Time course and differential responses of the major heat shock protein families in human skeletal muscle following acute nondamaging treadmill exercise.

The exercise-induced expression of heat shock proteins (HSPs) in rodent models is relatively well defined. In contrast, comparable data from human studies are limited and the exercise-induced stress response of human skeletal muscle is far from understood. This study has characterized the time course and magnitude of the HSP response in the skeletal muscles of a healthy active, but untrained, young male population following a running exercise protocol. Eight subjects performed 45 min of treadmill running at a speed corresponding to their lactate threshold (11.7 +/- 0.5 km/h; 69.8 +/- 4.8% maximum O2 uptake). Muscle biopsies were obtained from the vastus lateralis muscle immediately before and at 24 h, 48 h, 72 h, and 7 days postexercise. Exercise induced a significant (P < 0.05) but variable increase in HSP70, heat shock cognate (HSC) 70, and HSP60 expression with peak increases (typically occurring at 48 h postexercise) to 210, 170, and 139% of preexercise levels, respectively. In contrast, exercise did not induce a significant increase in either HSP27, alphaB-crystallin, SOD 2 (MnSOD) protein content, or the activity of SOD and catalase. When examining baseline protein levels, HSC70, HSP27, and alphaB-crystallin appeared consistently expressed between subjects, whereas HSP70 and MnSOD displayed marked individual variation of up to 3- and 1.5-fold, respectively. These data are the first to define the time course and extent of HSP production in human skeletal muscle following a moderately demanding and nondamaging running exercise protocol. Data demonstrate a differential effect of aerobic exercise on specific HSPs.     

Delta Opioid Receptors: The Link between Exercise and Cardioprotection

This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response.     

Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans.

Increased levels of reactive oxygen and nitrogen species, as seen in response to exercise, challenge the cellular integrity. Important protective adaptive changes include induction of heat shock proteins (HSPs). We hypothesized that supplementation with antioxidant vitamins C (ascorbic acid) and E (tocopherol) would attenuate the exercise-induced increase of HSP72 in the skeletal muscle and in the circulation. Using randomization, we allocated 21 young men into three groups receiving one of the following oral supplementations: RRR-alpha-tocopherol 400 IU/day + ascorbic acid (AA) 500 mg/day (CEalpha), RRR-alpha-tocopherol 290 IU/day + RRR-gamma-tocopherol 130 IU/day + AA 500 mg/day (CEalphagamma), or placebo (Control). After 28 days of supplementation, the subjects performed 3 h of knee extensor exercise at 50% of the maximal power output. HSP72 mRNA and protein content was determined in muscle biopsies obtained from vastus lateralis at rest (0 h), postexercise (3 h), and after a 3-h recovery (6 h). In addition, blood was sampled for measurements of HSP72, alpha-tocopherol, gamma-tocopherol, AA, and 8-iso-prostaglandin-F2alpha (8-PGF2alpha). Postsupplementation, the groups differed with respect to plasma vitamin levels. The marker of lipid peroxidation, 8-iso-PGF2alpha, increased from 0 h to 3 h in all groups, however, markedly less (P < 0.05) in CEalpha. In Control, skeletal muscle HSP72 mRNA content increased 2.5-fold (P < 0.05) and serum HSP72 protein increased 4-fold (P < 0.05) in response to exercise, whereas a significant increase of skeletal muscle HSP72 protein content was not observed (P = 0.07). In CEalpha, skeletal muscle HSP72 mRNA, HSP72 protein, and serum HSP72 were not different from Control in response to exercise. In contrast, the effect of exercise on skeletal muscle HSP72 mRNA and protein, as well as circulating HSP72, was completely blunted in CEalphagamma. The results indicate that gamma-tocopherol comprises a potent inhibitor of the exercise-induced increase of HSP72 in skeletal muscle as well as in the circulation.     

