Eur J Appl Physiol (1992) 65 : 197-201
Applied Physiology and Occupational Physiology © Springer-Verlag 1992
Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle Shin-ya Kuno ~'2, Masayoshi Akisada 2, and Fumiyuki Mitsumori ~ i National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan 2 Department of Radiology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Accepted March 10, 1992
Summary. To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (31p NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The 3ap NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (Pi), adenosine 5'-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr : (PCr + P0 at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that 3~p NMR is a potent method for evaluating the effects of training in the same individuals. Key words: Phosphorus-31 nuclear magnetic resonance - Muscle metabolism - Phosphocreatine - Adenosine 5 'triphosphate - Endurance training - Muscle fibre type
Introduction Studies on homogenates of whole muscle and mitochondrial fraction have shown that endurance exercise increases the potency of oxidative metabolism in skeletal muscle (Holloszy 1967; Holloszy et al. 1970; Holloszy and Booth 1976; Gollnick 1986). The enzyme activities of succinic dehydrogenase and cytochrome oxidase have been shown to increase approximately twofold in hind-
Offprint requests to: F. Mitsumori
limb muscles of rat in response to training. Increases in the size and number of mitochondria have also been observed using electron microscopy (Davies et al. 1981). In contrast to above effects, endurance training has been found to cause minor changes in glycolytic enzyme activities in the skeletal muscle (Saltin and Gollnick 1983). It has been reported that the decrease in the concentration of phosphocreatine (PCr) during exercise was less in trained muscles (Constable et al. 1987). It is not easy, however, to determine the changes in these labile metabolites during exercise by conventional biochemical methods. Moreover, repeated measurements on the same individuals before and after training are almost impossible. The phosphorus-31 nuclear magnetic resonance (31p NMR) spectroscopy provides a powerful means of investigating changes in the phosphorus metabolites and intramuscular pH at rest and during exercise. From a comparison of 31p NMR spectra from cat biceps brachi (greater than 75% fast-twitch, glycolytic fibres) and soleus (greater than 92°7o slow-twitch, oxidative) muscles (Meyer et al. 1985) it has been suggested that the relative amounts of phosphorus metabolites are dependent on the muscle fibre types in the resting muscle. This finding was later confirmed in in vivo muscles by other workers (Clark et al. 1988; Challiss et al. 1988). Effects of chronic stimulation were also examined on canine latissimus dorsi muscle by 31p NMR. The markedly enhanced resistance to fatigue in the conditioned muscle has been discussed in relation to the transformation of muscle fibre types and the increased ability of oxidative phosphorylation (Clark et al. 1988). Since the electric stimulation was continuous over 24h.day -1 for 8 weeks, and constituted only local exercise, there is a possibility that the training effects of whole body intermittent exercise, such as running, might be different from electrical stimulation. No NMR investigation has been carried out so far following the metabolic changes in muscle in experimental animals before and after endurance training. In the present study, to evaluate the effects of training by en-
198 d u r a n c e r u n n i n g o n skeletal muscle, we have used 31p N M R to examine the same a n i m a l s at rest, d u r i n g exercise, a n d recovery, before a n d after 3 m o n t h s of end u r a n c e training.
Methods A n i m a l care and training. Male Wistar rats age, 13-weeks mass
about 400 g were housed in plastic cages at 22+ 1° C with a diet of standard chow and water ad libitum. Days were divided into 12-h periods of light and darkness. In a preliminary experiment, all animals were run on a motor-driven rodent treadmill (Natsume, Tokyo, Japan) for a short period to establish whether any of them were reluctant to exercise. Such animals were eliminated from further experiments. A group of 16 rats was randomly divided into a sedentary control group and a training group. Before the start of training 31p NMR spectra were obtained at rest, during exercise by electrical stimulation and recovery from both groups. Following a resting period of at least 5 days, the rats in the training group were run for 45 min 3 days.week-1 for 3 months on a motor-driven treadmill at a 5° gradient. Speed of the treadmill was set at 26.8 m . m i n - I to attain a endurance exercise of moderate intensity. After the above training period, the second set of 31p NMR measurements were performed on the same animals. Trained animals were measured 48 h after their last run. This experimental protocol was approved by the Animal Experimental Committee of Tsukuba University. ~ I P N M R measurement. The 3ap NMR measurements were performed at 40.6 MHz with a 2-cm diameter surface coil placed on the right gastrocnemius muscle using a Biospec 24/30 spectrometer (Bruker, Karlsruhe, FRG) equipped with a 30-cm horizontal bore magnet operating at 2.35 T. The NMR spectra were collected by accumulating 32 transients with a pulse length of 25 ~ts, and a recycling time of 3 s. Metabolites were quantified by integrating the NMR peaks. To avoid partial saturation due to a short recycling time the peak area was calibrated using the saturation factors obtained by a comparison of peak areas measured with recycling times of 3 s and 20 s at rest. This saturation factor was also used for the calibration of the peak area during exercise assuming that the longitudinal relaxation times (T1 values) of metabolites are unchanged during exercise. Changes in PCr and inorganic phosphate (Pi) were evaluated by the ratio PCr: (PCr + Pi). Intramuscular pH was calculated from the chemical shift difference between Pi and PCr using the following equation (Taylor et al. 1983)
in silicon tubing through which water at 37°C was circulated throughout the experiment. Statistics. The results from the groups were compared using Stu-
dent's t-test. A significance level of P < 0.05 was chosen.
