Departments of 1 Physiology and 2 Movement Sciences, Maastricht University, 6200 MD Maastricht, The Netherlands
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ABSTRACT |
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Creatine kinase (CK)
forms a small family of isoenzymes playing an important role in
maintaining the concentration of ATP and ADP in muscle cells. To
delineate the impact of a lack of CK activity, we studied contractile
performance during a single maximal tetanic contraction and during 12 repeated tetanic contractions of intact dorsal flexors of CK knockout
(CK/
) mice. To investigate the effect on ATP
regeneration, muscular high-energy phosphate content was determined at
rest, immediately after the contraction series, and after a 60-s
recovery period. Maximal torque of the dorsal flexors was significantly
lower in CK
/
mice than in wild-type animals, i.e.,
23.7 ± 5.1 and 33.3 ± 6.8 mN · m · g
1 wet wt, respectively. Lower
muscle ATP (20.1 ± 1.4 in CK
/
vs. 28.0 ± 2.1 µmol/g dry wt in controls) and higher IMP (1.2 ± 0.5 in
CK
/
vs. 0.3 ± 0.1 µmol/g dry wt in controls)
levels at the onset of contraction may contribute to the declined
contractility in CK
/
mice. In contrast to wild-type
muscles, ATP levels could not be maintained during the series of 12 tetanic contractions of dorsal flexors of CK
/
mice and
dropped to 15.5 ± 2.4 µmol/g dry wt. The significant increase
in tissue IMP (2.4 ± 1.1 µmol/g dry wt) content after the
contraction series indicates that ATP regeneration through adenylate
kinase was not capable of fully compensating for the lack of CK. ATP
regeneration via the adenylate kinase pathway is a likely cause of
reduced basal adenine nucleotide levels in CK
/
mice.
skeletal muscle; creatine kinase deficiency; repeated contractions
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INTRODUCTION |
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CREATINE KINASE (CK) consists of a small family of isoenzymes catalyzing the transphosphorylation between phosphocreatine (PCr) and ATP (2, 19). Skeletal muscle contains two distinct isoforms, which are localized either in the cytosol (MM-CK) or at the interior site of the outer mitochondrial membrane (Mib-CK) (2, 19, 23, 24). Both CK isoforms play an important role in maintaining muscular ATP and ADP concentrations, e.g., during strenuous exercise (3). Previously, it was shown that ATP levels are lower in resting skeletal muscle of mice with combined deficiency in MM-CK and Mib-CK (14). Interestingly, the resting skeletal muscle PCr and creatine content was found to be comparable with that of age-matched control mice despite the virtual lack of CK activity in the former (4, 14). Moreover, the resting inosine monophosphate (IMP) level was enhanced in CK-deficient mice, which strongly suggests a disturbance in ATP regeneration (14).
Skeletal muscle of MM-CK- and Mib-CK-deficient mice shows
impaired contractile properties, which are marked by diminished tetanic
force output and prolonged relaxation time (8, 14). The
relationship between depressed contractility and utilization of
high-energy phosphates in CK-deficient mice, however, has barely been
investigated. In the present study, we explored the hypothesis that
muscles lacking both CK isoforms are unable to properly handle their
high-energy phosphate stores, which results in an impaired muscular
functioning. To test this hypothesis, contractile performance during a
single tetanic contraction of dorsal flexors of CK/
mice was determined while keeping the muscle in its natural
surroundings. Possible differences in contractile behavior during a
single tetanic contraction between CK
/
and wild-type
mice were related to differences in resting content of high-energy
phosphates and associated compounds in the muscle group studied.
Furthermore, the rate of decline in maximal torque during a series of 12 repetitive isometric tetanic contractions was monitored. By analyzing the tissue content of high-energy phosphates and related compounds in resting muscle immediately after the series of tetanic contractions and after a recovery period of 60 s, we investigated the effect of the lack of CK activity on ATP regeneration in muscles subjected to this high-intensity exercise series.
