Review
Stress (Heat Shock) Proteins
Molecular Chaperones in Cardiovascular Biology and Disease
Ivor J. Benjamin, D. Randy McMillan
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Abstract—How a cell responds to stress is a central problem in cardiovascular biology. Diverse physiological stresses (eg,
heat, hemodynamics, mutant proteins, and oxidative injury) produce multiple changes in a cell that ultimately affect
protein structures and function. Cells from different phyla initiate a cascade of events that engage essential proteins, the
molecular chaperones, in decisions to repair or degrade damaged proteins as a defense strategy to ensure survival.
Accumulative evidence indicates that molecular chaperones such as the heat shock family of stress proteins (HSPs)
actively participate in an array of cellular processes, including cytoprotection. The versatility of the ubiquitous HSP
family is further enhanced by stress-inducible regulatory networks, both at the transcriptional and posttranscriptional
levels. In the present review, we discuss the regulation and function of HSP chaperones and their clinical significance
in conditions such as cardiac hypertrophy, vascular wall injury, cardiac surgery, ischemic preconditioning, aging, and,
conceivably, mutations in genes encoding contractile proteins and ion channels. (Circ Res. 1998;83:117-132.)
Key Words: stress protein n gene expression n molecular chaperone n transcription factor
n vascular biology n aging n ischemia
P
cell survival (necrosis and apoptosis), we will emphasize the
emerging evidence regarding chaperone functions in physiological adaptation during cardiac hypertrophy, ischemic preconditioning, vessel wall injury, oxidative stress, and aging.
Although we presently can only speculate about their roles in
specific cardiac diseases, we will discuss many potential
opportunities to establish whether HSP chaperones either
exert an effect on or play a direct physiological role in the
natural history of diseases resulting from mutations of cardiac
contractile apparatus (eg, hypertrophic cardiomyopathy) and
ion channels (eg, long-QT syndrome) in humans.
hysiological stresses ranging from myocardial ischemia
to genetic mutations produce disease states in which
protein damage and misfolded protein structures are a common denominator.1–9 How does the cell respond? Multiple
endogenous pathways are engaged in restoring cellular homeostasis, but one well-characterized mechanism that involves protein folding is the heat shock family of stress
proteins, or HSPs.10,11 Insight into the mechanisms underlying
HSP function is provided by 2 main lines of evidence: (1) the
correct folding of many proteins in a cell requires proteinfolding machinery, the molecular chaperones,12,13 and (2) HSP
chaperones repair denatured proteins or promote their degradation after heat shock.14,15
Genetic studies provide convincing proof in different phyla
that overexpression of stress proteins is a powerful means of
cytoprotection, even in the intact heart.10,16 –18 Similarly, biochemical studies have demonstrated that the Hsc70 chaperone
increases the productive folding of the common DF508
mutation of CFTR. This suggests a physiological role of an
HSP chaperone in human diseases.19,20 Divergent mechanisms
that produce abnormal or misfolded cellular proteins converge into a common pathway, leading to an increase in the
levels of cytoprotective stress proteins, which decrease or
neutralize the deleterious effects of acute or chronic stresses.
In this review, we summarize present knowledge about the
regulation and function of individual chaperones (eg, Hsp90,
Hsp70, Hsp60, Hsp47, Hsp27, and aB-crystallin) in the
cardiovascular system. Besides their well-established roles in
Definitions and Nomenclature
What Are Stress “Heat Shock” Proteins?
The term “heat shock” protein is a misnomer but remains as
a legacy of Ritossa’s serendipitous discovery21 that heat shock
produced chromosomal puffs of salivary gland cells in
Drosophila. Heat stress ('5° above normal growth temperature) upregulates the rapid synthesis of a multigene family of
proteins, originally called heat shock proteins,22 which are the
result of a response often referred to as the heat shock
response.10,21 Prior sublethal heat stress transiently increases
the ability of a cell to withstand an otherwise lethal subsequent heat challenge. This phenomenon, or thermotolerance,
played a key role in launching numerous studies in both in
vitro and in vivo experimental models in which a similar
association was found between the heat shock response and
protection against either simulated hypoxia or ischemia.
Received February 4, 1998; accepted March 25, 1998.
From the Department of Internal Medicine and Integrative Biology Graduate Program, Molecular Cardiology Research Laboratories, The University
of Texas Southwestern Medical Center, Dallas, Tex.
Correspondence to Ivor J. Benjamin, MD, Molecular Cardiology Research Laboratories and Integrative Biology Graduate Program, U.T. Southwestern
Medical Center, Dallas, TX 75235-8573. E-mail [email protected]
© 1998 American Heart Association, Inc.
117
118
Stress Proteins in the Cardiovascular System
CFTR
ER
ERK
Grp
HO
Hsc
HSF
HSP
Hsp
JNK
MAPK
MAPKAP
MW
ROS
SAPK
TRiC
VSMC
Selected Abbreviations and Acronyms
5 cystic fibrosis transmembrane conductance regulator
5 endoplasmic reticulum
5 extracellularly responsive kinase
5 glucose-related protein
5 heme oxygenase
5 heat shock cognate
5 heat shock transcription factor
5 heat shock family of stress proteins
5 heat shock protein
5 c-Jun N-terminal kinase
5 mitogen-activated protein kinase
5 MAPK-activated protein kinase
5 molecular weight
5 reactive oxygen species
5 stress-activated protein kinase
5 TCP-1 ring complex
5 vascular smooth muscle cell
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Indeed, diverse stresses, including heavy metals, amino acid
analogues, inflammation, and oxidative/ischemic stress, induce the expression of HSP genes. Consequently, the terms
“stress proteins” or “heat shock family of stress proteins” are
preferred, although many of these proteins have essential
functions during unstressed conditions.13
Stress proteins belong to multigene families that range in
molecular size from 10 to 150 kDa and are found in all major
cellular compartments. The convention is to name stress
proteins of various molecular sizes as follows: Hsp27, Hsp70,
and Hsp90; whereas heat shock protein genes are designated
as follows: hsp27, hsp70, and hsp90.23 The distinction between constitutively expressed (eg, Hsc70 and Hsp90b) or
cognate members of the HSP family and their inducible
isoforms (Hsp70 and Hsp90a, respectively) is arbitrary, since
accumulating evidence, in physiologically relevant in vivo
systems, now indicates that such relationships depend on celland tissue-restricted expression.
