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First published online March 12, 2009
Journal of Experimental Biology 212, 977-985 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.026625
Changes in efficiency and myosin expression in the small-muscle phenotype of mice selectively bred for high voluntary running activity
1 Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N
1N4, Canada
2 Department of Biology, University of California, Riverside, CA 92521,
USA
* Author for correspondence (e-mail: syme{at}ucalgary.ca)
Accepted 17 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: artificial selection, efficiency, energetics, experimental evolution, multiple solutions, muscle, myosin heavy chain, oxygen, work
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). In
total, this selection experiment is composed of four replicate control lines
(lab designated lines 1,2,4,5), which are bred without regard to wheel
running, and four replicate selected lines (lab designated lines 3,6,7,8),
which are subjected to the selection protocol. Over the first 16 generations
of such breeding, mice in the selected lines ran increasingly more revolutions
per day than controls, eventually reaching an apparent plateau at which they
run 2.5- to 3-fold more revolutions per day at nearly twice the average speed
of control mice (e.g. Swallow et al.,
1999; Girard et al.,
2001; Garland,
2003; Belter et al.,
2004; Rezende et al.,
2006c; Gomes et al.,
2009).
Of particular interest to the present study, two of the selected lines (lab
designations 3 and 6) express a high incidence of a `mini-muscle' phenotype
(Garland et al., 2002), in
which the plantar flexor muscle group is 44–50% the mass of mice with
normal (wild-type) muscles (Garland et
al., 2002; Houle-Leroy et al.,
2003; Belter et al.,
2004; Syme et al.,
2005; Guderley et al.,
2006). The mini-muscle phenotype results from a Mendelian
recessive allele (Hannon et al.,
2008) that occurred at a frequency of 
7% in the founder
population (Garland et al.,
2002). The dramatic increase in frequency of the mini-muscle
phenotype only in two of the selected lines, leading to fixation in line 3 by
generation 36 (Syme et al.,
2005), is best explained by a statistical model in which the
phenotype was favoured by the selection protocol
(Garland et al., 2002). That
the phenotype was lost in the other two selected lines (7 and 8) and in all
four control lines is explainable by random genetic drift
(Garland et al., 2002).
Efforts to identify the mini-muscle gene are underway
(Hartmann et al., 2008).
Aside from the mini-muscle phenotype, which is restricted to two of the
four selected lines, all four selected lines exhibit a number of phenotypes
that appear to represent adaptations to support sustained, aerobically
maintained endurance running. These phenotypes include elevated maximal oxygen
consumption (Rezende et al.,
2006a; Rezende et al.,
2006b; Rezende et al.,
2006c) and running capacity
(Meek et al., 2007) during
forced treadmill exercise, increased insulin-stimulated glucose uptake in
extensor digitorum longus (Dumke et al.,
2001), and larger femoral heads and increased symmetry of hindlimb
bones (Garland and Freeman,
2005; Kelly et al.,
2006). A number of other traits that evolved in the selected lines
may or may not represent adaptations that facilitate increased endurance
running, including reduced body mass
(Swallow et al., 1999),
elevated circulating corticosterone levels
(Girard and Garland, 2002;
Malisch et al., 2009), and
elevated circulating adiponectin levels [Vaanholt et al.
(Vaanholt et al., 2008) and
references therein)].
The mini-muscle phenotype has been associated with characteristics other
than just changes in muscle mass. Some reports indicate that mice possessing
the mini-muscle phenotype have reduced body mass
(Garland et al., 2002;
Guderley et al., 2006;
Guderley et al., 2008), while
others do not (Houle-Leroy et al.,
2003; Guderley et al.,
2008). Mice that possess mini-muscles generally do not differ from
those with normal muscles in time spent on exercise wheels, although they tend
to run significantly faster and, in some samples, further
(Garland et al., 2002;
Kelly et al., 2006;
Gomes et al., 2009). Mice with
mini-muscles exhibit larger heart ventricles
(Garland et al., 2002;
Swallow et al., 2005) and
higher 
O2 max
(maximum rates of oxygen uptake) in hypoxia
(Rezende et al., 2006a;
Rezende et al., 2006b), and
their triceps surae muscles have greater mitochondrial volume densities and
higher mass-specific hexokinase and cytochrome C oxidase activities
(Guderley et al., 2006) (see
also Houle-Leroy et al.,
2003). Also, their medial gastrocnemius muscles contain nearly
twice the mass-specific myoglobin concentration and mass-specific citrate
synthase activity as normal muscles
(Rezende et al., 2006b), and
their gastrocnemius muscles have greatly reduced amounts of myosin heavy chain
type IIb and a large number of very small, unidentifiable muscle
fibres (Guderley et al., 2006;
Guderley et al., 2008) (L. E.
