I. INTRODUCTION

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Since the first report (338) on the heat-induced appearance of chromosomal puffings in salivary gland tissue of Drosophila busckii in 1962, a new research domain has been intensively explored. This research resulted in the discovery of a large number of related proteins and their physiological role in many prokaryotic and eukaryotic organisms, tissues, and individual cells and at the level of subcellular structures. These proteins were originally called "heat shock proteins" (356), because they were discovered in salivary glands and other tissues of Drosophila melanogaster recovering from a so-called transient sublethal heat shock, during which body temperature was increased ~5°C above normal body core temperature (400). Such a mild heat shock elicited a heat shock response, characterized by the synthesis of new heat shock proteins normally almost absent in tissues of adult animals and by an increased synthesis of constitutively present or cognate heat shock proteins. This event was followed by a transient increased tolerance to high, normally lethal temperatures (thermotolerance). Later it was found that not only the tolerance to enhanced temperature increases, but also the resistance toward other events like hypoxia, ischemia, inflammation, and exposure to such cellular toxins as heavy metals, endotoxins, and reactive oxygen species (cross-tolerance), all imposing serious stress upon tissues and their composing cells.

The increased resistance toward stressful events has been described in a great variety of organisms, organs, and tissues, including the heart. During the last years, more and more information has become available on the specific role of individual heat shock proteins and on the mechanisms of transient activation of gene transcription, leading to the enhanced cardiac synthesis of these proteins. A better insight has also been acquired in such subcellular targets as molecules or organelles that can be protected by heat shock proteins. Now the phase is reached that upregulation of heat shock protein synthesis is considered as a powerful physiological, endogenous route for protecting crucial cellular homeostatic mechanisms against disturbing external factors. Therefore, it is propagated that they provide a new therapeutic tool to protect the human heart during and after transient ischemic attacks.

Over the last 5 years comprehensive reviews have been published on the chaperone activity of heat shock proteins (34, 150, 248, 283, 430) and on their role in specific tissues and pathological processes (34, 94, 104, 200, 281, 340, 445). The role of such individual heat shock proteins as the 70-kDa heat shock protein, small heat shock proteins and the heat shock factors has also been highlighted (293, 334, 360, 395, 439).

This survey concentrates on the transient protection of the cardiovascular system in various mammalian species when heat shock protein synthesis is upregulated. The present state of the art regarding the stimuli, leading to the heat shock response, the transduction pathways involved in the activation transcription of the various heat shock genes, and the cellular structures protected by heat shock proteins is presented. Such new experimental tools as transgenic animals and transfection techniques to enhance or inhibit the transcription of heat shock genes are discussed. Although the physiological role of heat shock proteins and their protective potential under pathophysiological circumstances have been studied in detail in animal models, knowledge of the role and effects of these proteins in humans is still limited. Where possible the relevance of the observations for the human situation is discussed.

	II. NOMENCLATURE AND FAMILIES OF HEAT SHOCK PROTEINS

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The applied nomenclature is primarily derived from the trigger leading to the synthesis of these proteins. Because heat shock was the first discovered trigger of the heat shock response leading to enhanced transcription of certain genes, the related products of this transcriptional activity have been called heat shock proteins. On the basis of the adopted nomenclature after the Cold Spring Harbor Meeting of 1996, throughout this survey heat shock genes will conventionally be designated as hsp-genes, while the related proteins are called Hsps (150). Accordingly, proteins the synthesis of which was increased upon glucose starvation are called glucose-regulated proteins (Grps). For the majority of these genes and proteins the name is associated with a molecular mass indication like for instance hsp27 and Hsp27, respectively. Some proteins that were first discovered in a nonrelated domain carry a particular name like the major structural ocular lens protein B-crystallin or those proteins targeting abnormal proteins for degradation, i.e., the ubiquitins. The name of some Hsps is often preceded by an indication of the compartment in which they reside, like mitochondrial mt-Hsp75. Furthermore, distinction has been made between the proteins almost absent under nonstressed conditions but synthesized immediately after cellular stress, and the proteins that are constitutively synthesized in the tissue. The first are called inducible proteins, while the second class is known as cognate proteins, like for instance Hsc70. This distinction may be arbitrary because in various cell types low but measurable concentrations have been found of so-called inducible Hsps, while on the other hand the synthesis of some cognate proteins is also increased upon stress.

Classification of various Hsps in families is based on their related function and size, which can vary from 10 to 170 kDa. Family names are conventionally written in capitals (150). The HSP70 family can stand for a typical example. The proteins of this family range in weight between 70 and 78 kDa (194). All HSP70 family members bind ATP (284). Constitutive or cognate members are Hsc70, Hsp75, and Grp75 (see below), while the inducible member is Hsp72, commonly called Hsp70.

The nomenclature and classification for related Hsps in prokaryotic and eukaryotic organisms is different, although the sequence of nucleic acids in genes and amino acids in proteins is often highly identical. Various hsp-genes are well conserved during evolution. For instance, among eukaryotic organisms the identity of the nucleotide sequence of the hsp70-gene varies from 60 to 78% (194). Compared with the human hsp70-gene, the Drosophila hsp70-gene and the prokaryotic Escherichia coli dnaK-gene reach a 72 and 50% identity, respectively (168).

In the spectrum of Hsps, the so-called Grps form a special group. As mentioned earlier, the synthesis of these proteins increases when extracellular glucose concentrations are low. Other triggers, however, can also lead to enhanced Grp synthesis, like depletion of intracellular calcium stores or inhibition of protein glycosylation. Grps reside in various HSP families and comprise Grp58, Grp78, Grp94, and Grp170. They are all localized in the endoplasmic reticulum.

In Table 1, only the representative eukaryotic Hsps are presented. The functional characterization of the members in each family is indicated in the last column. Members of the various families do have a specific localization within the unstressed cell, which is indicated in the fourth column. Upon cell stress, however, they can be translocated to other cellular compartments. Inducible Hsps are rapidly synthesized after the onset of the stress and can have a time-dependent, and thus dynamic, specific intracellular localization.

According to the animal species in which they have been discovered, Hsps may have different names, although their nucleotide and amino acid sequence can be highly identical. As an example, in Table 2, the various genes of the HSP70 family for mouse, rat, and human tissues as well as the chromosomal locus symbol in humans are presented. The nature of their expression is also indicated (after Refs. 97, 395). The same accounts for Hsp27. Throughout this survey only the terms Hsp27 and Hsp70 will be used to represent the family member.

	III. TRANSCRIPTION OF HEAT SHOCK PROTEIN GENES

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A variety of physical and chemical factors evoking environmental stress enhance the synthesis of Hsps in cells of various tissues, including those in the vascular wall and the heart. Such a process is initiated by the stimulation of membrane-bound receptors, changes in physical properties of the cell membrane, or in such intracellular changes as temperature and partial oxygen pressure. At present, numerous stimuli are know that lead to enhanced expression or activation of Hsps in cells belonging to the cardiovascular system. These triggers are summarized in Table 3.

Not all stimuli are evenly potent in the induction of expression of heat shock genes. For instance, heat shock is a more potent activator of hsp70-gene transcription than hypoxia (279). Otherwise, some of these stimuli have an additional effect when applied in combination. For instance, in intact rats in various tissues heat shock combined with intraperitoneally administered aspirin results in a significantly higher synthesis of such inducible Hsps as Hsp70 (117).