Traditionally, mechanical functioning of muscles of small rodents is determined under less physiological in vitro conditions, e.g., isolated muscles with impaired innervation and blood supply, which may influence the mechanical properties of the muscle under investigation. To study muscle performance under physiological conditions, we recently developed a sensitive and accurate mouse isometric dynamometer (6). This approach enabled us to assess maximal tetanic torque and temporal parameters, such as rise time and relaxation time, of intact muscles in anesthetized mice.
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METHODS |
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Animals.
Mice lacking both mitochondrial and cytosolic CK
(Mib-CK/
× MM-CK
/
,
denoted as CK
/
) (15) were used in the
present study. Male wild-type littermates (C57Bl/6 × 129/sv;
58-60 days old) served as controls. Halothane (Fluothane; Zeneca,
Ridderkerk, The Netherlands) was used as an anesthesia agent supplied
in O2 and N2O (3:1, 1.5-2.0%) via a facemask through a flowmeter system (4.0 l/min; Medec, Montvalle, NJ).
All experimental procedures were approved by the Institutional Animal
Care and Use Committee of the Maastricht University and complied with
the principles of laboratory animal care.
Surgical procedure. Mice were positioned on a thermostatic platform (38.5 ± 0.1°C). A small incision in the lateral part of the knee was made to expose the peroneal nerve. After the skin was locally depilated, a bipolar platinum hook electrode with an interpole distance of 0.8 mm was carefully attached to the nerve, and the dorsal flexor muscle complex, consisting of the tibialis anterior and extensor digitorum longus (EDL) muscles, was stimulated via a pulse generator (HSE 215/IZ, Freiburg, Germany). The position of the electrode was changed, if a current of >1.0 mA was needed, to obtain maximal tetanic muscle contraction. The electrode was fixed to the skin with cyanoacrylate glue to prevent electrode displacement during muscle contraction.
Experimental measurement setup. For mechanical analysis, the anesthetized mouse was fixed via the hip and foot to the measurement device. Dorsal flexor torque around the ankle joint was measured with a sensitive and accurate custom-built device, the details of which were published earlier (6). Data acquisition was performed at 1,000 Hz with an Apple Macintosh 7100 PowerPC with an eight-channel, 12-bit Lab-NB analog-to-digital conversion board (National Instruments, Austin, TX). Postprocessing of the torque data was performed with Matlab 5.2.1 (The Math Works, Natick, MA).
Exercise protocol. Supramaximal stimulation current necessary to obtain recruitment of all muscle fibers was first determined using 3-5 isometric twitch contractions with increasing current. Resting periods between twitches were kept constant at 60 s.
Optimal muscle length, at optimal ankle angle, was determined using 9 twitch contractions at ankle angles between 10° dorsal flexion and 30° plantar flexion. The ankle angle at which the muscle exerted maximal torque was not different between the wild-type and CKTissue sampling. Subsets of mice were used for muscular tissue sampling at two different time points during the contraction protocol for the analysis of tissue high-energy phosphates and related compounds, i.e., immediately after the series of 12 contractions (E; n = 6), and after 60 s of recovery (R) after E (E + R; n = 6).
Control samples (C; n = 8) were taken from the resting contralateral dorsal flexor complex. Muscular tissue was rapidly freeze clamped using a pair of aluminum tongs cooled in liquid nitrogen. The tissue samples were stored atNumber of animals and statistical procedures.
In total, 14 CK/
and 14 wild-type littermates were
included in this study. Mechanical activity was measured in eight
CK
/
and eight wild-type animals. The dorsal flexor
muscle complex was freeze clamped 60 s after the high-intensity
contraction series. Due to technical imperfections, the high-energy
phosphate content was assessed in only six of eight muscle complexes.
In a subset of six animals, the flexor muscle complex was freeze
clamped immediately after the high-intensity contraction series and
used for HPLC analysis (see Tissue sampling). The
contralateral, resting flexor muscle complex was freeze clamped in 14 CK
/
and wild-type animals. Eight and five tissue
samples were used for HPLC analysis and assessment of enzymatic
activity, respectively. One sample of the contralateral flexor complex
in each group was lost due to technical imperfections.
