Effect of exercise on sarcoplasmic reticulum Ca 2 + transport in muscle of mouse lines long-term selected for different Performance traits

Mouse lines used were long-term selected for high (DU-hTP) or low treadmill Performance (DU-ITP), or high locomotor open field activity (DU-hOF). The control line DU-Ks was maintained unselected. For each line 30 mice were used at 42 days of age. All lines were split into three groups: the first group was investigated without running exercise, the second had to run 500 m, and the third had to run until exhaustion on the treadmill. Compared to mice without running, in exhausted mice the maximal rate of SR Ca uptake of the m. rectus femoris decreases were only found in the mouse lines DU-ITP, and DU-hOF (28.5 %, and 36.3 %, respectively; p<0.05). The effect ofthe 500 m run was significant (p<0.05) in DU-hTP, but not in the other lines. An effect of exhausting exercise on Ca* ATPase was detected only in DU-hOF with increased values after the exhaustive run. The hypothesis, that exercise induced alterations in Ca* transport in muscle of mice selected for high activity (DU-hOF) or high running Performance (DU-hTP) are attenuated compared to mice which were not selected (DU-Ks) or selected for low Performance (DU-ITP), could not be verified.


Introduction
Prolonged physical activity of moderate intensity to high intensity leads to skeletal muscle fatigue.Several mechanisms have been proposed to be responsible for the force decline (GREEN, 1997).Muscle damage (BYRD, 1992), characterised by Zband Streaming, myofibrilar disOrganisation (FRIDEN et al., 1983), and membrane disruption (YASUDA et al., 1997) can all be exercise-induced.Reactive oxygen species produced during strenuous skeletal muscle work contribute to the development of muscle fatigue (DIAZ et al., 1998).The accumulaüon of metabolic by-products affects different cellular events (FAVERO et al., 1995), and glycogen depletion as well as an inappropriate low activity of the Na + ,-K + pump (CLAUSEN and NIELSEN, 1994) are associated with fatigue during prolonged exercise.There is evidence that alterations in intracellular Ca 2+ handling play a major role in the fatigue process (WILLIAMS and KLUG, 1995;FAVERO, 1999).Reduced Ca 2+ handling of the sarcoplasmic reticulum (SR) has been shown following high intensity exercise of horses (BYRD et al., 1989a), in stimulated frog muscles (WILLIAMS et al., 1998), in exhaustively exercised rats (SEMBROWICH and GOLLNICK, 1977), and exercised man (GOLLNICK et al., 1991).Ca 2+ handling is fibre-type specific (BYRD et al., 1989b) and depends on exercise duration (SEMBROWICH and GOLLNICK, 1977;BYRD et al., 1989b).Inhibited SR Ca 2+ release has been identified as a relevant factor in the fatiguing muscle (FAVERO et al., 1993;FAVERO, 1999), probably caused by metabolic end products (FAVERO et al., 1995).Although most investigations indicate a decline of the SR Ca 2+ transport by exercise, some investigators found no alterations.Ca 2+ ATPase and Ca 2T uptake of rat soleus and gastrocnemius homogenates were unaffected by intermittent running (DOSSETT-MERCER et al., 1994).A short term Stimulation elicited no change in Ca 2+ uptake in white or red rat gastrocnemius tissues but increased Ca 2+ ATPase activity in white gastrocnemius tissues (DOSSETT-MERCER et al., 1995).With eccentric exercise there were no differences before and after exercise in SR Ca 2+ uptake, whereas the ATPase activity was increased (ENNS et al., 1999).
Exercise training increases physical fitness (MADSEN et al., 1994), but the effect on the SR Ca 2+ transport is ambiguous.In trained and non trained rats there was no difference in SR Ca 2+ uptake either at rest or after exhaustive exercise, but the Ca + ATPase activity was increased in exhausted muscle of trained rats (BONNER et al., 1976).Intense training did not affect skeletal muscle Ca 2+ ATPase concentration in human (MADSEN et al., 1994).However, Sprint conditioning of horses attenuated the decrease in the calcium transfer rate and in the Ca 2+ ATPase activity (WILSON et al., 1998), and high-resistance training attenuated exercise induced decreases in SR Ca ATPase activity (GREEN et al., 1998).It is suggested that exercise training can retard the onset of fatigue by an attenuation ofa disturbance ofthe Ca 2+ transport (GREEN et al., 1998;WILSON et al., 1998).Selection is another way to influence characters of animals.The mouse lines used in this investigation were long-term selected for high or low running Performance on a treadmill, or high locomotor open field activity, respectively, with an appreciable selection success (indicated in Tab. 1).The objective of this study was to determine the effect of this selection on Ca 2+ transport and Ca 2+ ATPase activity of the skeletal muscle SR at rest, during defined run activity, and after exhaustive exercise with the hypothesis that the effect of exercise on the rate of SR Ca 2+ uptake in mice selected for high locomotor activity or high running Performance is lower than in mice unselected or selected for low locomotor activity.

