion by the UPS. N-terminal fusion of a modified Ub with a sub-stitution of its final Gly with Val (UbG76V) creates UbG76V-GFP,which is degraded by the UPS. Glycine (K) residues at positions3 and 17 of GFP are potential ubiquitination sites (italics).




investigate localized UPS changes that may affect nuclear orcytoplasmic UPS function differently.70,74 Both reporter micehave been used to investigate UPS proteolytic function inseveral mouse models of human disease, and more extensiveuse of these fluorescence protein reporters will facilitate thestudy of UPS proteolytic function in cardiac physiology andpathology.73,75–77

ER-Associated Quality ControlIn eukaryotic cells, the ER is the factory for processing andassembly of the secretory pathway proteins, which includeproteins destined to the extracellular space, plasma mem-brane, and the exo/endocytic compartments. Nascent un-folded polypeptides are translocated into the lumen of ER,where a specialized set of enzymes and chaperones controlstheir posttranslational modification, helps the newly importedpolypeptides to assume their native structures, and mediatestheir assembly into multimeric complexes. Although esti-mates have been made that this pathway processes �25% ofthe protein complement, it should be emphasized that, in thecardiomyocyte, the myofibrillar protein complement embod-ies the majority of the protein complement of the cell and iscomposed of proteins not processed in the ER. Thus, ER-independent PQC will play a particularly critical role in thesecells. Unfortunately, ER-independent PQC has not beenstudied intensively, and little is known about the specificpathways underlying ER-independent quality control. Be-cause the UPS and many molecular chaperones exist in thecytosol,30 it is reasonable to assume that ER-independentquality control is partially mediated by the collaborationbetween molecular chaperones and UPS-mediatedproteolysis.

On exposure to aqueous solvent, hydrophobic segments inunfolded or partially folded proteins tend to aggregate.BiP/Kar2p, a member of the Hsp70 family, and other ER-resident chaperones bind to the hydrophobic patches, preventaggregation, and preserve the folding competence of nascentpeptide chains.1 Protein folding and maturation in the ER isessential for their subsequent transport through the secretorypathway. To prevent misfolded or unassembled proteins frombeing secreted, the ER contains a quality control system thatrecognizes and disposes of these proteins. ER-associateddegradation is an important level of quality control in mostcells78 and depends on specific retrotranslocation of aberrantproteins across the ER membrane to the cytosol, where theyare degraded by the UPS. This subject has been the topic ofa number of recent reviews, and the reader is referred to thosefor discussion and schematic depictions of the processesinvolved.1,78

Alterations in homeostasis by various cellular stressors canlead to a condition known as ER stress, which causesaccumulation of misfolded or unfolded proteins in the ER. Inresponse, the cell initiates a series of fundamental changes ingene expression, protein synthesis, and protein degradation,termed the unfolded protein response (UPR).79 Componentsof the UPR include transcriptional induction of UPR targetgenes (eg, ER-resident chaperones), inhibiting translationalinitiation and degrading existing mRNAs, which attenuatesprotein synthesis. Activation of the ER-associated degrada-

tion pathway also occurs. An increased level of unfoldedproteins in the ER lumen is sensed via the luminal portions of3 ER resident transmembrane glycoproteins, activating tran-scription factor 6 (ATF6), inositol-requiring enzyme-1(IRE1), and PERK (Protein kinase R–like ER Kinase), whichare normally associated with the ER chaperone BiP. Seques-tration of BiP by misfolded proteins results in dissociation ofBiP from these ER sensor proteins, which, until recently, wasbelieved to be the activating mechanism.79 However, deletionof the region of the BiP binding domain of IRE1 did notimpair its regulation,80 leading to the hypothesis that IRE1can bind directly to misfolded polypeptides, resulting inoligomerization and activation of IRE1.1 The endoribonucle-ase activity at the C terminus of activated IRE1 excisesprecisely 26 bases from the XBP1 (X-box–Binding Protein 1)transcript, which, after religation, creates XBP1(s). XBP1(s)is a potent transcription activator that binds to the ERstress–responsive element (ERSE) in the promoters of ERstress–related genes, upregulating transcription of genes thatencode the ER chaperones, structural proteins of the secretorypathway, components of the ER-associated degradation ma-chinery, and components of membrane biosynthesis. Addi-tionally, activation of IRE1 recruits TRAF2 and leads to JunN-terminal kinase (JNK) activation and caspase-12 cleavage,both of which are linked to apoptosis.79 Accumulation ofmisfolded proteins also allows ATF6 to reach the Golgi,where it is processed such that it can enter the nucleus andmediate the transcription of ER chaperone genes and Xbp1.79

Coincident with these processes, translation is also affected.PERK is an eIF-2a kinase, and activated PERK phosphory-lates eIF-2a and transiently inhibits cap-dependent transla-tional initiation. Together with IRE1-mediated selective deg-radation of mRNA encoding secreted proteins,81 the net resultis to reduce the level of unfolded proteins in the ER andreprogram the ER-associated translational apparatus to ac-commodate the changes needed in gene expression forupregulation of the UPR.

Blockage of protein synthesis early in the UPR arrests thecell cycle at the G1 phase and activates both pro- andantiapoptotic factors.79 In general, the UPR allows the cell tosurvive reversible environmental stresses. However, if thesepersist, proapoptotic pathways are activated. The balancebetween the cytoprotective and destructive arms is poorlyunderstood. Persistent accumulation of misfolded proteinsbeyond the capacity of ER quality control causes cellulardysfunction and cell death.82 This process has been impli-cated in a variety of human disorders, including diabetesmellitus and neurodegenerative diseases such as Alzheimer’sand Parkinson’s.83–86 Sustained ER stress is a potentialcausative factor for cardiomyocyte apoptosis in congestiveheart failure (CHF).87 The KDEL receptor is a retrievalreceptor for luminal ER chaperones that bear a carboxyl-terminal Lys-Asp-Glu-Leu sequence and contribute to ERquality control. Transgenic overexpression of a mutant KDELreceptor leads to aberrant ER quality control and results indilated cardiomyopathy in mice.88 Chronic use of the cancertherapeutic agent imatinib mesylate (Gleevec), a small-molecule inhibitor for the cytosolic tyrosine kinase Bcr-Abl,causes severe CHF in human and left ventricular dysfunction




in mice, and prolonged ER stress is implicated as theunderlying cardiotoxic mechanism.89

Consequences of Protein Misfolding andCompromised PQC in the Heart

For structures like sarcomeres, where the stoichiometryamong their constituents is highly maintained, the synthesisand incorporation of protein must be accurately coupled withthe degradation of the existing protein. Myofibrillar proteindegradation is thought to occur via the UPS. Insufficiency inthe molecular chaperones needed to mediate folding of thelarge sarcomeric components or UPS malfunction couldaffect either nascent protein assembly or efficient removal ofabnormal proteins. These processes could then result inabnormal protein accumulation and aberrant protein aggrega-tion that could further impair the UPS and PQC. If the UPSis compromised or overloaded, what are the consequences fora cell that is terminally differentiated and cannot divide? Inneurons, the consequences are well defined and proteinmisfolding is known to underlie a number of the neurodegen-erative diseases.90,91 Very often, the common pathologicalcharacteristic is the presence of distinct bodies in the affectedregions. These bodies can be extracellular or intracellular andcontain aggregates of misfolded proteins that are oftenubiquitinated but have failed to be targeted to the proteasomefor degradation. The intracellular aggregates, which aregenerically referred to as inclusion bodies, are intractable torefolding and dissolution. These insoluble and kineticallystable entities contain common components such as desmin,ubiquitin, and tubulin. In some cell types such as neurons, themisfolded proteins are attached to dynein motors, transportedin a retrograde manner along the microtubules to a perinu-clear location and coalesce into well-defined, electron densebodies known as aggresomes.92,93

In the heart, the direct consequence of inadequate PQC isaccumulation of misfolded proteins and aberrant proteinaggregation, which also are characteristic for the desmin-related cardiomyopathies. Desmin-related cardiomyopathy isan important component of desmin-related myopathy (DRM),which is a heterogeneous group of human myopathies char-acterized by the presence of abnormal intrasarcoplasmicprotein aggregates that are desmin positive. Although abnor-mal desmin-reactive material in affected muscle cells is ahallmark of DRM, a number of other proteins also accumu-late, including dystrophin, ubiquitin, nestin, vimentin,CryAB, and lamin-B. These accumulations are associatedwith a variable degree of myofibrillar degeneration; therefore,DRM is also referred to as myofibrillar myopathy.94,95 Clin-ically, DRM can present as a generalized myopathy, andrestrictive, hypertrophic, and dilated cardiomyopathies haveall been reported.95 Mutations in the desmin, selenoprotein N,myotilin, and CryAB genes have now been characterized ascausative for DRM.96–100 The desmin and CryAB mutationshave been modeled in mice using cardiomyocyte-specifictransgenesis, and their expression led to cardiac disease.11,101

A defining characteristic of the human DRMs is the presenceof intracellular, electron-dense granulofilamentous bodiesdetectable via transmission electron microscopy, and both Tgmodels showed aberrant intrasarcoplasmic and electron-

dense aggregates that were desmin positive and characteristicof human DRM morphology.

