pathways provided an increasing and anaerobic pathways a decreasing fraction of

Transcription

1 J. Physiol. (1986), 374, pp With 1 text-figure Printed in Great Britain SKELETAL MUSCLE METABOLISM, CONTRACTION FORCE AND GLYCOGEN UTILIZATION DURING PROLONGED ELECTRICAL STIMULATION IN HUMANS BY ERIC HULTMAN AND LAWRENCE L. SPRIET From the Department of Clinical Chemistry II, the Karolinska Institute, Huddinge University Hospital, Huddinge, Sweden (Received 30 May 1985) SUMMARY 1. Muscle metabolism and contraction force were examined in the quadriceps femoris muscles of seven volunteers during 45 min of electrical stimulation. Intermittent stimulation was used, with tetanic trains at 20 Hz lasting 1P6 s, separated by pauses of 1P6 s. Muscle biopsies were taken at rest and during stimulation (80 s, 15, 30 and 45 min). 2. During the initial 80 s of stimulation contraction force decreased to 72 % of initial force. The glycogenolytic rate was 40 9 mmol glucosyl units kg-' dry muscle min- and glycolytic intermediate levels increased several fold. Muscle phosphocreatine decreased to 26 % of resting concentration and the ATP turnover rate from anaerobic sources was 4'99 mmol kg-' dry muscle s-l. 3. With continued stimulation from 80 s to 15 min, force decreased to 43 % of the initial value at 5 min and 31 % at 15 min. Glycogenolysis fell to 5-4 mmol kg-' dry muscle min1 and glycolytic intermediate levels decreased suggesting that anaerobic glycolysis contributed progressively less ATP for force production. 4. The final 30 min of stimulation was characterized by a low rate of glycogenolysis ( mmol kg-' dry muscle min') and a constant force production (25-5 % of initial). The ATP turnover rate, assuming glycogen was metabolized aerobically, was 1-86 mmol kg-' dry muscle s-1. Phosphocreatine, ATP and glycolytic intermediates returned to near resting levels, indicating that anaerobic energy pathways were not reactivated. 5. After the first 80 s of the prolonged period of electrical stimulation, aerobic pathways provided an increasing and anaerobic pathways a decreasing fraction of the energy supply. This change occurred in the absence of input from the central nervous system. INTRODUCTION Electrical stimulation of human quadriceps femoris muscles has been used to examine skeletal muscle metabolism, contraction force and fatigue characteristics (Edwards, Young, Hosking & Jones, 1977; Hultman, Sj6holm, Sahlin & Edstrdm, 1981). Electrical stimulation presents the muscle with a constant contraction

2 494 E. HULTMAN AND L. L. SPRIET stimulus and together with the muscle biopsy technique makes it possible to compare contractile and metabolic changes independent of volitional effort. Earlier work from this laboratory examined skeletal muscle energy metabolism during short-term electrical stimulation where the ATP production required for muscular contraction depended mainly on anaerobic pathways (Hultman & Sjoholm, 1983; Sj6holm, Sahlin, Edstrom & Hultman, 1983). It was the intent of this study to extend the electrical stimulation of skeletal muscle to 45 min. This model differs from normal exercise tasks of maximal intensity where the subject volitionally works until exhaustion. Anaerobic energy pathways are maximally activated but this type of work can only be sustained for a few minutes. With continued electrical stimulation, in order to maintain force production, the muscle must shift from an initial dominance of anaerobic ATP production to aerobic ATP production using intrinsic control mechanisms. From measurements of muscle force production, phosphagen and metabolite concentrations and glycogen utilization we hoped to further our understanding of the mechanisms controlling fatigue and the pathways of ATP production using prolonged electrical stimulation. METHODS Seven healthy volunteers (four men and three women, aged years) participated in the study. The subjects were not well trained but regularly took part in some form of physical activity. Voluntary consent was obtained from all subjects following an explanation of the experimental procedures and possible risks involved. This experiment was part of a larger project approved by the Ethical Committee of the Karolinska Institute. Each subject reported to the laboratory in the morning following an overnight fast and reclined in a semisupine position on a bed. Both lower legs were flexed to 90deg over the end of the bed and one leg, chosen at random, was attached to a strain gauge in the frame of the bed via an ankle strap. The maximal voluntary isometric force generated by the knee