Figure 1 shows the presently known triggers leading to the activation of the transcription of hsp-genes in the eukaryotic cell. Despite the fact that the transduction pathways involved in the activation of the transcription of hsp-genes have been investigated intensively, for some distinct members of the various families the mechanisms underlying these pathways are still matter of debate.

View larger version (49K): [in this window] [in a new window]   Fig. 1. Extracellular and intracellular stresses that can lead to the activation of the heat shock factor (HSF) and subsequent heat shock protein (hsp)-gene transcription. ROS, reactive oxygen species; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PKC, protein kinase C; Hsp~HSF, binding between Hsp molecules and the HSF. Dotted lines indicate that the transduction pathway is not completely known.

In many organisms the temperature at which they live and the rate of change of this temperature determine the set point and the intensity of the heat shock response, respectively. For instance, it is obvious that the temperature set point for the heat shock response in arctic fishes is totally different from that in bacteria living in hot springs. Furthermore, slow increases in temperature activate hsp-gene transcription less than abrupt hyperthermia.

Although for such stimuli as angiotensin II and phenylephrine membrane receptors have been identified, no conclusions have been reached regarding the nature of the "primary sensor" for abnormal high temperatures in the cell membrane or within the cell. In yeast it has been suggested that the physiological response to changes in environmental temperature is dependent on the lipid composition of the plasmalemma. In yeast cells with different membrane fatty acid composition, more specifically with different ratios of saturated to unsaturated fatty acids, the threshold temperature of transcription of the hsp70-gene was found to be significantly different (54).

In contrast to the lack of understanding of the nature of the primary sensor at the level of the cell membrane, more information is available on the intracellular signaling cascade ultimately leading to the activation of hsp-gene transcription and subsequent protein synthesis. In eukaryotic cells this transcription is effectuated via a transcription factor known as the heat shock factor (HSF; see below). Because the HSF is inactive under nonstressed conditions, binding to DNA occurs only upon stress, implying that the HSF is negatively regulated (290).

Several factors can lead to HSF activation (see Fig. 1). The central process in HSF activation is the equilibrium between the binding of such free Hsp molecules as Hsp70 to the HSF and to stress-mediated unfolding proteins. Any increase in the presence of unfolding proteins shifts this equilibrium to the Hsp-unfolded proteins side, thereby releasing free HSF monomers, which can be activated subsequently. This hypothesis has been forwarded by several authors (2, 291), but contested by others (332). More recently it was found that not only Hsp70 binds to the HSF, but also Hsp90. Hsp90 also acts as a repressor protein of HSF (see below) (6, 455).

Second, HSF can be activated indirectly by some stressors that activate phospholipase C, thereby enhancing intracellular concentrations of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, associated with subsequent activation of protein kinase C (PKC) (111). The fact that PKC-activating drugs like phorbol 12-myristate 13-acetate (PMA) provoke Hsp70 synthesis (111) and that the PKC inhibitor chelerythrine chloride reduces the heat shock-elicited Hsp70 synthesis (446) underlines the central role of PKC in the activation of hsp70-gene transcription.

On the basis of results obtained in cultured neonatal cardiomyocytes, it has been argued that tyrosine kinases are also involved in the signaling pathway of hsp-gene transcription. In these cultures, herbimycin-A, a benzoquinoid ansamycin antibiotic and reputed tyrosine kinase inhibitor, provokes Hsp70 synthesis and increased resistance against a subsequent lethal heat stress (292). This drug, however, does not activate the transcription of such other genes as hsp90, hsp60, hsp27, and grp78. Moreover, another tyrosine kinase inhibitor, genistein, had no effect on hsp70-gene transcription at all. Thus herbimycin-A induces Hsp70 transcription via a tyrosine kinase-independent mechanism, because an even more specific tyrosine kinase inhibitor, tyrphostin 23, also does not affect hsp70-gene transcription (111).

Whether changes in intracellular calcium concentration are involved in the activation of hsp-gene transcription is incompletely understood. In cardiac tissue a transient increase of diastolic intracellular free calcium was found after heat shock (52, 249, 417), increased wall stretch, and -adrenoreceptor stimulation. All three stresses led to enhanced hsp-gene transcription. Detailed observations during heat shock revealed that the initial rise of the intracellular calcium concentration is due to ion release from intracellular stores, presumably from the endoplasmic reticulum. The rise in calcium concentration is preceded by a rapid release of IP₃, occurring within the first minute after imposition of the heat shock (52). Subsequently, the ion concentration is further increased by a secondary calcium influx from the extracellular space. An interesting observation was made by Löw-Friedrich and Schoeppe (244), who found that a CdCl₂-elicited enhanced synthesis of Hsp70 in isolated cardiomyocytes was reduced when the cells were preincubated with calcium antagonists, like nifedipine, diltiazem, or verapamil. The interpretation of these results, however, is complex. Calcium antagonists could interfere directly with the toxic effects of CdCl₂, such as blocking its entry into the cell or reducing the CdCl₂-elicited calcium release from mitochondria. On the other hand, it cannot be excluded that a long-term incubation with high doses of calcium antagonists has a direct blocking effect on Hsp70 synthesis through its inhibitory effect on protein synthesis.

The mechanism of activation of such small Hsps (sHsps) as Hsp27 and B-crystallin is likely different from that of the inducible hsp70-gene (16, 360). Because they are constitutively present in cardiac tissue in appreciable amounts (174), activation occurs mainly through phosphorylation. This process takes place very rapidly in response to heat shock, growth factors, phorbol esters, calcium ionophores, interleukin-1 (IL-1), and tumor necrosis factor- (TNF-) (16, 428). With regard to TNF--elicited activation of Hsp27, it has been shown that the mitogen-activated protein kinase (MAPK) pathway is involved via the p38 MAP kinase cascade and the MAPKAP kinases 2 and 3 (110, 170). PKC is likely not involved in the activation of sHsps, because TNF--elicited phosphorylation of Hsp27 is not inhibited by such kinase inhibitors as staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide (H-8), N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), or chelerythrine chloride (418).

Ultimately, the transcription of several hsp-genes is governed by the so-called HSFs, which are constitutively synthesized cellular transcription factors. At present, four different HSFs have been identified, i.e., HSF1 (330), HSF2 (357), HSF3 (300), and HSF4 (302). HSFs are products of the transcription of four different genes. HSF1, HSF2, and HSF4 have been identified in human tissues. At present, HSF3 has only been described in the chicken, in which it is involved in the development of various tissues (300, 388).

All four factors are thought to be activated differently by various forms of cellular stress (300, 330). HSF1 activation is extremely sensitive to temperature changes, whereas HSF2 is not (74, 95). HSF1 is primarily involved in the stress response, while HSF2 and HSF3 regulate the transcription of hsp-genes in specific tissues, undergoing processes of differentiation and development. HSF2 (72 kDa) is present in two isoforms, i.e., HSF2- and HSF2-. The two isoforms are products of alternative splicing of HSF2 pre-mRNA. HSF2- is predominantly found in the testis (352), whereas HSF2- is synthesized in heart and brain. The HSF2- form is transcriptionally more active than the HSF2- form (128). Interestingly, HSF2 operates additionally to HSF1. When both heat shock factors are activated by hemin treatment and heat shock, respectively, transcription of the hsp70-gene is more potent than after activation of HSF1 alone (363).