Wong, T.G. and R. T. Hepple, manuscript submitted).
Analysis of contractile characteristics reveals no differences between
soleus muscles in normal and mini-muscle mice, but a shift toward a markedly
slower and more fatigue-resistant medial gastrocnemius in the mini-muscle
phenotype, with slower twitches, a 50–80% reduction in mass-specific
cyclic power production, and a 50% reduction in mass-specific isotonic power
production (Syme et al.,
2005). Collectively, these findings indicate a shift toward a
slower phenotype in some limb muscles. As mice with these mini-muscles may be
better suited to sustained, aerobic activity than their normal counterparts,
this constitutes further evidence that the increased incidence of the
mini-muscle phenotype in the selected lines is an adaptive response to the
selection regimen (see also Garland et
al., 2002; Gomes et al.,
2009).
Slower, more aerobic muscles are commonly held to be more efficient at
producing mechanical work than faster, glycolytic muscles (e.g.
Crow and Kushmerick, 1982;
Curtin and Woledge, 1993;
Barclay, 1994;
Barclay, 1996). Therefore, we
hypothesized that the increase in oxidative capacity and shift to a slower,
more fatigue-resistant phenotype in medial gastrocnemius muscles of
mini-muscle mice would be associated with an increase in efficiency of
contraction compared with the normal medial gastrocnemius. The soleus, showing
no notable difference in contractile properties between mini-muscle and normal
mice (Syme et al., 2005), is
conversely not expected to exhibit a change in fibre type or contraction
efficiency. Such changes, or lack thereof, in these muscles will have
important consequences for both running ability and economy of movement, as
these plantar flexors are key producers of power for both slow and fast
locomotion (Walmsley et al.,
1978; Gregor et al.,
1988; Prilutsky et al.,
1996).
We measured the efficiency of performing cyclic work in soleus and medial gastrocnemius muscles from normal mice (selected lines 7 and 8) and from mini-muscle mice (selected line 3). In addition, we measured the proportion of myosin heavy chain isoforms in the plantar flexor muscles (soleus, medial and lateral gastrocnemius, plantaris) using SDS-PAGE, allowing comparison of potential changes in efficiency with changes in myosin expression. The masses of the ventricles and several limb muscles were also measured to asses differences between the selected lines and relationships with efficiency and myosin expression.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Muscle preparation and apparatus
Mice were weighed and then killed by CO2 inhalation with
subsequent cervical dislocation. The left hindlimb was removed and placed in a
dish containing chilled, physiological saline [in mmol l–1:
144 NaCl, 10 glucose, 6 KCl, 2 CaCl2, 1 MgCl2, 1
NaH2PO4, 1 MgSO4, 10 Hepes, pH 7.4 at
20°C using Tris base: based on Daut and Elzinga
(Daut and Elzinga, 1989)]. Both
the soleus and the medial head of the gastrocnemius were isolated intact with
tendon on either end. The soleus of all mice and the medial gastrocnemius of
M3 mice were relatively small and used intact. The medial gastrocnemius of N7
and N8 mice were relatively large (normal sized) and so were dissected to
approximately one half of their original width and then muscle fibres from the
proximal end were cleared from the tendon to produce preparations comparable
in mass to those from M3 (see Results). Great care was taken to minimize
damage to the remaining muscle bundle, and to ensure the location of the
segment of muscle that remained was consistent across preparations. Further
efforts to assess consistency are described in the section discussing myosin
heavy chain sampling.
