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Muscle sarcoplasmic reticulum calcium regulation in humans during consecutive days of exercise and recovery

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada

Submitted 20 April 2007 ; accepted in final form 4 July 2007

	ABSTRACT

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The study investigated the hypothesis that three consecutive days of prolonged cycle exercise would result in a sustained reduction in the Ca²⁺-cycling properties of the vastus lateralis in the absence of changes in the sarcoplasmic (endoplasmic) reticulum Ca²⁺-ATPase (SERCA) protein. Tissue samples were obtained at preexercise (Pre) and postexercise (Post) on day 1 (E1) and day 3 (E3) and during recovery day 1 (R1), day 2 (R2), and day 3 (R3) in 12 active but untrained volunteers (age 19.2 ± 0.27 yr; mean ± SE) and analyzed for changes (nmol·mg protein^–1·min^–1) in maximal Ca²⁺-ATPase activity (V_max), Ca²⁺ uptake and Ca²⁺ release (phase 1 and phase 2), and SERCA isoform expression (SERCA1a and SERCA2a). At E1, reductions (P < 0.05) from Pre to Post in V_max (150 ± 7 vs. 121 ± 7), Ca²⁺ uptake (7.79 ± 0.28 vs. 5.71 ± 0.33), and both phases of Ca²⁺ release (phase 1, 20.3 ± 1.3 vs. 15.2 ± 1.1; phase 2, 7.70 ± 0.60 vs. 4.99 ± 0.48) were found. In contrast to V_max, which recovered at Pre E3 and then remained stable at Post E3 and throughout recovery, Ca²⁺ uptake remained depressed (P < 0.05) at E3 Pre and Post and at R1 as did phase 2 of Ca²⁺ release. Exercise resulted in an increase (P < 0.05) in SERCA1a (14% at R2) but not SERCA2a. It is concluded that rapidly adapting mechanisms protect V_max following the onset of regular exercise but not Ca²⁺ uptake and Ca²⁺ release.

short-term training; calcium-adenosinetriphosphatase; calcium uptake; calcium release; SERCA isoforms

Unclear are the effects of repetitive days of prolonged exercise on SR Ca²⁺-cycling responses. Two primary different responses could occur. On the one hand, the disturbances in SR Ca²⁺-cycling behavior could persist and, in fact, become exacerbated with additional exercise. Conversely, adaptive processes could be initiated, resulting in changes in protein abundance, potentially offsetting the exercise-induced disturbances in SR Ca²⁺-cycling properties. Evidence exists for both possibilities.

The acute effects of exercise have been shown to result in structural alterations to both the Ca²⁺ pump and the calcium release channel, commonly labeled the ryanodine receptor (RyR). In the case of the Ca²⁺ pump, both prolonged exercise (36) and chronic low-frequency electrical stimulation (CLFS) (14, 38, 40) result in changes in the region of the adenine nucleotide site, ostensibly due to oxidation and nitrosylation secondary to damage from the accumulation of reactive oxygen species (ROS) (32, 39). The accumulation of ROS during contractile activity also remains as an inviting theory to explain the structural abnormalities that occur to the RyR (16).

Recovery of Ca²⁺-ATPase activity appears to take considerable time after exercise. In muscles of both animals subjected to CLFS (38) and voluntary exercise in humans (57), recovery of V_max was not complete for at least 36 h following exercise. In the absence of adaptive changes, a failure of V_max to fully recover could lead to greater compromises in SR Ca²⁺-cycling during subsequent exercise.

Prolonged contractile activity is also known to act as a powerful stimulus for remodeling of the skeletal muscle cell, including the properties of the SR. Of the few nonhuman studies that have addressed training-induced effects on the SR in mammalian muscle, it has been found that reductions in Ca²⁺-ATPase protein (21), Ca²⁺ uptake (30, 31), V_max (30), and Ca²⁺ release (30) occur. Interestingly, at least for the training protocols employed for the rat, it appears that the adaptations are more pronounced in muscle composed of a large proportion of fast-twitch fibers (30, 31). In the case of Ca²⁺-ATPase, measurements of mRNA also support that the changes are specific to fast-twitch-based muscles and to the SERCA2a isoform (33).