Results Figure 1 shows 31p N M R spectra at rest, d u r i n g exercise a n d recovery. A decrease in P C r a n d a n increase in Pi were observed d u r i n g exercise. However, n o change was observed in a d e n o s i n e 5 ' - t r i p h o s p h a t e ( A T P ) resonances t h r o u g h o u t the m e a s u r e m e n t s . This result shows that the exercise b y electric s t i m u l a t i o n in the present study was n o t s t r e n u o u s e n o u g h to reduce the c o n c e n t r a t i o n of A T P . Figure 2 shows the m e a n a n d i n d i v i d u a l values o f P C r : (PCr + Pi) ratios at rest, d u r i n g exercise a n d recovery before t r a i n i n g . T h e ratio decreased d u r i n g exercise a n d recovered e x p o n e n t i a l l y after s t i m u l a t i o n was stopped. T h a t ratio reached 0.69 at the end o f exercise in b o t h groups. Figure 3 shows the m e a n a n d i n d i v i d u a l values o f P C r : ( P C r + Pi) ratios at rest, d u r i n g exercise a n d recovery after 3 m o n t h s of e n d u r a n c e training. T h e m e a n ratio at rest showed n o difference b e t w e e n the t r a i n e d a n d c o n t r o l g r o u p even after t r a i n i n g . D u r i n g exercise the ratio was similar to the p r e t r a i n i n g value in the c o n t r o l group, b u t was d r a m a t i c a l l y c h a n g e d in the t r a i n e d group, reaching a steady-state at 0.82 (Fig. 3). This stea-
PCr
p H = 6.75 + log [(a - 3.27)/(5.69 - a)]
where a represents the chemical shift of Pi compared with PCr in ppm. Electrical stimulation in situ in the N M R measurements. Experi-
mental animals were anaesthetized with 1.5% halothane in 50°70 02 and 50% N20 administered through a nose cone during NMR measurement. Each rat was mounted in a NMR probe with an Achilles tendon attached to a force transducer (TB-611T, Nihonkohden, Tokyo, Japan). Electrical stimulation was performed using a stimulator (SEN-3301, Nihon-kohden) with one electrode attached to the Achilles tendon and the other needled to the proximal part of the gastrocnemius muscle. Hindlimb muscles (the gastrocnemius-plantaris-soleus muscle group) were submaximally stimulated with a train of DC pulses of 2 ms duration at 1 Hz. Polarity of DC pulses was automatically reversed every 30 s. Intensity of the pulse was around 20 V but for each animal was adjusted to give the same tension before and after training. The isometric tension generated was monitored during stimulation by a force transducer. With this stimulation protocol the tension was maintained constant during the stimulation (12 min). To prevent a decrease in body temperature by anaesthesia, the rat was encased
ppm
Fig. 1. A series of phosphorus-31 nuclear magnetic resonance (3ip NMR) spectra obtained from rat hindlimb muscles in vivo at rest (lower 4 traces), during exercise (middle 6) and recovery (upper 4). The NMR spectra were measured by accumulating 32 transients with a pulse length of 25 gs, and a recycling time of 3 s. Spectral assignments: Pi, inorganic phosphate; PCr, phosphocreatine: y, ix, fl, three phosphate groups of adenosine triphosphate. Chemical shift values are in ppm from the resonance of PCr
199 A
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rest, during exercise and recovery before training. (A) Mean and SD values of the PCr: (PCr+P3 ratio in the rats for training (O), and control (Q) group; (B) individual plots of the ratio obtained with eight animals in the group for training. Each symbol represents one animal. The rat hindlimb muscles (gastrocnemius-plantaris-soleus muscle group) were directly stimulated by electrical stimulation through electrodes attached to either end of the hindlimb muscles. Electrical stimulation was performed with a train of 2 ms-duration DC pulses at 1 Hz. The performed tension was adjusted to give the same exercise intensity for each animal during the exercise. For definitions see Fig. 1
I
recovery
J
20
30
40
i
i
0
10
Time(min)
(min)
Fig. 2. Changes in the relative PCr concentration to PCr+Pi at
•
Fig. 3. Changes in the relative PCr concentration to PCr+PI at rest, during exercise and recovery after 3 months of training. (A) Mean and SD values of P C r : ( P C r + Pi) ratio in the trained (O), and control (O) group; (B) indivMual plots of the ratio obtained with eight animals in the trained group. Each symbol corresponds to the same animal as in Fig. 2. The conditions for exercise were the same as described in the legend to Fig. 2. Asterisks indicate significant differences in comparison with the control sedentary rats; * P < 0 . 0 5 . For definitions see Fig. 1
A 7.20
