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MUSCLE CELL BIOLOGY AND CELL MOTILITY
-actinin-3 knockout mouse1School of Medical Sciences, University of New South Wales, Sydney; 2Institute for Neuromuscular Research, The Children's Hospital at Westmead, Sydney; and 3Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney, New South Wales, Australia
Submitted 31 March 2008 ; accepted in final form 21 July 2008
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ABSTRACT |
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-actinin-3 is one of the two isoforms of -actinin that are found in the Z-discs of skeletal muscle. -Actinin-3 is exclusively expressed in fast glycolytic muscle fibers. Homozygosity for a common polymorphism in the ACTN3 gene results in complete deficiency of -actinin-3 in about 1 billion individuals worldwide. Recent genetic studies suggest that the absence of -actinin-3 is detrimental to sprint and power performance in elite athletes and in the general population. In contrast, -actinin-3 deficiency appears to be beneficial for endurance athletes. To determine the effect of -actinin-3 deficiency on the contractile properties of skeletal muscle, we studied isolated extensor digitorum longus (fast-twitch) muscles from a specially developed -actinin-3 knockout (KO) mouse. -Actinin-3-deficient muscles showed similar levels of damage to wild-type (WT) muscles following lengthening contractions of 20% strain, suggesting that the presence or absence of -actinin-3 does not significantly influence the mechanical stability of the sarcomere in the mouse. -Actinin-3 deficiency does not result in any change in myosin heavy chain expression. However, compared with -actinin-3-positive muscles, -actinin-3-deficient muscles displayed longer twitch half-relaxation times, better recovery from fatigue, smaller cross-sectional areas, and lower twitch-to-tetanus ratios. We conclude that -actinin-3 deficiency results in fast-twitch, glycolytic fibers developing slower-twitch, more oxidative properties. These changes in the contractile properties of fast-twitch skeletal muscle from -actinin-3-deficient individuals would be detrimental to optimal sprint and power performance, but beneficial for endurance performance. extensor digitorum longus
-ACTININS ARE A GROUP of actin-binding proteins. In skeletal muscle, they are found in the Z-disc, where they cross-link the thin actin filaments of adjacent sarcomeres. The two isoforms found in the Z-disc are -actinin-2 and -actinin-3. -Actinin-2 is the predominant isoform found in oxidative muscle fibers, whereas -actinin-3 is restricted mainly to fast glycolytic fibers (8).
It is estimated that around 1 billion individuals worldwide completely lack -actinin-3, due to homozygosity for a common polymorphism in the -actinin-3 gene (10). -Actinin-3 deficiency is not associated with any disease phenotype, suggesting that its absence may largely be compensated for by the closely related protein, -actinin-2 (11). However, the genomic region surrounding the polymorphism shows low levels of genetic variation and recombination in individuals of certain populations, consistent with strong, recent positive selection (10). This suggests that -actinin-3 deficiency does have an important effect on skeletal muscle, and that muscles lacking -actinin-3 must be different in some way from muscles that have the protein.
A study of athletes at the Australian Institute of Sport (17) found that those engaged in sprint or power activities had a lower incidence of -actinin-3 deficiency than the general population (6% compared with 18%). In fact, among Olympic sprint athletes, there were no cases of -actinin-3 deficiency. Endurance athletes, in contrast, tended to have a higher incidence of -actinin-3 deficiency, although this trend was only statistically significant in females. The reduced incidence of -actinin-3 deficiency among elite sprint and power athletes has since been observed in other independent studies (13–15). -Actinin-3 deficiency has also been associated with reduced muscle strength (5) and poorer sprinting performance (12) in nonathletes. These data strongly suggest that a lack of -actinin-3 affects skeletal muscle in a way that is detrimental to sprint and power performance but beneficial for endurance activities.
Studies on a specially generated -actinin-3 knockout mouse (10) lend support to these findings in humans. In an endurance test in which mice were run on a motorized treadmill, knockouts were found to run 33% further than wild types before exhaustion (10), supporting the finding of a higher incidence of -actinin-3 deficiency in female endurance athletes. Knockouts also had reduced grip strength, lower muscle weights, and smaller fast fiber diameters than wild types (9), supporting the finding that -actinin-3-deficient individuals are underrepresented in strength and power activities.
