Clinical Science (2002) 102, 531–539 (Printed in Great Britain)
Muscle function during repetitive moderateintensity muscle contractions in myoadenylate
deaminase-deficient Dutch subjects
C. J. DE RUITER*, A. M. MAY*, B. G. M. VAN ENGELEN†, R. A. WEVERS†,
G. C. STEENBERGEN-SPANJERS† and A. DE HAAN*
*Institute for Fundamental and Clinical Human Movement Sciences, Vrije Universiteit, Van der Boechorststraat 9, 1081 BT
Amsterdam, The Netherlands, and †Institute for Fundamental and Clinical Human Movement Sciences, Department of
Neurology, University Medical Centre Nijmegen, 6500 HB Nijmegen, The Netherlands
A
B
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We investigated whether the capacity for repetitive submaximal muscle contraction was
reduced in a group of subjects (n l 8) with a primary deficiency of myoadenylate deaminase
(MAD). Quadriceps femoris muscle fatigue was evaluated using voluntary and electrically
stimulated contractions during 20 min of repetitive voluntary isometric contractions at 40 % of
maximal force-generating capacity (MFGC). After 5 min of exercise, MFGC had declined
significantly to 70.6p4.1 % (meanpS.E.M.) and 87.2p1.6 % of baseline values in MAD-deficient
and sedentary control subjects (n l 8) respectively (P l 0.002 between groups). After 5 min of
exercise, the half-relaxation time had increased significantly to 113.4p6.1 % of basline in MADdeficient muscle, but had decreased significantly to 94.1p1.3 % in control subjects (P l 0.003
between groups). All control subjects completed the 20-min exercise test. Five of the MADdeficient subjects had to stop exercising due to early muscle fatigue ; however, three of the
MAD-deficient subjects were able to complete the 20-min exercise test. In conclusion, although
the capacity for repetitive submaximal isometric muscle contractions for the group of MADdeficient subjects was significantly decreased, it remains uncertain whether MAD deficiency is
the sole cause of pronounced muscle fatigue.
INTRODUCTION
Myoadenylate deaminase (MAD ; EC 3.5.4.6) is the
muscle-specific isoform of adenylate deaminase. During
exercise, MAD catalyses the deamination of AMP into
IMP and ammonia. IMP can subsequently be re-aminated
to AMP in two steps involving the other enzymes of the
purine nucleotide cycle : adenylosuccinate synthase and
adenylosuccinate lyase [1].
In cases of MAD deficiency [2], IMP formation is
strongly diminished, and consequently the purine nucleotide cycle is disrupted, which affects muscle metabolism during exercise in MAD-deficient subjects [3,4].
MAD deficiency has a relatively high incidence (1.5–
2.0 %) in the general population [5–7]. In the vast
majority of cases, MAD deficiency is caused by a
common homozygous point mutation (C34T) in the
second exon of the gene. The mutation causes a premature
termination codon, resulting in a severely truncated
protein [5,6].
In many of the reported cases of MAD deficiency,
subjects suffer from exercise intolerance, increased fatiguability, exercise-induced muscle pains, stiffness, cramping and delayed recovery of muscle strength [2,3,8,9].
However, the clinical relevance of MAD deficiency can
be questioned, since many MAD-deficient subjects are
symptom free [6,9–11] and therefore MAD could show
harmless genetic variation [6]. Moreover, during high-
Key words : electrical stimulation, fatigue, MAD deficiency, skeletal muscle.
Abbreviations : MAD, myoadenylate deaminase ; MFGC, maximal force-generating capacity ; MVC, maximal voluntary
contraction.
Correspondence : Dr C. J. de Ruiter (e-mail CIJIdeIRuiter!fbw.vu.nl).
# 2002 The Biochemical Society and the Medical Research Society
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C. J. de Ruiter and others
Table 1
Subject characteristics
In addition to the activity levels listed, all subjects also cycled and/or walked for less than 1 h per week.
Symptoms
Subject
1
2
3
4
5
6
7
8
9
10*
11*
12†
13
14
15
16
Control/MAD-deficient
Age (years)
Female/male
Activity level (h/week) (activity)
Early fatigue
Cramping muscle
Muscle weakness
Control
Control
Control
Control
Control
Control
Control
Control
27
30
33
37
61
37
45
52
m
m
m
m
m
f
f
f
0
0
0
0
2 (walking)
1 (field hockey)
1 (tennis)
1 (basketball)
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
MAD-deficient
MAD-deficient
MAD-deficient
MAD-deficient
MAD-deficient
MAD-deficient
MAD-deficient
MAD-deficient
28
28
30
39
63
33
47
51
m
m
m
m
m
f
f
f
0
4 (soccer)
2 (aerobics)
0
1 (swimming)
0
2 (table tennis)
0
j
k
j
j
j
j
j
j
j
k
j
j
k
j
k
j
j
k
j
j
j
j
j
j
* Subject nos. 10 and 11 are brothers.