Exercise,antioxidants, and HSP72: protection against myocardial ischemia/reperfusion

Endurance exercise is associated with protection against myocardial ischemia/reperfusion (I/R) injury and has been shown to increase heat shock protein 72 (HSP72). Dietary antioxidants have also been reported to decrease I/R-induced injury. Because exercise and antioxidants may provide cardioprotection via different mechanisms, combining these countermeasures could provide additive protection. Alternatively, because exercise-induced oxidant production may promote expression of HSP72, antioxidants could attenuate exercise-induced HSP72 expression and decrease exercise-related cardioprotection. These experiments examined the individual and combined effects of exercise and antioxidants on myocardial I/R injury (in vivo). Rats receiving a mixed antioxidant diet or control diet were assigned to exercise or sedentary groups and randomized to receive: (i) short I/R (myocardial stunning), (ii) long I/R (myocardial infarction), or (iii) sham surgery. Antioxidants significantly increased total antioxidant capacity and attenuated exercise-related HSP72 accumulation. Nonetheless, during short I/R, exercise-trained animals demonstrated improved left ventricular developed pressure (LVDP), independent of diet. Further, antioxidants alone resulted in improved LVDP. Finally, compared to control diet/sedentary animals, both exercise groups (control and antioxidant diets) and the antioxidant diet/sedentary group sustained smaller infarctions. We conclude that exercise and antioxidants can independently provide protection against myocardial contractile dysfunction and infarction, and the combination of these two strategies does not enhance or inhibit the protection observed with each individual countermeasure.     

Glutamine's protection against cellular injury is dependent on heat shock factor-1

Glutamine (GLN) has been shown to protect cells, tissues, and whole organisms from stress and injury. Enhanced expression of heat shock protein (HSP) has been hypothesized to be responsible for this protection. To date, there are no clear mechanistic data confirming this relationship. This study tested the hypothesis that GLN-mediated activation of the HSP pathway via heat shock factor-1 (HSF-1) is responsible for cellular protection. Wild-type HSF-1 (HSF-1^+/+) and knockout (HSF-1^–/–) mouse fibroblasts were used in all experiments. Cells were treated with GLN concentrations ranging from 0 to 16 mM and exposed to heat stress injury in a concurrent treatment model. Cell viability was assayed with phenazine methosulfate plus tetrazolium salt, HSP-70, HSP-25, and nuclear HSF-1 expression via Western blot analysis, and HSF-1/heat shock element (HSE) binding via EMSA. GLN significantly attenuated heat-stress induced cell death in HSF-1^+/+ cells in a dose-dependent manner; however, the survival benefit of GLN was lost in HSF-1^–/– cells. GLN led to a dose-dependent increase in HSP-70 and HSP-25 expression after heat stress. No inducible HSP expression was observed in HSF-1^–/– cells. GLN increased unphosphorylated HSF-1 in the nucleus before heat stress. This was accompanied by a GLN-mediated increase in HSF-1/HSE binding and nuclear content of phosphorylated HSF-1 after heat stress. This is the first demonstration that GLN-mediated cellular protection after heat-stress injury is related to HSF-1 expression and cellular capacity to activate an HSP response. Furthermore, the mechanism of GLN-mediated protection against injury appears to involve an increase in nuclear HSF-1 content before stress and increased HSF-1 promoter binding and phosphorylation. knockout cells; amino acid; heat stress mechanism     

HSP70 and other possible heat shock or oxidative stress proteins are induced in skeletal muscle, heart, and liver during exercise

Exercise causes heat shock (muscle temperatures of up to 45 degrees C, core temperatures of up to 44 degrees C) and oxidative stress (generation of O2- and H2O2), and exercise training promotes mitochondrial biogenesis (2-3-fold increases in muscle mitochondria). The concentrations of at least 15 possible heat shock or oxidative stress proteins (including one with a molecular weight of 70 kDa) were increased, in skeletal muscle, heart, and liver, by exercise. Soleus, plantaris, and extensor digitorum longus (EDL) muscles exhibited differential protein synthetic responses ([3H]leucine incorporation) to heat shock and oxidative stress in vitro but five proteins (particularly a 70 kDa protein and a 106 kDa protein) were common to both stresses. HSP70 mRNA levels were next analyzed by Northern transfer, using a [32P]-labeled HSP70 cDNA probe. HSP70 mRNA levels were increased, in skeletal and cardiac muscle, by exercise and by both heat shock and oxidative stress. Skeletal muscle HSP70 mRNA levels peaked 30-60 min following exercise, and appeared to decline slowly towards control levels by 6 h postexercise. Two distinct HSP70 mRNA species were observed in cardiac muscle; a 2.3 kb mRNA which returned to control levels within 2-3 h postexercise, and a 3.5 kb mRNA species which remained at elevated concentrations for some 6 h postexercise. The induction of HSP70 appears to be a physiological response to the heat shock and oxidative stress of exercise. Exercise hyperthermia may actually cause oxidative stress since we also found that muscle mitochondria undergo progressive uncoupling and increased O2- generation with increasing temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Loss of exercise-induced cardioprotection after cessation of exercise.

