sarcoplasmic reticulum was decreased at high stimulus frequencies. The forcefrequency

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1 Journal of Physiology (1992), 448, pp With 5 figures Printed in Great Britain METABOLIC CHANGES WITH FATIGUE IN DIFFERENT TYPES OF SINGLE MUSCLE FIBRES OF XENOPUS LAEVIS BY A. S. NAGESSER, W. J. VAN DER LAARSE AND G. ELZINGA From the Laboratory for Physiology, Free University, van der Boechorststraat 7, 181 BT Amsterdam, The Netherlands (Received 18 December 199) SUMMARY 1. Peak isometric force of single fast (type 1) and slow (type 3) muscle fibres of Xenopus decreased when fibres were stimulated intermittently above their predicted sustainable duty cycle at 2 'C. Type 1 fibres could be fatigued to zero force. In most type 3 fibres force did not decrease below 5 % of the original (P) before activation failure, as indicated by irregular contractions. 2. Fibres were rapidly frozen at different force levels and analysed by highperformance liquid chromatography (HPLC) for ATP, IMP, phosphocreatine (PCr) and creatine (Cr). Lactate was determined enzymatically in type 1 fibres only. The relationships between force and PCr, and between force and ATP during fatigue were, apart from the range of values obtained, the same for both fibre types. When force had fallen to about 6-8 % of original, PCr was fully reduced. At lower force levels, the ATP content decreased, and a concomitant rise of IMP content was found. At zero force, ATP had fallen to about 25 % of its value in rested type 1 fibres, and up to 2,umol lactate (g dry weight)-' had accumulated. 3. Recovery from fatigue was studied in fibres where force had fallen to -6 P (both fibre types) and -2 P (type 1 only). After 1 h of recovery ATP had in all cases returned to the level measured in rested fibres. In fibres fatigued to -6 P, force almost returned to its original value. However, in type 1 fibres fatigued to '2 P, it returned to only 3 P. After 1 h of recovery the PCr/Cr ratio in type 1 fibres was lower (probability, P < 5) than in control fibres, whereas in type 3 fibres it was not significantly different from controls. 4. The relationship between peak force and stimulus frequency, which had a sigmoid shape in fully rested fibres, was drastically changed by fatiguing stimulation. Immediately after fatiguing stimulation of type 1 fibres, force hardly increased with stimulus frequency, corresponding to the observation that calcium efflux from the sarcoplasmic reticulum was decreased at high stimulus frequencies. The forcefrequency relationship of type 3 fibres was the same before and after intermittent stimulation. INTRODUCTION Since the rate of ATP hydrolysis during contraction is higher than the maximum steady-state rate of ATP formation, muscle cannot contract continuously. The MS PHY 448

2 512 A. S. NAGESSER, W. J. VAN DER LAARSE AND G. ELZINGA fraction of time a muscle is active is called the duty cycle. From the maximum rate of ATP formation and the rate of ATP hydrolysis during contraction, the sustainable duty cycle can be predicted, i.e. the duty cycle which can just be maintained by a muscle without fatigue. We predicted the sustainable duty cycle for different fibre types isolated from the iliofibularis muscle of Xenopus laevis, and found good correspondence between predicted and measured value (van der Laarse, Diegenbach & Elzinga, 1989a). This confirms the notion that the cellular processes underlying fatigue are closely linked to the energetic properties of the muscle fibre (van der Laarse, Liinnergren & Diegenbach, 1991). Two different kinds of mechanisms have been suggested for the link between cellular energetics and loss of contractile performance. Firstly, fewer cross-bridges may be formed due to decreased sensitivity of the contractile machinery for calcium (Fabiato & Fabiato, 1978) and/or impaired release of calcium from the sarcoplasmic reticulum. Alternatively, cross-bridges may produce less force. Experiments on single fibres (Edman & Mattiazzi, 1981; Chase & Kushmerick, 1988; Cooke, Franks, Luciani & Pate, 1988; Godt & Nosek, 1989) have shown that the decrease of force per cross-bridge may be a consequence of the production of protons and inorganic phosphate (Pi) related to lactate production and the splitting of PCr. It is, however, unlikely that in amphibian muscle fibres this increase of protons and Pi can fully explain the loss of force with fatigue, because severely fatigued fibres produce the same force as fully rested fibres when exposed to caffeine (Nassar-Gentina, Passonneau & Rapoport, 1981; Liinnergren & Westerblad, 1989). This suggests that in fatigue cytosolic calcium is diminished. In fact, a decrease of cytosolic calcium during