† This subject also participated in a previous study of ours [11].
intensity maximal exercise, when MAD becomes very
active in healthy muscle [12,13], several studies did not
find a reduction of performance in MAD-deficient
subjects [4,11,14].
On the other hand, in addition to the exercise-related
symptoms reported in many studies [3,5,15,16], reduced
performance has been found during cycle ergometer
exercise of rather moderate intensity [3,16]. Indeed, the
symptoms reported by MAD-deficient subjects often
develop during daily tasks of only moderate, submaximal
intensity [11]. Moreover, in isolated rat muscle, disruption of the purine nucleotide cycle reduced force
output during repetitive submaximal contractions [17,18].
Therefore the purpose of the present study was to
investigate whether the capacity for repetitive submaximal muscle contractions is reduced in MADdeficient subjects.
Voluntary exercise has the limitation that force production depends to a large degree on the subject’s
motivation, and consequently the extent to which the
decrease in power\force output is of central origin is
uncertain. This is always a point of concern, but especially
when working with untrained subjects and patients
during fatiguing exercise. In the present study electrical
stimulation was applied to assess muscle function, in
order to bypass the central nervous system. Four 5-min
blocks of repetitive voluntary isometric contractions of
the quadriceps muscle at 40 % of the muscle’s maximal
force-generating capacity (MFGC) (see the Methods
# 2002 The Biochemical Society and the Medical Research Society
section) were interposed with electrically stimulated contractions and a maximal voluntary contraction (MVC)
with superimposition of electrical stimulation. For
three of the eight MAD-deficient subjects, muscle fatigue
and changes in contractile characteristics during and
following the 20-min exercise test were similar to those
of the control subjects. The other five MAD-deficient
subjects could not complete the exercise protocol due
to early exhaustion. Possible reasons for the dichotomy
within the exercise response of MAD-deficient group
are discussed.
METHODS
Subjects
Eight subjects with primary MAD deficiency and eight
healthy untrained subjects matched for gender and age
took part in the experiments (Table 1). All but one (no. 10
in Table 1) of the MAD-deficient subjects experienced
early muscle fatigue in daily life, but besides the deficiency they had no other (known) disorders. All subjects
were interviewed to establish the amount of daily activity,
specifically hours per week spent walking, swimming and
cycling (which is very popular in the Netherlands). The
approval of the Ethical Committee of the Vrije Universiteit was obtained, and all subjects provided informed
consent.
Muscle function and myoadenylate deaminase deficiency
Diagnosis of MAD deficiency was established in all
eight subjects by an ischaemic forearm test [19] and by
immunohistochemical testing using muscle material.
Moreover, genomic DNA was extracted from blood
samples from deficient and control subjects to demonstrate respectively the presence and absence of homozygosity for the C34T mutation at the DNA level [6].
Following exercise testing, one of the control subjects
was found to be a heterozygote. However, all data from
this subject were within 1 S.D. of the mean control group
values. Moreover, the subject was symptom-free, and
therefore the data from this subject were included in the
control group.
Experimental set-up
Isometric force was recorded during voluntary and
electrically evoked contractions of the quadriceps muscle
of the left leg. Subjects sat in a specially designed chair,
which was adjustable for upper and lower leg length,
with 90m hip and knee angles. Straps restrained hip and
shoulders. The anterior lower leg was strapped to a force
transducer, which was mounted to the rigid aluminium
frame of the chair. The distance between the knee axis
and the transducer was kept constant (28.5 cm) for all
subjects. The knee extension force was displayed on a
screen, digitized (1000 Hz) and stored on disk.
Electrical stimulation was used to evaluate fatigueinduced changes in muscle function and to obtain a
measure for any central component of fatigue. The
quadriceps femoris muscle was stimulated transcutaneously with rectangular pulses of 200 µs using a pair of
80 mmi130 mm self-adhesive electrodes (Schwamedico, 283100 ; Nederland B.V.). The anode was placed
over the proximal anterior thigh just distal to the inguinal
ligament, and the cathode was placed with its distal edge
5 cm proximal to the superior border of the patella. The
electrical stimulation was applied from a constant-current
stimulator (model DS7 ; Digitimer Ltd, Welwyn Garden
City, Herts., U.K.).
Figure 1
Protocol
Baseline values
The MVC force of the quadriceps femoris was determined as the highest value from three maximal
voluntary knee extensions each lasting approx. 2 s, with
2 min of rest between. Subsequently the subjects were
familiarized with the electrical stimulation, and the
stimulation current was increased until 50 % of MVC
force was elicited during a tetanic pulse train (200 Hz) of
700 ms duration. This was followed by the determination
of the MFGC : a brief tetanic train (100 ms, 200 Hz) was
applied to the resting muscle, followed by superimposition of the tetanic train on an MVC (see Figure 2).