dy-state value was much higher than that of the control group. Although the ratio at the end of stimulation in the control group did not appear to show a steady-state clearly in Fig. 2, separate measurements with a prolonged stimulation period showed no further decrease in the ratio even with the prolonged exercise. During recovery the ratio returned to the value at rest more rapidly in the trained group compared with the control group. The mean vaIues of intramuscular p H decreased by approximately 0.1 p H unit during exercise but did not show a significant difference between the two groups even after training (Fig. 4).
Discussion
The ratio of PCr : (PCr + Pi) in the fast-twitch fibres has been shown to be higher than that in the slow-twitch fibres in animals (Meyer et al. 1985; Clark et al. 1988; Challiss et al. 1988). The ratio of 0.91 at rest in the present study showed that our spectra were obtained predominantly f r o m fast-twitch fibres. Although the
7.10 7.00 6.90
6.80
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--= 7.00 6.90 6.80
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0
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[
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1'0
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[
20
3o
4'0
Time(rain)
Fig. 4. Changes in intracellular pH at rest, during exercise and recovery (A) before and (B) after 3 months of training. Values are
mean and SD. O, trained group; O, control group
200 P C r : ( P C r + P~) ratio during exercise and recovery had changed significantly after 3 months of training, the ratio at rest showed no difference or change in either group even after training. This result would suggest that the transformation of the muscle fibre type was not brought about by endurance training of the intensity and duration used in the present study. Constable et al. (1987) have subjected rats to endurance training for 3 months and measured metabolites in the muscle during submaximal exercise by a freeze clamping method. They have reported that the decrease of PCr and increase of P~ were smaller in the trained group than in the untrained group even at the same exercise intensity. They have also reported that concentrations of PCr and Pi reached steady-states after 3 rain of exercise in both groups. Our results were consistent with most of their observations; however, the P C r : ( P C r + P i ) ratio in the trained group reached a steady-state much faster than in the control group (Fig. 3). This discrepancy may have been caused by the difference in the time resolution of the measurement. By conventional biochemical methods it is difficult to measure serially muscle metabolites during exercise and recovery with a good time resolution. In fact, Constable et al. (1987) have obtained only two data points during exercise (3 and 8 min) compared with the time resolution of 1.5 rain in the present work. The decrease in pH during exercise was only approximately 0.1 pH unit in the present study. Kushmerick and Meyer (1985) have reported a similar small decrease in pH to our results during exercise in the rat muscle. The pH change during exercise has also been shown to be insignificant in human flexor digitorum superficialis muscle when the ratio of P C r : ( P C r + P 0 was in the same range as in the present study (Taylor et al. 1983). They have suggested that glycolysis was only significantly activated when the concentration of PCr had decreased to about 40°7o of its original value. If this is also the case in rat muscle, the exercise induced by electrical stimulation in the present study can be regarded as oxidative. It has been shown that the oxygen or ATP consumption in the trained muscle is the same as in the untrained one at the same exercise intensity (Constable et al. 1987). In the present study the same exercise intensity was achieved by electrical stimulation for both trained and control groups during exercise. Therefore, the ATP consumption in the hindlimb muscle should have been the same in the two groups during exercise. After 3 months of training the ratio of P C r : ( P C r + P0 reached a steady-state at a much higher value of 0.82 in the trained group than in the control group (Fig. 3). An increase in muscle volume could have been the reason for the change in P C r : ( P C r + P I ) ratio during exercise. However, this was unlikely, since it has been reported repeatedly that hindlimb muscle mass has not been changed by endurance training the intensity of which was even higher than ours (Fuller and Nutter 1981; McAllister and Terjung 1991). Thus, the change in the P C r : ( P C r + Pi) ratio observed in the trained group has been attributed to the change in energy metabolism. The
