Exercise-heat acclimation in humans alters baseline levels and ex vivo heat inducibility of HSP72 and HSP90 in peripheral blood mononuclear cells

The induction of cellular acquired thermal tolerance (ATT) during heat acclimation (HA) in humans is not well described. This study determined whether exercise-HA modifies the human heat shock protein (HSP)72 and HSP90 responses and whether changes are correlated with physiological adaptations to HA. Using a 10-day HA protocol comprising daily exercise (treadmill walking) in a hot environment (T(a) = 49 degrees C, 20% RH), we analyzed baseline and ex vivo heat-induced expression of HSP72 and HSP90 in peripheral blood mononuclear cells (PBMCs) isolated prior to exercise from eight subjects on day 1 and 10 of the HA protocol. Classical physiological responses to HA were observed, including significantly reduced heart rate and core body temperature, and significantly increased sweating rate. Baseline levels of HSP72 and HSP90 were significantly increased following acclimation by 17.7 +/- 6.1% and 21.1 +/- 6.5%, respectively. Ex vivo induction of HSP72 in PBMCs exposed to heat shock (43 degrees C) was blunted on day 10 compared with day 1. A correlation was identified (r(2) = 0.89) between changes in core temperature elevation and ex vivo HSP90 responses to heat shock between days 1 and 10, indicating that volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. In summary, 1) exercise-HA resulted in increased baseline levels of HSP72 and HSP90, 2) ex vivo heat inducibility of HSP72 was blunted after HA, and 3) volunteers demonstrating the greatest physiological HA tended to exhibit the greatest blunting of ex vivo HSP induction in response to heat shock. These data demonstrate that physiological adaptations in humans undergoing HA are accompanied by both increases in baseline levels and changes in regulation of cytoprotective HSPs.     

Effect of heat acclimation on heat shock protein 72 and interleukin-10 in humans.

Heat acclimation (HA) results in whole body adaptations that increase heat tolerance, and in addition, HA may also result in protective cellular adaptations. We hypothesized that, after HA, basal intracellular heat shock protein (HSP) 72 and extracellular IL-10 levels would increase, while extracellular HSP72 levels decrease. Ten male and two female subjects completed a 10-day exercise/HA protocol (100-min exercise bout at 56% of maximum O(2) uptake in a 42.5 degrees C DB, 27.9% RH environment); subjects exhibited classic adaptations that accompany HA. Peripheral blood mononuclear cells (PBMCs) were isolated before and after each acclimation session on days 1, 6, and 10; plasma and serum were collected before and after exercise on the 1st and 10th day of HA. SDS-PAGE was used to determine PBMC HSP72 levels during HA, and ELISA was used to measure plasma IL-10 and serum HSP72 concentrations. The increase in PBMC HSP72 from pre- to postexercise on the 1st day of HA was not significant (mean +/- SD, 1.0 +/- 0 vs. 1.6 +/- 0.6 density units). Preexercise HSP72 levels on day 1 were significantly lower compared with the pre- and postexercise samples on days 6 and 10 (mean +/- SD, day 6: 2.1 +/- 1.0 and 2.2 +/- 1.0, day 10: 2.0 +/- 1.3 and 2.2 +/- 1.0 density units, respectively, P < 0.05). There were no differences in plasma IL-10 and serum HSP72 postexercise or after 10 days of HA. The sustained elevation of HSP72 from days 6 to 10 may be evidence of a cellular adaptation to HA that contributes to improved heat tolerance and reduced heat illness risk.     

Overexpression of F0F1-ATP synthase α suppresses mutant huntingtin aggregation and toxicity in vitro

Huntington’s disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F₀F₁-ATP synthase α belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F₀F₁-ATP synthase α may play a role in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recently, ATP synthase α was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase α in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase α suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase α is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase α suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase α has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase α.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

Response of mice to continuous 5-day passive hyperthermia resembles human heat acclimation

