Skeletal muscles are dynamic units capable of changing their phenotype under various physiological conditions (1). One of the most potent physiological stimuli is endurance training which may induce a number of adaptive responses in the skeletal muscles including changes in muscle capillary density (2), mitochondrial biogenesis (for review see e.g. 3) and transitions of myosin heavy chains and regulatory proteins (4).

The influence of the endurance training on calcium homeostasis in muscle fibers was shown as early as in 1981 by Kim et al. (5) who reported a decrease in the maximum sarco(endo)plasmic reticulum Ca2+ uptake and Ca2+ affinity measured after training in rats. Sarco(endo)plasmic reticulum (SR) is a principal organelle governing calcium homeostasis by ryanodine receptors (RyR) involved in calcium release into the cytosol and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) responsible for ATP dependent Ca2+ transport from cytosol to the lumen of SR (6). It is also known that SR characteristic differs between muscle fiber types (7). The fiber type differences comprising Ca2+ release and uptake by SR arise primarily from greater SR total surface area in fast twitch muscle fibers (8) as well as from faster kinetics of SERCA isoform present in the fast muscle fibers (7, 9). In adult human skeletal muscles two main isoforms of SERCA are present i.e. SERCA1 isoform, which is expressed exclusively in fast-twitch skeletal muscle fibers and SERCA2 present in cardiomyocytes and slow-twitch muscle fibers (6, 10).

The data concerning the influence of short term training program on the SR Ca2+ ATPase expression in humans are sparse (11 - 13). Green et al. (13) have found a significant decrease in the SERCA1 protein content in the vastus lateralis muscle of trained versus control leg after 10 week period single-leg submaximal cycle training, while SERCA2 was not altered. On the other hand Ørtenblad et al. (11) reported a significant large increase in both SERCA1 and SERCA2 isoforms in human vastus lateralis muscle following five week high-intensity intermittent cycling training. Therefore, the training induced changes in SERCA pumps are specific to the nature of training. It is suggested, that the influence of endurance training on SR function is related mainly to the muscle fiber type transition induced by training (12). In endurance trained subjects Ca2+ release, SR Ca2+ ATPase activity and Ca2+ uptake rate are lower when compared to that in untrained subjects (12), which is consistent with the lower percentage of type II muscle fibers in endurance trained athletes vs. untrained subjects (14).

So far the training induced factor(s) responsible for remodeling SERCA pumps is/are unknown. It is well established however, that SERCA are highly energy consuming pumps responsible for about 25-40% of ATP usage during muscle contraction (15, 16). Moreover, it was shown that ATP utilization by SR Ca2+ ATPase in fast muscle fibers is higher than that in slow muscle fibers (9), therefore one may expect that training induced remodeling of SERCA pumps may influence the oxygen cost of work. It is well known that relatively short period of endurance training can significantly reduce oxygen cost of cycling (17). The training induced decrease of oxygen cost was often postulated to be caused by lesser involvement of less efficient type II muscle fibers to the power output generation after training (18). Indeed, lower oxygen cost of cycling at the same work rate was reported in well trained athletes possessing higher proportion of MyHCI vs. athletes with lower MyHCI proportion in the vastus lateralis muscle (19). However, as mentioned above a substantial training induced decrease in oxygen cost may occur already within two weeks of training (17), which is to early to see any significant changes in MyHC composition (20).

In the present study, we have hypothesized that training induced decrease in oxygen cost of cycling may be accompanied by down-regulation of SERCA expression before any measurable changes in myosin heavy chain composition. Therefore, in this study we have evaluated the effect of 5 week endurance training program involving continuous and intermittent cycling of moderate intensity mainly, on the expression of SERCA1 and SERCA2 proteins as well as on the myosin heavy chain composition in human vastus lateralis muscle in relation to the oxygen cost of cycling.


SUBJECTS AND METHODS
Subjects

Fifteen untrained but physically active male volunteers (mean ± SE: age 22.7 ± 0.5 years; body mass (BM) 76.4 ± 2.3 kg; body mass index (BMI) 23.5 ± 0.6 kg · m-2; VO2max 46.0 ± 1.0 mL · kg-1 · min-1) took part in this study after giving informed consent. The study was approved by the Local Ethical Committee and performed under the guidelines of the Declaration of Helsinki.

