INTRODUCTION

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MANY CELLS AND TISSUES are able to survive a potentially lethal stress after an acute preconditioning stimulus such as heat shock or ischemia. Although the exact basis for this protective effect is unclear, several studies have revealed that these preconditioning stimuli induce the synthesis of a group of proteins known as heat shock (HSP) or, more generally, stress proteins (SPs; 3, 6, 16). The increased expression of SP72, an inducible isoform of one of these proteins from the 70-kDa family of SPs, has been correlated with reduced myocardial infarct size (5, 16) and less damage and enhanced contractile recovery after ischemia-reperfusion (6). The observation that transgenic mice, expressing high levels of SP72, also demonstrate enhanced contractile (22) and metabolic (24) recovery after ischemia-reperfusion suggests that SP72 plays a direct role in the observed cardioprotection.

It has been clearly established that exercise training reduces the risk of cardiovascular heart disease (17, 18, 30). Interestingly, exercise also induces the expression of SPs (10, 14), and it has been demonstrated by Locke and colleagues (14) that, after only 3 days of exercise training, an exercise-induced increase in SP72 is associated with myocardial protection. However, the forced-treadmill-run training protocol employed in this study (14) represents a severe exercise stress (2). In the present investigation, therefore, we examined whether voluntary exercise training could also induce potentially cardioprotective SPs to a degree similar to that observed after high-intensity treadmill run training.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals. This study was approved by the University of Colorado Committee on Animal Care and performed in accordance to the guiding principles of the Guide for the Institutional Care and Use of Laboratory Animals [Institute of Laboratory Resources, NRC, DHEW Publ. No. (NIH) 85-23, revised 1985, Office of Science and Health Reports, DRR/NIH]. Thirty-six 10-wk-old male Sprague-Dawley rats were individually housed and randomly assigned to groups that exercise trained on a motor-driven treadmill (TM-Tr; n = 10) or a free wheel attached to the holding cage (FW-Tr; n = 12) or to sedentary control groups that were caged and/or handled (TM-Sed; n = 8 and FW-Sed; n = 6, respectively). Rats in the TM-Sed group were caged and handled identically to their TM-Tr counterparts, except that, when they were placed on the treadmill, the apparatus was not turned on. Those in the FW-Sed group were housed in cages with a free wheel run compartment, except that the wheel was rendered immobile. Animals were maintained on a 12:12-h dark-light cycle, at 20 ± 1°C, 50% relative humidity, and were fed and watered ad libitum.

Training procedures. Rats in the TM-Tr group were subjected to a progressive exercise training program, 5 days/wk, on a motor-driven treadmill. Initially, rats ran at 17.4 m/min up a 10% grade for 15 min, with the training duration being increased by 5 min/day until the animals were running for 60 min/session. Thereafter, velocity was incrementally increased until animals were running 8,282 m/wk by the seventh week of training. At this time, each training session consisted of a warm-up of 5 min each at 21.5 and 25.5 m/min, followed by 45 min at 29.2 m/min, and finished with a 5-min warm-down at 21.5 m/min.

Euthansia. Animals from the TM-Tr group were killed 18 h after completion of the last training bout, whereas those from the FW-Tr group had continuous access to the training wheel up to the time of death. Both trained groups and their respective sedentary controls were killed by decapitation. On removal, hearts were divided into right (RV) and left (LV) ventricles, immediately frozen in liquid nitrogen, and stored at 80°C until subsequent analysis.