Evidence is accumulating that the latest discovered HSF, i.e., HSF4, is structurally related to the other three HSFs (302). This particular protein, however, is functionally distinct from the three others since it does not respond to stresses like heat. The precise role of HSF4 is still unknown, aside from its repressing effect of hsp-gene activation. Indeed, the transcription of the hsp70-, hsp90-, and hsp27-genes is reduced when HSF4 is overexpressed. Nakai et al. (302) suggested that HSF4 is not a transcription factor because it lacks the carboxy-terminal repeat shared by HSF1, HSF2, and HSF3 (302).

HSF1 is the major stress responsive HSF mediating the heat shock response in mammalian cells (330). The mechanism of action and its regulation and activation have been extensively reviewed by Wu (439). HSF1 is a 75-kDa protein that is constitutively synthesized and localized in the cellular cytosol and nucleus and often associated as a group of inactive monomers in a complex of ~200 kDa (21). In addition, in the resting state, the HSF1 monomers seem to be bound by Hsp70 and Hsp90, the latter being only liberated upon activation by kinases (6, 455).

Upon activation, HSF1 shifts from the cytosolic to the nuclear compartment and becomes organized in an active, DNA-binding homotrimer of ~700 kDa (129, 439). Trimerization is established by arrays of -helical residues (leucine zippers) in the amino-terminal domain of the protein and is negatively regulated by another leucine zipper domain at the carboxy-terminal site (331, 439). Crystal structure analysis has revealed that the DNA-binding domain of HSF1 contains a bundle of three helices capped by a four-strand antiparallel -sheet, forming a globular domain of ~90 residues (136). HSF1-DNA binding also depends on the presence of the so-called heat shock elements (HSEs) in the upstream promoter site of the hsp-genes. These HSEs are multiple and have variable numbers of alternating oriented arrays of 5'-nGAAn-3' (n stands for less conserved nucleotides) (33, 166, 211, 233, 236). The binding of the HSF homotrimers to HSE requires at least two nGAAn units, arranged head-to-head or tail-to-tail (441). Upon binding, the synthesis of new mRNA molecules encoded by related hsp-gene can be initiated.

The HSF1 activity is controlled by multiple regulatory mechanisms, including suppression by Hsp70 and Hsp90 and activation by different cellular signaling cascades (199). Interestingly, HSF1 can be activated into an intermediate state in which it binds to HSE sequences without stimulating gene transcription. For this ultimate process, HSF1 hyperphosphorylation seems to be indispensable (351, 440). Furthermore, trimerization and phosphorylation seem to be far more important for activation of hsp-gene transcription than the absolute cellular content of HSF1 (96).

Many stresses aside from heat activate HSF1. In cultured cells it was found that hypoxia, ethanol, and sodium arsenite increase HSF1-DNA binding and Hsp70 levels (33, 287). The prostaglandins A₁ and A₂ (PGA₁ and PGA₂, respectively) also enhance HSF1-mediated transcription of hsp70- and hsp90-genes (8, 9). PGA₁ inhibits nuclear factor-B (NF-B) by blocking phosphorylation and subsequent degradation of the NF-B inhibitor protein IB-. This inhibition of NF-B is associated with HSF1 activation (342). The hsp70-gene transcription through PGA₂ only occurs in proliferating but not in confluent cell layers in culture (156, 350) and seems to be cell type dependent. Our group found that hsp70-gene transcription is also stimulated by PGA₁ and PGA₂ in cultured myogenic cells and adult cardiomyocytes, although this expression does not lead to protection against a severe stress (unpublished data). Hyposmotic stress also elicits HSF1-DNA binding, which, however, does not result in hsp70-gene transcription (163). Because salicylates and arachidonic acid also activate HSF1 (178, 186-188), it is tempting to speculate that inflammation and degradation of cellular membranes, resulting in the release of arachidonic acid from membrane phospholipids, lead to enhanced Hsp concentrations in the cell. However, to the best of our knowledge, no enhanced Hsp levels have been found upon treatment with these compounds.

The role of the precise intracellular concentration of individual Hsps on the stress-mediated activation of HSF1 has been investigated by artificially reducing the levels of such Hsps as Hsp70 and Hsp90. Only Hsp90 reduction resulted in HSF1 activation pointing to its negative regulation by this particular Hsp. Heating leads to the dissociation of the constitutive heterocomplexes between HSF1 and Hsp90, because stress-denatured proteins compete for Hsp90 (6, 455). Inversely, by artificially reducing concentrations of nascent polypeptides, the HSF1-elicited hsp70-gene transcription was inhibited, indicating that cells are capable of registration of concentrations of not yet folded nascent proteins or stress-denatured proteins (22).

More recently, investigations have been performed to elucidate the mechanism of HSF1 deactivation, which seems to be regulated by multiple factors. First of all, increasing levels of inducible Hsps repress HSF1 activation in an inverse way, pointing to an autoregulatory mechanism for HSF1 activity (138, 236, 295). Furthermore, phosphorylation has both positive and negative effects on the activity of HSF1 (64, 199, 295). Administration of okadaic acid, a specific and potent inhibitor of serine/threonine phosphatases 2A and 1 to cultured cells, leads to inhibition of HSF1 dissociation and to prolonged activation of hsp70-gene transcription (57, 440). Inversely, blocking the serine/threonine kinase activity inhibits HSF1 phosphorylation and hsp70-gene transcription (440). Second, it was found that HSF1 activity can also be inhibited through phosphorylation of HSF1 serine residues (155), evoked by MAPKs (64). Third, it has been shown that the nuclear heat shock factor binding protein 1 (HSBP1) interacts with the active homotrimeric state of HSF1 during heat shock. More specifically, HSBP1 negatively affects the HSF1-DNA coupling by binding to the HSF1 heptad repeat. Overexpression of HSBP1 blocks the HSF1 activation (354).

Only recently, the HSE in the promoter of hsp-genes was found to be part of a so-called "composite response element" (CRE), consisting of various elements and integrating multiple regulatory signals. For instance, HSEs form CREs with promoter binding elements for two transcription factors, i.e., nuclear factor IL-6 (NF-IL-6) and the signal transducer and activator of transcription-3 (STAT-3). Activation of the STAT-3 transcription factor through the IL-6 receptor attenuates HSF1 activity, while NF-IL-6 consolidates its activity (375). Recently it has been found that interferon- (IFN-)-mediated activation of another transcription factor, i.e., STAT-1, also synergistically activates HSF1 (376).

	IV. FUNCTIONS OF HEAT SHOCK PROTEINS

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Most likely, the primary physiological function of Hsps is to fulfill chaperoning activity (28). Molecular chaperones have been defined as a nonrelated class of proteins that mediate the correct folding of other proteins, but do not take part in the final assembly of new structures (109). Although every newly synthesized protein contains within its amino acid sequence the necessary information for ultimate correct folding, this process can be hampered by several factors. For instance, during their synthesis, incomplete amino acid sequences may already associate with other unfinished parts of peptide chains or with totally completed peptides. For correct association and/or folding the amino acid chains need to be entirely synthesized and therefore have to be kept in the unfolded monomer state (348). Second, proteins that need to be translocated to other cellular compartments should also be kept in an unfolded or semi-folded state to pass intracellular membranes. Such physical state can be achieved through binding to chaperone proteins.