Muscles were secured by their tendons in a glass chamber for measurement of
work and oxygen consumption; see Trinh and Syme
(Trinh and Syme, 2007) for
details of the measuring apparatus and calculation of energy use from oxygen
consumed by the muscles. One end of the muscle was secured to a rigid pin and
the other end to an ergometer (model 350, Cambridge Technology, Cambridge, MA,
USA), which controlled muscle length and measured force production. The length
of the muscle was adjusted to remove visible slack. A bipolar stimulus pulse,
1 ms duration, was applied to the muscle via the attachment pins, and
the stimulus voltage was set to 150% of that required to elicit maximal twitch
force. Muscle fibre length was then varied systematically until the length
giving maximal, isometric force (using double stimulus pulses, 10 ms spacing)
was found. This length was measured using a calibrated ocular micrometer on a
stereomicroscope. The temperature of the chamber was maintained at
20°C.
Measurement of work and oxygen consumption
Different measurement protocols were employed for the soleus and medial
gastrocnemius muscles, each designed to result in the muscles producing near
maximal power and consuming oxygen in support of aerobic work without causing
fatigue. Work and power output were assessed using the work-loop technique
(Josephson, 1985). For the
soleus, muscle length was cycled in a sinusoidal trajectory at a frequency of
2 Hz. For the medial gastrocnemius, muscle length was cycled at a frequency of
4 Hz. The amplitude of muscle strain was 10% peak-to-peak. These frequencies
and strains were selected to approximately maximize power output from the
muscles at 20°C based on preliminary experiments and predictions from
previous studies on mouse muscles at 35°C
(James et al., 1995) and
27°C (Syme et al., 2005).
The muscles were stimulated phasically during the length cycles with a
stimulus pulse frequency of 100 Hz using stimulus durations and phases that
maximized net work production for each preparation: typically 140 ms duration
(28% duty cycle) and 15% phase for the soleus, and 70–90 ms duration
(32% duty cycle) and 10% phase for the medial gastrocnemius.
To assess efficiency of work production, the muscles were made to work under the conditions described above while their oxygen consumption was simultaneously measured. Before measurements began, the muscle chamber was flushed with saline with a partial pressure of oxygen (PO2) of about 70 kPa. The PO2 of the saline in the chamber then declined gradually over the course of the experiment as the muscle consumed oxygen, typically ending near 35 kPa. The PO2 of the saline bathing the muscle was measured every second (PSt3 fibre-optic oxygen probe connected to a Fibox 3 oxygen metre, PreSens Precision Sensing GmbH, Regensburg, Germany) and logged to a computer.
At the beginning of each experiment, the decline in PO2 of the saline due to resting muscle metabolism was measured for about 20 min to obtain a reliable baseline. The muscle was then activated to perform work, causing an increase in the rate of decline of PO2 in the chamber. For the medial gastrocnemius of N7 and N8 mice, the muscle was subjected to three cycles of work in sequence followed by a 90 s rest; this was repeated 10 times for a total of 30 work cycles. For the soleus muscles of all lines and the medial gastrocnemius of M3 mice, the muscle was subjected to three cycles of work in sequence followed by a 60 s rest; this was repeated 20 times for a total of 60 work cycles. These protocols were devised to ensure deflections of the oxygen trace that could be resolved reliably, yet not result in fatigue. After the bouts of work were completed and the muscle was again resting, the rate of decline of PO2 recovered back to the baseline level over a period of approximately 10 min for the soleus muscle and about 20 min for the medial gastrocnemius, and was measured for an additional 10–20 min to ensure a resting rate had been re-attained.
The change in PO2 in the saline as a result of the muscle being active and performing work was then calculated from the oxygen records, and converted to joule equivalents of energy released by oxidizeable substrates in a mixed diet, assuming 450 kJ mol–1 O2 consumed [see Syme and Trinh (Syme and Trinh, 2007) for details and rationale]. Muscle efficiency was then calculated by dividing the net work done during the entire series of contractions by the energy released via oxidation of substrates, as assessed from oxygen consumption.
Adequate diffusion of oxygen into the muscle is important to ensure that
oxidative metabolism supports muscle contraction and that measures of oxygen
consumption are a faithful representation of energy used by the muscle during
contraction. A number of precautions were taken to ensure this was the case.
