Protein Unfolding in Cardiomyopathies

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Protein Unfolding in Cardiomyopathies

Luisa Gorza, MDa , Federica del Monte, MD, PhD b,*

a University of Padova, Padova, Italy b Massachusetts General Hospital, Charlestown, MA, USA

Molecular chaperones are a heterogeneous group

of proteins involved in assisting and controlling the

folding of nascent polypeptides. Although in vitrostudies have shown that secondary and tertiary

structures of proteins are dictated by the linear poly-

peptide sequence, the auxiliary role in folding played

by chaperones becomes obligatory in the cellular

environment, where crowding of different macro-

molecules may favor unwanted intermolecular inter-

actions with nascent polypeptides. Their aggregation

with other unfolded protein species jeopardizes stable

protein structure and would be responsible for toxic

consequences. Molecular chaperones play a crucial

role against such toxicity in physiologic conditions.

Furthermore, their importance has been recognized in

several pathologic conditions in which destabiliza-

tion of protein folding may occur concomitantly to

upregulation of many chaperone genes. A large body

of evidence emphasizes the role of chaperones in

enhancing cell resistance to different stresses, al-

though the protective effect is apparently attributed

to the preferential, if not exclusive, interaction with

specific partner proteins.

The multifaceted properties of several molecular

chaperones have been recognized for simple eukary-

otic organisms and for organisms that are morecomplex. The nomenclature for the more relevant

protein homologs among bacteria, yeast, and mam-

malian cells is listed in Tables 1–3. In this article,

the authors first review the role and mechanisms of

molecular chaperones in protein folding in the dif-

ferent cellular compartments and attempt to coordi-nate the nomenclature of the proteins as they have

been described in different organisms with the cor-

responding proteins in mammals. Recent knowledge

of the relevance of the unfolding protein response

(UPR) and degradation pathways and the role of the

chaperone proteins in the development of human

diseases is explored. Although most of the disease

entities caused by protein misfolding have been de-

scribed for other organs, this article more specifically

addresses the consequences of protein misfolding for

cardiovascular diseases.

Overview of protein folding in the mammalian

heart

Folding of cytosolic proteins

Appropriate folding of cytosolic proteins is a

relevant event in cardiomyocytes in which cellular

architecture relies on myofibril assembly and align-

ment and on its anchorage by cytoskeleton to the

sarco(endo)plasmic reticulum (SR) and sarcolemmalmembranes. Three main chaperone systems operate at

this level: heat shock protein (HSP)70 and HSP90,

TCP1 ring complex (TRiC) (Fig. 1), and small HSPs

(see Table 1). Detailed references can be found in two

recent extensive reviews [1,2].

Heat shock protein 70 and heat shock protein

90 chaperone machinery

The constitutively expressed cytosolic HSP70

protein is the heat shock cognate protein (Hsc70)

and, like bacterial and yeast homologs, is a mono-

1551-7136/05/$ – see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.hfc.2005.03.009 heartfailure.theclinics.com

This work was supported by FIRB2001 grant RBAU01-

FYPJ and ISS grant CS45 to Dr. Gorza and by NIH grant

NIH-NHLBI 5K08HL069842 to Dr. del Monte.

* Corresponding author. Cardiovascular Research

Center and Cardiac Unit, Massachusetts General Hospital,

149 13th Street CNY-4, Charlestown, MA 02129.

E-mail address: [email protected] (F. del Monte).

Heart Failure Clin 1 (2005) 237 – 250

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meric protein with an N-terminal ATPase domain and

a C-terminal polypeptide-binding domain. Nucleotide

binding (ADP or ATP) regulates the kinetics of

Hsc70 interaction with nascent polypeptides, in that

when ATP is bound, the exchange of polypeptide

substrate is rapid, whereas polypeptide binding is

much more stable in the ADP-bound state. Hsc70

may bind directly to nascent polypeptides after they

emerge from the ribosome tunnel or after their re-

lease from the ribosome; in some cases, polypeptide binds to HSP70 chaperones after association with the

cochaperone HSP40. HSP40 is homologous to bac-

terial DnaJ and transfers the bound polypeptide to

Hsc70 after ATP hydrolysis.

Several isoforms of HSP40/DnaJ-like molecules

that differ by tissue distribution and substrate

interaction are known. In the mammalian heart, the

homolog Dj4/DjA4 isoform constitutes about the 1%

of total protein [3] and a cardiac-specific isoform of

Hdj2/DjA1, named pDJA1, has recently been iden-

tified in the pig heart [4]. It has been proposed that

HSP40 homologs, due to marked differences in theC-terminal region, may interact with HSP70 chaper-

one machinery in different subcellular compartments

and could be involved in protein targeting or as-

sembly of a specific intermediate filament [1]. In

contrast to Hsp40 and Hsc70 mRNA levels, pDJA1

transcript levels differ among heart chambers and

between the subepicardial and subendocardial layers

of the left ventricular wall [4]. Although comparable

evidence at the protein level is still lacking, a four-

fold increase in the expression of this latter cochaper-

one occurs after 1-hour reperfusion following acute

ischemia. This evidence and the transmural gra-dient in the expression of pDJA1 transcripts in the

left ventricular wall suggest sensitive oxygen- and

stretch-sensing mechanisms in upregulation of this

cochaperone gene [4].