Excellent reviews have been written on the chaperone function of Hsps in general (109, 123-125, 137, 145, 343, 379) and on Hsp70 in particular (194). The chaperone role of Hsps in the cardiovascular system has recently been reviewed by Benjamin and McMillan (34).

Chaperonins consist of a class of Hsps that assist in correct protein assembly at a later stage than the chaperones. This process occurs when completed protein chains are released from the ribosomes or are transported to such cell organelles as mitochondria. In eukaryotic cells, a distinction is made between two groups of chaperonins. Group I consists of Hsp60 and Hsp10, both residing in the mitochondria. Group II comprises the TCP1 T-complex polypeptide (TCP1) subfamily, the members of which can be found in the cytosol (107, 108). TCP1 is, among others, involved in the folding of actin and tubulin and thus indispensible for proper functioning of the cytoskeleton (341).

Hsp70 and Hsc70 act as single entities to exert their chaperone function. A peptide-binding site and enzymatic catalytic site characterize both protein chains. The peptide-binding domain resides in the vicinity of the evolutionary less-conserved carboxy-terminal domain (231), while the catalytic site is situated near the highly conserved amino-terminal domain of the protein (size 44 kDa). The latter site carries the ATPase activity (58, 227, 229), necessary for binding to and release from other protein chains (346). Furthermore, both proteins carry nuclear localization signals (NLS). In the human Hsp70 gene sequence, originally described by Hunt and Morimoto (168), a NLS sequence was presumed to be located in the 246-262 amino acid (AA) region. Coupling of protein kinase to this region indeed led to protein kinase transfer into nucleus and nucleolus (87). However, various sites in the Hsp70 protein can probably affect its nuclear targeting. Milarski and Morimoto (284) showed that an extensive deletion of another region (AA 351-414) inhibits nuclear Hsp70 localization. This result is difficult to interpret because the synthesized protein also lost other physiological properties, like binding of proteins and ATP. In contrast, site-directed mutations in the 246-251 AA sequence confirmed that this region is necessary for nuclear import, but not for viability of the cell after severe stress (201). Furthermore, by transfection of cells with mutants for the tyrosine-524 location, it was found that phosphorylation of this particular amino acid is necessary for both nuclear import and increased resistance against severe stress (205).

Hsc70 also carries a prototype basic NLS in the 246-262 AA region. Partial deletion of this region, however, does not affect Hsc70-mediated nuclear protein import, suggesting that another nonclassical NLS is present in the protein sequence. Lamian et al. (217) found that such an alternative signal is located in the amino terminus of Hsc70.

Under normal, nonstressed conditions Hsp70 chaperones a variety of processes in the cytosol. The protein stabilizes unfolded nascent precursor peptides (30, 137) and keeps them unfolded until they reach the final cellular compartment (18). More specifically, Hsp70 has been found to associate with cytoskeletal proteins (208, 348, 402), cell surface glycoproteins (165), calmodulin (66, 377), and saturated long-chain fatty acids, like palmitate, stearate, and myristate. Several functional explanations have been proposed to explain the interaction with fatty acids. Although Hsp70 could act as intracellular fatty acid transport carrier, it cannot be excluded that fatty acids are required for the proper protein folding by Hsp70 (131). The chaperone also assists in the translocation process across the membranes of the endoplasmic reticulum and mitochondrion (18, 75, 127, 158, 189, 407). Furthermore, Hsp70 guides misfolded proteins to lysosomes (3, 61) or the outer membrane of peroxisomes (424).

Figure 2 presents a model for the cellular chaperone function of Hsc70. Aside from Hsc70, Hsp40 can bind unfolded proteins and couple them to Hsc70 or binds directly to the already formed Hsc70-unfolded protein complex (119). In an ATP hydrolysis-dependent process, Hsc70 folds and releases the newly formed protein. According to the type of the folding process, the complex can have a different fate. Hsp40 is replaced by other proteins such as Hsc70-interacting protein (Hip), the Hsc70-organizing protein (Hop), or the Hsc70-accessory protein (Hap), leading to different interactions of the Hsc70-unfolded protein complex. Coupling to Hip conducts transport of the complex throughout the cell, to Hop the linkage to Hsp90, while binding to Hap stimulates the dissociation of Hsc70 and the unfolded protein (59, 154, 326). The dissociation of Hsc70 from newly folded or refolded proteins occurs after the conversion of ADP to ATP at the surface of the Hsc70 protein. This implies that the Hsc70 protein binding becomes more stable in ischemic/hypoxic periods when cellular ATP concentrations are relatively low (see Fig. 1) (29, 30).

View larger version (25K): [in this window] [in a new window]   Fig. 2. Scheme of interactions between the chaperones Hsc70 and native peptide chains or unfolding proteins. Binding to the chaperone protein and other associated proteins (not shown, see text) determines the fate of the folded protein. Grp78, glucose-regulated protein 78; mt-Hsp75, mitochondrial 75-kDa heat shock protein.

Hsp90, one of the most abundant cytosolic Hsps, binds steroid receptors, protein kinases, intermediate filaments, microtubules, and actin microfilaments in a very specific manner (208). Hsp90 is an essential component of the glucocorticoid receptor, assembled in a complex of several proteins (14, 327). Without the association to Hsp90, intracellular hormone receptors were found to be inactive. Via its interaction with filamentous actin in the cytosol, Hsp90 is thought to target steroid receptors to the nucleus and various kinases to their site of action (86, 327).

It is generally accepted that Hsp90 possesses ATPase activity and can be autophosphorylated on serine and threonine residues (299). It has been found that immobilized Hsp90 molecules can be dissociated from actin by ATP (193).

The 60-kDa chaperonin TCP1 can be seen as the cytosolic counterpart of the mitochondrial Hsp60 chaperonin. It forms a large (970 kDa) hetero-oligomeric protein complex called TriC (TCP1 ring complex), containing TCP1 and several other proteins, among which Hsp70 (122, 145, 228).

Hsp27 is very active in assisting the assembly of macroglobular protein complexes, such as F-actin polymerization. This function, however, is highly dependent on the phosphorylation state and monomeric or multimeric state of Hsp27. In the nonphosphorylated, monomeric state, Hsp27 inhibits F-actin polymerization via specific binding to the plus-end of the filaments. In the phosphorylated, multimeric state it does not and thus promotes the polymerization process (37). The other sHsp, B-crystallin, is more involved in protecting actin and desmin filaments under acidic conditions (36). By binding to the Z-line in the sarcomeres of cardiomyocytes, the protein prevents aggregation of the actin filaments (36, 242, 243).

Protein chaperoning also occurs within the intracellular compartments. To reach or to remain in these compartments, Hsps are equipped with organelle-targeting sequences. For instance, both Grp78 and Hsp75 possess an amino-terminal extension to reach the endoplasmic reticulum and mitochondrion, respectively (379). In addition, Grp78 contains a carboxy-terminal sequence for retention in the endoplasmic reticulum (132).

In mitochondria, mt-Hsp75 keeps mitochondrially encoded proteins in an assembly competent state (146). Cytosolic Hsc70 ensures efficient presentation of proteins to be transported across the mitochondrial membranes (396). Furthermore, in this organelle the chaperonin Hsp60 prevents aggregation of misfolded proteins and together with Hsp10 aids in the refolding of mitochondrial proteins (252). This process occurs in the so-called Anfinsen or folding cage. This cage consists of two stacked rings, each of them containing seven Hsp60 molecules. A protein chain can be bound inside these stacks, which is then closed on the upper side by one stack of Hsp60 molecules. When ATP is bound to the cage, the protein chain is released within the cage and allowed to fold. The folded chain is released after unbinding of the Hsp10 complex from the cage. The whole process is finished in the seconds time order (107).