Adaptations in the SR with training also appear to extend to humans. In the only longitudinal study published to date, we (19) have reported that 10 wk of prolonged submaximal exercise results in reductions in V_max, Ca²⁺ uptake, Ca²⁺ release, and SERCA1a in vastus lateralis. No changes occurred in SERCA2a. The adaptation in the SERCA isoforms appears to begin early. Sixteen sessions of heavy intermittent exercise performed over 16 h resulted in a small but significant increase in SERCA2a and a decrease in SERCA1a (57). Expectations of large increases in SERCA expression during the first few days of exercise may be unrealistic given the relatively slow turnover rate of the protein (37).

The purpose of this study was to investigate the effects of repeated days of prolonged exercise and recovery on SR Ca²⁺-cycling properties in skeletal muscle. We have hypothesized that the acute reductions in Ca²⁺-ATPase activity, Ca²⁺ uptake, and Ca²⁺ release observed during the first day of exercise would persist throughout the third consecutive day of exercise, resulting in greater disturbances in these properties following exercise. By the third day of inactivity, all SR properties would recover to preexperimental levels. The changes in SR Ca²⁺-ATPase will not be accompanied by changes in SERCA1a or SERCA2a protein abundance.

	METHODS

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Participants

The volunteers consisted of 12 active students with a mean age and body mass of 19.2 ± 0.27 yr and 71.1 ± 3.4 kg, respectively. Peak aerobic power (O_2peak) measured during a progressive cycle protocol to fatigue was 44.8 ± 2.0 ml·kg^–1·min^–1. All volunteers were healthy and free of any medication as determined by questionnaire. Although participants were recreationally active, they did not participate in prolonged exercise involving large muscle group action on a regular basis. The overall description of the study, the specific protocols to be employed, and the associated risks were described during a familiarization session before obtaining written consent. The study was approved by the Office of Research Ethics at the University of Waterloo before written consent was obtained from the volunteers.

Experimental Design

Each participant was required to visit the laboratory on at least seven different occasions to complete all testing and measurements. On the first visit, which occurred 2 wk before the start of the experiment, O_2peak was measured. The next six visits were used to examine the SR Ca²⁺-cycling responses to consecutive days of exercise and recovery. Prolonged submaximal exercise was performed on day 1 (E1), day 2 (E2), and day 3 (E3) in the morning at approximately the same time. The exercise task consisted of cycling at 60% O_2peak for a maximum of 2 h. For subjects who were not able to complete 2 h at E1, the same time was used for exercise at E2 and E3. Recovery characteristics were examined 24 h following E3 (R1) and at similar times on day 2 (R2) and day 3 (R3). No exercise was performed on the recovery days. Participants were also requested to refrain from exercise for at least 48 h before E1.

No dietary manipulation was applied during the days leading into the initial training session or during the experimental period. During these periods, the participants were requested to maintain their normal dietary habits. However, 2 h before reporting to the laboratory on the exercise days, participants were provided with and instructed to ingest a can of Ensure (250 kcal) meal replacement (Ross Products Division, Saint-Laurent, PQ, Canada). This practice was followed to standardize the nutrient intake before the prolonged exercise that occurred in the morning. No other nutrients (solid foods or beverage) was allowed before or during the testing. All participants were requested to abstain from caffeine and alcohol for at least 24 h before exercise at E1 and throughout the 6-day experimental period. On the experimental days, both exercise and recovery, temperatures ranged between 23 and 24°C and relative humidity between 50 and 60%. We have selected the three-consecutive-day model of prolonged exercise on the basis of a number of previous studies that have demonstrated rapid adaptations in muscle metabolic behavior (18, 22) and Na⁺-K⁺-ATPase concentration (20) within the first few days of regular exercise. The 3 days of inactivity selected for study was designed to allow for recovery of the changes in SR Ca²⁺-cycling properties induced by the last session of exercise and the expression of SERCA protein isoforms.

Exercise Protocols

The progressive exercise test for measurement of O_2peak was as previously employed in our laboratory (27) and included a 4-min period of cycling at 25 W followed by 15-W step increases in power output (PO) each minute until fatigue. Gas exchange was monitored throughout the protocol (28) and used to both characterize the relationship between PO and O₂ consumption (O₂) and to assess O_2peak. The PO-O₂ relationships were used to establish the PO designed to elicit 60% O_2peak in each volunteer. All exercise was performed upright on an electrically braked cycle ergometer (Quinton 870) that was calibrated on a daily basis. The seat height was standardized for each participant for both the progressive and prolonged exercise protocols.