A relevant component of the HSP70 chaperone

machinery is the HSP90 chaperone family. Hsp90

exists as a homodimer: dimerization occurs at the

C-terminus, whereas ATP binding domains localize

at the N-terminus. Binding and hydrolysis of ATP

change Hsp90 conformation and promote loading and

release of the polypeptide substrate, respectively.

Cooperation of Hsp90 with Hsc70 occurs for somesubstrates and is mediated by cochaperones that

physically link Hsc70 and Hsp90 and allow the

Table 1

Nomenclature of major cytosolic molecular chaperone families

Mammals Yeast Prokaryotic homolog

Chaperones

HSP70 Hsc70, constitutive Ssa1 DnaK

Hsp70, inducible Ssa2 Ssb

HSP90 Hsp90a/Hsp90/Hsp84 Hsp82/Hsc82 HtpG

Hsp90b/Hsc90/Hsp86

TCP1 ring complex TRiC TRiC GroEL/GroES

GimC/prefoldin GimC

Small HSPs Alpha A-/B-crystallin

Hsp25/27

HSP70 and HSP90 cochaperones

HSP40 Hdj1/Hsp40/DjB1 Djp1 DnaJ

Hdj2/DjA1

Hdj3/DjA2

Hdj4/DjA4

Tetratricopeptide repeat clamp domain

HOP Hop Sti1

UNC-45 CG-UNC45

SM-UNC45

RAR1/SGT1 Melusin? Sgt1

PPIase FKBP52

Cyclophilin Cyp40

Others

p23 p23 Sba1

CDC37 Cdc37 cdc37

Uppercase letters indicate chaperone families, whereas lowercase refer to single members.

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transfer of the polypeptide substrate (like the Hsp-organizing protein, Hop) or assist Hsp90 for the final

folding steps (like p23) [2].

Additional substrate-specific cochaperones may

be required. Hsp70/Hsp90 chaperone machinery is

involved, together with the cochaperone UNC-45,

in folding and assembly of conventional and non-

conventional myosins in a wide variety of organisms,

from yeast to humans [5]. The specific striated

muscle SM-UNC45 isoform is expressed in skeletal

muscle and in the heart [6], where it presumably

participates with Hsc70 and Hsp90 to myosin motor maturation, giving rise to complexes that act as a

checkpoint for thick filament assembly [7]. Inhibition

of Hsp90 ATPase activity by geldanamycin blocks

sarcomeric myosin maturation, resulting in the accu-

mulation of all newly synthesized myosin as a par-

tially folded intermediate [7].

Additional knowledge about proteins that interact

with Hsc70/Hsp90 folding complexes in the heart

is awaited, especially in light of the finding that

melusin, a novel cardiac protein involved in trans-

ducing mechanical stretch of myocardial cells into

physiologic hypertrophy (see the article by Selvetellaand Lembo in this issue for a review of this topic) [8],

displays more than 50% similarity with the zinc-

binding domain of resistance proteinase 1, an Hsp90

cochaperone protein family identified in plants [9].

TCP1 ring complex

Certain nascent polypeptides such as actin and

tubulin interact with GimC/prefoldin protein complexduring their translation and are then assisted in their

folding by the ATP-dependent multimeric chaperonin

TRiC. TRiC shows a double ring structure and, in

contrast to bacteria, lacks a capping cofactor (GroES

is the cap of the cavity formed by the GroEL

chaperonin complex). The two rings of TRiC each

contain eight different subunits that bind the protein

substrate with the apical domain and release it into

the enclosed central cavity where the folding reac-

tion takes place.

Other polypeptides bind Hsc70 first and becomesubstrates of TRiC later on, as occurs in bacteria.

In these cases, Hsc70 binds extended polypeptides

during translation and retains binding until TRiC-

mediated folding is completed.

Small heat shock protein chaperones

This heterogeneous group of low molecular mass

HSPs is more involved in control of the structural

integrity of the cytoskeleton than in protein folding,

despite the demonstration of in vitro chaperone ac-

tivity [10]. Nevertheless, for these reasons, they play

a central role in the preservation of cardiomyocytearchitecture and, thus, of mechanical function.

The most relevant small HSP expressed in car-

diomyocytes is the 22-kd protein alpha B-crystallin,

a member of the crystallin family of lens proteins.

The alpha crystallin domain is highly conserved

within the small HSP family and is thought to be

more important in the formation of the functional

Table 2

Nomen clature of majo r sarco(endo)plasmic reti culum

chaperones

Mammals Yeast

Chaperones

HSP70 Grp78/BiP Kar2p

Grp170 Lhs1p

HSP90 Grp94/gp96/endoplasmin

ORP150

Lectin Calnexin

Calreticulin

UDP-GT

Oxidoreductase PDI Pdi1p

ERp72

CaBP1

ERp29

Small HSPs Hsp47

Cochaperones J domain

HSP40 ERdj 1 – 5 Djp1

Calnexin– calreticulin complex

Oxidoreductase ERp57


PPIase Cyclophilin B

Uppercase letters indicate chaperone families, whereas

lowercase refer to single members.

Table 3

Nomenclature of major mitochondrial chaperones

Mammals Yeast

Chaperones

HSP70 Grp75/mtHsp75 Ssq

TCP1 ring complex Hsp60

Hsp10

Small HSPs Hsp22

Cochaperones J domain

HSP40 Mdj MDj1


TOM Tom70 Tom70

Tom34

Uppercase letters indicate chaperone families, whereas

lowercase refer to single members.

protein unfolding in cardiomyopathies 239

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oligomeric complex than in chaperone activity [11].