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RESULTS |
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Mean body mass of CK/
mice (21.9 ± 1.7 g) was significantly lower than wild-type body mass (24.0 ± 1.0 g). Dorsal flexor mass of CK
/
mice amounted to
52.5 ± 6.1 mg and was significantly lower than that of the
age-matched wild-type mice (62.1 ± 8.2 mg).
Figure 1 shows a representative maximal
dorsal flexor tetanic torque pattern for wild-type and
CK/
mice at 125-Hz stimulation frequency. The mean
values of isometric contractile parameters of dorsal flexors measured
in CK
/
and wild-type mice are shown in Table
1.
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Maximal tetanic torque at a stimulation frequency at 125 Hz was
significantly higher in wild-type mice compared with
CK/
mice, which could be partially explained by the
decline in muscle mass in CK
/
mice. However, after
normalization of the values of the maximal tetanic torque on muscle
mass, maximal tetanic torque was still significantly lower in
CK
/
. The rate of relaxation after tetanic contraction
was significantly slower in CK
/
than in control mice,
whereas the rise time was not affected in CK
/
dorsal
flexors (Table 1).
A representative torque output signal of a series of 12 consecutive
tetanic contractions of dorsal flexors of wild-type and CK/
mice is given in Fig.
2. In both cases, the maximal torque
declined during the contraction series. Figure 2 also shows that the
rates of torque buildup and relaxation are prolonged in
CK
/
mice during the course of the 12 consecutive
contractions.
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The mean maximal torque of each individual contraction during the
series of 12 tetanic contractions is depicted in Fig.
3. The maximal torque of both the
wild-type and CK/
mice showed an approximately linear
decrease. The maximal torque of the 12th contraction was 12.0 ± 3.3% lower than that of the 1st contraction of wild-type mice. The
maximal torque output of CK
/
mice tended to show a
larger decrease at the last contraction (22.9 ± 9.8%), but due
to the relatively high interindividual variation, this value was not
significantly different from the corresponding value of wild-type mice.
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The higher relaxation time, as observed in a single tetanic
contraction, was even more conspicuous in the series of 12 tetanic contractions. The half-relaxation time at the 12th contraction of
CK/
mice was significantly longer compared with that of
the wild-type mice, i.e., 26.2 ± 2.8 vs. 12.9 ± 3.5 ms.
Furthermore, in CK
/
mice, the half-relaxation time of
the 12th contraction was significantly longer than the half-relaxation
time measured after a single tetanic contraction; this phenomenon was
not present in experiments of wild-type mice. Moreover, the rise time
of the first contraction was comparable to the values calculated during
a single tetanic contraction, i.e., 11.1 ± 1.2 and 10.6 ± 1.2 ms for wild-type and CK
/
mice, respectively.
Wild-type muscles showed no difference in rise time during the 12 consecutive contractions compared with the initial rise time in
contrast to the CK
/
, which showed a substantial
increase in rise time in the 2nd and 3rd contraction, i.e., 19.6 ± 3.0 and 29.4 ± 3.7 ms, respectively. The rise time of the 4th
and succeeding contractions could not be calculated, because the
relaxation was prolonged to such an extent that torque did not fall
below 10% of the maximal torque value.
Table 2 shows the high-energy phosphate
content of dorsal flexors at three different time points of the
repeated contraction protocol for both wild-type and
CK/
mice.
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Basal PCr and Cr levels were unaffected in CK/
muscles,
which resulted in comparable PCr-to-Cr ratios between
CK
/
and wild-type muscles. Resting ATP values of
CK
/
mice were significantly lower than those of
wild-type mice, whereas the ADP and AMP content was significantly
higher in CK
/
mice. The total of adenine nucleotides
(TAN) was significantly lower in CK
/
dorsal flexors
compared with wild-type mice. Furthermore, resting IMP levels showed a
significant fourfold increase in CK
/
mice compared with
wild-type control muscles.