Cellular Consequences of Heat and Ischemic
Stress Are Similar
Like experimental ischemia/reperfusion, heat shock is a stress
that disrupts numerous metabolic processes and cellular
structures and that culminates in cell death when a critical
threshold is exceeded.10,24,25 Both heat stress and ischemia
cause extensive damage to the cytoskeleton, including collapse of the threadlike intermediate filament network into
large perinuclear aggregates, reorganization of the cytoplasmic network, relocalizaton of actin-containing fibers around
the nucleus, and disruption of microtubules and the mitotic
spindle.26,27 Mitochondrial swelling, loss of mitochondria, and
uncoupling of oxidative phosphorylation are similarly shared
features of heat shock and early reversible ischemic
injury.28 –30
Characteristically, general protein synthesis is inhibited
after extreme heat challenge as a result of phosphorylation of
initiation factors such as eIF2a, which disrupts ribosomal
assembly and inactivates cap-binding proteins.31–33 In contrast, HSP genes are efficiently expressed after heat challenge, in part, as a result of the absence of introns in several
inducible (eg, hsp70) genes. In addition, alterations of mRNA
splicing and heat-induced stabilization of mRNA are adaptive
mechanisms used to efficiently translate stress proteins,
which can reach 15% to 25% of total intracellular protein
within minutes after physiological stress under these conditions.10,34 Coincidentally, several cytosolic chaperones translocate into the nucleus,35 where heat-induced inhibition of
DNA chromatin assembly exposes a nuclease-sensitive conformation, a pathognomonic feature of both heat- and ischemia-induced apoptosis.36 Less dramatic changes are seen in
integral membrane proteins, the lipid bilayer, and cellular
surface morphology. Cessation of noxious stimuli is followed
by rapid and efficient degradation of Hsp mRNAs.37–39
As mentioned previously, prior sublethal heat stress or
“hyperthermic preconditioning” profoundly attenuates all of
the heat-induced cellular changes to a subsequent severe heat
challenge. Moreover, pretreatment with heat produces “cross
tolerance” to varying types of physiological stress. For
example, protection of the intact ischemic heart after heat
pretreatment may last for hours to days.40,41 Insight gained
from the physiological roles of Hsp expression during the
heat shock response has contributed to current thoughts about
chaperone functions in pathological states likely to result in
abnormal protein folding.
Attention has primarily focused on the induction of HSP
chaperones and the potential repair mechanisms involved in
mitigating ischemia/reperfusion injury. Figure 1 schematically summarizes many of these concepts and illustrates the
multiple well-recognized mechanisms implicated in ischemic
myocardial injury, including oxidative stress/damage, calcium overload and activated proteases, release of proteolytic
and lysosomal enzymes, alterations of the cytoskeleton, and
complement activation.
Diverse Physiological Stresses Induce HSP Gene
Expression Through a Common Mechanism
Rapid induction of stress protein expression is accomplished
through mechanisms of transcriptional activation and preferential translation.10,42 HSFs (HSF1 through HSF4) regulate
the inducible synthesis of HSPs during development, growth,
and adaptation.42– 44 Whereas essential single-copy genes encode HSF in Saccharomyces cerevisiae and Drosophila,45,46
multiple HSFs have been identified in chicks, plants, mice,
and humans.47–51 Two HSFs (HSF1 and HSF2, encoding
proteins of 75 and 72 kDa, respectively) have been identified
in the mouse.49 Neither HSF1 nor HSF2 is heat inducible, but
HSF1 is hyperphosphorylated in a ras-dependent manner by
members of the MAPK subfamilies (ERK1, JNK/SAPK, and
p38 protein kinase) during physiological stress.52,53 During
unstressed conditions, both DNA-binding activity and transcriptional activity of vertebrate HSF1 are under tight negative control (reviewed in Reference 44). However, it remains
controversial whether repression by chaperone Hsp70, sequestration of constitutive phosphorylation on serine residues, or unknown inhibitory regulators are the principal
mechanisms underlying stress-inducible activation and rapid
deactivation of HSF1.43,54 –56
Previous studies have demonstrated that in response to
both heat and simulated ischemia, the mechanism(s) for
Benjamin and McMillan
July 27, 1998
119
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Figure 1. A summary of the major pathophysiological signals that activate HSP synthesis (left of vertical solid line) and the potential
functions of HSPs (right of vertical solid line). Cellular injury is manifested by increased generation of ROS, availability of redox-active
iron, peroxidation of membrane lipids, and protein damage, among others.92 The extracellular sources of ROS (A) could be endothelial
cells, VSMCs, and even myocytes in the surrounding tissue; mitochondrial electron transport of molecular oxygen (B) may be the main
intracellular source for ROS generation. Intracellular free calcium (C), which increases 10-fold within 10 minutes of reperfusion,228,229 has
been implicated in the activation of proteases (D), which, in turn, are thought to contribute to intracellular myocardial injury.230 –232 Damage to the cytoskeleton (E) is an early event of ischemic injury, which causes ventricular dysfunction or “myocardial stunning” when the
injury is reversible.87 Myocardial injury could also occur from recruitment of polymorphonuclear leukocytes (PMNs) by multiple mechanisms into the ischemic territory and by the release of oxygen-derived free radicals (A), cytotoxic lysosomal enzymes (F), extracellular
proteases (G), and complement activation (H) (reviewed in Reference 233). Diverse physiological signals are thought to activate the
stress-inducible HSF1 (I), which binds to the sequence-specific heat shock element (HSE) (J), contained in the promoters of all HSPs
(K). Although the precise mechanism of Hsp70-mediated ischemic protection is poorly understood, it is widely attributed to the biological property of “molecular chaperones,” which are proposed to assist in the assembly or repair of newly synthesized or damaged proteins.11,234,235 In physiological terms, potential functions for molecular chaperones in the ischemic heart include the following: protein
folding of newly synthesized polypeptides essential for maintaining oxidative metabolism after myocyte damage (L), protection of mitochondria from ROS and cytokines such as TNFa204,236 and translocation of newly synthesized proteins during organelle repair (M), repair
of critical structural proteins after ischemia-induced cytoskeletal alterations (N),237 recycling of membrane vesicles (Hsc70) (O), transport
of potential toxic byproducts for degradation by the proteasome (P),78,238,239 suppression of proinflammatory cytokines such as pro–
interleukin-1b (Q),204,236,240 suppression of NADPH oxidase and the oxidative burst by the heat shock response (R),241 protection by NO
production from inducible synthesis of HSP expression (S),61,242–246 prevention of apoptosis either through the mitochondrial chaperone
Hsp60 binding of cytochrome c and/or Hsp70 binding of cytosolic targets (T),36,204 repair of ion channel (U), collagen synthesis by
Hsp47 chaperone for reparative fibrosis (V),180 and modulation of the immune-mediated ischemic injury (W).241
HSF1 activation is similar, if not identical, in myogenic
cells57 and that depletion of intracellular ATP plays an
important role in triggering the HSF1-DNA binding activity.58 In disease conditions, inducers of HSF1 activation, such
as oxidized LDL and reactive nitrogen intermediates, are
thought to increase protein damage, which triggers upregulation of HSP gene expression.59 – 61 However, transcriptional
activation of the HSF1 pathway does not require new protein
synthesis, since the preexisting transactivator (HSF1) is
inactive in the unstressed state.43,56,57 Physiological stresses,
such as heat and ischemia, induce HSF1 monomers to
oligomerize as homotrimers, which then bind to an upstream
sequence-specific motif, heat shock element, in the promoter
of all stress-inducible HSP genes62,63 (Figure 1I through 1K).