In the heart, alpha B-crystallin binds actin and desmin

filaments [12]. The latter require alpha B-crystallin to

remain functional and to prevent the aggregation of

abnormally folded desmin subunits [13].

Mitogen-activated protein kinase (MAPK)-

mediated phosphorylation of Hsp25/27 (Hsp25 in mice

and Hsp27 in humans), as it occurs after hypoxia-

reoxygenation, redistributes the protein from the cyto-

sol to the actin cytoskeleton, where it multimerizes andcontributes to microfilament stabilization [14]. In the

nonphosphorylated, monomeric state, Hsp25/27 inhibits

F-actin polymerization by binding to the plus end of

the filaments [15].

The endoplasmic reticulum folding machinery

Protein folding is a relevant function in the en-

doplasmic reticulum (ER): approximately one third

of all the proteins in eukaryotic cells are translocated

into the ER, where the unique oxidizing potentialsupports disulphide bond formation during protein

folding. In addition, protein concentration in the ER

is high; therefore, efficient folding requires that chap-

erones and folding enzymes outnumber the newly

synthesized polypeptides. An additional factor influ-

encing the equilibrium of such a gel-like protein ma-

trix is represented by the amount of ER-sequestered

Ca2+: most of the ER proteins involved in protein

folding bind large amounts of Ca2+, with variable

affinity [16]. This latter aspect, which has often been

neglected when generally considering the ER fold-

ing machinery, appears to be of specific relevance tocells like cardiomyocytes for which a tight control of

rapid changes of Ca2+ levels in the SR is required to

regulate the contractile activity, while stable levels of

Ca2+ are required for the protein folding function [17].

Similar to what is described for the cytosol, ER

chaperones work in complexes—the lectin complex

and the Grp78/Grp94 complex (Fig. 2) —that may

interact sequentially or alternatively with the protein

substrate (see Table 2) [18]. Nascent polypeptides,

entering the ER through the protein-conducting chan-

nel Sec61, are bound by Grp78/BiP, the ER homolog

of Hsc70, which localizes on the luminal surface of the pore. Polypeptides then recruit the ER chaperone

complex, depending on their intrinsic characteristics.

Fig. 1. Model of the chaperone-assisted folding of newly synthesized polypeptides in the cytosol. ( A) The nascent chain

polypeptide interacts with Hsc70 and its cofactor Hsp40. Folding proceeds by recruiting Hsp90 and its cochaperones, among

which is Hsp-organizing protein (Hop), or by binding to prefoldin and the multimeric chaperonin TRiC. ( B) Proteins like actin

and tubulin interact immediately with prefoldin and the chaperonin TRiC. ( Adapted from Barral JM, Broadley SA, Schaffar G,

et al. Roles of molecular chaperones in protein misfolding diseases. Semin Cell Dev Biol 2004;15:18; with permission.)


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The lectin (calreticulin–calnexin) complex

This complex is relevant for the folding of most

secretory and membrane proteins. Binding of the na-

scent polypeptides to the lectin domain of calnexin

(a type I transmembrane protein) or calreticulin (asoluble ER luminal protein) is possible only in the

presence of monoglucosylated N-linked glycans,

which derive from glucosidase activity on mannose

residues. Despite the homology between calnexin and

calreticulin, each being monomeric Ca2+ binding ER

proteins, their folding activity and substrate specific-

ity are different [19]. Calreticulin appears to play

a major role in heart development and in cardiomyo-

cytes of the cardiac conduction system [20,21],

whereas it is expressed at very low levels in adult

cardiomyocytes [22]. The question concerning how

calreticulin chaperone function is supplied in adult cardiomyocytes remains unanswered. In the process

of newly synthesized proteins, calnexin primarily

binds folding intermediates preferentially, but not

uniquely from glycoproteins, assisting folding and

the assembly of protein subunits. Calnexin also plays

a role in quality control because it prevents the ex-

cretion of misfolded glycoproteins and helps inrefolding glycoproteins. Folding by the lectin com-

plex occurs through the interaction with several other

accessory proteins whose number and function ap-

parently depend on substrate specificity, except for

the oxidoreductase ERp57, which is specifically re-

cruited by the complex.

The function of calnexin and calreticulin is to

retain the unfolded glycoprotein, whereas the third

lectin involved in the complex, UDP-glucose:glyco-

protein glucosyltransferase (UDP-GT), represents a

folding sensor. UDP-GT adds a single glucose after

complete deglucosylation of N-glycans bound in still-unfolded or partially folded protein regions. Deglu-

cosylation determines the release of the protein

Fig. 2. Involvement of the ER lectin chaperone complex in folding and in targeting ER-associated degradation (ERAD). The

nascent chain polypeptides are translocated across the ER membrane through a pore (Sec61p), interact with Grp78, and after

incomplete deglucosylation, bind to calnexin (CNX) and calreticulin (CRT). The cochaperone ERp57 is recruited to assist in

disulphide bridge formation. UDP-GT provides the addition of one glucose to N-glycans bound to the unfolded region of the

polypeptide and favors re-entering of the partially folded molecule in the cycle again. Misfolded polypeptides are bound by

calnexin and EDEM and retrotranslocated through Sec61p into the cytosol by interaction with p97 ATPase. Here, they are

ubiquitinated and degraded by the proteasome machinery. PDI, protein disulfide isomerase. ( Adapted from Kaufman RJ,

Scheuner D, Schroder M, et al. The unfolded protein response in nutrient sensing and differentiation. Nat Rev Mol Cell Biol

2002;3:412; with permission.)


