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J Physiol (2003), 547.2, pp. 395-403
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.034793
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ABSTRACT |
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Viable genetically engineered animals generally exhibit adaptations to the altered genotype, which may mask the role of the protein of interest. We now describe a novel method by which the direct effects of the altered genotype can be distinguished from secondary adaptive changes in isolated adult skeletal muscle cells. We studied contractile function and intracellular Ca2+ handling in single skeletal muscle fibres that are completely deficient of creatine kinase (CK; CK-/-) before and after microinjection of purified CK (injected together with the fluorescent Ca2+ indicator indo-1). The mean total CK activity after CK injection was estimated to be ~ 4 mM s-1, which is ~ 5 % of the activity in wild-type muscle fibres. After CK injection, CK-/- fibres approached the wild-type phenotype in several aspects: (a) the free myoplasmic [Ca2+] ([Ca2+]i) increased and force showed little change during a period of high-intensity stimulation (duty cycle, i.e. tetanic duration divided by tetanic interval = 0.67); (b) [Ca2+]i did not decline during a brief (350 ms) tetanus; (c) during low-intensity fatiguing stimulation (duty cycle = 0.14), tetanic [Ca2+]i increased over the first 10 tetani, and thereafter it decreased; (d) tetanic [Ca2+]i and force did not display a transient reduction in the second tetanus of low-intensity fatiguing stimulation. Conversely, tetanic force in the unfatigued state was lower in CK-/- than in wild-type fibres, and this difference persisted after CK injection. Injection of inactivated CK had no obvious effect on any of the measured parameters. In conclusion, microinjection of CK into CK-/- fibres markedly restores many, but not all, aspects of the wild-type phenotype.
(Received 22 October 2002; accepted after revision 12 December 2002; first published online 17 January 2003)
Corresponding author H. Westerblad: Department of Physiology and Pharmacology, Karolinska Institute, SE-171 77 Stockholm, Sweden. Email: hakan.westerblad{at}fyfa.ki.se
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INTRODUCTION |
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Skeletal muscle performance decreases during strenuous physical activity, a phenomenon known as fatigue. The mechanism underlying muscle fatigue is multifactorial, but changes in the high-energy phosphates ATP, ADP and phosphocreatine (PCr) have been suggested to play a major role (Westerblad et al. 1998a; Duke & Steele, 2001). Changes in the [ATP]:[ADP] ratio, which may occur during periods of intense activity, are minimized by the creatine kinase (CK) reaction:
(Bessman & Carpenter, 1985). CK may act as both a temporal energy buffer and as a spatial energy buffer, facilitating the communication between intracellular sites of ATP production and consumption (Wallimann et al. 1992). In the absence of CK, a decreased [ATP]:[ADP] ratio may develop during periods of high energy turnover and this may result in decreased contractile performance. Bearing this in mind, we recently studied fatigue properties in mouse skeletal muscle fibres lacking both cytosolic and mitochondrial creatine kinase (CK-/-; Steeghs et al. 1997). CK-/- muscle fibres showed a marked decline of [Ca2+]i and force during high-intensity stimulation (Steeghs et al. 1997; Dahlstedt et al. 2000). During prolonged stimulation at a lower intensity, on the other hand, CK-/- muscle fibres were markedly more fatigue resistant than wild-type fibres (Dahlstedt et al. 2000).
Genetically modified mice with either one or more genes deleted ('knock-out' mice) or with genes added or overexpressed (transgenic mice) are used extensively in studies of protein function. However, adaptations generally occur in genetically engineered animals, and scientists frequently find themselves studying the adaptation to gene modification rather than the specific function of the protein of interest. For instance, several proteins involved in energy metabolism show major adaptive changes in CK-/- skeletal muscle (Steeghs et al. 1998; de Groof et al. 2001). To distinguish between adaptive changes and direct CK effects, we have now compared contractile performance and Ca2+ handling during high-intensity and low-intensity fatiguing stimulation in isolated CK-/- muscle fibres before and after microinjection of purified CK. With this novel technique, we show that CK is important during rapid energy turnover, and the normal wild-type phenotype is partially reinstated in CK-injected CK-/- fibres.