These bodies were reminiscent of aggresomes, as theycontained desmin, CryAB, and other aggresomal pro-teins.98,102–104 Subsequently, the electron-dense bodies pres-ent in the cardiomyocytes expressing CryABR120G were con-firmed as being aggresomes, based on protein composition,time course of formation, and the dependence of perinuclearlocalization on microtubule-mediated transport.103 However,whether these bodies were inherently cytotoxic, benign, oreven protective was unclear. In some systems, aggresomesare clearly associated with pathogenesis,92,105 but there areequally compelling data that argue for cytoprotective ef-fects.106,107 Concentrations of aggregated, misfolded proteinscould easily interfere with either cell metabolism or theinherent function of a cardiomyocyte, which depends onrepeated cycles of unimpeded contraction and relaxation. Asthe aggregates can fill a significant volume of the cytosol anddeform the cell and/or nucleus, it is not difficult to imaginethese bodies could be inherently toxic. On the other hand,aggresomal formation could represent an attempt by the cellto sequester potentially cytotoxic, misfolded proteins fromthe general cytoplasm. Consistent with the latter hypothesis,preventing aggresome formation actually resulted in in-creased cytotoxicity in cardiomyocytes expressingCryABR120G.108

Aggresomes are commonly found in neurodegenerativedisorders that are caused by aberrant protein conformationand misfolding.109 Diseases that fall into this category includeAlzheimer’s disease, Huntington’s disease, Parkinson’s dis-ease, amyotrophic lateral sclerosis, Alexander’s disease, andthe prion-based disorders. Despite some commonality ofpathology, the etiologies are unique and location of theaggregates can differ. In Huntington’s disease, expansion ofpolyglutamine tracts results in large accumulations of intra-cellular aggregates containing the peptides, whereas in Alz-heimer’s disease, A� peptide plaques and misfolded tauneurofibrillar tangles are predominantly extracellular in re-stricted neuronal populations. Despite the unique primarymisfolded proteins responsible for these discrete neurodegen-erative diseases, the pathologies are linked by the accumula-tion of abnormal aggregates containing a �-sheet structure,which form because of either the intrinsic mutation(s) of aprotein(s) or alterations in its processing. The widespreaddistribution of aggresomes in diverse neurodegenerative dis-eases, and the appearance of aggresomes in a cardiomyocyte-based disease, prompted an examination of the possibleparallels between the 2 cell types and the pathological insultsto which they are subjected. Although the inability of neuronsand cardiomyocytes to divide has been challenged in recentyears,110,111 it is generally accepted that, for both cell types,the majority of cells are terminally differentiated and incapa-ble of division. Thus, both might be particularly sensitive toa chronic primary pathogenic stimulus, leading to proteindenaturation or misfolding if they are unable to clear theaggregates, as even a slow accumulation of misfolded pro-teins would eventually result in high cytoplasmic levels ofpotentially cytotoxic entities.




Aggresomes are typically thought of as part of the cellularresponse to either excess protein accumulation or as a way ofsequestering misfolded proteins from the general cytoplasmicmilieu if the proteasomal pathways are compromised oroverloaded. UPS malfunction has long been hypothesized asan important pathogenic mechanism for the protein confor-mational diseases. Support for this hypothesis was buttressedby the discovery that loss-of-function mutations in genesencoding UPS components can cause neurodegenerative dis-eases in humans and rodents.112–114 Using a HEK cell linestably expressing the UPS reporter, GFPu, Kopito and col-leagues first demonstrated that aberrant protein aggregationcaused by polyglutamine-expanded huntingtin (pQ-htt), or amutant cystic fibrosis membrane conductor protein, canimpair the UPS.69 Almost simultaneously, Jana et al showedthat cultured neuro2a expressing pQ-htt displayed aberrantprotein aggregation, depressed proteasomal activity, mito-chondrial dysfunction, and activation of caspases 9 and 3.115

Expression of a truncated myosin-binding protein C mutantlinked to human familial hypertrophic cardiomyopathy alsoresulted in the formation of abnormal protein aggregates andimpaired UPS function in cultured rat neonatal cardiomyo-cytes.116 Taken together, these reports indicate that aberrantprotein aggregation caused by expression of a mutant proteincan impair the UPS in cultured cells, includingcardiomyocytes.

PQC and the Formation of SolublePreamyloid Oligomers in Cardiomyocytes

In addition to aggresome accumulation, the neurodegenera-tive disorders are characterized by peptide accumulations thatcontain amyloid, defined as a substance with distinct ultra-structural (10-nm fibrils with �-pleated sheets) and tinctorial(apple green birefringence and Congo red–positive staining)properties.117 Amyloid formation is a common theme in manyneurodegenerative and protein conformational disorders, andtheir deposition has formed the framework for a unifyingtheory across the diverse disease types. Amyloidoses areassociated with the formation of extracellular plaques ortangles, or intracellular inclusion bodies with amyloid-likecharacteristics.91 The neurodegenerative amyloidoses, inwhich the deposits are localized to diverse or specificpopulations of neurons, have been studied most intensively,but other nonneuropathic localized amyloidoses exist.118 Sys-temic amyloidoses are well characterized in many tissues,including the heart, and are generally thought of as aheterogeneous syndrome characterized by the formation andaccumulation of extracellular proteinaceous fibrils. However,aggregate and plaque accumulation can be site specific,depending on the particular protein and for some of theneurodegenerative diseases such as Parkinson’s and Hunting-ton’s diseases, amyloid accumulation is intracellular. In theheart, systemic nonhereditary amyloidosis is not uncommon.The syndrome is known as AL amyloidosis, in which extra-cellular amyloid fibrils composed of monoclonal immuno-globulin light chains accumulate. Secondary amyloidoses,termed AA amyloidosis, normally develop as a complicationof chronic inflammatory disease such as occurs in patientswith rheumatoid arthritis, although, for this syndrome, car-

diac involvement is less common. Finally, there are a numberof inherited forms of systemic amyloidoses, a common formbeing associated with multiple mutations in the serum proteintransthyretin.119 The systemic amyloidoses have diverse ef-fects on cardiac function and can result in dilated or restric-tive cardiomyopathy or diastolic dysfunction.120–122

These cardiac and neurodegenerative diseases are linkedby the formation of amyloid-positive deposits, and the amy-loid hypothesis, which states that amyloid accumulation iscytotoxic, has been widely accepted.123 However, recent datahave cast doubt on the hypothesis,124 as there is a poorcorrelation between the concentration of amyloid plaques andthe degree of dementia in Alzheimer’s patients.125,126 Al-though the inherent toxicity of the amyloid-containing aggre-gates has been assumed in a wide range of human conditionssuch as light chain amyloidosis, the spongiform encephalop-athies, Alzheimer’s disease, Parkinson’s disease, and oth-ers,127,128 it is now generally thought that amyloid plaques area consequence of a long pathogenic process and represent“tombstones,” rather than being directly cytotoxic.129,130 Con-sistent with the pathogenicity of these early events in aggre-gate formation, significant UPS impairment occurs before thecoalescence of aggregated proteins into inclusion bodies.74

Thus, in the search for the primary cytotoxic entity, thefocus has shifted away from extracellular accumulation of theamyloid-positive plaques131 to the soluble amyloidogenicpeptides and the intracellular events that precede visibleplaque accretion.130,132 This shift in focus is explicitly rootedin the observation that disease can precede the appearance ofthe classically insoluble amyloid plaques and tangles. Al-though many different proteins can participate in, or initiatethe formation of, amyloid (see below), there are importantcommonalities in the amyloidogenic event, which starts withthe production of a native soluble protein or peptide fragmentthat is inherently prone to misfolding, yielding the precursorfor fibril formation. The misfolded but soluble protein canthen self-associate to form soluble preamyloid oligomers(PAOs), protofibrils, and other intermediates in the amyloidfibril pathway.133 For example, cleavage of amyloid �-proteinprecursor via the action of the � and � secretases producesA� protein, consisting of 40- to 42-aa residues. This peptidefragment is highly amyloidogenic and can assume an ordered,� sheet–containing conformation. These structures then con-tribute to the formation of small soluble oligomers that can goon to develop into protofibrils, which, in turn, self-associateinto the mature amyloid fibril found in the characteristicplaques and tangles (Figure 3).