higher P C r : ( P C r + Pi) ratio in the trained group would suggest a lower adenosine diphosphate (ADP) concentration in the muscle on the assumption that creatine kinase was working at equilibrium during exercise. The estimated lower ADP concentration during exercise in the trained group was presumably due to the increased capacity for oxidative phosphorylation. This was consistent with the findings that endurance training has increased the volume of the mitochondrial fraction (Holloszy et al. 1970; Karlsson et al. 1974; Holloszy and Booth 1976). After 3 months of training the standard deviations of the P C r : ( P C r + Pi) ratios during exercise were larger in the trained group than in the control group (Fig. 3A). However, individual values show that the PCr :(PCr + Pi) ratios in the steady-state during exercise were not fluctuating in each individual animal (Fig. 3B). This would indicate that the larger deviations in PCr: (PCr + Pi) during exercise were not due to larger errors in the measurements but were due to a larger variation in the steady-state levels from one animal to another. This variation could be an indicator in evaluating a training effect individually. In conclusion 31p NMR was demonstrated to be a potent method in the evaluation of training on the metabolism of resting and exercising muscle in the same individuals. The P C r : ( P C r + Pi) ratios during exercise were significantly higher in the trained group than in the control group. The ratio also recovered to the resting value more rapidly after exercise in the trained group. These results would indicate that endurance training improved the A T P supply system in skeletal muscle.
References Baldwin KM, Klinkerfuss GH, Terjung RL, Mole PA, Holloszy JO (1972) Respiratory capacity of white, red and intermediate muscle: adaptative response to exercise. Am J Physiol 222: 373-378 Challiss RAJ, Blackledge MJ, Radda GK (1988) Spatial heterogeneity of metabolism in skeletal muscle in vivo studied by 31p-NMR spectroscopy. Am J Physiol 254:C417-C422 Clark III BJ, Acker MA, McCully K, Subramanian HV, Hammond RL, Salmons S, Chance B, Stephenson LW (1988) In vivo 31P-NMR spectroscopy of chronically stimulated canine skeletal muscle. Am J Physiol 254:C258-C266 Constable SH, Favier RJ, McLane JA, Fell RD, Chert M, Holloszy JO (1987) Energy metabolism in contracting rat skeletal muscle: adaptation to exercise training. Am J Physiol 253 : C316-C322 Davies KJA, Packer L, Brooks GA (1981) Biochemical adaptation of mitochondria, muscle, and whole-animal respiration to endurance training. Arch Biochem Biophys 209:539-554 Fuller EO, Nutter DO (1981) Endurance training in the rat II. Performance of isolated and intact heart. J Appl Physiol 51:941947 Gollnick PD (1986) Metabolic regulation in skeletal muscle. Influence of endurance training as exerted by mitochondrial protein concentration. Acta Physiol Scand 128 [Suppl 556] :5366 Holloszy JO (1967) Biochemical adaptations in muscle. Effects of exercise on mitochondrial oxygen uptake and respiratory enzyme activity in skeletal muscle. J Biol Chem 242:2278-2282
201
Holloszy JO, Booth FW (1976) Biochemical adaptations to endurance exercise in muscle. Annu Rev Physiol 38:273-291 Holloszy JO, Oscai LB, Don IJ, Mole PA (1970) Mitochondrial citric acid cycle and related enzymes: adaptive response to exercise. Biochem Biophys Res Commun 40:1368-1373 Karlsson J, Nordesjo LO, Saltin B (1974) Muscle glycogen utilization during exercise after physical training. Acta Physiol Scand 90:210-217 Kushmerick M J, Meyer RA (1985) Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance. Am J Physiol 248 : C542-C549 McAllister RM, Terjung RL (1991) Training-induced muscle adaptations: increased performance and oxygen consumption. J Appl Physiol 70: 1569-1574
Meyer RA, Brown TR, Kushmerick MJ (1985) Phosphorus nuclear magnetic resonance of fast- and slow-twitch muscle. Am J Physiol 248 : C279-C287 Saltin B, Gollnick PD (1983) Skeletal muscle adaptability: significance for metabolism and performance. In: Peachey LD, Adrian RH, Geiger SR (eds) Handbook of physiology-skeletal muscle. American Physiology Society, Baltimore, pp 551-631 Saltin B, Henriksson J, Nygaard E, Jansson E, Andersen P (1977) Fiber types and metabolic potentials of skeletal muscles in sedentary man and endurance runners. Ann NY Acad Sci 301 : 329 Taylor DJ, Bore PJ, Styles P, Gadian DG, Radda GK (1983) Bioenergetics of intact human muscle. A 3~p nuclear magnetic resonance study. Mol Biol Med 1 : 77-94