intermittent, fatiguing stimulation has recently been measured (Allen, Lee & Westerblad, 1989). The link between reduced calcium efflux from the sarcoplasmic reticulum and cellular energetics is as yet unclear. The present study was undertaken to relate force production to cytosolic ATP, IMP, PCr, Cr and lactate in single muscle fibres contracting at duty cycles higher than the sustainable one. Such information is required in understanding the chain of events linking muscle energetics and fatigue. We studied type 1 and type 3 fibres isolated from the iliofibularis muscle of Xenopus laevis. Type 1 fibres split ATP at a high rate during contraction but have a low aerobic capacity while type 3 fibres are highly aerobic and use less ATP to produce force (Elzinga, Lannergren & Stienen, 1987; van der Laarse, Diegenbach & Elzinga, 1989 b). We related the change of these compounds to the force produced during fatigue, as well as to the force after 1 h of recovery. Preliminary reports of parts of this work have been presented (van der Laarse, Lammertse, Zaremba & Elzinga, 199; Nagesser, van der Laarse & Elzinga, 199). METHODS Preparation Xenopus laevis (females, 8-12 cm) were kept at room temperature and fed mealworms once a week. The animals were killed bv decapitation and both iliofibularis muscles were excised and transferred to a dissection trough similar to the one described by Liinnergren & Smith (1966). Single muscle fibres were isolated by dissection under a microscope with dark-field illumination, using small forceps and scissors. The fibres were stored overnight in Ringer solution (mm: NaCl, 116-5; KCl, 2-; CaCl2, 1.9; NaH2PO, 2-; EGTA. 1; adjusted to ph = 72 with NaOH) at 4 C to be used for the experiment the next day.

3 METABOLITES IN FATIGUED MUTSCLE FIBRES In m. iliofibularis five types of fibres have been recognized (Lannergren & Smith, 1966). During dissection these fibre types can be selected on the basis of the location in the muscle. For the present study two fibre types were selected: type 1 fibres, large (1-15 4am diameter), clear fibres found in the outer zone of the muscle, and type 3 fibres, usually thinner (4-7,um), opaque fibres, located in the tonus bundle of the muscle. A small platinum hook (5,m diameter) was tied to each trimmed-down tendon, using thin (2,m) nylon thread. The fibre was then transferred to the experimental chamber, filled with circulating, oxygenated Ringer solution. The solution was maintained at 2 C by a water jacket surrounding the chamber. Measurements Force Force was measured by mounting the fibre between a force transducer (AE 81, SensoNor) and a fixed rod; fibre length was adjusted to a resting sarcomere length of 2-3,m as judged by laser diffraction. For stimulation 4 ms monophasic current pulses, 12 times above threshold were applied via platinum plate electrodes on either side of the fibre. Force records were displayed on a storage oscilloscope for visual inspection and recorded on a penwriter (Gould 22). The crosssectional area of the fibres was determined from the dimensions of the fibre as described previously (van der Laarse et al. 1989b). Metabolites When the force reached a preselected level during the development of fatigue, it was rapidly frozen. This was done by lowering the experimental chamber, quickly placing a layer of solid carbonic acid with a groove underneath the fibre, and covering the groove and frozen fibre with another layer of solid carbonic acid. The freezing procedure took at most 3 s. After freezing, the fibres were freeze-dried. ATP, IMP, PCr and Cr were determined by HPLC analysis as described by Juengling & Kammermeier (198). Freeze-dried fibres typically weighed 2-4,tg as determined with a Cahn 29 electrobalance. The freeze-dried fibres were homogenized on ice in -1 M-perchloric acid solution for 1 min. Addition of K2C3/Tris (2-8 M/-1 M) to the homogenate caused precipitation of KCl4, which was spun down for 2 min at 24 g. Then 1,ul of the 4,ul supernatant was injected into the HPLC. The detection limits for PCr and IMP corresponded to about 1 and 1,umol (g dry weight)-', respectively. Lactate was determined in type 1 fibres as described by Lowry & Passonneau (1972). Glycogen content of type 1 and 3 fibres was determined on parts of freeze-dried muscles using the method of Krebs, Bennett, de Gasquet, Gascoyne & Yoshida (1963). The glycogen content of bundles of type 1 fibres (mean + S.D., n = 6) was mmol glycosyl (g dry weight)-' while that of bundles of type 3 fibres was mmol glycosyl (g dry weight)-'. Change of the glycogen content of fatigued type 1 fibres was assayed histochemically (periodic acid-schiff reaction according to Graumann, 1953) on cross-sections cut from a part of