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substrate, whereas glycosylation by UDP-GT permits

subsequent interaction with calnexin or calreticulin

to complete the folding.

The Grp78/Grp94 complex

Nonglycosylated proteins are more efficientlyretained by Grp78 and subsequently folded by the

Grp78/Grp94 complex, which shows several analo-

gies to the HSP70/HSP90 folding machinery [23].

Except for the presence of ERdj3 (the ER homolog of

HSP40 cochaperone), the members of the complex

interact with each other even in the absence of the

protein substrate. The ER homolog of Hsp90, Grp94,

participates in the complex, together with Grp170,

several oxidoreductase enzymes, UDP-GT, and the

immunophilins. Despite the analogy to the cytosolic

chaperone machinery, the function of Grp94 remainsobscure. In contrast to Hsp90, which is involved in

the maturation of several substrates and requires

specific cochaperones, few substrates and almost

no specific cochaperones have been identified for

Grp94. Grp94 is abundantly expressed in cardiomyo-

cytes, although higher levels of expression were ob-

served in cardiomyocytes of the heart conduction

system and during development [24].

Special mention should be given to the ER

resident protein Hsp47, a member of the serin

protease – inhibitor protein family. Hsp47 is speci-

fically involved in procollagen processing and transport [25] and is highly induced in pathologic

conditions associated with increased collagen synthe-

sis, such as end-stage dilated cardiomyopathy [26].

Endoplasmic reticulum chaperone function and Ca2+

homeostasis

As mentioned earlier, Grp78, Grp94, calnexin,

calreticulin, and protein disulfide isomerase (PDI)

bind significant amounts of Ca2+ in vitro [16]. Total

ER Ca2+ is commonly estimated to range up to

millimolar concentrations; however, the free Ca

2+

concentration of the ER is much lower because the

cation is largely bound to matrix proteins of high

capacity, but of relatively low affinity [27]. Indeed,

changes in the amount of calreticulin, Grp78, or

Grp94 have been shown to affect ER Ca2+ storage or

release [22,28–32]. On the other hand, the folding

machinery of the ER may require a Ca2+- enriched

environment for activity. Ca2+ binding alters calnexin

and calreticulin conformation in vitro, and inactive

calreticulin– calnexin complexes are formed in the

Ca2+-depleted ER [17]. Calreticulin and/or ERp57

can also affect ER Ca2+ content through other mechanisms; namely, by direct interaction with the

SR Ca2+ pump SERCA and by modulation of its

activity [33]. However, the role played by calreticulin

in the maintenance of cardiomyocyte Ca2+ homeo-

stasis is relevant only during development and is

supplied by calsequestrin in the postnatal heart. An

additional contribution in the maintenance of Ca2+

homeostasis could then derive from the most abun-dant SR chaperones, Grp78 and Grp94. Although

information concerning a role for Grp78 in main-

tenance of cardiomyocyte Ca2+ homeostasis is lack-

ing, it was hypothesized that Grp94 upregulation in

chronically fibrillating atrial cardiomyocytes may

counteract the Ca2+ overload secondary to the

arrhythmia because overexpression of Grp94 delays

the rise in free intracellular Ca2+ and the necrotic

death of cardiomyocytes exposed in vitro to Ca2+

overload and simulated ischemia [22,31].

Folding of mitochondrial proteins

Chaperones localized in the mitochondrial matrix

are involved in the folding of locally synthesized

polypeptides and those arriving from the cytosol (see

Table 3).

TOM is an HSP90 cochaperone protein family

involved in post-traslational import of mitochondrial

proteins having a nonclassic internal targeting se-

quence. Tom70 is inserted in the cytosolic face of

the mitochondrial outer membrane by way of the

N-terminus. After binding to Tom70 and cyclingof ATP by the chaperones, the preprotein is trans-

ported through the outer membrane by the import

machinery [1].

In addition to the mitochondrial Hsc70 homolog

mtHsp75, which keeps mitochondrial-encoded pro-

teins in assembly-competent state, mitochondria use a

multimeric chaperonin formed by Hsp60 and Hsp10.

The Hsp60/Hsp10 chaperonin is a homolog to bac-

terial GroEL/GroES; it is involved in the prevention

of aggregation and refolding of mitochondrial pro-

teins, as shown in experimental hearts exposed tocardioplegia and ischemia-reperfusion [34].

Destiny of unfolded proteins: to rescue or to

degrade?

Protein folding is far from being a successful

event. It has been calculated that for a given poly-

peptide of 27 amino acids, the number of possible

starting configurations to achieve the native-state

configuration corresponds to 1016. This astronomic

number is reduced to 1010 by the nonsystematic,stochastic search of the best configuration, which

usually corresponds to the more stable, lower-energy


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structure [35]. Despite intervention of the chaperone

machinery, about 30% of newly synthesized proteins

never reach a fully folded conformation [36]. Because

these polypeptides with incompletely folded chains

expose regions of the molecule that are buried in the

native state, such species are prone to inappropriatecontacts with other molecules. To prevent cytotox-

icity secondary to protein aggregation, cells express

diverse arrays of chaperones to optimize protein

folding and, at the same time, promote turnover of

newly synthesized proteins.