There are various hypotheses as to why -actinin-3 deficiency might adversely affect power performance and benefit endurance performance. One hypothesis is that -actinin-3 serves to stabilize the sarcomere when muscles are exercised to maximal or near-maximal capacity, as in sprinting. In its absence, the sarcomere may be weakened and more likely to be damaged during extreme athletic activity. Such a role is suggested by the protein's location in the Z-disc and its actin-binding properties.
Another hypothesis is that -actinin-3 influences fiber-type differentiation toward a fast-twitch, glycolytic profile that is beneficial for sprint performance, while its absence would lead to differentiation toward a slower oxidative profile that is beneficial for endurance performance. Such a role is suggested by the restricted distribution of the protein, which is confined to fast glycolytic fibers, and by molecular studies that indicate that the sarcomeric -actinins interact with signaling proteins involved in fiber-type differentiation and with metabolic enzymes involved in glycogenolysis (8). Under this hypothesis, one might expect that fast glycolytic fibers would adopt slower, more oxidative properties when -actinin-3 is absent. Indeed, the activity of key oxidative enzymes in the -actinin-3 knockout mouse is significantly higher than in wild-type controls, although there is no change in fiber types as defined by myosin heavy chain composition (9, 10).
In the present study we examined some physiological properties of isolated, whole -actinin-3-deficient muscles from the -actinin-3 knockout mouse to gain greater insight into the likely functions of this protein. For comparison, we used littermate wild-type controls that were homozygous for the -actinin-3 gene. We chose to analyze the extensor digitorum longus (EDL) muscle from the hindlimb, because -actinin-3 is found predominantly in fast glycolytic fibers (8), and the mouse EDL muscle contains a high proportion of these fibers. Thus any consequences of -actinin-3 deficiency will be most apparent in this muscle.
To see whether -actinin-3 plays a mechanical role in stabilizing the sarcomere, we measured the muscle damage resulting from eccentric contractions of 20% strain and also examined the morphology of individual fibers for evidence of repetitive muscle injury. To see whether -actinin-3 influences fiber-type differentiation and metabolism, we measured some basic contractile properties and also examined the responses of the muscles to a fatigue protocol.
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METHODS |
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6 mo, were used. All mice were males.
Muscle preparation.
All animals were anesthetized with halothane and killed by cervical dislocation. The EDL muscle was dissected from the hindlimb and tied by its tendons to a force transducer (Fort 10, World Precision Instruments) at one end and a linear tissue puller (University of New South Wales) at the other, using silk suture (Deknatel 6.0). The EDL muscle was placed in a bath continuously superfused with Krebs solution, with composition (in mM) 4.75 KCl, 118 NaCl, 1.18 KH2PO4, 1.18 MgSO4, 24.8 NaHCO3, 2.5 CaCl2, and 10 glucose, with 0.1% fetal calf serum; it was continuously bubbled with 95% O2-5% CO2 to maintain pH at 7.4. The muscle was stimulated by delivering a supramaximal current between two parallel platinum electrodes, using an electrical stimulator (A-M Systems). At the start of the experiment, the muscle was set to the optimum length LO that produced maximum twitch force. All experiments were conducted at room temperature (22°C to 24°C).
Force-frequency curve.
A force-frequency curve was then obtained by delivering 500-ms stimuli of different frequencies (2, 15, 25, 37.5, 50, 75, 100, 125, and 150 Hz) and measuring the force produced at each frequency of stimulation. A 30-s rest was allowed between each frequency. A curve relating the muscle force P to the stimulation frequency f was fitted to these data. The curve had the following equation:
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Eccentric contractions. The muscle was then subjected to a series of eccentric (lengthening) contractions. At time = 0 ms, the muscle was stimulated by supramaximal pulses of 1-ms duration and 125-Hz frequency. At time = 250 ms, after it had attained its maximum isometric force, the muscle was stretched at a speed of 0.2 LO/s until it was 20% longer than its optimum length, was held at this length for 0.5 s, and was then returned at the same speed to its original position. The electrical stimulus was stopped at time = 2,000 ms. This eccentric contraction was performed 5 times, at intervals of 2 min. After a 15-min recovery period, the optimum length was reset and a second force-frequency curve was obtained.