Immediately after the series of 12 consecutive tetanic contractions,
the PCr level in wild-type mice was significantly lower (PCr =
16.7 µmol/g dry wt), whereas the Cr level increased to a comparable
extent (
Cr = 14.9 µmol/g dry wt). The PCr-to-Cr ratio of
wild-type mice dropped from 1.6 ± 0.3 in resting conditions to
0.9 ± 0.2 after the contraction series.
In contrast, PCr and Cr levels in CK/
dorsal flexors
were not affected during the series of 12 tetanic contractions, which resulted in an unchanged PCr-to-Cr ratio. At the end of the contraction protocol, the CK
/
ATP level was significantly lower
compared with the resting values. ADP and AMP levels did not change,
whereas TAN significantly decreased. This contraction-induced decline
in ATP content was absent in wild-type mice. Compared with resting
values, CK
/
muscles showed a significant twofold
increase in IMP content at the end of the series of tetanic
contractions. In contrast, no accumulation of IMP occurred in wild-type muscles.
During the 60-s recovery period after the series of 12 tetanic
contractions, PCr and Cr levels and the PCr-to-Cr ratio in wild-type
mice returned to precontraction levels. In CK/
muscles,
recuperation of ATP levels did not occur during the recovery period,
which was also the case for TAN. IMP levels in CK-mutant mice remained
elevated during the 60-s recovery period.
The maximal activity of total CK in CK/
muscle was on
the order of 0.7% that of wild-type mice. No differences between
wild-type and CK
/
dorsal flexors in maximal activity of
adenylate kinase and AMP deaminase were found (Table
3).
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DISCUSSION |
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Total CK activity in dorsal flexors of the double CK-knockout mice
was reduced to ~0.7% that of wild-type animals. The residual activity can most likely be attributed to the CK- isoform present in
vascular endothelium and muscle satellite cells. Using nuclear magnetic
resonance analysis, Steeghs et al. (15) confirmed that PCr
no longer could be used to regenerate hydrolyzed ATP in
CK
/
mice. Hence, double-CK-deficient mice are a useful
model to study the impact of a lack of CK on muscle contractile
properties and adenine nucleotide handling during high-intensity contractions.
Single isometric tetanic contraction.
The present study clearly shows that the maximal tetanic torque output
of CK/
mice is significantly declined. This finding
implies that the lack of CK activity substantially affects contractile
function of intact muscles investigated in their natural environment.
Our in situ observation corroborates earlier findings obtained with isolated muscles, showing a decline in the force of muscles lacking CK
activity (4, 14, 16). Theoretically, alterations in the
maximal value of muscle torque can be caused, among other factors, by
variation in muscle mass, altered length of the muscle lever arm,
and/or changes in energy metabolism. We observed a 16% decline in
dorsal flexor muscular mass of the CK
/
mouse. After
normalization of maximal tetanic torque on muscle mass, torque per gram
of tissue was still 29% lower in CK
/
mice than that in
age-matched controls. It is of interest to note that Steeghs et al.
(14) previously observed a 30% decline in maximal force
output in isolated gastrocnemius medialis muscle of CK
/
mice. These combined findings strongly suggest that alterations in
lever arm do not likely occur in CK-mutant mice, indicating that
additional factors are involved in the decline of maximal torque.
Repetitive tetanic contractions.
When dorsal flexor muscles of CK/
and wild-type mice
were energetically challenged by a series of 12 high-intensity tetanic contractions, tour duty cycle 0.64, several interesting differences in
torque output became apparent. Although the decline in maximal torque
during the course of 12 tetanic contractions tended to be greater in
CK
/
muscles, the difference between CK
/
and wild-type muscles did not reach the level of significance. The
relaxation time significantly increased in CK
/
mice
during the series of 12 contractions.
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ACKNOWLEDGEMENTS |
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We thank Dr. B. Wieringa and F. Oerlemans (Dept. of Cell Biology and Histology, University of Nijmegen, The Netherlands) for kindly providing us with the CK knockout mice and wild-type littermates.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. J. van der Vusse, Dept. of Physiology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands (E-mail: vandervusse{at}fys.unimaas.nl).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 October 2000; accepted in final form 30 April 2001.
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