This was repeated three times, with 2 min periods of rest
in between. The amount of extra electrically evoked force
on top of the highest MVC was used to calculate the
MFGC of the muscle ([20] ; see also Data analysis
section). Subsequently the stimulation current was adjusted (increased slightly) such that, during a 700 ms
tetanic train (200 Hz), force reached 50 % of the muscle’s
MFGC. Thus, during electrical stimulation in each
subject, 50 % of the total quadriceps muscle mass was
maximally activated. In addition to the 700 ms 200 Hz
tetanus, the muscle was also activated with 10, 20, 50, 100
and 150 Hz (all for 700 ms, with 1 s in between), applied
in random order.
Exercise and recovery
After a 3 min rest, the subjects started to exercise (Figure
1, t l 0). Because we wanted the exercise protocol to be
selective, we aimed for a test that would be difficult to
complete for subjects with a sedentary lifestyle. During
pilot experiments, healthy subjects did not develop
noticeable fatigue during repetitive isometric contractions performed at 30 % of MFGC (1.5 s on, 1 s off). At
40 % of MFGC there was loss of force, but all subjects
could exercise for over 20 min. In contrast, at 50 % of
MFGC some subjects became exhausted during the first
Exercise protocol
Voluntary exercise started (following 3 min of rest) at t l 0, and consisted of four 5 min-blocks of repetitive isometric contractions of the quadriceps muscle at 40 %
of MFGC. These blocks were followed by a recovery period (t l 20–35 min). At 3 min before exercise (left arrow) and at t l 5, 10, 15, 20, 25, 30 and 35 min,
MFGC and tetanic force (700 ms at 200 Hz) were obtained (see arrows). At 3 min before exercise and at the end of exercise (t l 20 min), in addition to the 200 Hz
tetanic stimulation, the muscle was also activated with 10, 20, 50, 100 and 150 Hz (all 700 ms), applied in random order.
# 2002 The Biochemical Society and the Medical Research Society
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C. J. de Ruiter and others
on an MVC. The MFGC of the muscle was calculated
using the following formula :
MFGC l o1\[1k(ckb)\a]q:b
Figure 2
MFGC of the quadriceps muscle
Shown is the force in response to brief electrical activation (100 ms train and
200 Hz pulse frequency) applied to the fully relaxed quadriceps femoris muscle
(arrow at t l 500 ms) and superimposed on the force plateau during an MVC
(arrow at t l 2760 ms). The MFGC of the muscle was calculated using the
formula : MFGC l o1/[1k(ckb)/a]q:b, where ckb is the extra
electrically induced force on top of the MVC (b) and a is the peak force evoked
by the 100 ms pulse train applied on the fully relaxed muscle.
5–10 min of exercise. For these reasons, exercise intensity
was set at 40 % of MFGC during the main experiments,
and subjects produced isometric contractions (1.5 s, with
1 s of rest in between) during four consecutive exercise
blocks of 5 min duration each (Figure 1). The subjects
were guided by a sound signal (1.5 on, 1 s off), and 40 %
of MFGC was marked as a target force on an oscilloscope
placed in front of them.
The four exercise blocks were interposed (arrows in
Figure 1) with an MVC and (superimposed) electrically
stimulated contractions (200 Hz) to determine MFGC,
maximal tetanic force, the maximal rate of force development and half-relaxation time (see Data analysis
section). A 200 Hz stimulation frequency was used,
because pilot experiments showed that 200 Hz resulted
in slightly higher force values during recovery compared
with lower frequencies.
In cases where it became obviously difficult for a
subject to continue the exercise at the required intensity,
the subject was encouraged verbally to complete the
current exercise block. Immediately following the last
completed exercise block (usually at t l 20 min), MFGC
was assessed and, in addition to the 700 ms, 200 Hz
tetanus, the muscle was also activated with 10, 20, 50, 100
and 150 Hz (all for 700 ms, with 1 s in between), applied
in random order.
Recovery of MFGC and tetanic force (700 ms, 200 Hz)
were examined at 5, 10 and 15 min following the last
completed exercise block (Figure 1, t l 25, 30 and
35 min).
Data analysis
Figure 2 shows an example of the procedure used to
calculate MFGC. A brief (100 ms) tetanic train (200 Hz)
was applied to the fully relaxed muscle and superimposed
# 2002 The Biochemical Society and the Medical Research Society
where ckb is the extra electrically induced force on top
of the MVC (b) and a is the peak force evoked by the
100 ms pulse train applied on the fully relaxed muscle.
Half-relaxation time from the 700 ms contractions
(200 Hz) was determined as the time taken for force to
fall 50 % from its maximum value at the end of stimulation.
The maximal rate of force rise was calculated from the
positive filtered force signal of the 700 ms pulse (200 Hz)
train [20]. It has been defined as the maximum velocity of
force development, and was expressed as a percentage
of the maximal force reached within the same contraction.