Chronic repeated exposure to hyperthermia in humans results in heat acclimation (HA), an adaptive process that is attained in humans by repeated exposure to hyperthermia and is characterized by improved heat elimination and increased exercise capacity, and acquired thermal tolerance (ATT), a cellular response characterized by increased baseline heat shock protein (HSP) expression and blunting of the acute increase in HSP expression stimulated by re-exposure to thermal stress. Epidemiologic studies in military personnel operating in hot environments and elite athletes suggest that repeated exposure to hyperthermia may also exert long-term health effects. Animal models demonstrate that coincident exposure to mild hyperthermia or prior exposure to severe hyperthermia can profoundly affect the course of experimental infection and injury, but these models do not represent HA. In this study, we demonstrate that CD-1 mice continuously exposed to mild hyperthermia (ambient temperature ~37°C causing ~2°C increase in core temperature) for 5 days and then exposed to a thermal stress (42°C ambient temperature for 40 min) exhibited some of the salient features of human HA, including (1) slower warming during thermal stress and more rapid cooling during recovery and (2) increased activity during thermal stress, as well as some of the features of ATT, including (1) increased baseline expression of HSP72 and HSP90 in lung, heart, spleen, liver, and brain; and (2) blunted incremental increase in HSP72 expression following acute thermal stress. This study suggests that continuous 5-day exposure of CD-1 mice to mild hyperthermia induces a state that resembles the physiologic and cellular responses of human HA. This model may be useful for analyzing the molecular mechanisms of HA and its consequences on host responsiveness to subsequent stresses.     

Effects of thermal acclimation on transcriptional responses to acute heat stress in the eurythermal fish Gillichthys mirabilis (Cooper)

The capacities of eurythermal ectotherms to withstand wide ranges of temperature are based, in part, on abilities to modulate gene expression as body temperature changes, notably genes encoding proteins of the cellular stress response. Here, using a complementary DNA microarray, we investigated the sequence in which cellular stress response-linked genes are expressed during acute heat stress, to elucidate how severity of stress affects the categories of genes changing expression. We also studied how prior acclimation history affected gene expression in response to acute heat stress. Eurythermal goby fish (Gillichthys mirabilis) were acclimated to 9 ± 0.5, 19 ± 0.5, and 28 ± 0.5°C for 1 mo. Then fish were given an acute heat ramp (4°C/h), and gill tissues were sampled every +4°C to monitor gene expression. The average onset temperature for a significant change in expression during acute stress increased by ～2°C for each ～10°C increase in acclimation temperature. For some genes, warm acclimation appeared to obviate the need for expression change until the most extreme temperatures were reached. Sequential expression of different categories of genes reflected severity of stress. Regardless of acclimation temperature, the gene encoding heat shock protein 70 (HSP70) was upregulated strongly during mild stress; the gene encoding the proteolytic protein ubiquitin (UBIQ) was upregulated at slightly higher temperatures; and a gene encoding a protein involved in cell cycle arrest and apoptosis, cyclin-dependent kinase inhibitor 1B (CDKN1B), was upregulated only under extreme stress. The tiered, stress level-related expression patterns and the effects of acclimation on induction temperature yield new insights into the fundamental mechanisms of eurythermy.     

The typically mitochondrial DNA-encoded ATP6 subunit of the F1F0-ATPase is encoded by a nuclear gene in Chlamydomonas reinhardtii.

The atp6 gene, encoding the ATP6 subunit of F(1)F(0)-ATP synthase, has thus far been found only as an mtDNA-encoded gene. However, atp6 is absent from mtDNAs of some species, including that of Chlamydomonas reinhardtii. Analysis of C. reinhardtii expressed sequence tags revealed three overlapping sequences that encoded a protein with similarity to ATP6 proteins. PCR and 5'- and 3'-RACE were used to obtain the complete cDNA and genomic sequences of C. reinhardtii atp6. The atp6 gene exhibited characteristics of a nucleus-encoded gene: Southern hybridization signals consistent with nuclear localization, the presence of introns, and a codon usage and a polyadenylation signal typical of nuclear genes. The corresponding ATP6 protein was confirmed as a subunit of the mitochondrial F(1)F(0)-ATP synthase from C. reinhardtii by N-terminal sequencing. The predicted ATP6 polypeptide has a 107-amino acid cleavable mitochondrial targeting sequence. The mean hydrophobicity of the protein is decreased in those transmembrane regions that are predicted not to participate directly in proton translocation or in intersubunit contacts with the multimeric ring of c subunits. This is the first example of a mitochondrial protein with more than two transmembrane stretches, directly involved in proton translocation, that is nucleus-encoded.     