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METHODS |
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Animals
CK-/- mice were generated as described previously (Steeghs et al. 1997). They were housed at room temperature with a 12 h:12 h light-dark cycle. Food and water were provided ad libitum. Female and male CK-/- and wild-type mice (age ~ 12 months) were used; they were killed by rapid dislocation of the neck, after which the flexor digitorum brevis muscles of the hindlimb were isolated. All procedures were approved by the Stockholm North local ethical committee.
Solutions
Muscle fibres were superfused with a Tyrode solution of the following composition (mM): NaCl 121, KCl 5.0, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.4, NaHCO3 24.0, EDTA 0.1 and glucose 5.5; 0.2 % fetal calf serum was added to the solution to improve muscle fibre survival. The solution was bubbled with 5 % CO2/95 % O2, which gives a pH of 7.4. Experiments were performed at room temperature (24 °C). An inhibitor of CK, 2,4-dinitro-1-fluorobenzene (DNFB; 10 µM), was used in some experiments. The fibre was exposed to DNFB for 5 min and tetanic stimulation started after 2 min washout. DNFB was added to the Tyrode solution from a stock solution (100 mM) in DMSO; addition of the same amount of DMSO had no effect on tetanic force or [Ca2+]i (Dahlstedt & Westerblad, 2001).
Single-fibre dissection, mounting and stimulation
Intact, single fibres were dissected following a procedure described elsewhere (Lännergren & Westerblad, 1987). The isolated fibre was mounted between an Akers 801 force transducer (SensoNor, Horten, Norway) and an adjustable holder in a chamber that was superfused continuously with Tyrode solution. The fibre length was adjusted to that giving maximum tetanic force and the fibre diameter was measured. Tetanic stimulation was achieved by supramaximum current pulses (duration 0.5 ms) at 70 Hz delivered via platinum plate electrodes lying parallel to the fibres.
[Ca2+]i and force measurements
[Ca2+]i was measured with the fluorescent Ca2+ indicator indo-1 (Molecular Probes Europe, Leiden, The Netherlands). Indo-1 was mixed in a buffer (150 mM KCl, 10 mM Hepes, pH 7.1) to a final concentration of 10 mM and microinjected into fibres (together with purified CK; see below). The fluorescence of indo-1 was measured with a system consisting of a xenon lamp, a monochromator and two photomultiplier tubes (PTI, Photo Med, Wedel, Germany). The excitation light was set to 360 ± 5 nm and the light emitted at 405 ± 5 and 495 ± 5 nm was measured. The ratio of the light emitted at 405 nm to that at 495 nm was converted to [Ca2+]i, as described elsewhere (Andrade et al. 1998).
During contractions, fluorescence and force signals were sampled on-line and stored in a desktop computer for subsequent data analysis. The mean fluorescence ratio during tetanic stimulation was measured and translated to [Ca2+]i. Force is expressed in kPa (i.e. the peak tetanic force (kN) divided by the cross sectional area (m2)). Relative [Ca2+]i and forces are expressed as the percentage of the values obtained in 70 Hz, 350 ms control tetani.