Endurance exercise provides cardioprotection against ischemia-reperfusion (I/R) injury. Exercise-induced cardioprotection is associated with increases in cytoprotective proteins, including heat shock protein 72 (HSP72) and increases in antioxidant enzyme activity. On the basis of the reported half-life of these putative cardioprotective proteins, we hypothesized that exercise-induced cardioprotection against I/R injury would be lost within days after cessation of exercise. To test this, male rats (4 mo) were randomly assigned to one of five experimental groups: 1). sedentary control, 2). exercise followed by 1 day of rest, 3). exercise followed by 3 days of rest, 4). exercise followed by 9 days of rest, and 5). exercise followed by 18 days of rest. Exercise-induced increases (P < 0.05) in left ventricular catalase activity and HSP72 were evident at 1 and 3 days postexercise. However, at 9 days postexercise, myocardial HSP72 and catalase levels declined to sedentary control values. To evaluate cardioprotection during recovery from I/R, hearts were isolated, placed in working heart mode, and subjected to 20.5 min of global ischemia followed by 30 min of reperfusion. Compared with sedentary controls, exercised animals sustained less I/R injury as evidenced by maintenance of a higher (P < 0.05) percentage of preischemia cardiac work during reperfusion at 1, 3, and 9 days postexercise. The exercise-induced cardioprotection vanished by 18 days after exercise cessation. On the basis of the time course of the loss of cardioprotection and the return of HSP72 and catalase to preexercise levels, we conclude that HSP72 and catalase are not essential for exercise-induced protection during myocardial stunning. Therefore, other cytoprotective molecules are responsible for providing protection during I/R.     

Short-term exercise training can improve myocardial tolerance to I/R without elevation in heat shock proteins

We examined the effects of 3 days of exercise in a cold environment on the expression of left ventricular (LV) heat shock proteins (HSPs) and contractile performance during in vivo ischemia-reperfusion (I/R). Sprague-Dawley rats were divided into the following three groups (n = 12/group): 1) control, 2) exercise (60 min/day) at 4 degrees C (E-Cold), and 3) exercise (60 min/day) at 25 degrees C (E-Warm). Left anterior descending coronary occlusion was maintained for 20 min, followed by 30 min of reperfusion. Compared with the control group, both the E-Cold and E-Warm groups maintained higher (P < 0.05) LV developed pressure, first derivative of pressure development over time (+dP/dt), and pressure relaxation over time (-dP/dt) throughout I/R. Relative levels of HSP90, HSP72, and HSP40 were higher (P < 0.05) in E-Warm animals compared with both control and E-Cold. HSP10, HSP60, and HSP73 did not differ between groups. Exercise increased manganese superoxide dismutase (MnSOD) activity in both E-Warm and E-Cold hearts (P < 0.05). Protection against I/R-induced lipid peroxidation in the LV paralleled the increase in MnSOD activity whereas lower levels of lipid peroxidation were observed in both E-Warm and E-Cold groups compared with control. We conclude that exercise-induced myocardial protection against a moderate duration I/R insult is not dependent on increases in myocardial HSPs. We postulate that exercise-associated cardioprotection may depend, in part, on increases in myocardial antioxidant defenses.     

Regulatory Coordination between Two Major Intracellular Homeostatic Systems: HEAT SHOCK RESPONSE AND AUTOPHAGY*