(1) The muscles were activated for only three cycles in succession, followed
by 60–90 s of rest before the next contractions. Based on previous
experience with these preparations, this is a very modest work rate that could
be sustained for hours without signs of fatigue suggestive of anaerobic
metabolism. (2) Measures of oxygen consumption following termination of
stimulation continued for about 30 min, considerably longer than required to
attain rates of oxygen consumption similar to those in resting muscle before
stimulation. This ensured that recovery processes were complete and that any
potential glycolytic activity during contractions would be accounted for in
oxidative recovery. (3) The temperature at which the experiments were
conducted was reduced to 20°C, which will markedly reduce metabolic
demand, both resting and active. Although a reduced temperature may alter the
power from what might occur under normal body temperatures in these mice
(Rhodes et al., 2000),
evidence suggest that such temperature changes will have little, if any,
effect on efficiency (Smith et al.,
2005), and only a relative comparison of efficiencies between the
normal and mini-muscle phenotype was required for the purposes of this study.
(4) Finally, the sizes of the muscle preparations were relatively small, and
the gastrocnemius preparations of normal mice were further reduced to
approximately half their original mass (mini-muscles were already relatively
small). Barclay (Barclay, 2005)
estimated the maximal radii of muscles that could sustain aerobic activity to
the core relying on diffusion alone when bathed in saline perfused with 95%
oxygen at 20°C and subjected to an activation duty cycle of 30% (as in the
present study): for mouse soleus, this radius was about 0.4 mm, and for mouse
EDL (gastrocnemius in our study being not quite as fast and thus allowing a
larger maximal radius) it was about 0.25 mm. Based on the masses and lengths
of the preparations used in the present study (see Results), and assuming a
cylindrical cross-section as did Barclay
(Barclay, 2005), we estimate
the radii to be M3 soleus=0.15 mm, N7 and N8 soleus=0.11 mm for both, M3
medial gastrocnemius=0.35 mm, and experimental segments of N7 and N8 medial
gastrocnemius=0.39 mm for both. Thus, the soleus samples were easily small
enough to support oxidative metabolism through diffusion alone, whereas the
gastrocnemius muscles were slightly large. However, the muscles were exposed
to a stimulation duty cycle of about 30% only during three consecutive cycles
and then were allowed to rest for 60–90 s before subsequent
contractions. Therefore, the functional activation duty cycles were about 7%
for the soleus and 3% for the medial gastrocnemius, placing the radii of the
experimental samples well below those required to satisfy aerobic activity
based on diffusion alone.
At the end of the experiments, muscles were removed from the chamber, the tendons were cut from the muscle, and the muscles were cleared of visible fatty tissue. To obtain consistent masses of these small preparations, they were stored in centrifuge tubes at –70°C, then upon completion of all experiments the muscles were freeze dried, weighed using a Mettler MT5 microbalance, and associated wet masses of the muscles were calculated using an experimentally determined wet/dry mass ratio measured from samples of larger muscles, where measurements of wet mass are more consistent. Work is expressed relative to this muscle wet mass (J kg–1).
Myosin heavy chain isoform analysis
To provide further insight into potential differences in running ability
and muscle efficiency among lines and between mini-muscle and normal
phenotypes, the proportions of myosin heavy chain (MHC) isoforms in the
plantaris, soleus and the medial and lateral heads of the gastrocnemius were
determined using SDS-PAGE. Furthermore, to determine if the segments of medial
gastrocnemius used for measurement of muscle efficiency were representative of
the entire medial head of the gastrocnemius, the MHC isoforms were analysed in
both the segments and intact medial head.
MHC was isolated and purified based on established procedures
(Talmadge and Roy, 1993), with
all homogenization and purification steps being performed on ice. The muscles
were homogenized using a motor-driven mortar and pestle in a solution of 250
mmol l–1 sucrose, 100 mmol l–1 KCl, 5 mmol
l–1 EDTA and 10 mmol l–1 Tris base, pH 6.8.