extensors was measured with a strain gauge (Bofors). The strain gauge signal was amplified (d.c. amp. Medelec AD6), displayed on an oscilloscope (Medelec M) and recorded on U.V. sensitive paper. Electrical stimulation of human skeletal muscle has been previously described in detail (Hultman et al. 1981; Hultman, Sj6holm, JUderholm-Ek & Krynicki, 1983). Briefly, two large (9 x 6 cm) aluminum foil electrodes were applied proximally and distally to the anterolateral aspect of the thigh. The underlying muscles were stimulated to contract with square-wave pulses of 0 5 ms duration at a frequency of 20 Hz (Medelec 15/v stimulator). Stimulation was intermittent with trains lasting 1-6 s, separated by pauses of 1-6 s. The voltage range used ( V) produced an initial force corresponding to 26 % of the maximal voluntary isometric force. Since stimulation at 20 Hz produces a fused tetanus that represents % of the maximal tetanic force (Sjbholm et al. 1983), approximately % ofthe musculature that extends the knee was activated. We used a 20 Hz stimulation frequency to avoid the fatigue reported to occur at higher frequencies ( Hz). This high frequency fatigue is thought to be the result of impaired neuromuscular transmission or failure of membrane excitation (Edwards, 1981). The first leg was stimulated for 30 min while isometric force production was measured continuously. Needle biopsies were taken from the quadriceps femoris muscle (Bergstrom, 1962) at rest and following 80 s (twenty-five contractions) and 30 min of stimulation. The other leg was then prepared for electrical stimulation and force measurement and stimulated for 45 min. Muscle biopsies were taken at rest and following 15 and 45 min of stimulation. The muscle biopsy samples were immediately frozen (3-5 s from the insertion of the needle) in liquid Freon maintained at its melting point (-150 'C) by liquid nitrogen. Samples were freeze-dried and dissected free from blood and connective tissue. A portion of the powdered muscle (2-5 mg) was used for the determination of glycogen (Harris, Hultman & Nordesjd, 1974). The remainder of the muscle (6-10 mg) was extracted with 0 5 M-HClO4. The neutralized extracts were

3 MUSCLE METABOLISM DURING PROLONGED STIMULATION 495 analysed enzymically (Bergmeyer, 1965) for ATP, phosphocreatine (PCr), creatine (Cr), glucose, glucose-i -phosphate (G-I -P), glucose-6-phosphate (G-6-P), fructose-6-phosphate (F-6-P), glycerol- 1-phosphate (Glyc-1-P) and lactate as described by Harris et al. (1974). Resting glycogen and metabolite concentrations represent the average of measurements performed on biopsies from both legs of each individual. Concentrations of muscle metabolities were expressed per kilogram dry muscle to avoid changes in concentration due to water shifts. In addition, the metabolite concentrations have been adjusted to the highest content of total creatine found in the biopsies from each subject to compensate for remaining admixture of connective tissue and non-muscular elements. This correction averaged % in seven subjects. The data are presented as mean+ S.E. of mean ~~~~~~~~~~~~~~~~~o E 0 ~~~~~~~~~~~~~~~~~~~E 40 C uf40 Mt 0~ 20 < 20 '1 0 L II I Stimulation time (min) Fig. 1. Muscle contraction force and concentrations of phosphocreatine (PCr), ATP and lactate during 45 min of intermittent stimulation with an open circulation. Stimulation consisted of repeated tetanic contractions at 20 Hz lasting 1-6 s, separated by pauses of 1-6 s. Data points represent the mean of seven subjects. Standard error bars for ATP, phosphocreatine and lactate are not included for clarity but appear in Table 1. 0~ RESULTS Isometric force production by the activated muscle declined rapidly during the initial 15 min of stimulation, falling to 90 % of peak after 40 s and 72 0/0 after 80 s (Fig. 1). The respective values at 5, 10 and 15 min were 43, 35 and of initial force. Over the final 30 min the decline in force was minimal with 25 % of peak force maintained after 30 min and 24 % after 45 min of stimulation. Muscle glycogen concentrations at rest and during stimulation are presented in Table 1. During only 80 s of intermittent stimulation, glycogen concentration decreased from to mmol glucosyl units kg-1 dry muscle, corresponding to a glycogenolytic rate of 40 9 mmol kg-' dry muscle min- (Table 2). During the subsequent 13-7 min the rate of glycogenolysis was considerably lower at 5-4 mmol kg-' dry muscle min-. Glycogen utilization decreased even further during the final 30 min of stimulation, averaging 1P mmol kg-' dry muscle min- (Table 2).

