Constitutive Expression Of Hsp70 Research Articles

Age-dependent expression profiles of two adaptogenic systems and thermotolerance in Drosophila melanogaster.

Here, we monitored the expression of three genes (hsp70, hsp22, and hsf1) involved in heat shock response in Drosophila melanogaster in males and females of different age. Also, we investigated age- and sex-dependent expression of three major genes participating in the production of hydrogen sulfide (H2S) (cse, cbs, and mst), implicated in stress resistance and aging. In addition to the control strain, we monitored the expression of all of these genes in a cbs knockout strain (cbs-/-) generated using the CRISPR technique. The tested strains differ in the induction capacities of the studied genes. Relative to the control strain, under normal conditions, the cbs-/- strain expresses all of the studied genes more abundantly, especially hsp22. In the control strain, aging leads to a dramatic increase in hsp22 synthesis, whereas in the cbs-/- strain, hsp22 induction is not pronounced. Furthermore, in 30-day-old cbs-/- flies, the constitutive expression of hsp70 and mst is decreased. Surprisingly, in the cbs-/- strain, we detected an upregulation of hsf1 transcription in the 30-day-old females. After heat shock in the control strain, hsp70 and hsp22 induction decreased with age in males and hsp22 decreased in females, while in the cbs-/- strain, a pronounced drop in the induction capacity of both hsp genes was seen in 30-day-old males and females. However, in most cases, the expression levels of hsf1 and H2S-producing genes do not exhibit pronounced changes depending on sex, age, or heat shock. Flies of control and cbs-/- strain exhibited strong reduction in basal thermotolerance with age. Our data suggest a cross-talk between the two studied ancient and universal adaptive systems.

Molecular, behavioral, and performance responses of juvenile largemouth bass acclimated to an elevated carbon dioxide environment.

Aquatic hypercarbia, either naturally occurring or anthropogenically induced, can have extensive impacts on aquatic environments and resident organisms. While the impact of acute hypercarbia exposure on the behavior and physiology of fishes has been well studied, relatively little work has examined the physiological impact and acclimation capacity of fishes to chronic hypercarbia. To better understand the impacts of prolonged hypercarbia exposure, largemouth bass were held at ambient CO2 (13 mg L(-1)) and elevated CO2 (31 mg L(-1); ≈ 21,000 µatm) for 58 days. Following this acclimation period, fish were subjected to three separate, yet complementary, experiments: (1) acute hypercarbia challenge of 120 mg L(-1) CO2 for 1 h to quantify physiological and molecular responses; (2) hypercarbia avoidance challenge to compare CO2 agitation and avoidance responses; and (3) swim performance challenge to quantify burst swimming performance. Acclimation to 31 mg L(-1) CO2 resulted in a significant constitutive upregulation of c-fos expression in erythrocytes, combined with significant constitutive expression of hsp70 in both gill and erythrocytes, relative to controls. Largemouth bass acclimated to elevated CO2 also had a reduced glucose response (relative to controls) following an acute CO2 exposure, indicating a reduced stress response to CO2 stressors. In addition, largemouth bass acclimated to elevated CO2 conditions required 50 % higher CO2 concentrations to illicit agitation behaviors and displayed prolonged burst swimming abilities in high CO2 environments relative to controls. Together, results demonstrate that largemouth bass exposed to chronic hypercarbia may possess a physiological advantage during periods of elevated CO2 relative to naïve fish, which may permit increased performance in hypercarbia.

Hsp70-Dependent Antiviral Immunity against Cytopathic Neuronal Infection by Vesicular Stomatitis Virus

The major inducible 70-kDa heat shock protein (hsp70) protects against measles virus (MeV) neurovirulence in the mouse that is caused by a cell-associated noncytolytic neuronal infection. Protection is type I interferon (IFN) dependent, and we have established a novel axis of antiviral immunity in which hsp70 is released from virus-infected neurons to induce IFN-β in macrophages. The present work used vesicular stomatitis virus (VSV) to establish the relevance of hsp70-dependent antiviral immunity to fulminant cytopathic neuronal infections. In vitro, hsp70 that was constitutively expressed in mouse neuronal cells caused a modest increase in VSV replication. Infection induced an early extracellular release of hsp70 from viable cells, and the release was progressive, increasing with virus-induced apoptosis and cell lysis. The impact of this VSV-hsp70 interaction on neurovirulence was established in weanling male hsp70 transgenic and nontransgenic mice. Constitutive expression of hsp70 in neurons of transgenic mice enhanced viral clearance from brain and reduced mortality, and it was correlated with enhanced expression of type I IFN mRNA. Nontransgenic mice were also protected against neurovirulence and expressed increased type I IFN mRNA in brain when hsp70 was expressed by a recombinant VSV (rVSV-hsp70), indicating that hsp70 in the virus-infected cell is sufficient for host protection. In vitro data confirmed extracellular release of hsp70 from cells infected with rVSV-hsp70 and also showed that viral replication is not enhanced when hsp70 is expressed in this manner, suggesting that hsp70-mediated protection in vivo is not dependent on stimulatory effects of hsp70 on virus gene expression.