Statistical analysis
Unless indicated otherwise, the results are presented as
meanspS.E.M. A non-parametric Wilcoxon Signed
Ranks test was used to test for statistical differences
(P 0.05) between contractile characteristics of unfatigued and fatigued muscle. The non-parametric Mann–
Whitney U test was applied to test for significant
differences (P 0.05) between the groups. Pearson’s
correlation coefficient was calculated to establish the
significance (P 0.05) of a correlation.
RESULTS
Baseline values
There were no differences between the groups with
regard to the MFGC of the quadriceps muscles, the
percentage of the MFGC that could be recruited by
the subjects during an MVC, the half-relaxation time
or the maximal rate of force rise (Table 2).
Fatigue
As was expected from pilot experiments with untrained
subjects, all control subjects were able to complete the
20-min fatigue test without problems. They did, however,
indicate that it was hard work, particularly during the
first two exercise blocks (0–10 min). In contrast, only
three of the MAD-deficient subjects (nos. 10, 13 and 14 in
Table 1) completed the full 20 min of exercise. The other
five MAD-deficient subjects had to stop earlier. Due
to muscle weakness and aches, they were no longer able to
deliver the required force output : subject no. 12 managed
to finish the second exercise block (10 min), whereas
subjects 9, 11, 15 and 16 could barely complete the first
5 min of exercise, and only succeeded under strong verbal
encouragement. Consequently a complete data set for all
16 subjects was only available following the first 5-min
exercise block, and therefore these data were used for the
Muscle function and myoadenylate deaminase deficiency
Table 2
Baseline values for control and MAD-deficient subjects
Fmax is defined as the maximal force reached within the same contraction. Data are meanspS.E.M. P values indicate that
there are no significant differences between the groups.
Figure 3
Parameter
Control (n l 8)
MAD-deficient (n l 8)
P value
MFGC (N)
MVC (% of MFGC)
Half-relaxation time (ms)
Max. rate of force rise (%Fmax:ms−1)
660.4p69.7
90.4p3.7
122.3p4.9
1.4p0.1
643.0p72.2
92.6p1.9
113.9p3.0
1.5p0.1
1.00
0.67
0.14
0.40
Contractile properties of quadriceps muscle during fatigue and recovery
Shown are MFGC (A), tetanic force (B), half-relaxation time (C) and maximal rate of force rise (D) of the quadriceps femoris muscle, expressed as a percentage
(meanspS.E.M.) of baseline values (l 100 %) and as a function of time, for control subjects ($) and for MAD-deficient subjects who exercised for the full 20 min
(# ; n l 3), 10 min (= ; n l 1) or 5 min ( ; n l 4). The vertical dashed line at (2) 0 min, which denotes the start of the recovery period (0 min), separates
the exercise period on the left from the recovery period on the right. Recovery was studied at 5, 10 and 15 min after the end of the exercise protocol. *Denotes a
significant difference (P 0n05) between the control (n l 8) and MAD-deficient (n l 8) subjects.
statistical analyses. It should be noted that, despite the
severe fatigue experienced by five of the MAD-deficient
subjects during the first 5 min of exercise, they delivered
a force and a force–time integral comparable with that of
the other subjects. The mean force–time integrals during
the first 5 min of exercise were 345p44 Ns and 365p
35 Ns for the MAD-deficient and control groups respectively, which was not different between the groups
(P l 0.67).
deficient subjects who had to give up after this first
exercise block had the lowest MFGC values (61.8p3.9%),
followed by the MAD-deficient subject who had to stop
after 10 min (71.8 %). For the three MAD-deficient
subjects that were able to exercise for the full 20 min,
MFGC after 5 min of exercise (81.9p0.8 %) was within
the range of the control subjects (87.2p1.6 %). However,
at 10 and 15 min of exercise, MFGC for these three
MAD-deficient subjects was lower than the lowest value
of the control subjects (Figure 3A).
Changes in MFGC
Following the first 5 min of exercise, MFGC had declined
significantly to 70.6p4.1 % and 87.2p1.6 % of baseline
values in the MAD-deficient and control subjects respectively (Figure 3A). At 5 min MFGC was different
between the groups (P l 0.002). The four MAD-
Force response to 200 Hz stimulus train
Similar to the baseline situation (Table 2), and despite the
greater fatigue in most of the MAD-deficient subjects,
subjects in both groups were able to voluntarily activate
their muscles to a comparable extent (P l 0.34 between
# 2002 The Biochemical Society and the Medical Research Society
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C. J. de Ruiter and others
groups) after 5 min of exercise. During an MVC, the
MAD-deficient and control subjects generated 85.2p
3.4 % and 88.2p3.8 % of their muscles’ MFGC respectively. To our surprise, maximal tetanic force was not
a reliable parameter for monitoring changes in force
output during the exercise protocol. The variation in
maximal tetanic force (Figure 3B) was greater than the
variation in MFGC (Figure 3A). After the first exercise
block, tetanic force had declined significantly to 76.5p
6.0 % and 77.8p4.2 % in the MAD-deficient and control
groups respectively ; the decline was similar in the
two groups (P l 1.00). In addition, and in contrast with
MFGC (Figure 3A), maximal tetanic force could not be
used to separate the MAD-deficient subjects who completed the test from those that failed to complete the full
20-min test (Figure 3B). Moreover, and equally unexpectedly, there was no correlation between the decreases
in the muscles’ MFGC and electrically induced force
responses during the 700 ms tetanic contraction (r l
0.23, P l 0.39).