We recently established a gene knockout model of Hsf1 and
demonstrated in in vitro studies the essential requirement of
this regulatory pathway in cellular defense and thermotolerance.36 In addition, stress protein expression has been implicated in promoting tumor cell survival64 and protection of the
ischemic heart.16 –18
Stress Protein Overexpression Enhances the Speed
of Physiological Recovery of the Ischemic Heart
A substantial literature describes the induction of Hsp70 by
ischemia,57,65,66 the potential role of Hsp70 in ischemic preconditioning,40,67 and an inverse correlation between expression of Hsp70 induced by ischemic or thermal preconditioning and infarct size in animal models.41,68 –70 In addition,
forced expression of Hsp70 conveys a cytoprotective effect in
cultured cells, including cardiac myocytes subjected to simulated ischemia.71,72 Specifically, overexpression of the major
70-kDa heat shock protein (Hsp70) in transgenic mice improves myocardial function,16 –18 preserves metabolic functional recovery,18 and reduces infarct size73 after ischemia/
reperfusion. In addition to Hsp70, Hsp27 and aB-crystallin
can protect primary cardiomyocytes against ischemic damage.74 Although the precise mechanisms are insufficiently
understood, stress proteins are thought to mediate cardioprotection through their biological functions as molecular
chaperones.
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Stress Proteins in the Cardiovascular System
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Figure 2. A proposed scheme for the
reaction cycle of chaperone Hsc70 complex in folding of a hypothetical substrate in the cell. Starting at the bottom
of the figure, chaperone Hsc70 (A) consists of an N-terminal ATPase and
C-terminal domain for binding target
substrate. Hsp40 (B), the eukaryotic
homologue of DnaJ, either binds
unfolded proteins (UPs) first (C) and then
binds Hsc70.ADP, or it binds to the
Hsc70.ADP.UP complex (D), which has a
low on/off rate for UPs. Several different
fates for the Hsc70.ADP.UP complex are
envisioned, each of which depends on
the replacement of Hsp40 by different
accessory partners, specifically, Hip
(Hsc70-interacting protein), Hop (Hsc70/
Hsp90-organizing protein), and Hap
(Hsc70 accessory protein).166,247,248 After
Hsp40 is released, Hip binds to the
ATPase domain of Hsc70 (E), stimulates
its activity, and then remains bound to
stabilize the ADP-Hsc70-substrate complex, suggesting that it may play a role
in the transport of substrates within cells
(F).166 Hop serves as a physical linker
between Hsc70 and Hsp90 (G), presumably to facilitate the transfer of UPs
between these chaperones. Whether or
not Hop changes the ATPase activity of
Hsc70 is controversial.249 The function of
Hup, which stimulates the release of UPs from Hsc70 (H) and converts oligomers of Hsc70 into monomers (I), remains to be established. The energy transferred from ATP hydrolysis, the rate-limiting step of the eukaryotic chaperone cycle, is used in successive
cycles to assist folding and prevent aggregation and degradation. To this end, two related proteins, BAG-1 (not shown) and Hap (J),
which bind to the Hsc70 ATPase domain, also inhibit binding of Hsp70 to unfolded protein substrate in vitro, suggesting that these
proteins may play roles in maintaining quality control by directing nonproductive interactions toward degradation250 (illustration kindly
provided by L.E. Hightower, University of Connecticut, Storrs).
Stress Proteins Can Function as
Molecular Chaperones
Molecular chaperones, like Hsp70 and aB-crystallin, are proteins that facilitate the folding, assembly, and disassembly of
other proteins but are not part of the finished product.75 Since
many proteins require chaperones to fold, these proteins are
essential components in the final stage of the central dogma of
molecular biology: DNA7RNA3polypeptide3folded protein.11,75 In vitro chaperones function to prevent aggregation of
other proteins under conditions of stress and to promote restoration of enzymatic activity of denatured protein substrates or
enzymes (eg, citrate synthase, b-galactosidase, or luciferase) on
removal of the stress.11,13,76
Figure 2 shows schematically the reaction cycle of chaperone Hsc70 in relation to recently identified cochaperones
and molecular substrates in the cell. For example, chaperone
Hsp40 plays a major catalytic function in loading target
substrates onto the Hsc70 binding/release cycle.77 Although
the mechanisms of these functions are still emerging, the
major functions of molecular chaperones are to (1) transiently
bind and delay the folding of nascent polypeptide chains until
synthesis is complete, (2) maintain polypeptide chains in an
appropriate conformation suitable for translocation across
organelle membranes, (3) prevent aggregation from intramolecular or intermolecular interactions, (4) actively disassemble clathrin-coated vesicles, (5) hold steroid aporeceptor
complexes in ligand competent states (Hsp90 and cochaperone), and (6) assist in degrading toxic metabolites by pro-
moting ubiquination and proteasome lysis78 (Figure 1L
through 1P).
The Case for Molecular Chaperones in
Cardiac Diseases
Widespread clinical interest into the biological functions of
molecular chaperones extends over a range of human pathologies from degenerative conditions such as Alzheimer’s
disease, prions, amyloidosis, cataract formation, sickle cell
disease, cystic fibrosis, and various cardiac diseases including
myocardial ischemia. Early events that reestablish timely
reflow of the ischemic myocardium either through
thrombolytics, direct angioplasty, or spontaneous clot lysis
are essential for enhancing myocardial salvage and reducing
morbidity and mortality.79,80 Nonetheless, recurrent ischemia
either from rupture of an unstable plaque or congestive heart
failure can complicate the clinical course of an acute myocardial infarction. In uncomplicated acute myocardial infarction, physiological recovery at the cellular level begins within
minutes but could last for weeks to months before myocardial
repair is complete. Thus, endogenous protective mechanisms
have clinical relevance in mitigating the effects of ischemic
heart disease.
Biochemical Activities of HSP Regulatory Pathway
and Chaperones During Myocardial Ischemia
In previous work undertaken to define the proximate stimulus
to HSF activation, we observed that severe intracellular
Benjamin and McMillan
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acidosis (pH 6.7) was insufficient to induce the DNA binding
of HSF1 in cultured myogenic cells exposed to simulated
ischemia, if ATP stores were preserved.58 In contrast, severe
ATP depletion (65%) stimulated DNA binding of HSF1, even
if pH was maintained within the normal range.58 In the intact
ischemic heart, 15 minutes of ischemia, which produces
reversible injury, is associated with a similar reduction (65%)
of high-energy ATP stores, whereas lethal injury is found
with prolonged ischemia (.40 minutes) and .90% depletion
of high-energy pools.81 The Km for the weak ATPase activity
of bovine Hsc70 is 1 to 2 mmol/L, 3 orders of magnitude
below the millimolar concentrations of intracellular adenine
nucleotide pools.75 Therefore, ATP-dependent activation of
the HSF1 regulatory pathway and the biochemical properties
of molecular chaperones are unlikely to be adversely affected
during periods of transient ischemia or reversible myocardial
ischemic injury.
Proof of principle indicating a cardioprotective effect of
Hsp70 in transgenic animals subjected to ischemia/reperfusion suggests that pharmacological or genetic methods to
increase stress protein expression in the myocardium of
patients at risk of acute ischemic events might limit ischemic
injury.82 However, additional basic knowledge is needed
regarding (1) their relationships to other endogenous pathways involved in myocardial protection from oxidative stress/
damage, (2) functional specificity among chaperone members
of the HSP multigene family, and (3) the contribution of this
pathway during acute ischemia and other physiological states
that trigger the heat shock response, before clinical
application.
