Acta Physiol Scand 1990, 139, 459-466
ATP content in single fibres from human skeletal muscle after electrical stimulation and during recovery K. S O D E R L U N D and E. H U L T M A N Department of Clinical Chemistry 11, Huddinge University Hospital, Karolinska Institutet, Huddinge, Sweden SODERLUND, K. & HULTMAN, E. 1990.ATP content in single fibres from human skeletal muscle after electrical stimulation and during recovery. Acta Physiol Scand 139, 459-466. Received 26 January 1990,accepted 23 February 1990.ISSN 0001-6772. Department of Clinical Chemistry 11, Huddinge University Hospital, Karolinska Institutet, Huddinge, Sweden. The ATP content was measured in type I and type I1 fibres from human vastus lateralis muscle at rest, after electrical stimulation and during recovery. At rest the mean values were 25.2 f 4.02 and 25.92 3.62 mmol kg-l dry muscle (mean 2 SD) for type I and type I1 fibres respectively. Normal distribution curves were found for both types I and I1 fibres. After intermittent electrical stimulation for 83 s (1.6s stimulation, 1.6s pause) with occluded blood flow, the force generation decreased to 22% of the initial value and the muscle tissue showed a mean decrease in ATP to 14.8and in phosphocreatine to 5.44mmol kg-' dry muscle; lactate increased to 128.9mmol kg-' dry muscle. The ATP content in isolated fibres was equally decreased in both fibre types to 16mmol kg-' dry muscle. In I I yoof the fibres the ATP content was lower than 10mmol kg-' dry muscle. After 15 min rest with intact blood circulation ATP was completely resynthesized in type I fibres and to 91 yo in type I1 fibres. Key words :ATP concentration, ATP resynthesis, electrical stimulation, human muscle, single fibres.
I t is known that human skeletal muscle consists of a mixture of fibres with different contractile and metabolic properties (Eberstein & Goodgold 1968). T y p e I fibres are slow-contracting and have a high oxidative capacity, whereas type I1 fibres are fast-contracting and have a high glucolytic capacity. T h e A T P concentration in the two fibre types was reported to be equal both at rest and after continuous excercise to exhaustion (Essen 1978). Similar results were obtained by Jansson et al. (1987)but with tendency for a higher A T P concentration in type I1 than in type I fibres at rest and a greater decrease in type I1 fibres after maximal voluntary isometric exercise with the knee extensors. Previous studies in man have shown a 50% Correspondence : Karin Soderlund, Department of Clinical Chemistry 11, Huddinge University Hospital, S-14186 Huddinge, Sweden.
decrease in the A T P in whole-muscle samples from the quadriceps femoris during intermittent electrical stimulation (Harris & Hultman 1985, Hultman & Sjoholm 1986).I n these studies the blood flow to the muscles was occluded during the stimulation. T h e decrease in ATP was matched by a corresponding increase in inosine monophosphate (IMP), and by the end of the contraction the force generation had declined to about 20% of the initial value. T h e present study was undertaken to extend these earlier observations by examining ATP depletion and resynthesis in isolated, characterized fibres.
M A T E R I A L S A N D METHODS Seven subjects, four women and three men, volunteered to participate in the study. The character-
459
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K. Soderlund and E . Hultman
istics of the subjects were as follows : age 30 7 years, height 173+9 cm and weight 68+ 1 2 kg. The subjects were informed about the nature and purpose of the experiment before they gave their voluntary consent to participate. This experiment was part of a larger project approved by the Ethical Committee of the Karolinska Institute. T h e subjects reclined in a semisupine position on a bed. T h e lower legs were flexed over the end of the bed to 90' and one leg, chosen at random, was attached to a strain gauge on the frame of the bed via an ankle strap. The subject then performed three maximal voluntary contractions to determine the maximal voluntary isometric force (MVC) of the knee extensor muscles. The leg was then prepared for electrical stimulation of the quadriceps femoris muscle as previously described by Hultman et a/. (1983). T h e vastus lateralis muscle of the thigh was stimulated to contract via surface electrodes at a frequency of 20 Hz with square-wave pulses of 0.5 ms duration. The stimulation was intermittent with trains lasting 1.6 s and separated by pauses of 1.6 s for a total of 52 contractions. The total time was 165 s and contraction time was 83 s. Approximately 30% of the musculature that extends the knee was activated to 70% of maximum (Hultman et al. 1983). In the experiment with occluded circulation a cuff around the proximal portion of the thigh was inflated (250 mmHg) to occlude the blood flow 30 s prior to the onset of stimulation and remained inflated during the stimulation and biopsy sampling. The force produced was recorded throughout the stimulation by a strain gauge attached to a strap around the ankle. Biopsies were taken from the vastus lateralis muscle (Bergstrom 1962) at rest, following -52 contractions (83 s contraction time) and after 1 5 min of recovery.