The prolonged exercise protocol was designed to allow for the investigation of exercise-induced changes in the SR Ca²⁺-cycling properties and the response of these properties during the recovery days. Before beginning the exercise, preparations were made for tissue sampling from the vastus lateralis muscle. For tissue sampling, seven separate sites were prepared, randomized between legs. Four sites were used for tissue sampling during the exercise days (Pre, Post on E1 and E3) and three sites for sampling during the recovery days, one per day. The location and preparation of the sites and the technique used for tissue sampling were as previously employed by our group (18, 22). In brief, on a given experimental day, the sites were selected (2 in the case of the exercise days, 1 per leg), and incisions were made after local anesthesia (2% xylocaine with epinephrine) was applied. Two separate samples were extracted from each site using suction to increase tissue yield. The tissue samples were obtained within 5 min of beginning exercise (Pre) and immediately following the exercise (Post).

Analytical Techniques

SR Ca²⁺-ATPase properties.   Whole muscle homogenates prepared from fresh tissue following extraction from the vastus lateralis and stored (80°C) were used to determine the catalytic properties of the SR Ca²⁺-ATPase. Homogenates were prepared using 30 mg tissue diluted 1:11 (wt/vol) in ice-cold buffer containing (in mM) 250 sucrose, 5 HEPES, 10 NaN₃, and 0.2 phenylmethysulfonyl fluoride (PMSF) (pH 7.5) by using a handheld glass homogenizer (Duall 20, Kontes). The catalytic activity was measured in duplicate at 37°C by using a spectrophotometric method originally developed by Simonides and van Hardeveld (51) with modifications (8, 45). The assay mixture (pH 7.0) contained (in mM) 200 KCl, 20 HEPES, 15 MgCl₂, 1 EGTA, 10 NaN₃, 5 ATP, 0.3 NADH, 10 phosphoenolpyruvate (PEP), 18 U/ml lactate dehydrogenase (LDH), 18 U/ml pyruvate kinase (PK), and 25 µl homogenate. Ca²⁺-dependent ATPase activity was measured by using 0.5-µl additions of 100 mM CaCl₂. The additions were continued until a plateau and subsequent decline in Ca²⁺-ATPase activity were observed. Ca²⁺-independent or basal ATPase activity was determined by using 40 µM of the Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) (49). The Ca²⁺-ATPase activity was calculated as the difference between the total ATPase activity measured without CPA and the basal ATPase measured with CPA. Ca²⁺-ATPase activity was measured with and without 1 µM of the Ca²⁺ ionophore A-23187 (Sigma C-7522). The Ca²⁺ ionophore was added to prevent intraluminal Ca²⁺ accumulation in SR, which is inhibitory to Ca²⁺-ATPase activity (52). By calculating the ratio of Ca²⁺-ATPase activity, measured with and without the ionophore A-23187, it is possible to gain an indirect measure of the effects of exercise on changes in SR membrane integrity.

Measurement of [Ca²⁺]_f, used to determine the Ca²⁺ dependency of the Ca²⁺-ATPase reaction, was performed with dual-wavelength spectrofluorometry and the Ca²⁺-fluorescent dye indo-1. The excitation wavelength was 355 nm, and the emission was measured at 405 and 485 nm for Ca²⁺-bound and Ca²⁺-free forms of the dye, respectively. The dissociation constant used for the interaction between Ca²⁺ and indo-1 was 250 nM. Further details of the assay appear in earlier publications from our laboratory (55).

The kinetic properties measured included maximal Ca²⁺-ATPase activity (V_max), the [Ca²⁺]_f needed to produce 50% V_max (Ca₅₀), and the slope of the relationship between Ca²⁺-ATPase activity and [Ca²⁺]_f (Hill coefficient, n_H). V_max was defined as the peak Ca²⁺-ATPase activity; Ca₅₀ represented the [Ca²⁺]_f obtained from a sigmoid fit of the data that yields 50% V_max; and n_H was obtained through nonlinear regression, using a portion of the curve that corresponded to between 10 and 90% V_max.

To assess whether phosphatase activation may occur during tissue preparation, potentially biasing out measurement of V_max, we have used a phosphatase inhibitor cocktail (Sigma P2850) added during the homogenization procedure using tissue obtained from a related experiment on humans. A comparison of V_max with and without the phosphatase inhibitor indicated no systematic difference between conditions either at rest or during exercise (differences ranging between 2.0% and 6.3%) (H. J. Green, unpublished observation).

