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Anaerobic energy release in skeletal muscle
during electrical stimulation in men
LAWRENCE L. SPRIET, KARIN SODERLUND,
MATS BERGSTROM, AND ERIC HULTMAN
Department
of Clinical Chemistry II, Karolinska Institute,
Huddinge University Hospital, S-141 86 Huddinge, Sweden
SPRIET, LAWRENCE ., KARIN WDERLUND, MATS BERG-
STROM,AND ERIC HULTMAN.Anaerobic energy reZease in skel-
etal muscle during electrical stimulation in men. J. Appl. Physiol.
62(Z): 611-615, 1987.-The quadriceps femoris muscles of
seven men were electrical ly stimulated under extended anaer-
obic conditions to quantitate anaerobic energy release and the
contribution of the glyco lytic system to total ATP production.
Muscles were intermittently stimulated 64 times at 20 Hz while
leg blood flow was occluded. Each contraction lasted 1.6 s and
was followed by 1.6 s of rest. The total contraction time was
102.4 s. Muscle biopsies were taken at rest and following 16,
32,48, and 64 contractions. The ATP turnover rates during the
four 16-contraction periods were 6.12, 2.56, 2.17, and 0.64
mmol l kg dry muscle-l ws- contraction time. Glyco lysis pro-
vided 58 , phosphocreatine 40 and a decreased ATP store
2 of the consumed energy during the initial 16 contractions.
Glycolysis was responsible for 90 of the total ATP production
beyond contraction 16. Absolute glyco lytic ATP production
decreased to 60, 55, and
17%
of the amount in the initial
16
contractions during the final three periods, respect ively. In
conclusion glycolysis produced -195 mmol ATP/kg dry muscle
during the initial 48 contractions (76.8 s) and only -15 mmol
ATP/kg dry muscle during the final 16 contractions. Equivalent
values for total ATP turnover were 278 and 16.5 mmol/kg dry
muscle.
adenosine triphosphate turnover rate; glycolysis; phosphocrea-
tine; lactate; isometric force production; adenosine diphos-
phate; adenosine monophosphate; inosine monophosphate
THEREHAS BEEN CONSIDERABLE nterestinhumanmus-
cle metabolism and performance during short-term max-
imal or near-maximal exercise (3, 5, 6,
12, 16).
The high
rate of ATP production required to support intense iso-
metric or dynamic exercise lasting Cl min is largely
dependent on the muscles ability to regenerate ATP
anaerobically. In the muscle cell, ATP is produced an-
aerobically in the glycolytic pathway with the formation
of lactate and hydrogen ions and through the degradation
of phosphocreatine (PCr). Approximately 30-40 of the
muscle ATP store may also be directly used to support
muscular contraction, although this contribution is
quantitatively very small.
In recent studies, where muscle biopsies were obtained
before and following 30-50 s of maximal isokinetic cy-
cling (16), sprinting (6) or electrical stimulation (12, 14),
substantial PCr degradation and lactate accumulation
occurred. Estimates of the ATP turnover rates during
dynamic exercise were between 6-8 mmol
l
kg dry mus-
cle-l l s-l, with glycolysis accounting for 6080 and PCr
utilization ZO-40 of the total ATP produced. Hultman
and Sjoholm (12) obtained biopsies every 10 s from the
quadriceps femoris muscle group during 50 s of continu-
ous electrical stimulation. The ATP turnover rate in the
isometrically contracting muscles decreased from 5.6
mmol l kg dry muscle- l s-l during the initial
10 s
to
4.0
mmol l kg dry muscle- l s-l in the final
10
s. The fraction
of ATP produced by anaerobic glycolysis increased from
40 to 90 during the same time periods. Collectively
these studies demonstrate that the regeneration from
PCr is limited to 30 s or less during intense muscular
contraction. The energy required to sustain contractions
beyond this time under anaerobic conditions must be
derived from glycolysis.
The present investigation was designed to quantitate
the ATP turnover rate during short-term intense mus-
cular activity in men and to determine the contribution
of the glycolytic system to ATP production under ex-
tended anaerobic conditions. To accomplish this the
quadriceps femoris muscles were electrically stimulated
for 100 s of contraction time during circulatory occlusion
with muscle biopsies taken every 25 s.