Analytical methods T h e biopsy samples were immediately frozen in liquid nitrogen (N,) and freeze-dried. One part was used for analysing muscle metabolites (Harris et al. 1974) in the whole piece, and one part was used for single-fibre dissection. Single fibres were separated and stained according to the method described by EssCn et a/. (1975). The fibres were weighed on a customized quartzfibre fishpole balance (Lowry & Passonneau 1972). Two pieces (0.1 pg) from each fibre were excised and stained for myofibrillar ATPase to classify type I and type I1 fibres. T h e fibres were extracted in zoo ,dof 2.5% trichloroacetic acid (TCA) for 2-3 min and stored at -70 "C until analysed. T h e ATP content in single fibres (weighing 0.5-4pg) was measured using the bioluminescence technique and an ATP-monitoring kit (1243-102, LKB-Wallace) (Lundin et al. 1976). The analyses were performed on a LKB-Wallace 1250 luminorneter (Turku, Finland).
Statistical treatment and precision of the method Statistics. Student's t-test was used to compare group means. T h e SDtOtwas calculated from all fibres at rest ( n = 137).The SDindfor all fibres from each individual was determined, and the mean of the SD from seven subjects gives the SD,,,,. The ATP concentration in individual fibre fragments did not show any systematic variation with sample weight. The precision of the ATP measurement was 4% (coefficient of variation, CV) determined from 52 duplicate analyses on TCA extracts. The CV in ATP between fragments of the same fibre was 3 %. An attempt was made to calculate the number of fibres needed to obtain a reliable mean. Means of the first five and 10fibres from 22 biopsy samples were compared with the mean of the total number of fibres analysed from the same biopsy sample of a specific fibre type. The number of fibres from each biopsy ranged from I I to 22. The mean of five fibres gave a difference of 504,and the mean of 10 fibres gave a difference of zyo, from the mean of the total number of fibres analysed of a specific fibre type of the biopsy. This finding suggests that 5-10 fibres will be enough to obtain a reliable mean value. Comparison between individual fibre analyses and analyses of whole-muscle samples was performed by comparing the mean of all individual contents with the content in the whole sample.
RESULTS T h e ATP content at rest
The distribution of ATP in the resting state in the two fibre types is shown in Fig. I . The distribution frequencies followed a normal distribution curve in both fibre types and there was no difference in ATP level in the two fibre types. This was also evident when the individual results were examined (Table I ; Fig. 2a and b). In six out of seven subjects there was close agreement between the average fibre ATP and the content determined in the whole-muscle sample (Table I). I n subject 5 , the whole-sample content was noticeably lower. This may have been due to the inclusion of blood in the biopsy sample. Muscle contractions with occluded circulation
After 5 2 contractions with occluded circulation the force had decreased to 22% of the initial value (Fig. 3). Simultaneously there was a decrease in ATP and phosphocreatine (PCr)
ATP content in single fibres n
25
20
15
10
5
0 15
20
25
30
35
ATP: mmol kg" d.m.
l r
Fig. I . The distribution of ATP in the resting state of all type I (0) and type I1 (0) fibres from seven subjects.
contents of 42 and 93% respectively, and an increase in lactate to 128.2 mmol kg-' dry muscle (d.m.) (Table 2). The ATP in single fibres showed an equal decrease in both fibre types from approximately 25 to 16 mmol kg-' d.m. (Table 3 ) . The lowest ATP value was 4.5 mmol kg-' d.m. (subject I , type I1 fibre). In this subject a further eight fibres out of a total of 33 analysed had an ATP content of less than 1 0 mmol kg-' d.m. In the remaining three subjects 0 / 2 3 , 2 / 1 8 and 1 / 2 4 fibres had an ATP concentration lower than 1 0 mmol kg-' d.m. In total, 1 1 % of the fibres had an ATP concentration lower than 1 0 mmol kg-l d.m.
3 c3
0
a c-" dF
Recowry after occhded circulation 3
After 15 min recovery the ATP in whole muscle was significantly decreased compared with the resting value (Table 2). PCr was back to normal but lactate concentration remained increased (Table 2). In single fibres the ATP was completely resynthesized in type I fibres, but only to 91 yo in type I1 (Table 4).
c)
.-DW
c 2 i
46 1
462
K. Soderlund and E . Hultman n 15-
a
10 -
5-
0-
, . ....' ... I
15
20
25
.
30
35
.
151 b
10-
5-
0-
2. Normal distribution curves of (a) type I (0) and (b) type I1 (0) fibres at rest. The continuous curve describes the distribution of all fibres measured at rest using SD,,, 3.81 mmol kg-' dry muscle (d.m.). The dashed line describes the expected distribution in a single individual using SD,,, 3.22 mmol kg-' d.m.
Fig.
Figure 4 presents a summary of single-fibre distribution at rest, following 5 2 contractions and after I j min recovery. I t is clear that type I1 fibres show a larger variation in A T P concentration after 5 2 contractions than type I fibres. After I j min recovery with intact blood flow the A T P level was completely normalized in type I fibres, but only to 91% in type 11.