Data that A� protein and other amyloidogenic proteinsexert their cellular toxicity as soluble PAO intermediates andnot as insoluble aggregates or fibrils have been gath-ered.127,134,135 In situ experiments, in which small solubleprefibrillar A� was added to mouse brain slice cultures,confirmed the neurotoxicity of the soluble protein.132 A�amyloid oligomers were found in the cerebrospinal fluid ofAlzheimer’s patients, and the soluble A� oligomer concen-tration in the human brain more accurately predicted diseaseseverity than plaque accumulation.126,136,137 In Huntington’sdisease, the cellular toxicity of soluble amyloid induced bythe expanded glutamine repeats present in the mutated hun-




tingtin protein has also been linked to neural pathogenesis. Infact, rather than being a predictor for disease, the visibleinclusion bodies that accrete as a result of mutant huntingtinexpression were associated with improved cell survival, at thesame time, leading to decreased soluble mutant huntingtinlevels in the neurons and suggesting that inclusion bodyformation is actually protective against the toxic soluble formof the mutant protein.138 These results, along with extensiveother data,129,132,139–142 indicate that the soluble PAO proba-bly has a larger cytotoxic role than the insoluble fibrillaramyloid deposits.

Hundreds, if not thousands, of peptide domains haveamyloidogenic potential,143–146 a concept underscored by thelack of homology or any obvious sequence identity in theamyloidogenic peptides. The commonality underlying PAOformation is the ability of a peptide or peptide fragmentdomain to form cross-� structures, and the A-11 antibody,which recognizes the (presumably) toxic conformer, hasrecently been characterized.134 Although these soluble oli-gomers are presumably intermediates in mature fibril forma-tion,136 it became apparent that the oligomers of the amyloi-dogenic proteins were, in fact, long lived. Subsequently,oligomers of A� were found in cell cultures transfected withA�1–42, in Tg models of Alzheimer’s disease, and in postmor-tem Alzheimer’s brain specimens. Other amyloidogenic pro-teins such, as �-synuclein (Parkinson’s), transthyretin, insu-lin, or polyglutamine-containing peptides (Huntington’s) alsoform these oligomers.140,147–149

These data imply that PAO has a shared conformation thatis conserved among diverse proteins, although it is also clearthat amyloidogenic peptides differ in their degree of toxicityin a sequence-dependent manner.150 Whether these differentproteins also share a common mechanism of pathogenicaction is unknown at this time, although there are some dataindicating that the soluble PAOs, at least those recognized bythe A-11 antibody, can affect membrane integrity and cal-

cium homeostasis,139 both of which are critical parameters inmaintaining cardiomyocyte function and viability. Consistentwith this concept, PAO appears to be present in at least somemodels of heart disease, particularly where protein misfoldinghas occurred. Using the A-11 antibody, significant PAOconcentrations were detected in the CryABR120G heart failuremodel and widespread distribution in cardiomyocytes derivedfrom various forms of human heart failure was noted aswell.103

Abnormal Protein Metabolism in theDiseased Heart

Regardless of the primary cause(s), CHF is often precededand accompanied by increased cardiomyocyte protein synthe-sis and hypertrophy. Invariably, this leads to increasedproduction of abnormal proteins, which are cotranslationallydegraded by the UPS.151 Ischemic heart disease is the mostcommon cause of CHF. Ischemia/reperfusion injury, hypoxia,and oxidative stress all can stress the heart, affect the foldingand assembly of nascent and mature polypeptides, andincrease compromised protein levels. Thus, the UPR isactivated in cardiac myocytes in response to hypoxia andglobal ischemia/reperfusion,152,153 whereas sustained ERstress inhibits UPS-mediated proteolysis of reporter pro-teins.154 Consistent with the physiological significance ofthese data, Martindale et al recently reported that cardiomyo-cyte-restricted overexpression of a tamoxifen-regulated formof ATF6 could induce ER stress–related gene expression andprotect against ischemia/reperfusion injury in ex vivo mouseheart preparations.153 Adding to the pathological load, re-moval of the abnormal proteins by the proteasome can also bedecreased in heart failure. The removal of abnormal proteinsrelies on the collaboration between chaperones and the UPS,and multiple lines of evidence (see below) suggest that UPSproteolytic function is compromised in heart failure.

Figure 3. Consequences of protein misfolding. Shown is a schematic diagram of the temporal consequences of protein misfolding.Nascent proteins interact with chaperones or small HSPs and fold correctly. However, because of genetic mutation or environmentalstimuli such as mechanical stress, hypoxia or ischemia, misfolded proteins, or peptide fragments accumulate. These can aggregate intokinetically stable, insoluble entities or be recognized by the PQC, ubiquitinated, and degraded by the proteasome. Isolated regions insome of these unfolded or cleaved proteins are able to assume a �-pleated sheet structure and interact with each other to form aseries of intermediate but stable structures, resulting in soluble preamyloid oligomers, which are cytotoxic. These entities can, undercertain circumstances, go on to form protofibrils and may coalesce, resulting in the classic amyloid fibrils, plaques, and tangles. Thearrangements of the �-strands perpendicular to the fiber axis and the �-sheets parallel to the axis in the mature amyloid fibrils are rep-resented by the red arrows. The reversibility of many of the processes leading to mature amyloid fibril deposition is emphasized by thebidirectional arrows.




To dissect molecular mechanisms underlying cardiovascu-lar disease induced by CryABR120G, Chen et al determinedtemporal changes in UPS proteolytic function in CryABR120G

Tg mouse hearts.77 Ubiquitinated proteins in both the solublefraction and total protein extracts from the heart progressivelyincreased in CryABR120G Tg mice, but not wild-type (WT)CryAB Tg mice, at 1 and 3 months of age, whereas freeubiquitin remained unchanged, compared with normal litter-mates. Ubiquitinated proteins are normally degraded effi-ciently by 26S proteasomes.12 An increase in ubiquitinatedproteins indicates either proteasomal malfunction or en-hanced ubiquitination. Cytosolic protein levels of both totaland phosphorylated �-catenin, an endogenous substrate of theUPS, were increased in CryABR120G hearts. To demonstrateUPS impairment, GFPdgn reporter mice were crossed withCryABR120G or WT-CryAB Tg mice. As observed in heartstreated with specific proteasome inhibitors,73 GFPdgn proteinwas dramatically increased in CryABR120G/GFPdgn double Tghearts compared with GFPdgn single Tg littermates. Theincrease in GFPdgn protein in CryABR120G/GFPdgn double Tghearts was greater at 3 months than at 1 month. The increasewas attributable to decreased degradation rather than in-creased synthesis of GFPdgn, as the steady-state GFPdgntranscript levels were not increased. Furthermore, the abilityof crude protein extracts from CryABR120G Tg hearts todegrade immunoprecipitated GFPdgn protein in vitro wassignificantly compromised. These data indicate that UPSproteolytic function in the heart is significantly impaired byexpression of CryABR120G protein, which misfolds and aggre-gates.104,108 The function of 20S proteasomes is unlikely theprimary cause of observed proteasomal malfunction as theirpeptidase activities were actually increased, not reduced, asthe hearts entered congestive failure. Further analyses sug-gested that the primary defect can be attributed to thecompromised entry of ubiquitinated proteins into the 20Sproteasome and that aberrant protein aggregation is necessaryfor CryABR120G to impair UPS proteolytic function.77 Similarfindings were obtained with another mouse model of desmin-related cardiomyopathy produced by cardiac expression of ahuman DRM-linked desmin mutation.71,76

The proteasome is also compromised in animal models ofpressure-overload cardiomyopathy and myocardial ischemia/reperfusion, 2 common causes of CHF. In mice with thoracicaortic constriction–induced pressure overload, Tsukamoto etal measured significant decreases in proteasomalchymotrypsin-like, trypsin-like, and caspase-like peptidaseactivities 2 weeks after surgery but before any discernablecardiac dysfunction. These decreases became more substan-tial at 4 weeks when cardiac failure became evident. Consis-tent with proteasome functional depression, ubiquitinatedproteins significantly and progressively increased between 2and 4 weeks after thoracic aortic constriction.155 Using a ratmodel, Bulteau et al demonstrated that myocardial ischemia/reperfusion resulted in decreased cytosolic proteasomal pep-tidase activity and oxidative modification of the componentsof the 20S proteasome.52 This study, along with a previousreport by Okada et al,156 confirmed that 20S proteasomalsubunits are modified under these pathological conditions,although the pathophysiological significance of altered UPS

function in either pressure-overloaded cardiomyopathy orischemia/reperfusion injury remains to be determined.