The chaperone Hsp104, a member of the HSP100/

Clp subfamily of AAA ATPase, is able to resolubilize

insoluble protein aggregates but has been identified

only in bacteria and yeast [2]. Thus, efficient turn-

over of the proaggregating folding intermediates re-

mains the main resource for mammalian cells.

Protein degradation in the cytosol: the

ubiquitin-proteasome system

The ubiquitin-proteasome system represents the

major mechanism of intracellular protein breakdown.

Mostly underestimated until 1980 [37], the process

of protein degradation is a highly specific and timely

controlled event that is carried out by the coupling of

subsequent steps. The ubiquitin-proteasome system is

an energy-requiring process in which the C-terminus

of a polyubiquitin chain is covalently linked to ane-amino group of an internal lysine of the protein

substrate. This event requires the participation of an

enzymatic cascade involved in the synthesis of the

polyubiquitin chain (ubiquitin-activating enzyme E1

and ubiquitin carrier protein E2) and ligation to the

target protein (ubiquitin protein ligase E3). The

interaction between the E3 enzyme and the substrate

protein is dictated by the presence of more or less

destabilizing N-terminal residue (N-end rule path-

way). In some cases, the destabilizing N-terminus is

generated from the existing N-terminus by direct intervention of specific enzymes. The multiubiquitin

chain represents a signal for binding to the protea-

some. Indeed, ubiquitin protein ligase E3 itself

physically interacts with specific subunits of the

26S proteasome [11], a large (2 Md) complex of a

multicatalytic protease composed of a catalytic core

(20S) linked to two 19S regulatory complexes. The

19S components carry the recognition site for the

ubiquitinated substrate and the unfolding site for

the substrate protein to enter the 20S catalytic chan-

nel. HSP70 or chaperonelike proteins act to facilitate

the binding of specific proteins to the ubiquitincomplex. Degradation of partially folded proteins

still bound to Hsc70 may be promoted through the

interaction of the chaper one with specific cochaper-

ones (Bag-1 and CHIP) [2]. Bag-1 contains a ubiq-

uitinlike domain and CHIP promotes ubiquitination

through its type-E3 ubiquitin protein ligase activity.

Endoplasmic reticulum– associated proteindegradation

Proteins that are unfolded or that cannot fold are

removed from the folding pathway by ER-associated

degradation (ERAD) (see Fig. 2). ERAD implies ret-

rotranslocation of the misfolded or incompletely

assembled glycoprotein back in the cytosol, where

it is ubiquitinated and degraded by the protea-

some. Unfolded or partially folded monoglucosy-

lated N-glycan glycoproteins are again cycled by

calreticulin – calnexin. The proteins lose one mannoseresidue by a-mannosidase, interact with the lectin

endoplasmic reticulum degradation – enhancing 1,2

mannosidase-like protein (EDEM) and calnexin, and

are targeted to ERAD, whereas unfolded or partially

unfolded monoglucosylated N-glycan glycoproteins

are again cycled by calreticulin/calnexin [38]. The

transfer to the cytosol requires interaction with a

complex of substrate-specific ER proteins, which

accompany the protein substrate through the mem-

brane and recruit the p97ATPase at the cytosolic side.

This ATPase is a member of the AAA ATPase family

and represents the driving force that pulls theretrotranslocating protein into the cytosol [39]. Other

proteins undergo deglycosylation in the ER and

recycle between ER, intermediate, and Golgi com-

partments, where they undergo partial proteolysis

before ERAD [38].

Cellular responses to unfolded protein

accumulation

Conditions that lead to accumulation of inter-

mediate folding species, dysfunction of protein deg-

radation, or both are responsible for upregulation of

chaperone genes and increased expression of chaper-

ones. These events concern the cytosolic chaperone

machinery, the ER quality control system, or the

mitochondrial protein folding machinery. They may

occur independently and involve specific signaling

systems that correspond to the cytosolic heat shock

response (HSR), the ER unfold protein response

(UPR), or a recently described putative mitochondrial

UPR [40]. Pathways involved in the first two re-sponses have been carefully described and are the

subject of excellent reviews [41–43].


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Fig. 3. Schematic diagram of the ER stress response. (Upper panel ) Unstressed ER. ( Lower panel ) After misfolding of proteins

occurs, chaperones are recruited to bind the misfolded proteins. The recruitment sequesters the chaperones, which release binding

to IRE1-ATF6-PERK. IRE1 and PERK undergo dimerization and phosphorylation. Dimerized IRE1 cleaves XBP1 mRNA and

generates a transcription factor that translocates to the nucleus. An additional transcription factor derives from the release of

ATF6 cytosolic domain by proteolitic cleavage in the Golgi apparatus. Binding of the transcription factors leads to increased

translation of genes for the UPR proteins (chaperones, CHOP). Phosphorylation and dimerization of PERK results in

phosphorylation of the transcription factor elF2a and the blocking of protein translation.



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Hsc70 for the heat shock response and Grp78 for

the UPR play comparable roles in both pathways.