The eukaryotic cell depends on multitiered homeostatic systems ensuring maintenance of proteostasis, organellar integrity, function and turnover, and overall cellular viability. At the two opposite ends of the homeostatic system spectrum are heat shock response and autophagy. Here, we tested whether there are interactions between these homeostatic systems, one universally operational in all prokaryotic and eukaryotic cells, and the other one (autophagy) is limited to eukaryotes. We found that heat shock response regulates autophagy. The interaction between the two systems was demonstrated by testing the role of HSF-1, the central regulator of heat shock gene expression. Knockdown of HSF-1 increased the LC3 lipidation associated with formation of autophagosomal organelles, whereas depletion of HSF-1 potentiated both starvation- and rapamycin-induced autophagy. HSP70 expression but not expression of its ATPase mutant inhibited starvation or rapamycin-induced autophagy. We also show that exercise induces autophagy in humans. As predicted by our in vitro studies, glutamine supplementation as a conditioning stimulus prior to exercise significantly increased HSP70 protein expression and prevented the expected exercise induction of autophagy. Our data demonstrate for the first time that heat shock response, from the top of its regulatory cascade (HSF-1) down to the execution stages delivered by HSP70, controls autophagy thus connecting and coordinating the two extreme ends of the homeostatic systems in the eukaryotic cell.     

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.     

Exercise-induced HSP-72 elevation and cardioprotection against infarct and apoptosis.

Successive bouts of endurance exercise are associated with both increased cardiac levels of heat shock protein-72 (HSP-72) and improved cardioprotection against ischemia-reperfusion (I/R)-induced cardiac cell death. Although overexpression of HSP-72 has been shown to be cardioprotective in transgenic animals, it is unclear whether increased levels of HSP-72 are essential for exercise-induced cardioprotection against I/R-mediated cell death. We tested the hypothesis that exercise-induced increases in myocardial levels of HSP-72 are required to achieve exercise-mediated protection against I/R-induced cardiac cell death. To test this postulate, we investigated the effect of preventing the exercise-induced increase in cardiac HSP-72 on myocardial infarction and apoptosis after 50 min of in vivo ischemia and 120 min of reperfusion. Adult male rats remained sedentary or performed successive bouts of endurance exercise in cold (8 degrees C) or warm (22 degrees C) environments. We found that, compared with sedentary control animals, exercise in a warm environment significantly increased myocardial HSP-72 content. In contrast, exercise in the cold environment prevented the exercise-induced increase in myocardial HSP-72 levels. After in vivo myocardial I/R, infarct size was reduced in both exercised groups compared with sedentary animals. Furthermore, compared with sedentary rats, I/R-induced myocardial apoptosis (as indicated by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive nuclei and caspase-3 activity) was attenuated in both groups of exercised animals. Therefore, although HSP-72 has cardioprotective properties, our results reveal that increased myocardial levels of HSP-72 (above control) are not essential for exercise-induced protection against I/R-induced myocardial infarction and apoptosis.     

Attenuation of exercise-induced heat shock protein 72 expression blunts improvements in whole-body insulin resistance in rats with type 2 diabetes

Heat shock proteins (HSPs) play an important role in insulin resistance and improve the cellular stress response via HSP induction by exercise to treat type 2 diabetes. In this study, the effects of exercise-induced HSP72 expression levels on whole-body insulin resistance in type 2 diabetic rats were investigated. Male 25-week-old Otsuka Long-Evans Tokushima Fatty rats were divided into three groups: sedentary (Sed), trained in a thermal-neutral environment (NTr: 25 °C), and trained in a cold environment (CTr: 4 °C). Exercise training was conducted 5 days/week for 10 weeks. Rectal temperature was measured following each bout of exercise. An intraperitoneal glucose tolerance test (IPGTT) was performed after the training sessions. The serum, gastrocnemius muscle, and liver were sampled 48 h after the final exercise session. HSP72 and heat shock cognate protein 73 expression levels were analyzed by Western blot, and serum total cholesterol, triglyceride (TG), and free fatty acid (FFA) levels were measured. NTr animals exhibited significantly higher body temperatures following exercise, whereas, CTr animals did not. Exercise training increased HSP72 levels in the gastrocnemius muscle and liver, whereas, HSP72 expression was significantly lower in the CTr group than that in the NTr group (p < 0.05). Glucose tolerance improved equally in both trained animals; however, insulin levels during the IPGTT were higher in CTr animals than those in NTr animals (p < 0.05). In addition, the TG and FFA levels decreased significantly only in NTr animals compared with those in Sed animals. These results suggest that attenuation of exercise-induced HSP72 expression partially blunts improvement in whole-body insulin resistance and lipid metabolism in type 2 diabetic rats.     