Stress Proteins and Antioxidant Pathways
for Cardioprotection
Since the 1970s, the hypothesis that free radical scavengers
can ameliorate oxidative damage during ischemia/reperfusion
has been pursued by clinicians and researchers.83 In model
systems ranging from transgenic Drosophila to mice, overexpression of catalase, superoxide dismutase, or glutathione
peroxidase tends to be protective against oxidative stress.84 – 86
Oxidative stress, from ischemia/reperfusion, also plays a
central role in the injury of vital organs such as the brain,
kidney, and heart. ROS are thought to contribute to ventricular dysfunction, or “myocardial stunning,” arrhythmias, and
progressive cell damage or death after ischemic injury (Figure 1).87–90
Discrepant results have been reported from attempts to
deliver antioxidants during and after myocardial ischemia/
reperfusion.91,92 Exogenous antioxidants, which are restricted
to the interstitial spaces, may have limited ability to protect
intracellular proteins against ROS. For example, the hydroxyl
free radical (zOH), believed to be the main agent of oxidative
damage, is so highly reactive with a typical substrate that its
half-life at 37°C is 7310210 seconds.93 Thus, it is difficult to
envision that the administration of exogenous antioxidants, at
concentrations that are physiologically feasible, can effectively prevent zOH-induced macromolecular damage. A potentially more effective strategy may be to physiologically
minimize the production of zOH. Indeed, overexpression of
members of the HSP family may provide one such avenue.
July 27, 1998
121
Several studies have reported that during protection against
myocardial ischemia, upregulation of stress protein levels
correlates with increases in the enzymatic activity of catalase,
suggesting potential additive or synergistic interactions of
these endogenous pathways against oxidative stress.40,94,95 An
important unanswered question is whether functions of stress
proteins, as molecular chaperones, complement the unique
functions of antioxidant enzymes in protecting against oxidative stress/damage.
Molecular Chaperones of the
Cytosol/Nuclear Compartments
The Table shows the major classes of HSPs, the intracellular
compartments, their putative functions, and potential significance in cardiovascular biology.
Hsp70 Chaperones
Members of the Hsp70 family are the most widely studied
and abundant group in eukaryotic cells.96 In the cytosol,
Hsp70 binds to nascent polypeptides before their release from
the ribosome.97 All members of the Hsp70 chaperone class
possess two distinct domains: a highly conserved N-terminal
ATPase domain and a more divergent C-terminal domain,
which binds short hydrophobic peptides of target substrates98,99 (Figure 2A). Hsp70 chaperone function requires
the N-terminal ATPase domain, which, interestingly, is similar structurally to rabbit skeletal muscle actin despite little
sequence similarity.100
These structure/functional relationships of Hsp70 likely
confer in vivo chaperone activity in cardioprotection. In this
regard, virtually nothing is known about the constitutive
Hsc70 chaperone, which shares .80% sequence homology
with Hsp70. Conceivably, modest upregulation of the constitutive Hsc70 could promote substantial cardioprotective benefit. However, upregulation of HSPs beyond a critical threshold may have deleterious cellular consequences.101 Distinct
functions between Hsp70 members were reported recently to
exist in regions outside the peptide binding domain, suggesting additional levels of complexity to chaperone functions in
vivo.102
Hsp90 Chaperones
Figure 2 shows that the chaperone Hsp90 is a component of
the reaction cycle involving the chaperone Hsc70 complex
and newly synthesized proteins. In addition, members of the
Hsp90 family, Hsp90a and Hsp90b, which constitute 1% to
2% of the total soluble cytoplasmic proteins, have the best
characterized in vivo functional relationships with target
proteins, the steroid hormone receptors.103 Hsp90 with chaperone partners, Hsp70 and Hsp56, directly binds, stabilizes,
and maintains the aporeceptor complex in an inactive conformation. Ligand binding (eg, estrogen) to the aporeceptor
complex triggers ATP hydrolysis by Hsp90, which dissociates from an “activated” receptor that can now bind to the
sequence-specific recognition motif and induce the transcription of target genes.104 In addition, elevated levels of Hsp90
expression destabilize the estrogen receptor/estrogen responsive element complex and downregulate estrogen-responsive
122
Stress Proteins in the Cardiovascular System
Major Classes of HSPs in Cardiovascular Biology
Major Stress Proteins
and/or Family
Cellular/Organelle
(Tissue)
Cochaperones/
(Protein Targets)
Cellular Function
(Inducer)
Potential Cardiovascular
Significance
(Pathophysiological
Mechanisms)
Hsp110
Hsp110 (human)
Nucleolus/cytoplasm
Hsc70, Hsp40
Thermotolerance (heat stress)
(Ischemic cross tolerance)
Apg-1 (mouse)
Cytoplasm
...
Protein refolding (heat stress)
...
Hsp105
Cytoplasm
...
...
...
Osp94
(Renal medulla)
...
(Osmotic and heat stress)
(Hyperosmolar stress,
dehydration)
Hsp90a (Hsp86)
Cytoplasm
Hsp70, Hsp56, p23
Aporeceptor function (heat
stress)
(Estrogens and atherosclerosis)
Hsp90b (Hsp84)
Cytoplasm
Immunophilins, cyclophilins
(steroid receptors)
...
(Immunosuppressive therapy
and cardiac transplantation)
ER
94-Kinase, Grp78
Calcium binding chaperone
(glucose starvation)
(Ischemia)
Hsc70, cognate
Cytoplasm,
peroxisome
Hsp40
Protein folding
CFTR binding
Hsp70, inducible
Cytoplasm/nucleus
RAD46, platelets, Hsp40
Protein folding (heat
stress/unfolded proteins)
Cytoprotection (ischemic
events)
Mitochondria
...
Translocation and protein
folding
(Cytochrome c and apoptosis)
ER
Grp94
Protein folding (unfolded
proteins)
(CFTR binding)
(Myocardium)
zzz
(Self-antigen)
Chagas’ disease (molecular
mimicry)
Cytoplasm
...
(Hypoxia, HIF-1 transcription
factor)
Antioxidant properties
Cytoplasm/nucleus
p38 kinase, aB-crystallin
Actin dynamics
Cross tolerance
(preconditioning)
Cytoplasm
Hsp27
Cytoskeletal stabilization at Z
bands
Cross tolerance
(preconditioning)
Hsp90
Grp94
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Hsp70
mHsp75
Grp78/BiP
...
Small Hsp
Heme oxygenase (HO-1,
HO-2, Hsp32)
Hsp27
aB-Crystallin
p20
Cytoplasm
Hsp27, aB-crystallin
Vasorelaxation
...
MKBP
Cytoplasm
Myotonic dystrophy protein
kinase
Protection of Z bands and
neuromuscular junction
Myotonic dystrophy
ER
Procollagen I and III
Quality control of collagen
synthesis
(Reactive and reparative
interstitial fibrosis)
Cytoplasm
Hsp70, Hsc70
Chaperone functions
...
Mitochondria
Chaperonin 10
Protein import/folding
(Cytochrome c and apoptosis)
Cytoplasm
Actin/tubulin
Protein folding
...