Injection of creatine kinase
A commercial preparation of CK purified from skeletal muscle (0.35 mol min-1 g-1 lyophilizate at 25 °C; Boehringer Mannheim) was solubilized in a buffer (10 mM Hepes, 150 mM KCl, pH 7.1) to yield an activity of 1.0 M s-1. This CK solution was injected into fibres together with the indo-1 solution. The myoplasmic CK activity after injection was quantified from the amount of indo-1 injected, as described previously (Westerblad & Allen, 1996), assuming that in CK-/- fibres ~ 30 % of the cell volume is accounted for by the sarcoplasmic reticulum (SR), t-tubules, mitochondria and lipid droplets (Luff & Atwood, 1971; Steeghs et al. 1998). Different mixtures of CK and indo-1 were tried. On the one hand, a high CK:indo-1 ratio resulted in rapid obstruction of the microelectrodes, possibly because the negatively charged protein stuck to the glass, and only small volumes could be injected. Hence the intracellular concentration of indo-1 was small and reliable measurements of [Ca2+]i could not be performed. On the other hand, with a low CK:indo-1 ratio, large injections were required to get a satisfactory myoplasmic CK activity. The indo-1 concentration might then be so high that significant buffering of [Ca2+]i occurred. The most suitable CK:indo-1 ratio ranged between 1:1 and 2:1. In one control experiment, purified CK was injected together with mag-indo-1 instead of indo-1. Since mag-indo-1 has a markedly lower Ca2+ affinity than indo-1, no noticeable increase in [Ca2+]i buffering will occur with mag-indo-1 injection. The force changes induced by CK injection in this experiment were well within the range obtained with CK-indo-1 injections.
Control experiments were performed with injection of inactivated CK; the quantities injected were similar to those used for injection of active CK. In these experiments, purified CK was denatured by adding a small volume of 1 M NaOH. The protein was then precipitated with 70 % ethanol and centrifuged (23 000 g). The ethanol wash was repeated 2-3 times to completely remove the NaOH. Thereafter, the protein was dried, dissolved and injected as above.
Experimental procedure
The muscle fibre was allowed to rest for at least 60 min after dissection. Thereafter some tetanic contractions (350 ms duration) were produced at 1 min intervals to ensure that force was stable. The fibre was then exposed to a high-intensity stimulation protocol consisting of 20 cycles of 200 ms tetanic stimulation given every 300 ms (i.e. with a duty cycle (tetanic duration divided by tetanic interval) of 0.67). After this series of tetani, the fibre was allowed to rest for at least 30 min, by which time tetanic force had fully recovered. Then followed a short low-intensity stimulation protocol consisting of three 350 ms tetanic stimulations given at an interval of 2.5 s (i.e. with a duty cycle of 0.14). The fibre was then injected with either active or inactive CK, allowed to rest for at least 60 min, and then the series of contractions described above were repeated. In some experiments, fibres initially injected with inactive CK were subsequently injected with active CK and the series of contractions repeated once more.
Some fibres injected with active CK were fatigued by the low-intensity stimulation protocol (350 ms stimulation every 2.5 s). These fibres were stimulated for 100 tetani or until force was reduced to 30 % of the original level. For comparison, we used single wild-type and CK-/- (not injected with CK) fibres fatigued by the same protocol.
Statistics
Values are presented as means ± S.E.M. Paired and unpaired t tests were used as appropriate. Statistical significance was set at P < 0.05.
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RESULTS |
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High-intensity stimulation and rapid changes in tetanic [Ca2+]i and force
The force produced in 70 Hz tetani by unfatigued CK-/- fibres after CK injection (219 ± 20 kPa, n = 7) was not significantly different from that produced before injection (244 ± 22 kPa). Also, tetanic force was not significantly altered by injection of inactive CK (data not shown).
We have shown previously that during a high-intensity stimulation protocol, wild-type fibres show an increase of tetanic [Ca2+]i and no significant change in force, whereas CK-/- fibres display a transient decline of both tetanic [Ca2+]i and force (Dahlstedt et al. 2000). Figure 1A shows representative records from high-intensity stimulation of a CK-/- fibre injected with inactive CK, and a decline in both tetanic [Ca2+]i and force can be observed. The decline reached its maximum in the second to third stimulation train and then some recovery took place. Thus, injection of inactive CK had no obvious effect on the characteristics seen in CK-/- fibres. However, after the subsequent injection of active CK, tetanic [Ca2+]i increased and there were only minor changes in tetanic force (Fig. 1B; i.e. the pattern was now similar to that of a wild-type fibre, Fig. 1C).