the freeze-dried fibre. Protocols Intermittent stimulation The relationship between peak force of 25 ms tetani and stimulus frequency (5-1 Hz) was determined in type 1 and 3 fibres. The time interval between tetani was 3 min. On the basis of these measurements, it was decided to fatigue type 1 fibres by intermittent stimulation at 4 Hz, and type 3 at 3 Hz. At these stimulus frequencies, peak force (mean+ s.d._ n = 5) was and % of peak force at 1 Hz in type 1 and type 3 fibres, respectively. (In initial experiments 3 and 7 Hz were also used for type 1 fibres.) To induce fatigue, type 1 fibres were stimulated for 25 ms every 5 s, and type 3 fibres for 25 ms every 5 ms. Recovery from fatigue When the type 1 and type 3 fibres had been fatigued to -6 P, fatiguing stimulation was stopped and the recovery of the fibres was monitored by determining the force-frequency relationship. In this case the time interval between tetani was 3 s. (It took about 7 min to measure one series.) During the recovery period, four such series were performed with intervals of 1 min during which the fibre was not stimulated. Thus, this protocol lasted 55 min. About 5 min after the fourth series of measurements, the fibres were rapidly frozen and freeze-dried as described above in order to investigate metabolic recovery. A similar procedure was followed for type 1 fibres which were fatigued to -2 P

4 514 A. S. NAGESSER, W. J. VAN DEB LAARSE AND G. ELZINGA Statistics Values are given as the mean+standard deviation (S.D.). Analysis of variance was used for statistical evaluation of the data. Multiple regression was carried out as described by Sokal & Rohlf (1981). RESULTS Fatigue For 25 ms tetani the sustainable duty cycles of type 1 and type 3 fibres are -2 and -2, respectively (van der Laarse et al. 1989a; Elzinga & van der Laarse, 199). To fatigue the fibres we imposed duty cycles of.5 and 5, respectively. Examples of the force response to intermittent stimulation are shown in Fig. 1. Both fibre types show initially a rapid decrease of peak force, down to about 75% of the original, during the first minute of intermittent stimulation. In type 1 fibres (Fig. IA), following the initial decrease, force remains fairly constant for a few minutes and then falls rapidly to a new, fairly steady level. The rate ofrelaxation slows down with increasing duration of the intermittent stimulation (result not shown, but see e.g. Liinnergren & Westerblad, 1989). Slowing of relaxation was also found in type 3 fibres: these fibres had insufficient time to relax completely after 3-5 s of intermittent stimulation, resulting in an upward shift of the baseline of the force record (Fig. 1B). Except in one of the type 3 fibres, force did not decrease to levels below 5% of the original. Attempts to fatigue type 3 fibres to a lower force level by increasing the duty cycle resulted in irregular contractions, probably indicative of activation failure. To study metabolite contents, fatigued type 1 and 3 fibres were rapidly frozen after different durations of intermittent stimulation. Figure 2A shows that in type 1 fibres PCr is reduced with decreasing force levels. Phosphocreatine is undetectable below force levels of about P. The decrease of ATP with the fall in force (Fig. 2B) is less pronounced, reflecting the high equilibrium constant of the creatine kinase reaction. When the PCr store is depleted, ATP falls and IMP rises by a similar amount (cf. Fig. 2B and D), i.e. the sum of ATP and IMP does not correlate with relative force, suggesting that the rise of ADP and AMP is relatively small. The correlations between relative force and ATP content (r = 86) and between relative force and lactate content (Fig. 2C, r = - 85) were highly significant (probability, P < 1). Lactate and ATP contents also correlated (r = - 72, P < -1). The coefficient of multiple correlation for relative force with lactate and ATP contents was -93. Thus, taken together, lactate and ATP content explain 86% of the variation of peak force. The correlations between relative force and PCr (r = 66, for relative forces > 75) and relative force and IMP (r = --89, for relative forces < 75) were also significant (P < 1 and P < 1, respectively). Glycogen was depleted only in the two most severely fatigued fibres (Fig. 2). Phosphocreatine in type 3 fibres was also almost fully reduced when force had fallen to about 6-8% of control (Fig. 3A). Although type 3 fibres could not be fatigued to force levels as low as those attained by type 1 fibres, it appears that the reduction of ATP in type 3 (Fig. 3B) and type 1 fibres follows a similar pattern. In only three of the fatigued type 3 fibres (n = 14) small amounts of IMP (< 6 gmol (g dry weight)-' were found.