Cardiac proteasomal function decreases with ageing andmay contribute to the temporal pathogenesis of some CHF.Although ubiquitination enzyme activity does not consis-tently change with age, accumulation of oxidized proteins andhighly ubiquitinated proteins are associated with ageing andsome ageing-related diseases, raising the possibility that theproteasome degradation pathway is impaired.157,158 Protea-some peptidase activities in cytosolic proteins extracted fromthe hearts of Fisher 344 rats decreased from 8 to 26 months,and this could be partially attributed to decreases in 20Sproteasome abundance in the heart.159 The ability of cardiac20S proteasomes purified from aged rats to degrade [14C]-methyl casein in vitro was also decreased significantly,compared with activity from younger rats.159 These decreasesin proteolytic activity of senescent proteasomes are correlatedwith alterations in subunit composition and/or posttransla-tional modifications of the proteasomal subunits. Takentogether, these lines of evidence are consistent with thehypothesis that the removal of abnormal proteins by theproteasome is insufficient in failing human hearts, and, in thefuture, it may be productive to examine the status of UPSproteolytic function in the heart during progression to heartfailure.

However, the potential therapeutic manipulation of protea-somal activity is fraught with unknowns. Although acute andshort-term proteasome inhibition with pharmacological inhib-itors reduced myocardial ischemia/reperfusion injury, possi-bly through inducing heat-shock responses and preventingnuclear factor �B activation,13,160,161 pharmacological inhibi-tion of 20S proteasomes caused necrotic and apoptotic celldeath in cardiomyocyte cultures.72,155 Interestingly, a recentcase report from a lung cancer patient suggests that chemo-therapy using the proteasome inhibitor bortezomib causesreversible severe cardiac failure.162 In a separate report onrelapsed and refractory multiple myeloma patients receiving aregimen containing bortezomib, heart failure is described as acause of death unrelated to cancer progression.163 Theseobservation and reports are sporadic and preliminary but raisethe possibility that proteasome malfunction may be sufficientto cause heart failure in humans.

Future DirectionsThe importance of these basic cellular processes in normalcardiac function and the development of cardiovasculardisease is clearly established. However, our understanding ofthe molecular details and relative importance of nascentprotein folding, maturation, aggregation, and degradation inthe healthy and diseased heart is in its infancy. PQC doesappear to be compromised in a range of cardiac disease, asdoes chaperone and proteasome activity, and accumulation ofpresumably toxic PAOs in failing animal and human hearts isintriguing. Inadequate PQC can lead to the accumulation ofmisfolded and damaged proteins that, in turn, further impairsPQC in the heart, forming a vicious cycle that can lead tocompromised cardiomyocyte function and cell death. Never-theless, the necessity and sufficiency of these processes have,with rare exceptions, not been established in human cardio-




vascular disease. The impact of cardiomyocyte-restrictedUPS inhibition on the development of heart failure should beaddressable in animal models using gain- and loss-of-function studies restricted to the relevant cell types. Similarapproaches can be used to address whether and how UPSmalfunction causes heart failure, the role proteasome mal-function plays in the process, and whether acute or chronicmanipulation of proteasomal activity is a viable therapeuticapproach. The provocative parallels between the neurodegen-erative and cardiovascular diseases, which can be drawnusing the commonalities of protein conformation-based pa-thology, need to be explored in detail, and any potentialtherapeutics based on these processes evaluated in bothsystems.

Sources of FundingWe gratefully acknowledge support from the National Heart, Lung,and Blood Institute/NIH for grants R01HL072166 andR01HL085629 (to X.W.) and NIH P01HL69779, R01 HL56370, P50HL074728, P50 HL077101 (to J.R.).

DisclosuresNone.

References1. Bukau B, Weissman J, Horwich A. Molecular chaperones and protein

quality control. Cell. 2006;125:443–451.2. Ciechanover A. The ubiquitin proteolytic system: from a vague idea,

through basic mechanisms, and onto human diseases and drug targeting.Neurology. 2006;66:S7–S19.

3. Kampinga HH. Chaperones in preventing protein denaturation in livingcells and protecting against cellular stress. Handb Exp Pharmacol.2006;1–42.

4. Sghaier H, Le Ai TH, Horiike T, Shinozawa T. Molecular chaperones:proposal of a systematic computer-oriented nomenclature and con-struction of a centralized database. In Silico Biol. 2004;4:311–322.

5. Morange M. HSFs in development. Handb Exp Pharmacol. 2006;153–169.

6. Multhoff G. Heat shock proteins in immunity. Handb Exp Pharmacol.2006:279–304.

7. Taylor RP, Benjamin IJ. Small heat shock proteins: a new classificationscheme in mammals. J Mol Cell Cardiol. 2005;38:433–444.

8. Birnie DH, Vickers LE, Hillis WS, Norrie J, Cobbe SM. Increased titresof anti-human heat shock protein 60 predict an adverse one yearprognosis in patients with acute cardiac chest pain. Heart. 2005;91:1148–1153.

9. Pantos C, Malliopoulou V, Mourouzis I, Thempeyioti A, Paizis I, DimopoulosA, Saranteas T, Xinaris C, Cokkinos DV. Hyperthyroid hearts display a phe-notype of cardioprotection against ischemic stress: a possible involvement ofheat shock protein 70. Horm Metab Res. 2006;38:308–313.

10. Bennardini F, Wrzosek A, Chiesi M. Alpha B-crystallin in cardiactissue. Association with actin and desmin filaments. Circ Res. 1992;71:288–294.

11. Wang X, Osinska H, Klevitsky R, Gerdes AM, Nieman M, Lorenz J,Hewett T, Robbins J. Expression of R120G-alphaB-crystallin causesaberrant desmin and alphaB-crystallin aggregation and cardiomyopathyin mice. Circ Res. 2001;89:84–91.

12. Glickman MH, Ciechanover A. The ubiquitin-proteasome proteolyticpathway: destruction for the sake of construction. Physiol Rev. 2002;82:373–428.

13. Powell SR. The ubiquitin-proteasome system in cardiac physiology andpathology. Am J Physiol Heart Circ Physiol. 2006;291:H1–H19.

14. Li HH, Kedar V, Zhang C, McDonough H, Arya R, Wang DZ, PattersonC. Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependentcardiac hypertrophy by participating in an SCF ubiquitin ligase complex.J Clin Invest. 2004;114:1058–1071.

15. Powell SR. The cardiac 26S proteasome: regulating the regulator. CircRes. 2006;99:342–345.

16. Koegl M, Hoppe T, Schlenker S, Ulrich HD, Mayer TU, Jentsch S. Anovel ubiquitination factor, E4, is involved in multiubiquitin chainassembly. Cell. 1999;96:635–644.

17. Kaneko-Oshikawa C, Nakagawa T, Yamada M, Yoshikawa H,Matsumoto M, Yada M, Hatakeyama S, Nakayama K, Nakayama KI.Mammalian E4 is required for cardiac development and maintenance ofthe nervous system. Mol Cell Biol. 2005;25:10953–10964.

18. Li W, Chanda SK, Micik I, Joazeiro CA. Methods for the functionalgenomic analysis of ubiquitin ligases. Methods Enzymol. 2005;398:280–291.

19. Semple CA. The comparative proteomics of ubiquitination in mouse.Genome Res. 2003;13:1389–1394.

20. Pickart CM. Mechanisms underlying ubiquitination. Annu Rev Biochem.2001;70:503–533.

21. Huibregtse JM, Scheffner M, Beaudenon S, Howley PM. A family ofproteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci U S A. 1995;92:2563–2567.