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Figure 1. Injection of creatine kinase (CK) markedly improves the performance of CK-/- skeletal muscle fibres during high-intensity stimulation Typical records of [Ca2+]i (upper) and force (lower) from a CK-/- fibre injected with inactive CK (A) and subsequently with active CK (B). Note that the initial decline in tetanic [Ca2+]i and force was clearly smaller after CK injection. For comparison, records from a wild-type fibre are shown in C. Fibres were activated by 20 cycles of 200 ms, 70 Hz stimulation and 100 ms rest. | ||
The amount of CK injected into the myoplasm was calculated from the increase in the indo-1 signal. The enzyme activity in the myoplasm was then found to range between 0.22 and 6.35 mM s-1 (mean = 3.90 ± 0.74 mM s-1, n = 8). The effect of different CK activities was investigated by plotting the relative restoration of force in the 20th (last) stimulation train of high-intensity stimulation vs. the calculated myoplasmic CK activity (Fig. 2). Note that the fibre that received the smallest CK injection exhibited a markedly smaller force restoration than the rest of the fibres and this fibre was excluded from calculations of mean values of force and [Ca2+]i after CK injection. Conversely, for the other seven fibres there was no correlation between the degree of force restoration and CK activity.
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Figure 2. CK injection results in a marked force restoration in CK-/- fibres during high-intensity stimulation Relative force restoration in the last (20th) stimulation train of high-intensity stimulation plotted against the estimated myoplasmic CK activity. Calculations of the relative force restoration were based on the assumption that with active CK, force remains constant during this type of stimulation, which was the result obtained in wild-type fibres (Dahlstedt et al. 2000). Thus, the percentage force restoration was calculated as (Pa - Pb)(100 - Pb)-1, where Pa and Pb are the relative forces during the last stimulation train after and before injection of CK, respectively. | ||
The results from high-intensity stimulation are summarized in Fig. 3. When comparing forces in fibres before injections and after injection of inactive CK, there was no significant difference at any time point (data not shown) and only fibres injected with inactive CK are plotted in Fig. 3. With the exception of the first stimulation train, tetanic [Ca2+]i and force were clearly higher after injection of active CK than after injection of inactive CK. For comparison, mean data from wild-type fibres exposed to the same stimulation protocol are also shown in Fig. 3 (dashed line; data from Dahlstedt et al. 2000). Clearly [Ca2+]i and force of CK-injected CK-/- fibres and wild-type fibres exhibited a similar pattern during this high-intensity stimulation, with the only major difference being a lower force during the initial part of the stimulation period in the CK-injected CK-/- fibres.
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Figure 3. The performance of CK-/- fibres injected with CK approaches the wild-type phenotype Mean data (± S.E.M.) of tetanic [Ca2+]i (upper) and force (lower) during high-intensity stimulation after injection of inactive CK (open triangles; n = 3) and active CK (filled circles; n = 5 for [Ca2+]i and n = 7 for force). For comparison, mean tetanic [Ca2+]i and force (dashed lines) from wild-type fibres are also shown (data from Dahlstedt et al. 2000). | ||
Three CK-/- fibres injected with active CK were subsequently exposed to DNFB. Original records from one of these experiments are shown in Fig. 4A-C. DNFB exposure resulted in a rapid drop of both tetanic [Ca2+]i and force during high-intensity stimulation, thus resembling the pattern before CK injection. However, mean results showed a larger decrease of tetanic force during high-intensity stimulation after DNFB exposure as compared to non-injected CK-/- fibres (Fig. 4E). The decrease in tetanic [Ca2+]i was also slightly larger after DNFB exposure (cf. Fig. 4D and Fig. 3). This difference may be due to unspecific inhibitory effects of DNFB on myofibrillar function (Fryer et al. 1995; Dahlstedt & Westerblad, 2001) and/or SR Ca2+ release (Hadad et al. 1999). Two wild-type fibres stimulated with the high-intensity stimulation protocol after being exposed to DNFB exhibited changes similar to those observed after DNFB exposure of CK-injected CK-/- fibres: in the last contraction, tetanic [Ca2+]i and force were reduced to about 70 % and 10 % of the original levels, respectively.