Exercise protocol

An incremental exercise was performed on the cycloergometer Ergo-Line GmbH & Co KG 800s (Bitz, Germany) following a 6 minute resting period allowed to establish basic cardio-respiratory parameters. The exercise test was performed at pedaling rates of 60 rev · min-1 starting from power output 30 W and followed by gradual increase by 30 W every 3 min until exhaustion. The incremental test was performed two days before and two days after five-weeks of endurance training.

Gas exchange variables

Gas exchange variables were recorded continuously breath by breath using the Oxycon Champion, Mijnhardt BV (Bunnik, The Netherlands) starting from 6th minute before exercise until the end of test. The gas analyzer was calibrated with certified calibration gases as previously described (21).

Plasma lactate concentration [La-]

For plasma lactate measurement blood samples were collected via Abbot Int-Catheter, Ireland (18G/1.2 × 45 mm) inserted into the antecubital vein and connected to an extension set using “T” Adapter SL Abbot, Ireland (the tube 10 cm in length). Blood samples (0.5 mL each) were taken before the exercise test and at the end of exercise protocol. Samples were transferred to 1.8 mL Eppendorf tubes containing 1 mg ammonium oxalate and 5 mg sodium fluoride, mixed for about 20 s and centrifuged. The supernatants containing blood plasma (about 0.2 mL) were stored at minus 40°C until further analysis of lactate concentration ([La-]pl), using automatic analyzer Vitros 250 Dry Chemistry System, Kodak (Rochester, NY, USA).

Lactate threshold (LT)

Lactate threshold in this study was defined as the highest power output above which plasma lactate concentration [La-] showed a sustained increase of more than 0.5 mmol · L-1 · step-1 (21).

Plasma free thyroid hormone concentration [fT3]

For plasma fT3 measurement blood samples (0.5 mL) were taken at rest in the morning hours 7:30 – 8:00 a.m. in fasting state, twice: before and after five weeks of endurance training. Samples were centrifuged and stored at minus 40°C until further analysis. fT3 concentration was determined using electrochemiluminescence immunoassay (ECLIA) using automatic analyzer Elecsys 2010 (Roche Diagnostics).

Endurance training program

Five-weeks endurance training program was performed on cycloergometers Monark 874 E at pedaling rates 60 rev · min-1 according to two different protocols. Continuous endurance cycling protocol was applied for 40 min on Tuesdays and Fridays at the power output (PO) corresponding to 90% of oxygen consumption measured at previously determined lactate threshold (90% VO2 at LT). Intermittent endurance cycling protocol was performed on Mondays and Thursdays and comprised 6 minutes cycling without resistance (unloaded cycling) and 3 minutes cycling at the power output corresponding to 50% . This intermittent training was repeated four times and ended with 4 minutes of unloaded cycling. The power output corresponding to 50% was calculated for each subject according to the formula: 50% = PO at LT + 0.5 (POmax – POLT) (22). There was no training on Wednesdays, Saturdays and Sundays. In total each volunteer performed on average 20.8 ± 0.56 training sessions for 13.9 ± 0.37 hours. The training workload was predominantly of moderate intensity since 85% of workload (expressed in time) was performed below the LT and only 15% above the LT (at 50% , see above). The scaling of the training intensity was based on the principle that the main training workload should be performed in moderate exercise intensity domain (see 23) in order to recruit predominantly the type I muscle fibers (24). However, some of the training workloads were planned to be performed in the heavy intensity domain (23) in order to recruit some of the type II muscle fibers as well (24).

Muscle biopsy

Muscle biopsies were obtained under local anesthesia (1% lidocaine) from the right vastus lateralis m. quadricipitis femoris approximately 15 cm above the upper margin of patella with 2 mm ø biopsy needle (Pro-Mag™ I 2.2, MDTECH). The specimens were frozen immediately in liquid nitrogen and used for electrophoresis and Western immunoblotting. Muscle biopsies were taken before and after five-weeks of endurance training. The second muscle biopsy (i.e. after training) was obtained about 24 ± 2 h after the last training session.