22. Yang Y, Lorick KL, Jensen JP, Weissman AM. Expression and eval-uation of RING finger proteins. Methods Enzymol. 2005;398:103–112.

23. Beaudenon S, Dastur A, Huibregtse JM. Expression and assay of HECTdomain ligases. Methods Enzymol. 2005;398:112–125.

24. Deshaies RJ. SCF and Cullin/Ring H2-based ubiquitin ligases. Annu RevCell Dev Biol. 1999;15:435–467.

25. Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C,Koepp DM, Elledge SJ, Pagano M, Conaway RC, Conaway JW, HarperJW, Pavletich NP. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCFubiquitin ligase complex. Nature. 2002;416:703–709.

26. Kipreos ET, Lander LE, Wing JP, He WW, Hedgecock EM. cul-1 isrequired for cell cycle exit in C. elegans and identifies a novel genefamily. Cell. 1996;85:829–839.

27. Wei N, Deng XW. The COP9 signalosome. Annu Rev Cell Dev Biol.2003;19:261–286.

28. Jiang J, Ballinger CA, Wu Y, Dai Q, Cyr DM, Hohfeld J, Patterson C.CHIP is a U-box-dependent E3 ubiquitin ligase: identification of Hsc70as a target for ubiquitylation. J Biol Chem. 2001;276:42938–42944.

29. McDonough H, Patterson C. CHIP: a link between the chaperone andproteasome systems. Cell Stress Chaperones. 2003;8:303–308.

30. Qian SB, McDonough H, Boellmann F, Cyr DM, Patterson C. CHIP-mediated stress recovery by sequential ubiquitination of substrates andHsp70. Nature. 2006;440:551–555.

31. Younger JM, Chen L, Ren HY, Rosser MF, Turnbull EL, Fan CY,Patterson C, Cyr DM. Sequential quality-control checkpoints triagemisfolded cystic fibrosis transmembrane conductance regulator. Cell.2006;126:571–582.

32. Zhang C, Xu Z, He XR, Michael LH, Patterson C. CHIP, a cochaperone/ubiquitin ligase that regulates protein quality control, is required formaximal cardioprotection after myocardial infarction in mice. Am JPhysiol Heart Circ Physiol. 2005;288:H2836–H2842.

33. Centner T, Yano J, Kimura E, McElhinny AS, Pelin K, Witt CC, BangML, Trombitas K, Granzier H, Gregorio CC, Sorimachi H, Labeit S.Identification of muscle specific ring finger proteins as potential regu-lators of the titin kinase domain. J Mol Biol. 2001;306:717–726.

34. Witt SH, Granzier H, Witt CC, Labeit S. MURF-1 and MURF-2 targeta specific subset of myofibrillar proteins redundantly: towards under-standing MURF-dependent muscle ubiquitination. J Mol Biol. 2005;350:713–722.

35. Kedar V, McDonough H, Arya R, Li HH, Rockman HA, Patterson C.Muscle-specific RING finger 1 is a bona fide ubiquitin ligase thatdegrades cardiac troponin I. Proc Natl Acad Sci U S A. 2004;101:18135–18140.

36. Arya R, Kedar V, Hwang JR, McDonough H, Li HH, Taylor J, PattersonC. Muscle ring finger protein-1 inhibits PKCe activation and preventscardiomyocyte hypertrophy. J Cell Biol. 2004;167:1147–1159.

37. Razeghi P, Taegtmeyer H. Cardiac remodeling: UPS lost in transit. CircRes. 2005;97:964–966.

38. Laing JG, Tadros PN, Green K, Saffitz JE, Beyer EC. Proteolysis ofconnexin43-containing gap junctions in normal and heat-stressedcardiac myocytes. Cardiovasc Res. 1998;38:711–718.

39. Qin H, Shao Q, Igdoura SA, Alaoui-Jamali MA, Laird DW. Lysosomaland proteasomal degradation play distinct roles in the life cycle of Cx43in gap junctional intercellular communication-deficient and -competentbreast tumor cells. J Biol Chem. 2003;278:30005–30014.

40. Leithe E, Rivedal E. Ubiquitination and down-regulation of gap junctionprotein connexin-43 in response to 12-O-tetradecanoylphorbol13-acetate treatment. J Biol Chem. 2004;279:50089–50096.




41. Willis MS, Patterson C. Into the heart: the emerging role of theubiquitin-proteasome system. J Mol Cell Cardiol. 2006;41:567–579.

42. Gomes AV, Zong C, Edmondson RD, Li X, Stefani E, Zhang J, JonesRC, Thyparambil S, Wang GW, Qiao X, Bardag-Gorce F, Ping P.Mapping the murine cardiac 26S proteasome complexes. Circ Res.2006;99:362–371.

43. Rechsteiner M, Hill CP. Mobilizing the proteolytic machine: cell bio-logical roles of proteasome activators and inhibitors. Trends Cell Biol.2005;15:27–33.

44. Li X, Lonard DM, Jung SY, Malovannaya A, Feng Q, Qin J, Tsai SY,Tsai MJ, O’Malley BW. The SRC-3/AIB1 coactivator is degraded in aubiquitin- and ATP-independent manner by the REGgamma pro-teasome. Cell. 2006;124:381–392.

45. Zhou P. REGgamma: a shortcut to destruction. Cell. 2006;124:256–257.46. Foucrier J, Bassaglia Y, Grand MC, Rothen B, Perriard JC, Scherrer K.

Prosomes form sarcomere-like banding patterns in skeletal, cardiac, andsmooth muscle cells. Exp Cell Res. 2001;266:193–200.

47. Stuhmer T, Chatterjee M, Hildebrandt M, Herrmann P, Gollasch H,Gerecke C, Theurich S, Cigliano L, Manz RA, Daniel PT, Bommert K,Vassilev LT, Bargou RC. Nongenotoxic activation of the p53 pathwayas a therapeutic strategy for multiple myeloma. Blood. 2005;106:3609–3617.

48. Ahmad K. Proteasome inhibitor for treatment of multiple myeloma.Lancet Oncol. 2005;6:546.

49. Divald A, Powell SR. Proteasome mediates removal of proteins oxidizedduring myocardial ischemia. Free Radic Biol Med. 2006;40:156–164.

50. Wilk S, Orlowski M. Cation-sensitive neutral endopeptidase: isolationand specificity of the bovine pituitary enzyme. J Neurochem. 1980;35:1172–1182.

51. Reinheckel T, Sitte N, Ullrich O, Kuckelkorn U, Davies KJ, Grune T.Comparative resistance of the 20S and 26S proteasome to oxidativestress. Biochem J. 1998;335(pt 3):637–642.

52. Bulteau AL, Lundberg KC, Humphries KM, Sadek HA, Szweda PA,Friguet B, Szweda LI. Oxidative modification and inactivation of theproteasome during coronary occlusion/reperfusion. J Biol Chem. 2001;276:30057–30063.

53. Powell SR, Wang P, Katzeff H, Shringarpure R, Teoh C, Khaliulin I,Das DK, Davies KJ, Schwalb H. Oxidized and ubiquitinated proteinsmay predict recovery of postischemic cardiac function: essential role ofthe proteasome. Antioxid Redox Signal. 2005;7:538–546.

54. Zong C, Gomes AV, Drews O, Li X, Young GW, Berhane B, Qiao X,French SW, Bardag-Gorce F, Ping P. Regulation of murine cardiac 20Sproteasomes: role of associating partners. Circ Res. 2006;99:372–380.

55. Mason GG, Hendil KB, Rivett AJ. Phosphorylation of proteasomes inmammalian cells. Identification of two phosphorylated subunits and theeffect of phosphorylation on activity. Eur J Biochem. 1996;238:453–462.

56. Castano JG, Mahillo E, Arizti P, Arribas J. Phosphorylation of C8 andC9 subunits of the multicatalytic proteinase by casein kinase II andidentification of the C8 phosphorylation sites by direct mutagenesis.Biochemistry. 1996;35:3782–3789.

57. Bose S, Stratford FL, Broadfoot KI, Mason GG, Rivett AJ. Phosphor-ylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26Sproteasome and plays a role in the regulation of proteasome complexesby gamma-interferon. Biochem J. 2004;378:177–184.