SDS-PAGE. Frozen samples of ~60-mg mass were homogenized in 19 volumes of 600 mM NaCl and 15 mM Tris, pH 7.5. Muscle protein concentrations were determined by using the technique described by Lowry and colleagues (15) with BSA as the standard. One hundred micrograms of protein from cardiac muscle homogenates diluted in 2× sample buffer and boiled for 5 min were loaded onto one-dimensional SDS 12% polyacrylamide gels. PAGE was performed as described by Laemmli (8). Proteins were transferred from the gels to nitrocellulose membranes (0.2-µm thickness; Bio-Rad Laboratories) according to the method described by Towbin and colleagues (29), using the Bio-Rad mini-protein II gel-transfer system. After transfer, nitrocellulose membranes were treated in a manner similar to that described by Locke et al. (11), and Western blots were probed for inducible (SP72, StressGen SPA-812), constitutive (SP73, StressGen SPA-815), and mitochondrial (SP75, StressGen SPA-825) isoforms of the SP70 family. An alkaline phosphatase-conjugated secondary antibody (goat anti-mouse IgG, Bio-Rad) reacted in a carbonate buffer (100 mM Na₂CO₃, 1 mM MgCl₂, pH 9.8) containing 3% (wt/vol) p-nitro blue tetrazolium chloride p-toluidine salt in 70% N,N-dimethylformamide and 15% (wt/vol) 5-bromo-4-chloro-3-indolyl phosphate in 100% N,N-dimethylformamide was used to visualize the signal intensity. Quantification of relative protein content was made by using an LKB Ultroscan XL laser densitometer equipped with an LKB 2220 recording integrator. For all blots, one sample of RV and LV from each of the groups was loaded. Relative concentrations were tabulated as a percentage of the RV value for the FW-Sed group on the same blot. Relative migration of the protein of interest was determined by simultaneously running molecular weight markers (Bio-Rad Kaleidoscope prestained standards) on each gel. Gels run as above but stained in Coomassie brilliant blue G in 50% methanol and 10% glacial acetic acid and destained in 50% methanol and 10% glacial acetic acid were employed to further ensure that all lanes were equally loaded.

Citrate synthase (CS) activity. CS activity in both cardiac and soleus samples was conducted according to Srére (28) and measured at 30°C.

Statistical analysis. For each of the SPs assessed, 12 individual gels were run, with 1 sample (both LV and RV) from each of the 4 groups/gel. For samples that were run more than once, as a result of differences in individual group numbers, the average of duplicate values was employed in the subsequent statistical analysis. All values were reported as means ± SE and compared by using a two-way ANOVA. On determination of significant main effects or interaction, pairwise comparisons employing a Student-Newman-Keuls post hoc test were conducted. Differences were considered significant at P  0.05.

	RESULTS

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Characterization of animals and exercise training. At the start of the study, rats in all groups had similar body weights. Animals assigned to the FW-Tr group ran an average of 4,401 ± 755 m during the first week of training, and then the distance leveled off at ~5,600 m over the next 6 wk before declining to 4,415 ± 662 m in week 8. In contrast, as noted above, rats in the TM-Tr group continuously increased their weekly running distance and exercise intensity throughout most of the 8-wk training period (Fig. 1). Although all animals in the TM-Tr group ran the same distance, average weekly distances run for the FW-Tr group ranged from 2,404 ± 312 to 9,899 ± 575 m.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Weekly running distance for exercise-trained rats. See MATERIALS AND METHODS for details.

Effect of exercise training on relative cardiac SP content. Figure 2 represents a complete immunoblot probed with SP72 (Fig. 2A) and a duplicate Coomassie blue-stained gel (Fig. 2B). Figure 2 not only indicates the relative migration of the protein of interest but also demonstrates the equal loading of all lanes (100 µg protein). Comparable immunoblots assessed for relative content of SP72 (Fig. 3A), SP73 (Fig. 4), and SP75 (Fig. 5B) revealed similar levels in the myocardium of both RV and LV of the hearts examined. However, these isoforms exhibited a differential response to exercise training. For SP72, hearts from the TM-Tr group exhibited a significant training-induced (>4-fold) increase. Despite running an average of ~5,300 m/wk, the FW-Tr group did not differ from the sedentary control animals in cardiac SP72 content (Fig. 3A).