5 427 kpai METABOLITES IN FATIGUED MUSCLE FIBRES 4 7 k a A kpai 2 s B I L-j 1 s Fig. 1. Fatigue curves for a type 1 (A) and a type 3 fibre (B). Type 1 fibres were stimulated intermittently at 4 Hz, one 25 ms tetanus every 5 s. Type 3 fibres were stimulated at 3 Hz to contract for 25 ms every 5 ms. 1. A 2 - I * I 1 * B - 1 S I1 S +. > - -.2? 1 - E S O a I- o.5 m a) Cu E. n! -o -W II cm 5- S I. 1 I *1 S * -5 C 1 o I- o * CD 2- n 1- E ) ' 1 Relative force *.5 Fig. 2. Relationships between relative force and PCr (A), ATP (B), lactate (C) and IMP (D) in type I fibres. * denotes fully rested fibres. indicates fibres in which glycogen was depleted. D

6 516 A. S. NAGESSER, W. J. VAN DER LAARSE AND G. ELZINGA 4- A 'a a) 5 - Ē._ V E I- - * 1-5 Relative force 1.5 Relative force Fig. 3. Relationship between relative force and PCr (A) and ATP (B) in type 3 fibres. * denotes fully rested fibres B C.) o a) O a) CC k I :) C 1 25 Stimulus frequency (Hz) a) o.> 5-.n_ Stimulus frequency (Hz) 1 Fig. 4. The force-frequency relationship of fully rested fibres () compared with those measured at the onset (U) and the end () of the recovery period for a type 1 fibre fatigued to -6 P (A), a type 1 fibre fatigued to -2 P (B), and a type 3 fibre fatigued to -6 P (C). The arrows indicate the order in which the measurements were performed.

7 METABOLITES IN FATIGUED MUSCLE FIBRES 517 Recovery from fatique Recovery of the force-frequency relationship of a type 1 fibre which was fatigued to -6 P is illustrated in Fig. 4A. Immediately after the intermittent stimulation, twitch force was potentiated while tetanic force was decreased at higher frequencies: 15- -ce ~~~Tp 1re yp fbe Fig. 5. n= 5)..... fromitige t -6es typ 1and type PoRes)n-2 typ 1,nP=3) the steep paxt of the force-frequency relationship disappeared. The force-frequency relationship recovered almost completely in I h: at a stimulus frequency of 1 Hz force recovered to 9+ 7 %/ of the original (n = 4). An example of recovery of type I fibres which were fatigued to -2 Po is given in Fig. 4B. Immediately after the intermittent stimulation, the force-frequency relationship was almost flat, and even twitch force was reduced. There was only a moderate recovery of the force-frequency relationship in I h; force remained severely depressed at % of the original at 1 Hz (n = 3). The force-frequency relationship of a type 3 fibre after fatigue to -6 Po is shown in Fig. 4C. In these fibres twitch force hardly changed while at the end of the recovery period force at higher frequencies had almost returned to the control level (9 + 4 % of the original at 1 Hz, n = 3). ATP, PCr and Cr content of fully rested and recovered type I and 3 fibres are given in Fig. 5. The results should be compared with those of Figs 2 and 3 where it can be seen that at the end of the fatigue protocol PCr was undetectable and ATP was significantly reduced. The comparison reveals that after almost I h of recovery ATP and PCr had returned to the levels found in fully

8 518 A. S. NAGESSER, W. J. VAN DER LAARSE AND G. ELZINGA rested fibres. However, the ratio PCr/Cr of recovered type 1 fibres was significantly decreased (from , n = 5, in controls, to 1P9+-8, n = 7, i.e. the pooled value for all recovered type 1 fibres; P < 5). The decrease of the PCr/Cr ratio in type 3 fibres (from 28+16, n = 4 in controls, to 13+ 1, n = 4, after recovery) is not significant. Note that the ATP content and the total creatine content (PCr + Cr) in type 3 fibres are significantly less than in type 1 fibres (P < 5 and P < -1, respectively). Total creatine contents of fully rested and recovered fibres are the same. DISCUSSION Metabolite contents of rested fibres The two fibre types studied show significant differences in ATP, Cr and total creatine contents in the resting state (Fig. 5). This variation in metabolite levels is similar to that found in mammalian muscle (Saltin & Gollnick, 1983). The PCr contents of type 1 and type 3 fibres of Xenopus are the same. Consequently, the PCr/Cr ratio of rested fibres is higher in type 3 than in type 1. Taking into account the different ATP contents of type 1 and type 3 fibres, and assuming that the equilibrium constant of the creatine kinase reaction and ph are the same for both fibre types, this implies that the level of free ADP is about nine times lower in type 3 than