58. Mason GG, Murray RZ, Pappin D, Rivett AJ. Phosphorylation ofATPase subunits of the 26S proteasome. FEBS Lett. 1998;430:269–274.

59. Satoh K, Sasajima H, Nyoumura KI, Yokosawa H, Sawada H. Assemblyof the 26S proteasome is regulated by phosphorylation of the p45/Rpt6ATPase subunit. Biochemistry. 2001;40:314–319.

60. Schwechheimer C. The COP9 signalosome (CSN): an evolutionaryconserved proteolysis regulator in eukaryotic development. BiochimBiophys Acta. 2004;1695:45–54.

61. Zhou C, Wee S, Rhee E, Naumann M, Dubiel W, Wolf DA. Fissionyeast COP9/signalosome suppresses cullin activity through recruitmentof the deubiquitylating enzyme Ubp12p. Mol Cell. 2003;11:927–938.

62. Groisman R, Polanowska J, Kuraoka I, Sawada J, Saijo M, Drapkin R,Kisselev AF, Tanaka K, Nakatani Y. The ubiquitin ligase activity in theDDB2 and CSA complexes is differentially regulated by the COP9signalosome in response to DNA damage. Cell. 2003;113:357–367.

63. Huang X, Hetfeld BK, Seifert U, Kahne T, Kloetzel PM, Naumann M,Bech-Otschir D, Dubiel W. Consequences of COP9 signalosome and26S proteasome interaction. FEBS J. 2005;272:3909–3917.

64. Hetfeld BK, Helfrich A, Kapelari B, Scheel H, Hofmann K, GutermanA, Glickman M, Schade R, Kloetzel PM, Dubiel W. The zinc finger of

the CSN-associated deubiquitinating enzyme USP15 is essential torescue the E3 ligase Rbx1. Curr Biol. 2005;15:1217–1221.

65. Luker GD, Pica CM, Song J, Luker KE, Piwnica-Worms D. Imaging26S proteasome activity and inhibition in living mice. Nat Med. 2003;9:969–973.

66. Lindsten K, Menendez-Benito V, Masucci MG, Dantuma NP, KumarapeliAR, Horak KM, Zheng H, Wang X. GFP reporter mouse models of UPSproteolytic function. FASEB J. 2006;20:1027.

67. Menendez-Benito V, Heessen S, Dantuma NP. Monitoring of ubiquitin-dependent proteolysis with green fluorescent protein substrates. MethodsEnzymol. 2005;399:490–511.

68. Lindsten K, Dantuma NP. Monitoring the ubiquitin/proteasome systemin conformational diseases. Ageing Res Rev. 2003;2:433–449.

69. Bence NF, Sampat RM, Kopito RR. Impairment of the ubiquitin-proteasome system by protein aggregation. Science. 2001;292:1552–1555.

70. Bence NF, Bennett EJ, Kopito RR. Application and analysis of theGFP(u) family of ubiquitin-proteasome system reporters. MethodsEnzymol. 2005;399:481–490.

71. Liu J, Tang M, Mestril R, Wang X. Aberrant protein aggregation isessential for a mutant desmin to impair the proteolytic function of theubiquitin-proteasome system in cardiomyocytes. J Mol Cell Cardiol.2006;40:451–454.

72. Dong X, Liu J, Zheng H, Glasford JW, Huang W, Chen QH, Harden NR,Li F, Gerdes AM, Wang X. In situ dynamically monitoring the pro-teolytic function of the ubiquitin-proteasome system in cultured cardiacmyocytes. Am J Physiol Heart Circ Physiol. 2004;287:H1417–H1425.

73. Kumarapeli RKA, Horak KM, Glasford JW, Li J, Chen Q, Liu J, ZhengH, Wang X. A novel transgenic mouse model reveals deregulation of theubiquitin-proteasome system in the heart by doxorubicin. FASEB J.2005;19:2051–2053.

74. Bennett EJ, Bence NF, Jayakumar R, Kopito RR. Global impairment ofthe ubiquitin-proteasome system by nuclear or cytoplasmic proteinaggregates precedes inclusion body formation. Mol Cell. 2005;17:351–365.

75. Bowman AB, Yoo SY, Dantuma NP, Zoghbi HY. Neuronal dysfunctionin a polyglutamine disease model occurs in the absence of ubiquitin-proteasome system impairment and inversely correlates with the degreeof nuclear inclusion formation. Hum Mol Genet. 2005;14:679–691.

76. Liu J, Chen Q, Huang W, Horak KM, Zheng H, Mestril R, Wang X.Impairment of the ubiquitin-proteasome system in desminopathy mousehearts. FASEB J. 2006;20:362–364.

77. Chen Q, Liu J-B, Horak KM, Zheng H, Kumarapeli ARK, Li J, Li F,Gerdes AM, Wawrousek EF, Wang X. Intrasarcoplasmic amyloidosisimpairs proteolytic function of proteasomes in cardiomyocytes by com-promising substrate uptake. Circ Res. 2005;97:1018–1028.

78. Meusser B, Hirsch C, Jarosch E, Sommer T. ERAD: the long road todestruction. Nat Cell Biol. 2005;7:766–772.

79. Brewer JW, Hendershot LM. Building an antibody factory: a job for theunfolded protein response. Nat Immunol. 2005;6:23–29.

80. Kimata Y, Oikawa D, Shimizu Y, Ishiwata-Kimata Y, Kohno K. A rolefor BiP as an adjustor for the endoplasmic reticulum stress-sensingprotein Ire1. J Cell Biol. 2004;167:445–456.

81. Hollien J, Weissman JS. Decay of endoplasmic reticulum-localizedmRNAs during the unfolded protein response. Science. 2006;313:104–107.

82. Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress.Trends Cell Biol. 2004;14:20–28.

83. Harding HP, Ron D. Endoplasmic reticulum stress and the developmentof diabetes: a review. Diabetes. 2002;51(suppl 3):S455–S461.

84. Oyadomari S, Koizumi A, Takeda K, Gotoh T, Akira S, Araki E, MoriM. Targeted disruption of the Chop gene delays endoplasmic reticulumstress-mediated diabetes. J Clin Invest. 2002;109:525–532.

85. Katayama T, Imaizumi K, Sato N, Miyoshi K, Kudo T, Hitomi J,Morihara T, Yoneda T, Gomi F, Mori Y, Nakano Y, Takeda J, Tsuda T,Itoyama Y, Murayama O, Takashima A, St George-Hyslop P, TakedaM, Tohyama M. Presenilin-1 mutations downregulate the signallingpathway of the unfolded-protein response. Nat Cell Biol. 1999;1:479–485.

86. Imai Y, Soda M, Inoue H, Hattori N, Mizuno Y, Takahashi R. Anunfolded putative transmembrane polypeptide, which can lead to endo-plasmic reticulum stress, is a substrate of Parkin. Cell. 2001;105:891–902.

87. Okada K, Minamino T, Tsukamoto Y, Liao Y, Tsukamoto O, TakashimaS, Hirata A, Fujita M, Nagamachi Y, Nakatani T, Yutani C, Ozawa K,




Ogawa S, Tomoike H, Hori M, Kitakaze M. Prolonged endoplasmicreticulum stress in hypertrophic and failing heart after aortic con-striction: possible contribution of endoplasmic reticulum stress tocardiac myocyte apoptosis. Circulation. 2004;110:705–712.

88. Hamada H, Suzuki M, Yuasa S, Mimura N, Shinozuka N, Takada Y,Suzuki M, Nishino T, Nakaya H, Koseki H, Aoe T. Dilated cardiomy-opathy caused by aberrant endoplasmic reticulum quality control inmutant KDEL receptor transgenic mice. Mol Cell Biol. 2004;24:8007–8017.

89. Kerkela R, Grazette L, Yacobi R, Iliescu C, Patten R, Beahm C, WaltersB, Shevtsov S, Pesant S, Clubb FJ, Rosenzweig A, Salomon RN, VanEtten RA, Alroy J, Durand JB, Force T. Cardiotoxicity of the cancertherapeutic agent imatinib mesylate. Nat Med. 2006;12:908–916.

90. Baglioni S, Casamenti F, Bucciantini M, Luheshi LM, Taddei N, ChitiF, Dobson CM, Stefani M. Prefibrillar amyloid aggregates could begeneric toxins in higher organisms. J Neurosci. 2006;26:8160–8167.