Applied Physiology and Occupational Physiology © Springer-Verlag 1992
Phosphorus-31 nuclear magnetic resonance study on the effects of endurance training in rat skeletal muscle Shin-ya Kuno ~'2, Masayoshi Akisada 2, and Fumiyuki Mitsumori ~ i National Institute for Environmental Studies, Tsukuba, Ibaraki 305, Japan 2 Department of Radiology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305, Japan Accepted March 10, 1992
Summary. To evaluate changes in muscle energetics following endurance training, we measured phosphorus-31 nuclear magnetic resonance (31p NMR) spectra on rat muscle in vivo before and after training in the same animals. The endurance training lasted for 3 months. The 3ap NMR spectra were obtained serially at rest, during exercise by electrical stimulation, and during recovery. Intramuscular phosphocreatine (PCr), inorganic phosphate (Pi), adenosine 5'-triphosphate (ATP) and pH were determined from the NMR spectra. The ratio of PCr : (PCr + P0 at rest showed no difference between the trained and control groups even after 3 months of training. During exercise, however, this ratio was significantly higher in the trained group than in the control group. The ratio also recovered more rapidly after exercise in the trained group. The intramuscular pH decreased slightly by approximately 0.1 pH unit during exercise but did not show a significant difference between the groups. These results indicated that endurance training of 3 months duration improved the ATP supply system in the muscle. They also demonstrated that 3~p NMR is a potent method for evaluating the effects of training in the same individuals. Key words: Phosphorus-31 nuclear magnetic resonance - Muscle metabolism - Phosphocreatine - Adenosine 5 'triphosphate - Endurance training - Muscle fibre type
Introduction Studies on homogenates of whole muscle and mitochondrial fraction have shown that endurance exercise increases the potency of oxidative metabolism in skeletal muscle (Holloszy 1967; Holloszy et al. 1970; Holloszy and Booth 1976; Gollnick 1986). The enzyme activities of succinic dehydrogenase and cytochrome oxidase have been shown to increase approximately twofold in hind-
Offprint requests to: F. Mitsumori
limb muscles of rat in response to training. Increases in the size and number of mitochondria have also been observed using electron microscopy (Davies et al. 1981). In contrast to above effects, endurance training has been found to cause minor changes in glycolytic enzyme activities in the skeletal muscle (Saltin and Gollnick 1983). It has been reported that the decrease in the concentration of phosphocreatine (PCr) during exercise was less in trained muscles (Constable et al. 1987). It is not easy, however, to determine the changes in these labile metabolites during exercise by conventional biochemical methods. Moreover, repeated measurements on the same individuals before and after training are almost impossible. The phosphorus-31 nuclear magnetic resonance (31p NMR) spectroscopy provides a powerful means of investigating changes in the phosphorus metabolites and intramuscular pH at rest and during exercise. From a comparison of 31p NMR spectra from cat biceps brachi (greater than 75% fast-twitch, glycolytic fibres) and soleus (greater than 92°7o slow-twitch, oxidative) muscles (Meyer et al. 1985) it has been suggested that the relative amounts of phosphorus metabolites are dependent on the muscle fibre types in the resting muscle. This finding was later confirmed in in vivo muscles by other workers (Clark et al. 1988; Challiss et al. 1988). Effects of chronic stimulation were also examined on canine latissimus dorsi muscle by 31p NMR. The markedly enhanced resistance to fatigue in the conditioned muscle has been discussed in relation to the transformation of muscle fibre types and the increased ability of oxidative phosphorylation (Clark et al. 1988). Since the electric stimulation was continuous over 24h.day -1 for 8 weeks, and constituted only local exercise, there is a possibility that the training effects of whole body intermittent exercise, such as running, might be different from electrical stimulation. No NMR investigation has been carried out so far following the metabolic changes in muscle in experimental animals before and after endurance training. In the present study, to evaluate the effects of training by en-
198 d u r a n c e r u n n i n g o n skeletal muscle, we have used 31p N M R to examine the same a n i m a l s at rest, d u r i n g exercise, a n d recovery, before a n d after 3 m o n t h s of end u r a n c e training.