4 496 E. HULTMAN AND L. L. SPRIET Following 80 s of intermittent stimulation muscle ATP decreased to 830 and phosphocreatine to 26 % of rest values while lactate concentration increased to 718 mmol kg-1 dry muscle (Table 1, Fig. 1). With continued stimulation, the metabolite concentrations gradually returned towards resting levels. Following 15, 30 and 45 min of stimulation, muscle ATP increased to 86, 97 and 98 % of resting levels, respectively (Table 1). Similar values for phosphocreatine regeneration during TABLE 1. Muscle glycogen and metabolite concentrations during 45 min of intermittent stimulation at 20 Hz Rest 80 s 15 min 30 min 45 min ATP A PCr Cr P P5+5-8 Glycogen Glucose G-1-P G-6-P F-6-P Glyc-1 -P P Lactate Values are means + S.E. of mean in mmol kg-1 dry muscle, except glycogen where units are mmol glucosyl units kg-' dry muscle. n = 7. TABLE 2. Glycogen utilization during 45 min of electrical stimulation Rate of Glycogen utilized glycogenolysis Time period (mmol glucosyl units kg-1 dry muscle) (mmol kg-' dry muscle min-') 0-80 s s-15 min 73' min min min min continued stimulation were 50, 65 and 73 %. Muscle lactate concentration decreased to 36-4, 12-8 and 6-7 mmol kg-' dry muscle at the same time points. Changes in creatine content were reciprocal to changes in phosphocreatine at all time points and total creatine content (uncorrected for admixture of non-muscular elements) was constant during prolonged stimulation within biopsies from individual subjects. Changes in the concentrations of the glycolytic intermediates G-1-P, G-6-P, F-6-P and Glyc-1-P were similar to the pattern seen in lactate, increasing at 80 s and returning to rest levels by 30 or 45 min (Table 1). Muscle glucose concentration increased from 1'79 mmol kg-' dry muscle at rest to 5-08 mmol kg-1 dry muscle at 80 s and remained elevated throughout the stimulation period (Table 1).

5 MUSCLE METABOLISM DURING PROLONGED STIMULATION 497 DISCUSSION Previous studies from this laboratory have examined human skeletal muscle metabolism and contraction force during short-term electrical stimulation where ATP production is predominantly anaerobic (Hultman & Sj6holm, 1983, 1986). Intermittent stimulation for 80 s at 20 Hz with an open or occluded circulation resulted in similar reductions in muscle force production and PCr and ATP levels and large increases in lactate concentration (Hultman & Sjdholm, 1986). The ATP turnover rate with an occluded circulation averaged 5-35 mmol kg-' dry muscle s-1 of stimulation as calculated from the muscle phosphagen utilization and accumulation of metabolites. TABLE 3. Calculated ATP turnover rates in the quadriceps femoris muscle during electrical stimulation at 20 Hz ATP turnover ratet (mmol kg-' dry muscle s-') Average force (% of peak) 0-80 s Glycolysis* 3-41 PCr, ATP utilization 1-58 Total min Glucose oxidation from glycogent * Calculated from the measured glycogen breakdown minus the hexose monophosphate and Glyc-1-P accumulation (1 mol glycogen produces 3 mol ATP). t Assumes that all glucose derived from glycogen was metabolized aerobically (1 mol glycogen produces 37 mol ATP; McGilvery, 1975). 1 ATP turnover rates are expressed per second of actual contraction time and calculations are based on mean values in Table 1. In the present study we were able to estimate the ATP turnover rate during 80 s of stimulation with an open circulation by measuring muscle glycogen utilization in addition to phosphagen and metabolite changes. The calculated value of 4-99 mmol kg-1 dry muscle s-1 of stimulation (Table 3) was similar to the rate with an occluded circulation and accounted for the comparable force production in the two conditions. With an open circulation anaerobic glycolysis contributed 680% and phosphagen utilization 32 % of the produced ATP. Of the 54-5 mmol kg-' dry muscle of glycogen utilized in the first 80 s (Table 2), 42-3 were accounted for by the measured increases in muscle lactate, Glyc-1-P and hexose monophosphates (Table 1). Assuming the majority of the remaining glycogen (12-2 mmol kg-' dry muscle) was metabolized anaerobically, approximately 24-4 mmol lactate kg-1 dry muscle escaped the muscle during 80 s of intermittent stimulation. Since muscle lactate increased by 66-5 mmol kg-' dry muscle during this period, lactate efflux represented approximately 27 % of the total lactate produced. Aerobic metabolism of muscle glycogen would be minimal during this period due to the lack of blood flow during contractions and the delay in the increased delivery of oxygen at the onset of stimulation. However, even assuming a maximal rate of aerobic glycogen utilization (McGilvery, 1975), less than 2 mmol of glycogen kg-' dry muscle could be utilized in only 80 s.

