303
J. Physiol. (1975), 251, pp. 303-315 With 5 text-ftgures Printed in Great Britain
HEAT PRODUCTION AND CHEMICAL CHANGES DURING ISOMETRIC CONTRACTIONS OF THE HUMAN QUADRICEPS MUSCLE
BY R. H. T. EDWARDS, D. K. HILL AND D. A. JONES From the Departments of Medicine and Medical Physics, Royal Postgraduate Medical School,
Hammersmith Hospital, London W12 OHS (Received 13 February 1975) SUMMARY
1. Development of a new thermal probe and use in conjunction with chemical analysis of needle biopsy samples, has made possible a thermodynamic study of the energetic of muscular contraction in the human quadriceps. 2. The observed rate of muscle temperature rise was proportional to the force of the contraction. During maximal contractions the rate of heat production was 54 + 8-5 W/kg wet muscle (mean + S.D.). 3. The observed rates of muscle temperature rise agreed well with the rates calculated from the measured metabolite changes when standard values for the enthalpy changes of the reactions involved were used. 4. During prolonged stimulation of the quadriceps at 15/sec via the femoral nerve, the rate of heat production per unit force fell to nearly half the initial value. It is estimated that this represented a two- to fourfold increase in economy of ATP turn-over required to maintain a given force. 5. Relaxation becomes progressively slower during prolonged contractions and it is suggested that the slowing of relaxation and the increased economy of force maintenance may both be due to an increased cross-bridge cycle time in the fatigued muscle. INTRODUCTION
The study of muscle energetics is a well established discipline which has provided much information concerning the mechanisms of contraction (Wilkie, 1960; Hill, 1965; Woledge, 1971). Most of this work has been with isolated amphibian muscles but recent developments now permit the extension of this type of investigation to human skeletal muscle in situ.
304 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES The temperature of human muscle was first measured by Becquerel & Breschet (1835) with a thermocouple and simple galvanometer. These workers did not determine metabolic heat production but correctly noted that muscle temperature is greatly dependent on the local circulation. Barcroft & Millen (1939) exploited this fact when they used a thermocouple to study changes in the local circulation during sustained isometric contractions of the human gastrocnemius/soleus muscle group. These authors showed that the circulation in this muscle group is arrested at forces greater than 20 % of the force of a maximum voluntary contraction (MVC). They also made estimates of the energy exchanges that had occurred by the end of a contraction held to fatigue (Millen, 1939; Barcroft & Millen, 1947) but were not in a position to confirm their estimates by chemical analysis of muscle samples. Changes in intramuscular pressure and temperature during isometric contractions of the quadriceps (Sylvest & Hvid, 1959; Edwards, Hill & McDonnell, 1972) indicate that the circulation in this muscle is arrested at about the same force (%MVC) as in the gastrocnemius/soleus group. There have been a few isolated measurements of metabolic heat production in human muscle during isometric contractions with arrested circulation (Buchthal, H0nke & Lindhard, 1944; Akre & Aukland, 1970). The quadriceps muscle has been chosen because it is large and free from important blood vessels and nerves, permitting the necessary invasive procedures to be safely performed. A further advantage is that the intramuscular pressure rises and arrests the circulation during contractions. During isometric contractions no external work is done and the muscle, deprived of its blood supply, therefore behaves as a 'closed system' for the study of temperature and chemical changes. In the present work we have made use of a muscle biopsy technique first used by Duchenne (1868) and re-introduced by Bergstr6m (1962) together with enzymic micro-analytical methods to measure a range of muscle metabolites. This approach had been used in the study of muscle metabolism during several forms of physical activity in man (Hultman, 1967; Bergstr6m, Harris, Hultman & Nordesj6, 1971; Edwards, Harris, Hultman, Kaijser, Koh & Nordesj6, 1972; Karlson & Ollander, 1972.) METHODS
Subject. Informed consent was obtained in writing from all subjects before any invasive procedure was undertaken. Ten were normal males (aged 25-36 years), the remainder (nine males, two females) were patients complaining of weakness and fatigue but with no abnormality shown by electromyography and routine chemical analysis of blood and muscle samples. Histology, histochemistry and electron microscopy of needle biopsies of the quadriceps were also normal in these subjects. The results from two patients with abnormal muscle (one hypothyroid,
MYOTHERMAL MEASUREMENTS IN MAN
305
one alcoholic) have been included in the comparison between chemical change and heat production. Measurement of muscle force. Subjects sat on a muscle testing chair (Tornvall, 1963) and the force of the isometrically contracting quadriceps was measured with a strain gauge attached to a strap placed around the ankle. The knee was flexed to a right angle. The MVC force was defined as the greatest force held for 2 see in the best of three 5 see maximal efforts. Submaximal forces were held constant by the subject with the aid of a target line on an oscilloscope screen placed in front of him. Electrical stimulation. The lateral part of the quadriceps was stimulated by surface electrodes (10 cm square) soaked in isotonic saline and placed about 10 and 30 cm from the patella on the lateral side of the leg. Stimulation was by square wave pulses of 50 ,sec duration, 40-70 V. Comparison with the force of the MVC showed that up to 50 0 of the whole muscle was activated; this involved the greater part, if not all, of the vastus lateralis. Maximum force was obtained by stimulating at about 50/sec. In one subject the entire quadriceps was activated by stimulation of the femoral nerve. The anode was a 10 cm square electrode, as described above, placed high on the thigh and the femoral nerve was located by moving a 1-5 cm diameter cathode over an area 3-5 cm lateral to the femoral artery, just below the superior iliac spine. Pulses were of 50 /tsec duration, 30-70 V. The maximum tetanic contraction obtained was equal to the MVC. The jerk caused by the sudden onset of electrical stimulation involves the risk of lateral displacement of the patella. This method of stimulation has therefore to be used cautiously, raising the voltage to a maximum during the initial second or two of contraction. Measurement of relaxation time. Submaximal voluntary contractions were briefly interrupted at intervals to measure the t1 of the approximately exponential late