INTRODUCTION

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SEVERAL STRESSORS induce production of the so-called heat shock protein (HSP), which is also called a stress protein (18). The HSP family contains a series of proteins with different molecular weights, and one of the prominent inducible HSPs in humans is HSP70 with a molecular weight of 72 (17). The production of HSP70 depends on the intensity and/or duration of a stressor (15). Exercise is one such stressor that can cause HSP70 production (23).

It has been demonstrated that exercise-induced HSP70 production can take place in blood (21), liver, heart, and muscle (22), but whether exercise can increase HSP70 in human muscle remains unclear (20). Exercise-mediated HSP70 induction could be independent of the elevation of temperature (23). Exercise-induced muscular ischemia, glycogen depletion, and oxidative free radicals might also be involved in HSP induction. Because changes in the exercise intensity or its duration may exert different effects on the exercise-induced muscular ischemia, glycogen depletion, and oxidative radicals, etc., it might be assumed that different exercise forms, intensities, and volumes have different impacts on HSP70.

Rowing is a force- and endurance-demanding type of exercise, and the training in rowing is complex (24), varying in both intensity and duration. During adaptation to exercise, training not only may cause muscular hypertrophy and change of the muscular composition (9) but also may induce HSP70 production. This study investigated the human skeletal muscle HSP70 response to 4 wk of rowing training.

	METHODS

Subjects. Ten male rowers, aged 18 yr, qualified for the German National Team, with eight chosen to be coxswains and the other two serving as replacements. Their height and body mass were 192 ± 5 cm and 85 ± 7 kg, respectively. After being informed about the study purpose and procedure, including the risks of a muscle biopsy, the subjects gave their written informed consent. This study was approved by the ethics committee of the Medical Faculty of the University of Ulm (Ulm, Germany).

Training. A 4-wk training program was conducted in preparation for the World Championships. This program consisted of four 1-wk phases (Table 1). Before and at the end of the training, a multiple-step rowing ergometry was performed until subjective exhaustion so that the power at 4 mmol/l of lactate (11) and relative maximum power (the maximum power output reached during this exercise test) could be obtained.

Muscle biopsy. The muscle biopsy taken at rest in the morning was performed in the middle of the belly of the right musculus vastus lateralis before the training and at the end of each training phase. A 16-gauge biopsy needle (Manan Medical Products, Northbrook, IL) was punctured 2 cm into the muscle belly, and the biopsy gauge was shot twice to attain ~3 mg of muscle tissue for each sample. The tissue was immediately frozen in liquid nitrogen and stored at 80°C.

SDS-PAGE. Frozen muscle tissue was homogenized in 200 µl of protein-extraction buffer (5) with help of an ultrasonic homogenizator, and protein concentration was determined (19). The sample solution was added to the sample buffer according to Laemmli (16). The discontinuous gel system was used for the SDS-PAGE: 4.5% stacking gel and 8.75% separation gel. The 5 µg of protein from each muscle sample were electrophoretically separated by using Bio-Rad Miniprotean II gel system at 60 V/gel for 45 min in running buffer (16). A series of standard HSP70 (H 8778, Sigma Chemical, St. Louis, MO) was prepared in the same way.

Protein transfer and immunodetection. Proteins were transfered from gels to polyvinylidenefluoride membranes (8) by using the LKB 2117 Multiphor II-Electrophoresis-Chamber (Pharmacia Biosystem, Freiburg, Germany) and a so-called semidry system at 200 V and 1.2 mV/cm² membrane for 60 min. The membranes were then blocked by 5% bovine serum albumin. Immunodetection for HSP70 was performed according to the protocol of enhanced chemiluminescence (ECL) Detection System (Amersham Life Science, Amersham International). Primary monoclonal antibody (mouse anti-human IgM) against inducible HSP70 (HSP72) diluted 1:2,500 (Clone 2A4, Affinity BioReagents, Golden, CO) and secondary antibody (goat anti-mouse anti-IgM, 115-035-075, Jackson ImmunoResearch Laboratories, West Grove, PA) conjugated with horseradish peroxidase diluted 1:20,000 were used. Membranes were reacted for 1 min with ECL reagents containing H₂O₂, and ECL films were exposed for 2 min.

