Time course of responses of human skeletal muscle to oxidative stress induced by nondamaging exercise.

Previous studies in animals have demonstrated that a single period of aerobic exercise induces a rise in the skeletal muscle activity of the antioxidant enzymes superoxide dismutase and catalase and an increase in the muscle content of heat shock proteins (HSPs). The purpose of this study was to examine the time course of response of human skeletal muscle superoxide dismutase and catalase activities and the content of HSP60 and HSP70 after a period of exhaustive, nondamaging aerobic exercise. Seven volunteers undertook one-legged cycle ergometry at 70% maximal oxygen uptake for 45 min. Biopsies were obtained from the vastus lateralis muscle 7 days before and at 1, 2, 3, and 6 days after exercise. Muscle superoxide dismutase activity increased to a peak at 3 days postexercise, muscle catalase activities were unchanged, and muscle content of HSP60 and the inducible HSP70 increased by variable amounts to reach means of 190% and 3,100% of preexercise values, respectively, by 6 days postexercise. These data indicate that human skeletal muscle responds to a single bout of nondamaging exercise by increasing superoxide dismutase activity and provide the first evidence of an increase in HSP content of human skeletal muscle after a submaximal exercise bout.     

Lifelong training preserves some redox-regulated adaptive responses after an acute exercise stimulus in aged human skeletal muscle

Several redox-regulated responses to an acute exercise bout fail in aged animal skeletal muscle, including the ability to upregulate the expression of antioxidant defense enzymes and heat shock proteins (HSPs). These findings are generally derived from studies on sedentary rodent models and thus may be related to reduced physical activity and/or intraspecies differences as opposed to aging per se. This study, therefore, aimed to determine the influence of age and training status on the expression of HSPs, antioxidant enzymes, and NO synthase isoenzymes in quiescent and exercised human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis before and 3 days after an acute high-intensity-interval exercise bout in young trained, young untrained, old trained, and old untrained subjects. Levels of HSP72, PRX5, and eNOS were significantly higher in quiescent muscle of older compared with younger subjects, irrespective of training status. 3-NT levels were elevated in muscles of the old untrained but not the old trained state, suggesting that lifelong training may reduce age-related macromolecule damage. SOD1, CAT, and HSP27 levels were not significantly different between groups. HSP27 content was upregulated in all groups studied postexercise. HSP72 content was upregulated to a greater extent in muscle of trained compared with untrained subjects postexercise, irrespective of age. In contrast to every other group, old untrained subjects failed to upregulate CAT postexercise. Aging was associated with a failure to upregulate SOD2 and a downregulation of PRX5 in muscle postexercise, irrespective of training status. In conclusion, lifelong training is unable to fully prevent the progression toward a more stressed muscular state as evidenced by increased HSP72, PRX5, and eNOS protein levels in quiescent muscle. Moreover, lifelong training preserves some (e.g., CAT) but not all (e.g., SOD2, HSP72, PRX5) of the adaptive redox-regulated responses after an acute exercise bout. Collectively, these data support many but not all of the findings from previous animal studies and suggest parallel aging effects in humans and mice at rest and after exercise that are not modulated by training status in human skeletal muscle.     

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.     

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)     

Acute heat stress prior to downhill running may enhance skeletal muscle remodeling