Hsp70 and a Novel Axis of Type I Interferon-Dependent Antiviral Immunity in the Measles Virus-Infected Brain

The major inducible 70-kDa heat shock protein (hsp70) is host protective in a mouse model of measles virus (MeV) brain infection. Transgenic constitutive expression of hsp70 in neurons, the primary target of MeV infection, abrogates neurovirulence in neonatal H-2(d) congenic C57BL/6 mice. A significant level of protection is retained after depletion of T lymphocytes, implicating innate immune mechanisms. The focus of the present work was to elucidate the basis for hsp70-dependent innate immunity using this model. Transcriptome analysis of brains from transgenic (TG) and nontransgenic (NT) mice 5 days after infection identified type I interferon (IFN) signaling, macrophage activation, and antigen presentation as the main differences linked to survival. The pivotal role of type I IFN in hsp70-mediated protection was demonstrated in mice with a genetically disrupted type I IFN receptor (IFNAR(-/-)), where IFNAR(-/-) eliminated the difference in survival between TG and NT mice. Brain macrophages, not neurons, are the predominant source of type I IFN in the virus-infected brain, and in vitro studies provided a mechanistic basis by which MeV-infected neurons can induce IFN-β in uninfected microglia in an hsp70-dependent manner. MeV infection induced extracellular release of hsp70 from mouse neuronal cells that constitutively express hsp70, and extracellular hsp70 induced IFN-β transcription in mouse microglial cells through Toll-like receptors 2 and 4. Collectively, our results support a novel axis of type I IFN-dependent antiviral immunity in the virus-infected brain that is driven by hsp70.

Hsp70 is required for optimal cell proliferation in mouse A6 mesoangioblast stem cells

Mouse Hsp70 (70 kDa heat shock protein) is preferentially induced by heat or stress stimuli. We previously found that Hsp70 is constitutively expressed in A6 mouse mesoangioblast stem cells, but its possible role in these cells and the control of its basal transcription remained unexplored. Here we report that in the absence of stress, Ku factor is able to bind the HSE (heat shock element) consensus sequence in vitro, and in vivo it is bound to the proximal hsp70 promoter. In addition, we show that constitutive hsp70 transcription depends on the co-operative interaction of different factors such as Sp1 (specificity protein 1) and GAGA-binding protein with Ku factor, which binds the HSE consensus sequence. We used mRNA interference assays to select knockdown cell clones. These cells were able to respond to heat stress by producing a large amount of Hsp70, and produced the same amount of Hsp70 as that synthesized by stressed A6 cells. However, severe Hsp70 knockdown cells had a longer duplication time, suggesting that constitutive Hsp70 expression has an effect on the rate of proliferation.

Expression of Heat Shock Protein 70 in Human Skin Cells as a Photoprotective Function after UV Exposure.

Human skin is exposed to various environmental stresses, such as heat, cold, and ultraviolet (UV) radiation. Heat shock proteins (HSPs) induced by temperature elevations, as a physiologic response to mediate repair mechanisms and reduce cellular damage. The purpose of this study was to investigate the induction of HSPs in human skin cells after UV exposure. We performed immunoblotting using a specific monoclonal antibody to the HSP70 family, one of the best-conserved stress proteins in humans, with cultured normal human keratinocytes, A431 cells, human melanocytes, SK30 cells, and human dermal fibroblasts (HDF). Our results indicated that high expression of HSP70 in the unstressed state was noted in epidermal cells, including normal human keratinocytes, A431 cells, human melanocytes, and SK30 cells, but epidermal cells showed no additional up-regulation of HSP70 after UV irradiation. On the other hand, HDF expressed very small amounts of HSP70 at baseline, but significantly higher amounts of HSP70 after UV exposure. These findings suggest that constitutive expression of HSP70 in epidermal cells may be an important mechanism for protection of the human epidermis from environmental stresses, such as sunlight exposure.