DISCUSSION The ATP content at rest There was no significant difference in A T P level between type I and type I1 fibres in human mixed muscle, a result which bears out the studies by Essen (1978) and Rehunen & Harkonen (1980). Earlier estimates in various animal species have indicated a higher ATP
ATP content in single3bres
463
10080-
.cE
6040-
0
s a 2
20-
e
,
o!
l
12.8
I
20
40
64.0
128.0
0 4
1
52 no of contr.
164.8 time, s
Fig. 3. Force in per cent of initial value during 52 contractions with occluded circulation. Tabl e 2. Muscle metabolites in muscle samples obtained at rest, after 52 contractions and after 15 min recovery. The blood circulation to the leg during the contraction was occluded. Values are means k SD in mmol kg-' dry muscle. Force is given as per cent of the initial value Occluded circulation Rest (n = 4)
52 contractions (n = 4)
1 5 min recovery (n = 4)
25.6f 1.39 84.5 f6.36 47.7 f 7.49 132.0f 13.00 2.8 f0.64
14.8f 2.96 5.4+ 1.12 123.6f 18.00 128.7f 17.70 128.9& 24.50
20.9f 1.52" 90.0 I I .30 38.7 k 8.55 129.1 t 18.80 12.0f5.60
_ ~ _ ._ .
ATP PCr Cr TC r Lactate Force (yo)
22.5 f9.5
I00
Statistics: Student's t-test was used to compare the ATP content at rest and after 1 5 min recovery. *;
P < 0.05.
Tabl e 3. ATP content in single muscle fibres after 52 contractions with occluded circulation. Values are mean SD in mmol kg-' dry muscle. n = number of single fibres
+
I 2
3 4 Total
Type I + type I1
Type I1
Type 1 Subject no.
ATP
n
ATP
n
15.1f5.03 16.2f2.98 15.8f1.20 18.3f3.06 16.1f3.82
22
12.2f5.88 19.6f4.62 I2.4k3.17 20.0 k 5.43 15.9f6.19
11
16 10 12
60
concentration in predominantly fast-twitch or mixed muscle compared with slow-twitch muscles (Edstrom et al. 1982, Hintz et al. 1982). A broad distribution of fibre ATP content at rest ranging from 16 to 34 mmol kg-' d.m. has also been noted by Jansson et al. (1987).
5 8 I2
36
ATP
n
14.1 f 5 . 4 0 17.0f3.63 14.3f2.81 19.1f4.40 16.0f4.82
33 21
18 24 96
Contraction with occluded circulation
With occluded circulation the force generation declined rapidly and reached 2 2 % of the initial value after 52 contractions. An almost complete loss of PCr occurred, and lactate increased to
464
K. Soderlund and E. Hultman
L 5
m
0
'i_
10
5l
r-
0
0
5
10
15 20 AT? mmol kg-' d.m.
25
30
L 35
Fig. 4. The ATP content in single type I (0) and type I1 (0) fibres from four subjects (a) at rest, (b) following 5 2 contractions and (c) after 15 min recovery.
128.7 mmol kg-' d.m. (Table 2). I n this situation, where energy demand is greater than the capacity for glucolytic ATP resynthesis, a decrease in ATP is seen when the PCr store is depleted. In the whole-muscle sample the ATP level decreased by 42 %. The mean ATP decrease was equal in both fibre types after the intense electrical stimulation. The individual fibres showed a range of ATP
concentration from 7 to 28 and from 4 to 26 mmol kg-' d.m. for type I and type I1 fibres respectively. This is in agreement with the findings of Jansson et al. (1987). The lowest ATP concentration reported in their study was 10mmol kg-' d.m. In the present study we found fibres with a concentration as low as 4.5 mmol kg-' d.m. The difference could be due to a different exercise protocol. Jansson et al.
ATP content in singleJbres
465
Table 4. ATP content in single muscle fibres after 1 5 rnin recovery following contraction with occluded circulation. Values are mean+SD in mmol kg-l dry muscle. n = number of single fibres Type I1
Type 1 Subject no. I
2
3 4 Total
ATP
n
27.4i2.38 25.8 k 0.93 26.1f2.95 21.4 f3-27 25.6k3.29
II
5 19
7 42
Type I+type I1
ATP
n
ATP
n
2 1.3
I1
24.4f3.96 q . 3 f 1.72 25.2f2.77 19.9f3.27 24.3k3.46
22
2.62 25.1 f 1 . 9 1 24.5 f2.45 18.5k2.71 23.3 k 3.27*
'5 22
7 55
20
41
14 97
Statistics: Student's t-test was used for groups of unequal sample sizes to compare the ATP content in type 15 min recovery. * P < 0.05.