91. Chiti F, Dobson CM. Protein misfolding, functional amyloid, and humandisease. Annu Rev Biochem. 2006;75:333–366.

92. Garcia-Mata R, Gao YS, Sztul E. Hassles with taking out the garbage:aggravating aggresomes. Traffic. 2002;3:388–396.

93. Kopito RR. Aggresomes, inclusion bodies and protein aggregation.Trends Cell Biol. 2000;10:524–530.

94. Goldfarb LG, Vicart P, Goebel HH, Dalakas MC. Desmin myopathy.Brain. 2004;127:723–734.

95. Selcen D, Ohno K, Engel AG. Myofibrillar myopathy: clinical, mor-phological and genetic studies in 63 patients. Brain. 2004;127:439–451.

96. Dalakas MC, Park KY, Semino-Mora C, Lee HS, Sivakumar K,Goldfarb LG. Desmin myopathy, a skeletal myopathy with cardiomy-opathy caused by mutations in the desmin gene. N Engl J Med. 2000;342:770–780.

97. Kumarapeli AR, Wang X. Genetic modification of the heart: chaperonesand the cytoskeleton. J Mol Cell Cardiol. 2004;37:1097–1109.

98. Vicart P, Caron A, Guicheney P, Li Z, Prevost MC, Faure A, Chateau D,Chapon F, Tome F, Dupret JM, Paulin D, Fardeau M. A missensemutation in the alphaB-crystallin chaperone gene causes a desmin-related myopathy. Nat Genet. 1998;20:92–95.

99. Goldfarb LG, Park KY, Cervenakova L, Gorokhova S, Lee HS, Vasconcelos O,Nagle JW, Semino-Mora C, Sivakumar K, Dalakas MC. Missense mutations indesmin associated with familial cardiac and skeletal myopathy. Nat Genet.1998;19:402–403.

100. Munoz-Marmol AM, Strasser G, Isamat M, Coulombe PA, Yang Y,Roca X, Vela E, Mate JL, Coll J, Fernandez-Figueras MT, Navas-Palacios JJ, Ariza A, Fuchs E. A dysfunctional desmin mutation in apatient with severe generalized myopathy. Proc Natl Acad Sci U S A.1998;95:11312–11317.

101. Wang X, Osinska H, Dorn GW 2nd, Nieman M, Lorenz JN, Gerdes AM,Witt S, Kimball T, Gulick J, Robbins J. Mouse model of desmin-relatedcardiomyopathy. Circulation. 2001;103:2402–2407.

102. Bova MP, Yaron O, Huang Q, Ding L, Haley DA, Stewart PL, HorwitzJ. Mutation R120G in alphaB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defectivechaperone-like function. Proc Natl Acad Sci U S A. 1999;96:6137–6142.

103. Sanbe A, Osinska H, Saffitz JE, Glabe CG, Kayed R, Maloyan A,Robbins J. Desmin-related cardiomyopathy in transgenic mice: a cardiacamyloidosis. Proc Natl Acad Sci U S A. 2004;101:10132–10136.

104. Wang X, Klevitsky R, Huang W, Glasford J, Li F, Robbins J. AlphaB-crystallin modulates protein aggregation of abnormal desmin. Circ Res.2003;93:998–1005.

105. Mishra RS, Bose S, Gu Y, Li R, Singh N. Aggresome formation bymutant prion proteins: the unfolding role of proteasomes in familialprion disorders. J Alzheimers Dis. 2003;5:15–23.

106. French BA, van Leeuwen F, Riley NE, Yuan QX, Bardag-Gorce F, GaalK, Lue YH, Marceau N, French SW. Aggresome formation in liver cellsin response to different toxic mechanisms: role of the ubiquitin-proteasome pathway and the frameshift mutant of ubiquitin. Exp MolPathol. 2001;71:241–246.

107. Taylor JP, Tanaka F, Robitschek J, Sandoval CM, Taye A,Markovic-Plese S, Fischbeck KH. Aggresomes protect cells byenhancing the degradation of toxic polyglutamine-containing protein.Hum Mol Genet. 2003;12:749–757.

108. Sanbe A, Osinska H, Villa C, Gulick J, Klevitsky R, Glabe CG, KayedR, Robbins J. Reversal of amyloid-induced heart disease in desmin-related cardiomyopathy. Proc Natl Acad Sci U S A. 2005;102:13592–13597.

109. Kopito RR, Ron D. Conformational disease. Nat Cell Biol. 2000;2:E207–E209.

110. Anversa P, Kajstura J, Leri A, Bolli R. Life and death of cardiac stemcells: a paradigm shift in cardiac biology. Circulation. 2006;113:1451–1463.

111. Shin S, Rao MS. Large-scale analysis of neural stem cells and progenitorcells. Neurodegener Dis. 2006;3:106–111.

112. Mizuno Y, Hattori N, Mori H, Suzuki T, Tanaka K. Parkin and Par-kinson’s disease. Curr Opin Neurol. 2001;14:477–482.

113. Saigoh K, Wang YL, Suh JG, Yamanishi T, Sakai Y, Kiyosawa H,Harada T, Ichihara N, Wakana S, Kikuchi T, Wada K. Intragenicdeletion in the gene encoding ubiquitin carboxy-terminal hydrolase ingad mice. Nat Genet. 1999;23:47–51.

114. He L, Lu XY, Jolly AF, Eldridge AG, Watson SJ, Jackson PK, BarshGS, Gunn TM. Spongiform degeneration in mahoganoid mutant mice.Science. 2003;299:710–712.

115. Jana NR, Zemskov EA, Wang Gh, Nukina N. Altered proteasomalfunction due to the expression of polyglutamine-expanded truncatedN-terminal huntingtin induces apoptosis by caspase activation throughmitochondrial cytochrome c release. Hum Mol Genet. 2001;10:1049–1059.

116. Sarikas A, Carrier L, Schenke C, Doll D, Flavigny J, Lindenberg KS,Eschenhagen T, Zolk O. Impairment of the ubiquitin-proteasome systemby truncated cardiac myosin binding protein C mutants. Cardiovasc Res.2005;66:33–44.

117. Westermark P, Benson MD, Buxbaum JN, Cohen AS, Frangione B,Ikeda S, Masters CL, Merlini G, Saraiva MJ, Sipe JD. Amyloid: towardterminology clarification. Report from the Nomenclature Committee ofthe International Society of Amyloidosis. Amyloid. 2005;12:1–4.

118. Hoppener JW, Lips CJ. Role of islet amyloid in type 2 diabetes mellitus.Int J Biochem Cell Biol. 2006;38:726–736.

119. Comenzo RL. Primary systemic amyloidosis. Curr Treat Options Oncol.2000;1:83–89.

120. Hassan W, Al-Sergani H, Mourad W, Tabbaa R. Amyloid heart disease.New frontiers and insights in pathophysiology, diagnosis, and man-agement. Tex Heart Inst J. 2005;32:178–184.

121. Lie JT. Pathology of amyloidosis and amyloid heart disease. ApplPathol. 1984;2:341–356.

122. Falk RH. Diagnosis and management of the cardiac amyloidoses. Cir-culation. 2005;112:2047–2060.

123. Sisodia SS, Price DL. Role of the beta-amyloid protein in Alzheimer’sdisease. FASEB J. 1995;9:366–370.

124. Hardy J. Testing times for the “amyloid cascade hypothesis”. NeurobiolAging. 2002;23:1073–1074.

125. Lue LF, Kuo YM, Roher AE, Brachova L, Shen Y, Sue L, Beach T,Kurth JH, Rydel RE, Rogers J. Soluble amyloid beta peptide concen-tration as a predictor of synaptic change in Alzheimer’s disease. Am JPathol. 1999;155:853–862.

126. McLean CA, Cherny RA, Fraser FW, Fuller SJ, Smith MJ, BeyreutherK, Bush AI, Masters CL. Soluble pool of Abeta amyloid as a deter-minant of severity of neurodegeneration in Alzheimer’s disease. AnnNeurol. 1999;46:860–866.

127. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J,Taddei N, Ramponi G, Dobson CM, Stefani M. Inherent toxicity ofaggregates implies a common mechanism for protein misfoldingdiseases. Nature. 2002;416:507–511.

128. Trojanowski JQ, Mattson MP. Overview of protein aggregation insingle, double, and triple neurodegenerative brain amyloidoses. Neuro-molecular Med. 2003;4:1–6.