Heat shock proteins (HSPs) are chaperones that are known to have important roles in facilitating protein synthesis, protein assembly and cellular protection. While HSPs are known to be induced by damaging exercise, little is known about how HSPs actually mediate skeletal muscle adaption to exercise. The purpose of this study was to determine the effects of a heat shock pretreatment and the ensuing increase in HSP expression on early remodeling and signaling (2 and 48 h) events of the soleus (Sol) muscle following a bout of downhill running. Male Wistar rats (10 weeks old) were randomly assigned to control, eccentric exercise (EE; downhill running) or heat shock + eccentric exercise (HS; 41°C for 20 min, 48 h prior to exercise) groups. Markers of muscle damage, muscle regeneration and intracellular signaling were assessed. The phosphorylation (p) of HSP25, Akt, p70s6k, ERK1/2 and JNK proteins was also performed. As expected, following exercise the EE group had increased creatine kinase (CK; 2 h) and mononuclear cell infiltration (48 h) compared to controls. The EE group had an increase in p-HSP25, but there was no change in HSP72 expression, total protein concentration, or neonatal MHC content. Additionally, the EE group had increased p-p70s6k, p-ERK1/2, and p-JNK (2 h) compared to controls; however no changes in p-Akt were seen. In contrast, the HS group had reduced CK (2 h) and mononuclear cell infiltration (48 h) compared to EE. Moreover, the HS group had increased HSP72 content (2 and 48 h), total protein concentration (48 h), neonatal MHC content (2 and 48 h), p-HSP25 and p-p70s6k (2 h). Lastly, the HS group had reduced p-Akt (48 h) and p-ERK1/2 (2 h). These data suggest that heat shock pretreatment and/or the ensuing HSP72 response may protect against muscle damage, and enhance increases in total protein and neonatal MHC content following exercise. These changes appear to be independent of Akt and MAPK signaling pathways.     

Heat stress facilitates stretch-induced hypertrophy of cultured muscle cells

Increased mechanical stress induced by stretch is an important growth stimulus in skeletal muscle. Heat shock proteins (HSPs) are an important family of endogenous, protective proteins. HSP90 and HSP70 families show elevated levels under beat stress. Mechanical stress, such as physical exercise, is known to induce not only muscular hypertrophy but also the elevation of HSPs expression in skeletal muscle. The purpose of this study was to determine whether heat stress facilitates the stretch-induced hypertrophy of skeletal muscle cells. Cultured rat myotubes (L6) were plated on collagenized Silastic membranes and incubated at 41 degrees C for 60 and 75 minutes (heat shock). Following the incubation, the cells were subjected two-second stretching and four-second releasing for 4 days at 37 degrees C. Protein concentrations in the homogenates and pellets of the cultured skeletal muscle cells increased under heat shock and/or mechanical stretching. The protein concentration of cells following mechanical stretching following heat shock was significantly higher than that following either heat shock or mechanical stretching alone. HSP72 in supernatants and HSP90 in pellets increased under heat shock and/or mechanical stretching. HSP90 in supernatants decreased following heat shock and/or mechanical stretching. Changes in HSPs and cellular protein concentrations in stressed cells suggest that the expression of HSPs may be closely related with muscular hypertrophy.     

Quantitative RT-PCR analysis and immunohistochemical localization of HSP70 in sea bass Dicentrarchus labrax exposed to transport stress

In aquaculture, fish are exposed to stressful conditions, which cause an increased synthesis of heat shock proteins (HSPs) at the cellular level. In this work we considered the expression of the constitutive and inducible forms of HSP70 as an indicator of stress caused by transport, during development of the sea bass (Dicentrarchus labrax), a teleost fish of high value for aquaculture. Qualitative RT-PCR analysis revealed expression of inducible HSP70 gene in larvae and fry (25, 40 and 80 days) as well as in adult tissues (liver, brain, muscle, gills, kidney, gonads, heart, spleen and skin) of both control and stressed animals. Expression of inducible HSP70 mRNA examined in different adult tissues by Real-Time PCR, was significantly higher in skin and skeletal muscle of stressed animals than in controls. Immunolocalization of inducible and constitutive forms of heat shock protein 70 (HSP70 and HSC70), reported here for the first time, demonstrated an ubiquitous distribution of HSC70 protein in several tissues of both stressed and control animals (at all stages), while inducible HSP70 protein was found only in skeletal muscle of stressed animals. In all stressed animals, regardless of their developmental stage, cortisol levels were higher than in control animals.     