Methods A n i m a l care and training. Male Wistar rats age, 13-weeks mass
about 400 g were housed in plastic cages at 22+ 1° C with a diet of standard chow and water ad libitum. Days were divided into 12-h periods of light and darkness. In a preliminary experiment, all animals were run on a motor-driven rodent treadmill (Natsume, Tokyo, Japan) for a short period to establish whether any of them were reluctant to exercise. Such animals were eliminated from further experiments. A group of 16 rats was randomly divided into a sedentary control group and a training group. Before the start of training 31p NMR spectra were obtained at rest, during exercise by electrical stimulation and recovery from both groups. Following a resting period of at least 5 days, the rats in the training group were run for 45 min 3 days.week-1 for 3 months on a motor-driven treadmill at a 5° gradient. Speed of the treadmill was set at 26.8 m . m i n - I to attain a endurance exercise of moderate intensity. After the above training period, the second set of 31p NMR measurements were performed on the same animals. Trained animals were measured 48 h after their last run. This experimental protocol was approved by the Animal Experimental Committee of Tsukuba University. ~ I P N M R measurement. The 3ap NMR measurements were performed at 40.6 MHz with a 2-cm diameter surface coil placed on the right gastrocnemius muscle using a Biospec 24/30 spectrometer (Bruker, Karlsruhe, FRG) equipped with a 30-cm horizontal bore magnet operating at 2.35 T. The NMR spectra were collected by accumulating 32 transients with a pulse length of 25 ~ts, and a recycling time of 3 s. Metabolites were quantified by integrating the NMR peaks. To avoid partial saturation due to a short recycling time the peak area was calibrated using the saturation factors obtained by a comparison of peak areas measured with recycling times of 3 s and 20 s at rest. This saturation factor was also used for the calibration of the peak area during exercise assuming that the longitudinal relaxation times (T1 values) of metabolites are unchanged during exercise. Changes in PCr and inorganic phosphate (Pi) were evaluated by the ratio PCr: (PCr + Pi). Intramuscular pH was calculated from the chemical shift difference between Pi and PCr using the following equation (Taylor et al. 1983)
in silicon tubing through which water at 37°C was circulated throughout the experiment. Statistics. The results from the groups were compared using Stu-
dent's t-test. A significance level of P < 0.05 was chosen.
Results Figure 1 shows 31p N M R spectra at rest, d u r i n g exercise a n d recovery. A decrease in P C r a n d a n increase in Pi were observed d u r i n g exercise. However, n o change was observed in a d e n o s i n e 5 ' - t r i p h o s p h a t e ( A T P ) resonances t h r o u g h o u t the m e a s u r e m e n t s . This result shows that the exercise b y electric s t i m u l a t i o n in the present study was n o t s t r e n u o u s e n o u g h to reduce the c o n c e n t r a t i o n of A T P . Figure 2 shows the m e a n a n d i n d i v i d u a l values o f P C r : (PCr + Pi) ratios at rest, d u r i n g exercise a n d recovery before t r a i n i n g . T h e ratio decreased d u r i n g exercise a n d recovered e x p o n e n t i a l l y after s t i m u l a t i o n was stopped. T h a t ratio reached 0.69 at the end o f exercise in b o t h groups. Figure 3 shows the m e a n a n d i n d i v i d u a l values o f P C r : ( P C r + Pi) ratios at rest, d u r i n g exercise a n d recovery after 3 m o n t h s of e n d u r a n c e training. T h e m e a n ratio at rest showed n o difference b e t w e e n the t r a i n e d a n d c o n t r o l g r o u p even after t r a i n i n g . D u r i n g exercise the ratio was similar to the p r e t r a i n i n g value in the c o n t r o l group, b u t was d r a m a t i c a l l y c h a n g e d in the t r a i n e d group, reaching a steady-state at 0.82 (Fig. 3). This stea-
PCr
p H = 6.75 + log [(a - 3.27)/(5.69 - a)]
where a represents the chemical shift of Pi compared with PCr in ppm. Electrical stimulation in situ in the N M R measurements. Experi-