An inducible 70 kDa-class heat shock protein is constitutively expressed during early development and diapause in the annual killifish Austrofundulus limnaeus

The annual killifish Austrofundulus limnaeus inhabits ephemeral ponds in regions of northern South America, where they survive the periodic drying of their habitat as diapausing embryos. These diapausing embryos are highly resistant to a number of environmental insults such as high temperature, dehydration, anoxia, and increased salinity. Molecular chaperones are known to play a role in stabilizing protein structure and function during events of cellular stress. Relative levels of heat shock protein (Hsp)70 were measured in developing and diapausing embryos of A. limnaeus using quantitative Western blots. An inducible or embryo-specific form of Hsp70 is expressed during embryonic development in A. limnaeus and is elevated during diapause II in this species. Constitutive expression of Hsp70 during development may afford these embryos protection from environmental stresses during development more quickly than relying on the induction of a classic heat shock response.

Heat shock factor 1 is required for constitutive Hsp70 expression and normal lens development in embryonic zebrafish

Heat shock factors (HSFs) are the major transcription factors responsible for heat-induced upregulation of heat shock protein (Hsp) genes. All three mammalian HSFs (HSF1, HSF2, HSF4) have also been shown to be required for normal mammalian development. It is currently unknown if HSFs play similarly important roles during normal development of non-mammalian vertebrates. In the present study, a morpholino modified antisense oligonucleotide (MO) approach targeted against hsf1 mRNA (hsf1-MO) was used to examine the requirement of HSF1 in zebrafish development. Embryos depleted of HSF1 displayed a reproducible small eye phenotype characterized by an immature lens and a disorganized retinal structure. These defects were strikingly similar to those observed when constitutive, lens specific Hsp70 expression was reduced through the microinjection of MO targeting hsp70. The data suggest that HSF1 is involved in regulating constitutive lens specific expression of hsp70 in the embryonic zebrafish. This conclusion is supported by a marked reduction in Hsp70 protein in hsf1-MO injected embryos. Microinjection of MO targeted to hsf2 mRNA (hsf2-MO) did not result in a small eye phenotype in a significant number of embryos. These data also suggest that HSF1 and HSF2 play distinct roles in non-mammalian vertebrates, similarly to what has been demonstrated previously in mouse.

Acquisition of resistance to butyrate induces resistance to luminal components and other types of stress in human colon adenocarcinoma cells

Butyrate, naturally produced by anaerobic fermentation of diet-fiber, is the major nutrient of colonocytes and also an important regulator of colonic epithelium renewal and physiology. Other luminal components, such as bile acids or bacterial products, influence these processes. The model system we used to analyze the influence of several luminal stressors is composed of a previously established cell line resistant to the apoptotic effects of butyrate and their parental butyrate-sensitive cells. Viability of butyrate-resistant cells is unaffected by mild heat-shock (2 h, 42 °C) and only slightly reduced by severe heat-shock (2 h, 45 °C) in contrast to their butyrate-sensitive counterparts. The higher constitutive expression of HSP70 and HSP60 could contribute to this resistance. In addition, expression of HSP70 follows a different pattern after heat-shock in both cell lines. Butyrate-resistant cells are quite unaffected by treatment with deoxycholic acid but apoptosis is induced by chenodeoxycholic acid although to a lower extent than in butyrate-sensitive cells. These resistant cells are also less sensitive to lipopolysaccharide and show differences regarding the activation of ERK following osmotic stress. Thus, the cell model herein reported is a useful tool for investigating the molecular mechanisms of resistance to apoptosis, as well as to analyze specific targets for butyrate-resistant tumors.