Secretion of the heat shock proteins HSP70 and HSC70 by baby hamster kidney (BHK‐21) cells

The HSPs (heat‐shock proteins) of the 70‐kDa family, the constitutively expressed HSC70 (cognate 70‐kDa heat‐shock protein) and the stress‐inducible HSP70 (stress‐inducible 70‐kDa heat‐shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK‐21) cells released HSP70 and HSC70 in a serum‐free medium and that this process was the result of an active secretion of HSPs rather than the non‐specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non‐classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl‐β‐cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK‐21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK‐21 cells in a serum‐free medium through a non‐classical pathway in which lipid rafts play an important role.     

Muscle type-specific response of HSP60, HSP72, and HSC73 during recovery after elevation of muscle temperature.

An original method to induce heat stress was used to clarify the time course of changes in heat shock proteins (HSPs) in rat skeletal muscles during recovery after a single bout of heat stress. One hindlimb was inserted into a stainless steel can and directly heated by raising the air temperature inside the can via a flexible heater twisted around the steel can. Muscle temperature was increased gradually and maintained at 42 degrees C for 60 min. Core rectal and contralateral muscle temperatures were increased <1.5 degrees C during the heat stress. HSP60, HSP72, and heat shock cognate (HSC) 73 content in the slow soleus and fast plantaris in both limbs were determined immediately (0 h) and 2, 4, 8, 12, 24, 36, 48, or 60 h after heat stress. Within 0-4 h, all HSPs were approximately 1.5- to 2.2-fold higher in heat-stressed than contralateral soleus. Compared with the contralateral plantaris, the heat-stressed plantaris had a higher (1.5-fold) HSP60 content immediately and 2 h after heat stress and a higher (2.5- to 6.8-fold) HSP72 content between 24 and 48 h after heat stress. Plantaris HSC73 content was not affected by heat stress. This unique heat-stress method provides advantages over existing systems; muscle temperature can be controlled precisely during heating and the HSP response can be compared between muscles in heat-stressed and contralateral limbs of individual rats. Results show a differential response of HSPs in the soleus and plantaris during recovery after heat stress; soleus demonstrated a more rapid and broader HSP response to heat stress than plantaris.     

Expression of small heat shock protein alphaB-crystallin is induced after hepatic stellate cell activation

Using the differential PCR display method to select cDNA fragments that are differentially expressed after hepatic stellate cell (HSC) activation, we have isolated from activated HSCs a cDNA that corresponds to rat alphaB-crystallin. Northern blots confirmed expression of alphaB-crystallin in culture-activated HSCs but not in quiescent HSCs. Western blot analysis and immunocytochemical staining confirmed expression of alphaB-crystallin protein in activated but not quiescent HSCs. alphaB-crystallin is induced as early as 6 h after plating HSCs on plastic and continues to be expressed for 14 days in culture. Expression of alphaB-crystallin was also induced in vivo in activated HSCs from experimental cholestatic liver fibrosis. Confocal microscopy demonstrated a cytoplasmic distribution of alphaB-crystallin in a cytoskeletal pattern. Heat shock treatment resulted in an immediate perinuclear redistribution that in time returned to a normal cytoskeletal distribution. The expression pattern of alphaB-crystallin was similar to that of HSP25, another small heat shock protein, but differed from the classic heat shock protein HSP70. Therefore, alphaB-crystallin represents an early marker for HSC activation.     

Exercise-induced elevation of HSP70 is intensity dependent.