Hyperthermia increases interleukin-6 in mouse skeletal muscle

Skeletal muscles produce and contribute to circulating levels of IL-6 during exercise. However, when core temperature is reduced, the response is attenuated. Therefore, we hypothesized that hyperthermia may be an important and independent stimulus for muscle IL-6. In cultured C2C12 myotubes, hyperthermia (42°C) increased IL-6 gene expression 14-fold after 1 h and 35-fold after 5 h of 37°C recovery; whereas exposure to 41°C resulted in a 2.6-fold elevation at 1 h. IL-6 protein was secreted and significantly elevated in the cell supernatant. Similar but reduced responses to heat were seen in C2C12 myoblasts. Isolated soleus muscles from mice, exposed ex vivo to 41°C for 1 h, yielded similar IL-6 gene responses (>3-fold) but without a significant effect on protein release. When whole animals were exposed to passive hyperthermia, such that core temperature increased to 42.4°C, IL-6 mRNA in soleus increased 5.4-fold compared with time matched controls. Interestingly, TNF-α gene expression was routinely suppressed at all levels of hyperthermia (40.5-42°C) in the isolated models, but TNF-α was elevated (4.2-fold) in the soleus taken from intact mice exposed, in vivo, to hyperthermia. Muscle HSP72 mRNA increased as a function of the level of hyperthermia, and IL-6 mRNA responses increased proportionally with HSP72. In cultured C2C12 myotubes, when heat shock factor was pharmacologically blocked with KNK437, both HSP72 and IL-6 mRNA elevations, induced by heat, were suppressed. These findings implicate skeletal muscle as a "heat stress sensor" at physiologically relevant hyperthermia, responding with a programmed cytokine expression pattern characterized by elevated IL-6.     

HSP72 as a complementary protection against oxidative stress induced by exercise in the soleus muscle of rats

Given the potential of reactive oxygen species to damage intracellular proteins during subsequent bouts of muscle contractions, it was suggested that, when this production exceeds the antioxidant capacity, the preexisting antioxidant pathways may be complemented by the synthesis of the defense mechanism represented by heat shock proteins (HSPs), stress proteins with the function of repair and maintaining protein folding. To test this hypothesis, we analyzed reactive carbonyl derivatives in plasma and the expression of HSP72 and activities of enzymes from the oxidative and antioxidant defense systems in the soleus muscle of sedentary rats and rats trained by two protocols: continuous and intermittent. We analyzed all three groups at rest and 2 h after acute exercise. After 8 wk of training, the animals from both groups clearly demonstrated higher resistance to exercise. Both trained groups showed significantly higher citrate synthase, catalase, and glutathione reductase activities than the control group (P < 0.01). After acute exercise, catalase and glutathione reductase activities significantly decreased (P < 0.01) and plasma reactive carbonyl derivatives significantly increased (P < 0.05) in the sedentary group, suggesting an oxidative-stress condition as responsible for exhaustion in this group. Finally, after acute exercise, the induction of HSP72 expression occurred only in the sedentary group, suggesting that HSP72 acts as a complementary protective mechanism in exercise-induced oxidative stress.     

Heat-induced increases in endothelial NO synthase expression and activity and endothelial NO release

Endothelial nitric oxide (NO) synthase (eNOS) is regulated by heat shock protein 90 (HSP90), a heat-inducible protein; however, the effect of heat shock on eNOS expression and eNO release is unknown. Bovine aortic endothelial cells were incubated for 1 h at 37 degrees C, 42 degrees C, or 45 degrees C and cell lysates were evaluated with the use of Western blotting. We observed a 2.1 +/- 0.1-fold increase in eNOS protein content, but no change in HSP90 content, HSP70 content, or HSP90/eNOS association, 24 h after heat shock at 42 degrees C. We also observed a 7.7 +/- 1.5-fold increase in HSP70 protein content, but did not observe a change in eNOS or HSP90 24 h after heat shock at 45 degrees C. eNOS activity and maximal bradykinin-stimulated NO release was significantly increased 24 h after heat shock at 42 degrees C. Heat shock in rats (core temperature: 42 degrees C, 15 min) resulted in a significant increase in aortic eNOS, HSP90, and HSP70 protein content. The aorta from heat-shocked rats exhibited a decreased maximal contractile response to phenylephrine, which was abolished by preincubation with NG-nitro-l-arginine. We conclude that prior heat shock is a physical stimulus of increased eNOS expression and is associated with an increase in eNOS activity, agonist-stimulated NO release, and a decreased vasoconstrictor response.     