Assorted
Hsp47
Hsp40 (Hdj-1)
Hsp60 (chaperonin system)
Hsp60
TRiC (also called CCT)
mHsp75 indicates mitochondrial Hsp75; HIF-1, hypoxia-inducible transcription factor 1; and MKBP, myotonic dystrophy protein kinase.
target gene expression, indicating a regulatory feedback
loop.105
Hsp90 chaperone functions are mediated by signal transduction pathways involving various protein kinases of both
tyrosine and serine-threonine types, casein kinase II, the
heme-regulated eIF-2a, and a variety of other cellular proteins, such as calmodulin, actin, and tubulin (reviewed in
Reference 106). Finally, Hsp90 chaperones of Saccharomyces cerevisiae are essential for survival under all conditions,
supporting their important physiological roles in lower
eukaryotes.107
Cytosolic Chaperones of Special Interest in
Cardiac and Vascular Biology
Unlike the ubiquitous Hsp70 and Hsp90 counterparts, specific members of the small MW HSPs (HO-1 or Hsp32,
Hsp27, aB-crystallin, and Hsp20 chaperones) exhibit tissue-
Benjamin and McMillan
restricted expression, suggesting potential specialized properties in the cardiovascular system.
Inducible HO (Hsp32)
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HO is the rate-limiting enzyme in the degradation of heme to
biliverdin (a potent antioxidant), molecular iron, and carbon
monoxide. Three related single-copy genes encode HO isoforms: HO-1, HO-2, and HO-3.108–110 HO-1 is a bona fide
32-kDa stress protein (Hsp32) that is induced by diverse physiological stresses, including hypoxia, ischemia/reperfusion, hemin, hydrogen peroxide, and several heavy metals (selenium,
arsenite, cobalt, cadmium, and stannous ions).108–110 Inducible
Hsp32 (HO-1), the most widely expressed isoform, is present in
myocardial cells.111
Hsp32, like the inducible form of NO synthase mediates
guanylyl cyclase– dependent platelet inhibition and vasodilation of VSMCs.112–114 However, physiologically relevant hemodynamic forces (shear stress and cyclic strain) induce
HO-1 mRNA expression but not inducible NO synthase
expression, suggesting specificity of this stress response
pathway to physiological signals.115 Either endogenously
released or exogenous administered NO induces a 3- to 6-fold
increase in Hsp32 (HO-1) gene expression and CO production in VSMCs 116 ; similarly, the inhibitor tin
protoporphyrin-IX prevents platelet aggregation through induction of HO-1 gene expression and CO production in aortic
VSMCs.115 In rat VSMCs, angiotensin II treatment decreases
Hsp32 (HO-1) mRNA expression in a calcium-dependent
manner.117 However, angiotensin II–induced hypertension
increases Hsp32 (HO-1) mRNA expression in rat aorta,
suggesting the overriding influences of hemodynamic factors
in vivo.118
Several regulatory pathways are involved in the induction
of Hsp32 gene expression, most notably the HSF1 and the
hypoxia-inducible transcription factor-1 pathways.119 –122 Although direct evidence of a chaperone function of Hsp32 is
lacking, its overexpression in several cell types protects
against oxidative stress/damage.123–125 Accordingly, studies to
define the precise regulatory pathways stimulated by heavy
metals, hypoxia, and oxidative stress could provide novel
insight into the biological roles of Hsp32 (HO-1) in modulating oxidative stress/damage during ischemia/reperfusion,
vascular tone (eg, hypertension), and inhibition of platelet
aggregation and/or VSMC proliferation after balloon
angioplasty.
Hsp25/27 Chaperone
After its discovery as an inhibitor of actin polymerization,126
chaperone Hsp 25/27 (Hsp25 in mice and Hsp27 in humans)
has been demonstrated to play a major role in actin filament
dynamics in diverse cell types. Physiological stimuli (oxidative stress, cytokines, and growth factors) dramatically increase the phosphorylation of human Hsp27 at Ser15, Ser78,
and Ser83 residues, which is essential for acquired tolerance.127–129 Hsp25/27 phosphorylation is catalyzed by the
MAPKs (p38-MAPKs, JNKs, or SAPKs) and ERKs.130 In the
perfused adult heart, both p38-MAPK and JNK/SAPK are
activated after ischemia/reperfusion.131 In response to ROS
July 27, 1998
123
treatment, activation of p38-MAPK increases MAPKAP
kinase 2 activity, which phosphorylates Hsp27.132
In human endothelial cells, inhibition of vascular endothelial growth factor–induced p38-MAPK activation abolishes
Hsp27 phosphorylation, actin polymerization, and cell migration, suggesting a possible link between Hsp27 and angiogenesis.133 Together, available evidence places the p38MAPK as an upstream activator of stress-inducible Hsp25/27
phosphorylation, and this pathway underlies the effect of
p38-MAPK on the reorganization of filamentous actin, accumulation of stress fibers, and the recruitment of vinculin at
focal adhesion sites.134 It will be important next to determine
whether Hsp25/27 exerts vasoprotective actions in response
to hemodynamic forces or vessel wall injury. However, direct
analysis will likely require an Hsp27 gene knockout model.
aB-Crystallin Chaperone (Hsp22)
Whereas Hsp27 is detected in endothelial cells, VSMCs, and
cardiomyocytes, the aB-crystallin chaperone is expressed in
cardiomyocytes only.135 Both Hsp27 and aB-crystallin are structurally related bona fide HSPs with in vitro chaperone activity
but, unlike Hsp70, are not ATP-binding proteins.136 –139 Heightened interest into the regulation and function of aB-crystallin
protein (Hsp22), a major structural protein of the ocular lens, is
related to its tissue-restricted expression in striated myogenic
lineages with high oxidative capacity, such as the heart and type
I skeletal muscle fibers.140 In nonlenticular tissues, aB-crystallin
postnatal expression increases and reaches its highest levels in
the adult heart ('1% to 3% of the total soluble protein),
followed by skeletal muscle and the kidney.140 Previous immunohistochemical studies have localized the highest aB-crystallin
expression in the cardiac conduction fibers of the adult heart.141
Whether an alteration of aB-crystallin expression could lead to
abnormalities of the conduction system is presently unknown
but raises an intriguing possibility.
Although aB-crystallin expression has been localized to Z
bands of the cytoskeleton, in a pattern similar to desmin and
actin,142 recent studies suggest that this interaction is much
more transient and dynamic with respect to intracellular
targets, depending on the physiological conditions. In unstimulated cardiac myocytes, biochemical studies indicate
that aB-crystallin is highly soluble and remains in the
cytosolic fraction; heat or ischemia triggers rapid translocation of aB-crystallin into the insoluble cytoskeletal/nuclear
fractions, aggregation, and specific interactions at Z bands
(Reference 142 and I.J. Benjamin, unpublished data, 1998).
The physiological significance for the tendency of both
aB-crystallin and Hsp25/27 to form large hetero-oligomeric
complexes (500 to 800 kDa) both in vivo and in vitro after
physiological stresses remains a mystery.143,144 Although aBcrystallin chaperone provides cytoprotection to cardiomyocytes,74 the regulatory mechanisms of posttranslation modifications such as phosphorylation, glycation, and deacetylation
on aB-crystallin function await direct analysis in the cardiovascular system.