View larger version (54K): [in this window] [in a new window]   Fig. 2.   A: complete sample immunoblot, probed with stress protein (SP) 72 primary antibody and visualized with an alkaline phosphatase-linked secondary antibody, indicates a single band migrating at ~72 kDa. Mol Wt Stds, molecular weight standards. B: Coomassie blue-stained gel, loaded equivalently to those used for transfer to immunoblots, demonstrates equal loading of all lanes (100 µg protein). RV, right ventricle; LV, left ventricle.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   A: cardiac SP72 content is increased only in rats subjected to high-intensity treadmill running. SP72 was detected by immunoblotting [100 µg protein/well (inset)] and expressed relative to content in RV of free wheel-trained sedentary group. B: even when free wheel-trained group is separated into high and low responders, cardiac SP72 content increases only in treadmill-trained group. Values for sedentary groups are provided for comparison. Distance run represents average distance (km) covered over 8-wk training period. SP72 was detected by immunoblotting and expressed relative to content in RV of free wheel sedentary group. SP72 values in B represent pooled data from both RV and LV. * Significantly larger than in all other groups, P  0.05. a Significantly less than in all other groups, P  0.05.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   SP73 (constitutive isoform of SP70 family) is refractory to exercise training in rat myocardium. SP73 was detected by immunoblotting [100 µg protein/well (inset)] and is expressed relative to content in RV of free wheel sedentary group.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Neither citrate synthase (CS) activity (A) nor SP75 (mitochondrial isoform of SP70 family) content (B) is altered by exercise training in rat myocardium. CS activity was measured at 30°C. SP75 was detected by immunoblotting [100 µg protein/well (inset)] and is expressed relative to content in RV of free wheel sedentary group.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Exercise is known to activate the induction of SPs (7, 10, 11) and to be associated with enhanced myocardial recovery during ischemia-reperfusion (14). The nature of the exercise stimulus required to elicit an increase in tissue SP levels is unclear. Hence, in the present study, this question was addressed by employing two forms of exercise training, treadmill and free wheel running. The results indicate that, unlike the treadmill run training protocol of the present study, chronic wheel running does not increase the expression of the cardioprotective SP72 in rat cardiac muscle.

When allowed free access to exercise wheels, rats will voluntarily run long distances daily (9, 25). Unlike rats forced to run on a treadmill, however, animals allowed access to training wheels run several discrete running bouts over a prolonged period (primarily during the daily 12-h dark cycle). Although individual bouts may be of high intensity, they are of short duration (25) and do not invoke the same cardiovascular responses as similar exercise bouts on the treadmill (31). In the present study, although FW-Tr rats ran total distances that were similar to those run by their TM-Tr counterparts (Fig. 1) and demonstrated complementary reductions in body weight gain (Table 1), CS activity in the soleus was increased only in TM-Tr animals (Table 1). This was so, even when animals in the FW-Tr group were divided into high and low responders (16.4 ± 0.6 and 15.1 ± 1.5 µmol · g wet wt¹ · min¹, respectively). This could be because rats in the present investigation averaged weekly running distances of only 2.4-9.9 km/wk, values that would place them at the low end of the continuum compared with previous studies (9, 25). Nevertheless, others have noted that despite average running distances of as much as 88 km/wk, several mitochondrial oxidative enzymes were unaltered (when expressed as micromoles per gram wet wt per minute) after FW-Tr (25). This suggests that free wheel training is less intense than treadmill training and hence is insufficient to invoke comparable adaptations in mitochondrial enzyme activity in soleus muscle (4).

The most important observation in the present study was that when changes in the relative protein content of SP72 with exercise training were assessed, only the TM-Tr group demonstrated a response (Fig. 3A). This suggests that not only are the previously noted elevations in SP72 in response to acute exercise (11, 14) maintained during 8 wk of a progressive exercise program but also a high volume of exercise does not necessarily result in an increase in the cardioprotective SP72 unless the exercise is sufficiently intense. The observation that induction of SP72 expression appears to exhibit a threshold is not new. In muscle extracted from rats passively heated for 15 min, activation of the transcription factor responsible for activating transcription of the SP72 gene (heat shock factor) in skeletal (12) and cardiac muscle (11) only occurs when rectal temperature is elevated above 41 and 42°C, respectively. Although exercise activates the signaling cascade, leading to elevated SP72 expression at lower core temperatures than this (11), to date most studies demonstrating an elevation in SP72 after exercise (7, 10, 11, 27) have employed high-intensity exercise protocols (2). On the basis of the present data (Fig. 3), it appears that any training threshold for SP72 expression is related more to exercise intensity than to total quantity of exercise accomplished. Even the cohort of the FW-Tr group, which ran farther than their treadmill-trained counterparts, failed to exhibit a change in cardiac SP72 levels. Interestingly, although the levels of SP72 in human cardiac muscle have never been investigated after exercise training, humans do respond to exercise with increased SP70 mRNA in skeletal muscle after exercise (23). Furthermore, Morris and colleagues (17, 18) have noted that the cardioprotective effect of exercise in middle-aged men was most influenced by the intensity of the exercise rather than the total physical activity. These observations have recently been confirmed in a cross-sectional study employing both men and women (30). Thus it may well be that increases in SP72 in response to intense physical activity may play a role in the positive effects of such exercise.