METHODS
Seven male subjects agreed to participate in the study
(age 28.4 t
1.2
yr; height,
184
t 3 cm; weight, 82.6 t
2.2
kg). The subjects were not well trained but were healthy
and active as they 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.
Subjects reported to the laboratory in the postprandial
state and reclined in a semisupine position on a bed. The
lower legs were flexed over the end of the bed to 90 and
one leg, chosen at random, was attached to a strain gauge
in the frame of the bed via an ankle strap. The subject
then performed three maximal voluntary contractions
(MVC) to determine the maximal voluntary isometric
force of the knee extensors. Isometric force production
was measured with a strain gauge (AB Bofors, Karlskoga,
Sweden) and the signal was amplified (direct-current
0161-7567/87 $1.50 Copyright
0
1987
the American Physiological
Society 611
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612 SKELE TAL MUSCLE ANAEROBIC ENERGY RELEASE
amp Medelec AD6, Surrey, UK), displayed on an oscil-
loscope (Medelec M) and recorded on ultraviolet-sensi-
tive paper.
The leg was then prepared for electrical stimulation of
the quadriceps femoris muscles as previously described
(13, 15). Briefly, two large (9 X 6 cm) aluminum foil
electrodes were applied proximally and distally to the
anterolateral aspect of the thigh. The under lying muscles
were stimulated to contract with square-wave pulses of
0.5 ms duration at a frequency of 20 Hz (Medelec 15-V
stimulator). Stimulat ion was intermittent with trains
lasting 1.6 s and separated by rest pauses of 1.6 s. The
voltage used (120-180 V) produced an ini tia l force cor-
responding to 22 of the maximal voluntary isometric
force. Since stimulation at 20 Hz produces a fused teta-
nus representing 70-75 of the maximal tetanic force
obtained at higher frequencies (20), -29-32 of the
musculature that extends the knee was activated. The
muscle activated in this study was limited to the antero-
lateral aspect of the thigh as muscle needle biopsies were
taken from the vastus lateralis muscle as described by
Bergstrom (2).
The experimental protocol used in this study is sche-
matically presented in Fig. 1. Before the stimulation of
leg one, three incisions in the skin were performed fol-
lowing local anesthesia and a resting biopsy was ob-
tained. Thirty seconds before stimulation a pneumatic
cuff around the proximal portion of the thigh was inflated
to a pressure of 250 Torr. In this manner blood flow was
occluded and a predominantly anaerobic situation cre-
ated throughout the stimulation period. The muscles
were then stimulated at 20 Hz for a total of 64 contrac-
tions, each lasting 1.6 s and separated by 1.6 s of rest.
The total stimulation time was 204.8 s and the contrac-
tion t ime 102.4 s (Fig. 1). Muscle biopsies were taken in
the rest periods following contractions 16 and 48. These
rest periods were elongated to -3-5 s to permit time for
biopsy sampling.
The second leg was then prepared for stimulation and
force measurement as described above and stimulated to
contract 64 times. The time between stimulation of legs
one and two was 30-40 min. Muscle biopsies were taken
from leg two at rest and following 32 and 64 contractions.
The isometric force production by the activated knee
extensor muscles was continuously recorded and the
reported data represents the average of measurements
obtained from both legs in seven subjects. The peak force
obtained during each tetanic contraction was used in the
presentation of the force data.
-
Muscle biopsy samples were immediately frozen (3-5
s from the insertion of the needle) in liqu id freon main-
Biopsy ($1, ILeg 1
1
Leg 2
1
1
1
No. of contract ions
No. of contractions 0 16
32 48 64
I
1 I I
r
I
1 i
Contraction time , set 0 25.6 51.2 76.8 102.4
Stimulation time , set
0 51.2 102.4 153.6 204.8
FI G. 1. Schem atic representation of experimental design. Each con-
traction lasted 1.6 s at 20 Hz and w as followed by 1.6 s of rest. Blood
flow was occlude d throughout experiment.