The homogenate was centrifuged at 3000 g for 10 min at 3°C
and the supernatant was discarded. The pellet was resusupended in a solution
of 150 mmol l–1 KCl, 10 mmol l–1 Tris base
and 0.5% Triton X-100, pH 6.8, homogenized, centrifuged as above, and the
supernatant discarded. The pellet was resusupended in a solution of 150 mmol
l–1 KCl and 10 mmol l–1 Tris base, pH 7.0,
homogenized, centrifuged as above, and this final procedure repeated three
more times. Upon final centrifugation, the supernatant was discarded and the
pellet was suspended in enough 150 mmol l–1 KCl and 10 mmol
l–1 Tris base pH 7.0 to cover the pellet, along with
approximately 300 µl of protein sample buffer. The sample was boiled for 5
min, and 25 µl was loaded into the wells of the gel [prepared as per
Talmadge and Roy (Talmadge and Roy,
1993)]. Gel electrophoresis was performed using a Bio-Rad
Mini-Protean III gel unit in a refrigerator at 4°C for 48 h at a constant
100 V (Accu Power model 500, VWR Scientific Products, West Chester, PA, USA).
Gels were stained using Coomassie Blue; some gels with a low protein
concentration were silver stained following directions in the kit (Bio-Rad
silver stain kit, Hercules, CA, USA). Gels were photographed using Bio-Rad Gel
doc 2000, and images stored as tiff files using Quantity One (Bio-Rad). Band
(MHC isoform) density was quantified using Scion Image for Windows (based on
NIH Image, National Institute of Health, Frederick, MA, USA). Band densities
were then expressed as a percentage of the sum of all MHC bands in a lane. In
some instances it was not possible to clearly resolve the separation between
MHC types IIa and IIx, which run in very close proximity
on the gel. Thus, results are also presented as type IIa+x for all
samples, where the two bands were treated as one. Where it was possible to
resolve these bands reliably, the data for type IIa and
IIx MHC are also presented separately.
Statistical analyses
Data were transformed when necessary to satisfy the requirement of normal
residuals from the statistical model (e.g. arcsine for the MHC data). Most
comparisons among lines employed one-way analysis of variance followed by
Holm–Sidak tests. However, for organ masses, which would be expected to
correlate positively with overall body mass, we used analysis of covariance
(ANCOVA) with log10 body mass as the covariate and a planned
contrast of line 3 mini-muscle versus lines 7 and 8 normal (using SAS
Procedure Mixed). For some of the organ mass ANCOVAs, the partial regression
coefficient for body mass was negative, apparently because of one or two heavy
mice with a large amount of body fat noted at dissection. In these cases, we
re-ran the analysis without body mass as the covariate. We also re-ran
analyses without the single heaviest individual to verify that results did not
change in any appreciable way. As they did not, all individuals were included
in statistical analyses.
Differences were considered significant at P<0.05. As the Holm–Sidak procedure adjusts the level of significance for repeated comparisons, and because we stress only differences that are large and clear and are not attempting to tease out small or marginal effects, we did not apply further adjustments to the level of significance for multiple comparisons. All data are presented as means with s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Masses of the experimental samples used for measurement of efficiency were: M3 medial gastrocnemius 13.9±1.46 mg, M3 soleus 8.91±0.960 mg, N7 medial gastrocnemius 16.1±2.21 mg, N7 soleus 5.64±0.191 mg, N8 medial gastrocnemius 17.0±0.767 mg, N8 soleus 6.03±0.203 mg.
Muscle efficiency
Examples of work loops as recorded during measures of efficiency for soleus
and medial gastrocnemius muscles from each line are shown in
Fig. 1. There were no notable
differences in the shapes of the loops between soleus preparations from the
different lines, suggesting similar abilities to produce force and rates of
contraction and relaxation. While the shapes of the loops between medial
gastrocnemius preparations from the different lines did not differ
substantially, again suggesting similar rates of contraction and relaxation,
muscles from line 3 tended to produce less force than muscles from lines 7 and
8. The net work done per cycle by the medial gastrocnemius averaged
2.32±0.291 J kg–1 for M3 (N=6),
7.48±1.24 J kg–1 for N7 (N=9) and
9.17±0.933 J kg–1 for N8 (N=10). The
mass-specific work output of the M3 gastrocnemius was significantly less
(F2,25=9.455, P<0.001) than that done by N7
and N8, with no difference between N7 and N8. The equivalent power output of
the medial gastrocnemius with a cycle frequency of 4 Hz was 9.3 W
kg–1 for M3, 29.9 W kg–1 for N7 and 36.7 W
kg–1 for N8. The mass-specific net work done per cycle by the
soleus averaged 4.42±0.436 J kg–1 for M3
(N=9), 4.69±0.441 J kg–1 for N7
(N=9) and 4.52±0.477 J kg–1 for N8
(N=10). There were no statistically significant differences among
lines (F2,28=0.092, P=0.912). The equivalent
power output for the soleus with a cycle frequency of 2 Hz was 8.8 W
kg–1 for M3, 9.4 W kg–1 for N7 and 9.0 W
kg–1 for N8.