I1 fibres at rest and after
(1987) used voluntary dynamic knee exercise with open circulation, whereas we used electrical stimulation with occluded circulation. The symmetrical distribution of the ATP contents after 52 contractions (Fig. 4) seems to indicate that ATP is not limiting as an energy substrate. This is in accordance with the studies by Tokiwa & Tonomura (1965) showing a K , for myosine ATPase corresponding to an ATP level below I mmol kg-' d.m. The decline in ATP during this type of contraction is quantitatively matched by an increase in I M P (Spriet et al. 1987). I M P was not analysed in single fibres, but whole-muscle samples showed the expected increase in I M P after 5 2 contractions. As shown in Table 2 the ATP resynthesis from' I M P was not complete after 15 min recovery in the whole sample. Single-fibre analyses showed that type I fibres were normalized, while the resynthesis was incomplete in type I1 fibres. Similar results have been published by Meyer & Terjung (1979) comparing the resynthesis rate in slow-twitch soleus and fast-twitch gastrocnemius muscle of rat after electrical stimulation. The soleus muscle showed complete ATP resynthesis after I min while gastrocnemius after 30 min recovery still had a concentration below the resting concentration. T h e reason for this slow resynthesis of ATP in fast-twitch muscle is unclear. The nearly complete restitution of ATP in the human muscle fibres within 15 min recovery indicates that no damage has occurred due to the intense muscle stimulation This work was supported by the Medical Research Council (grant 02647). The authors wish to thank the
entire staff of the Department of Clinical Chemistry I1 for excellent collaboration in this investigation.
REFERENCES 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 3 Clan Lab Invest 14, Suppl. 68, 1-1 1 0 .
EBERSTEIN, A. & GOODGOLD, J. 1968. Slow and fast twitch fibers in human skeletal muscle. Am 3 Physiolz15, 535-541. EDSTROM, L., HULTMAN, E., SAHLIN, K. & SJOHOLM, H. 1982. T h e contents of high-energy phosphates in different fibre types in skeletal muscles from rat, guinea-pig and man. 3 Physiol 332, 47-58. ESSEN,B. 1978. Studies on the regulation of metabolism in human skeletal muscle using intermittent exercise as an experimental model. Acta Physiol Scand Suppl. 454, 1 4 4 . ESSEN,B., JANSSON, E., HENRIKSSON, J., TAYLOR, A.W. & SALTIN, B. 1975. Metabolic characteristics of fibre types in human skeletal muscle. Acta Physiol Scand 95, 153-165. HARRIS, R.C. & HULTMAN, E. 1985. Adenine nucleotide depletion in human muscle in response to intermittent stimulation in situ (abstract). Proc Physiol Sac C59, 78P. HARRIS, R.C., HULTMAN, E. & NORDESJO, L.-0, 1974. Glycogen, glucolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand 3 Clin Lab Invest 33, 109-1 20. HINTZ,C.S., CHI,M.M.-Y., FELL,R.D., IVY,J.L., KAISER,K.K., LOWRY,C.V. & LOWRY,O.H. (1982). Metabolite changes in individual rat muscle
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fibers during stimulation. A m 3 Physiol 242, LUNDIN, A,, RICKARDSSON, A. & THORE, A. 1976. C218-C228. Continuous monitoring of ATP converting reactions by purified firefly luciferase. Anal Biochem 75, HULTMAN, E. & SJOHOLM, H. 1986. Biochemical causes of fatigue. I n : N.L. Jones, N. McCartney & 611 4 2 0 . A.J. McComas (eds.) Human Muscle Power, pp. MEYER,R.A. & TERJUNG, R.L. 1979. Differences in 215-238. Human Kinetic Publ., Champaign, Illiammonia and adenylate metabolism in contracting nois. fast and slow muscle. AmJPhysiol237, C I I I-CI 18. HULTMAN, E., SJOHOLM, H., JADERHOLM-EK, I. & REHUNEN,S. & HARKONEN, M . 1980. High-energy phosphate compounds in human slow-twitch and KRYNICKI,J. 1983. Evaluation of methods for fast-twitch muscle fibres. Scand 3 Clin Lab Invest electrical stimulation of human skeletal muscle tn situ. Pfugers Arch 398, 139-141. 4% 45-54. K., BERGSTROM, M. & JANSSON, E., DUDLEY, G.A., NORMAN,A. & TESCH,SPRIET, L.L., SODERLUND, P.A. 1987. ATP and I M P in single human muscle HULTMAN, E. 1987. Anaerobic energy release in fibres after high intensity exercise. Clin Physiol 7 , skeletal muscle during electrical stimulation in man. 3 Appl Physiol62, 61 1 4 1 5 . 337-345T. & TONOMURA, Y. 1965. The pre-steady LOWRY,O.H. & PASSONNEAU, J.V. 1972. A Flexible TOKIWA, state of the myosin-adenosine triphosphate system. System of Enzymatic Analysis, pp. 189-193. Academic Press, New York. 3 Biochem 57, 616-626.