mental animals were anaesthetized with 1.5% halothane in 50°70 02 and 50% N20 administered through a nose cone during NMR measurement. Each rat was mounted in a NMR probe with an Achilles tendon attached to a force transducer (TB-611T, Nihonkohden, Tokyo, Japan). Electrical stimulation was performed using a stimulator (SEN-3301, Nihon-kohden) with one electrode attached to the Achilles tendon and the other needled to the proximal part of the gastrocnemius muscle. Hindlimb muscles (the gastrocnemius-plantaris-soleus muscle group) were submaximally stimulated with a train of DC pulses of 2 ms duration at 1 Hz. Polarity of DC pulses was automatically reversed every 30 s. Intensity of the pulse was around 20 V but for each animal was adjusted to give the same tension before and after training. The isometric tension generated was monitored during stimulation by a force transducer. With this stimulation protocol the tension was maintained constant during the stimulation (12 min). To prevent a decrease in body temperature by anaesthesia, the rat was encased
ppm
Fig. 1. A series of phosphorus-31 nuclear magnetic resonance (3ip NMR) spectra obtained from rat hindlimb muscles in vivo at rest (lower 4 traces), during exercise (middle 6) and recovery (upper 4). The NMR spectra were measured by accumulating 32 transients with a pulse length of 25 gs, and a recycling time of 3 s. Spectral assignments: Pi, inorganic phosphate; PCr, phosphocreatine: y, ix, fl, three phosphate groups of adenosine triphosphate. Chemical shift values are in ppm from the resonance of PCr
199 A
1.0
a.- 0.9
0.~ ¢C-
+ o
(3_ L;
on
A
1.0
0
c3 0.8
a. 0.8
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0.7
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+, B
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o
e •
I rest I exercise 0
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[,
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[
20
30
40
Time
[
rest
I
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rest, during exercise and recovery before training. (A) Mean and SD values of the PCr: (PCr+P3 ratio in the rats for training (O), and control (Q) group; (B) individual plots of the ratio obtained with eight animals in the group for training. Each symbol represents one animal. The rat hindlimb muscles (gastrocnemius-plantaris-soleus muscle group) were directly stimulated by electrical stimulation through electrodes attached to either end of the hindlimb muscles. Electrical stimulation was performed with a train of 2 ms-duration DC pulses at 1 Hz. The performed tension was adjusted to give the same exercise intensity for each animal during the exercise. For definitions see Fig. 1
I
recovery
J
20
30
40
i
i
0
10
Time(min)
(min)
Fig. 2. Changes in the relative PCr concentration to PCr+Pi at
•
Fig. 3. Changes in the relative PCr concentration to PCr+PI at rest, during exercise and recovery after 3 months of training. (A) Mean and SD values of P C r : ( P C r + Pi) ratio in the trained (O), and control (O) group; (B) indivMual plots of the ratio obtained with eight animals in the trained group. Each symbol corresponds to the same animal as in Fig. 2. The conditions for exercise were the same as described in the legend to Fig. 2. Asterisks indicate significant differences in comparison with the control sedentary rats; * P < 0 . 0 5 . For definitions see Fig. 1
A 7.20
dy-state value was much higher than that of the control group. Although the ratio at the end of stimulation in the control group did not appear to show a steady-state clearly in Fig. 2, separate measurements with a prolonged stimulation period showed no further decrease in the ratio even with the prolonged exercise. During recovery the ratio returned to the value at rest more rapidly in the trained group compared with the control group. The mean vaIues of intramuscular p H decreased by approximately 0.1 p H unit during exercise but did not show a significant difference between the two groups even after training (Fig. 4).
Discussion
The ratio of PCr : (PCr + Pi) in the fast-twitch fibres has been shown to be higher than that in the slow-twitch fibres in animals (Meyer et al. 1985; Clark et al. 1988; Challiss et al. 1988). The ratio of 0.91 at rest in the present study showed that our spectra were obtained predominantly f r o m fast-twitch fibres. Although the
7.10 7.00 6.90
6.80
B -rc3.