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Efficiency was calculated as the ratio of net work performed by the muscle to energy released by oxidizeable substrates based on the amount of oxygen consumed. The efficiency of soleus muscles from M3 (15.2±1.03%), N7 (14.4±1.08%) and N8 mice (14.3±1.29%) did not differ among the three lines studied (P=0.843; Fig. 2). Perhaps surprisingly, the efficiency of medial gastrocnemius from M3 mice (13.0±1.67%) was significantly lower than the efficiency of medial gastrocnemius from N7 (19.3±0.97%) and N8 mice (19.5±0.83%; P<0.001; Fig. 2).
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The MHC isoform profile of the plantaris muscle in the different lines was similar to that of the medial and lateral heads of the gastrocnemius, with a shift toward a slower profile in M3 mice (Fig. 7). M3 mice showed a large decrease in type IIb MHC (P<0.001) and increases in type I (P<0.001) and types IIa+x (P<0.001). The increases in type IIx and IIa MHC in M3 mice were more modest than observed in the gastrocnemius, being statistically significant only from N8 mice. Of interest, N7 mice had significantly less type IIb MHC and more type IIa+x MHC than N8 mice (P<0.001).
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Comparison of experimental samples with whole muscle
As discussed above, it was necessary to use segments of the larger medial
gastrocnemius muscles from N7 and N8 mice rather than the entire muscle when
measuring efficiency. Although care was taken to ensure that samples were
removed from the same anatomical region of the muscle for each experiment,
this did not ensure that the dissected segments were representative of the
whole muscle. To assess this, MHC analysis was performed on both the
experimental segments and whole medial gastrocnemius muscles taken from the
contralateral leg of N7 and N8 mice. No significant differences in MHC content
were found for any comparisons between experimental and whole muscles for type
I, IIa+x, IIa, IIx or IIb isoforms
from N7 mice (P>0.37–0.74) or N8 mice
(P>0.15–0.84). This demonstrates that the experimental
segments of medial gastrocnemius used in the efficiency experiments were
representative of the whole muscles.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). The lack
of change in the soleus probably reflects the very small change in myosin
expression toward faster isoforms (Fig.
4), while the slowing of contractile performance in the
gastrocnemius muscles of mini-muscle mice probably reflects the loss of type
IIb myosin and increased type IIa,x and I myosin
(Guderley et al., 2006;
Guderley et al., 2008) (Figs
5 and
6). The shift toward a
marginally faster phenotype (and larger mass) in soleus muscles of mini-muscle
mice may endow an enhanced ability to run faster on the wheels than normal
mice (Kelly et al., 2006;
Gomes et al., 2009), while
still retaining the putative benefits of a slower phenotype (discussed below).
The shift toward a much slower phenotype in the gastrocnemius and plantaris
muscles of mini-muscle mice may reflect selection for sustained, but not
necessarily high-speed, running. Of interest, lines 7 and 8, which lack the
mini-muscle phenotype, have responded similarly to the selection protocol as
has line 3 in terms of their amount of wheel running. This indicates that high
voluntary running can be accomplished in more than one way with respect to
muscle anatomy and physiology, which is an example of multiple adaptive
solutions in response to selection (see also
Garland, 2003;
Houle-Leroy et al., 2003;
Swallow et al., 2009).