phase of relaxation, as previously described (Edwards, Hill & Jones, 1972). In stimulated contractions relaxation time was determined as the time from the last stimulus to 50 % loss of force (Edwards, Hill & McDonnell, 1973). Temperature measurements. In most of the experiments a thermistor probe was used as previously described (Edwards, Hill & McDonnell, 1974). In a few experiments a thermocouple probe was used. This consists of a stainless-steel needle (diameter 0 5 mm), into which a constantan-copper thermocouple (NK 19, supplied by Sierex Ltd, London) was placed. The tip of the probe was inserted to a depth of 3-5 cm into the muscle. Recording. The amplified currents from the force transducer and thermal probes were recorded with a U.V. oscillograph (SE Laboratories Ltd). Muscle biopsies and analyses of metabolites. Biopsy studies were carried out as described elsewhere (Edwards, 1971; Edwards et al. 1972) and analyses were performed only after dissection of the freeze-dried muscle samples to remove fat and connective tissue. The accuracy of the analyses (coefficient of variation) was about 5 %. Details of the biochemical methods are given by Harris, Hultman & Nordesj6 (1974) and Edwards, Jones, Maunder & Batra (1975). It has been mentioned that the quadriceps normally becomes a closed system for contractions stronger than 20 % MVC because the hydrostatic pressure arrests the blood supply. However, in weaker subjects and at lower forces the precaution was taken of inflating a pneumatic cuff around the upper thigh. This has the added advantage of avoiding the small errors in interpretation caused by the utilization of oxygen stored in solution or in combination with myoglobin. Three min of ischaemia at rest was taken as the time necessary to use the stored oxygen (McArdle & Verel, 1956; Hultman, 1973). Biopsies from a structure as large as
R. H. T. EDWARDS, D. K. HILL AND D. A. JONES the vastus lateralis would be expected to be subject to sampling errors. Care was 306
taken to minimize these by taking biopsies from about the same depth (3-5 cm below the skin) and in the same vicinity as the thermal probe.
RESULTS
Temperature records during sustained contractions showed an initial fall, due to a small movement of the probe towards the surface of the muscle at the start of the contraction (Edwards et at. 1974), followed by a steady rise in temperature (Fig. 1). The rate of change of temperature at rest, measured with the circulation arrested by a cuff inflated around the thigh, was of the order of 0.005° C/min. During contractions the overall rise in muscle temperature was no more than 1° C/min, which would have produced only a marginal increase in the temperature gradient between the interior of the muscle and the outside environment. Heat loss during contractions was therefore likely to be of the same magnitude as that observed at rest. 25 20
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In a series of fifty-eight voluntary contractions in eight subjects, the rate of temperature rise was found to be proportional to the force held when this was expressed as a percentage of the MVC (Fig. 2). The rate of temperature rise in forty-eight maximal voluntary and stimulated contractions of unfatigued muscle in twelve subjects was 0 91 + 0 14' Cl min (mean +S.D.). Using the value for the specific heat of muscle given
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308 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES below, this is equivalent to a heat production of 54 + 8-5 W/kg wet muscle (mean + S.D.). Changes in relaxation during prolonged contractions were measured in six subjects who held a force of 50 % MVC until the force could no longer be maintained. The contractions were briefly interrupted at intervals of approximately 10 sec and the relaxation recorded. As reported previously (Edwards, Hill & Jones, 1972) relaxation time (tj) increased progressively throughout the contractions, relaxation being nearly four times slower at the end of the longest contractions than in the fresh muscle (Fig. 3). In nine subjects muscle biopsy samples were taken before and at the end of contractions when myothermal measurements were also made. Metabolite contents for the individual subjects are given in Table 1, together with the duration of the contractions and the observed rates of temperature rise. With the exception of subjects M. N. (i) and J. B., all contractions were made with a cuff inflated around the thigh. Thermal measurements are reported as rates of temperature rise as the movement artifact on the thermal records makes it difficult to establish an initial value to measure the total rise in temperature. The expected temperature rise as the result of the metabolic changes was calculated making the following assumptions. (1) Enthalpy values (R. C. Woledge, personal communication):
ATP-+ADP+P,
PC+ADP-+ATP+C
Net reaction PC- C + Pi glycosyl unit-> lactate + 1 ATP
i
AH kJ/mol at 370 C -46 +13 -33 -26
(2) Specific heat of muscle: 3-75 kJ/kg wet wt. (Hill & Woledge, 1962). (3) Water content: 77 % wet weight of muscle (Karlsson, 1971). Changes due to any oxidative metabolism that may have occurred, in subjects where the circulation to the leg was not arrested before the contraction, were almost certainly completed during the first 5 sec of the contraction. Since temperature measurements were not made until after this period, in order to avoid the movement artifacts, the measured rate of temperature rise reflects only the anaerobic processes. This is also true of the temperature measurements made during voluntary contractions at different forces (Fig. 2). The effects of oxidative metabolism on the metabolic changes would be to spare the depletion of phosphagen and delay the onset of anaerobic glycolysis. It has been calculated that the maximum ATP yielded by the stored oxygen is about 15 ,umol/g dry wt. (Edwards, Nordesj6, Koh, Harris & Hultman, 1971), which might delay the onset of lactate formation by 5 sec. This is unlikely to lead to
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310 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES more than a 15 % error in the calculated rates of temperature rise in the two subjects, M.N. (i) and J.B. The rates of temperature rise calculated from the data in Table 1 using the net value of -33 kJ/mol for the enthalpy change associated with splitting of phosphorylcreatine (see above) agree well with the rates observed (Fig. 4). Also shown in Fig. 4 is the regression line for the same data when the expected temperature rise was calculated using the value of -46 kJ/mol for the enthalpy change associated with the 1.0