Muscle mass. Finally, the muscle was removed from the bath. The tendons were trimmed and the muscle was lightly blotted on filter paper and then weighed. An estimate of the cross-sectional area was obtained by dividing the muscle's mass by the product of its optimum length and the density of mammalian muscle (1.06 mg/mm3) (3).
Muscle stiffness. To estimate muscle stiffness, we divided the change in muscle force (as a percentage of isometric force) by the change in muscle length (as a percentage of optimum length) during the first eccentric contraction. We only measured up to the point where the muscle reached 109% of optimum length; beyond this length, many muscles developed forces that exceeded the measurement capacity of the force transducer.
Fatigue. In a separate set of experiments using different muscles from those used for eccentric contractions, muscles were examined for their responses to a fatiguing protocol. Muscles were set up as described above. A force-frequency curve was obtained as described above, except that the duration of stimulation at each frequency was only 250 ms. After 5 min, the fatigue protocol was started. The muscle was given a 1-s, 100-Hz tetanus every 2 s over a period of 30 s. The muscle was then allowed to recover for a period of 30 min, during which force recovery was monitored with a brief (250 ms) 100-Hz tetanus every 5 min. Additional force-frequency curves were obtained 90 s after the end of the fatigue protocol and 1 min after the final recovery tetanus.
The 30-min recovery period was chosen because of time constraints. At this time, recovery in the muscles ranged from 74% to 92% of prefatigue force. The experiment would have been too prolonged if the muscle had been left to recover to 100%; in any case, it is unlikely that complete recovery would have occurred because of the possible development of an anoxic core during the very vigorous stimulation protocol.
Muscle fiber morphology.
Individual fiber morphology was examined in the EDL muscles of one wild-type and one knockout mouse aged 6 mo. Animals were anesthetized with halothane and killed by cervical dislocation. The EDL muscle was dissected from the hindlimb. Following dissection, the muscles were digested to yield individual fibers. The solution used for the digestion was Krebs solution containing 3 mg/ml collagenase Type I (Sigma) and 1 mg/ml trypsin inhibitor (Sigma), continuously bubbled with 95% O2-5% CO2 and maintained at 37°C. After about 30 min, the muscles were removed from this solution, rinsed in Krebs solution, and placed in a relaxing solution with the following composition (concentrations in mM): 117 K+, 36 Na+, 1 Mg2+, 60 HEPES, 8 ATP, 50 EGTA2–, and free Ca2+ of 10–7 M. The muscle was gently agitated with pipette suction, releasing some individual fibers from the muscle mass.
Individual fibers were examined either with a light microscope (Olympus BX60) or a laser-scanning confocal microscope (Leica TCS SP).
Statistical analyses. Data are presented as means ± SE. For the contractile properties and eccentric contraction data, two-tailed t-tests were used. For the fatigue data, the Mann-Whitney U-test was used because of smaller sample sizes. All tests were conducted at a significance level of 5%. All statistical tests and curve fitting were performed using a standard statistical software package (GraphPad Prism version 4.00 for Windows, GraphPad Software, San Diego, CA).
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RESULTS |
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The fiber morphology was performed on EDL muscles from one wild-type and one knockout mouse, both males aged about 6 mo.
The fatigue experiments were performed on six wild-type muscles and eight knockout muscles. Each muscle was taken from a different mouse. Animals were male mice aged 8 to 10 wk.
General physical properties.
The general physical properties of wild-type and knockout muscles are shown in Table 1. While their optimum lengths were virtually identical to those of wild-type muscles, -actinin-3-deficient muscles were 9% lighter than -actinin-3-positive muscles, with a corresponding 9% reduction in cross-sectional area.
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To see whether there were any differences at submaximal levels of stimulation, we analyzed the forces produced at 100 Hz (about 67% of maximum frequency) by the cohort of muscles used in the fatigue experiments (described in Fatigue below). The 100-Hz absolute forces of knockouts were 10.9% lower than wild types (P = 0.008, by Mann-Whitney test), but there was no difference in 100-Hz specific forces.