Modulation of α(2C) adrenergic receptor temperature-sensitive trafficking by HSP90

Decreasing the temperature to 30°C is accompanied by significant enhancement of α(2C)-AR plasma membrane levels in several cell lines with fibroblast phenotype, as demonstrated by radioligand binding in intact cells. No changes were observed on the effects of low-temperature after blocking receptor internalization in α(2C)-AR transfected HEK293T cells. In contrast, two pharmacological chaperones, dimethyl sulfoxide and glycerol, increased the cell surface receptor levels at 37°C, but not at 30°C. Further, at 37°C α(2C)-AR is co-localized with endoplasmic reticulum markers, but not with the lysosomal markers. Treatment with three distinct HSP90 inhibitors, radicicol, macbecin and 17-DMAG significantly enhanced α(2C)-AR cell surface levels at 37°C, but these inhibitors had no effect at 30°C. Similar results were obtained after decreasing the HSP90 cellular levels using specific siRNA. Co-immunoprecipitation experiments demonstrated that α(2C)-AR interacts with HSP90 and this interaction is decreased at 30°C. The contractile response to endogenous α(2C)-AR stimulation in rat tail artery was also enhanced at reduced temperature. Similar to HEK293T cells, HSP90 inhibition increased the α(2C)-AR contractile effects only at 37°C. Moreover, exposure to low-temperature of vascular smooth muscle cells from rat tail artery decreased the cellular levels of HSP90, but did not change HSP70 levels. These data demonstrate that exposure to low-temperature augments the α(2C)-AR transport to the plasma membrane by releasing the inhibitory activity of HSP90 on the receptor traffic, findings which may have clinical relevance for the diagnostic and treatment of Raynaud Phenomenon.     

Heat acclimation: phenotypic plasticity and cues to the underlying molecular mechanisms

Acclimation, in contrast to evolutionary adaptation, is a “within life time phenotypic adaptation” resulting in a widening of the dynamic regulatory range of body temperature. Increased efficiency and capacity of the thermoregulatory effectors, and delayed onset of the temperature threshold for thermal injury, contribute to the beneficial effects of acclimation. Reprogrammed gene expression and changes in cellular signaling underlie these responses. Constitutive elevation of the inducible heat shock protein (HSP) 72 kDa provides cytoprotection and delays thermal injury without the need for de novo HSP synthesis upon thermal stress. The time window for evocation of heat acclimation is the early phase of acclimation, the short-term heat acclimation (STHA), with accelerated sympathetic excitability and a drop in plasma thyroxin playing an essential role. An important consequence of thermal acclimation is the development of cross-tolerance between heat acclimation and ischemia/reperfusion insults. The beneficial implications of this feature are discussed.     

Each yeast mitochondrial F1F0-ATP synthase complex contains a single copy of subunit 8

The stoichiometry of subunit 8 in yeast mitochondrial F(1)F(0)-ATP synthase (mtATPase) has been evaluated using an immunoprecipitation approach. Single HA or FLAG epitopes were introduced at the N-terminus of subunit 8. Expression of each tagged subunit 8 variant in yeast cells lacking endogenous subunit 8 restored a respiratory phenotype and had little measurable effect on ATP hydrolase activity of the isolated enzyme. Moreover, the two epitope-tagged subunit 8 variants could be stably co-expressed in the same host cells and both of HA-Y8 and FLAG-Y8 could be detected in ATP synthase complexes isolated by native gel electrophoresis. Mitochondria isolated from each yeast strain were solubilized to release ATP synthase complexes in either the monomeric or dimeric forms. In each case, monoclonal antibodies directed against either the FLAG or HA epitope could immunoprecipitate intact ATP synthase complexes. When both HA-Y8 and FLAG-Y8 were co-expressed in cells, monomeric ATP synthases contained only a single subunit 8 variant after immunoprecipitation, corresponding to the particular antibody used (HA or FLAG). By contrast, both subunit 8 variants were recovered in samples of immunoprecipitated dimeric ATP synthase complexes, irrespective of the antibody used. We conclude that each monomeric yeast mitochondrial ATP synthase complex contains a single copy of subunit 8.     