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break-down of phosphorylereatine. This is the value found using intact muscles by Wilkie (1968). This regression does not differ significantly from that obtained using the value of -33 kJ/mol. The proportion of the total heat production derived from glycolytic reactions is estimated as 66 + 11 % from the data in Table 1. In one subject temperature records were made during very prolonged stimulation of the muscle via the femoral nerve at different frequencies of stimulation. Force was well maintained at frequencies between 8 and
31 311 MYOTHERMAL MEASUREMENTS IN MAN 20/sec, however, stimulating at above this frequency resulted in a rapid Joss of force. Because of the uncertainties associated with results from muscle that is losing force, we report here oniy data concerning lowfrequency stimulation where there was no appreciable loss of force. At 15/sec force was maintained for about 60 sec (Fig. 5A) and the heat rate per unit force was calculated every 6 sec throughout the contraction. There was a progressive decline in these rates which reach nearly half 37.0
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the original value after 60 sec stimulation (Fig. 5B). Relaxation time was nearly three-times longer at the end of the long contraction than when fresh muscle was tested immediately preceding the contraction. DISCUSSION
The comparison between the chemical changes and the observed temperature rise (Fig. 4) indicates that values for the enthalpy changes that have been used are consistent with the observed rises in temperature.
312 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES There is no evidence of 'unknown reactions' such as may occur in frog muscle (Gilbert, Kretschmar, Wilkie & Woledge, 1971; Canfield, Lebacq & Mar6chal, 1973) although, because of the relatively slow time resolution of the temperature measurements and the prolonged contraction times involving glycolytic reactions, it is unlikely that any effects such as reported by the authors cited above would be detected. Using a variety of patterns of stimulation with poisoned muscles Wilkie (1968) obtained a value of -46 kJ/mol for the enthalpy change associated with phosphorylcreatine break-down. Woledge (1972), however, on the basis of in vitro calorimetric measurements, favours a value of -34 kJ/mol (at 0° C). Although, by eye, the value of -33 kJ/mol (at 370 C) appears to give the best fit to the line of equality (Fig. 4) there is no significant difference between the regression line for this set of points and the regression obtained from the same data using the value of -46 kJ/mol. Again the contractions in this work were long and the major source of heat production (66 %) was ATP splitting associated with glycolytic reactions. The present experimental model is not appropriate to differentiate between the two values for the enthalpy change. The comparison between observed and calculated rates of temperature rise indicates that temperature measurements may be reliably used as an indicator of metabolic processes occurring during contraction of human muscle. The economy of force maintenance. At low frequencies isometric force was well maintained in a prolonged contraction (40-60 sec) and it was found that the rate of temperature rise per unit force decreased during the contraction (Fig. 5A, B). Where there is a change in the rate of heat production per unit force it is of interest to consider what this means in terms of ATP turn-over. The key to this question lies in knowing, at any given time during the contraction, the proportions of ATP hydrolysed that are derived from phosphorylereatine and from glycolytic reactions. If the ratio of the rates of phosphorylcreatine splitting and of lactate production remained constant throughout the contraction a given decrease in heat production would indicate an equal reduction in ATP turn-over. But if, as is likely, the proportion of energy derived from glycolysis increased during the later phase of the contraction, the heat associated with the hydrolysis of ATP would have increased. Thus the progressive decrease in heat production per unit force probably indicates a relatively greater increase in economy of ATP turn-over. Taking the extreme case, if the early heat production were entirely due to energy derived from phosphorylereatine, and later the energy came entirely from glycolysis, the enthalpy values given above indicate that the twofold reduction in heat-rate (Fig. 5)
MYOTHERMAL MEASUREMENTS IN MAN 313 would represent a fourfold increase in economy of ATP turn-over required to maintain a given force. Further work is required to determine the proportions of energy derived from phosphorylcreatine and from glycolysis during different stages of the contraction. Whatever the exact proportions the present findings indicate that there is a substantial decrease in ATP turn-over required to maintain force in the later part of the contraction. Feng (1931) demonstrated a similar effect with prolonged stimulation of frog muscles; he reported a fourfold decrease in heat production per unit force. Recently, in collaboration with Dr K. M. Kretzschmar, we have recorded a twofold decrease in this ratio when stimulating a rat soleus at 201sec for 75 sec under anaerobic conditions at 250 C. Feng (1931) was in no doubt that the slowing of the muscle with the onset of fatigue was the cause of the economy he observed. Relaxation became very much slower during prolonged submaximal voluntary contractions (Fig. 3), and as the result of the prolonged femoral nerve stimulation relaxation became nearly three-times slower: an indication of this is seen in the reduction of the oscillation on the force record (Fig. 5A). Such slowing appears to be characteristic of muscle fatigue and is also seen with isolated mouse muscle (Edwards et al. 1975). Slow muscles are known to maintain isometric contractions more efficiently than fast muscles (Awan, Frearson, Goldspink & Waterson, 1972). It seems reasonable, therefore, to suppose that the slowing of relaxation is closely connected with the increased economy. We have suggested (Edwards, Hill & Jones, 1975) that the slowing of relaxation as the result of fatigue in isolated mouse muscles is a consequence of an increased cross-bridge cycle time, and that this also accounts for the observed decrease in ATP turn-over during prolonged contractions. The increased economy of force maintenance associated with a slowing of relaxation in human muscle described in this work may also be explained on this basis. The very size of the apparent economy of ATP turn-over (between two- and fourfold) strongly suggests that the processes that are changing in function are associated with the major ATP utilizing processes, namely the force-generating mechanism. We would like to thank the subjects for volunteering to take part in these studies. Support from the Wellcome Trust and Muscular Dystrophy Group of Great Britain is gratefully acknowledged.