As the mini-muscle phenotype appears to be favoured by selection for high,
sustained activity (Garland et al.,
2002; Gomes et al.,
2009), we hypothesized that the slower, mini-muscle phenotype of
gastrocnemius may be accompanied by greater muscle efficiency and favour more
economical running as compared to the normal muscle condition. Based on the
lack of change in contractile performance in the soleus of mini-muscle mice
(Syme et al., 2005) and given
the modest differences in the proportions of the MHC isoforms in the mini and
normal soleus (Fig. 4), we did
not anticipate a notable change in efficiency of the soleus. In support of the
latter, the efficiency of soleus muscle was not statistically different
between the normal and mini-muscle phenotypes
(Fig. 2). However, in contrast
to expectations for the medial gastrocnemius, we noted a reduced efficiency of
medial gastrocnemius from M3 mice compared with that from N7 and N8 mice
(Fig. 2), despite the
considerably slower contractile phenotype
(Syme et al., 2005) and
differences in MHC isoforms (Fig.
5) in M3 compared with N7 and N8 gastrocnemius (see also
Guderley et al., 2006;
Guderley et al., 2008).
Other observations comparing efficiency of fast and slow muscles also lead
to questions about the functional and adaptive basis for the slower
mini-muscle phenotype. Muscle efficiencies measured from the slower soleus and
faster medial gastrocnemius of mice expressing the normal phenotype (N7 and
N8) were likewise not consistent with a notion that slower muscles are more
efficient than faster muscles (Fig.
2). Our observations were conspicuously similar to those from
studies using slow and fast rat muscle during shortening contractions and
using oxygen analysis to assess energy use; 19% efficient for the relatively
fast EDL shortening at 1.0 muscle length s–1, and 15%
efficient for the slow soleus shortening at 0.5 muscle lengths
s–1 (Heglund and Cavagna,
1987). Barclay and Weber
(Barclay and Weber, 2004) also
argue, based on initial and recovery processes in mouse muscle, that net
efficiencies of fast and slow muscles do not appear to be different.
Furthermore, Smith et al. (Smith et al.,
2005) provide a preliminary rationale for why an unfailing
relationship between efficiency and fibre types may not exist. Thus, despite
well-established expectations that slow muscles are inherently more efficient
than fast (see Introduction), there are clearly either exceptions or
conditions under which this is not the case.
One potential complication in interpreting and comparing measures of
efficiency between fast and slow muscle (soleus versus gastrocnemius
and/or normal versus mini-muscle) is that each possesses a different
(at the time unknown) MHC profile, and thus has different force–velocity
properties and capacities for contraction kinetics. Yet the cycle frequency
for work measurements (i.e. rate of shortening and extension) was maintained
constant across all gastrocnemius preparations (4 Hz) and all soleus
preparations (2 Hz) to facilitate direct comparisons between the muscles.
Thus, faster and slower preparations would be operating at different relative
velocities of shortening and load, and with different abilities to contract
and relax at the rates imposed. As efficiency is affected by load and velocity
(Smith et al., 2005), these
relative and absolute differences between conditions under which efficiency
was measured may confound our ability to make direct comparisons. However,
when muscles are operating at the loads and velocities near which they produce
maximal power, efficiency is relatively insensitive to either load or velocity
(Smith et al., 2005). Our
protocols were designed to produce near-maximal power, so we expect efficiency
to be near maximal for each preparation. Furthermore, the modest changes in
MHC composition between normal and mini soleus would probably minimize the
effect of this complication. The larger changes in MHC composition of the
medial gastrocnemius would result in the M3 muscles working at relatively
faster velocities than the N7 and N8 muscles, but efficiency is maximized over
a much broader range of velocities in faster than in slow muscles
(Smith et al., 2005;
Reggiani et al., 1997), so the
impact will be lessened. Activation phase and duration were optimized
individually for each preparation, and so changes in MHC composition between
preparations was accounted for. However, it cannot be argued with certainty
that differences in efficiency between normal and mini gastrocnemius
preparations are due entirely to inherent differences in the physiology of the
muscle itself. Furthermore, the present study only describes the efficiency of
the muscles when operating under conditions for near maximal power output,
which in an absolute sense will probably be different from the efficiency when
operating under conditions as experienced in vivo.