SDS-PAGE and Western immunoblotting

Muscle biopsies (8 - 28 mg) were minced with a scissors and ultrasonicated (UP 50H sonicator, Dr. Hielscher GmbH) on ice with 150 - 200 µL of buffer containing 62.5 mM Tris pH 6.8, 10% glycerol, 2.5% SDS. Samples were centrifuged and supernatants assayed for protein with Bicinchoninic Acid Protein Assay Reagent (Sigma) and Bovine Serum Albumin as a standard. 2-mercaptoethanol was added to the remaining samples to 2.5% final concentration. For myosin heavy chains analysis SDS-PAGE was performed with 4% stacking and 6% separating gels containing 37.5% glycerol as previously described (25). For SERCA1 and SERCA2 analysis SDS-PAGE was carried out in 4.5% stacking and 12.5% separating gels. Proteins resolved by electrophoresis were transferred onto immobilon P transfer membranes (Millipore Corporation, Bedford, USA), blocked with 10% non-fat dry milk in TBS, 0.1% Tween 20 and then incubated for 1 h with primary mouse monoclonal antibody against SERCA1 ATPase (MA3-912) or SERCA2 ATPase (MA3-910) (both antibodies from Affinity BioReagents, Inc., CO, USA) diluted 1:2500. Bound primary antibody was detected with goat anti-mouse IgG alkaline phosphatase conjugate (31323, Pierce Chemical Co., Rockford, IL, USA) diluted 1:2000 and followed by BCIP/NBT treatment. Molecular mass of SERCA1 and SERCA2 was estimated by reference to standard proteins (ProSieve Protein Markers, Cambrex Corporation). The relative amounts of SERCA proteins investigated in muscle biopsies were assayed using CCD camera (Fotodyne Incorporated) and Gel Pro Analyzer software and expressed as integrated optical density units (IOD). The optical density of each protein band was related to that of a internal standard (SERCA in human muscle extract of known protein concentration), which was run on the same gel.

Statistics

The results are expressed as mean and standard error (x ± SE). Significance was set at P<0.05. Statistical significance between two paired samples was tested using nonparametric Wilcoxon signed-rank test and non-asymptotic, exact, two-sided P-values are presented (see Results section). Correlation between two variables was tested with Spearman’s correlation analysis. Moreover, analysis of covariance (ANCOVA) was used to analyse oxygen uptake (VO2) during incremental cycling exercise in the range of moderate intensity power outputs i.e. 30-120 W before and after the endurance training for the group of fifteen subjects. First we tested equality of slopes (parallelism test) of the linear dependencies between power outputs (in the range 30-120 W) and VO2 before and after the endurance training. Since the hypotheses of identical slopes of this dependencies have not been rejected, ANCOVA was then used to test the equality of the intercepts. The statistics was done using the statistical packet STATISTICA 8.0 and StatXact 6.1.


RESULTS
Body mass, maximal oxygen uptake and power output before and after endurance training

The data including body mass (BM), myosin heavy chain type I content (MyHCI) in the vastus lateralis muscle, maximal oxygen uptake (VO2max), power output reached at VO2max (PO at VO2max) and plasma lactate concentration [La-] at VO2max as well as plasma free thyroid hormone concentration [fT3] at rest in the fasting state are presented in Table 1.

Table 1. Characteristics of subjects (n = 15) before and after 5 weeks of endurance training.

Values are means ± SE. BM, body mass; MyHCI, myosin heavy chain type I in the vastus lateralis muscle; VO2max, the maximal oxygen uptake; PO at VO2max, power output reached at VO2max; [La-] at VO2max, plasma lactate concentration determined at VO2max; [fT3], plasma thyroid hormone concentration determined at rest after overnight fasting. Non-asymptotic, exact P–values (Wilcoxon-signed-rank test) are given.

There were no significant changes in the BM (P = 0.20) as well as in MyHCI content in the vastus lateralis muscle (P = 0.67) after training. Plasma [fT3] determined at rest, after overnight fasting was significantly reduced following endurance training (P<10-4). Moreover, the endurance training resulted in a significantly higher VO2max (P = 0.048), PO at VO2max (P = 0.0001) as well as in plasma [La-] at VO2max (P = 0.008). Moreover, VO2 to power output ratio at VO2max was significantly reduced (P = 0.003) after the training (see Fig. 1).

Fig. 1. The oxygen uptake to power output ratio at VO2max before and after the endurance training. Data presented as mean value ± SE for fifteen subjects.