Force-frequency characteristics. Table 1 shows various contractile properties derived from the force-frequency curves of individual muscles. The half-frequency is the stimulation frequency at which the muscle develops a force which is halfway between its minimum and maximum forces. The Hill coefficient is a measure of the slope of the curve. The half-frequency and Hill coefficient indicate the sensitivity of the contractile proteins to calcium. The lower the half-frequency and the higher the Hill coefficient, the greater the sensitivity. The twitch-to-tetanus ratio measures the minimum force as a proportion of the maximum force.
The half-frequency in wild-type muscles was not significantly different from the half-frequency in knockouts. The Hill coefficient was significantly higher in knockouts than in wild types. The twitch-to-tetanus ratio in knockouts was significantly lower than the ratio in wild types.
The effects of these differences on the shape of the force-frequency curve are shown in Fig. 1, in which individual force-frequency data for wild types and knockouts have been aggregated into single curves. At low frequencies, the curve for knockouts is depressed slightly compared with wild types, reflecting the lower twitch-to-tetanus ratio in knockouts. Over middle frequencies, where the curves are rising steeply, the curve for knockouts has a slightly steeper slope than the curve for wild types, reflecting the higher Hill coefficient in knockouts.
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The extent of each of these three changes was used to assess the degree of muscle damage in wild types and knockouts, and the results are shown in Fig. 3. Fig. 3A shows the force deficit, or the percentage fall in maximum force. This was 1.6 ± 2.0% in wild types and 2.6 ± 1.5% in knockouts. Fig. 3B shows the percentage increase in half-frequency, or the extent of the rightward shift of the curve. This was 15.2 ± 0.6% in wild types and 12.9 ± 1.1% in knockouts. Fig. 3C shows the percentage decrease in the Hill coefficient, or the extent of the reduction in the curve's steepness. This was 10.1 ± 1.5% in wild types and 12.4 ± 1.4% in knockouts. There were no statistically significant differences between wild types and knockouts in any of these measures.
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A muscle which was particularly susceptible to eccentric injury might be expected to develop a large number of branched fibers over time. We thus examined the individual fiber morphology from muscles of older mice (6 mo old) to see whether there was any fiber branching indicative of repetitive muscle damage.
Individual fibers from the EDL muscles of one wild-type and one knockout mouse were examined by laser-scanning confocal microscopy. Examples of fibers are shown in Fig. 5. Almost all fibers were normal, with no branches and with peripherally located nuclei, as shown in Fig. 5A. Branching was detected in a very small number of knockout fibers, such as the one shown in Fig. 5B. All the branched fibers had peripherally located nuclei. Hence any morphological evidence of fiber damage was minimal and was not sufficient to suggest that knockouts were any more susceptible than wild types to eccentric muscle injury.
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Figure 6 shows the results obtained from six wild-type muscles and eight knockout muscles. The descending part of the curve shows the decline in 100-Hz force with each successive tetanus during the 30-s fatigue protocol. By the end of this fatigue protocol, 100-Hz force had declined to 45.1 ± 1.3% of original in wild types and to 42.9 ± 4.5% of original in knockouts. These values were not significantly different (Mann-Whitney test). The ascending part of the curve shows the recovery in 100-Hz force over the 30 min following the fatigue protocol. By the end of this period, knockouts had recovered to 86.1 ± 1.1% of their original force, but wild types recovered to only 78.4 ± 1.9% of original. These values were significantly different (P = 0.013, by Mann-Whitney test), indicating that recovery of 100-Hz force following fatigue was better in knockouts than in wild types. The difference in 30-min recovery has previously been presented by MacArthur et al. (9), but the full data are included here so as to show the time course of the changes in force during fatigue and recovery.
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These force-frequency curves are shown in Fig. 7, A and B. Here, data from individual muscles have been aggregated and forces expressed as a percentage of the prefatigue maximum to facilitate comparison. Wild-type muscles are shown in Fig. 7A and knockouts are shown in Fig. 7B. The fatiguing protocol produced a rightward shift of the force-frequency curve, as is evident from the rightward displacement of FF2 compared with FF1 in both wild types and knockouts. Right-shifting of the force-frequency curve is commonly found after fatigue and may indicate some impairment of the excitation-contraction coupling mechanism. The magnitude of the rightward shift can be quantified as the percentage increase in half-frequency between FF1 and FF2. This percentage increase is shown in Fig. 7C. There was a 33.7 ± 2.8% increase in half-frequency for wild types and a 23.3 ± 4.0% increase for knockouts, but this difference was not statistically significant (Mann-Whitney test).