6 498 E. HULTMAN AND L. L. SPRIET Anaerobic pathways of ATP production are activated within the first few seconds of tetanic electrical stimulation (Hultman & Sj6holm, 1983). However, in the present study muscle force production decreased to 72 % of peak in only 80 s of intermittent stimulation (Fig. 1) when ATP production from glycolysis is known to be constant (Hultman & Sjoholm, 1986). Consequently, the decline in force production appeared to be the result of muscle phosphocreatine depletion and the inability of this pathway to maintain its initial high ATP turnover rate. In this investigation electrical stimulation was continued beyond 80 s to 45 min to examine muscle metabolism, force production and glycogen utilization during a prolonged period where oxidative metabolism provides the majority of ATP. With this model, all muscle fibres continue to receive a constant stimulus for contraction at a regular frequency. Glycogen utilization averaged 5-4 mmol kg-' dry muscle min-' from 80 s to 15 min and mmol kg-' dry muscle min-' from 15 to 45 min (Table 2). Initially force declined rapidly to 43 % of peak at 5 min and then more slowly to 31 % at 15 min and 24 % at 45 min (Fig. 1). The ATP turnover rate during the final 30 min was 1-86 mmol kg-' dry muscle s-1 of stimulation, assuming that the glycogen used was metabolized aerobically. This value compares favourably with the maximal ATP production rate from aerobic glycogen metabolism during exercise reported by McGilvery (1975). This suggests that muscle glycogen may have provided all the required ATP during this period assuming a constant ratio between ATP turnover rate and force production. Although exogenous glucose and free fatty acids (FFA) may also provide substrate for aerobic metabolism, their contribution in this model would be reduced. With intermittent tetanic contractions above 30 % maximal voluntary contraction force, blood flow reaches the muscle only during the pauses between contractions (Richardson, 1981) or one-half the total stimulation time. Also, an increase in the plasma FFA concentration generally mediated through increased circulating catecholamines would not be expected from the local stimulation of approximately 1 kg of muscle. The lower glycogenolytic rates coupled with decreases in muscle lactate and glycolytic intermediates suggested that anaerobic glycolysis contributed progressively less ATP with continued stimulation beyond 80 s. Through some mechanism a transition from a predominance of ATP production from anaerobic glycolysis to aerobic ATP production occurred. At the same time muscle force generation was reduced in accordance with the lower rate of ATP production from aerobic pathways. In an attempt to explain the muscle force and metabolic findings we first consider the possibility that not all of the muscle fibres initially stimulated to contract were activated with continued stimulation. A rapid accumulation of H+ would accompany the large build-up of muscle lactate during tetanic stimulation (Fig. 1). Numerous investigations have shown that increased extra- and intracellular H+ concentrations adversely affect membrane action potential propagation (Campbell & Hille, 1976; Fink, Hase, Luttgau & Wettwer, 1982), the release, binding and uptake of Ca2+ by the sarcoplasmic reticulum (Fabiato & Fabiato, 1978) and the Ca2+ sensitivity of contractile elements (Donaldson, Hermansen & Bolles, 1978; Blanchard, Pan & Solaro, 1984). A large H+ build-up would be expected in the highly glycolytic, fast-twitch fibres early in stimulation. Progressive failure to activate the fast-twitch