SR Ca²⁺ uptake and Ca²⁺ release.   Ca²⁺ uptake and Ca²⁺ release were determined in a common assay using homogenates prepared from fresh tissue that was frozen and stored in conjunction with the homogenates used for Ca²⁺-ATPase determinations. Ca²⁺-uptake and Ca²⁺-release rates were determined in duplicate at 37°C. The reaction buffer consisted of (in mM) 200 KCl, 20 HEPES, 15 MgCl₂, 10 NaN₃, 0.005 N,N_,N¹N¹-tetrakis(2-pyridlymethyl)-ethylanediamine, 5 oxalate, 10 PEP, 18 U/ml LDH, 18 U/ml PK, and 0.0015 indo-1. To produce a constant starting [Ca²⁺]_f of 3.5 µM, 2.5 µl of CaCl₂ (10 mM) was added to the cuvette containing 2 ml of the reaction buffer. Ca²⁺ uptake was initiated by adding 5 mM ATP. The rate of Ca²⁺ uptake was determined at 2,000 nM by differentiating a linear fit curve with [Ca²⁺]_f measured using indo-1, as earlier described.

After Ca²⁺ uptake had stabilized, Ca²⁺ release was initiated by adding 20 µM of 4-chloro-m-cresol (4-CMC), a highly specific Ca²⁺-releasing agent (23). As described previously (56), we have measured two phases of Ca²⁺ release, namely phase 1 and phase 2. With our procedures, we consistently find that 4-CMC induces a biphasic Ca²⁺ release, which can be detected by distinct differences in slope. The rapid early phase (phase 1) produces the most marked slope, while the more delayed later phase (phase 2) has a lower slope. Maximal Ca²⁺-release rates for each phase were calculated using the same method as for Ca²⁺ uptake and differentiating a linear fit curve.

The ratio between Ca²⁺ uptake (2,000 nM) and V_max was determined and labeled the apparent coupling ratio (12). We have called this the apparent coupling ratio because of the different assay conditions used to determine each property. For Ca²⁺ uptake, a precipitating agent (oxalate) had to be employed to bind Ca²⁺ inside the SR to prevent back-inhibition (41). For Ca²⁺-ATPase activity, a membrane permeabilizing agent was necessary to also prevent back-inhibition (52). We also emphasize that given the limited sensitivity of indo-1 for Ca²⁺, it is not clear if 2,000 nM was sufficient to produce a maximal Ca²⁺-uptake rate.

For all SR properties, protein was determined in duplicate by the method of Lowry as modified by Schacterle and Pollock (47).

Ca²⁺-ATPase isoforms.   Detection of Ca²⁺-ATPase isoform distribution (SERCA1a and SERCA2a) was performed using electrophoresis and Western blotting techniques, consistent with procedures previously detailed in our laboratory (19). In brief, 10 µg protein, obtained from a postnuclear homogenate, was electrophoresed with 7.5% SDS-polyacrylamide gels (Bio-Rad Mini-PROTEAN II), equilibrated (15 min) in cold transfer buffer (25 mM Tris, 192 mM glycine, and 20% vol/vol methanol), and transferred to a polyvinylidine difluoride (PVDF) membrane (Bio-Rad) by placing the gels in transfer buffer and applying high voltage (23 V) for 40 min (Trans-Blot Cell, Bio-Rad). The postnuclear homogenate was obtained from the supernatant of frozen tissue, which had been initially prepared in a buffer consisting of 5 mM HEPES (pH 7.5), 250 mM sucrose, 0.2% NaN₃, and 0.1 µM PMSF in a ratio of 15:1 (vol/wt) and then extracted by adding an equal volume of buffer containing 10 mM sodium phosphate (pH 7.4), 150 mM NaCl, 2% Triton X-100, 2% deoxycholate, 0.2% SDS, 0.2 mM PMSF, and 1,000 IU aprotonin. To obtain the postnuclear homogenate, this mixture was centrifuged at 10,000 g for 15 min, and the supernatant was withdrawn and stored at –80°C pending analysis. It has been reported that 95–99% of the SERCA protein remains in the supernatant after this procedure (58).

The PVDF membranes were blocked for nonspecific binding by using a 10% skim milk powder in Tris-buffered saline (pH 7.5) applied overnight at room temperature, before a 60-min incubation at room temperature with the primary monoclonal antibodies for SERCA1a (A52) and SERCA2a (7E6) obtained from Affinity Bioreagents using dilution of 1:2,500 (SERCA1a) and 1:1,000 (SERCA2a).