129. Carrell RW. Cell toxicity and conformational disease. Trends Cell Biol.2005;15:574–580.

130. Glabe C. Intracellular mechanisms of amyloid accumulation and patho-genesis in Alzheimer’s disease. J Mol Neurosci. 2001;17:137–145.

131. Howlett DR, Jennings KH, Lee DC, Clark MS, Brown F, Wetzel R,Wood SJ, Camilleri P, Roberts GW. Aggregation state and neurotoxicproperties of Alzheimer beta-amyloid peptide. Neurodegeneration.1995;4:23–32.

132. Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, LiosatosM, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C,Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derivedfrom Abeta1–42 are potent central nervous system neurotoxins. ProcNatl Acad Sci U S A. 1998;95:6448–6453.

133. Caughey B, Lansbury PT. Protofibrils, pores, fibrils, and neurodegen-eration: separating the responsible protein aggregates from the innocentbystanders. Annu Rev Neurosci. 2003;26:267–298.




134. Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW,Glabe CG. Common structure of soluble amyloid oligomers impliescommon mechanism of pathogenesis. Science. 2003;300:486–489.

135. Walsh DM, Klyubin I, Fadeeva JV, Rowan MJ, SelkoeDJ. Amyloid-beta oligomers: their production, toxicity and therapeuticinhibition. Biochem Soc Trans. 2002;30:552–557.

136. Kuo YM, Emmerling MR, Vigo-Pelfrey C, Kasunic TC, Kirkpatrick JB,Murdoch GH, Ball MJ, Roher AE. Water-soluble Abeta (N-40, N-42)oligomers in normal and Alzheimer disease brains. J Biol Chem. 1996;271:4077–4081.

137. Pitschke M, Prior R, Haupt M, Riesner D. Detection of single amyloidbeta-protein aggregates in the cerebrospinal fluid of Alzheimer’spatients by fluorescence correlation spectroscopy. Nat Med. 1998;4:832–834.

138. Arrasate M, Mitra S, Schweitzer ES, Segal MR, Finkbeiner S. Inclusionbody formation reduces levels of mutant huntingtin and the risk ofneuronal death. Nature. 2004;431:805–810.

139. Demuro A, Mina E, Kayed R, Milton SC, Parker I, Glabe CG. Calciumdysregulation and membrane disruption as a ubiquitous neurotoxicmechanism of soluble amyloid oligomers. J Biol Chem. 2005;280:17294–17300.

140. Glabe CG. Common mechanisms of amyloid oligomer pathogenesis indegenerative disease. Neurobiol Aging. 2006;27:570–575.

141. Glabe CG, Kayed R. Common structure and toxic function of amyloidoligomers implies a common mechanism of pathogenesis. Neurology.2006;66:S74–S78.

142. Kayed R, Sokolov Y, Edmonds B, McIntire TM, Milton SC, Hall JE,Glabe CG. Permeabilization of lipid bilayers is a common conformation-dependent activity of soluble amyloid oligomers in protein misfoldingdiseases. J Biol Chem. 2004;279:46363–46366.

143. Galzitskaya OV, Garbuzynskiy SO, Lobanov MY. Is it possible topredict amyloidogenic regions from sequence alone? J BioinformComput Biol. 2006;4:373–388.

144. Saiki M, Konakahara T, Morii H. Interaction-based evaluation of thepropensity for amyloid formation with cross-beta structure. BiochemBiophys Res Commun. 2006;343:1262–1271.

145. Yoon S, Welsh WJ. Detecting hidden sequence propensity for amyloidfibril formation. Protein Sci. 2004;13:2149–2160.

146. Yoon S, Welsh WJ. Rapid assessment of contact-dependent secondarystructure propensity: relevance to amyloidogenic sequences. Proteins.2005;60:110–117.

147. Conway KA, Lee SJ, Rochet JC, Ding TT, Williamson RE, Lansbury PTJr. Acceleration of oligomerization, not fibrillization, is a sharedproperty of both alpha-synuclein mutations linked to early-onset Par-kinson’s disease: implications for pathogenesis and therapy. Proc NatlAcad Sci U S A. 2000;97:571–576.

148. Jimenez JL, Nettleton EJ, Bouchard M, Robinson CV, Dobson CM,Saibil HR. The protofilament structure of insulin amyloid fibrils. ProcNatl Acad Sci U S A. 2002;99:9196–9201.

149. Sousa MM, Cardoso I, Fernandes R, Guimaraes A, Saraiva MJ. Depo-sition of transthyretin in early stages of familial amyloidotic polyneu-

ropathy: evidence for toxicity of nonfibrillar aggregates. Am J Pathol.2001;159:1993–2000.

150. Duennwald ML, Jagadish S, Muchowski PJ, Lindquist S. Flankingsequences profoundly alter polyglutamine toxicity in yeast. Proc NatlAcad Sci U S A. 2006;103:11045–11050.

151. Schubert U, Anton LC, Gibbs J, Norbury CC, Yewdell JW, Bennink JR.Rapid degradation of a large fraction of newly synthesized proteins byproteasomes. Nature. 2000;404:770–774.

152. Terai K, Hiramoto Y, Masaki M, Sugiyama S, Kuroda T, Hori M,Kawase I, Hirota H. AMP-activated protein kinase protects cardiomyo-cytes against hypoxic injury through attenuation of endoplasmicreticulum stress. Mol Cell Biol. 2005;25:9554–9575.

153. Martindale JJ, Fernandez R, Thuerauf D, Whittaker R, Gude N, SussmanMA, Glembotski CC. Endoplasmic reticulum stress gene induction andprotection from ischemia/reperfusion injury in the hearts of transgenicmice with a tamoxifen-regulated form of ATF6. Circ Res. 2006;98:1186–1193.

154. Menendez-Benito V, Verhoef LG, Masucci MG, Dantuma NP. Endo-plasmic reticulum stress compromises the ubiquitin-proteasome system.Hum Mol Genet. 2005;14:2787–2799.

155. Tsukamoto O, Minamino T, Okada K, Shintani Y, Takashima S, Kato H,Liao Y, Okazaki H, Asai M, Hirata A, Fujita M, Asano Y, Yamazaki S,Asanuma H, Hori M, Kitakaze M. Depression of proteasome activitiesduring the progression of cardiac dysfunction in pressure-overloadedheart of mice. Biochem Biophys Res Commun. 2006;340:1125–1133.

156. Okada K, Wangpoengtrakul C, Osawa T, Toyokuni S, Tanaka K, UchidaK. 4-Hydroxy-2-nonenal-mediated impairment of intracellular prote-olysis during oxidative stress. Identification of proteasomes as targetmolecules. J Biol Chem. 1999;274:23787–23793.

157. Ciechanover A, Orian A, Schwartz AL. The ubiquitin-mediated pro-teolytic pathway: mode of action and clinical implications. J CellBiochem. 2000;77:40–51.

158. Carrard G, Bulteau AL, Petropoulos I, Friguet B. Impairment of pro-teasome structure and function in aging. Int J Biochem Cell Biol.2002;34:1461–1474.

159. Bulteau AL, Szweda LI, Friguet B. Age-dependent declines in pro-teasome activity in the heart. Arch Biochem Biophys. 2002;397:298–304.

160. Pye J, Ardeshirpour F, McCain A, Bellinger DA, Merricks E, Adams J,Elliott PJ, Pien C, Fischer TH, Baldwin AS Jr, Nichols TC. Proteasomeinhibition ablates activation of NF-kappa B in myocardial reperfusionand reduces reperfusion injury. Am J Physiol Heart Circ Physiol. 2003;284:H919–H926.

161. Luss H, Schmitz W, Neumann J. A proteasome inhibitor confers car-dioprotection. Cardiovasc Res. 2002;54:140–151.

162. Voortman J, Giaccone G. Severe reversible cardiac failure after bort-ezomib treatment combined with chemotherapy in a non-small cell lungcancer patient: a case report. BMC Cancer. 2006;6:129.

163. Ciolli S, Leoni F, Gigli F, Rigacci L, Bosi A. Low dose Velcade,thalidomide and dexamethasone (LD-VTD): an effective regimen forrelapsed and refractory multiple myeloma patients. Leuk Lymphoma.2006;47:171–173.




Xuejun Wang and Jeffrey RobbinsHeart Failure and Protein Quality Control

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Heart failure and protein quality control

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