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Figure 4. The performance after pharmacological inhibition of CK is similar to that before CK injection Original records of [Ca2+]i (upper) and force (lower) from a CK-/- fibre exposed to high-intensity stimulation (200 ms, 70 Hz given every 300 ms) before CK injection (A), after CK injection (B) and subsequently after application of 10 µM 2,4-dinitro-1-fluorobenzene (DNFB; C). Mean data (± S.E.M.; n = 3) of relative [Ca2+]i (D) and force (E) obtained before CK injection (open triangles in E), after CK injection (filled circles) and finally after DNFB exposure (open circles). Data are expressed relative to [Ca2+]i and force in a standard 350 ms, 70 Hz tetanus produced before the high-intensity stimulation protocol after CK injection. | ||
It has been shown previously that CK-/- fibres exhibit a transient decline of tetanic [Ca2+]i and force in the second tetanus of low-intensity stimulation (350 ms stimulation every 2.5 s; Dahlstedt et al. 2000). In line with this, before CK injection the present fibres showed a force reduction in the second tetanus to 79 ± 7 % of the control, and force then increased, being 99 ± 2 % in the third tetanus (n = 8). After injection of active CK, there was no transient force reduction at the onset of low-intensity stimulation, and in the second tetanus, tetanic [Ca2+]i was 108 ± 5 % (n = 5) and force was 102 ± 1 % (n = 7) of the control.
The shape of tetanic [Ca2+]i transients differs between wild-type and CK-/- fibres: [Ca2+]i increases during the tetanus in wild-type fibres, whereas it tends to fall during the tetanus in CK-/- fibres (Dahlstedt et al. 2001). Bearing this in mind, we compared the shape of tetanic [Ca2+]i records after injection of inactive and active CK. A representative example is shown in Fig. 5, from which it can be seen that [Ca2+]i was better maintained after injection of active CK. Mean data show a slope of 0.56 ± 0.35 µM s-1 (n = 5; measured in each fibre as the difference between mean [Ca2+]i during the last and the first 100 ms stimulation) after injection of active CK, a value that was significantly higher (P < 0.05) than after injection of inactive CK, -0.50 ± 0.16 µM s-1 (n = 3).
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Figure 5. [Ca2+]i is better maintained in CK-/- fibres after CK injection Representative [Ca2+]i records obtained from 350 ms, 70 Hz tetanic contractions elicited before (dashed line) and after (continuous line) injection of CK into a CK-/- fibre. Note the fast [Ca2+]i decline during the contraction before CK injection (rate of decline shown by straight lines). | ||
Low-intensity fatiguing stimulation
In the unfatigued state, tetanic [Ca2+]i was not significantly different between wild-type fibres (1.23 ± 0.08 µM, n = 10), unmodified CK-/- fibres (1.24 ± 0.08 µM, n = 11), and CK-injected CK-/- fibres (1.58 ± 0.16 µM, n = 5). However, tetanic force was markedly higher in wild-type fibres (317 ± 26 kPa) than in unmodified CK-/- fibres (217 ± 15 kPa) and CK-injected CK-/- fibres (206 ± 27 kPa). Figure 6A shows [Ca2+]i and force records from selected tetani during fatiguing stimulation of a CK-injected CK-/- fibre. In this fibre, tetanic [Ca2+]i increased during the initial 10 tetani, subsequently falling to a level slightly below that measured at the start. Tetanic force, on the other hand, exhibited a minor decrease during the first 10 tetani and then remained stable. Mean data show the same picture. In CK-injected CK-/- fibres, tetanic [Ca2+]i was increased by about 30 % in the 10th tetanus (P < 0.05) and reduced by about 20 % in the last (100th) tetanus (P < 0.05; Fig. 6B). Thus, the changes in tetanic [Ca2+]i in these injected fibres were quite different from those of unmodified CK-/- fibres, which did not show any significant changes during fatigue. Instead, the pattern observed in injected fibres resembled that of wild-type fibres, although the rate of decline after the 10th tetanus was markedly lower in the injected fibres.