SERCA1 and SERCA2 proteins expression in the vastus lateralis muscle before and after endurance training

An example of Western immunoblotting analysis of SERCA1 and SERCA2 in vastus lateralis muscle before and after the endurance training in the same subject is presented in Fig. 2.

Fig. 2. An example of Western immunoblotting analysis of SERCA1 and SERCA2 in vastus lateralis muscle before and after the endurance training in one subject.

In the studied group of fifteen subjects there was a significant decrease (P = 0.03) in SERCA2 isoform content after training. A clear tendency (P = 0.055) towards lowering SERCA1 content after five weeks of endurance training was also observed (Fig. 3).

Fig. 3. Relative SERCA1 and SERCA2 proteins expression in the vastus lateralis muscle before and after the endurance training. Data presented as mean value ± SE for fifteen subjects. All values in integrated optical density units (IOD).

Moreover, we have found a significant positive correlation between relative concentrations of the electrophoretically separated MyHCI and SERCA2 proteins before training (r = 0.66, P = 0.006) as well as after training (r = 0.80, P = 0.0003, see Fig. 4).

Fig. 4. Correlation between relative SERCA2 protein expression and relative MyHCI expression in the vastus lateralis muscle before and after the endurance training (n = 15).

The oxygen uptake to power output ratio (VO2/PO) during incremental cycling (in the range of power outputs 30-120 W) before and after training

Oxygen uptake during moderate intensity cycling before and after five weeks of endurance training is presented in Fig. 5. In a group of fifteen subjects the oxygen uptake determined during cycling in the range of power outputs 30 - 120 W was significantly lower (ANCOVA, F = 5.9; P = 0.02) after the endurance training when compared to the pre-training values.

Fig. 5. The oxygen uptake / power output relationship during incremental cycling in the range 30 - 120 W performed at 60 rev · min-1. Data presented as mean value ± SE at rest and at each power output for the group of fifteen subjects before ( ) and after ( ) the endurance training.

DISCUSSION
The main finding of this study is that 5 week endurance training of moderate intensity induced significant (P = 0.03) decrease in SERCA2 content and tended to lower (P = 0.055) SERCA1 level in the human vastus lateralis muscle (Fig. 3). This effect was accompanied by a significantly lower (P = 0.02) oxygen uptake to power output ratio occurring during moderate intensity cycling i.e. 30-120 W (Fig. 5) as well as by lower (P = 0.003) VO2 to power ratio at VO2max (Fig. 1). A significant reduction (P<10-4) in plasma free thyroid hormone concentration measured at rest after overnight fasting was observed following training (Table 1). No significant changes (P = 0.67) in the MyHC composition in vastus lateralis muscle after endurance training was observed (Table 1).

Our results regarding the decrease in the VO2 to power ratio are in agreement with the previous study showing that a relatively short period of training significantly decreases oxygen cost of cycling at high intensity (17). The training induced decrease of oxygen cost was often postulated to be caused by lesser involvement of less efficient type II muscle fibers to the power output generation after training (18). Indeed, lower oxygen cost of cycling at the same work rate was reported in well trained athletes possessing higher proportion of MyHCI vs. athletes with lower MyHCI proportion in the vastus lateralis muscle (19). However, as mentioned above in the present study no significant changes in MyHC content was found after the training (P = 0.67), therefore the observed decrease in the oxygen cost of cycling (Fig. 5, P = 0.02) can not be related to the changes in muscle fiber composition in any simple way.

Our finding regarding the effect of training on the SERCA pumps in general are in agreement with the notion that the prolonged training of submaximal intensity in humans causes a reduction in the protein content and diminishes the processes involved in Ca2+ handling (12, 13). This finding is also in agreement with several other studies involving low frequency chronic stimulation resulting in a decrease of SERCA proteins in nonhuman muscles (26, 27). Little is known on the molecular mechanisms involved in regulation of varied SERCA isoforms related to training. It was shown however, that the changes in thyroid hormone can affect SERCA expression (for review see e.g. 28). In the present study, the decrease in SERCA pumps after training (Fig. 3) was indeed accompanied by a significant decrease in free T3 concentration measured at rest after overnight fasting (Table 1), which is in line with the earlier findings showing the effect of changes in T3 on SERCA expression (28).