In lysosomes, Hsc70 assists in protein degradation by transferring them into this organelle (3, 397). Furthermore, Hsc70 is associated with the cellular centrosome and is required for repair of this organelle after damage (48), probably in collaboration with accessory proteins like p16, a member of the Nm23/nucleoside diphosphate (NDP) kinase family (224).

Compared with the nonstressed situation, much less is known about the various Hsps that exert their chaperone function during and after stress. This lack of insight is in part due to the fact that under these circumstances general protein synthesis is completely disturbed and new Hsps appear in various cellular compartments. In addition, many constitutive members redistribute within the cell as newly synthesized inducible Hsps translocate to other cell compartments immediately upon synthesis at the ribosomal site. The role of stress-mediated translocation of Hsps is poorly understood but seems to be pivotal in the protection by these proteins (201, 284). Probably best studied is the stress-mediated translocation of both Hsc70 and Hsp70 into the cellular nucleus, in particular to the nucleolus (403, 432). Upon heat shock, translocation occurs within 60 min and terminates ~3 h later, the time at which the highest content of these proteins is reached (308).

The cellular stress response, especially as a consequence of heating, has been extensively studied in the past 20 years, and excellently reviewed in 1992 by Welch (429). It is well known that heat shock negatively affects the organization of several functional structures in the cell. The Golgi apparatus is disrupted and fragmented. Mitochondria swell and cristae packing changes. Striking alterations occur in the cytoskeleton, more specifically in intermediate filaments. The microtubular network is not affected, but intermediate filaments aggregate to form a tight perinuclear network. This phenomenon, however, is reversible since normal distribution is gradually reappearing after the stress is relieved (435). Within the nucleus, rod-shaped bodies occur consisting of actin filaments. Nucleoli look less condensed, the number and size of the granular ribonucleoprotein components are changed, and the nuclear fibrillar reticulum is reorganized (433). Upon stress the most prominent Hsps present in the nucleolus are the inducible Hsp70 and Hsp110 (433).

The fact that some Hsps translocate to the nucleolus suggests a specific and unique role in the repair and protection of these cellular structures (67). The immediate translocation of newly synthesized Hsp70 into the cell nucleus and nucleolus occurs in various cell types and tissues, including the heart (49, 65, 313, 365, 383, 415, 432). Nuclear Hsp70 accumulation occurs in close vicinity of the preribosomal-containing granular region (434). Normally soluble proteins such as topoisomerases I and II and DNA polymerases  and  become more tightly bound after heat shock and coisolate with the nuclear matrix (45). In such precipitates large amounts of Hsp70 can be found, suggesting binding to matrix-precipitated proteins (65, 412, 427). Recently, the consequence of inhibition of translocation of Hsp70 to the nucleolus has been investigated in more detail. Knowlton (201) mutated the Hsp70 nuclear targeting sequence and investigated nucleolar translocation and cellular viability after heat shock. A relatively small mutation of only six amino acids mitigated nuclear Hsp70 accumulation, but surprisingly did not affect the protection of cellular viability following a severe heat shock. Only when a second mutation was induced, leading to loss of Hsp70 ATPase activity, protection of viability was completely lost. Unfortunately, no definite conclusions can be drawn concerning interactions of both protein regions, because viability was not tested in cells transfected with only mutation of the ATP-binding region. These results suggest that nuclear Hsp70 translocation is not the sole factor responsible for proper cellular protection. Hsps, like Hsc70 (436) and the sHsps (see below), that redistribute to the nucleus have also been considered to play a role in the protection of nucleolar protein structures (65, 412, 427). Hsc70 was found to associate with topoisomerase I and refolded this nuclear protein upon cessation of heat stress. For proper refolding, however, Hsc70 was likely assisted by other proteins, because in vitro refolding with purified Hsc70 alone were not successful (65, 224).

Upon recovery after heat shock, both Hsp70 and Hsc70 exit the nucleolus to accumulate back in the cytoplasm, more specifically in the perinuclear region, along the perimeter of the cell, and in association with large cytosolic phase-dense structures (432, 434). Perinuclear condensation of Hsp70 seems to coincide with reassembly of the centrosome and microtubuli, and also with the cytoplasmic distribution of ribosomes. This suggests that Hsp70 plays a crucial role in the function of these organelles immediately after heat shock and during the subsequent recovery phase (48, 434).

A particular role in the protection of cells against various stresses is played by the constitutively present sHsps, i.e., Hsp27 and B-crystallin. Both proteins are phosphorylated upon heat stress and associate with structural proteins in sarcomeres, cytoskeleton, and nucleus (17, 196, 410). In sarcomeres, Hsp27 colocalizes with actin in the I band as shown in isolated hyperthermically perfused rat hearts and cultured cardiomyocytes (153). It has been suggested that association with Hsp27 increases the resistance against oxidative stress-induced actin fragmentation and cell death (171). In rat hearts after ischemia, Barbato et al. (24) found that the other sHsp, i.e., B-crystallin, bound to the cytosolic proteins troponin T and troponin I. The trigger for this binding seems to be the evolving acidosis in the ischemic heart. This interaction may affect postischemic myocardial contractility (24). It cannot be excluded that B-crystallin also binds to the actin and desmin filaments and prevents their aggregation during and after an ischemic insult (36). These two sHsps can also govern refolding of other proteins. Under in vitro circumstances heat shock- or urea-mediated denaturation of citrate synthase and -glucosidase could be completely prevented by adding Hsp27 or B-crystallin (179).

As pointed out above, proteins that lose their normal three-dimensional conformation provoke Hsp synthesis through the activation of HSF1 (13). During and after heat shock, cytosolic proteins normally aggregate and have a reduced solubility (101, 416). Transient binding to unfolding proteins is caused by the high affinity of Hsps for hydrophobic sites, which are normally covered inside the protein core (101, 317, 319). For the dissociation of Hsp70 from unfolded proteins, ATP is indispensable, whereas high concentrations of ADP or inorganic phosphate slow down the rate of dissocation and increase the rate of Hsp70-protein complex formation and stability of this complex (318).

Although under nonstressed conditions Hsp90 already comprises 1-2% of the total protein content, its synthesis is further stimulated upon heat shock (215). Normally Hsp90 is diffusely distributed in both cytoplasm and nucleus. After heat shock a transient translocation of Hsp90 to the nucleus occurs, reaching a plateau after 15 h (5). In this case, Hsp90 binds to and stabilizes unfolding proteins (180). There are indications that Hsp90 is also involved in thermotolerance, because artificial reduction of the cellular Hsp90 content is associated with lower survival at high temperatures (23).

In samples of mouse liver and brain and in cultured cells it has been shown that Hsp110 is closely associated with the nucleolus under control circumstances. Interestingly, treatment of the cells with ribonuclease results in the loss of the Hsp110 from the nucleus, indicating binding of Hsp110 to RNA, either directly or indirectly via another protein or protein complex (382).