After washing with Tris-buffered saline and 0.1% Tween 20 (TBS-T), the PVDF membranes were incubated for 60 min at room temperature with a secondary antibody (anti-mouse IgG₁ conjugated to horseradish peroxidase) and protein determined by densitometry and an enhanced chemiluminescence immunodetection procedure (Amersham ECL-RPN2106 P1), and visualized using the Chemi Genius imaging system and Gene Snap software (SynGene). Densitometry measurements were performed using Gene Tools software (Syngene). Signal intensity of the blots were found to be linear over the protein range employed in our experimental conditions.

All samples obtained at rest (before exercise in the case of E1 and E3) for a given subject were run in duplicate on separate gels along with the standard on the same day. The average value of the duplicate measurements were initially expressed as a percentage of the standard and then as a percentage of the initial control value obtained before exercise on E1. A sample of tissue obtained from human vastus lateralis was used as the standard. Multiple aliquots obtained from the postnuclear homogenate were stored at –80°C and used as a standard during each analytical session.

Statistics

The data were analyzed using one-way ANOVA procedures for repeated measures. The one-way ANOVA was used to investigate the effects of the exercise (Pre, Post) at E1 and E3 and the effect of the recovery days (R1, R2, R3). Where significant differences were found, the Neuman-Keuls technique was applied to determine which means were different. Significance was set at the 0.05 level. Throughout the text, data are expressed as means ± SE. Where a difference between means is indicated in the text, significance is implied.

	RESULTS

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SR Changes

Exercise at E1 but not E3 resulted in a reduction in V_max. The reduction in V_max occurred in the absence of changes in Ca²⁺ sensitivity as measured by both Ca₅₀ and n_H (Fig. 1). Before the third day of exercise, V_max had recovered to preexperimental levels. No differences were observed in V_max between R1, R2, and R3 or between the recovery days and E1 and E3 at Pre. Exercise, either on E1 and E3, did not alter the ionophore ratio. Similarly, the ionophore ratios were not different before exercise on the exercise days or between any of the recovery days (Table 1).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Muscle sarcoplasmic reticulum Ca2+-ATPase kinetic characteristics during consecutive days of exercise and recovery. Values are means ± SE (n = 12). E1, E3, exercise on days 1 and 3, respectively; R1, R2, and R3, recovery on days 1, 2, and 3, respectively; Pre, preexercise; Post, postexercise. A: maximal Ca2+-ATPase activity (Vmax; nmol·mg protein–1·min–1). B: Ca2+ concentrations needed to elicit 50% Vmax (Ca50). C: Hill coefficient (nH) obtained by using the relationship between Ca2+ concentration and Ca2+-ATPase activity over a section of the curve that corresponded to 10–90% of Vmax. *Significantly different (P < 0.05) from Pre E1. Significantly different (P < 0.05) from E1 Post.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Muscle sarcoplasmic reticulum Ca2+ uptake (nmol·mg protein–1·min–1) during consecutive days of exercise and recovery. Values are means ± SE (n = 12). *Significantly different (P < 0.05) from Pre E1. Significantly different (P < 0.05) from Post E1. Significantly different (P < 0.05) from Post E3.

Ca²⁺ release phase 1 and phase 2 were reduced by 25.1% and 35.2%, respectively, during exercise on E1 (Fig. 3). Exercise at E3 failed to elicit changes in either phase 1 or phase 2 Ca²⁺ release. However, unlike phase 1, which showed no difference between preexercise levels at E1 and E3, phase 2 Ca²⁺ release before exercise on E3 remained depressed. Although there was a trend for phase 2 Ca²⁺ release to remain depressed throughout the recovery days compared with E1, the difference was not significant.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Muscle sarcoplasmic reticulum Ca2+ release (nmol·mg protein–1·min–1) during consecutive days of exercise and recovery. Values are means ± SE (n = 12). Phase 1, early rapid phase of Ca2+ release; phase 2, slower, more delayed phase of Ca2+ release. *Significantly different (P < 0.05) from Pre E1. Significantly different (P < 0.05) from Post E1.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Representative Western blots (A) and relative changes in muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA) concentration (B) during consecutive days of exercise and recovery. Values are means ± SE. Values are means ± SE in relative units. The values for E1 was set at 1.00; n = 12. Measurements at E1 and E3 were made before exercise. Western blot analyses of fast (SERCA1a; left) and slow (SERCA2a; right) Ca2+-ATPase were made with the use of monoclonal antibodies A52 and 7E6. *Significantly different (P < 0.05) from E1. #Significantly different (P < 0.05) from R2.