Slower skeletal muscle phenotypes are critical for constitutive expression of Hsp70 in overloaded rat plantaris muscle

Heat shock protein 72 (Hsp70) is constitutively expressed in rat hindlimb muscles, reportedly in proportion to their content of type I myosin heavy chain. This distribution pattern has been suggested to result from the higher recruitment and activity of such muscles and/or a specific relationship between myosin phenotype and Hsp70 content. To differentiate between these possibilities, the fiber-specific distribution of Hsp70 was examined in male Sprague-Dawley rat plantaris under control conditions, following a fast-to-slow phenotypic shift in response to surgically induced overload (O) and in response to O when the phenotypic shift was prevented by 3,5,3'-triiodo-dl-thyronine administration. Constitutive expression of Hsp70 was restricted to type I and IIa fibers in plantaris from control rats, and this fiber-specific pattern of expression was maintained following O of up to 28 days, although Hsp70 content in the O muscle doubled. When O (for 40 days) of the plantaris was combined with 3,5,3'-triiodo-dl-thyronine administration, despite typical hypertrophy in the overloaded plantaris, prevention of the normal phenotypic transformation also blocked the increased expression of Hsp70 observed in euthyroid controls. Collectively, these data suggest that chronic changes in constitutive expression of Hsp70 with altered contractile activity appear critically dependent on fast-to-slow phenotypic remodeling.

The role of heat shock proteins Hsp70 and Hsp27 in cellular protection of the central nervous system

Heat shock proteins (Hsps) are highly conserved and under physiological conditions act as molecular chaperones and/or have anti-apoptotic activities. Expression in the brain of two heat shock proteins, the70 kDa Hsp (Hsp70) and the 27 kDa Hsp (Hsp27), is notable because both proteins are highly inducible in glial cells and neurons following a wide range of noxious stimuli including ischemia, epileptic seizure and hyperthermia. In the central nervous system, constitutive expression of Hsp27 is limited to many (but not all) sensory and motor neurons of the brain stem and spinal cord, while there is little or no constitutive expression of Hsp70. However, inducible expression of both Hsp70 and Hsp27 is present in many areas of the brain and retina and is associated with cellular resistance to a variety of insults. The potential for manipulating the expression levels of Hsps for therapeutic advantage in neurodegenerative diseases such as Alzheimer's disease, stroke and glaucoma will be explored.

Targeted disruption of hsf1 leads to lack of thermotolerance and defines tissue-specific regulation for stress-inducible Hsp molecular chaperones.

The rapid synthesis of heat shock proteins (Hsps) in cells subjected to environmental challenge is controlled by heat shock transcription factor-1 (Hsf1). Regulation of Hsps by Hsf1 is highly complex and, in the whole organism, remains largely unexplored. In this study, we have used mouse embryo fibroblasts and bone marrow progenitor cells from hsf1-/- mice as well as hsp70.3-lacZ knock-in mice bred on the hsf1deficient genetic background (hsf1-/--hsp70.3+/--lacZ), to further elucidate the function of Hsf1 and its participation as a transcriptional activator of Hsp70 synthesis under normal or heat-induced stress conditions in vitro and in vivo. The results revealed that heat-induced Hsp70 expression in mouse tissue is entirely controlled by Hsf1, whereas its activity is not required for tissue-specific constitutive Hsp70 expression. We further demonstrate that Hsf1 is critical for maintaining cellular integrity after heat stress and that cells from hsf1-/- mice lack the ability to develop thermotolerance. This deficiency is explained by the elimination of stress-inducible Hsp70 and Hsp25 response in the absence of Hsf1 activity, leading to a lack of Hsp-mediated inhibition of apoptotic cell death via both caspase-dependent and caspase-independent pathways. The pivotal role of the Hsf1 transactivator in regulating rapid synthesis of Hsps as a critical cellular defense mechanism against environmental stress-induced damage is underlined.

Treatment with 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin.

We previously reported that skin closure is improved by photoirradiation of the wound margins with an 815-nm diode laser system. To determine whether the beneficial effects of laser treatment involve the overexpression of the inducible 72-kDa heat shock protein, Hsp70. Expression of Hsp70 was investigated by immunocytochemistry in normal hairless rat dorsal skin and compared with its expression after laser photoirradiation. Constitutive expression of Hsp70 was mainly confined to the upper epidermal layer. Laser irradiation further increased epidermal expression of Hsp70 while inducing de novo synthesis of the protein in dermal structures, particularly around blood vessels, hair follicles and sebaceous glands. Laser-induced expression of Hsp70 was still present 7 days after photoirradiation. Laser-induced expression of Hsp70 might contribute to improved tissue regeneration and wound healing.