Stress Proteins and Striated Muscle Development
and Differentiation
Increased small MW HSP chaperone expression has been
described during periods associated with increased protein
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synthesis, protein degradation, and cellular reorganization,
such as myogenic differentiation and embryogenesis.145 The
tissue-restricted expression of aB-crystallin during skeletal
muscle myogenesis may require the MyoD family of basic
helix-loop-helix transcription factors, which bind the essential E-box enhancer contained in aB-crystallin promoter.146,147
We recently reported that expression of aB-crystallin, but not
Hsp27, is directly linked to increases in oxidative metabolism
in skeletal muscle after chronic nerve stimulation.148 However, the physiological role of upregulation of Hsp27 expression, which precedes early differentiation of murine embryonic stem cells, remains to be established in myogenic
lineages.149
Much less is known about the regulatory mechanisms
involved in aB-crystallin–restricted expression in cardiac
myocytes. aB-Crystallin is abundantly expressed in early
cardiac development beginning at embryonic day 8.5, suggesting a role either as a structural protein or as a molecular
chaperone in myofiber stabilization.150 Since Myo-D–like
factors are absent in the heart, in vitro binding studies of
cardiac nuclear extracts have implicated the transcriptional
activities of an upstream stimulating factor at the E-box
element and the serum response factor at a reverse CArG box
in the aB-crystallin promoter.151 So far, an in vivo developmental survey reveals that aB-crystallin expression is unaffected in skeletal muscle of myf 5 null mice or the heart of
d-HAND null mice at embryonic day 9.0 (I.J. Benjamin,
unpublished data, 1998).152,153
Other Cytosolic/Nuclear Chaperones
Several other HSPs that exist in the cytosolic/nuclear compartment are of potential interest in cardiovascular biology.
For example, the cytosolic 20-kDa protein, p20, is abundantly
expressed in heart, skeletal, and smooth muscle and copurifies with the chaperones aB-crystallin and Hsp27.154,155 Although p20 expression is induced by neither heat nor chemical stress, it contains the homologous C-terminal “acrystallin domain” shared by all members of the small MW
HSPs.138 In VSMCs, p20 is a substrate for both cAMP and
cGMP protein kinases, suggesting a regulatory role to postulated functions in physiological maintenance of vascular tone
and adaptation to vessel wall injury.156
A fourth member of the small HSPs (besides Hsp27, aBcrystallin, and p20), a myotonic dystrophy protein kinase–
binding protein associates and increases the activity of dystrophy
protein kinase and prevents its heat-induced denaturation in
vitro.157 Myotonic dystrophy protein kinase, unlike Hsp27 or
aB-crystallin, is upregulated in the skeletal muscle of patients
with myotonic dystrophy, suggesting that this novel protein may
be involved in the pathogenesis of this disease.157
Members of the Hsp110 family exhibit chaperone and
cytoprotective functions, although details about their relative
expression in myocardial cells and regional distribution in the
cardiovascular system await further characterization.158 Additional Hsp110 family members include Hsp105, Apg-1, and
Osp94.159 –161 There is intense interest in identifying a mammalian homologue of yeast Hsp104, which, instead of preventing protein aggregation, seems to resolubilize insoluble
protein aggregates.162
Mitochondrial Hsp70 Chaperone System
All organisms possess ATP-dependent mechanisms for protein folding and assembly within organelles.11,13 Translocation
of proteins across the mitochondrial membrane requires
mitochondrial chaperone Hsp70 in the matrix, where folding
into the native state is completed13 (see Figure 1).
Mitochondrial Chaperonin System
Besides Hsp70-like chaperones, mitochondrial chaperonins
Hsp60 and Hsp10 constitute a separate system that provides
a sequestered environment for folding a subset of proteins in
vivo.163 These 7-membered rings are arranged as cylindrical
structures in which ATP-dependent protein folding occurs in
their central cavity.164 Evidence from in vitro studies suggests
the Hsp70 chaperones and chaperonin systems function
cooperatively in protein folding and assembly in eukaryotes
(reviewed in Reference 13).
Cytosolic Chaperonin System
TRiC chaperonin is considered the functional equivalent of
chaperonin Hsp60/Hsp10 in the eukaryotic cytosol. The TRiC
complex, which consists of either 8- or 9-membered double
rings of '55 to 65 kDa subunits, is required for folding of
actin and tubulin in vivo.165 Chaperonin TRiC requires additional components, such as Hsp40, which stimulates the
Hsc70 ATPase, for protein folding in the cytosol77,166 (see
Figure 2). Available evidence suggests that TRiC chaperonin
functions in the final stages of folding during translation of a
limited number of polypeptide domains.13
Implications
Mitochondrial chaperones and chaperonins are only modestly
induced by physiological stress in cardiomyocytes and the
heart.167 However, the location of mitochondrial Hsp70 chaperones and chaperonins at major sites of ROS production
could serve to complement both enzymatic and nonenzymatic
defense mechanisms to diminish oxidative injury and increase the rate of physiological recovery after ischemic
injury.168,169 Whether overexpression of either mitochondrial
Hsp75 chaperone or chaperonin can provide equivalent or
superior protection against ischemic injury is presently unknown. Another important question concerns whether the
overlapping functions of Hsp70 chaperones and Hsp60 or
TRiC/Hsp40 chaperonin systems are coordinated for de novo
protein folding and potential cytoprotection during the pathogenesis of heart disease.
Molecular Chaperones in ER
Glucose-Regulated Proteins
Analysis of ER chaperones may be of particular clinical
interest, because the ER functions to degrade or repair
proteins damaged after myocardial ischemia or after mutant
proteins (eg, CFTR or thyroglobulin) fail to fold properly.11,170,171 Anoxia, glucose starvation, and calcium ionophores
induce members of the Ca21-binding glucose-regulated class
of stress proteins, Grp170, Grp94, and Grp78/BiP.11,172 Variable changes in the expression of Grp78 protein have been
reported after ischemia.173,174 Although “guilt by association”
with cytosolic chaperones is the presumed role of chaperone
Benjamin and McMillan
Grp function in protein surveillance and quality control, more
studies of their expression are needed in pathophysiological
states associated with the expression, posttranslational glycosylation, and secretion of abnormal proteins via the ER-Golgi
pathway.
July 27, 1998
125
costs from the longer gestation periods, longer time to reach
sexual maturity, and smaller litter sizes of such larger species.
However, investigators with expertise in molecular biology
and molecular physiology undertaking collaborative projects
increase the chances of success, while avoiding the duplication of efforts.