ATP content in single fibres from human skeletal muscle after electrical stimulation and during recovery K. S O D E R L U N D and E. H U L T M A N Department of Clinical Chemistry 11, Huddinge University Hospital, Karolinska Institutet, Huddinge, Sweden SODERLUND, K. & HULTMAN, E. 1990.ATP content in single fibres from human skeletal muscle after electrical stimulation and during recovery. Acta Physiol Scand 139, 459-466. Received 26 January 1990,accepted 23 February 1990.ISSN 0001-6772. Department of Clinical Chemistry 11, Huddinge University Hospital, Karolinska Institutet, Huddinge, Sweden. The ATP content was measured in type I and type I1 fibres from human vastus lateralis muscle at rest, after electrical stimulation and during recovery. At rest the mean values were 25.2 f 4.02 and 25.92 3.62 mmol kg-l dry muscle (mean 2 SD) for type I and type I1 fibres respectively. Normal distribution curves were found for both types I and I1 fibres. After intermittent electrical stimulation for 83 s (1.6s stimulation, 1.6s pause) with occluded blood flow, the force generation decreased to 22% of the initial value and the muscle tissue showed a mean decrease in ATP to 14.8and in phosphocreatine to 5.44mmol kg-' dry muscle; lactate increased to 128.9mmol kg-' dry muscle. The ATP content in isolated fibres was equally decreased in both fibre types to 16mmol kg-' dry muscle. In I I yoof the fibres the ATP content was lower than 10mmol kg-' dry muscle. After 15 min rest with intact blood circulation ATP was completely resynthesized in type I fibres and to 91 yo in type I1 fibres. Key words :ATP concentration, ATP resynthesis, electrical stimulation, human muscle, single fibres.
I t is known that human skeletal muscle consists of a mixture of fibres with different contractile and metabolic properties (Eberstein & Goodgold 1968). T y p e I fibres are slow-contracting and have a high oxidative capacity, whereas type I1 fibres are fast-contracting and have a high glucolytic capacity. T h e A T P concentration in the two fibre types was reported to be equal both at rest and after continuous excercise to exhaustion (Essen 1978). Similar results were obtained by Jansson et al. (1987)but with tendency for a higher A T P concentration in type I1 than in type I fibres at rest and a greater decrease in type I1 fibres after maximal voluntary isometric exercise with the knee extensors. Previous studies in man have shown a 50% Correspondence : Karin Soderlund, Department of Clinical Chemistry 11, Huddinge University Hospital, S-14186 Huddinge, Sweden.
decrease in the A T P in whole-muscle samples from the quadriceps femoris during intermittent electrical stimulation (Harris & Hultman 1985, Hultman & Sjoholm 1986).I n these studies the blood flow to the muscles was occluded during the stimulation. T h e decrease in ATP was matched by a corresponding increase in inosine monophosphate (IMP), and by the end of the contraction the force generation had declined to about 20% of the initial value. T h e present study was undertaken to extend these earlier observations by examining ATP depletion and resynthesis in isolated, characterized fibres.
M A T E R I A L S A N D METHODS Seven subjects, four women and three men, volunteered to participate in the study. The character-
459
460
K. Soderlund and E . Hultman
istics of the subjects were as follows : age 30 7 years, height 173+9 cm and weight 68+ 1 2 kg. The subjects were informed about the nature and purpose of the experiment before they gave their voluntary consent to participate. This experiment was part of a larger project approved by the Ethical Committee of the Karolinska Institute. T h e subjects reclined in a semisupine position on a bed. T h e lower legs were flexed over the end of the bed to 90' and one leg, chosen at random, was attached to a strain gauge on the frame of the bed via an ankle strap. The subject then performed three maximal voluntary contractions to determine the maximal voluntary isometric force (MVC) of the knee extensor muscles. The leg was then prepared for electrical stimulation of the quadriceps femoris muscle as previously described by Hultman et a/. (1983). T h e vastus lateralis muscle of the thigh was stimulated to contract via surface electrodes at a frequency of 20 Hz with square-wave pulses of 0.5 ms duration. The stimulation was intermittent with trains lasting 1.6 s and separated by pauses of 1.6 s for a total of 52 contractions. The total time was 165 s and contraction time was 83 s. Approximately 30% of the musculature that extends the knee was activated to 70% of maximum (Hultman et al. 1983). In the experiment with occluded circulation a cuff around the proximal portion of the thigh was inflated (250 mmHg) to occlude the blood flow 30 s prior to the onset of stimulation and remained inflated during the stimulation and biopsy sampling. The force produced was recorded throughout the stimulation by a strain gauge attached to a strap around the ankle. Biopsies were taken from the vastus lateralis muscle (Bergstrom 1962) at rest, following -52 contractions (83 s contraction time) and after 1 5 min of recovery.