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By comparing FF3 with FF1 in Fig. 7, A and B, it can be seen that after 30 min, the force at very low and very high frequencies is very close to prefatigue levels; it is only over the middle frequencies that force remains significantly depressed. This is illustrated in Fig. 8, where the force at each frequency following 30-min recovery is expressed as a percentage of the force generated at that frequency before the muscle was fatigued. At very low and very high frequencies, the postrecovery force is 90% of the prefatigue force. However, over middle frequencies, this percentage is considerably lower. Another feature of the graph is that, at very high and very low frequencies, the percentages for both wild types and knockouts are similar, but over intermediate frequencies, wild types have not recovered to the same degree as knockouts. At 37.5, 50, 75, and 100 Hz, the recovery in wild types is significantly less than in knockouts (2-way ANOVA with Bonferroni posttests). As maximum force was usually reached at 150 Hz, these frequencies represent a range that is 25% to 67% of maximum stimulation frequency.
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DISCUSSION |
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-actinin-3 is still in an early stage. We do know that its expression is confined largely to fast glycolytic muscle fibers and that its presence is somehow beneficial for power and sprint athletes at the elite level. One of the proposed roles for this protein is as a mechanical stabilizer of the sarcomere, while another possible function may be as an influencer of signaling and metabolic pathways, enabling a muscle fiber to differentiate toward a fast-twitch, glycolytic profile. The present study and the one by MacArthur et al. (9) represent the first investigations of the physiological properties of isolated skeletal muscle in the -actinin-3 knockout mouse and provide some data against which to evaluate these hypotheses about the protein's functions.
Our findings in the eccentric contraction studies do not appear to support the hypothesis that -actinin-3 mechanically strengthens the sarcomere during extreme muscle activity. Following an eccentric contraction protocol with a large (20%) strain, knockout muscles did not show any more damage than wild-type muscles in any of the measures of damage. Moreover, morphological analysis of muscle fibers of older mice did not reveal any major evidence of repetitive injury in knockout muscles, apart from a very small number of split fibers.
One complicating factor in interpreting these results is the size of the muscle fibers. Fast glycolytic fibers in knockout mice have reduced fiber diameters compared with wild types (9), so there would be less shear stress on the fast fibers in knockouts, thus protecting them from eccentric damage (7). Hence it is still possible that sarcomeres are inherently weaker when they lack -actinin-3, but this is masked by the protective effect of smaller fiber diameter.
Our measurements of muscle stiffness concur with our eccentric contraction studies because no difference was found in muscle stiffness between wild types and knockouts, suggesting that -actinin-3 deficiency did not affect the strength or stiffness of the muscle as a whole.
Besides their interactions with actin, the sarcomeric -actinins are also known to interact with cell-signaling proteins that are involved in fiber type differentiation and with enzymes involved in metabolic pathways. This raises the possibility that one role of -actinin-3 could be to influence fiber type differentiation toward a fast-twitch, glycolytic profile. While there is no evidence of a shift in myosin heavy chain composition from IIB to other isoforms in -actinin-3-deficient muscle, there is evidence that -actinin-3-deficient fibers have higher levels of oxidative enzymes than would be expected in a fast glycolytic fiber (10). Enzyme assays reveal that the activity of key enzymes involved in oxidative metabolism, such as citrate synthase, succinate dehydrogenase, and cytochrome-c oxidase, is significantly higher in knockouts than in wild types, while the anaerobic pathway enzyme, lactate dehydrogenase, has significantly lower activity in knockouts (9). Thus it may also be hypothesized that -actinin-3 somehow helps fast glycolytic fibers to use more anaerobic pathways, and in its absence these fibers resort to more oxidative pathways.
In the present study, we found that some features of the knockout muscle that were consistent with the hypothesis that -actinin-3-deficient fibers are more oxidative than normal. These features were smaller cross-sectional areas, lower twitch-to-tetanus ratios, and better recovery from fatigue.