Mice from the selected lines primarily run faster than control lines, and
only in males is the increase in amount of time spent running statistically
significant (Swallow et al.,
1998; Girard et al.,
2001; Garland,
2003; Belter et al.,
2004; Swallow et al.,
2005; Rezende et al.,
2006c). Likewise, mini-muscle individuals run faster than normal
mice (Kelly et al., 2006;
Hannon et al., 2008;
Gomes et al., 2009). In the
context of selective breeding for higher sustained running activity, the
evolution of a slower, more economical muscle phenotype would seem to be
beneficial. Yet the apparent lack of an increase in efficiency of the
functionally and physiologically slower gastrocnemius muscle of mini-muscle
mice lends support to the notion that the benefit of slower muscle may not be
an increase in efficiency per se. In mice with ad-libitum
access to food and water, living in a controlled and hospitable caged
environment, and where enhanced fecundity is not a trait under direct
selection (Girard et al.,
2002), increased muscle efficiency for routine running might not
be an important selective advantage
(Swallow et al., 2001) [see
also Vaanholt et al. and references therein
(Vaanholt et al., 2007)],
particularly given that selected mice with normal muscles show similar
enhancements in running behaviour (wheel revolutions per day).
Rather, perhaps the advantage of slower muscle is a reduced overall rate of
energy use (Barclay and Weber,
2004), making them more suitable for sustained tasks or for
completing a task with less reliance on anaerobic metabolism. Reduced energy
use may have permissive effects on the behaviour of high voluntary wheel
running. For example, a slower, more oxidative mini-muscle phenotype may allow
the mice to run faster without significant accumulation of anaerobic
by-products, which appear to promote discomfort
(Pan et al., 1999;
Immke and McCleskey, 2001;
Yagi et al., 2006), and may
inhibit volition to exercise (see also Li
et al., 2004; Keeney et al.,
2008). In other words, the mini-muscle phenotype may confer an
advantage by enhancing the ability of the mice to run on wheels through
reduced reliance on glycolytic metabolism. If the mini-muscle phenotype
confers such an advantage in wheel-running ability, then mice with the normal
phenotype in the selected lines, which also show enhanced running ability over
control mice, must have adopted an alternate adaptive response. Perhaps normal
mice in the selected lines have overcome inhibitory effects related to the use
of faster, more glycolytic muscles through increased behavioural tolerance of
discomforts associated with endurance running. Although Li et al.
(Li et al., 2004) did not find
significant differences in pain tolerance between the control lines and those
selected for high activity, it is not known if such differences might exist
among the four selected lines (see also
Keeney et al., 2008). In
addition, the observation that selected mice with the normal phenotype run
slower than those with the mini-muscle phenotype
(Kelly et al., 2006;
Hannon et al., 2008;
Gomes et al., 2009) may be of
consequence in this context.
With regard to masses of the muscles
(Table 1), previous studies
(Garland et al., 2002;
Houle-Leroy et al., 2003;
Syme et al., 2005;
Guderley et al., 2006;
Guderley et al., 2008;
Gomes et al., 2009) have
reported differences in masses of the plantar flexor muscles between
mini-muscle and normal mice. We further note that the mini-muscle phenotype is
not restricted to the plantar flexors, with muscles of the upper forelimb
being reduced by about one third in mass in the M3 mice compared with normal,
and the quadriceps of M3 mice being approximately half the mass of N7 and N8
mice. Unexpectedly, in N7 mice the mass of the lower forelimb muscles was less
compared with both N8 and M3 (Table
1). Such observations of variability not clearly associated with
selective breeding or the normal/mini phenotypes point to the importance of
random genetic drift and/or alternate responses to uniform and well-defined
selection, especially with such complex traits as voluntary activity levels
(Swallow et al., 2005;
Swallow et al., 2009;
Garland and Kelly, 2006).
Future studies will address whether the identified differences in mass,
efficiency (this study) and contractile properties
(Syme et al., 2005) of
mini-muscles are reflected in whole-animal cost of transport
(Rezende et al., 2006c),
maximal sprint-running speed (Djawdan and
Garland, 1988) or endurance capacity.
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Significantly different (P<0.01) from
N8.