Analysis. Quantification of HSP70 was performed by using a laser densitometer and an analysis software (Mecam, Labor für Bildverarbeitung, Fachhochschule Ulm, Ulm, Germany) referring to the standard series. The densitometric integral derived from each sample band, i.e., the integral of a mean density over a measured area, was taken to calculate the amount in each sample according to the known standard values (Fig. 1). Samples were run in replicate. The coefficient of variance of the HSP70 measurements was 26%, and the recovery rate of HSP70 added to muscle homogenates was 81.96%. HSP70 levels were expressed as means ± SE, and differences between two biopsies were tested with paired t-test and assumed to be significant at P < 0.05. 

View larger version (32K): [in this window] [in a new window]   Fig. 1.   Heat shock protein 70 (HSP70) blot depicting standard HSP70 and HSP70 obtained from subject 1. Lanes 1-5 represent standard HSP70 of 0, 0.05, 0.1, 0.15, and 0.20 µg, respectively. Lanes 6-10 are HSP70 taken from biopsies 1-5; HSP70 of each muscle sample (5 µg of total protein) was calculated to be 0.047, 0.096, 0.182, 0.217, and 0.193 µg, respectively.

	RESULTS

A linear relationship was found between the known HSP70 and the densitometric integrals: densitometric integral = 37.4 · HSP70 + 1.1 (r = 0.98, P < 0.01; Fig. 2A), whereas the relationship between the known HSP70 and the optical densities of the protein bands was curvilinear (Fig. 2B).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Relationship of standard HSP70 to densitometric integrals (A) and to densities of each band (B). While a hyperbolic relationship between HSP70 and densities is shown in B, a more linear relationship between both variables can be demonstrated in A, showing the following: densitometric integral = 37.4 · HSP70 (µg) + 1.1 (r = 0.98, P < 0.01).

On the basis of this linear relationship, HSP70 of each muscle sample was calculated. In comparison with the pretraining level (biopsy 0), HSP70 derived from the subsequent biopsies was elevated. With the pretraining HSP70 taken as 100%, the maximum level reached in the third week of training demonstrated an increase of about fivefold (Fig. 3A). The differences between two consecutive biopsies are depicted in Fig. 3B, which may serve as an indirect indicator of HSP70 production.

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Schematic illustration of HSP70 changes during the entire rowing training period. A: compared with before training, HSP70 increased after training began (P < 0.01), reached maximum level in third phase, and decreased in fourth phase. Values are means ± SE. B: differences () between 2 consecutive biopsies, serving as indirect indicator of HSP70 production, show that maximum HSP70 increase happened at end of second training week.

	DISCUSSION

The present study was conducted to observe the human skeletal muscle HSP70 response to training in rowing, and a clear increase of HSP70 in exercised human skeletal muscle was demonstrated.

Quantification of HSP70. Most studies on HSP70 are performed semiquantitatively (22). In this study, HSP70 was quantified by referring to a series of known amounts of HSP70. A prerequisite for comparing the HSP70 of each muscle sample is the determination of the protein concentration. We used a well-established method (19) and examined the reproducibility (coefficient of variance = 0.72%).

HSP70 response to training. Exercise can induce HSP70 production in muscles of animals (18), whereas no significant increase of HSP70 was found within 3 h after a bout of exercise in muscles of humans, although the concentration of HSP70 mRNA did increase (20). In the present study, HSP70 production increased to reach a maximum of five times the pretraining level.

Mechanisms of HSP70 induction. Hyperthermia causes a HSP70 response (25). Because hyperthermia occurs during exercise (2), a "heat-shock" effect might play a role, but exercise-induced HSP70 can be independent of changes in the body temperature (23). Metabolic stress induces HSP70 (13), which may be at least partly responsible for HSP70 production in exercised muscles. During the training program, blood lactate frequently reached 4-8 mmol/l, causing a decrease of blood pH, which can induce HSP70 production (27). High-volume exercise demands a high-energy supply provided by substrate oxidation, producing glycogen depletion and oxidative free radicals, which induce HSP70 production (17).

Meanings of HSP70 production. HSP70 production is considered to protect the vital function and to prevent damage (12). Currie et al. (3) demonstrated that in myocardium pretreated with HSP70 induction (by hyperthermia) not only was the cardiac function less impaired by ischemia and could recovery more rapidly but also a reduction of myocardial infarct size could be produced (4). Conversely, failure to produce HSP70 leads to an exaggerated organic damage (10). We observed the change of creatine kinase (CK) during the training period. At the beginning of training, CK was elevated, and with the training CK decreased. Because CK is associated with muscular injury, HSP70 accumulation might be partly responsible for lowering CK level (6) and provide a protective effect on musculature (14).