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Figure 6. CK-injected CK-/- fibres approach the wild-type phenotype during low-intensity fatiguing stimulation A, selected records of [Ca2+]i and force from a CK-injected CK-/- fibre fatigued by 350 ms, 70 Hz tetani given at 2.5 s intervals. Mean data of relative tetanic [Ca2+]i (B) and force (C) are in each fibre expressed as a percentage of the first fatiguing tetanus. CK-injected CK-/- fibres (filled circles, n = 5); wild-type fibres (open circles, n = 10); unmodified CK-/- fibres (open triangles, n = 11). | ||
CK-injected CK-/- fibres showed a tendency towards decreased force production during fatigue (Fig. 6C). In the 10th tetanus, tetanic force was slightly reduced in four of the five fibres studied, but the overall decrease was not statistically significant (P = 0.13). In the last tetanus of the fatigue run, tetanic force was reduced in all fibres, but the decrease was not significant (P = 0.08). Thus, the force changes in injected fibres were more similar to unmodified CK-/- fibres, which showed very limited changes, than wild-type fibres, which showed a major force decrease.
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DISCUSSION |
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In the present study we show that relatively small injections of CK markedly restore many aspects of the wild-type phenotype in mouse CK-/- skeletal muscle fibres. This was not due to the injection of protein per se, since injection of inactive CK did not have the same effect. Thus, this novel method to inject the missing protein into adult knock-out muscle cells may be used in a general context to distinguish between the direct effects of gene manipulation and secondary adaptive changes.
High-intensity stimulation and rapid changes in tetanic [Ca2+]i and force
The following functional properties were clearly improved towards the wild-type phenotype after CK injection: (a) [Ca2+]i and force production during a period of high-intensity fatiguing stimulation; (b) tetanic [Ca2+]i and force in the second tetanus of low-intensity stimulation; (c) the shape of [Ca2+]i during a 350 ms tetanus. All of these improvements after CK injection can be explained by a better maintenance of the [ATP]:[ADP] ratio during periods of high energy turnover. Thus, during periods of high ATP consumption in the absence of CK, the reduction of tetanic [Ca2+]i is likely to be attributable to inhibition of the SR Ca2+-release channels due to reduced [ATP], an associated increase in [Mg2+]i and accumulation of ATP breakdown products (Blazev & Lamb, 1999; Laver et al. 2001). The accompanying reduction of force would then simply be a consequence of the reduced [Ca2+]i.