in type 1 fibres, a difference which may be related to the five- to sevenfold greater mitochondrial density in type 3 fibres (Smith & Ovalle, 1973; van der Laarse et al. 1989b). This would make sense if basal metabolic rates were about the same for both fibre types, as has been shown for mouse extensor digitorum longus (fast) and soleus (slow) muscle (Crow & Kushmerick, 1982). In this case, the mitochondrial driving force in resting muscle is inversely related to the volume density of mitochondria. The ATP content of different Xenopus fibres correlates with the cross-sectional area of the fibre, irrespective of the fibre type (Diegenbach, Elzinga, Gnodde, van Hardeveld & van der Laarse, 1988). The significance of the variation of ATP content is not known. Metabolites during fatigue A large difference exists in fatiguability of type 1 and type 3 fibres (Liinnergren & Smith, 1966; van der Laarse et al. 1989a). We applied duty cycles of 2-5 times the sustainable duty cycle of each fibre type, i.e. 5 and 5 for type 1 and type 3 respectively. When for type 1 fibres the chosen duty cycle was maintained for a sufficiently long period, force fell eventually to zero. In type 3 fibres the tenfold higher duty cycle resulted in forces usually above 5 P. This difference in response to the imposed duty cycle may be explained by a higher glycolytic ATP production in type 3 fibres. Histochemical assays of glycolytic enzymes indicate that the activity of these enzymes is higher in type 3 than in type 1 fibres (Spurway & Rowlerson, 1989). An alternative explanation may be that the rate of ATP hydrolysis decreases more in type 3 than in type 1 fibres during the development of fatigue. In spite of the large differences in the maximum rates of ATP production and hydrolysis in type 1 and 3 fibres, the mechanism underlying the drop of force may be similar for the two fibre types. The relationships between force and PCr and ATP (Figs 2A and B, and 3) show, apart from the range of data, no systematic differences.

9 METABOLITES IN FATIGUED MUSCLE FIBRES Also the way peak tetanic force changes during the fatigue protocol (Fig. 1) appears to be similar in spite of the rise of force between contractions in type 3 fibres. The time course of the development of fatigue is similar to that of fast mammalian motor units (Burke, Levine, Tsairis & Zajac, 1973), and also the relationships between force and metabolite contents (Figs 2 and 3) are similar to those reported for mammalian muscle (e.g. Edwards, Hill & Jones, 1975; Hultman & Sjbholm, 1986; Taylor, Styles, Matthews, Arnold, Gadian, Bore & Radda, 1986). Our results are somewhat different from those reported previously for metabolic change during fatiguing stimulation of frog muscle (Dawson, Gadian & Wilkie, 1978; Nassar-Gentina, Passonneau, Vergara & Rapoport, 1978; Nassar-Gentina et at. 1981): in our preparation a considerable decrease of the ATP content is found, whereas only a slight reduction of ATP was apparent in the previous studies. It is possible that this difference is due to a species difference (between Rana and Xenopus) or to experimental conditions like temperature or stimulation pattern. Effects of metabolites on force production When fatigued fibres are exposed to caffeine, which opens the calcium release channels of the sarcoplasmic reticulum, about 8 % of the original tetanic force induced by electrical stimulation is produced (Lannergren & Westerblad, 1989). A decreased calcium concentration during fatigue has been demonstrated (Allen et at. 1989), which explains a major part of the reduced force. The present results also suggest that calcium efflux from the sarcoplasmic reticulum is diminished, because the shape of the force-frequency relationships is changed considerably as a result of the intermittent stimulation (Fig. 4A and B). However, reduced calcium concentration cannot explain completely the reduced force production because an increased calcium concentration was found during the first phase of the intermittent stimulation while force already decreased (Allen et al. 1989). Thus, a part of the force reduction must be due to effects of the metabolic changes on cross-bridge kinetics. Furthermore, the cause of the decreased calcium concentration is not known, but may well be a consequence of the metabolic changes. It can be deduced from