Hsp47 Chaperones
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The 47-kDa collagen-binding glycoprotein Hsp47 is a member of the serpin (serine protease inhibitor) superfamily and is
highly induced by heat stress or pathophysiological states (eg,
hepatic fibrosis) associated with increased collagen synthesis.175 Hsp47 resides in the ER and contains the C-terminal
Arg-Asp-Glu-Leu ER retention signal.176 Hsp47 binds transiently to collagen types I to IV and avidly to denatured
collagen substrates; thus, its role in procollagen processing
and transport seems firmly established.177 Future studies must
now address the likely biological and clinical relevance of
Hsp47 expression at the onset and progression of pathophysiological states, such as myocardial infarction, idiopathic and
hypertrophic cardiomyopathies, and hypertension, in which
myocardial fibrosis is prominently featured.178
Current Challenges and Future Directions
Physiological recovery of the ischemic heart begins within
minutes, but the rate of cellular ischemic repair, which may
last for days to weeks, is critical for the reduction of
subsequent cardiovascular morbidity and mortality. The idea
that stress proteins can speed the physiological recovery of
reversible myocardial injury is based on experimental evidence indicating that multiple proximate “signals” can activate the heat shock response and, thereby, evoke the endogenous protective mechanisms of Hsps. Major clinical events
such as unstable angina, recurrent occlusion after
thrombolytic therapy, and acute exacerbation of chronic
angina are potential physiological inducers of cytoprotective
stress proteins. The next logical step would be to consider the
possibility that related members of the multigene stress
protein family confer similar or additional functional benefits.
Because ischemia and other physiological perturbations disrupt the normal structure-function relationships of intracellular proteins, future studies must establish whether molecular
chaperones, either alone or in combination, alleviate ischemic
damage by accelerating physiological recovery of the myocardial cell in the intact organism.
Experimental Models for Stress Protein Research
Transgenic models of Hsp70 overexpression (gain of function) and hsf1-deficient mice (loss of function), and their
subsequent characterization, are beginning to illuminate their
physiological roles in vivo.16 –18,36 One exciting direction of
such efforts could lead to integrated approaches into the
physiological roles of entire systems or regulatory networks
in genetically modified animal models of human diseases.
However, the wealth of existing knowledge of physiological
studies in animals larger than the mouse should not be
disregarded. Studies to define the role of HSP expression in
myocardial stunning using conscious animals could justify
the development of transgenic rat and rabbit models. Potential
limitations of such strategies include substantially greater
Does Stress-Inducible Expression of HSP
Genes Affect Infarct Size, Arrhythmogenesis,
and Myocardial Remodeling After Acute
Myocardial Infarction?
Previous studies have demonstrated that pretreatment (24
hours) with heat stress attenuates free radical release in the
isolated rat heart95 and reduces the amount of arrhythmias, a
major functional hallmark of ischemia/reperfusion injury
(reviewed in Reference 92). Together, these studies correlate
potential interactions between these protective pathways but
are inadequate to establish a cause and effect relationship.
The development of an HSF1 gene knockout mouse model
provides a powerful experimental approach to determine
whether a deficiency in stress-inducible HSP synthesis, during physiological perturbations such as myocardial ischemia,
affects the outcome of postinfarction injury and the repair
mechanisms in the intact organism. Results of such studies
can help establish causality, elucidate potential mechanisms
by which HSP synthesis relates to myocardial ischemia, and
evaluate the participation of stress proteins during early and
late ischemic preconditioning. Timely characterization of
several knockout models, currently in development in laboratories worldwide, should continue to foster fruitful collaborations among investigators.179 Further progress should accelerate the flow of new knowledge into related areas such as
postischemic reactive and reparative fibrosis,180 postreperfusion inflammation,181 and myocardial stunning.89,182
Stress Protein Expression and Mechanisms of
Ischemic Preconditioning
Ischemic preconditioning is the most powerful experimental
maneuver that reproducibly protects the heart against subsequent ischemic challenge.183 However, controversy exists
about the precise roles of stress proteins in this wellcharacterized phenomenon.184 Several mechanisms involving
protein kinase C, adenosine receptors, and their relationships
to signal transduction pathways have been implicated in
ischemic preconditioning.185 Sufficient evidence indicates a
lack of correlation between stress-inducible expression (eg,
Hsp70) and early preconditioning, which is short-lived and
lasts between 1 and 3 hours, depending on the model and
species.186
Is the Chaperone Family of Stress Proteins
Unlikely to Have Physiological Relevance in
Early Preconditioning?
In our opinion, studies in this area have either prematurely
dismissed or paid inadequate attention to the potential importance of the small MW Hsps, such as aB-crystallin and
Hsp27. These chaperones seem likely to be candidates for the
“first line of defense” against nonlethal stress. Whether
oligomerization of small MW Hsps contributes to the me-
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chanical stability or speculated role of the cytoskeleton in
ischemic protection of the cardiac myocyte is presently
unknown.187,188 Both aB-crystallin and Hsp27 gene knockout
models, currently in development, could directly address this
hypothesis.
Similarly, studies are needed to address the correlation
between late preconditioning and stress protein expression.
Twenty-four hours after an initial ischemic stress, several
studies have reported increases in Hsp60 and Hsp70 synthesis
in rabbits,41 Hsp70 in pigs,182 and manganese-SOD in
dogs189,190 and delayed ischemic tolerance or late preconditioning. Therefore, a fundamental question is whether a direct
relationship exists between stress protein expression and late
preconditioning.191–193 A more detailed assessment of the
specific role of individual Hsp in cardioprotection is also
required. Attempts to determine the mechanism of the cardioprotective ATP-sensitive potassium channel by use of
inhibitors have found a lack of correlation with stress protein
Hsp70 expression.194 However, approaches using specific
inhibitors are inadequate for establishing specificity, to any
degree of certainty, beyond the proteins under
investigation.195
Molecular Chaperones and Cell
Death Pathways
Stress proteins are ideal candidates to play key regulatory roles
in cell survival and death pathways involving DNA damage and
protein synthesis, repair, and degradation. Maneuvers that increase Hsp70 expression after heat shock, sodium butyrate
exposure, and constitutive or regulated overexpression inhibit
apoptosis in a variety of cell types.196–198 The proto-oncogene
c-myc potentiates heat shock–induced apoptosis199,200; in contrast,
Bcl-2 overexpression augments thermotolerance-induced cellular survival.201 Tissue-specific expression of Hsp70-2 prevents
apoptosis in certain mitotic cells through a mechanism involving
cell cycle control.202 Hsp70 chaperone function in regulating
apoptosis may be at the level of signal transduction, as it has
been implicated in the stress-activated kinase pathway.203
Recent studies in our laboratory using hsf1-deficient cultured cells have established the role of stress-inducible Hsps
to render cells thermotolerant to heat-induced apoptosis.36
The present study provides a genetic model to examine
potential interdependent relationships between stressinducible of Hsps and the mechanisms involved in cell
survival and/or cell death pathways. Sublethal heat stress
protects mitochondria against oxidative stress and prevents
cell death by apoptosis.204 Given their strategic locations in all
major organelles, it is tempting to speculate that multiple
Hsps may combat oxidative stress/damage by refolding damaged repressors of the cell death pathway or preventing their
degradation. Alternatively, repressing the release of suicide
activators such as cytochrome c could occur through interactions with mitochondrial chaperones and chaperonins
(Figure 1).
In addition, results of several recent studies have implicated the small MW Hsp25/27 in cell survival pathways
involving cell differentiation and oxidative stress/damage.