314
R. H. T. EDWARDS, D. K. HILL AND D. A. JONES
REFERENCES AKUE, S. & AuKAND, K. (1970). Energy turnover in contracting muscle. Acta physiol. 8cand. 79, 20-21A. AwAN, M. Z., FREARSON, N., GOLDSPINK, K. & WATERSON, S. E. (1972). Biochemical efficiencies of smooth muscle and different types of striated muscle. J. Mechanochem. Cell Motility 1, 225-232. BARCROFT, H. & MuTTTaN, J. L. E. (1939). The blood flow through muscle during sustained contraction. J. Physiol. 97, 17-31. BARcROFT, H. & MILL N, J. L. E. (1947). On the heat production in human muscle during voluntary contraction. J. Physiol. 106, 13-14P. BERGSTROM, 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. Scand. J. clin. Lab. Invest. suppl. 68. BERGSTROM, J., HARRIS, R. C., HULTMAN, E. & NORDESJO, L.-O. (1971). Energy rich phosphagens in dynamic and static work. In Muscle Metabolism during Exercise, ed. PERNOW, B. & SALTIN, B., pp. 341-355. New York: Plenum Press. BECQUEREL & BRESCHET (1835). An apparatus for indicating the internal temperature of animals and vegetables and of the mode of using it. English translation in The Magazine of Popular Science and Journal of the Useful Arts, vol. 1, pp. 257-264. London: John W. Parker. BUCHTIEALT, F., HoNcKE, P. & LINDHARD, J. (1944). Temperature measurements in human muscles in situ at rest and during muscular work. Acta physiol. scand. 8, 230-258. CANFIELD, P., LEBACQ, J. & MARtCHAL, G. (1973). Energy balance in frog sartorius muscle during an isometric tetanus at 20° C. J. Physiol. 232, 467-483. DuciHNNE, G. B. (1968). Recherches sur la paralysis musculaire pseudo-hypertrophique ou paralysis myosclerosique. Archs gen. Med. 11, 203-207. EDWARDS, R. H. T. (1971). Percutaneous needle biopsy of skeletal muscle in diagnosis and research. Lancet ii, 593-596. EDWARDS, R. H. T., HARRIS, R. C., HULTMAN, E., KAIJSER, L., KOH, D. & NORDESJO, L.-O. (1972). Effect of temperature on muscle energy metabolism and endurance during successive isometric contractions, sustained to fatigue of the quadriceps muscle in man. J. Physiol. 220, 335-352. EDWARDS, R. H. T., HILL, D. K. & JONES, D. A. (1972). Effect of fatigue on the time course of relaxation from isometric contractions of skeletal muscle in man. J. Physiol. 227, 26-27P. EDWARDS, R. H. T., HmLL, D. K. & JONES, D. A. (1975). Metabolic changes associated with the slowing of relaxation in fatigued mouse muscle. J. Physiol. 251, 287-301. EDWARDS, R. H. T., HILL, D. K. & McDONNELL, M. (1972). Myothermal and intramuscular pressure measurements during isometric contractions of the human quadriceps muscle. J. Physiol. 224, 58-59P. EDWARDS, R. H. T., HILL, D. K. & McDONNELL, M. (1973). Metabolic heat production and relaxation rate of electrically stimulated contractions of the quadriceps muscle in man. J. Physiol. 231, 81-83P. EDWARDS, R. H. T., HnaL, D. K. & McDONNELL, M. (1974). A thermistor probe for myothermal measurements in man. J. apple. Physiol. 36, 511-513. EDWARDS, R. H. T., JONES, D. A., MAUNDER, C. A. & BATRA, G. (1975). Muscle biopsy studies in patients and normal subjects. Lancet i, 736-740. EDWARDS, R. H. T., NORDESJ6, L.-O., KOH, D., HARRIS, R. C. & HULTMAN, E. (1971). Isometric exercise - factors influencing endurance and fatigue. In Muscle Metabolism during Exercise, ed. PERNOW, B. & SALTIN, B., pp. 357-360. New York: Plenum Press.