7.20 7.10
--= 7.00 6.90 6.80
I
0
rest
[
exercise
1'0
I
recovery
[
20
3o
4'0
Time(rain)
Fig. 4. Changes in intracellular pH at rest, during exercise and recovery (A) before and (B) after 3 months of training. Values are
mean and SD. O, trained group; O, control group
200 P C r : ( P C r + P~) ratio during exercise and recovery had changed significantly after 3 months of training, the ratio at rest showed no difference or change in either group even after training. This result would suggest that the transformation of the muscle fibre type was not brought about by endurance training of the intensity and duration used in the present study. Constable et al. (1987) have subjected rats to endurance training for 3 months and measured metabolites in the muscle during submaximal exercise by a freeze clamping method. They have reported that the decrease of PCr and increase of P~ were smaller in the trained group than in the untrained group even at the same exercise intensity. They have also reported that concentrations of PCr and Pi reached steady-states after 3 rain of exercise in both groups. Our results were consistent with most of their observations; however, the P C r : ( P C r + P i ) ratio in the trained group reached a steady-state much faster than in the control group (Fig. 3). This discrepancy may have been caused by the difference in the time resolution of the measurement. By conventional biochemical methods it is difficult to measure serially muscle metabolites during exercise and recovery with a good time resolution. In fact, Constable et al. (1987) have obtained only two data points during exercise (3 and 8 min) compared with the time resolution of 1.5 rain in the present work. The decrease in pH during exercise was only approximately 0.1 pH unit in the present study. Kushmerick and Meyer (1985) have reported a similar small decrease in pH to our results during exercise in the rat muscle. The pH change during exercise has also been shown to be insignificant in human flexor digitorum superficialis muscle when the ratio of P C r : ( P C r + P 0 was in the same range as in the present study (Taylor et al. 1983). They have suggested that glycolysis was only significantly activated when the concentration of PCr had decreased to about 40°7o of its original value. If this is also the case in rat muscle, the exercise induced by electrical stimulation in the present study can be regarded as oxidative. It has been shown that the oxygen or ATP consumption in the trained muscle is the same as in the untrained one at the same exercise intensity (Constable et al. 1987). In the present study the same exercise intensity was achieved by electrical stimulation for both trained and control groups during exercise. Therefore, the ATP consumption in the hindlimb muscle should have been the same in the two groups during exercise. After 3 months of training the ratio of P C r : ( P C r + P0 reached a steady-state at a much higher value of 0.82 in the trained group than in the control group (Fig. 3). An increase in muscle volume could have been the reason for the change in P C r : ( P C r + P I ) ratio during exercise. However, this was unlikely, since it has been reported repeatedly that hindlimb muscle mass has not been changed by endurance training the intensity of which was even higher than ours (Fuller and Nutter 1981; McAllister and Terjung 1991). Thus, the change in the P C r : ( P C r + Pi) ratio observed in the trained group has been attributed to the change in energy metabolism. The
higher P C r : ( P C r + Pi) ratio in the trained group would suggest a lower adenosine diphosphate (ADP) concentration in the muscle on the assumption that creatine kinase was working at equilibrium during exercise. The estimated lower ADP concentration during exercise in the trained group was presumably due to the increased capacity for oxidative phosphorylation. This was consistent with the findings that endurance training has increased the volume of the mitochondrial fraction (Holloszy et al. 1970; Karlsson et al. 1974; Holloszy and Booth 1976). After 3 months of training the standard deviations of the P C r : ( P C r + Pi) ratios during exercise were larger in the trained group than in the control group (Fig. 3A). However, individual values show that the PCr :(PCr + Pi) ratios in the steady-state during exercise were not fluctuating in each individual animal (Fig. 3B). This would indicate that the larger deviations in PCr: (PCr + Pi) during exercise were not due to larger errors in the measurements but were due to a larger variation in the steady-state levels from one animal to another. This variation could be an indicator in evaluating a training effect individually. In conclusion 31p NMR was demonstrated to be a potent method in the evaluation of training on the metabolism of resting and exercising muscle in the same individuals. The P C r : ( P C r + Pi) ratios during exercise were significantly higher in the trained group than in the control group. The ratio also recovered to the resting value more rapidly after exercise in the trained group. These results would indicate that endurance training improved the A T P supply system in skeletal muscle.
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Holloszy JO, Booth FW (1976) Biochemical adaptations to endurance exercise in muscle. Annu Rev Physiol 38:273-291 Holloszy JO, Oscai LB, Don IJ, Mole PA (1970) Mitochondrial citric acid cycle and related enzymes: adaptive response to exercise. Biochem Biophys Res Commun 40:1368-1373 Karlsson J, Nordesjo LO, Saltin B (1974) Muscle glycogen utilization during exercise after physical training. Acta Physiol Scand 90:210-217 Kushmerick M J, Meyer RA (1985) Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance. Am J Physiol 248 : C542-C549 McAllister RM, Terjung RL (1991) Training-induced muscle adaptations: increased performance and oxygen consumption. J Appl Physiol 70: 1569-1574
Meyer RA, Brown TR, Kushmerick MJ (1985) Phosphorus nuclear magnetic resonance of fast- and slow-twitch muscle. Am J Physiol 248 : C279-C287 Saltin B, Gollnick PD (1983) Skeletal muscle adaptability: significance for metabolism and performance. In: Peachey LD, Adrian RH, Geiger SR (eds) Handbook of physiology-skeletal muscle. American Physiology Society, Baltimore, pp 551-631 Saltin B, Henriksson J, Nygaard E, Jansson E, Andersen P (1977) Fiber types and metabolic potentials of skeletal muscles in sedentary man and endurance runners. Ann NY Acad Sci 301 : 329 Taylor DJ, Bore PJ, Styles P, Gadian DG, Radda GK (1983) Bioenergetics of intact human muscle. A 3~p nuclear magnetic resonance study. Mol Biol Med 1 : 77-94