	DISCUSSION

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The recovery characteristics of the different Ca²⁺-cycling properties during and following the three consecutive days of exercise also demonstrate novel insights. As postulated, all three properties recovered by the third day of inactivity. However, differences were observed between the properties in the time course of recovery between the consecutive days of exercise and the consecutive days of inactivity. Depressions in Ca²⁺ uptake persisted during the first 2 days of exercise and during the first day of recovery, while the depression in Ca²⁺ release (phase 2) was only evident during days of exercise. A strong trend was observed for phase 1 to be depressed during the period as well, but the differences were not significant. V_max was not different at rest between any of the exercise or recovery days. Collectively, these results indicate a dissociation between the recovery kinetics of V_max and Ca²⁺ uptake and Ca²⁺ release following an exercise protocol. It should be emphasized that the changes in V_max and Ca²⁺ uptake either with exercise or recovery were not accompanied by changes in membrane permeability as measured by the ionophore ratio. A cumulative effect of the repetitive days of exercise appears to change the efficiency of Ca²⁺ sequestration as measured by the apparent coupling ratio since the value following exercise on day 3 was lower than the value observed before exercise on day 1.

Our results indicate that in response to three consecutive days of prolonged submaximal exercise, an increase in the fast isoform of the Ca²⁺-ATPase, namely SERCA1a but not SERCA2a, occurs. Moreover, the increase is transient, not evident until 2 days following the last exercise session before reversing by 3 days of recovery. To our knowledge this is only the third study investigating isoform adaptability in SR Ca²⁺-cycling proteins in human skeletal muscle in response to regular exercise. In a previous study from our laboratory, we (19) reported a decrease in the SERCA1a isoform and no change in the SERCA2a isoform in response to a progressive 10-wk program of prolonged heavy cycle exercise. In the additional study, a 5-wk program of sprint training resulted in an increase in the relative density of the SERCA1a isoform (44). These two studies suggest that at least over a more extended training period, the Ca²⁺-ATPase may adapt according to the demands placed on SR Ca²⁺ cycling. High-intensity exercise resulting in a demand for rapid Ca²⁺ cycling, both Ca²⁺ release and Ca²⁺ uptake, promotes increases in both the relative densities of RyR and the SERCA1a isoform (44). These adaptations promote a phenotype approaching that observed in type II or fast-twitch fibers, which possess much higher densities of these proteins, consistent with the need for a rapid regulation of the Ca²⁺ transient given the rapid velocities that these fibers are capable of generating (4). However, it must be emphasized that since the vastus lateralis is composed of 50% of type I and type II fibers (46), it is not clear if changes observed in SERCA1a and RyR following training was specific to one fiber type or altered the abundance in both fiber types. In this regard, it was reported that the sprint training also resulted in increases in SERCA2a (44), with the distribution by fiber type also unclear.

Given the demands imposed by our prolonged submaximal cycling protocol, we had expected that the isoform response would be in the direction of increased SERCA2a, typical of what has been reported in animals subjected to regular prolonged contractile activity. The unexpected increase that we have reported in SERCA1a with our exercise model could occur as a result of decreases in degradation, increases in synthesis, or a combination of both mechanisms. The increases in SERCA1a may be a response to the strain imposed early by the dramatic and sustained increase in contractile activity. Given the results of a previous study in which we (19) reported increases in SERCA2a with an extended training program, it might be expected that the SERCA isoform changes may display a time dependency, ultimately culminating in a shift toward the slow isoform.

It might be expected that increases in SERCA1a protein levels as we have observed in this study would result in an increase in both V_max and Ca²⁺ uptake, specifically at R2, the point at which the increase was detected. However, we found no change in either V_max or Ca²⁺ uptake at R2 compared with preexercise at E1. Furthermore, at R3, when SERCA1a regressed to preexercise levels, no differences existed between R2 and R3 for either V_max or Ca²⁺ uptake. The failure to find a coupled response of V_max and Ca²⁺ uptake with changes in SERCA1a could occur as a result of the insensitivity of our analytical measurements to detect change of the magnitude expected or because of incomplete recovery from the disturbing effects of exercise at E3. To examine the latter probability, we have incorporated a 3-day recovery period into our experimental design. Our results clearly demonstrate that V_max recovered from the exercise-induced reduction observed at E1 and E3 within 24 h and then remained stable throughout the recovery days. Ca²⁺ uptake, although displaying a slower recovery than V_max, was recovered to preexercise levels by R2 with no further change at R3. These results provide evidence that the dissociation that exists between the changes in SERCA1a and the Ca²⁺-sequestering properties cannot be attributed to a failure to recover from the last exercise session.