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FENG, T. P. (1931). The heat tension ratio in prolonged tetanic contractions. Proc. R. Soc. B. 108, 522-537. GILBERT, C., KRETZSCHMAR, K. M., WILKIE, D. R. & WOLEDGE, R. C. (1971). Chemical change and energy output during muscular contraction. J. Physiol. 218, 163-193. 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. Scand. J. clin. Lab. Invest. 33, 109-120. HILL, A. V. (1965). Trails and Trials in Physiology, pp. 38-82. London: Edward Arnold. HILT, A. V. & WOLEDGE, R. C. (1962). An examination of absolute values in myothermic measurements. J. Physiol. 162, 311-333. HULTMAN, E. (1967). Studies on muscle metabolism of glycogen and active phosphate in man with special reference to exercise and diet. Scand. J. clin. Lab. Invest. 19, suppl. 94. HULTMAN, E. (1973). Energy metabolism in human muscle. J. Physiol. 231, 56P. KARLSSON, J. (1971). Lactate and phosphagen concentrations in working muscle of man. Acta physiol. scand. suppl. 358. KARLSSON, J. & OLLANDER, B. (1972). Muscle metabolites with exhaustive static exercise of different duration. Acta physiol. scand. 86, 309-314. McARDLE, B. & VEREL, D. (1956). Responses to ischaemic work in the human forearm. Clin. Sci. 15, 305-318. MILLEN, J. L. E. (1939). Intramuscular temperature and bloodflow through muscle. M.D. Thesis, Queens University, Belfast. SYLVEST, 0. & HVID, N. (1959). Pressure measurements in human striated muscles during contraction. Acta rheum. sand. 5, 216-222. TORNYALL, G. (1963). Assessment of physical capabilities with special reference to the evaluation of maximal voluntary isometric muscle strength and maximal working capacity. Acta physiol. scand. suppl. 201. WILKIE, D. R. (1960). Thermodynamics and the interpretation of biological heat measurements. Prog. Biophys. biophys. Chem. 10, 259-298. WXILKIE, D. R. (1968). Heat, work and phosphorylcreatine break-down in muscle. J. Physiol. 195, 157-183. WOLEDGE, R. C. (1971). Heat production and chemical change in muscle. Prog. Biophys. molec. Biol. 22, 39-74. WOLEDGE, R. C. (1972). In vitro calorimetric studies relating to the interpretation of muscle heat experiments. Cold Spring Harbor Symp. quant. Biol. 37, 629634.
J. Physiol. (1975), 251, pp. 303-315 With 5 text-ftgures Printed in Great Britain
HEAT PRODUCTION AND CHEMICAL CHANGES DURING ISOMETRIC CONTRACTIONS OF THE HUMAN QUADRICEPS MUSCLE
BY R. H. T. EDWARDS, D. K. HILL AND D. A. JONES From the Departments of Medicine and Medical Physics, Royal Postgraduate Medical School,
Hammersmith Hospital, London W12 OHS (Received 13 February 1975) SUMMARY
1. Development of a new thermal probe and use in conjunction with chemical analysis of needle biopsy samples, has made possible a thermodynamic study of the energetic of muscular contraction in the human quadriceps. 2. The observed rate of muscle temperature rise was proportional to the force of the contraction. During maximal contractions the rate of heat production was 54 + 8-5 W/kg wet muscle (mean + S.D.). 3. The observed rates of muscle temperature rise agreed well with the rates calculated from the measured metabolite changes when standard values for the enthalpy changes of the reactions involved were used. 4. During prolonged stimulation of the quadriceps at 15/sec via the femoral nerve, the rate of heat production per unit force fell to nearly half the initial value. It is estimated that this represented a two- to fourfold increase in economy of ATP turn-over required to maintain a given force. 5. Relaxation becomes progressively slower during prolonged contractions and it is suggested that the slowing of relaxation and the increased economy of force maintenance may both be due to an increased cross-bridge cycle time in the fatigued muscle. INTRODUCTION
The study of muscle energetics is a well established discipline which has provided much information concerning the mechanisms of contraction (Wilkie, 1960; Hill, 1965; Woledge, 1971). Most of this work has been with isolated amphibian muscles but recent developments now permit the extension of this type of investigation to human skeletal muscle in situ.
304 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES The temperature of human muscle was first measured by Becquerel & Breschet (1835) with a thermocouple and simple galvanometer. These workers did not determine metabolic heat production but correctly noted that muscle temperature is greatly dependent on the local circulation. Barcroft & Millen (1939) exploited this fact when they used a thermocouple to study changes in the local circulation during sustained isometric contractions of the human gastrocnemius/soleus muscle group. These authors showed that the circulation in this muscle group is arrested at forces greater than 20 % of the force of a maximum voluntary contraction (MVC). They also made estimates of the energy exchanges that had occurred by the end of a contraction held to fatigue (Millen, 1939; Barcroft & Millen, 1947) but were not in a position to confirm their estimates by chemical analysis of muscle samples. Changes in intramuscular pressure and temperature during isometric contractions of the quadriceps (Sylvest & Hvid, 1959; Edwards, Hill & McDonnell, 1972) indicate that the circulation in this muscle is arrested at about the same force (%MVC) as in the gastrocnemius/soleus group. There have been a few isolated measurements of metabolic heat production in human muscle during isometric contractions with arrested circulation (Buchthal, H0nke & Lindhard, 1944; Akre & Aukland, 1970). The quadriceps muscle has been chosen