The effect of CK injection was small in the CK-/- fibre receiving the smallest injection, whereas an almost full restoration of the wild-type phenotype was observed after larger injections (Fig. 2). The mean CK activity in injected fibres, calculated from the activity measured under maximal velocity conditions in vitro, was about 4 mM s-1. It is somewhat surprising that this modest activity, which is only about 5 % of the in vitro CK activity of fast-twitch muscle of wild-type mice (Brosnan et al. 1993), had such marked effects on contractile function and [Ca2+]i handling. However, the rate of PCr breakdown measured in vivo with 31P magnetic nuclear resonance during brief tetanic contractions in wild-type mice was 6.3 µM s-1 (g wet weight)-1 (Roman et al. 1996), which translates to about 3 mM s-1 at the temperature used in the present study (assuming a Q10 of 2.9; He et al. 1997). Furthermore, an ATPase activity rate of ~ 4 mM s-1 was obtained from a recent study on actomyosin ATPase of skinned fibres during isometric contractions (He et al. 1999; calculated from the actomyosin ATPase rate at 12 °C assuming a Q10 of 2.9 (He et al. 1997) and that the actomyosin ATPase activity is 75 % of the total ATPase activity; Woledge et al. 1985). In a recent study from our laboratory, luciferin/luciferase was used to measure ATP in isolated mouse muscle fibres under conditions similar to those used in the present study (Allen et al. 2002). In this study an ATPase activity rate of 5 mM s-1 was calculated from the decline of ATP during tetanic contractions performed after pharmacological inhibition of CK. Taken together, these results indicate that the intracellular CK activity of the CK injected fibres is sufficient to support energy turnover during high-intensity isometric contractions. Moreover, the CK activity of wild-type skeletal muscle appears to be markedly higher than that required for normal contractile function and [Ca2+]i handling during isometric contractions. Two separate studies suggest that this is indeed the case. First, no major change in isometric contractile function was observed when comparing wild-type and transgenic mice with increased CK activity (Roman et al. 1996). Second, mouse mutants with cytosolic CK activity reduced to 16 % of the wild-type level showed a decrease in mean force production of only 13 % (van Deursen et al. 1994).
Nevertheless, a high CK activity might be required for maintaining a high shortening velocity, and hence power production, during repeated contractions. An important function of CK is to keep the myoplasmic [ADP] low during periods of rapid energy turnover, and with a low CK activity, [ADP] might increase during contractions. While increased [ADP] has a limited effect on isometric force production, it markedly reduces the shortening velocity (Cooke & Pate, 1985). In line with this, we have shown that pharmacological inhibition of CK markedly reduced the maximum shortening velocity in prolonged tetani, whereas the effect on isometric force was small (Westerblad & Lännergren, 1995; Westerblad et al. 1998b).
Low-intensity fatiguing stimulation and long-lasting changes in tetanic [Ca2+]i and force
The present study shows that CK-/- muscle fibres produce significantly less tetanic force than wild-type fibres, which agrees with previous results (Steeghs et al. 1997; Dahlstedt et al. 2000). The medial gastrocnemius muscles of CK-/- mice studied in situ produced less force than wild-type muscles when stimulated at frequencies below 140 Hz, but this difference disappeared at higher stimulation frequencies (de Haan et al. 1999). This would indicate that the lower force in CK-/- muscles is due to impaired Ca2+ activation of the contractile elements. However, this is not the cause of the lower CK-/- force observed under the present experimental conditions, because force is also lower in tetanic contractions produced in the presence of caffeine during which [Ca2+]i is supramaximal (Dahlstedt et al. 2001). The lower force in CK-/- fibres may be partially attributed to a reduced concentration of myofibrils, secondary to an increased fraction of the cell volume being occupied by mitochondria and lipid droplets (Steeghs et al. 1998; de Groof et al. 2001). Moreover, the myoplasmic concentration of inorganic phosphate ([Pi]) is increased in rested fast-twitch CK-/- muscles (Steeghs et al. 1997; Dahlstedt et al. 2000) and increased [Pi] reduces crossbridge force production (Pate & Cooke, 1989; Millar & Homsher, 1990; Dahlstedt et al. 2001).
CK injection did not increase the tetanic force produced by CK-/- fibres. Obviously, these injections cannot increase the concentration of myofibrils. However, it might be expected that CK injections would reduce myoplasmic [Pi] but, as judged from the unaffected tetanic force, this probably did not occur. One possible explanation of this is that the cellular localization of the injected CK may be different from that of wild-type fibres and the localization of CK has been suggested to be of major importance for muscle cell function (Wallimann et al. 1992; Korge et al. 1993). However, the contractile function was normal in skeletal muscle where the muscle isoenzyme of CK was replaced by the brain isoform, which indicates that the subcellular localization is of limited importance in this aspect (Roman et al. 1997). Another possible explanation behind the apparent lack of a reduction in myoplasmic [Pi] after CK injection is that CK has little immediate effects on the metabolic state of CK-/- muscle cells at rest, where the net flux through CK is small. Thus, under resting conditions, the duration of the present experiments (up to about 2 h) might be too short for CK to have any obvious effect on myoplasmic [Pi] at rest and consequently the force produced in a brief tetanus.