In our previous training study (19), we found that the SR Ca²⁺-sequestering properties, measured in tissue samples extracted 4 days following the last training session, displayed an expected reduction in V_max and Ca²⁺ uptake consistent with the reduction in SERCA1a concentration. Two previous studies (30, 31) employing prolonged training in rats have also reported reductions in resting Ca²⁺ uptake following training. However, the reduction was only observed in plantaris (30) and the superficial vastus lateralis (31) and not the soleus (30, 31) or the deep vastus lateralis (31), where no training changes were detected. Given the fiber-type composition of these muscles and regions (7), it would appear that the training effect was highly specific to muscles containing a predominance of type IIB or fast-glycolytic, low-oxidative based fibers. Interestingly, reductions in V_max were observed with training in the plantaris but not the soleus muscle (30). Since acute exercise also resulted in reductions in V_max and Ca²⁺ uptake in both the plantaris and soleus muscles (30), it is not clear if the 48-h period provided from the last training session was sufficient to allow for full recovery of the enzyme. In one other study investigating training at SR properties in humans, it was found that resting V_max and Ca²⁺ uptake was depressed in endurance-trained subjects compared with untrained subjects (35). Since this was a cross-sectional study dealing with elite athletes, the differences could have existed before training.

In this study, we demonstrate a dissociation between V_max and Ca²⁺ uptake during recovery, with the recovery of Ca²⁺ uptake being much more delayed. Several possibilities exist to explain this apparent contradiction. Conceivably, the dissociation could occur as a product of the different conditions used to measure each property. In the case of Ca²⁺-ATPase activity, the Ca²⁺ ionophore A-23187 was employed to prevent the accumulation of Ca²⁺ in the lumen of the SR. The use of oxalate has been shown to increase Ca²⁺-uptake rates in homogenates, supposedly by preventing the increases in SR luminal [Ca²⁺]_f (29). We also emphasize that while Ca²⁺-ATPase was measured during maximal activating conditions, producing a clear V_max, Ca²⁺ uptake was not. This is because of limitations in the sensitivity of the indo-1, the fluorescent dye used to measure [Ca²⁺]_f. We routinely measure Ca²⁺ uptake at Ca²⁺ concentrations up to 2,000 nM, as we have in this study, but it is not clear how close these measurements are to the maximal Ca²⁺ uptake.

We do not feel that these technical issues were significant, however, since the discrepancy between V_max and Ca²⁺ uptake only occurred under very specific conditions, namely during recovery. It is possible that the slower recovery observed for Ca²⁺ uptake following exercise could be explained by leakage of Ca²⁺ during uptake through the SR membrane or the RyR channel, resulting in a net reduction in Ca²⁺ uptake. Thermal stress, as an example, is known to increase leakage of Ca²⁺ from the lumen of the SR in the cytosol (6, 48, 50). In the absence of changes in V_max, decreases in Ca²⁺ uptake should result in a reduction in the coupling ratio. This ratio, which we have defined as the apparent coupling ratio to reflect differences in the protocols used for measurement of V_max and Ca²⁺ uptake, was not observed to change, Pre to Post at E1 and E3 or at rest between the exercise and recovery days. However, a trend is clearly evident as indicated by the reduction in the apparent coupling ratio with exercise at E1 and E3. At Post E3, the apparent coupling ratio was lower than Pre E1, suggesting a cumulative effect of the consecutive days of exercise. In this regard, we would emphasize that in our many previous studies using prolonged exercise, we have not observed any change in the apparent coupling ratio when a normal diet is followed (8, 9, 19). However, exercise plus a 4-day period of a low-carbohydrate diet elicits modifications in the apparent coupling ratio (10, 11). In this study, since participants followed a normal carbohydrate diet, it would appear that the repeated sessions of exercise culminate in a lower apparent coupling ratio.

An inviting possibility to explain the apparent protection offered to V_max with consecutive days of exercise is via heat shock protein 70 (HSP70). This heat shock protein has been shown to bind to SERCA1a and prevent thermal activation (54). Given the intensity and prolonged nature of the exercise, substantial increases in muscle temperature would be expected (17). This would not seem a credible possibility, however, since the 12% increase observed in HSP at R1 was not significant (P = 0.11) (data not shown). Moreover, V_max recovered to resting levels before there was any indication of a change in HSP70.