One of the most obvious factors that could account for the divergent response in cardiac SP72 content between TM-Tr and FW-Tr rats, despite similar total distance run, is a differential increase in body temperature for the two groups during training. Although in the present study, rectal temperatures of the exercising rats were not recorded, it is likely that the TM-Tr animals achieved higher sustained exercising body temperatures than did the FW-Tr group. Naive rats running on a motor-driven treadmill under similar conditions were found to reach temperatures >40°C after 60 min of running (10, 11). Peak body temperature elevations in female FW-Tr rats were less during nocturnal running periods, reaching a peak of just under 39.5°C (19). Although a greater temperature increase during exercise for the TM-Tr group compared with the FW-Tr animals likely played a major role in the present observations, it is possibly not the only factor associated with the exercise-induced elevation in SP72 in TM-Tr rats. Increased cardiac SP72 expression still occurs during high-intensity exercise when increases in rectal temperature are clamped at resting levels (27). SP72 content measured immediately after exercise increased 1.5-fold in LV of temperature-clamped runners vs. 4.7-fold in rats in which body temperature was allowed to increase during exercise (27). Heat shock factor activation has also been observed early in exercise when rectal temperature is elevated to <39°C (11).

Because SPs play an essential role in protein turnover and targeting in both cytoplasm and mitochondria (1, 26), the elevated SP72 in the TM-Tr group could have been a consequence of increased protein turnover. For example, Ornatsky et al. (20) recently demonstrated a proportional relationship between mitochondrial cytochrome oxidase activity and SP75. When skeletal muscle mitochondrial biogenesis was induced by chronic electrical stimulation, both cytochrome oxidase activity and SP75 content were increased. Presumably, with mitochondrial biogenesis and elevated protein turnover, an increase in the mitochondrial protein chaperone SP75 was necessitated. In the present study, as assessed from CS activities, cardiac mitochondrial biogenesis was not stimulated with any of the exercise training protocols. This was anticipated from previous work (21) that suggested that the mitochondrial capacity of hearts from sedentary rats is sufficient to meet the metabolic demands accompanying exercise. Interestingly, SP75 was also unchanged in the exercise-trained groups (Fig. 5B), thereby maintaining a proportional relationship between SP75 and cardiac mitochondrial components. This not only indicates minimal change in the rate of myocardial protein turnover but also could suggest that SP75 does not perform specific stress-related functions in the mitochondria similar to those conducted by SP72 in the cytoplasm. Alternatively, as with enzymes involved in mitochondrial respiration, there may be sufficient SP75 in the heart to meet those challenges imposed on cardiac mitochondria by exercise. The observation that SP73 was also refractory to exercise training (Fig. 4) further suggests that the increases in cardiac SP72 levels in the TM-Tr group were not simply a consequence of increased protein turnover.

One interesting observation was that despite four- to fivefold differences in developed systolic pressure, the relative content of all SPs was similar in both LV and RV of the rat heart (Figs. 3A, 4, and 5B). This had been observed previously in the hearts of sedentary swine for SP72 (13) and suggests that whatever the factor(s) controlling SP expression in the myocardium, it is coordinated in magnitude across both ventricles.

In conclusion, the expression of cardiac SP72 is increased after treadmill, but not free wheel, run training. This is true even in FW-Tr rats that accumulated greater average weekly running distances than did their TM-Tr counterparts. This could suggest that exercise intensity may be a critical factor in evoking an increase in the cardioprotective SP72. The observation that the relative content of SPs in both RV and LV of the myocardium is the same suggests that absolute developed pressure is not a primary determinant of SP level. The lack of change in SP73, CS activity, and SP75 after any of the exercise protocols suggests that the SP72 response was not simply due to altered protein turnover in the heart. Furthermore, it suggests that SPs may be differentially regulated and that SP72 may play a specific, perhaps protective, role in cardiac muscle stressed by high-intensity exercise. The reason(s) for a specific effect of exercise intensity on SP72, however, has yet to be determined.