the results presented in Fig. 2 that during fatigue a large number of alterations occur in the composition of the sarcoplasm, which may affect both the number of force-producing cross-bridges and the force produced per crossbridge. First, PCr is split, resulting in a rise of Pi and Cr, which are both known to depress force in skinned fibres (Chase & Kushmerick, 1988; Cooke et al. 1988; Godt & Nosek, 1989). Then, ATP is converted to IMP, further increasing the amount of Pi. Because ATP forms a complex with magnesium and has a higher binding constant for magnesium than the other relevant nucleotides (ADP, AMP and IMP, for review, see Curtin & Woledge, 1978) it is expected that the free magnesium concentration in the sarcoplasm increases as a result of the conversion of ATP to IMP. This is of particular interest because magnesium inhibits calcium release from the sarcoplasmic reticulum (e.g. Lamb & Stephenson, 1991). Thus it can be hypothesized that the decreased calcium concentration during fatiguing stimulation may result from an increased sarcoplasmic magnesium concentration. In addition to these changes, lactate accumulates in the fibre, which leads to acidification (to ph at 4 P; Westerblad & LUnnergren, 1988) despite the increase of buffer power as a result of the formation of Pi and, possibly, ammonia. 519

10 52 A. S. NAGESSER, W. J. VAN DER LAARSE AND G. ELZINGA The acidification reduces the force produced by individual cross-bridges and reduces the calcium sensitivity of the filaments (Fabiato & Fabiato, 1978). Thus the conversion of ATP to IMP and the acidification lead to a reduction of the number of force-producing cross-bridges and reduced force production per cross-bridge. These mechanisms may underly the observed multiple correlation coefficient (r = '93, cf. Results) which indicates that the force production during fatiguing intermittent stimulation is almost completely explained by ATP and lactate content of the fibre. In addition to these effects, it can be deduced from Fig. 2 that the metabolic changes increase the osmolarity of the sarcoplasm and this leads to an increase of fibre volume (by about 35 % at 4 P; Liannergren, 199). The effect of osmotic swelling on force production, at constant extracellular osmolarity, is probably small (Edman & Hwang, 1977). It should be noted, however, that the swelling will reduce the concentration of at least some sarcoplasmic constituents. It is thus unlikely that any of the metabolic changes individually can explain fully the force response to intermittent stimulation (cf. Godt & Nosek, 1989). However, it is difficult to estimate the magnitude of the separate effects especially since it can be expected that the various changes interact. One of the possible interactions is worth noting, because it may relate to the increase of calcium concentration observed during the first phase of intermittent stimulation (Allen et al. 1989). This phenomenon can be explained by the binding of magnesium to HP42- as follows: the transient increase of the HPO42- concentration during the first phase of intermittent stimulation as a result of the splitting of PCr and progressive acidification (Wilkie, 1986), may cause a transient decrease of free magnesium and thus a transient increase of calcium efflux from the sarcoplasmic reticulum. Recovery from fatigue The force did not recover completely in any of the fibres during 1 h following the fatigue protocol of the fibre types 1 and 3. The difference between the control force and the final force found in the recovery phase was largest in the type 1 fibres which were fatigued to -2 P (Fig. 4B), despite the fact that in these fibres ATP and PCr content were not significantly different from control (Fig. 5). However, at the end of the recovery period, the PCr/Cr ratio in type 1 fibres was significantly lower than control. This decrease was independent of whether the fibres were fatigued to -6 or to -2 P, but it was not significant in type 3 fibres. The lower PCr/Cr ratio found after 1 h of recovery suggests that the free energy of ATP hydrolysis is not recovered to the value reached in control fibres, which can be seen as a sign of impaired mitochondrial function. In fact, mitochondria in fatigued lumbrical fibres ofxenopus are disrupted after 1 min of recovery (Liinnergren, Westerblad & Flock, 199). The reason for these mitochondrial changes is not known. It may be that accumulation of calcium and Pi in the mitochondria plays a role (Gunter & Pfeiffer, 199), although accumulation of calcium in mitochondria of fatigued frog muscle fibres has been excluded previously as a possible cause of the fall of force; Gonzalos-Serratos, Somlyo, McClellan, Shuman, Borrero & Somlyo (1978) found no calcium accumulation in mitochondria, but, in fatigued fibres, calcium in the sarcoplasmic reticulum was higher than control. The mitochondria in their preparations, which were not allowed to recover, showed no sign of damage. This differs from the result