Exercise induces expression of the protective heat shock protein, HSP70, in striated muscle. To characterize the relationship between induction of this protein and exercise intensity in muscles exhibiting different recruitment patterns, male Sprague-Dawley rats were assigned to a sedentary control or one of seven exercise groups for which treadmill running speed varied between 15 and 33 m/min (n = 8/group). Twenty-four hours after a single 60-min exercise bout, hearts, red and white portions of the vastus (RV and WV, respectively) muscles, and soleus (Sol) muscles were harvested and analyzed for both relative and absolute HSP70 content. Cardiac HSP70 was significantly elevated only when animals were exercised at 24 m/min and beyond. Similarly, HSP70 was elevated in RV at running speeds above 24 m/min but did not increase in WV until 27 m/min. In contrast, HSP70 content was initially elevated in the Sol but subsequently declined at the highest running speeds. The observed patterns of HSP70 expression in skeletal muscle were in general accordance with known muscle recruitment patterns and suggest that alterations in muscle loading, resulting from changes in exercise intensity, are an important component of exercise-induced increases in HSP70 content.     

Myocardial accumulation and localization of the inducible 70-kDa heat shock protein, Hsp70, following exercise

Exercise increases the 70-kDa heat shock protein (Hsp70) in the myocardium, and this exercise-induced increase is associated with significantly improved cardiac recovery following insult. However, while heat shock has been shown to elevate Hsp70 primarily in the cardiac vasculature of the myocardium, the localization following exercise is unknown. Male Sprague-Dawley rats performed continuous treadmill running at 30 m/min for 60 min (2% incline) on either 1 or 5 consecutive days. At 30 min and 24 h following exercise, hearts were extirpated, and the left ventricle was isolated, OCT-cork mounted, and sectioned for immunofluorescent analysis. Whereas immunofluorescent analysis revealed little to no Hsp70 in control hearts and 30 min postexercise, the accumulation of Hsp70 24 h after a single exercise bout or 5 days of training was predominantly located in large blood vessels and, in particular, colocalized with a marker of smooth muscle. Furthermore, higher core temperatures attained during exercise led to more abundant accumulation in smaller vessels and the endothelium. It is concluded that the accumulation of myocardial Hsp70 following acute exercise predominantly occurs in a cell type-specific manner, such that changes in the cardiac vasculature account for much of the increase. This accumulation appears first in the smooth muscle of larger vessels and then increases in smaller vessels and the endothelium, as core temperature attained during exercise increases. This finding supports the observations after heat shock and further suggests that the vasculature is a primary target in exercise-induced cardioprotection.     

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.     

Maximal eccentric exercise induces a rapid accumulation of small heat shock proteins on myofibrils and a delayed HSP70 response in humans

In this study the stress protein response to unaccustomed maximal eccentric exercise in humans was investigated. Eleven healthy males performed 300 maximal eccentric actions with the quadriceps muscle. Biopsies from vastus lateralis were collected at 30 min and 4, 8, 24, 96, and 168 h after exercise. Cellular regulation and localization of heat shock protein (HSP) 27, alpha B-crystallin, and HSP70 were analyzed by immunohistochemistry, ELISA technique, and Western blotting. Additionally, mRNA levels of HSP27, alpha B-crystallin, and HSP70 were quantified by Northern blotting. After exercise (30 min), 81 +/- 8% of the myofibers showed strong HSP27 staining (P < 0.01) that gradually decreased during the following week. alpha B-Crystallin mimicked the changes observed in HSP27. After exercise (30 min), the ELISA analysis showed a 49 +/- 13% reduction of the HSP27 level in the cytosolic fraction (P < 0.01), whereas Western blotting revealed a 15-fold increase of the HSP27 level in the myofibrillar fraction (P < 0.01). The cytosolic HSP70 level increased to 203 +/- 37% of the control level 24 h after exercise (P < 0.05). After 4 days, myofibrillar-bound HSP70 had increased approximately 10-fold (P < 0.01) and was accompanied by strong staining on cross sections. mRNA levels of HSP27, alpha B-crystallin, and HSP70 were all elevated the first day after exercise (P < 0.01); HSP70 mRNA showed the largest increase (20-fold at 8 h). HSP27 and alpha B-crystallin seemed to respond immediately to maximal eccentric exercise by binding to cytoskeletal/myofibrillar proteins, probably to function as stabilizers of disrupted myofibrillar structures. Later, mRNA and total HSP protein levels, especially HSP70, increased, indicating that HSPs play a role in skeletal muscle recovery and remodeling/adaptation processes to high-force exercise.     