170 65
i
150 75
d
;
m i,
i 120 601%
1 I 1 I 1
0
16 32 48 64
Number of cant ract ions
0 25.6
51.2 76.8 102.
Contracton tme, set
FI G. 2. Muscle contraction force and phosphocrea tine (PCr), ATP,
and lactate concen trations during intermittent electrical stimulation
with an occlude d circulation. Data points represent mean of 7 subjec ts.
SEs for phospho creatine (PC,), ATP, and lactate are not included but
appear in Table 1. On left y-axis, PCr, and ATP data points correspond
to O-85 scale and lactate data points correspond to O-170 sca le.
tained at its melting point (-150C) with l iquid nitrogen.
Samples were freeze-dried, dissected free of blood and
connective tissue, and extracted with 0.5 M HClO, (1.0
mM EDTA). The neutralized extracts (2.3 M KHCOs)
were analyzed enzymatically (1) for ATP, ADP, PCr, Cr,
and lactate as described by Harris et al. (11). Adenosine
monophosphate (AMP) and inosine monophosphate
(IMP) were measured with high-pressure liquid chro-
matography (HPLC) (18). Resting metabolite concentra-
tion represents the average of measurements performed
on biopsies from both legs in seven subjects. Muscle
metabol ite concentrations are expressed per kilogram
dry muscle and al l data are presented as means t SE.
RESULTS
Maximal voluntary contractions of the knee extensor
muscles in the left and right legs produced mean forces
of 651 t 45 and 657 t 49 N, respectively. Approximately
30 of the knee extensor muscle mass was electrically
stimulated to contract at 20 Hz (see
METHODS),
produc-
ing initial forces of 144 t 13 N in the left leg and 145 t
15 N in the right leg. These forces represented 22 of
MVC in both legs.
Isometric force production was well maintained during
the early contractions as 87.6 t 2.5 of the initial force
was held during contraction 16 (Fig. 2). In the subsequent
32 contractions force production decreased rapidly to
55.6 t 2.5 and 26.7 t 2.7 of initial at contractions 32
and 48, respectively. During the final 16 contractions
force production was extremely low, amounting to 16.8
t 2.3 of the initial force at contraction 64.
Resting metabolite concentrations were in the normal
range for human skeletal muscle (Tables 1 and 2). Muscle
ATP decreased progressively with continued stimulation
under anaerobic conditions to a low of 56.6 of the
resting concentration following 64 contractions (Table
2, Fig. 2). The muscle PCr store was 80 depleted
following 16 contractions (Table 1, Fig. 2). Continued
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SKEL ETAL MUSCLE ANAEROBIC ENERGY RELEASE
613
TABLE 1. Muscle metabolite concentrations
during intermittent electrical stimulation
with an occluded circulation
No. of Contractions
Rest
16 (25.6) 32 (51.2) 48 (76.8) 64 (102.4)
PCr 78.7t2.4 15.7t3.9 9.0t2.7 5.4t1.1 5.5t0.9
Cr 42.4t2.6 103.3t5.7 109.5t4.7 116.2t2.2 11 8.2t2.7
Total Cr 121.1k1.6 119.0t2.8 118.5t3.2 121.6t1.9 123.7t3.2
Lactate 5.lkl.O 65.627.4 101.7k9.4 135.2k13.5 145.3k9.5
Values are means & SE in mmol/kg dry muscle; n = 7. Values in
parentheses indicate duration (in s). Quadriceps femoris m uscle s were
stimulated 64 times at 20 Hz with each contraction lasting 1.6 s and
followed by 1.6 s of rest. PCr, phosphocreatine; Cr, creatine.