11 METABOLITES IN FATIGUED MUSCLE FIBRES of Lainnergren et al. (199), which may indicate that mitochondria are damaged during the recovery period rather than during intermittent fatiguing stimulation. In spite of the effect of fatigue on the PCr/Cr ratio, the lack of correlation between force and ATP content at the end of the recovery period indicates that reduced energy stores cannot explain the depressed force. This conclusion, in combination with the finding of Allen et al. (1989) that during recovery calcium release is sufficiently diminished to explain the force reduction, suggests that a (partial) block in excitation-contraction coupling at the level of the T-tubules or the triads (Liinnergren & Westerblad, 1989) may play an important role during the recovery period. We thank R. Zaremba for expert technical assistance. 521 REFERENCES ALLEN, D. G., LEE, J. A. & WESTERBLAD, H. (1989). Intracellular calcium and tension during fatigue in isolated single muscle fibres from Xenopus laevis. Journal of Physiology 415, BURKE, R. E., LEVINE, D. N., TSAIRIS, P. & ZAJAC, F. E. (1973). Physiological type and histochemical profiles in motor units of the cat gastrocnemius. Journal of Physiology 234, CHASE, P. B. & KUSHMERICK, M. J. (1988). Effects of ph on contraction of rabbit fast and slow skeletal muscle fibers. Biophysical Journal 53, COOKE, R., FRANKS, K., LUCIANI, G. B. & PATE, E. (1988). The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate. Journal of Physiology 395, CROW, M. & KUSHMERICK, M. J. (1982). Chemical energetics of slow- and fast-twitch muscles of the mouse. Journal of General Physiology 79, CURTIN, N. A. & WOLEDGE, R. C. (1978). Energy changes and muscular contraction. Physiological Reviews 58, DAWSON, M. J., GADIAN, D. G. & WILKIE, D. R. (1978). Muscular fatigue investigated by phosphorus nuclear magnetic resonance. Nature 274, DIEGENBACH, P. C., ELZINGA, G., GNODDE, M. W. J., VAN HARDEVELD, C. & VAN DER LAARSE, W. J. (1988). Variation of adenosine triphosphate and phosphocreatine levels from isolated muscle fibers of Xenopus laevis. Journal of Physiology 4, 26P. EDMAN, K. A. P. & HWANG, J. C. (1977). The force-velocity relationship in vertebrate muscle fibres at varied tonicity of the extracellular medium. Journal of Physiology 269, EDMAN, K. A. P. & MATTIAZZI, A. R. (1981). Effects of fatigue and altered ph on isometric force and velocity of shortening at zero load in frog muscle fibres. Journal ofmuscle Research and Cell Motility 2, EDWARDS, R. H. T., HILL, D. K. & JONES, D. A. (1975). Metabolic changes associated with the slowing of relaxation in fatigued mouse muscle. Journal of Physiology 251, ELZINGA, G., LXNNERGREN, J. & STIENEN, G. J. M. (1987). Stable maintenance heat rate and contractile properties of different single muscle fibres from Xenopus laevis at 2 'C. Journal of Physiology 393, ELZINGA, G. & VAN DER LAARSE, W. J. (199). Oxygen consumption related to tetanic contractions of single slow-twitch muscle fibres of Xenopus laevis. Journal of Physiology 42, 113P. FABIATO, A. & FABIATO, F. (1978). Effects of ph on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. Journal ofphysiology 276, GODT, R. E. & NoSEK, T. M. (1989). Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle. Journal of Physiology 412, GONZALES-SERRATOS, H., SOMLYO, A. V., MCCLELLAN, G., SHUMAN, H., BORRERO, L. M. & SOMLYO, A. P. (1978). Composition of vacuoles and sarcoplasmic reticulum in fatigued muscle: Electron probe analysis. Proceedings of the National Academy of Sciences of the USA 75,