Contractile speed
Half-relaxation time after the first 5 min of the fatigue
protocol was significantly different between the control
and MAD-deficient subjects (P l 0.003 ; Figure 3C).
Half-relaxation time decreased significantly to 94.1p
1.3 % of the baseline value in the control group. In
contrast, half-relaxation time increased significantly (to
113.4p6.1 %) in the MAD-deficient group. However,
similar to the data on MFGC, there was a differentiated
response within the MAD-deficient group. Half-relaxation time increased significantly to 121.4p7.4 % for the
five subjects who did not complete the test, whereas that
for the three MAD-deficient subjects who exercised for
the full 20 min had not changed after the first 5 min of
exercise (100.2p5.2 % ; Figure 3C).
Following 5 min of exercise, the maximal rate of force
rise had increased significantly in both groups, to
114.0p7.2 % and 124.3p4.6 % of the baseline value in
the MAD-deficient and control subjects respectively
(P l 0.172 between groups). The maximal rate of force
rise for the three MAD-deficient subjects who exercised
for the full 20 min continued to increase during the
remainder of the test in a pattern similar to that of
the control subjects (Figure 3D).
Recovery
There was incomplete recovery of MFGC (Figure 3A)
and tetanic force (Figure 3B). After 5 min of recovery, the
subjects who had exercised for the full 20 min (MADdeficient and control subjects taken together ; n l 11) had
regained only 10.2p12.2 % and 26.1p4.3 % of the lost
(l 100 %) MFGC and tetanic force respectively. The five
subjects who could not complete the full test had regained
50.2p8.5 % and 69.4p56.8 % of the lost MFGC and
# 2002 The Biochemical Society and the Medical Research Society
tetanic force respectively. These five MAD-deficient
subjects tended to recover faster than the other 11 subjects (P l 0.04 and P l 0.07 for MFGC and tetanic
force respectively).
In the five MAD-deficient subjects who exercised for
only 5 min and displayed an increase in half-relaxation
time during exercise, the half-relaxation time decreased
to values similar to those of the other subjects during
recovery (Figure 3C). In all subjects, half-relaxation time
was significantly lower after 15 min of recovery than
baseline values. The maximal rate of force rise was still
increased at 15 min following exercise in those subjects
who had increased values at the end of exercise
(Figure 3D).
DISCUSSION
Whether MAD deficiency is a true disease [21] or just a
harmless genetic variant [6] remains unclear. The present
study used electrical stimulation to calculate the MFGC
of the quadriceps muscle, as well as half-relaxation times.
The changes in these muscle parameters during repetitive
submaximal isometric contractions were significantly
greater in MAD-deficient subjects than control subjects.
Therefore the present study is the first to show that the
exercise intolerance reported by primary MAD-deficient
subjects is due to fatigue at the level of the muscle, and is
not associated with decreased drive from the central
nervous system. However, the present study also shows
a diverse response among MAD-deficient subjects.
The MAD-deficient subjects in the present study had
the primary (inherited) form of the defect, and were
shown to be homozygous for the C34T mutation [6].
There is also a secondary (acquired) form of the defect,
which is associated with other neuromuscular diseases
[21], but in the present study we only included MADdeficient subjects with no other known diseases or health
problems. The control subjects were selected from the
very sedentary part of the population and carefully
matched for age and gender. Therefore we are confident
that the greater fatigue of the MAD-deficient subjects in
the present study is not the consequence of a lower
training status relative to that of the control subjects.
During maximal all-out exercise, MAD becomes highly activated in healthy muscle [12,13]. Therefore it is
rather surprising that, during maximal all-out exercise,
power and work output are not reduced in the absence of
functional MAD [4,11,14]. In the present study the
subjects exercised for a longer duration, and capacity for
repetitive submaximal isometric muscle contractions was
found to be lower in MAD-deficient subjects. It should
be noted that the only MAD-deficient subject (no. 12)
who previously during maximal short-term exercise [11]
had a fully normal response, in the present study suffered
from early fatigue. The present results are in line with the
Muscle function and myoadenylate deaminase deficiency
limited data indicating reduced work output in MADdeficient patients during submaximal exercise [3,16].
One has to be very cautious in applying the present
results to all subjects with MAD deficiency. It is
important to realize that all the MAD-deficient subjects
in the present study (except no. 10) were patients who
sought medical attention for their problems, whereas the
majority of MAD-deficient subjects in the general
population appear to be symptom-free [6]. Therefore it
may not be surprising that the symptom-free MADdeficient subject (no. 10) indeed managed to complete the
exercise test without difficulty, although he suffered
from muscle soreness for 6 days following the test.