It was reported in nonhuman muscle, that the relative SERCA2a content strongly positively correlates with relative MyHCI content during chronic electrical stimulation (26) as well as in denervated soleus muscle (29), which suggests a co-ordinated expression of slow myosin and SERCA2a. In the present study, we have found a similar correlation before and after training (Fig. 4). However, in our study the decrease in SERCA2 expression after training (Fig. 3) was not accompanied by a significant changes in MyHCI content in the vastus lateralis muscle (P = 0.67). This clearly indicates, that the training induced modification in SERCA expression precede the changes in myosin heavy chain transition, which was suggested previously (see e.g. 30). A similar pattern of changes was also observed in animal model involving chronic low frequency stimulation (27). Those authors postulated that the observed force reduction in fast muscles in the early stage of chronic low frequency stimulation is related to the alteration in Ca2+ handling and excitation-contraction coupling, which precede the changes in myofibryllar proteins (27).

A clear picture emerging from our study is that the mechanism of SERCA regulation is more sensitive to endurance training than that responsible for transitions of MyHC isoforms. It was postulated that the decrease in phosphorylation potential (GATP) of muscle fibers might be responsible for the fast to slow transition in chronic low frequency stimulation (31) or after the endurance training (32). It is known that fast muscle fibers posses higher GATP than slow muscle fibers (31, 33). Recently, it was reported that indeed the GATP determined in the calf muscles of highly trained endurance athletes at rest is significantly lower when compared to the muscles of untrained subjects (34). Therefore, the decrease in the GATP during muscle contraction as well as training induced decrease in GATP at rest can be responsible for the functional impairment of SERCA pumps after training (see Ref. 1) as well as for the adaptive down-regulation of SERCA expression. This may be due to exercise induced decrease in availability of muscle cell energy, unabling to maintain pretraining muscle protein profile status (for discussion see 35).

SERCA is a highly energetically dependent pump consuming during muscle contraction about 25-40% of the total ATP turnover (9, 15, 16, 36). Interestingly, in our study the applied moderate intensity training had stronger effect on down-regulation of SERCA2 than on SERCA1 expression in the previously untrained subjects (Fig. 3). Since SERCA2 isoform is specific for type I muscle fibers, this direction of changes in SERCA proteins after training suggests that the training induced decrease in oxygen cost of moderate intensity exercise (30-120 W) (Fig. 5) might be related primarily to the increase of type I muscle fibers efficiency. Moreover, we postulate that the training induced decrease in SERCA pumps may be responsible for the decrease of the magnitude of the slow component of the VO2 kinetics occurring during heavy constant power output exercise, at least in the early stage of training. This suggestion can be justified by the fact that in the present study the VO2/PO ratio determined at the end of exercise i.e. at VO2max was also significantly reduced (Fig. 1) in the presence of the decrease in SERCA pumps (for discussion of this point see 37).

The training induced down-regulation of SERCA could be a strategy for the muscle providing greater metabolic stability i.e. lesser decrease in PCr, lesser increase in ADPfree, Pi, H+ and NH3 concentrations etc. during fatiguing prolonged exercise (see e.g. 20, 32) even for the price of compromising Ca2+ sequestration and presumably also muscle relaxation capability (38). On the other hand it should be mentioned that the training induced increase in the intracellular Ca2+ concentration due to down-regulation of SERCA pumps (27, 30) might be responsible for the upregulation of Ca2+ sensitive genes in skeletal muscles (among others mitochondrial proteins) and for the fast to slow transition of muscle fibers (see e.g. 1, 30, 39).

We have concluded that the increase in the mechanical efficiency of cycling occurring during first weeks of endurance training is not related to changes in MyHC composition but it may be due to the down-regulation of SERCA pumps accompanied by a significant decrease in plasma thyroid hormone concentration. We postulate that down-regulation of energy consuming SERCA pumps is an early muscle adaptive response to endurance training providing higher mechanical efficiency during sustained cycling exercise presumably for the price of compromising muscle relaxation rate.

Acknowledgements: The authors thank to dr Piotr Pierzchalski from the Department of Clinical Physiology, Jagiellonian University Medical College for analyzing SERCA expression level.
The study was supported by grants from the University School of Physical Education Krakow, Poland (grant 186/IFC/2005 and funds for the statutory research in 2008 for the Department of Physiology and Biochemistry).


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