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First published online September 14, 2007
Journal of Experimental Biology 210, 3356-3360 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007088
Chronic electrical stimulation drives mitochondrial biogenesis in skeletal muscle of a lizard, Varanus exanthematicus
1 Department of Zoology, Miami University, Oxford, OH 45056, USA
2 Department of Biological Sciences, Northern Arizona University Flagstaff,
AZ, USA
3 Department of Physiology, University of Arizona School of Medicine,
Tucson, AZ, USA
* Author for correspondence (e-mail: schaefpj{at}muohio.edu)
Accepted 29 May 2007
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Summary |
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Key words: muscle plasticity, sarcoplasmic reticulum, intracellular lipid, exercise, activity
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Introduction |
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). In their
groundbreaking report, Buller et al. demonstrated that the functional
characteristics of muscle, rather than being genetically fixed, reflect the
type of nervous input that the muscle receives
(Buller et al., 1960). Later,
Salmons and Vrbova developed the technique of using chronic electrical
stimulation (CES). By eliminating normal neuronal input in exogenously
activated muscle, they were able to critically differentiate between secreted
neural factors and the pattern of activation. They found that stimulation of
extensor digitorum longus and tibialis anterior muscle by implanted electrodes
in both cats and rabbits led to characteristic fast-to-slow shifts in its
metabolic and contractile properties
(Salmons and Vrbova, 1969).
Through the intervening 40 years, mammalian muscle is now considered among the
most malleable tissues in the body, capable of dramatic changes in both
structure and function in response to changes in the nature as well as
magnitude of the demand placed on it.
Skeletal muscle phenotypic plasticity involves structural and functional
changes in both the metabolic and contractile properties of skeletal muscle.
Evidence of the capacity for muscle plasticity is provided by the dramatic
transformations seen in skeletal muscle in response to strength and endurance
training (Flück, 2006),
disuse atrophy (Jackman and Kandarian,
2004), and CES (Pette and
Vrbova, 1999; Pette,
2001). The `fast-to-slow' transition in muscle type seen following
CES leads to a muscle with a well characterized assemblage of proteins that
are associated with slower contraction velocities as well as higher metabolic
capacity and endurance (Booth and Baldwin,
1996). In other words, they possess the complete suite of
characteristics common to slow fibers.
The study of muscle plasticity has been very active for more than 40 years
and much knowledge has been gained regarding the nature of the changes that
occur in response to altered use. Sufficient demonstration of phenotypic
plasticity in mammalian skeletal muscle now exists to invite the question as
to whether this is a unique evolved property of the mammals. Studies of avian
muscle plasticity are less extensive; however, evidence suggests that
plasticity is an avian feature as well. The mass of flight muscle relative to
body mass increases both in preparation for migration
(Piersma, 1998) as well as
during moult, when relative lift is reduced
(Lind and Jakobsson, 2001).
The cellular phenotype of these hypertrophying muscles remains unknown.
However, when muscle hypertrophy was induced by stretch overload, contractile
properties slowed, suggesting a phenotypic shift
(Alway, 1994). In addition to
shifts in contractile properties, metabolic properties of avian muscle also
appear to be plastic. In ducklings, shivering induced by cold exposure
resulted in increases in cytochrome oxidase activity
(Barré et al., 1987) and
increased reliance on fatty acid utilization by skeletal muscle
(Bénistant et al.,
1998). Studies of amphibian and reptilian muscle show that
skeletal muscle properties correlate well with life history and behavior
(Bonine et al., 2001), but
there remains little information on muscle plasticity within an individual
lifespan in these animals. There are considerable experimental challenges in
altering muscle activity in amphibians and reptiles, not only due to
motivation to exercise but also the limited capacity of the lungs and heart to
supply sufficient oxygen to support increased aerobic activity. Endurance
exercise training of Amphibolurus nuchalis, an Australian agamid
lizard, failed to result in an adaptive response
(Garland et al., 1987).