Despite the fact that, as a group, the MAD-deficient
subjects clearly had a reduced capacity for repetitive
submaximal muscle contractions, it remains uncertain
whether MAD deficiency is the primary cause of the
problems reported by our subjects, since three of them
did not fatigue. It may be suggested that the early fatigue
in five of the subjects could simply be due to a lower
training status and\or a greater percentage of quickly
fatiguing fibres compared with the control subjects.
However, activity levels in terms of hours per week of
walking and cycling, in addition to other locomotor
activities, were very low in all of our subjects (Table 1).
Moreover, muscle biopsies were taken from five of the
MAD-deficient subjects, and the percentage of type II
fibres varied between 43 % and 66 %, which is within the
normal range. Therefore differences in training status
and\or fibre-type composition between the groups are
highly unlikely to account for the results. The large
variation within the MAD-deficient group suggests that
there were mechanisms in the muscles of three of the
eight subjects which compensated for the potentially
increased fatiguability [21].
The presented data show that, during exercise, five of
the MAD-deficient subjects failed to reach a ‘ steady
state ’ and that aerobic metabolism was insufficient to
supply the energy for muscle contraction. Consequently,
anaerobic glycolysis and phosphocreatine breakdown
were probably increased, and the slowing of muscle
relaxation and the loss of force most probably resulted
from a reduced intramuscular pH and increased levels of
inorganic phosphate [22]. Moreover, since during the
first 5 min of exercise, relative exercise intensity increased
from 40 % to about 65 % of their muscles’ MFGC, these
five subjects were probably forced to recruit additional
(less fatigue-resistant) motor units to maintain the required force production.
The fast recovery of half-relaxation time, tetanic force
and MFGC in the early-fatiguing MAD-deficient subjects compared with the other subjects also indicates that
the fatigue in the early-fatiguing subjects was of metabolic origin, because recovery from short-term fatiguing
exercise usually occurs within minutes following exercise,
and in parallel with phosphocreatine re-synthesis [23,24].
Nevertheless, recovery was incomplete in all subjects,
which has been found previously following repetitive
submaximal voluntary isometric knee extensor exercise
[25]. Moreover, Saugen et al. [25] found a clear dissociation between metabolic and mechanical changes
during and following repetitive voluntary submaximal
contractions. They suggested this kind of long-lasting
fatigue to be associated with an impairment of excitation–
contraction coupling, which is consistent with the greater
fall in stimulated force at low compared with high
stimulation frequencies during fatigue in this previous
study [25] and in the present study (results not shown).
Such low-frequency fatigue is probably caused by lower
calcium release by the sarcoplasmic reticulum per action
potential [26].
An unexpected finding in the present study was that
tetanic force (Figure 3B) decreased to a greater extent
than MFGC (Figure 3A) in the muscles of control
subjects. The most likely explanation is that, in most
subjects, the muscle mass activated by percutaneous
electrical stimulation decreased as the exercise progressed. Moreover, in both groups the variation in tetanic
force was much greater than the variation in MFGC, and
there was no correlation between changes in tetanic force
and in MFGC. We have no explanation for the possible
decline in activated muscle mass, but it may have been
caused by some stimulation- and\or exercise-induced
changes in the fluid under the skin and\or around the
muscle. Interestingly, Saugen et al. [25] also found greater
decreases in stimulated compared with voluntarily exerted force (their Figure 1), but they did not comment on
those findings.
In subjects that did not suffer from early exhaustion,
the half-relaxation time decreased (Figure 3C) and the
maximal rate of force rise increased (Figure 3D), illustrating an increase in isometric contractile speed
during exercise. This is in agreement with previous
findings during similar forms of exercise [27], and may be
caused by an increase in muscle temperature [28].
The present results suggest that, somehow, aerobic
energy provision was limited in our MAD-deficient
subjects, particularly during the first 5–10 min of exercise. What might have caused the reduced capacity for
repetitive submaximal contractions of MAD-deficient
muscle, if we assume that the impaired muscle function in
our subjects was indeed due to the deficiency ? MAD is
an enzyme of the purine nucleotide cycle, and an
important role of this cycle could be the provision of
tricarboxylic acid cycle intermediates, which would be
important for optimal aerobic energy production [1,18,
29,30]. Therefore it could be hypothesized that failure to
provide sufficient tricarboxylic acid cycle intermediates
in MAD-deficient muscle, particularly during the first
5–10 min of exercise when levels of these intermediates
have been found to increase substantially in healthy
muscle [31–33], would lead to a shift of energy pro# 2002 The Biochemical Society and the Medical Research Society
537
538
C. J. de Ruiter and others
duction towards anaerobic sources and early fatigue.