In conclusion, Hsp chaperoning is a permanent cellular event during both nonstressed and stressed conditions. The role of Hsc70, Hsp90, Hsp40, and the sHsps is relatively well understood during nonstressed conditions. However, upon heat shock or other stresses, upregulation of the synthesis and translocation of the various Hsps to other cellular compartments suggest that during evolution tissues developed intrinsic defense mechanisms for rescuing unfolding proteins in various cellular compartments. Further investigations are needed to elucidate the precise role of each of the involved Hsps during and after stress in the various cell organelles.

	V. HEAT SHOCK PROTEINS IN THE CARDIOVASCULAR SYSTEM

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To date, acute and chronic ischemic heart disease is one of the major causes of death among people in the Western world, despite numerous exogenous pharmacological protective measures like calcium antagonists, coronary vasodilators, and blocking agents of the angiotensin converting enzyme and -adrenoreceptors. Especially during ischemic disease, the heart could benefit from protective measures from an endogenous source. Upregulation of the synthesis of Hsps is one such phenomenon leading to improved tolerance to ischemia in experimental models. In humans, however, investigations regarding the protective potential are in their infancy.

In both the vascular and cardiac compartment, heat shock proteins are present and can be induced by specific stressors. The type of proteins expressed in the vascular compartment is somewhat different from that expressed in the heart. In nonstressed, adult mice such Hsps as Hsp27, Hsc70, Hsp70, and Hsp84 are constitutively expressed in a variety of tissues, including the heart (392). In the heart of this species these four Hsps are clearly present, but at low levels compared with other tissues. In contrast, in the adult rabbit, cardiac Hsp70 levels are similar to those in other tissues (253; see Table 4). In unstressed rats the heart contains relatively high B-crystallin levels, whereas intermediate levels are found for Hsp27 (Table 5). In both rat and human heart, Hsp27 can be found in endothelial cells, smooth muscle cells, and cardiomyocytes, whereas B-crystallin is only present in cardiomyocytes (247). Although specific investigations of the various HSFs in the heart are scarce, it has been shown that HSF1, HSF2, and HSF4 are present in cardiac tissue (128, 302).

View this table: [in this window] [in a new window]   Table 5. Hsp27 and B-crystallin concentrations in various rat organs at various time points after heat stress

In rabbits after whole body heat shock, cardiac Hsp70 concentrations are much higher than in neural tissues, but still lower than in other, nonneural tissues as liver and kidney (253). As shown in Table 4, in heat-shocked rats the Hsp70 content is significantly lower in heart and brain than in colon, liver, kidney, and spleen (27). In these tissues, stress-induced synthesis of new Hsps occurs very rapidly. Within minutes Hsp70 mRNA transcripts are present (84), whereas protein accumulation reaches its maximum at ~12 h after stress induction (70, 329). In the later time domain, the cardiac Hsp70 content slowly decreases but remains detectable up to 192 h after the initial stimulus (190). Immunohistologically, Hsp70 was found to be present in the nucleus of cardiomyocytes, in fibroblasts, and in endothelial cells in the coronary vessel wall within 3 h after stress induction (365). HSF1 was found to be activated upon an ischemic insult, leading to prominent mRNA signals coding for Hsp70 and Hsp90. Under these circumstances, however, HSF2 is not activated (309).

In the adult nonstressed heart, B-crystallin is constitutively abundant (198, 278) and comprises 1-3% of total soluble protein (192). The protein can be found in high concentrations in the conduction system (222). Inaguma et al. (174) compared the levels of B-crystallin and Hsp27 in various rat tissues, including the heart during the first 16 h after heat shock (Table 5). The striking finding was the great variability of the tissue Hsp concentrations and the differences between the B-crystallin and Hsp27 concentrations within the same tissue. In the heart, Hsp27 concentrations double during the first 16 h after heat shock, whereas B-crystallin increases by 20%.

Although constitutive expression of the various Hsps has been well documented in the adult heart, only limited information is available on the regulation of the constitutive expression and concentrations of Hsps in the embryonic, newborn, and developing heart. Only recently have expression patterns of Hsp70 and Hsc70 in the immature ovine myocardium during the perinatal transition phase and the juvenile phase been described. In the heart, the Hsp70 synthesis seems to be developmentally regulated in both left and right ventricles. In the fetal heart very low Hsp70 levels are found. These levels, however, increase upon development and peak after the first 2 wk after birth. In contrast, the Hsc70 protein contents remain unchanged during left ventricular development, whereas they decrease with age in the right ventricle (378).

B-Crystallin plays an exceptional role in normal cardiac development, activating genetic programs responsible for cardiac morphogenesis. As early as 8.5 days postconception, B-crystallin can be detected in the mouse heart and is uniformly distributed in atria and ventricles. In the endothelial cushion, pulmonary trunk, aorta, and endothelium, however, the protein seems to be absent (35).

Upon exposure to environmental stress, all cell types in the blood vessel wall respond with the synthesis of Hsps (11, 152, 364). Aside from heat (207), vascular Hsp synthesis is induced by such triggers as circulating hormones (288, 443), reactive oxygen species (ROS) (245), and sodium arsenite (426). Nitric oxide (NO) is probably involved in heat shock-mediated Hsp70 synthesis in blood vessels, because the NO synthase (NOS) inhibitor N-nitro-L-arginine (L-NNA) also inhibits Hsp70-gene transcription. The signal transduction pathway for the activation of Hsp70-gene expression through NO is still unclear, because either increased calcium influx or heat shock-mediated production of ROS could be involved, respectively activating the constitutive NOS and the inducible NOS pathway (251).

In the aorta, as in cardiomyocytes, two isozymes of Hsp32, i.e., heme-oxygenase-1 (HO-1) and HO-2 are constitutively synthesized (1, 114, 314). HO-1 is involved in the degradation of heme to biliverdin, iron, and carbon monoxide. In the aorta, aside from hypoxia and heat, HO-1-gene transcription can be activated rapidly by severe physical stress (112), hemin, hydrogen peroxide (H₂O₂), heavy metals (76, 113, 266), and postischemic myocardial reperfusion (263). Interestingly, enhanced HO-1 synthesis is associated with significant elevation of intracellular cGMP levels so that smooth muscle cell relaxation and concomitant vasodilation occur (63, 114). Postischemic HO-1 synthesis can be blocked by the addition of such scavenging enzymes as superoxide dismutase and catalase to the perfusate. Inversely, when enhanced concentrations of HO-1 are present in the vascular wall, free radical formation upon severe stress is reduced, pointing to its protective role against oxidative stress-mediated vascular cell damage.

In 1993, Amrani et al. (12) discovered that some essential functions in rat coronary artery endothelial cells are protected against ischemia and reperfusion after preceding whole body heat shock, associated with enhanced Hsp70 levels. In these hearts, endothelial cell-mediated vasodilation elicited through 5-hydroxytryptamine infusion is completely preserved during and after a 4-h lasting ischemic insult combined with intracoronary cardioplegia. The same authors found that protection of the endothelial cell-mediated vasodilation is completely abolished by blocking catalase activity through 3-amino-1,2,4-triazole (3-AT). Later these investigators proposed that the improved postischemic recovery of cardiac mechanical function depends exclusively on the protection by Hsp70 of the coronary endothelium because protection of postischemic cardiac function could be abolished by removing endothelial cells by saponin (11). It can be argued as to whether this hypothesis is valid, because saponin may be so damaging to the coronary vessels that this in itself alters the response to ischemia.