7 MUSCLE METABOLISM DURING PROLONGED STIMULATION 499 fibres could account for the decreasing force during the initial 15 min and the lower force (25-30 % of peak) in the final 30 min of stimulation. This interpretation is consistent with the findings of Edwards, Hill, Jones & Merton (1977) who reported the existence of a long-lasting, low-frequency fatigue that appeared to affect only fast-twitch fibres. It was suggested that excitation-contraction coupling was impaired so less force was produced for each excitation of the muscle membrane (Edwards, 1981). Slow-twitch fibres constitute approximately 50 % of the total in untrained human quadriceps muscle (Sahlin & Henriksson, 1984) and could provide aerobic energy to maintain % ofpeak force during the final 30 min ofstimulation. The regeneration of phosphocreatine and the low lactate levels in muscle beyond 15 min establishes that anaerobic energy pathways were not reactivated. A persistent inability to stimulate fast-twitch fibres would explain these metabolic findings. An increased blood flow to these fibres as a result of the lack of force production would permit the aerobic regeneration of ATP and phosphocreatine found in this study. Future studies examining single muscle fibres will be required to determine if selective inactivation of fibre types exists during prolonged stimulation in this model. It is also possible that force generation was dependent on the rate of ATP production. High concentrations of H+ are also known to interfere with the activity of phosphofructokinase (PFK) and the transformation of phosphorylase b to a in skeletal muscle (Trivedi & Danforth, 1966; Chasiotis, Hultman & Sahlin, 1983). Control of the regulatory glycolytic enzymes in this manner would prevent a continued high rate of muscle glycogen utilization and accumulation of H+, leaving aerobic metabolism as the only major ATP producing pathway for muscular contraction. In addition, increased oxidative metabolism during the first few minutes of stimulation would produce a number of cellular changes which favour continued low glycolytic activity. At the onset of stimulation, decreased muscle ATP and phosphocreatine levels and increased free AMP, ADP and inorganic phosphate (Pi), F-6-P and NH4 + concentrations activate PFK. As aerobic metabolism was established ATP and phosphocreatine concentrations increased (Table 1) while free AMP, ADP and Pi concentrations must be decreased tending to reverse the initial activation of PFK. In addition, a build-up of the Kreb 's cycle intermediate citrate has been shown to inhibit PFK in heart muscle (Parmeggiani & Bowman, 1963) and to correlate with decreased glycolytic activity in skeletal muscle (Rennie & Holloszy, 1977). Finally, if H+ were involved in the selective inhibition of fibre activation early in the stimulation, an additional unknown mechanism was responsible for maintaining this inhibition when muscle lactate and H+ returned to near normal concentrations between 15 and 30 min of stimulation. This work was supported by grants from the Swedish Medical Research Council (02647) and the Swedish Work Environment Fund ( ). Dr Spriet is a recipient of a Post-doctoral Research Fellowship from the Medical Research Council of Canada. The excellent assistance of Dr Mats Bergstrom is gratefully acknowledged. The authors also wish to thank the entire staff at the Department of Clinical Chemistry II for excellent collaboration in this investigation.