Exercise at E1 resulted in pronounced reductions in both phase 1 and phase 2 Ca²⁺ release. Both phases of Ca²⁺ release appear to remain depressed before exercise at E3; however, only in the case of phase 2 was significance found. At E3, exercise failed to elicit further declines regardless of phase. Although a clear trend exists for Ca²⁺ uptake to remain depressed throughout the first 2 days of recovery and in the case of phase 1, throughout the 3-days of recovery, significance was not found. The depression in Ca²⁺ release during exercise was expected given the many previous studies that have reported a similar finding in humans (3, 8, 9, 12, 13, 25, 34, 35). The persistent reduction in Ca²⁺ release is a novel finding and suggests that recovery processes are not complete during the 24-h period between exercise sessions. Given that the reduction in Ca²⁺ release appears to be structural damage to the RyR (16), it would appear that repair of the damaged RyR or the synthesis of new RyRs is a more delayed process.

In previous training studies in humans, using a prolonged exercise protocol for 5 wk, a pronounced reduction in Ca²⁺ release was observed (19). A similar finding has been reported in a cross-sectional study employing trained endurance athletes and untrained controls (35). However, in rats subjected to a 10-wk endurance program, resting Ca²⁺ release was not altered either in the plantaris or soleus, two muscles composed of a predominance of fast- and slow-twitch fibers, respectively (30). The contradictory effect of training on Ca²⁺ release may be due to differences between species and/or the muscles examined.

In this study, unlike others (34), we have assessed Ca²⁺ release in two phases, namely phase 1 and phase 2, based on clear biphasic Ca²⁺-release rate. We have examined the different phases in an earlier study using both 4-CMC and AgNO₃ and found that with 4-CMC, which is highly specific to RyR, a sixfold higher release occurred (56). Moreover, we have found that ruthenium red, a known inhibitor of the RyR, reduced phase 1 by 59% and completely blocked phase 2 (56). The differential effects observed have led us to speculate that different mechanisms may be involved in the two phases of release. Consequently, and given that a clear demarcation exists between the two phases of Ca²⁺ release, we routinely report on both phases. Although most of our studies report a similar pattern of change for response to exercise for both phases, some differences have been reported. In this study, in general, the changes in phase 1 and phase 2 Ca²⁺ release are similar. However, there is a suggestion of a more delayed normalization of phase 2 Ca²⁺ release in recovery and particularly at E3. It is unclear what the physiological significance of this difference is, if any.

In summary, we have shown that, as expected, prolonged exercise in the unacclimatized results in a disturbance in SR Ca²⁺-cycling function, as evidenced by the decrease in V_max, Ca²⁺ uptake, and Ca²⁺ release. A similar session of exercise performed on the third day failed to reduce any of the SR properties examined. With the exception of V_max, which recovered to preexercise levels, the failure to find an exercise effect occurred in the presence of incomplete recovery. Although Ca²⁺ release recovered to levels observed before exercise at day 1, during the first day of recovery, Ca²⁺ uptake was more delayed and did not recover until day 2. The consecutive days of exercise also increased SERCA1a expression, an adaptation not evident until recovery day 2. The differences in time-course responses in the different SR properties examined emphasizes the limitation of examining acute and/or adaptive responses of the SR to exercise at the same time point. In a related study, we examined the effects of the consecutive-day protocol of exercise and recovery on mechanical function (53). As expected, we have observed that the first and final days of exercise resulted in a pronounced reduction in vastus lateralis force at low frequencies of electrical stimulation, with recovery observed between days. However, after three consecutive days of exercise, incomplete recovery of force was observed at both high and low frequencies of stimulation during all 3 recovery days. The reduction in force at low frequencies of stimulation has been labeled low-frequency fatigue (LFF) (15), with postcontractile depression (PCD) used to indicate the existence of fatigue at both low and high frequencies of stimulation (5). Force loss under these conditions has been shown to persist for hours and even days (5, 15) and appears to be due to reductions in [Ca²⁺]_f, possibly as a direct result of disturbances in SR Ca²⁺ cycling (1). It is possible that the reduction that we have observed in Ca²⁺ release and Ca²⁺ uptake on the first and third day of exercise may be involved in LFF. However, the persistence of fatigue during the recovery days while SR Ca²⁺-cycling properties have recovered to levels not different from preexercise on day 1 indicates that other factors are involved.

	GRANTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Financial support for this study was received from the Natural Sciences and Engineering Council of Canada (H. J. Green).

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. J. Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, Ontario, Canada N2L 3G1 (e-mail: green{at}healthy.uwaterloo.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

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