Analytical methods T h e biopsy samples were immediately frozen in liquid nitrogen (N,) and freeze-dried. One part was used for analysing muscle metabolites (Harris et al. 1974) in the whole piece, and one part was used for single-fibre dissection. Single fibres were separated and stained according to the method described by EssCn et a/. (1975). The fibres were weighed on a customized quartzfibre fishpole balance (Lowry & Passonneau 1972). Two pieces (0.1 pg) from each fibre were excised and stained for myofibrillar ATPase to classify type I and type I1 fibres. T h e fibres were extracted in zoo ,dof 2.5% trichloroacetic acid (TCA) for 2-3 min and stored at -70 "C until analysed. T h e ATP content in single fibres (weighing 0.5-4pg) was measured using the bioluminescence technique and an ATP-monitoring kit (1243-102, LKB-Wallace) (Lundin et al. 1976). The analyses were performed on a LKB-Wallace 1250 luminorneter (Turku, Finland).
Statistical treatment and precision of the method Statistics. Student's t-test was used to compare group means. T h e SDtOtwas calculated from all fibres at rest ( n = 137).The SDindfor all fibres from each individual was determined, and the mean of the SD from seven subjects gives the SD,,,,. The ATP concentration in individual fibre fragments did not show any systematic variation with sample weight. The precision of the ATP measurement was 4% (coefficient of variation, CV) determined from 52 duplicate analyses on TCA extracts. The CV in ATP between fragments of the same fibre was 3 %. An attempt was made to calculate the number of fibres needed to obtain a reliable mean. Means of the first five and 10fibres from 22 biopsy samples were compared with the mean of the total number of fibres analysed from the same biopsy sample of a specific fibre type. The number of fibres from each biopsy ranged from I I to 22. The mean of five fibres gave a difference of 504,and the mean of 10 fibres gave a difference of zyo, from the mean of the total number of fibres analysed of a specific fibre type of the biopsy. This finding suggests that 5-10 fibres will be enough to obtain a reliable mean value. Comparison between individual fibre analyses and analyses of whole-muscle samples was performed by comparing the mean of all individual contents with the content in the whole sample.
RESULTS T h e ATP content at rest
The distribution of ATP in the resting state in the two fibre types is shown in Fig. I . The distribution frequencies followed a normal distribution curve in both fibre types and there was no difference in ATP level in the two fibre types. This was also evident when the individual results were examined (Table I ; Fig. 2a and b). In six out of seven subjects there was close agreement between the average fibre ATP and the content determined in the whole-muscle sample (Table I). I n subject 5 , the whole-sample content was noticeably lower. This may have been due to the inclusion of blood in the biopsy sample. Muscle contractions with occluded circulation
After 5 2 contractions with occluded circulation the force had decreased to 22% of the initial value (Fig. 3). Simultaneously there was a decrease in ATP and phosphocreatine (PCr)
ATP content in single fibres n
25
20
15
10
5
0 15
20
25
30
35
ATP: mmol kg" d.m.
l r
Fig. I . The distribution of ATP in the resting state of all type I (0) and type I1 (0) fibres from seven subjects.
contents of 42 and 93% respectively, and an increase in lactate to 128.2 mmol kg-' dry muscle (d.m.) (Table 2). The ATP in single fibres showed an equal decrease in both fibre types from approximately 25 to 16 mmol kg-' d.m. (Table 3 ) . The lowest ATP value was 4.5 mmol kg-' d.m. (subject I , type I1 fibre). In this subject a further eight fibres out of a total of 33 analysed had an ATP content of less than 1 0 mmol kg-' d.m. In the remaining three subjects 0 / 2 3 , 2 / 1 8 and 1 / 2 4 fibres had an ATP concentration lower than 1 0 mmol kg-' d.m. In total, 1 1 % of the fibres had an ATP concentration lower than 1 0 mmol kg-l d.m.
3 c3
0
a c-" dF
Recowry after occhded circulation 3
After 15 min recovery the ATP in whole muscle was significantly decreased compared with the resting value (Table 2). PCr was back to normal but lactate concentration remained increased (Table 2). In single fibres the ATP was completely resynthesized in type I fibres, but only to 91 yo in type I1 (Table 4).
c)
.-DW
c 2 i
46 1
462
K. Soderlund and E . Hultman n 15-
a
10 -
5-
0-
, . ....' ... I
15
20
25
.
30
35
.
151 b
10-
5-
0-
2. Normal distribution curves of (a) type I (0) and (b) type I1 (0) fibres at rest. The continuous curve describes the distribution of all fibres measured at rest using SD,,, 3.81 mmol kg-' dry muscle (d.m.). The dashed line describes the expected distribution in a single individual using SD,,, 3.22 mmol kg-' d.m.
Fig.
Figure 4 presents a summary of single-fibre distribution at rest, following 5 2 contractions and after I j min recovery. I t is clear that type I1 fibres show a larger variation in A T P concentration after 5 2 contractions than type I fibres. After I j min recovery with intact blood flow the A T P level was completely normalized in type I fibres, but only to 91% in type 11.