We found that -actinin-3-deficient muscles had a 9% smaller cross-sectional area than -actinin-3-positive muscles. This is consistent with the fact that the diameter of fast glycolytic fibers is smaller in knockouts than in wild types (9) and supports the hypothesis that these fibers take on more oxidative characteristics when -actinin-3 is absent, because oxidative fibers have smaller diameters than fast glycolytic fibers.
Slow-twitch motor units tend to have lower twitch-to-tetanus ratios than fast-twitch motor units (4). Our finding that knockout muscles had lower twitch-to-tetanus ratios than wild-type muscles is therefore consistent with the -actinin-3-deficient fast glycolytic fibers changing to a more slow-twitch, oxidative profile.
Knockout muscles also recovered better from fatigue than wild-type muscles. At the end of the 30-min recovery period, knockouts had recovered significantly more of their original 100-Hz force than wild types, and the rightward shift of the force-frequency curve, thought to represent impairment of excitation-contraction coupling due to fatigue, was much less pronounced in knockouts than in wild types. The difference in recovery between wild types and knockouts was most apparent at stimulation frequencies that were between 25% and 67% of maximum. At these frequencies, knockouts were able to develop a much higher percentage of their prefatigue force than wild types. The improved recovery from fatigue in knockouts is consistent with the notion that -actinin-3-deficient fibers may change toward the properties of more oxidative fiber types, which are more fatigue-resistant than glycolytic fibers. This also lends support to earlier findings that knockout mice can run 33% further than wild types before exhaustion when subjected to treadmill running (9), and may be one factor behind the increased incidence of -actinin-3 deficiency in endurance athletes (17).
Although wild types and knockouts showed differences in their rates of recovery from fatigue, it can be seen from Fig. 6 that there were no differences in their rates of force decline during the 30 s of fatiguing stimulation. One explanation for this may be the accumulation of extracellular K+ that occurs when muscles are subjected, as they were in the present study, to intense, repeated stimulation in vitro (1). Although only small amounts of K+ leave the muscle fiber during each action potential, repeated action potentials can significantly increase the [K+] inside the lumen of the t-tubules, which comprise only 1% of the total fiber volume but 80% of the total membrane surface area. The increased extracellular [K+] and reduced intracellular [K+] result in membrane depolarization and a reduction of membrane excitability (1).
It is possible that this impaired excitability is the predominant factor underlying the reduction in force during the fatigue protocol in our study. This may have masked any differences in metabolic efficiency between wild types and knockouts during the fatiguing stimulation, so that their forces declined at similar rates. However, [K+] and membrane potential rapidly return to normal once stimulation is stopped (1), so the differences we observed between wild types and knockouts during recovery are likely to be due to metabolic factors, and these differences are consistent with -actinin-3 deficient fibers developing more oxidative properties, enabling knockout muscles to recover more quickly from fatigue.
There is one further piece of evidence suggesting that the contractile properties of -actinin-3-deficient fibers may change toward those of a slower-twitch, more oxidative fiber type. This is the 2.6-ms increase in the twitch half-relaxation time of knockout muscles compared with wild types, as we reported previously in experiments on the same set of muscles (9). Such a change is consistent with the observation that elite sprinters have a very low incidence of -actinin-3 deficiency. Lack of this protein would significantly prolong the time taken for muscles to relax and would thus be detrimental to activities requiring repeated rapid contractions, such as sprinting (2).
To summarize, the present study has provided an overview of some basic contractile properties of the EDL muscle in a new knockout mouse, together with data on its responses to eccentric contractions and fatiguing stimulation. While these data do not appear to support the hypothesis that -actinin-3 provides mechanical protection to the muscle fiber during strenuous physical activity, some results are consistent with the hypothesis that -actinin-3 plays an important role in the differentiation of the fiber toward a fast-twitch, glycolytic profile, and that in its absence the fiber may tend toward a slower-twitch, more oxidative profile. The present study demonstrates that -actinin-3 deficiency does have important effects on physiological function in muscle, and since -actinin-3 deficiency is common in humans, it will be important to determine the effects of such deficiency in situations such as ageing and congenital muscle disease.
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FOOTNOTES |
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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.
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REFERENCES |
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, Significant difference in force between WT and KO at 30 min (P = 0.013). Shaded regions represent 4-min time intervals during which force-frequency (FF) curves were obtained for each muscle. The three curves (FF1, FF2, and FF3) are shown in 