α-Lipoic Acid Inhibits Endotoxin-stimulated Expression of iNOS and Nitric Oxide Independent of the Heat Shock Response in RAW 264.7 Cells

The heat shock response protects against sepsis-induced mortality, organ injury, cardiovascular dysfunction, and apoptosis. Several inducers of the heat shock response, such as hyperthermia, sodium arsenite, and pyrollidine dithiocarbonate, inhibit NF-κB activation and nitric oxide formation. The antioxidant lipoic acid (LA) has recently been found to inhibit NF-κB activation and nitric oxide formation. We therefore tested the hypothesis that LA induces a heat shock response. To test this hypothesis, we determined whether exposure to LA affects expression of both heat shock protein 70 (HSP-70) and nuclear heat shock factor-1 (HSF-1) in lipopolysaccharide (LPS) stimulated macrophages. LA and hyperthermia attenuated LPS-induced increases in nuclear NF-κB, iNOS protein, and media nitrite concentrations. LPS and hyperthermia increased HSP-70 concentrations 8-fold and 20-fold, respectively. No effect of LA treatment alone on HSP-70 protein expression was detected. Likewise, no effect of LA on HSF-1 protein expression was detected. These data suggest that LA inhibits LPS-induced activation of iNOS in macrophages independent of the heat shock response.     

alpha-lipoic acid inhibits endotoxin-stimulated expression of iNOS and nitric oxide independent of the heat shock response in RAW 264.7 cells

The heat shock response protects against sepsis-induced mortality, organ injury, cardiovascular dysfunction, and apoptosis. Several inducers of the heat shock response, such as hyperthermia, sodium arsenite, and pyrollidine dithiocarbonate, inhibit NF-κB activation and nitric oxide formation. The antioxidant lipoic acid (LA) has recently been found to inhibit NF-κB activation and nitric oxide formation. We therefore tested the hypothesis that LA induces a heat shock response. To test this hypothesis, we determined whether exposure to LA affects expression of both heat shock protein 70 (HSP-70) and nuclear heat shock factor-1 (HSF-1) in lipopolysaccharide (LPS) stimulated macrophages. LA and hyperthermia attenuated LPS-induced increases in nuclear NF-κB, iNOS protein, and media nitrite concentrations. LPS and hyperthermia increased HSP-70 concentrations 8-fold and 20-fold, respectively. No effect of LA treatment alone on HSP-70 protein expression was detected. Likewise, no effect of LA on HSF-1 protein expression was detected. These data suggest that LA inhibits LPS-induced activation of iNOS in macrophages independent of the heat shock response.     

Short-term exercise improves myocardial tolerance to in vivo ischemia-reperfusion in the rat.

These experiments examined the independent effects of short-term exercise and heat stress on myocardial responses during in vivo ischemia-reperfusion (I/R). Female Sprague-Dawley rats (4 mo old) were randomly assigned to one of four experimental groups: 1) control, 2) 3 consecutive days of treadmill exercise [60 min/day at 60-70% maximal O2 uptake (VO2 max)], 3) 5 consecutive days of treadmill exercise (60 min/day at 60-70% VO2 max), and 4) whole body heat stress (15 min at 42 degrees C). Twenty-four hours after heat stress or exercise, animals were anesthetized and mechanically ventilated, and the chest was opened by thoracotomy. Coronary occlusion was maintained for 30-min followed by a 30-min period of reperfusion. Compared with control, both heat-stressed animals and exercised animals (3 and 5 days) maintained higher (P < 0.05) left ventricular developed pressure (LVDP), maximum rate of left ventricular pressure development (+dP/dt), and maximum rate of left ventricular pressure decline (-dP/dt) at all measurement periods during both ischemia and reperfusion. No differences existed between heat-stressed and exercise groups in LVDP, +dP/dt, and -dP/dt at any time during ischemia or reperfusion. Both heat stress and exercise resulted in an increase (P < 0.05) in the relative levels of left ventricular heat shock protein 72 (HSP72). Furthermore, exercise (3 and 5 days) increased (P < 0.05) myocardial glutathione levels and manganese superoxide dismutase activity. These data indicate that 3-5 consecutive days of exercise improves myocardial contractile performance during in vivo I/R and that this exercise-induced myocardial protection is associated with an increase in both myocardial HSP72 and cardiac antioxidant defenses.