DISCUSSION The ATP content at rest There was no significant difference in A T P level between type I and type I1 fibres in human mixed muscle, a result which bears out the studies by Essen (1978) and Rehunen & Harkonen (1980). Earlier estimates in various animal species have indicated a higher ATP
ATP content in single3bres
463
10080-
.cE
6040-
0
s a 2
20-
e
,
o!
l
12.8
I
20
40
64.0
128.0
0 4
1
52 no of contr.
164.8 time, s
Fig. 3. Force in per cent of initial value during 52 contractions with occluded circulation. Tabl e 2. Muscle metabolites in muscle samples obtained at rest, after 52 contractions and after 15 min recovery. The blood circulation to the leg during the contraction was occluded. Values are means k SD in mmol kg-' dry muscle. Force is given as per cent of the initial value Occluded circulation Rest (n = 4)
52 contractions (n = 4)
1 5 min recovery (n = 4)
25.6f 1.39 84.5 f6.36 47.7 f 7.49 132.0f 13.00 2.8 f0.64
14.8f 2.96 5.4+ 1.12 123.6f 18.00 128.7f 17.70 128.9& 24.50
20.9f 1.52" 90.0 I I .30 38.7 k 8.55 129.1 t 18.80 12.0f5.60
_ ~ _ ._ .
ATP PCr Cr TC r Lactate Force (yo)
22.5 f9.5
I00
Statistics: Student's t-test was used to compare the ATP content at rest and after 1 5 min recovery. *;
P < 0.05.
Tabl e 3. ATP content in single muscle fibres after 52 contractions with occluded circulation. Values are mean SD in mmol kg-' dry muscle. n = number of single fibres
+
I 2
3 4 Total
Type I + type I1
Type I1
Type 1 Subject no.
ATP
n
ATP
n
15.1f5.03 16.2f2.98 15.8f1.20 18.3f3.06 16.1f3.82
22
12.2f5.88 19.6f4.62 I2.4k3.17 20.0 k 5.43 15.9f6.19
11
16 10 12
60
concentration in predominantly fast-twitch or mixed muscle compared with slow-twitch muscles (Edstrom et al. 1982, Hintz et al. 1982). A broad distribution of fibre ATP content at rest ranging from 16 to 34 mmol kg-' d.m. has also been noted by Jansson et al. (1987).
5 8 I2
36
ATP
n
14.1 f 5 . 4 0 17.0f3.63 14.3f2.81 19.1f4.40 16.0f4.82
33 21
18 24 96
Contraction with occluded circulation
With occluded circulation the force generation declined rapidly and reached 2 2 % of the initial value after 52 contractions. An almost complete loss of PCr occurred, and lactate increased to
464
K. Soderlund and E. Hultman
L 5
m
0
'i_
10
5l
r-
0
0
5
10
15 20 AT? mmol kg-' d.m.
25
30
L 35
Fig. 4. The ATP content in single type I (0) and type I1 (0) fibres from four subjects (a) at rest, (b) following 5 2 contractions and (c) after 15 min recovery.
128.7 mmol kg-' d.m. (Table 2). I n this situation, where energy demand is greater than the capacity for glucolytic ATP resynthesis, a decrease in ATP is seen when the PCr store is depleted. In the whole-muscle sample the ATP level decreased by 42 %. The mean ATP decrease was equal in both fibre types after the intense electrical stimulation. The individual fibres showed a range of ATP
concentration from 7 to 28 and from 4 to 26 mmol kg-' d.m. for type I and type I1 fibres respectively. This is in agreement with the findings of Jansson et al. (1987). The lowest ATP concentration reported in their study was 10mmol kg-' d.m. In the present study we found fibres with a concentration as low as 4.5 mmol kg-' d.m. The difference could be due to a different exercise protocol. Jansson et al.
ATP content in singleJbres
465
Table 4. ATP content in single muscle fibres after 1 5 rnin recovery following contraction with occluded circulation. Values are mean+SD in mmol kg-l dry muscle. n = number of single fibres Type I1
Type 1 Subject no. I
2
3 4 Total
ATP
n
27.4i2.38 25.8 k 0.93 26.1f2.95 21.4 f3-27 25.6k3.29
II
5 19
7 42
Type I+type I1
ATP
n
ATP
n
2 1.3
I1
24.4f3.96 q . 3 f 1.72 25.2f2.77 19.9f3.27 24.3k3.46
22
2.62 25.1 f 1 . 9 1 24.5 f2.45 18.5k2.71 23.3 k 3.27*
'5 22
7 55
20
41
14 97
Statistics: Student's t-test was used for groups of unequal sample sizes to compare the ATP content in type 15 min recovery. * P < 0.05.