Expression of exercise-induced HSP70 in long-distance runner''s leukocytes

This study was designed to investigate the expression of heat shock protein 70 (HSP70), after acute moderate intensity exercise, in human peripheral blood leukocytes of trained runners and untrained controls. Ten male long-distance trained runners (TR) and untrained sedentary control subjects (SED) ran for 1 h at 70% of heart rate reserve (HRR). Basal HSP70 expression in TR was usually lower than that in SED, but basal HSP70 gene expression in TR was usually higher than that in SED. Although expression rates of exercise-induced HSP70 in both groups were similar, levels of HSP70 in SED were significantly higher than in TR. Significant increases in leukocytes, neutrophils, and lymphocytes after exercise were observed in both groups, but there were some differences between groups. We conclude that 1 h treadmill running at 70% HRR intensity is a sufficient stimulus to leukocytosis, neutrocytosis, lymphocytosis, and HSP70 proteins and gene expression in leukocytes. Adaptation to training was observed in TR.     

Effect of postexercise carbohydrate supplementation on glucose uptake-associated gene expression in the human skeletal muscle

We previously found that the exercise-induced elevation in GLUT4 mRNA of rat muscle can be rapidly down-regulated when glucose is given immediately following exercise. The purpose of this study was to determine the effect of postexercise carbohydrate diet on GLUT4 and hexokinase (HK) II mRNA levels in the human skeletal muscle. Eight untrained male subjects (age, 20.7+/-3.1 years) exercised for 60 min on a cycle ergometer at a 70-75% maximal oxygen consumption. The postexercise dietary treatment was performed in a crossover design. Immediately after the exercise, a diet with 70% carbohydrate content (1 g per kilogram of body weight; 356+/-19.8 kcal) was given to half of the subjects (eaten in 10 min) followed by a 3-h recovery, while the control subjects remained unfed for 3 h. Biopsies were performed on the deep portion of the vastus lateralis muscle of all subjects immediately after the exercise and 3 h after the carbohydrate ingestion. Blood glucose and serum insulin concentrations were measured every 30 min for 3 h. At the end of the 3-h recovery, blood glucose and serum insulin levels were not different from control levels, indicating that the oral carbohydrate was mostly disposed in the body within 3 h. In addition, GLUT4 and HK II mRNA levels were significantly lowered in the exercised human skeletal muscle in subjects receiving the carbohydrate diet. In conclusion, the present study demonstrates that GLUT4 mRNA and HK II mRNA in the exercised human skeletal muscle were significantly lowered by a high-carbohydrate diet.     

Exercise training reverses downregulation of HSP70 and antioxidant enzymes in porcine skeletal muscle after chronic coronary artery occlusion

Oxidative stress is associated with muscle fatigue and weakness in skeletal muscle of ischemic heart disease patients. Recently, it was found that endurance training elevates protective heat shock proteins (HSPs) and antioxidant enzymes in skeletal muscle in healthy subjects and antioxidant enzymes in heart failure patients. However, it is unknown whether coronary ischemia and mild infarct without heart failure contributes to impairment of stress proteins and whether exercise training reverses those effects. We tested the hypothesis that exercise training would reverse alterations in muscle TNF-alpha, oxidative stress, HSP70, SOD (Mn-SOD, Cu,Zn-SOD), glutathione peroxidase (GPX), and catalase (CAT) due to chronic coronary occlusion of the left circumflex (CCO). Yucatan swine were divided into three groups (n = 6 each): sedentary with CCO (SCO); 12 wk of treadmill exercise training following CCO (ECO); and sham surgery controls (sham). Forelimb muscle mass-to-body mass ratio decreased by 27% with SCO but recovered with ECO. Exercise training reduced muscle TNF-alpha and oxidative stress (4-hydroxynonenal adducts) caused by CCO. HSP70 levels decreased with CCO (-45%), but were higher with exercise training (+348%). Mn-SOD activity, Mn-SOD protein expression, and Cu,Zn-SOD activity levels were higher in ECO than SCO by 72, 82, and 112%, respectively. GPX activity was 177% greater in ECO than in SCO. CAT trended higher (P = 0.059) in ECO compared with SCO. These data indicate that exercise training following onset of coronary artery occlusion results in recovery of critical stress proteins and reduces oxidative stress.     