because it is large and free from important blood vessels and nerves, permitting the necessary invasive procedures to be safely performed. A further advantage is that the intramuscular pressure rises and arrests the circulation during contractions. During isometric contractions no external work is done and the muscle, deprived of its blood supply, therefore behaves as a 'closed system' for the study of temperature and chemical changes. In the present work we have made use of a muscle biopsy technique first used by Duchenne (1868) and re-introduced by Bergstr6m (1962) together with enzymic micro-analytical methods to measure a range of muscle metabolites. This approach had been used in the study of muscle metabolism during several forms of physical activity in man (Hultman, 1967; Bergstr6m, Harris, Hultman & Nordesj6, 1971; Edwards, Harris, Hultman, Kaijser, Koh & Nordesj6, 1972; Karlson & Ollander, 1972.) METHODS
Subject. Informed consent was obtained in writing from all subjects before any invasive procedure was undertaken. Ten were normal males (aged 25-36 years), the remainder (nine males, two females) were patients complaining of weakness and fatigue but with no abnormality shown by electromyography and routine chemical analysis of blood and muscle samples. Histology, histochemistry and electron microscopy of needle biopsies of the quadriceps were also normal in these subjects. The results from two patients with abnormal muscle (one hypothyroid,
MYOTHERMAL MEASUREMENTS IN MAN
305
one alcoholic) have been included in the comparison between chemical change and heat production. Measurement of muscle force. Subjects sat on a muscle testing chair (Tornvall, 1963) and the force of the isometrically contracting quadriceps was measured with a strain gauge attached to a strap placed around the ankle. The knee was flexed to a right angle. The MVC force was defined as the greatest force held for 2 see in the best of three 5 see maximal efforts. Submaximal forces were held constant by the subject with the aid of a target line on an oscilloscope screen placed in front of him. Electrical stimulation. The lateral part of the quadriceps was stimulated by surface electrodes (10 cm square) soaked in isotonic saline and placed about 10 and 30 cm from the patella on the lateral side of the leg. Stimulation was by square wave pulses of 50 ,sec duration, 40-70 V. Comparison with the force of the MVC showed that up to 50 0 of the whole muscle was activated; this involved the greater part, if not all, of the vastus lateralis. Maximum force was obtained by stimulating at about 50/sec. In one subject the entire quadriceps was activated by stimulation of the femoral nerve. The anode was a 10 cm square electrode, as described above, placed high on the thigh and the femoral nerve was located by moving a 1-5 cm diameter cathode over an area 3-5 cm lateral to the femoral artery, just below the superior iliac spine. Pulses were of 50 /tsec duration, 30-70 V. The maximum tetanic contraction obtained was equal to the MVC. The jerk caused by the sudden onset of electrical stimulation involves the risk of lateral displacement of the patella. This method of stimulation has therefore to be used cautiously, raising the voltage to a maximum during the initial second or two of contraction. Measurement of relaxation time. Submaximal voluntary contractions were briefly interrupted at intervals to measure the t1 of the approximately exponential late phase of relaxation, as previously described (Edwards, Hill & Jones, 1972). In stimulated contractions relaxation time was determined as the time from the last stimulus to 50 % loss of force (Edwards, Hill & McDonnell, 1973). Temperature measurements. In most of the experiments a thermistor probe was used as previously described (Edwards, Hill & McDonnell, 1974). In a few experiments a thermocouple probe was used. This consists of a stainless-steel needle (diameter 0 5 mm), into which a constantan-copper thermocouple (NK 19, supplied by Sierex Ltd, London) was placed. The tip of the probe was inserted to a depth of 3-5 cm into the muscle. Recording. The amplified currents from the force transducer and thermal probes were recorded with a U.V. oscillograph (SE Laboratories Ltd). Muscle biopsies and analyses of metabolites. Biopsy studies were carried out as described elsewhere (Edwards, 1971; Edwards et al. 1972) and analyses were performed only after dissection of the freeze-dried muscle samples to remove fat and connective tissue. The accuracy of the analyses (coefficient of variation) was about 5 %. Details of the biochemical methods are given by Harris, Hultman & Nordesj6 (1974) and Edwards, Jones, Maunder & Batra (1975). It has been mentioned that the quadriceps normally becomes a closed system for contractions stronger than 20 % MVC because the hydrostatic pressure arrests the blood supply. However, in weaker subjects and at lower forces the precaution was taken of inflating a pneumatic cuff around the upper thigh. This has the added advantage of avoiding the small errors in interpretation caused by the utilization of oxygen stored in solution or in combination with myoglobin. Three min of ischaemia at rest was taken as the time necessary to use the stored oxygen (McArdle & Verel, 1956; Hultman, 1973). Biopsies from a structure as large as
R. H. T. EDWARDS, D. K. HILL AND D. A. JONES the vastus lateralis would be expected to be subject to sampling errors. Care was 306
taken to minimize these by taking biopsies from about the same depth (3-5 cm below the skin) and in the same vicinity as the thermal probe.