Animals and exercise
The experiments were carried out in the Mouse Laboratory of the Research Institute for the Biology of Farm Animals in Dummerstorf and based on the outbred strain Fzt:DU, which has been obtained in 1969/1970 by systematic crossbreeding of 4 inbred and 4 outbred lines (SCHÜLER, 1985;RENNE et al., 1985).Four different mouse lines were used.Selection parameters were: high (DU-hTP) or low (DU-ITP) running Performance in a treadmill, and high locomotor activity in an open field (line DU-hOF).The selection for high/low treadmill Performance resulted in an increase/decrease ofthe running Performance of about 180%/71%, and the open field activity increased by about 110% (Tab.1).At the day of exercise all mice were 42 ± 2 days of age.Altogether 120 female mice were used, with 30 mice per selection line.One third of each mouse line was assigned as control without exercise (Con), the second had to run 500 m on a treadmill (500 m), and the third had to run until exhaustion (Exh).The rationale for the 500 m run was to compare the effects of identical loads (same running speed and interval) opposed to the exhaustive run (with expected different running distances) on the investigated parameters.In each of the three activity groups within the lines füll sibs were investigated.Because of their refusal to run six mice had to be exeluded from the evaluations.For standardising the treadmill test the mice were running only in the morning (8.00 to 10.00 a.m.).The treadmill test was a Computer controlled submaximal test.Mice ran on the treadmill (10 % incline) at a rate of about 22.8 m min' 1 (start speed: 15 m min" 1 , final speed: 38 m min" 1 ) aecording to a special test plan (RENNE et al., 1997).The test stop depended on remaining of the mice on the stimulating equipment of the treadmill.

Sample preparation
The animals were killed by cervical dislocation either without exercise (Con) or immediately after exercise (500 m; Exh), the skin was removed, and the m.rectus femoris from both legs were excised.The muscles were freed of fat and as far as possible of connective tissue, immediately frozen in liquid nitrogen, and stored at -70° C. The time interval from the end of exercise to sample freezing was no longer than 5 min.Later, the samples were thawed, finely minced with scissors, and homogenised with a glass/glass Potter-EIvejem type homogeniser in 9 vol of 100 mM KCl, buffered with 20 mM MOPS (pH 7.5), filtered through 3 layers of cheese cloth, and used for immediate measurements of Ca 2+ uptake and SR Ca 2+ ATPase.The protein content of the homogenates was estimated by a modified biuret method (GORNALL et al., 1949), using bovine serum albumin as Standard.