However, it is questionable whether the purine nucleotide cycle has an important anaplerotic function. First,
adenylosuccinate synthase and adenylosuccinate lyase
activities are very low compared with MAD activity [34],
and consequently high rates of IMP re-amination with
concomitant fumarate formation are not expected.
Secondly, the anaplerotic function of the purine nucleotide
cycle was found to be unimportant during the first
5–10 min of knee extensor exercise [32,33]. Instead, the
alanine aminotransferase reaction seems the most important source of tricarboxylic acid cycle intermediates
during the first 10 min of moderate-intensity exercise
[32,33]. However, since we did not measure muscle
metabolites, the exact mechanism behind the reduced
capacity for repetitive submaximal muscle contractions
in our MAD-deficient subjects remains to be established.
In conclusion, during repetitive submaximal voluntary
isometric contractions of the knee extensors, muscle
fatigue was significantly greater in a group of eight
MAD-deficient subjects compared with sedentary control subjects. However, similar to the control subjects,
three of the MAD-deficient subjects did not fatigue. This
suggests either that these three subjects were able to
compensate for the deficiency metabolically, or that
MAD deficiency was not the, or not the only, underlying
cause of the reduced capacity for repetitive submaximal
muscle contractions in the other five MAD-deficient
subjects. These alternatives would explain why many of
MAD-deficient subjects in the general population are
symptom-free.
8
9
10
11
12
13
14
15
16
17
18
19
REFERENCES
1
2
3
4
5
6
7
Lowenstein, J. M. (1972) Ammonia production in muscle
and other tissues : the purine nucleotide cycle. Physiol.
Rev. 52, 382–414
Fishbein, W. N. B., Armbrustmacher, V. W. and Griffin,
J. L. (1978) Myoadenylate deaminase deficiency : a new
disease of muscle. Science 200, 545–548
Sabina, R. L., Swain, J. L., Olanow, C. W. et al. (1984)
Myoadenylate deaminase deficiency. Functional and
metabolic abnormalities associated with disruption of the
purine nucleotide cycle. J. Clin. Invest. 73, 720–730
Sinkeler, S. P. T., Wevers, R. A., Binkhorst, R. A.,
Joosten, E. M. G., Coerwinkel, M. M. and Oei, T. L.
(1987) AMP deaminase deficiency : study of the human
skeletal muscle purine metabolism during ischaemic
isometric exercise. Clin. Sci. 72, 475–482
Morisaki, T., Gross, M., Morisaki, H., Pongratz, D.,
Zo$ llner, N. and Holmes, E. W. (1992) Molecular basis of
AMP deaminase deficiency in skeletal muscle. Proc. Natl.
Acad. Sci. U.S.A. 89, 6457–6461
Verzijl, H. T., van Engelen, B. G., Luyten, J. A. et al.
(1998) Genetic characteristics of myoadenylate deaminase
deficiency. Ann. Neurol. 44, 140–143
Norman, B., Mahnke-Zizelman, D. K., Vallis, A. and
Sabina, R. L. (1998) Genetic and other determinants of
AMP deaminase activity in healthy adult skeletal muscle.
J. Appl. Physiol. 85, 1273–1278
# 2002 The Biochemical Society and the Medical Research Society
20
21
22
23
24
25
26
Sabina, R. L., Swain, J. L., Patten, B. M., Ashizawa, T.,
O’Brien, W. E. and Holmes, E. W. (1980) Disruption of
the purine nucleotide cycle. A potential explanation for
muscle dysfunction in myoadenylate deaminase
deficiency. J. Clin. Invest. 66, 1419–1423
Sinkeler, S. P., Joosten, E. M., Wevers, R. A. et al. (1988)
Myoadenylate deaminase deficiency : a clinical, genetic,
and biochemical study in nine families. Muscle Nerve 11,
312–317
Shumate, J. B., Katnik, R., Ruiz, M., Kaiser, K., Brooke,
M. H. and Carroll, J. E. (1979) Myoadenylate deaminase
deficiency. Muscle Nerve 2, 213–216
de Ruiter, C. J., van, Engelen, B. G. M., Wevers, R. A.
and de Haan, A. (2000) Muscle function during fatigue in
myoadenylate deaminase-deficient Dutch subjects. Clin.
Sci. 98, 579–585
Dudley, G. A. and Terjung, R. L. (1985) Influence of
aerobic metabolism on IMP accumulation in fast-twitch
muscle. Am. J. Physiol. 248, C37–C42
de Haan, A. and Koudijs, J. C. (1994) A linear
relationship between ATP degradation and fatigue during
high-intensity dynamic exercise in rat skeletal muscle.