RESULTS
Temperature records during sustained contractions showed an initial fall, due to a small movement of the probe towards the surface of the muscle at the start of the contraction (Edwards et at. 1974), followed by a steady rise in temperature (Fig. 1). The rate of change of temperature at rest, measured with the circulation arrested by a cuff inflated around the thigh, was of the order of 0.005° C/min. During contractions the overall rise in muscle temperature was no more than 1° C/min, which would have produced only a marginal increase in the temperature gradient between the interior of the muscle and the outside environment. Heat loss during contractions was therefore likely to be of the same magnitude as that observed at rest. 25 20
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In a series of fifty-eight voluntary contractions in eight subjects, the rate of temperature rise was found to be proportional to the force held when this was expressed as a percentage of the MVC (Fig. 2). The rate of temperature rise in forty-eight maximal voluntary and stimulated contractions of unfatigued muscle in twelve subjects was 0 91 + 0 14' Cl min (mean +S.D.). Using the value for the specific heat of muscle given
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308 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES below, this is equivalent to a heat production of 54 + 8-5 W/kg wet muscle (mean + S.D.). Changes in relaxation during prolonged contractions were measured in six subjects who held a force of 50 % MVC until the force could no longer be maintained. The contractions were briefly interrupted at intervals of approximately 10 sec and the relaxation recorded. As reported previously (Edwards, Hill & Jones, 1972) relaxation time (tj) increased progressively throughout the contractions, relaxation being nearly four times slower at the end of the longest contractions than in the fresh muscle (Fig. 3). In nine subjects muscle biopsy samples were taken before and at the end of contractions when myothermal measurements were also made. Metabolite contents for the individual subjects are given in Table 1, together with the duration of the contractions and the observed rates of temperature rise. With the exception of subjects M. N. (i) and J. B., all contractions were made with a cuff inflated around the thigh. Thermal measurements are reported as rates of temperature rise as the movement artifact on the thermal records makes it difficult to establish an initial value to measure the total rise in temperature. The expected temperature rise as the result of the metabolic changes was calculated making the following assumptions. (1) Enthalpy values (R. C. Woledge, personal communication):
ATP-+ADP+P,
PC+ADP-+ATP+C
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(2) Specific heat of muscle: 3-75 kJ/kg wet wt. (Hill & Woledge, 1962). (3) Water content: 77 % wet weight of muscle (Karlsson, 1971). Changes due to any oxidative metabolism that may have occurred, in subjects where the circulation to the leg was not arrested before the contraction, were almost certainly completed during the first 5 sec of the contraction. Since temperature measurements were not made until after this period, in order to avoid the movement artifacts, the measured rate of temperature rise reflects only the anaerobic processes. This is also true of the temperature measurements made during voluntary contractions at different forces (Fig. 2). The effects of oxidative metabolism on the metabolic changes would be to spare the depletion of phosphagen and delay the onset of anaerobic glycolysis. It has been calculated that the maximum ATP yielded by the stored oxygen is about 15 ,umol/g dry wt. (Edwards, Nordesj6, Koh, Harris & Hultman, 1971), which might delay the onset of lactate formation by 5 sec. This is unlikely to lead to
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310 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES more than a 15 % error in the calculated rates of temperature rise in the two subjects, M.N. (i) and J.B. The rates of temperature rise calculated from the data in Table 1 using the net value of -33 kJ/mol for the enthalpy change associated with splitting of phosphorylcreatine (see above) agree well with the rates observed (Fig. 4). Also shown in Fig. 4 is the regression line for the same data when the expected temperature rise was calculated using the value of -46 kJ/mol for the enthalpy change associated with the 1.0
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break-down of phosphorylereatine. This is the value found using intact muscles by Wilkie (1968). This regression does not differ significantly from that obtained using the value of -33 kJ/mol. The proportion of the total heat production derived from glycolytic reactions is estimated as 66 + 11 % from the data in Table 1. In one subject temperature records were made during very prolonged stimulation of the muscle via the femoral nerve at different frequencies of stimulation. Force was well maintained at frequencies between 8 and
31 311 MYOTHERMAL MEASUREMENTS IN MAN 20/sec, however, stimulating at above this frequency resulted in a rapid Joss of force. Because of the uncertainties associated with results from muscle that is losing force, we report here oniy data concerning lowfrequency stimulation where there was no appreciable loss of force. At 15/sec force was maintained for about 60 sec (Fig. 5A) and the heat rate per unit force was calculated every 6 sec throughout the contraction. There was a progressive decline in these rates which reach nearly half 37.0
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the original value after 60 sec stimulation (Fig. 5B). Relaxation time was nearly three-times longer at the end of the long contraction than when fresh muscle was tested immediately preceding the contraction. DISCUSSION
The comparison between the chemical changes and the observed temperature rise (Fig. 4) indicates that values for the enthalpy changes that have been used are consistent with the observed rises in temperature.