TABLE 2. Muscle adenine nucleotide and IMP
concentrations during intermittent electrical
stimulation under anaerobic conditions
No. of Contractions
Rest
16 (25.6) 32 (51.2) 48 (76.8)
64 (102.4)
ATP 24.81t0.80 21.7621.23 17.2821.26 15.48rtO.61 14.04tl.19 -
ADP 2.92t0.06 3.59t0.25 3.71t0.40 3.92t0.19 3.59k0.24
AMP 0.33t0.05 0.44t0.09 0.4320.04 0.38t0.03 0.45t0.06
TAN 28.06t0.78 25.79t1.47 21.42t1.45 19.78k0.58 18.08 tl.25
IMP
0.54t0.08 2.39t1.09 7.90t1.52 10.61t1.90 11.63t1.60
Values are means t SE in mmol/kg dry mus cle; n = 7. Values in
parentheses indicate duration (in s). TAN, total adenine nucleo tides;
IMP, inosine m onophospha te. ATP and ADP were analyzed enzymat-
ically; AMP and IMP were analyzed with high-pressure liquid chro-
matography (see METHODS). TAN = ATP + ADP + AMP.
stimulation produced a slight additional decrease such
that 93 of the available PCr store was utilized following
48 and 64 contractions. Changes in Cr were reciprocal to
PCr changes and the total Cr content remained constant
throughout the stimulation period (Table 1). Muscle
lactate content increased -30-fold during the entire
stimulation period (Table 1, Fig. 2). The largest increase
occurred during the initial 16 contractions, whereas min-
imal lactate accumulated between contractions 48 and
64
Concentrations of ADP and AMP increased slightly
during the initial 16 contractions and remained elevated
for the entire stimulation period (Table 2). Increases in
IMP content were stoichiometrically equivalent to de-
creases in ATP, indicating a degradation of adenine
nucleotides via the AMP deaminase reaction. Conse-
quently, total adenine nucleotides decreased as a function
of the decreases in ATP, reaching 64 of the resting
concentration following 64 contractions.
The occlusion of muscle blood flow during electrical
stimulation provided a closed metabolic system, limiting
the production of ATP to anaerobic processes and pre-
venting the escape of metabolites from the muscle.
Therefore, the ATP turnover rate for each 16 contraction
periods (25.6 s) was calculated from the changes in
muscle metabolites; ATP turnover rate (mmol . kg dry
muscle-l l s-l) = lLj(A[lactate]) + A[PCr] + [Z(A[ATP])
- A[ADP]/25.6 s.The small amount of ATP production
or utilization associated with the accumulation of addi-
tional metabolites, such as pyruvate and glyc-3-P, were
neglected as they represented ~2 of the ATP turnover
rate in all cases.
The calculation of ATP turnover rate also makes no
allowance for the presence of 0, stored or trapped in the
occluded muscle. Harris et al. (10) suggested that the
upper limit for this store was 2 mmol/kg dry muscle,
enough to produce 12 mmol ATP/kg dry muscle. Assum-
ing that all of the stored OZ was used in the first 15 s of
contraction, the aerobic ATP production would amount
to 0.8 mmol. kg dry muscle-
l
s-l during this period.
Subsequent contractions would then rely purely on an-
aerobic metabolism.
The ATP turnover rate during the initial 16 contrac-
tions was 6.12 t 0.56 mmol. kg dry muscle- l s-l with
58 of the ATP derived from glycolysis, 40 from PCr
degradation, and 2 from partial utilization of the ATP
store (Fig. 3). During contractions 17-32 and 33-48 the
respective ATP turnover rates decreased to 2.56 t 0.76
and 2.19 t 0.83 mmol l kg dry muscle- es-l, representing
42 and 36 of the initial rate. Glycolysis produced 83-
90 of the total ATP during this time as the PCr store
was largely depleted and decreases in the ATP content
contributed minimal amounts of energy (Fig. 3). The
ATP turnover rate during the final 16 contractions was
extremely low (0.64 t 0.77 mmol. kg dry muscle-o s-l)
and represented only 11 of the initial rate. Glycolysis
was again responsible for the majority (92 ) of the
produced ATP.
DISCUSSION
This study estimated the ATP turnover rate in elec-
trically stimulated skeletal muscle under anaerobic con-
ditions. The stimulation period was extended to 100 s of
contraction time and the contribution of the glycolytic
system to total ATP production was determined.
Electrical stimulation was used to induce muscular
contraction, since a constant contraction stimulus could
be delivered to the muscles for a predetermined length
of time, independent of volitional effort. This minimized
the possibility of central fatigue-limiting muscular per-
formance and metabolism as may occur in normal exer-
cise tasks of maximal intensity where the subject vol-