Previous studies have demonstrated that overexpression of
Hsp25/27, like the antiapoptotic protein Bcl-2, increases the
levels of the antioxidant glutathione and resistance to Fas/
APO-1–mediated apoptosis, although whether this occurs
directly remains unclear.205 Withdrawal of mouse embryonic
stem cells from the cell cycle induces upregulation of
Hsp25/27 mRNA, which is accompanied by decreases in
phosphorylation and increases in oligomerization of
Hsp25/27 protein.149 Antisense reduction of Hsp25/27 reverses these changes through the prolongation of the cell
cycle, reduction in glutathione levels, and acceleration toward
apoptosis. Together, these provocative findings suggest that
during proliferation and differentiation of myocardial cells,
the Hsp25/27 chaperone, and others, can reduce oxidative
stress/damage and prevent apoptosis through a novel redoxdependent mechanism.206
Does Stress-Inducible Expression of HSP Genes
Affect the Natural History of Chronic
Cardiovascular Diseases, Including Aging?
Numerous studies have correlated the induction of Hsp
expression and pressure overload by aortic banding, acute
hypertension, exposure to vasoactive agents or left ventricular
hypertrophy, and growth factor expression in several cell
types.207–209 Decreases in the expression of HSP genes and the
DNA-binding activity of HSF1 are reported to occur during
the aging process in the rodent myocardium.210 There is
increasing evidence suggesting that oxidative stress/damage
may be a major causal factor in the aging process.211 The level
of oxidative stress and the susceptibility of tissues to experimentally induced oxidative stress seem to increase during the
aging process. Whether the increased amounts of molecular
oxidative damage, observed during the aging process, are
causally associated with a decreased activity of HSF1 and, by
implication, Hsp gene expression is presently unknown. The
availability of transgenic and gene knockout models will
enable future studies to establish the role of stress-inducible
heat shock gene expression during normal aging or physiological adaptation to disease-associated cardiac risk factor(s).
Molecular Chaperones in Cardiac Disease States
Caused by Expression of Mutant Proteins
Elevated levels of misfolded or denatured proteins, as well as
microinjection of abnormal proteins, are potent inducers of
HSP gene expression.14,59 Recent studies indicating that the
Hsc70 chaperone interacts indirectly with the CFTR harboring the common DF508 folding mutation support this general
notion of their biological role in human diseases.20
From the perspective of chaperone biology, cardiac diseases that arise from mutations in genes encoding components of the contractile apparatus or ion channel proteins are
essentially problems of abnormal proteins. Multiple mutations of sarcomeric proteins have been implicated in the
pathogenesis of familial hypertrophic cardiomyopathy, including myosin heavy and light chains, troponin I and T
subunits, myosin binding protein C, and tropomyosin (Reference 6 and reviewed in Reference 7). Whereas chaperone
Hsp27 plays a role in actin polymerization and chaperonin
TRiC is involved in actin and tubulin folding, the physiological role of chaperone and chaperonin systems in folding and
assembly of sarcomeric structures, under normal conditions
Benjamin and McMillan
or in disease, remains a mystery. Conceivably, chaperones
can influence either the repair or degradation of mutant
sarcomeric proteins, which, ultimately, affect the structurefunction relationships and phenotype of the disease.212 In vitro
analyses are first needed to determine whether chaperone
function can affect productive protein folding caused by the
relevant mutations, which can be validated in appropriate
animal models.18,213
Molecular Chaperones in Vascular Biology
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Numerous opportunities exist to dissect chaperone functions
during the synthesis and secretion of biological active peptides and proteins, cellular proliferation, intracellular signaling, and cytoskeletal rearrangement. Results of such studies
may identify the specific HSP protein targets and the specificity among the various cell types in promoting the vascular
protective effects of estrogen.214,215 The essential roles of
Hsp90 and cochaperones in steroid receptor biology suggest
that HSP chaperones play a clinically significant role in the
effects of hormonal replacement therapy in postmenopausal
women.216,217 Epidemiological studies have found that the
protective benefit against coronary heart disease in premenopausal women is abolished in the postmenopausal years.218 –220
Potential protective actions of estrogen have been attributed
to its antioxidative and vasoprotective properties, lowering of
blood lipids and lipoproteins, and direct effects on the vessel
wall.221 It will be important to define whether stress-inducible
Hsp expression in genetically modified mice plays a role in
the vascular response to injury in male and female animals.215
Molecular Chaperone and Immunological Diseases
In contrast to their well-established cytoprotective roles,
certain stress proteins have been implicated in the pathogenesis of cardiovascular diseases. For example, elevated serum
levels of antibodies against the bacterial homologue of
mammalian Hsp60 have been demonstrated in patients with
cardiomyopathy and diabetes, in asymptomatic individuals
with carotid stenosis, and in atherosclerotic lesions in rabbits
and humans (reviewed in Reference 222). One hypothesis is
that autoimmune disease results from cross-reactivity of
immunogenic peptides, which are derived from bacterial and
mitochondrial chaperones and Hsp60 (chaperonin) and are
recognized by activated g/d T lymphocytes.223,224 Humoral
immune responses and seropositive markers against the
Hsp70 family (especially ER Grp78 and Hsc70) of the
protozoan parasite Trypanosoma cruzi have been implicated
in the pathogenesis of Chagas’ disease,224a the most common
cause of congestive heart failure in Latin America (see
Table). Although these correlative studies have inherent
limitations, future research to establish causality could open
avenues to develop vaccines or other novel therapies for
treatment and prevention.
Potential Therapeutic Applications of
Molecular Chaperones
Strategies that could increase the rate of physiological recovery after postinfarction stunning and ventricular dysfunction
remain important goals in the management of patients with
acute myocardial infarction. Because maneuvers using either
July 27, 1998
127
tissue or whole body hyperthermia are cumbersome and
impractical in conscious humans, pharmacological strategies
that increase stress protein expression for isothermal protection have potential merit against ischemic damage to the
heart, kidney, and brain. Proteasome inhibitors, which transiently elevate the level of unfolded proteins inside cells, is
one potential approach.225 Alternative approaches may involve the development of small molecules and peptides226,226a
that mimic the in vivo actions of chaperones with therapeutic
benefits.
Perspectives
The role of stress proteins in cardioprotection has been
acknowledged as one of the most important future directions
of research in ischemic heart disease.227 Since the number of
affected individuals is so large, a therapeutic intervention that
contributes to a small change in post–myocardial infarction
morbidity and mortality, for example, can have dramatic
effects on overall clinical outcomes. Opportunities to address
the physiological roles of cytoprotective chaperones in cardiac diseases need to be expanded to include their likely roles
during chronic conditions (atherosclerotic, hypertension, diabetes, genetic disorders, and valvular heart disease) that
converge through common pathways, resulting in heart failure and sudden death. Strategic alliances among research
teams could forge new directions and accelerate progress in
this promising area, which, ultimately, could succeed in
exploiting endogenous pathways to enhance physiological
health and to reduce physiological attrition associated with
cardiovascular diseases.
Acknowledgments
Dr Benjamin was supported by the American Foundation for
Medical Research, the National Institutes of Health, and an Established Investigator Award from the American Heart Association. We
thank R. S. Williams, R. Meidell, A. Davis, P. Thomas, and R. Sohal
for their critical comments regarding our manuscript.
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Stress (Heat Shock) Proteins: Molecular Chaperones in Cardiovascular Biology and
Disease
Ivor J. Benjamin and D. Randy McMillan
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Circ Res. 1998;83:117-132
doi: 10.1161/01.RES.83.2.117
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