312 R. H. T. EDWARDS, D. K. HILL AND D. A. JONES There is no evidence of 'unknown reactions' such as may occur in frog muscle (Gilbert, Kretschmar, Wilkie & Woledge, 1971; Canfield, Lebacq & Mar6chal, 1973) although, because of the relatively slow time resolution of the temperature measurements and the prolonged contraction times involving glycolytic reactions, it is unlikely that any effects such as reported by the authors cited above would be detected. Using a variety of patterns of stimulation with poisoned muscles Wilkie (1968) obtained a value of -46 kJ/mol for the enthalpy change associated with phosphorylcreatine break-down. Woledge (1972), however, on the basis of in vitro calorimetric measurements, favours a value of -34 kJ/mol (at 0° C). Although, by eye, the value of -33 kJ/mol (at 370 C) appears to give the best fit to the line of equality (Fig. 4) there is no significant difference between the regression line for this set of points and the regression obtained from the same data using the value of -46 kJ/mol. Again the contractions in this work were long and the major source of heat production (66 %) was ATP splitting associated with glycolytic reactions. The present experimental model is not appropriate to differentiate between the two values for the enthalpy change. The comparison between observed and calculated rates of temperature rise indicates that temperature measurements may be reliably used as an indicator of metabolic processes occurring during contraction of human muscle. The economy of force maintenance. At low frequencies isometric force was well maintained in a prolonged contraction (40-60 sec) and it was found that the rate of temperature rise per unit force decreased during the contraction (Fig. 5A, B). Where there is a change in the rate of heat production per unit force it is of interest to consider what this means in terms of ATP turn-over. The key to this question lies in knowing, at any given time during the contraction, the proportions of ATP hydrolysed that are derived from phosphorylereatine and from glycolytic reactions. If the ratio of the rates of phosphorylcreatine splitting and of lactate production remained constant throughout the contraction a given decrease in heat production would indicate an equal reduction in ATP turn-over. But if, as is likely, the proportion of energy derived from glycolysis increased during the later phase of the contraction, the heat associated with the hydrolysis of ATP would have increased. Thus the progressive decrease in heat production per unit force probably indicates a relatively greater increase in economy of ATP turn-over. Taking the extreme case, if the early heat production were entirely due to energy derived from phosphorylereatine, and later the energy came entirely from glycolysis, the enthalpy values given above indicate that the twofold reduction in heat-rate (Fig. 5)
MYOTHERMAL MEASUREMENTS IN MAN 313 would represent a fourfold increase in economy of ATP turn-over required to maintain a given force. Further work is required to determine the proportions of energy derived from phosphorylcreatine and from glycolysis during different stages of the contraction. Whatever the exact proportions the present findings indicate that there is a substantial decrease in ATP turn-over required to maintain force in the later part of the contraction. Feng (1931) demonstrated a similar effect with prolonged stimulation of frog muscles; he reported a fourfold decrease in heat production per unit force. Recently, in collaboration with Dr K. M. Kretzschmar, we have recorded a twofold decrease in this ratio when stimulating a rat soleus at 201sec for 75 sec under anaerobic conditions at 250 C. Feng (1931) was in no doubt that the slowing of the muscle with the onset of fatigue was the cause of the economy he observed. Relaxation became very much slower during prolonged submaximal voluntary contractions (Fig. 3), and as the result of the prolonged femoral nerve stimulation relaxation became nearly three-times slower: an indication of this is seen in the reduction of the oscillation on the force record (Fig. 5A). Such slowing appears to be characteristic of muscle fatigue and is also seen with isolated mouse muscle (Edwards et al. 1975). Slow muscles are known to maintain isometric contractions more efficiently than fast muscles (Awan, Frearson, Goldspink & Waterson, 1972). It seems reasonable, therefore, to suppose that the slowing of relaxation is closely connected with the increased economy. We have suggested (Edwards, Hill & Jones, 1975) that the slowing of relaxation as the result of fatigue in isolated mouse muscles is a consequence of an increased cross-bridge cycle time, and that this also accounts for the observed decrease in ATP turn-over during prolonged contractions. The increased economy of force maintenance associated with a slowing of relaxation in human muscle described in this work may also be explained on this basis. The very size of the apparent economy of ATP turn-over (between two- and fourfold) strongly suggests that the processes that are changing in function are associated with the major ATP utilizing processes, namely the force-generating mechanism. We would like to thank the subjects for volunteering to take part in these studies. Support from the Wellcome Trust and Muscular Dystrophy Group of Great Britain is gratefully acknowledged.
314
R. H. T. EDWARDS, D. K. HILL AND D. A. JONES
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FENG, T. P. (1931). The heat tension ratio in prolonged tetanic contractions. Proc. R. Soc. B. 108, 522-537. GILBERT, C., KRETZSCHMAR, K. M., WILKIE, D. R. & WOLEDGE, R. C. (1971). Chemical change and energy output during muscular contraction. J. Physiol. 218, 163-193. 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. Scand. J. clin. Lab. Invest. 33, 109-120. HILL, A. V. (1965). Trails and Trials in Physiology, pp. 38-82. London: Edward Arnold. HILT, A. V. & WOLEDGE, R. C. (1962). An examination of absolute values in myothermic measurements. J. Physiol. 162, 311-333. HULTMAN, E. (1967). Studies on muscle metabolism of glycogen and active phosphate in man with special reference to exercise and diet. Scand. J. clin. Lab. Invest. 19, suppl. 94. HULTMAN, E. (1973). Energy metabolism in human muscle. J. Physiol. 231, 56P. KARLSSON, J. (1971). Lactate and phosphagen concentrations in working muscle of man. Acta physiol. scand. suppl. 358. KARLSSON, J. & OLLANDER, B. (1972). Muscle metabolites with exhaustive static exercise of different duration. Acta physiol. scand. 86, 309-314. McARDLE, B. & VEREL, D. (1956). Responses to ischaemic work in the human forearm. Clin. Sci. 15, 305-318. MILLEN, J. L. E. (1939). Intramuscular temperature and bloodflow through muscle. M.D. Thesis, Queens University, Belfast. SYLVEST, 0. & HVID, N. (1959). Pressure measurements in human striated muscles during contraction. Acta rheum. sand. 5, 216-222. TORNYALL, G. (1963). Assessment of physical capabilities with special reference to the evaluation of maximal voluntary isometric muscle strength and maximal working capacity. Acta physiol. scand. suppl. 201. WILKIE, D. R. (1960). Thermodynamics and the interpretation of biological heat measurements. Prog. Biophys. biophys. Chem. 10, 259-298. WXILKIE, D. R. (1968). Heat, work and phosphorylcreatine break-down in muscle. J. Physiol. 195, 157-183. WOLEDGE, R. C. (1971). Heat production and chemical change in muscle. Prog. Biophys. molec. Biol. 22, 39-74. WOLEDGE, R. C. (1972). In vitro calorimetric studies relating to the interpretation of muscle heat experiments. Cold Spring Harbor Symp. quant. Biol. 37, 629634.