In the endothelium, heat shock-mediated Hsp27 phosphorylation is probably also involved in the protection of specific intracellular structures against environmental stresses. In these cells, metabolic inhibition through a combination of glucose depletion and rotenone addition leads to early breakdown of such cytoskeletal structures as F-actin. A preceding heat shock-mediated increased Hsp27 phosphorylation leads to an improved stability of cytoskeletal F-actin and a better preserved ATP concentration. When dephosphorylation of Hsp27 is prevented through addition of such phosphatase inhibitors as okadaic acid, cantharidin, or sodium orthovanadate, the metabolic block-associated degradation of cytoskeletal F-actin is also prevented, pointing to the stabilizing effect of this particular heat shock protein on cytoskeletal structures (241).

C.  Protection of the Heart by Hsps

1.  Protection of cultured cardiomyocytes and fibroblasts

To demonstrate the unique protective characteristics of Hsps on the cardiac tissue, numerous studies have been performed on its composing cells in culture. Several cell types have been investigated, from freshly isolated neonatal or adult cardiomyocytes to such cell lines as myogenic C₂C12 (33) and H9c2 cells (197) and fibroblasts (432, 434). Studies performed on isolated cells should be interpreted with caution, because factors like the unnatural cell environment, the number of cell passages, the degree of culture confluency, and the chemical composition of the bathing medium may influence the outcome of the studies. It is known for instance that extracellular matrix proteins, like collagen, attenuate both the constitutive and heat-induced expression of several Hsps in cultured cardiomyocytes (372).

With these shortcomings taken into account, cell culture models have been very helpful in unraveling the expression patterns and mechanism(s) of protection of Hsps. Several reports describe the existence of protection in cultured adult or neonatal cardiomyocytes against a severe, lethal stress challenge after previous activation of hsp-gene transcription. Activation of hsp70- and hsp90-gene transcription can be achieved by heat or metabolic stress (307) and of the hsp70- and HO-1-gene after exposure to the antioxidant compound ebselen (159). That hypoxia and heat stress pretreatment can lead to unexpected and opposite effects has been shown in primary cultures of neonatal cardiomyocytes (297). Hypoxia induced the synthesis of such Hsps as Hsp70, associated with a subsequent improved tolerance to lethal heat stress. In contrast, hyperthermic pretreatment leading to comparable increased Hsp70 levels is not protective against lethal heat stress. From such experiments it could be concluded that enhanced Hsp70 contents are not the sole factor explaining improved stress tolerance (315). This finding could be associated with the cell type used, because Benjamin et al. (33) showed that hypoxia and heat shock offer equipotential protection in myogenic C₂C12 cells. These investigators also showed that ATP depletion alone is sufficient to induce HSF1-DNA binding when oxidative metabolism is impaired by hypoxia (32).

Hsp-mediated protection against the bacteria-derived lipopolysaccharide (LPS) endotoxin has been investigated, among others, in the H9c2 cell. This cell line was isolated from embryonic cardiac tissue, and certain features of cardiac specificity were retained (147, 197). Experimental H9c2 results have been criticized, because of the strong H9c2 homology to skeletal muscle. However, because the potential to upregulate Hsp synthesis is retained (141, 280), they have been explored intensively for characterizing Hsp-mediated protection. Survival of H9c2 cells transfected with the Hsp70-gene is significantly better upon exposure to endotoxin. Endotoxin toxicity is mediated through NO synthesis and cytokines like TNF-, IL-1, and IL-6. Therefore, it is very well possible that Hsp70 blocks, in part, the deleterious effects of one of these compounds or their production (60). However, at present no conclusive evidence has been presented that this is indeed the case. Su et al. (380) used H9c2 cells to investigate the resistance against H₂O₂ after heat pretreatment. Resistance to mild H₂O₂ toxicity already appeared in an early phase, i.e., between 10 and 14 h after heating. Full protection against moderate H₂O₂ concentrations, however, occurred only after 20-24 h (380).

The protective role of Hsps has also been investigated in cultured cells of other origin. In cultured fibroblasts, heat-induced redistribution of Hsp70 and Hsc70 from the cytosol to the cell nucleus has been described in detail by Welch and co-workers and, more recently, by Knowlton and colleagues (201, 205, 432, 434). The above-described increased tolerance toward H₂O₂ challenge depends on the heat shock-elicited Hsp70 and Hsc70 redistribution to the nucleus. Homogeneous distribution of both Hsps throughout nucleus and cytoplasm is required to obtain full protection against moderate H₂O₂ concentrations, occurring only 20-24 h after the challenge (380). This observation could imply that the antioxidant effect of the uniformly distributed Hsps is based on protection of both nuclear and cytoplasmic structures. Otherwise, it could also mean that deleterious effects of the preceding heat shock per se on cellular protein structures mask the improved defense mechanism evoked by Hsps in the early time domain.

A) TRANSIENT CARDIAC ISCHEMIA. In 1988, Currie et al. (82) for the first time reported that whole body heat shock in rats is associated with improvement of cardiac functional recovery after a global ischemic insult, applied 24 h later. A number of remarkable findings were reported: the early postischemic recovery of left ventricular contractility is significantly improved while the postischemic creatine kinase loss is significantly reduced compared with nonpretreated control hearts. Ultrastructural investigation of the heart revealed that mitochondrial morphology is better preserved. In addition, the postischemic activity of the scavenging enzyme catalase was found to be higher than in control hearts. As an indicator of the stress response, the cardiac Hsp70 concentration was significantly increased before the ischemic insult. The authors concluded that the beneficial effects of heat shock pretreatment are likely to be related to both increased radical scavenging activity and the presence of Hsp70 within the cardiac tissue. In the same model, it was shown that physical exercise 3 days before isolated heart perfusion leads to enhanced intracardiac Hsp70 levels and is associated with significantly improved functional recovery after a global ischemic insult. Moreover, the percentage recovery correlated with the degree of training intensity (240). Furthermore, it was found that the transient decay of the cardiac content of Hsp70 during the period following its heat-induced maximal accumulation coincides with the decay of ischemia tolerance (190, 446). This finding is in agreement with the disappearance of inducible Hsps and the loss of thermotolerance in whole animals (381). These and other observations led to the assumption that the degree of postischemic functional recovery correlates with the absolute cardiac Hsp70 tissue contents. Aside from later findings in transgenic animals, in isolated rat hearts it was confirmed that the degree of protection against ischemia is related to the absolute Hsp70 tissue content, that increased stepwise with heat pretreatment at 40, 41, and 42°C (173). Further support was found in isolated papillary muscles, in which the degree of postischemic mechanical recovery correlates with the Hsp70 content in the twin papillary muscle (259).

Hsp-mediated cardioprotection was also established in other mammal species. In isolated rabbit hearts, transient coronary flow reduction (60 min) is better tolerated when the animals are heat-pretreated 24 h earlier (450). The beneficial effect is demonstrated by a better recovery of diastolic and developed left ventricular pressure than in nonpretreated control hearts. Creatine kinase loss is significantly lower, and accumulation of oxidized glutathione, a marker for oxidative stress, is significantly reduced. Cardiac ATP and phosphocreatine levels were found to be better preserved. Furthermore, in intact pigs 1-h coronary artery ligation was significantly better tolerated after the injection of the body temperature-enhancing drug amphetamine 24 h earlier. Postischemic recovery of myocardial segment shortening, developed pressure, and global contractility in the left ventricle occurred significantly faster than in nonpretreated hearts. These phenomena were associated with a less prominent loss of creatine kinase and a higher activity of catalase and superoxide dismutase (262, 265).