These proteins work as stress sensors, in that the

destabilization of Hsc70 binding to transcription

factors of the heat shock factor (HSF) family and of

Grp78 binding to IRE1, ATF6, or PERK trans-membrane transducers initiates the signaling for

activation of HSF- and UPR-responsive genes,

respectively (Fig. 3). Grp78 normally binds to

IRE1-ATF6-PERK proteins, thus inhibiting their

activation by dimerization. The interaction with

unfolded proteins leads to Grp78 sequestration and

release from the binding to the IRE1-ATF6-PERK

complexes (see Fig. 3). These factors, largely studied

in yeast, show correspondent pathways in eukaryote

cells in which a more complex sensor system

translates the UPR.Three distinct events are induced in response to

UPR activation. All of these events are directed

to reduce the unfolded protein load or, ultimately, to

reduce cell death. One event is represented by the

upregulation of genes that encode various compo-

nents of the chaperone machinery and the ERAD,

resulting in increased folding capacity of the ER

and in enhancement of protein degradation. A second

event is the general reduction of gene transcription

and shift of protein translation. Finally, activation of

apoptotic pathways involves activation of caspase 12

and increased expression of the proapoptotic tran-

scription factor CHOP.

Protein misfolding in human diseases

The process of protein folding and misfolding

has recently gained attention in the medical field with

the discovery of human disease entities that directly

or indirectly result from an aberration in the folding

process or in the folding process response (Table 4).

Although the majority of the disease entities linked to

such alterations have been investigated in other

organs (mostly in the brain), evidence is emerging

for the involvement of protein misfolding in the

pathogenesis of cardiac diseases. As a result of

protein misfolding, aberrant proteins are retained insubcellular compartments and targeted for refolding

by the chaperone machinery or for degradation by

way of the ubiquitin-proteasome pathways or ERAD.

Genetic mutations or environmental factors leading to

the misfolding of proteins result not only in the loss

of proteins (retention and degradation) or protein

function (loss of function, dominant negative) but

also in the formation of products toxic to the cells,

which thus gain toxic functions. Among the recog-

nized diseases (see Table 4) in which protein

misfolding has been demonstrated to play a patho-

Table 4

Partial list of disease entities caused by defects in protein folding affecting different tissues/organs

Disease Organ Gene/protein

Transthyretin cardiomyopathy Heart TransThyretin

Desmin-related cardiomyopathy Heart Alpha B-crystallin

Down syndrome Multiorgan a-Synuclein, Ab peptide, tau

Fabry’s disease Heart and vessels a-Galactosidase A

Cystic fibrosis Lung Cystic fibrosis transmembrane conductance receptor

Nephrogenic diabetes insipitus Kidney Aquaporin

Diabetes mellitus Pancreas Proinsulin 2Marfan syndrome Connective tissue Fibrillin

Retinitis pigmentosa Eye Rhodopsin

Osteogenesis imperfecta Bone Collagen type I

Glanzmann thrombasthenia Blood GPIIb

von Willebrand’s disease Blood von Willebrand

Familial hypercholesterolemia II Systemic Low density lipoprotein receptor

Congenital goitrous hypothyroidism Thyroid Thyroglobulin

Atherosclerosis Systemic Apolipoprotein

Alzheimer’s disease Brain Amyloid precursor protein

Huntington’s disease Brain Huntington

Creutzfeldt-Jacob disease Brain Prion protein

Parkinson’s disease Brain a-synuclein

Tauopathies Brain Tau proteinAmyotrophic lateral sclerosis Skeletal muscle Superoxide dismutase

Protein folding defects affecting the heart are in italics.


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genetic role, only a few have been described to lead

to cardiomyopathy and heart failure.

Genetic mutations of chaperone proteins

Chaperone function has been shown to be important in the development of misfolding disease in

humans; however, mutations that involve the com-

plete loss of chaperone function causing human dis-

eases have been described and are limited to some

specialized chaperone molecules. Mutations in spe-

cific chaperone proteins may result in congenital

defects of various degrees. Mutations in a gene en-

coding to a protein similar to TRiC chaperonins has

been shown to be responsible for two congenital

syndromes: the McKusick-Kaufman (MKK) and the

Bardet-Biedl type 6 syndromes. Congenital cardiacdefects are part of the MKK syndrome in which a

missense mutation resides in the hMKK syndrome

gene. The mutation leads to a mild form of congenital

disease syndrome because the missense mutation

allows the maintenance of a partial function of the

protein. On the other hand, the Bardet-Biedl type 6

syndrome recognizes a frameshift mutation, whereby

the protein is completely nonfunctional, resulting in

more severe forms of congenital defects.

One of the first described cardiomyopathies

from mutations of chaperone proteins was a form

of desmin-related cardiomyopathy [44]. Described10 years before in skeletal muscle in a large French

family as an autosomal dominant disease, desmin-

related myopathies are inherited disorders in which

desmin, a type III intermediate filament protein,

accumulates over time in the muscle, leading to an

adult-onset muscle disease. In the heart, a desmin-

related cardiomyopathy results from a missense muta-

tion leading to a substitution of an arginine residue

in the protein core (R120G) of the B subunit of

alpha crystallin. As mentioned before, alpha cystal-

lin is a small HSPs that assists in the folding processof desmin. In this genetic-based disease, alpha

B-crystallin has a reduced or absent chaperone func-

tion [45]. Failure of the alpha B-crystallin to mediate

the proper folding of desmin into cytoskeleton struc-

tures leads to the precipitation of toxic aggregates

(8– 10 nm intermediate filaments) composed of

alpha B-crystallin, desmin, and ubiquitin [46]. The

toxic effect of the disaggregated fragments can

give rise to different forms of cardiomyopathy: hy-

pertrophic, dilated, and restrictive, with associated

rhythm disturbances.

