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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-