Other stress stimuli resulting in enhanced Hsp synthesis without an increase in body temperature, but which enhance Hsp synthesis, are also associated with subsequent transient cardioprotection. An ischemic insult was better tolerated 24 h after the intraperitoneal injection of norepinephrine than in control hearts, as evidenced by a significantly improved postischemic functional recovery of left ventricular developed pressure. Both the hsp70-gene transcription and improvement of postischemic functional recovery could be blocked by the ₁-adrenoceptor blocker prazosin, but not by the -receptor blocker propranolol. Because ₁-adrenoceptor stimulation activates PKC, it is conceivable that this protein kinase cascade may regulate the transcription of the hsp-genes, as pointed out earlier (276). This notion is further supported by the finding that the PKC inhibitor chelerythrine also blocks the beneficial effect of the delayed norepinephrine-mediated cardioprotection (286).

The report of Knowlton et al. (204) on the induction of Hsp70 synthesis by a short, nonpathological ischemic stress prompted other investigators to perform detailed studies on ischemia tolerance after a preceding ischemic episode. In an elaborated study on isolated hearts Myrmel et al. (298) showed that the Hsp70 mRNA synthesis increased when coronary flow is gradually reduced from normal to zero flow levels. Hsp70 synthesis could be correlated with the onset of anaerobic metabolism, but not with enzyme leakage from the tissue. The authors hypothesized that anaerobic metabolism is a strong stimulus for hsp70-gene transcription, since the cessation of anaerobic metabolism is associated with complete shutdown of Hsp70 mRNA synthesis. As pointed out by other investigators, a likely candidate for stimulating HSF1-DNA binding is ATP depletion during anaerobic metabolism, which results in enhanced hsp-gene transcription (32).

Investigations of the relationship between expression patterns of Hsp70 and ischemia tolerance within the first 24 h after heat shock led to the conclusion that no significant correlation exists between both parameters in the early phase after hyperthermia. Although Hsp70 was found to accumulate to maximal tissue levels within 6 h after heat shock, improvement of postischemic functional recovery (267) as well as worsening (70) or no effect at all (329) have been reported. It should be emphasized that heat shock itself has transient but profound negative effects on such cellular structures as intermediate filaments (435) and, hence, could mask the early beneficial effects of hsp-gene expression. Furthermore, it cannot be excluded that yet unknown circulating factors produced in the vascular compartment negatively affect cardiac function during the first hours after heat pretreatment (422).

In conclusion, studies on the protective potential of Hsps on the tolerance to transient ischemia reveal that postischemic function can be significantly improved in various mammal species in the time domain beyond 24 h after the induction of Hsp synthesis. Protection has been documented in both isolated hearts and intact animals. It is possible that the presence or absence of protection in the early time domain depends on a number of unknown factors, related to the trigger applied to elicit Hsp synthesis.

B) MYOCARDIAL INFARCTION. Aside from transgenic mice (see below), rats as well as rabbits have been used to investigate the potential of Hsps to limit myocardial infarct size (99, 255). Protection against myocardial infarction as mediated by enhanced Hsp synthesis following whole body heat shock or a short ischemic episode was found to be present but transient (449). For instance, in rabbits, myocardial infarct size significantly declines when infarction is induced 24 h, but not 40 h after heat pretreatment. In addition, infarct size is only reduced when coronary occlusion is kept relatively short. Whereas a 30-min period is well tolerated, a 45-min occlusion in heat-pretreated animals results in infarct sizes comparable to those in control animals. By using a brief ischemic episode as trigger to elicit Hsp70 and Hsp60 synthesis, increased resistance against myocardial infarction was found 24 h later (255). These experiments also shed some light on the mechanism of induction of Hsp synthesis during and/or after the brief ischemic episode. It is known that infusing such ROS-generating complexes as xanthine/xanthine oxidase, irradiated rose bengal, or H₂O₂ can mimic reperfusion-associated events. Such infusions lead to enhanced Hsp70 synthesis that can be blocked only by concomitant administration of superoxide dismutase (212). It is therefore conceivable that Hsp synthesis is evoked by reperfusion rather than the brief ischemic episode itself.

It has also been questioned whether or not Hsps are synthesized in the myocardium during and after myocardial infarction. In situ hibridization on left ventricular tissue revealed accumulation of hsp70 and hsc70 mRNA signals within the ischemic zone. Upon reperfusion, both messenger signals further increased in the ischemic zone but disappeared in the central necrotic region (322). Histological investigation of rat hearts within the first week after myocardial infarction revealed normal Hsp70 but increased Hsp75 and Grp78 levels in the borderzone around the infarcted area. In the infarct center no changes were found in the contents of either of these Hsps. In contrast, in the noninfarcted septum, concentrations of all three Hsps were significantly increased, suggesting that the increased load in this region served as a stress trigger. After 2-3 wk, Hsp concentrations completely normalized in all zones (195). The consequences of these findings are that Hsps may confer some protection to nonlethally injured zones in the heart, such as the zone immediately adjacent to the area completely devoid of perfusion, but also to remote areas performing more compensatory work.

Heat shock pretreatment also has beneficial effects on myocardial stunning occurring after a short ischemic insult. Because stunning is characterized by mild lysis of contractile proteins in combination with a transient decrease of calcium sensitivity of the sarcomeres, stunned hearts show reduced contractility despite a completely normal coronary perfusion (254). In nonpretreated dogs, 3 h of reperfusion following 15 min of coronary circumflex artery occlusion is associated with a significantly reduced preload-recruitable work area and myocardial segment length change in the former ischemic region. Under identical experimental conditions, heat pretreatment 24 h earlier completely prevents these phenomena (339). In mice hearts with genetically induced high Hsp70 levels, the recovery of regional epicardial strain after a short global ischemic insult is faster and better than in Hsp70-negative mice hearts. The maximal strain recovery measured amounted to 88% of the preischemic values and to 58% in wild-type control hearts (401). Because stunning has been described to result, among others, from intracellular ROS, it is conceivable that Hsps attenuate specifically these hazardous molecules through activation of scavenging enzymes (see below) (82, 289).

C) CARDIOPLEGIA. In a number of animal species, using different experimental approaches, the effects of stress pretreatment on the functional recovery following cardioplegic arrest have been investigated. In isolated rat hearts, arrested at 4°C with a cardioplegic solution during 4 h, a preceding whole body heat stress improves function upon return to normal temperatures. This beneficial effect is characterized by improved recovery of cardiac output and peak aortic pressure as well as by a better preserved response of the coronary endothelium to the vasodilatory agent 5-hydroxytryptamine (10). Pig hearts perfused with a warm (42°C) cardioplegic solution during 15 min before a 2-h hypothermic cardioplegic arrest showed significantly better recovery of developed left ventricular pressure, contractility, and segmental shortening upon reperfusion than nonpreheated control hearts. In addition, the pretreated hearts lost less creatine kinase, while the activity of superoxide dismutase (SOD) was significantly higher than in the control hearts (238). This study is one of the rare examples in which the beneficial effects of heat pretreatment can already be observed in a very short time frame after pretreatment.

D) ENDOTOXIN TOLERANCE. Hsp-mediate