A mice model of mutated alpha B-crystallincardiomyopathy has helped to dissect the mecha-

nisms of this form of chaperone-deficient cardio-

myopathy and the role of alpha B-crystallin in the

desmin aggregation. These experiments have helped

to demonstrate that mutated alpha B-crystallin is

sufficient for the development of cardiomyopathy

and heart failure [13] and support the involvement

of the ubiquitin pathways in the pathogenesis of thisdisease form [47]. Similarly, alpha B-crystallin was

shown to interact with an F-box protein (FBX4;

part of the ubiquitin pathway) to induce ubiquitin-

dependent degradation, a binding enhanced by the

mutated R120G form of alpha B-crystallin [48].

Toxic aggregates and amyloid

Ischemic injury was among the first conditions in

which protein unfolding and aggregation was recog-

nized in the pathogenesis of cardiac diseases.Following coronary occlusion and ischemic injury,

recovery is limited in terminally differentiated cells,

and protein aggregation represents a complication

after reperfusion. Reactive oxygen-free radicals are

produced following ischemia and reperfusion, induc-

ing oxidative stress and leading to, among others,

protein oxidative damage. In addition to the acute

coronary event, protein misfolding and aggregation

following oxidative injury are also part of the process

of aging; thus, in both conditions, the unraveling of

the mechanism of misfolding may also appropriately

address the role of molecular chaperones as atherapeutic tool.

As was mentioned earlier, the missense substitu-

tion of B subunit of alpha B-crystallin leads to

R120G desmin-related cardiomyopathy and heart

failure. Similarly, missense mutations of desmin

protein (Ile451Met) have been described to be

responsible for at least some cases of familial

idiopathic dilated cardiomyopathies [49], for which

the exact mechanism of disease development arising

from the mutation has not been dissected.

Accumulation of toxic aggregates can also deter-mine development of cardiac dysfunction and heart

failure. Characteristically described in the brain (ie,

in Alzheimer’s disease and other neurodegenerative

pathologies). Proteins of different origin can (under

specific, most often unknown circumstances) de-

posit as aggregates of b-sheet conformation, known

as amyloid.

Amyloidosis is a disorder of protein structure

that can recognize an acquired or inherited origin and

give rise to organ-specific and systemic multiorgan

diseases. Specific amyloidoses are named according

to the identity of the protein precursor constituting themain fibril component, and many different pro-

teins are known to deposit as amyloidotic fibrils in


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different organs. In addition to alpha B-crystallin in

the desmin-related myopathies, in the heart, a form

of amyloidotic cardiomyopathy from a folding defect

is secondary to deposits of a plasma protein: the thy-

roid hormone and retinal-binding protein (vitamin A)

blood carrier protein transthyretin (TTR). Severalmutations (more than 80) of the transthyretin gene

result in destabilization of the monomeric or the tet-

rameric structure of TTR that can assume an in-

termediate form prone to deposit in tissues. TTR

mutations, initially described as being transmitted as

an autosomal dominant trait, showed more complex

expression with incomplete penetration, anticipation

(prevalently in the endemic areas), and the occurrence

of sporadic cases, whereby environmental factors

may play an independent role. Of the more than 80

known mutations of TTR, about 50% affect the heart among other organs and 7 specifically form deposits

in the heart. The most common mutation causing

cardiomyopathy is the substitution of a valine residue

with isoleucine (TTR V122I), a variant most com-

monly recognized in the African American popula-

tion and in West Africa, giving rise to late-onset

cardiomyopathy without involvement of other or-

gans [50].

Similar to other types of amyloidotic cardiomy-

opathies, TTR cardiomyopathy is mostly character-

ized by thick hearts of a restrictive functional pattern

and by aggregate deposited in the extracellular space,leading to heart failure and fatal arrhythmias.

Of note, aging per se can lead to deposits of

TTR-originating fibrils, leading to a senile systemic

amyloidosis. The senile form of amyloidotic TTR,

however, derives from the unmodified normal se-

quence of the protein as opposed to the variant form

that leads to a less stable protein with earlier onset of

the disease. It is conceivable that other proteins can

become unstable over time or that overly low pene-

trance mutations can lead to senile or adult onset of

the amyloidotic diseases and to heart failure.

Misfolded proteins in the endoplasmic reticulum

Ca2+ dishomeostasis, perturbation of the redox

status of the cell, energy (ATP) or glucose depriva-

tion, altered protein post-translational modification

(glycosylation), the occurrence of misfolded proteins,

or increased unfolded protein load to the ER are

conditions that can induce ER ‘‘stress.’’ Failure of the

UPR response or in the ERAD over time can translate

into defects in chaperone-mediated protein folding

and diseased phenotypes.As described earlier, the ER response to stress is

mediated by the complex pathways of transcription

factor binding proteins IRE1, ATF6, and PERK. In

the mammalian system, the ER membrane proteins

IRE1a and -b undergo dimerization and phosphory-

lation upon stress, leading to cleavage of a 26-mer

(26 nucleotide oligomer) mRNA intron for the bZIP

transcription activator XBP1. XBP1 activates thetranscription of numerous genes for the UPR.

Experimentally, deletion of the XBP1 genes can

cause cardiomyopathy and cardiac cell death (see

Fig. 3). Myocyte necrosis leading to embryonic death

after deletion of an hXBP-1 gene (TREB5) in mice

was described by Masaki et al [51].