A hsp70-2 Mutation Recognized by CTL on a Human Renal Cell Carcinoma

We performed T cell cloning experiments with a tumor-infiltrating lymphocyte subpopulation derived from a renal cell carcinoma tumor site (RCC-7) in which the TCR clonotypic repertoire had been analyzed in terms of TCRBV complementarity-determining region 3 size distribution. We report in this work the characterization of one of the five RCC-specific MHC class I-restricted CTL clones isolated in RCC-7. This TCRBV6J1S1 CTL recognized only the autologous RCC-7 tumor cell line in the context of HLA-A*0201, and the Ag is encoded by a mutated form of the hsp70-2 gene found in the tumor cells, but not in autologous PBLs nor in 47 other tumors. The identification of this gene was achieved by cotransfecting into COS cells a cDNA library of RCC-7 together with HLA-A*0201. Transfectants expressing the Ag were identified by their ability to stimulate TNF release by the CTL clone. The antigenic peptide is a decamer with a mutated residue at position 8. Half-maximal lysis was obtained with only 5 x 10(-11) M of decapeptide in target sensitization assays compared with 5 x 10(-8) M for the wild-type decapeptide. This difference in recognition was not related to difference in binding HLA-A*0201-presenting molecules, as assessed in an immunofluorescence-based peptide-binding assay using T2 cells. Constitutive hsp70 expression in various tumors suggests that this stress-induced protein may be recognized in situ by tumor-infiltrating lymphocytes. The finding in the tumor of a mutated form of the stress-induced hsp70-2 gene whose product is specifically recognized by TILs with high avidity is discussed in view of the present use of mycobacteria or heterologous heat-shock proteins as immunomodulators or as subunit vaccine candidates.

Inhibition of hsp70-1 and hsp70-3 expression disrupts preimplantation embryogenesis and heightens embryo sensitivity to arsenic.

Mouse 70-kDa heat shock proteins Hsp70-1 and Hsp70-3 (Hsp70-1/3) are stress-inducible protein chaperones thought to protect embryonic cells and tissues from the effects of a wide range of environmental exposures. Hsp70-1/3 are expressed constitutively, and at times are stress-inducible during various stages of preimplantation embryogenesis. In order to elucidate the functions of constitutive and stress-inducible Hsp70 expression in mouse preimplantation embryos, the consequences of inhibiting expression with antisense oligonucleotides complementary to the mRNAs of hsp70-1 and hsp70-3 (A070-1/3) were evaluated. Transfection of preimplantation embryos (four-cell stage) with 2.5 microM A070-1/3 had no effect on in vitro blastocoel formation. However, transfection with 5 or 10 microM A070-1/3 reduced in vitro blastocyst development to 30% and 0%, respectively (approximately 90% control embryos developed to blastocyst). Thus constitutive expression of Hsp70-1/3 appears significant to preimplantation embryogenesis. Limiting expression of Hsp70-1/3 with 5 microM A070-1/3 also heightened embryo sensitivity to arsenic, resulting in less than 5% in vitro development to blastocyst in the presence of the subtoxic dose of 0.4 microM sodium arsenite. Whether the combined effect of A070-1/3 and arsenic is due to blocking inducible expression of the Hsp70s, or due to further reducing the amount of constitutively expressed Hsp70s available to the embryo is not known at this time. However, these results clearly indicate that some minimal amount of Hsp70-1 and/or Hsp70-3 is required for preimplantation embryogenesis, and that increasing the demand for Hsp70s by arsenic exposure heightens this requirement.

Suppression of Glial Nitric Oxide Synthase Induction by Heat Shock: Effects on Proteolytic Degradation of IκB-α

Rat C6 glioma cells were stably transfected with a human cDNA encoding heat shock protein (HSP) 70. Immunostaining revealed the presence of largely cytosolic HSP70 in C6-hsp70 cells, but not in control (vector transfected) C6-pTK cells. Induction of nitric oxide synthase (NOS-2) expression in C6-hsp70 cells, assessed by nitrite accumulation, was significantly reduced compared to control C6-pTK cells (25 ± 8% of control cell induction,P< 0.005), when induced with a maximally stimulatory combination of bacterial endotoxin lipopolysaccharide (LPS) plus a mixture of three cytokines (“CM:” TNF-α, IL1-β, and IFN-γ). Immunostaining for the transcription factor NFκB p65 subunit revealed decreased cytokine-dependent nuclear uptake in HSP70 expressing cells compared to control cells. Activation of C6 cell NFκB by LPS plus CM required IκB degradation by the 20S proteasome, since NOS-2 expression was blocked by a selective proteasome inhibitor. In parental C6 cells, the presence of LPS plus CM caused a rapid (within 30 min) decrease in inhibitory IκB-α protein levels, and this loss was abolished by prior heat shock of the cells. In contrast, IκB-α levels in transfected cells were not modified by the expression of HSP70. These results demonstrate that constitutive HSP70 expression in glial cells can reduce NOS-2 induction, presumably due to inhibition of NFκB nuclear uptake. Furthermore, whereas prevention of decreases in IκB-α can account for the suppressive effects of heat shock, the results suggest that HSP70 blocks NOS-2 induction by interfering at a later step in the NFκB activation pathway.