Ca 2+ uptake and Ca 2+ ATPase activity
The rate of Ca 2+ uptake was measured at 30°C with a calcium sensitive minielectrode in 0.5 ml medium containing 100 mM KCl, 5 mM K-oxalate, 5 mM MgCl 2 , 5 mM NaN 3 , 5 mM ATP, and 10 mM HEPES, pH 6.8 (KÜCHENMEISTER et al., 1999).The uptake was initiated by 10 ul homogenate and duplicate measurements were performed.The data acquisition system DaisyLab (DASYTEC GmbH, Mönchengladbach, Germany) was used to determine the highest rate of Ca 2+ uptake by the homogenate (nM min" 1 mg" 1 homogenate protein), i.e. the maximum value ofthe first deviation of the change in calcium concentration.This rate of Ca 2+ uptake will sometimes be referred to as uptake only.Ca ATPase activity of the homogenates was measured spectrophotometrically with a coupled enzyme assay at 30°C by a modified method described previously (SIMONIDES and VAN HARDEVELD, 1990).The 0.8 ml reaction mixture consisted of 20 mM HEPES (pH 7.5), 200 mM KCl, 2 mM ATP, 5 mM MgCl 2 , 1 mM EGTA, 1 mM CaCl 2 , 10 mM NaN 3 , 0.2 mM NADH, 16 IU lactate dehydrogenase, 10 IU pyruvate dehydrogenase, 10 mM phosphoenolpyruvate with 2 uM Ca 2+ ionophore A23187.As confirmed by variations ofthe Ca 2+ concentration, at the chosen Ca 2+ concentration the activity was maximal.The reaction was initiated by the addition of 10 ul homogenate.After about 1 min the change of absorbance was essentially linear and after a further + run of 3 minutes 2 uM cyclopiazonic acid (CPA) was added to inhibit the SR Ca + ATPase selectively.The absorbance was measured for another 3 min.The activity of the Ca 2+ ATPase was determined as the difference of activities before and after the addition of CPA.

Statistical Methods
The data were analysed with the procedure GLM of the Statistical Analysis Package SAS .The Statistical model included the fixed effect "mouse line" and the random effect "exercise level".Differences were considered to be significant if p < 0.05.The tables and figures contain least-squares means (LSM) and the Standard error (SE).