During low-intensity fatiguing stimulation, the introduction of CK into CK-/- fibres resulted in changes in tetanic [Ca2+]i that were similar to those observed in wild-type fibres (see Fig. 6). This indicates that these fatigue-induced changes in tetanic [Ca2+]i are related to some consequence(s) of CK activity and increasing [Pi], due to a gradual breakdown of PCr, would be a strong candidate (Allen & Westerblad, 2001; Dahlstedt et al. 2001). Rising [Pi] may cause the early increase in tetanic [Ca2+]i by, for instance, increasing the open probability of the SR Ca2+-release channels (Balog et al. 2000). The subsequent decrease in tetanic [Ca2+]i may also be due to increasing [Pi]. The mechanism would then be that some Pi enters the SR, where it precipitates with Ca2+, hence causing a reduction of the free Ca2+ available for release (Fryer et al. 1995; Dahlstedt & Westerblad, 2001).
During low-intensity fatiguing stimulation, wild-type fibres showed a markedly larger reduction of tetanic force than CK-injected CK-/- fibres, which in this context were more similar to unmodified CK-/- fibres. The force decline in wild-type fibres observed after only 10 fatiguing tetani has been ascribed to the inhibitory effects of increased [Pi] on crossbridge function (Dahlstedt et al. 2001). At this time there was only a tendency towards a reduction in tetanic force in CK-injected CK-/- fibres, which might argue against an increase of [Pi] in these. However, [Pi] appears to be increased already in the unfatigued state in these fibres (see above), and the relationship between [Pi] and force is logarithmic so that the effect of increased [Pi] becomes smaller as [Pi] increases (Pate & Cooke, 1989; Millar & Homsher, 1990). Thus, the Pi-induced depression of crossbridge force production in early fatigue would be expected to be small in CK-injected CK-/- fibres, and therefore a statistically significant decrease may not be obtained with the limited number of fibres studied. A similar explanation might apply to another apparent difference between wild-type fibres and CK-injected CK-/- fibres; that is, [Ca2+]i was decreased to a similar percentage at the end of fatigue, whereas force was markedly more reduced in wild-type fibres. At this stage, tetanic [Ca2+]i is on the steep part of the force-[Ca2+]i curve (Allen et al. 1995), and a larger fatigue-induced decrease in myofibrillar Ca2+ sensitivity due to increasing [Pi] (Millar & Homsher, 1990) would be expected in wild-type fibres if these have a lower resting [Pi] than in CK-injected CK-/- fibres. Finally, the higher fatigue resistance of CK-injected CK-/- fibres as compared to wild-type fibres would also reflect the higher mitochondrial content of the former (Steeghs et al. 1998; de Groof et al. 2001).
Conclusion
To study the role of CK in contractile function, we developed a novel method where purified CK was microinjected into adult CK-/- skeletal muscle cells. This novel technique may be used to differentiate between the direct effects of targeted proteins and adaptive responses in other genetically engineered animal models (e.g. when other enzymes involved in energy metabolism are altered). The results show that CK is important for preventing fatigue during high-intensity stimulation and at the onset of low-intensity stimulation. During prolonged low-intensity stimulation, on the other hand, CK may induce fatigue-associated changes in [Ca2+]i and force.
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REFERENCES |
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Acknowledgements
This work was supported by grants from the Swedish Research Council (Project 10842), the Swedish National Center for Sports Research, the Lars Hierta's Memorial Fund and funds at the Karolinska Institute. The authors thank Professor B Wieringa (Department of Cell Biology and Histology, University of Nijmegen) for donation of the CK-/- mice and helpful discussions.
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