Alpha-lipoic acid does not alter stress protein response to acute exercise in diabetic brain

Heat shock proteins (HSPs) are molecular chaperones which may act protective in cerebrovascular insults and peripheral diabetic neuropathy. We hypothesized that alpha-lipoic acid (LA), a natural thiol antioxidant, may enhance brain HSP response in diabetes. Rats with or without streptozotocin-induced diabetes were treated with LA or saline for 8 weeks. Half of the rats were subjected to exhaustive exercise to investigate HSP induction, and the brain tissue was analyzed. Diabetes increased constitutive HSC70 mRNA, and decreased HSP90 and glucose-regulated protein 75 (GRP75) mRNA without affecting protein levels. Exercise increased HSP90 protein and mRNA, and also GRP75 and heme oxygenase-1 (HO-1) mRNA only in non-diabetic animals. LA had no significant effect on brain HSPs, although LA increased HSC70 and HO-1 mRNA in diabetic animals and decreased HSC70 mRNA in non-diabetic animals. Eukaryotic translation elongation factor-2, essential for protein synthesis, was decreased by diabetes and suggesting a mechanism for the impaired HSP response related to translocation of the nascent chain during protein synthesis. LA supplementation does not offset the adverse effects of diabetes on brain HSP mRNA expression. Diabetes may impair HSP translation through elongation factors related to nascent chain translocation and subsequent responses to acute stress.     

Molecular identification and expression of heat shock cognate 70 (HSC70) in the Pacific white shrimp Litopenaeus vannamei

Heat shock protein 70s (HSP70s) are fundamental chaperone proteins that are indispensable to most living organisms. In order to investigate the function of HSP70 and heat shock response in shrimp, a heat shock cognate (HSC70) gene of the white shrimp (Litopenaeus vannamei), containing a 1959-bp open reading frame, was cloned and characterized. The amino acid sequence, 71.5 kDa of molecular weight, shares 80-99.6% homology with 12 diverse species' HSP70s and HSC70s. In fact, some segments of the eukaryotic HSC70 sequence, such as ATP/GTP-binding site, cytoplasmic HSP70 C-terminal sequence, and GGMP/GAP repeats, are also found in the putative shrimp HSC70. Moreover, multi-tissue RT-PCR was performed to assay the basal expressions of HSC70 in the heart, gill, hepatopancreas, stomach, gut, and muscle. The results demonstrate that the basal expressions of HSC70 in theses organs are similar to that of beta-actin. Furthermore, quantitative real-time experiments showed that HSC70 was up-regulated in hepatopancreas (4.6-fold), stomach (5.9-fold), gut (2.6-fold), and muscle (3.5-fold) but not in the heart (1.7-fold) and gill (1.6-fold) after 2 h of heat shock. Nevertheless, the HSC70 was found to be highly expressed in the heart and gill following 6 h of heat shock. This suggests that HSC70 in white shrimp possess both short-term and long-term responses to heat shock stress, indicating this HSC70 may be a heat-dependent HSC70 member. Finally, we constructed an expression vector to generate HSC70 in Escherichia coli BL21, which displayed immune cross-reactivity with mouse HSP70 antibody. In conclusion, the identification and expression of white shrimp HSC70 gene present useful data for studying the molecular mechanism of heat shock response and the effect of heat shock proteins in shrimps' cytoprotection.