Running Performance and weights
Table 2 shows the running distance until the mice were exhausted and characterizes the body weight and the weight of the muscle (m.rectus femoris) used for this investigation.Despite the long-term selection, within each mouse line there was a very high variability in the running distance.Only the running Performance of the line DU-hOF showed significantly higher values compared to the other lines.Even though the mean running distance of DU-hTP was more than twice of that DU-ITP no significance could be ascertained.The live body weight of DU-ITP was highest followed by DU-Ks.The mice with the high OF-activity and high treadmill Performance are the lightest.The weights ofthe m. rectus femoris were determined from other animals ofthe same lines to get information about differences between the hnes.Related to the body weight the percentage ofthe m. rectus femoris was lowest in DU-Ks but not significantly different from DU-ITP muscle and was not different between exercise groups (data not shown).Ca 2+ uptake and Ca 2+ ATPase activities Without exercise the rate of Ca 2+ uptake in muscle homogenates was lowest in DU-Ks mice followed by Du-ITP (Fig. 1).The uptake ofthe homogenate of DU-hOF and DU-hTP was not different at rest but was significantly higher than in the unselected line DU-Ks or in the line DU-ITP, selected for low treadmill Performance A significant effect of the 500 m run on the rate of uptake (Fig. 1) could only be detected in one line (DU-hTP).The exhaustive run reduced the rate of Ca uptake in DU-hOF (36.3 %), in DU-hTP (21.8 %), and in DU-ITP (28.5 %).Uptake reduction in DU-Ks did not attain Statistical significance.
Figure 2 illustrates the effect of exercise on the SR Ca 2+ ATPase activity (measured with inclusion of an ionophore) of the different mouse lines.Pre-exercise values of the activities of Ca 2+ ATPase between mouse lines were not different.The effect of exercise on the activity of the Ca 2+ ATPase was minor and not uniform.
An exercise for 500 m had no effect.Only the exhaustive run of DU-hOF mice augmented the Ca 2+ ATPase by 29 % whereas exercise for 500 m had no significant effect (Fig. 2).Discussion Using the same muscle of the species mouse our aim was to investigate the effect of long-term selection on the Ca 2+ transport at rest and following exercise.We originally hypothesized, that exercise induced alterations in SR Ca 2+ transport in muscle of mice long-term selected for high locomotor activity or running Performance are attenuated compared to mice which were not selected or selected for low Performance.This hypothesis could not be verified.Contradictory results conceming Ca 2+ uptake and Ca 2+ ATPase are intensely discussed to be at least in part a result of different sample preparation (CHIN et al., 1995;DOSSETT-MERCER et al, 1995;CHIN and GREEN, 1996).To circumvent uncertainties in the SR isolation technique, homogenates were used in this investigation.
At rest, the rate of Ca 2+ uptake was lowest in DU-Ks muscle (unselected line), which could be interpreted that low Performance is related to a low SR sequestering function.However, the line selected for low treadmill Performance (DU-ITP) had comparable low running Performances.Also, the minor differences in Ca 2+ ATPase activities at rest do not lead to the conclusion that the selection had the expected effect on SR Ca 2+ transport.This is in agreement with training experiments, showing an increased Performance but no change in Ca 2+ transport (SEMBROWICH et al., 1978;MADSEN et al., 1994;GREEN et al., 1998), but not with other investigations, indicating a reduced uptake and ATPase activity by long-term training (BELCASTRO, 1987), or an increased uptake and ATPase activity (WILSON et al., 1998), determined at rest.In agreement with the majority of investigations Ca 2+ uptake was affected by the level of exercise.However, whereas the overall (all mouse lines) decrease in the rate of uptake in our experiment by 14.3% for the 500 m run and 24.1% for the exhaustive run (data not shown) attained Statistical significance, the effect of exercise on the uptake between the different mouse lines is not as clear.Contrary to our hypothesis we can not recognize unequivocal differences in the uptake between the different Performance lines either by a 500 m run or the exhaustive run.Comparing training experiments with our selection experiments for different running Performances the effects are confusing in either treatment.Endurance training of rats decreased the Ca 2+ uptake at rest (BELCASTRO, 1987) and a fibre type dependence has been shown (KIM et al., 1981).Sprint conditioning of horses increased the SR Ca 2+ uptake at rest and attenuated an exercise induced decrease in Ca 2+ transport (WILSON et al., 1998).Following endurance training of rats the Ca 2+ uptake of isolated SR was reduced, but not so in muscle homogenates (SEMBROWICH et al., 1978).Training had no significant effect on SR Ca 2+ uptake either at rest or following exercise (BONNER et al., 1976).There was hardly any significant effect of exercise on SR Ca 2+ ATPase, except for the line DU-hOF with an increase in activity by 29.3 % following an exhaustive run (Fig. 2).The reason for this increase is not obvious.These animals run an average distance of 3005 m (corresponding to an average run period of 84 min).It can be speculated that this significantly longer running period compared to the other lines and exercise groups resulted in an activation of latent ATPase enzyme.The muscle used in our investigation, the m.rectus femoris, contains about 64 % white, and 36 % red and intermediate fibres (REHFELDT et al., 1987).An increase in the SR Ca 2+ ATPase activity has been found following a continuous intense run in the soleus and the red gastrocnemius, but not in white gastrocnemius muscle (GREEN et al., 1996).On the other hand, short term Stimulation increased Ca 2+ ATPase activity in white gastrocnemius, but not soleus or red gastrocnemius (DOSSETT-MERCER et al., 1995).An increase in Ca 2+ ATPase shortly after exercise following 2 h of running was suggested to be the result of increasing the proportion of functional SR Ca 2+ ATPase proteins (FERRINGTON et al., 1996).Ca 2+ ATPase activity was elevated in female vastus lateralis immediately following eccentric exercise (ENNS et al., 1999).Possibly, an insufficient supply of blood and energy may have initiated an ATPase activity increase in exhausted DU-hOF mice.Ischemia for 1 to 3 h induced elevations of the SR Ca + ATPase activity dependent on the fibre type composition with greater effects in white muscle, suggesting a recruitment of a latent pool of the enzyme (GREEN et al., 1996).The combination of a decreased uptake rate (Fig. 1) and increased Ca 2+ ATPase activity in exhaustively exercised DU-hOF mice (Fig. 2), means that there is at least in vitro an unexplained Ca 2+ ATPase activity not resulting in Ca 2+ pumping.A comparable effect has been found (DOSSETT-MERCER et al., 1995) which mechanistic basis remained unknown.In summary, previous results indicate a great variability in the effect of exercise and training on SR Ca + transport, depending on species, age, muscle type, exercise protocol, training, use of homogenates or purified SR, and analytical techniques.The long-term selection of mice, resulting in differences in running Performances of about 400 %, seems not to change muscle sarcoplasmic reticulum Ca 2+ transport properties to a corresponding extent.