12 522 A. S. NAGESSER, W. J. VAN DER LAARSE AND G. ELZINGA GRAUMANN, W. (1953). Zur standardisierung des Schiff'schen Reagens. Zeitschrift fur Wissenschaftliche Mikroskopie und fur mikroskopische Technik 61, GUNTER, T. E. & PFEIFFER, D. R. (199). Mechanisms by which mitochondria transport calcium. American Journal of Physiology 258, C HULTMAN, E. & SJ6HOLM, H. (1986). Biochemical causes of fatigue. In Human Muscle Power, ed. JONES, N. L., MCCARTNEY, N. & MCCOMAS, M. J., pp Human Kinetic Publ., Champaign, IL, USA. JUENGLING, E. & KAMMERMEIER, H. (198). Rapid assay of adenine nucleotides and creatine compounds in extracts of cardiac tissue by paired-ion reverse-phase high-performance liquid chromatography. Analytical Biochemistry 12, KREBS, H. A., BENNETT, D. A. H., DE GASQUET, P., GASCOYNE, T. & YOSHIDA, T. (1963). Renal gluconeogenesis. The effect of diet on the gluconeogenic capacity of rat kidney slices. Biochemical Journal 86, LAMB, G. D. & STEPHENSON, D. G. (1991). Effect of Mg2+ on the control of Ca2" release in skeletal muscle fibres of the toad. Journal of Physiology 434, LXNNERGREN, J. (199). Volume changes of isolated Xenopus muscle fibres associated with repeated tetanic contractions. Journal of Physiology 42, 116P. LXNNERGREN, J. & SMITH, R. (1966). Types of muscle fibres in toad skeletal muscle. Acta Physiologica Scandinavica 68, LXNNERGREN, J. & WESTERBLAD. H. (1989). Maximum tension and force-velocity properties of fatigued single Xenopus muscle fibres studied by caffeine and high K+. Journal of Physiology 49, LXNNERGREN, J., WESTERBLAD, H. & FLOCK, B. (199). Transient appearance of vacuoles in fatigued Xenopus muscle fibres. Acta Physiologica Scandinavica 14, LOWRY,. H. & PASSONNEAU, J. V. (1972). A Flexible System of Enzymatic Analysis, pp Academic Press, London. NAGESSER, A. S., VAN DER LAARSE, W. J. & ELZINGA, G. (199). Metabolic recovery of fatigued muscle fibres of Xenopus laevis. Journal of Physiology 426, 32P. NASSAR-GENTINA, V., PASSONNEAU, J. V. & RAPOPORT, S. I. (1981). Fatigue and metabolism of frog muscle fibres during stimulation and in response to caffeine. American Journal of Physiology 241, C NASSAR-GENTINA, V., PASSONNEAU, J. V., VERGARA, J. L. & RAPOPORT, S. I. (1978). Metabolic correlates of fatigue and of recovery from fatigue in single frog muscle fibres. Journal of General Physiology 72, SALTIN, B. & GOLLNICK, P. D. (1983). Skeletal muscle adaptability: significance for metabolism and performance. In Handbook of Physiology, section 1, Skeletal Muscle, pp American Physiological Society, Bethesda, MD, USA. SMITH, R. S. & OVALLE, W. K. (1973). Varieties of fast and slow extrafusal muscle fibres in amphibian hind limb muscles. Journal of Anatomy 116, SOKAL, R. R. & ROHLF, F. J. (1981). Biometry, pp Freeman and Company, San Francisco, USA. SPURWAY, N. C. & ROWLERSON, A. M. (1989). Quantitative analysis of histochemical and immunohistochemical reactions in skeletal muscle fibres of Rana and Xenopus. Histochemical Journal 21, TAYLOR, D. J., STYLES, P., MATTHEWS, P. M., ARNOLD, D. A., GADIAN, D. G., BORE, P. & RADDA, G. K. (1986). Energetics of human muscle: Exercise-induced ATP depletion. Magnetic Resonance in Medicine 3, VAN DER LAARSE, W. J., DIEGENBACH, P. C. & ELZINGA, G. (1989a). The duty cycle of single muscle fibres from Xenopus laevis. In Progress in Clinical and Biological Research, vol. 315, Muscle Energetics, ed. PAUL, R. J., ELZINGA, G. & YAMADA, K., pp A. R. Liss, New York. VAN DER LAARSE, W. J., DIEGENBACH, P. C. & ELZINGA, G. (1989b). Maximum rate of oxygen consumption and quantitative histochemistry of succinate dehydrogenase in single muscle fibres of Xenopus laevis. Journal of Muscle Research and Cell Motility 1, VAN DER LAARSE, W. J., LAMMERTSE, T. S., ZAREMBA, R. & ELZINGA, G. (199). Some metabolite contents of fatigued, single fast twitch muscle fibres from Xenopus laevis. Journal of Muscle Research and Cell Motility 11, 77.

13 METABOLITES IN FATIGUED MUSCLE FIBRES 523 VAN DER LAARSE, W. J., LXNNERGREN, J. & DIEGENBACH, P. C. (1991). Resistance to fatigue of single muscle fibres from Xenopus laevis related to succinate dehydrogenase and myofibrillar ATPase activities. Experimental Physiology 76, WESTERBLAD, H. & LXNNERGREN, J. (1988). The relation between force and intracellular ph in fatigued, single Xenopus muscle fibres. Acta Physiologica Scandinavica 133, WILKIE, D. R. (1986). Muscular fatigue: effects of hydrogen ions and inorganic phosphate. Federation Proceedings 45,