8 500 E. HULTMAN AND L. L. SPRIET REFERENCES BERGMEYER, H. U. (1965). Methods of Enzymatic Analysis. New York: Academic Press. BERGSTR6M, J. (1962). Muscle electrolytes in man. Determined by neutron activation analysis on needle biopsy specimens. A study on normal subjects, kidney patients, and patients with chronic diarrhoea. Scandinavian Journal of Clinical and Laboratory Investigation 14, suppl. 68. BLANCHARD, E. M., PAN, B.-S. & SOLARO, R. J. (1984). The effect of acidic ph on the ATPase activity and troponin binding of rabbit skeletal myofilaments. Journal of Biological Chemistry 259, CAMPBELL, D. T. & HILLE, B. (1976). Kinetic and pharmacological properties of the sodium channel of frog skeletal muscle. Journal of General Physiology 67, CHASIOTIS, D., HULTMAN, E. & SAHLIN, K. (1983). Acidotic depression of cyclic AMP accumulation and phosphorylase b to a transformation in skeletal muscle of man. Journal of Physiology 335, DONALDSON, S. K. B., HERMANSEN, L. & BOLLES, L. (1978). Differential direct effects of H+ on Ca2+-activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits. Pfluigers Archiv 376, EDWARDS, R. H. T. (1981). Human muscle function and fatigue. In Human Muscle Fatigue: Physiological Mechanisms, Ciba Foundation Symposium No. 82, pp London: Pitman Medical. EDWARDS, R. H. T., HILL, D. K., JONES, D. A. & MERTON, P. A. (1977). Fatigue of long duration in human skeletal muscle after exercise. Journal of Physiology 272, EDWARDS, R. H. T., YOUNG, A., HOSKING, G. P. & JONES, D. A. (1977). Human skeletal muscle function: description of tests and normal values. Clinical Science and Molecular Medicine 52, FABIATO, A. & FABIATO, F. (1978). Effects of ph on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. Journal of Physiology 276, FINK, R., HASE, S., LUTTGAU, H. C. & WETTWER, E. (1982). The effect of cellular energy reserves and internal calcium ions on the potassium conductance in skeletal muscle of the frog. Journal of Physiology 336, HARRIS, R. C., HULTMAN, E. & NORDESJ6, L.-O. (1974). Glycogen, glycolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scandinavian Journal of Clinical and Laboratory Investigation 33, HULTMAN, E. & SJ6HOLM, H. (1983). Energy metabolism and contraction force of human skeletal muscle in situ during electrical stimulation. Journal of Physiology 345, HULTMAN, E. & SJ6HOLM, H. (1986). Biochemical causes of fatigue. In Muscle Power: Factors Underlying Maximal Performance, ed. JONES, N. L. & MCCARTNEY, N. Champaign, IL: Human Kinetic Publishers (in the Press). HULTMAN, E., SJ6HOLM, H., JADERHOLM-EK, I. & KRYNICKI, J. (1983). Evaluation of methods for electrical stimulation of human skeletal muscle in situ. Pfluigers Archiv 398, HULTMAN, E., SJ6HOLM, H., SAHLIN, K. & EDSTR6M, L. (1981). Glycolytic and oxidative energy metabolism and contraction characteristics of intact human muscle. In Human Muscle Fatigue: Physiological Mechanisms, Ciba Foundation Symposium No. 82, pp London: Pitman Medical. MCGILVERY, R. V. (1975). The use of fuels for muscular work. In Metabolic Adaptation to Prolonged Physical Exercise, ed. HOWALD, H. & POORTMANS, J. R., pp Basel: Birkhiiuser Verlag. PARMEGGIANI, A. & BOWMAN, R. H. (1983). Regulation ofphosphofructokinase activity by citrate in normal and diabetic muscle. Biochemical and Biophysical Research Communications 12, RENNIE, M. J. & HOLLOSZY, J. 0. (1977). Inhibition of glucose uptake and glycogenolysis by availability of oleate in well-oxygenated perfused skeletal muscle. Biochemical Journal 168, RICHARDSON, D. (1981). Blood flow response of human calf muscles to static contractions at various percentages of MVC. Journal of Applied Physiology 51, SAHLIN, K. & HENRIKSSON, J. (1984). Buffer capacity and lactate accumulation in skeletal muscle of trained and untrained men. Acta physiological scandinavica 122,

9 MUSCLE METABOLISM DURING PROLONGED STIMULATION 501 SJ6HOLM, H., SAHLIN, K., EDSTR8M, L. & HULTMAN, E. (1983). Quantitative estimation of anaerobic and oxidative energy metabolism and contraction characteristics in intact human skeletal muscle during and after electrical stimulation. Clinical Physiology 3, TRIVEDI, B. & DANFORTH, W. H. (1966). Effect of ph on the kinetics of frog muscle phosphofructokinase. Journal of Biological Chemistry 211,