I1 fibres at rest and after
(1987) used voluntary dynamic knee exercise with open circulation, whereas we used electrical stimulation with occluded circulation. The symmetrical distribution of the ATP contents after 52 contractions (Fig. 4) seems to indicate that ATP is not limiting as an energy substrate. This is in accordance with the studies by Tokiwa & Tonomura (1965) showing a K , for myosine ATPase corresponding to an ATP level below I mmol kg-' d.m. The decline in ATP during this type of contraction is quantitatively matched by an increase in I M P (Spriet et al. 1987). I M P was not analysed in single fibres, but whole-muscle samples showed the expected increase in I M P after 5 2 contractions. As shown in Table 2 the ATP resynthesis from' I M P was not complete after 15 min recovery in the whole sample. Single-fibre analyses showed that type I fibres were normalized, while the resynthesis was incomplete in type I1 fibres. Similar results have been published by Meyer & Terjung (1979) comparing the resynthesis rate in slow-twitch soleus and fast-twitch gastrocnemius muscle of rat after electrical stimulation. The soleus muscle showed complete ATP resynthesis after I min while gastrocnemius after 30 min recovery still had a concentration below the resting concentration. T h e reason for this slow resynthesis of ATP in fast-twitch muscle is unclear. The nearly complete restitution of ATP in the human muscle fibres within 15 min recovery indicates that no damage has occurred due to the intense muscle stimulation This work was supported by the Medical Research Council (grant 02647). The authors wish to thank the
entire staff of the Department of Clinical Chemistry I1 for excellent collaboration in this investigation.
REFERENCES 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 3 Clan Lab Invest 14, Suppl. 68, 1-1 1 0 .
EBERSTEIN, A. & GOODGOLD, J. 1968. Slow and fast twitch fibers in human skeletal muscle. Am 3 Physiolz15, 535-541. EDSTROM, L., HULTMAN, E., SAHLIN, K. & SJOHOLM, H. 1982. T h e contents of high-energy phosphates in different fibre types in skeletal muscles from rat, guinea-pig and man. 3 Physiol 332, 47-58. ESSEN,B. 1978. Studies on the regulation of metabolism in human skeletal muscle using intermittent exercise as an experimental model. Acta Physiol Scand Suppl. 454, 1 4 4 . ESSEN,B., JANSSON, E., HENRIKSSON, J., TAYLOR, A.W. & SALTIN, B. 1975. Metabolic characteristics of fibre types in human skeletal muscle. Acta Physiol Scand 95, 153-165. HARRIS, R.C. & HULTMAN, E. 1985. Adenine nucleotide depletion in human muscle in response to intermittent stimulation in situ (abstract). Proc Physiol Sac C59, 78P. HARRIS, R.C., HULTMAN, E. & NORDESJO, L.-0, 1974. Glycogen, glucolytic intermediates and high-energy phosphates determined in biopsy samples of musculus quadriceps femoris of man at rest. Methods and variance of values. Scand 3 Clin Lab Invest 33, 109-1 20. HINTZ,C.S., CHI,M.M.-Y., FELL,R.D., IVY,J.L., KAISER,K.K., LOWRY,C.V. & LOWRY,O.H. (1982). Metabolite changes in individual rat muscle
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K. Soderlund and E. Hultman
fibers during stimulation. A m 3 Physiol 242, LUNDIN, A,, RICKARDSSON, A. & THORE, A. 1976. C218-C228. Continuous monitoring of ATP converting reactions by purified firefly luciferase. Anal Biochem 75, HULTMAN, E. & SJOHOLM, H. 1986. Biochemical causes of fatigue. I n : N.L. Jones, N. McCartney & 611 4 2 0 . A.J. McComas (eds.) Human Muscle Power, pp. MEYER,R.A. & TERJUNG, R.L. 1979. Differences in 215-238. Human Kinetic Publ., Champaign, Illiammonia and adenylate metabolism in contracting nois. fast and slow muscle. AmJPhysiol237, C I I I-CI 18. HULTMAN, E., SJOHOLM, H., JADERHOLM-EK, I. & REHUNEN,S. & HARKONEN, M . 1980. High-energy phosphate compounds in human slow-twitch and KRYNICKI,J. 1983. Evaluation of methods for fast-twitch muscle fibres. Scand 3 Clin Lab Invest electrical stimulation of human skeletal muscle tn situ. Pfugers Arch 398, 139-141. 4% 45-54. K., BERGSTROM, M. & JANSSON, E., DUDLEY, G.A., NORMAN,A. & TESCH,SPRIET, L.L., SODERLUND, P.A. 1987. ATP and I M P in single human muscle HULTMAN, E. 1987. Anaerobic energy release in fibres after high intensity exercise. Clin Physiol 7 , skeletal muscle during electrical stimulation in man. 3 Appl Physiol62, 61 1 4 1 5 . 337-345T. & TONOMURA, Y. 1965. The pre-steady LOWRY,O.H. & PASSONNEAU, J.V. 1972. A Flexible TOKIWA, state of the myosin-adenosine triphosphate system. System of Enzymatic Analysis, pp. 189-193. Academic Press, New York. 3 Biochem 57, 616-626.