Hsp70 and Hsc70 are preferentially expressed in differentiated epithelial cells in normal human endometrium and ectocervix.

Two highly related 70K heat shock proteins, encoded by the hsc70 and hsp70 genes, are located in the nucleocytoplasmic compartment of mammalian cells. In contrast to recent cell lines, which express Hsp70 only when stressed, many human cell lines constitutively express Hsp70. The degree to which this reflects constitutive expression of Hsp70 in normal human tissues has not been extensively examined. In this study, we show by immunoblotting that human Hsp70 is constitutively expressed in the ovary, cervix, and endometrium and, by immunohistochemical analysis using Hsp70- and Hsc70-specific antibodies, that Hsp70 and Hsc70 are expressed in distinctive and predominantly overlapping patterns in the cervix and endometrium. In these two tissues, the highest levels of both proteins are seen in differentiated, non-proliferating epithelial cells, which is surprising in light of previous studies suggesting growth stimulation of hsp70 gene expression. These observations suggest the possibility that in certain human tissues, basal expression of the hsp70 and hsc70 genes is co-regulated.

Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury.

Using transgenic mice constitutively expressing the human inducible Hsp70, we examined the role of Hsp70 on cell survival after focal cerebral ischemia. Twenty-four hours after permanent occlusion of the middle cerebral artery, no difference in infarct area was detected between Hsp70-transgenic and non-transgenic mice. In the non-transgenic mice, many pyramidal neurons of the ipsilateral hippocampus were observed to be pyknotic. However, in all Hsp70-transgenic mice, hippocampal pyramidal neurons showed normal morphology and no evidence of pyknosis. This suggests that constitutive expression of Hsp70 reduces the extent of damage following permanent middle cerebral artery occlusion.

Heat shock protein hsp70 overexpression confers resistance against nitric oxide

Heat stress is known to render rat islet cells resistant against the toxic effects of nitric oxide, reactive oxygen intermediates and the islet cell toxin streptozotocin. We report here for the first time that protection against nitric oxide is mediated by the major heat shock protein, hsp70, even in the absence of heat stress. The human hsp70 gene was stably transfected into the rat insulinoma cell line RINm5F. Constitutive expression of hsp70 caused protection from NO-induced cell lysis which was of the same extent as seen after heat stressing cells. Our results identify hsp70 as a defence molecule against nitric oxide.

Increased HSF activation in muscles with a high constitutive Hsp70 expression.

Stress-induced transcriptional regulation of the Hsps is mediated by trimerization and binding of a pre-existing heat shock transcription factor (HSF1) to a specific DNA sequence located in the 5' region of hsp genes, known as the heat shock element. Hsp70 has been implicated in regulating the activation of the HSF and, according to cell culture models, high steady-state levels of Hsp70 are inversely correlated with HSF activation. To determine if this applies in an intact animal, muscles of the rat hindlimb which differ in the constitutive expression of Hsp70, were assessed for HSF activation following heat shock. Mobility shift gel analyses demonstrated that HSF activation was detectable in extracts from all muscles following heat shock regardless of Hsp70 content. However, muscles comprised predominantly of slow/Type I fibers (soleus) demonstrated a greater HSF activation, as well as a faster HSF activation and inactivation, than muscles comprised predominantly of fast/Type II fibers (white gastrocnemius). In addition, muscles pretreated by two heat shocks (24 h apart) demonstrated a stronger HSF activation than muscles subjected to only one heat shock. Thus, results from cell culture models demonstrating that tissue levels of Hsp70 are inversely correlated with HSF activation, may not apply to the muscles of an intact animal.