Exp. Physiol. 79, 865–868
Norman, B., Sabina, R. L., Esbjo$ rnsson-Liljedahl, M. and
Jansson, E. (1998) ATP metabolism during sprint exercise
in subjects with different genotype for AMP deaminase in
skeletal muscle. J. Physiol. (Cambridge, U.K.) 506P,
PC32
Patten, B. M. (1982) Beneficial effect of D-ribose in
patient with myoadenylate deaminase deficiency. Lancet
i, 1071
Zo$ llner, N., Reiter, S., Gross, M. et al. (1986)
Myoadenylate deaminase deficiency : successful
symptomatic therapy by high dose oral administration of
ribose. Klin. Wochenschr. 64, 1281–1290
Swain, J. L., Hines, J. J., Sabina, R. L., Harbury, O. L.
and Holmes, E. W. (1984) Disruption of the purine
nucleotide cycle by inhibition of adenylosuccinate lyase
produces skeletal muscle dysfunction. J. Clin. Invest. 74,
1422–1427
Flanagan, W. F., Holmes, E. W., Sabina, R. L. and Swain,
J. L. (1986) Importance of purine nucleotide cycle to
energy production in skeletal muscle. Am. J. Physiol. 251,
C795–C802
Sinkeler, S. P., Wevers, R. A., Joosten, E. M. et al. (1986)
Improvement of screening in exertional myalgia with a
standardized ischemic forearm test. Muscle Nerve 9,
731–737
de Haan, A., de Ruiter, C. J., van der Woude, L. H. and
Jongen, P. J. (2000) Contractile properties and fatigue of
quadriceps muscles in multiple sclerosis. Muscle Nerve
23, 1534–1541
Sabina, R. L. and Holmes, E. W. (2000) Myoadenylate
deaminase deficiency. In Myoadenylate Deaminase
Deficiency, vol. II (Scriver, C. R., Beaudet, A. L., Sly,
W. S. and Valle, M. D., eds), pp. 2627–2638, McGrawHill, New York
Cooke, R., Franks, K., Luciani, G. B. and Pate, E. (1988)
The inhibition of rabbit skeletal muscle contraction by
hydrogen ions and phosphate. J. Physiol. (Cambridge,
U.K.) 395, 77–97
Bogdanis, G. C., Nevill, M. E., Boobis, L. H., Lakomy,
H. K. A. and Nevill, A. M. (1995) Recovery of power
output and muscle metabolites following 30 s of maximal
sprint cycling in man. J. Physiol. (Cambridge, U.K.) 482,
467–480
de Ruiter, C. J., De Haan, A. and Sargeant, A. J. (1995)
Repeated force production and metabolites in two medial
gastrocnemius muscle compartments of the rat. J. Appl.
Physiol. 79, 1855–1861
Saugen, E., Vøllestad, N. K., Gibson, H., Martin, P. A.
and Edwards, R. H. (1997) Dissociation between
metabolic and contractile responses during intermittent
isometric exercise in man. Exp. Physiol. 82, 213–226
Westerblad, H., Duty, S. and Allen, D. G. (1993)
Intracellular calcium concentration during low-frequency
fatigue in isolated single fibers of mouse skeletal muscle.
J. Appl. Physiol. 75, 382–388
Muscle function and myoadenylate deaminase deficiency
27
Vøllestad, N. K., Sejersted, I. and Saugen, E. (1997)
Mechanical behavior of skeletal muscle during
intermittent voluntary isometric contractions in humans.
J. Appl. Physiol. 83, 1557–1565
28 de Ruiter, C. J., Jones, D. A., Sargeant, A. J. and de Haan,
A. (1999) Temperature effect on the rates of isometric
force development and relaxation in the fresh and
fatigued human adductor pollicis muscle. Exp. Physiol.
84, 1137–1150
29 Scislowski, P. W., Aleksandrowicz, Z. and Swierczynski,
J. (1982) Purine nucleotide cycle as a possible anaplerotic
process in rat skeletal muscle. Experientia 38, 1035–1037
30 Tullson, P. C., Arabadjis, P. G., Rundell, K. W. and
Terjung, R. L. (1996) IMP reamination to AMP in rat
skeletal muscle fiber types. Am. J. Physiol. 270,
C1067–C1074
31
Sahlin, K., Katz, A. and Broberg, S. (1990) Tricarboxylic
acid cycle intermediates in human mucle during
prolonged exercise. Am. J. Physiol. 259, C834–C841
32 van Hall, G., Saltin, B., van der Vusse, G. J., So$ derlund,
K. and Wagenmakers, A. J. M. (1995) Deamination of
amino acids as a source for ammonia production in
human skeletal muscle during prolonged exercise.
J. Physiol. (Cambridge, U.K.) 489, 251–261
33 Gibala, M. J., MacLean, D. A., Graham, T. E. and Saltin,
B. (1997) Anaplerotic processes in human skeletal muscle
during brief dynamic exercise. J. Physiol. (Cambridge,
U.K.) 502, 703–713
34 Manfredi, J. P. and Holmes, E. W. (1984) Control of the
purine nucleotide cycle in extracts of rat skeletal muscle :
effects of energy state and concentrations of cycle
intermediates. Arch. Biochem. Biophys. 233, 515–529
Received 11 June 2001/22 November 2001; accepted 31 January 2002
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