Transgenic mice models for the ER unfolding

response helped to evaluate the role of pathways of

ER stress response. Naturally occurring proteins

travel in their native form to and from the ER and

Golgi to be secreted or incorporated in the membraneor in subcellular compartments. Specific signals

control the retention of misfolded proteins in the

ER. Among these signals, the tetrapeptide KDEL

(K = lysine, D = apartate, E = glutamate, L = leucine)

is bound to the C-terminus of secreted proteins and

interacts with the KDEL receptor to signal the

retention [52,53] in the ER and Golgi apparatus

through coated vesicles called COPI-I. Membrane

proteins are signaled to retrieve by KKXX or

KXKXX sequences (X = other amino acids). With

similar mechanisms, misfolded proteins undergo

r et ro tr an sp or t i n t he E R t o b e r ef ol de d b ythe chaperone machinery. Mutation of the KDEL re-

ceptor has been shown in vitro to disturb the circu-

lation of secreted proteins between the ER and Golgi

apparatus, resulting in misfolded protein accumula-

tion in the ER in stable cell lines [54]. The per-

turbation of ER quality control by mutation of KDEL

resulted in transgenic mice developing dilated car-

diomyopathy with accumulation of misfolded pro-

teins in the ER [55], suggesting the role of ER folding

defect as a pathogenetic factor in cardiac failure. In

addition, cell-specific ER stress – induced transcrip-tion factors have been described [56], indicating

the possibility of specialized pathways for the UPR

in mammalian cell types mediating differences in

pathologic outcome.

In contrast, Fabry’s disease is an example of

ER protein quality control being too efficient.

Farbry’s disease is an inherited disease linked to the

X chromosome, whereby a deficient activity of

a-galactosidase A, a lysosomal enzyme for glyco-

sphingolipids, most often leads to accumulations of

intracytoplasmatic lamellar inclusions in the vascu-

lar endothelium, with consequent systemic organdefects. In a subset of cases, a cardiac variant results

in cardiac hypertrophy, accounting for 3% to 9% of


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hypertrophic cardiomyopathies, and over time, in

death. In this form, the enzyme acquires a defective

conformational structure that prevents its transport to

the Golgi apparatus and export to the lysosomes.

More than 160 mutations leading to Fabry’s disease

have been identified; among those, few lead to themaintenance of partial enzymatic activity in the

cardiac variant of the disease. The misfolding origin

of the mutated protein is supported by the beneficial

effect of the administration of galactose or other

reversible competitive inhibitors that act as chemical

chaperones enabling the dissociation from the chap-

erone machinery and the dimerization of the enzyme

and reducing its degradation by the proteasome.

Human diseases can result from defects in the

UPR or in defects in the ERAD pathway. Genetic

mutations leading to misfolding of proteins that bindto Grp78/BiP influence the UPR. Among those

mutations, defects in the PERK gene, for example,

lead to the accumulation of procollagen type I and the

lack of formation of mature collagen in the bone in

osteogenesis imperfecta or proinsulin in pancreatic

b-cells in diabetes mellitus. Diseases of proteins that

do not bind Grp78/BiP, on the other hand, are

unlikely to influence the UPR, and a defect in the

ubiquitin-proteasome system and ERAD is likely to

be responsible for the defect and the clinical

phenotype. Examples of misfolding diseases of

proteins that do not bind Grp78/BiP and thus result from slow degradation from ERAD are cystic fibrosis

(mutations of the chloride channel) and emphysema

(a1-antitrypsin mutation). Alterations of the ubiqui-

tin-proteasome system have also mostly been

described in the brain in relation to neurodegenerative

diseases, but it has recently been shown how defects

in the system may play an important role in the

cardiovascular system. An animal model of dilated

cardiomyopathy [57] and human dilated cardiomy-

opathy [58] have showed significant increased

expression in some of the players of the ubiquitin- proteasome pathway and an overall increase of the

total protein-ubiquitin conjugation in failing hearts,

predominantly from dilated cardiomyopathy com-

pared with ischemic cardiomyopathies.

Of note, direct involvement of the ubiquitin-

proteasome system has recently been suggested in

the pathogenesis of different stages of development

of atherosclerotic plaque and its complication in the

cardiovascular system with respect to coronary circu-

lation [59]. In addition to its role in the protein deg-

radation process, the ubiquitin-proteasome system is

involved in important aspects of cell proliferation,inflammation, and apoptosis, which are all impor-

tant aspects of the pathogenesis of atherosclerosis.

These initial observations require further investiga-

tion to better understand the role of the ubiquitin-

proteasome system in the pathogenesis of the various

aspects of the atherosclerotic process and vascular

oxidative stress.

Summary

For many years, protein misfolding was the basis

for biochemical and biophysical studies in vitro or

in microorganisms such as yeast. Recently, clinically

related studies are merging the evidence collected

from microorganisms with human diseases. Thus, a

growing body of evidence is accumulating that iden-

tifies defects in protein folding or protein degradation

as pathogenetic hallmarks for many disease entities predominantly of late onset, including cardiomyopa-

thies and heart failure. Dissecting the pathogenetic

pathways opens new opportunities for therapy aimed

to re-equilibrate the folding capacities. The devel-

opment of chemical and pharmacologic chaperones

has helped to understand the mechanisms of some

aspects of protein misfolding and may find new ap-

plications to direct target-specific therapy. Further

understanding of the mechanisms of protein for-

mation and its defects will address the important as-

pects of modern medicine of directing early diagnosisand prevention.

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