The gamma subunit of F1 and the PVP protein of F0 (F0I) are components of the gate of the mitochondrial F0F1 H(+)-ATP synthase

The gamma subunit of the F1 moiety of the bovine mitochondrial H(+)-ATP synthase is shown to function as a component of the gate. Addition of purified gamma subunit to F0-liposomes inhibits transmembrane proton conduction. This inhibition can be removed by the bifunctional thiol reagent diamide. Immunoblot analysis shows that the diamide effect is likely due to disulphide bridging of the gamma subunit with the PVP protein of the F0 sector.     

HSP72 inhibits apoptosis-inducing factor release in ATP-depleted renal epithelial cells

Inhibition of the mitochondrial release and nuclear translocation of apoptosis-inducing factor (AIF) by heat stress protein (HSP)72 may ameliorate apoptosis in renal epithelial cells exposed to a metabolic inhibitor. To evaluate this hypothesis, cells were transiently exposed to 5 mM sodium cyanide in the absence of medium glucose, a maneuver known to induce apoptosis. ATP depletion for 1-2 h resulted in the progressive accumulation of mitochondrial AIF in the cytosol of samples obtained by selectively permeabilizing the plasma membrane with digitonin. During recovery from ATP depletion, time-dependent nuclear AIF accumulation (but not cytochrome c, an F₀F₁ ATP synthase subunit, or talin) was observed in isolated nuclei. Nuclear AIF accumulation was associated with peripheral chromatin condensation and DNA degradation. Prior heat stress (HS) significantly reduced AIF leakage into the cytosol, decreased nuclear accumulation of AIF, and inhibited DNA degradation. HS also increased the interaction between AIF and HSP72 detected by immunoprecipitation. In ATP depleted cells, selective overexpression of human HSP72 reduced the leakage of mitochondrial AIF in a dose-dependent manner (r = 0.997). This study suggests that mitochondrial membrane injury and subsequent AIF release contribute to nuclear injury and apoptosis in ATP-depleted renal cells. HSP72, an antiapoptotic protein, inhibits cell injury in part by preventing mitochondrial AIF release and perhaps by decreasing its nuclear accumulation. heat stress; adenovirus; metabolic inhibitors; heat stress protein 60; DNA degradation     

Activity of antioxidant enzymes in rat skeletal muscle and brown fat: effect of cold and propranolol

1. Effects of acute (6 h) and chronic (21 day) cold (6°C) exposure, as well as propranolol (15 mg/kg) on the activities of CuZnSOD, MnSOD and catalase in the rat skeletal muscle (SM) and brown adipose tissue (BAT), which are important sites of cold-induced thermogenesis, were investigated.

2. The changes in the activity of antioxidant enzymes were tissue specific and dependent on the duration of cold exposure. Thus, in the SM of acutely cold exposed rats, the activity of all antioxidant enzymes studied was elevated, whereas in the BAT the activity of both SOD_s decreased and that of catalase remained unchanged. In cold acclimated rats, the activity of all the three enzymes was increased in the BAT whereas in the SM, CuZnSOD activity was enhanced, MnSOD activity decreased and catalase activity returned to the control level.

3. Propranolol also differently altered the antioxidant enzyme activity in SM and BAT, alterations being dependent on the acclimation temperature. Thus, in room acclimated rats propranolol decreased the activity of all antioxidant enzymes in SM but did not affect those in BAT. However, in the SM propranolol prevented the elevation of MnSOD and catalase activities, induced by acute cold. In cold acclimated rats propranolol inhibited CuZnSOD activity in both SM and BAT but increased that of MnSOD.