Nonetheless, modest fast-to-slow shifts in myosin isoforms and ATPase activity
were described following `forced terrestrial stepping', a moderate exercise
stimulus, in an Urodelan amphibian (Launay
et al., 1998). Organismal limitations bypassed with short term,
direct electrical stimulation of the gastrocnemius muscle in the Indian green
frog (Rana hexadactyla) revealed increased mitochondrial protein
content and cytochrome c oxidase activity
(Moorthy et al., 1981). These
data suggest that phenotypic plasticity of muscle, rather than a derived
character reliant upon a high capacity oxygen delivery system, as found in
endotherms, may be ancestral.
In the present study we investigate the effects of chronic electrical stimulation on skeletal muscle mitochondrial biogenesis in the Savannah monitor lizard Varanus exanthematicus. V. exanthematicus is a wide-ranging active predator with relatively high metabolic capacity and thus represents a species within the Squamata in which skeletal muscle may be likely to be capable of acclimative plasticity in response to alterations in activity level.
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Materials and methods |
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Surgery
To implant the stimulating electrodes, animals were anesthetized with
halothane and the iliofibularis muscle of the right hind limb was isolated.
This muscle contains two distinct anatomical areas, which are unambiguously
divided into `white' and `red' regions. Two 32-gauge, multi-stranded stainless
steel wires (California Fine Wire, Grover Beach, CA, USA) were implanted into
the white section of the muscle approximately one quarter muscle length from
each end and secured using a small dab of tissue glue. The electrode wires
were sutured to the skin at their point of exit near the pelvis and then ran
to a 3 cmx3 cm `backpack' plug attached to the lizard's back.
Stimulation protocol
Electrical stimulation was begun the day following electrode implantation
by connecting the backpack plug with a Grass stimulator (Model 48, Grass
Instruments, West Warwick, RI, USA). Stimulating voltage was set visually to
be sufficient to cause a noticeable tremor of the hind leg. This was a voltage
of 1–3 V for a duration of 2 ms. Stimulation frequency was initially 4
Hz (the maximum without tetany) for the first few days and was gradually
increased over a period of 2 weeks until a maximum of 10 Hz was achieved.
Stimulation at 10 Hz proceeded for 8 h per day, 5 days a week, for 6 weeks.
During the 8-week period the voltage was adjusted daily to invoke the leg
tremor. In general, the voltage was increased during the course of the
experiment for each animal; the highest end-experiment voltage was 5 V.
Muscle samples
Following 6 weeks of muscle stimulation, the animals were sacrificed with a
lethal dose of sodium pentobarbitol. Both the stimulated iliofibularis and its
contralateral control muscle were immediately dissected out. Samples from the
white region of the muscle were cut into small strips, fixed in 6.25%
gluteraldehyde buffered with 0.1 mol l–1 sodium cacodylate,
pH 7.4 and processed for electron microscopy as described
(Schaeffer et al., 2003).
Transverse sections were cut at a thickness of 60–100 nm, stained in 2%
aqueous uranyl acetate for 20 min, alkaline lead citrate for 10 min and washed
in 50% ethanol. Examination and photography of the grids was done using a JEOL
1200 transmission electron microscope.
Stereology
Quantitative analysis of intracellular volume densities was accomplished
using published techniques (Weibel,
1979). Eleven resin blocks were made for each of the stimulated
and control muscles. Five blocks for each condition were chosen randomly and
sectioned. Four micrographs were taken from each grid at a magnification of
4000x. For stereological analysis, the micrographs were magnified
6.5x (final magnification 26 000x) and projected onto a 100-point
grid with an Ausjena projection unit. Because half of each micrograph covered
the grid, 200 points were counted for each micrograph for a total of 4000
points per sample using the Stepone stereology software
(Wainschtein and Cruz-Orive,
1994). Volume densities of mitochondria, myofibrils, sarcoplasmic
reticulum and lipid droplets were calculated following Weibel
(Weibel, 1979).
Statistical analysis
In all cases, statistical comparisons were made for each cellular structure
between electrically stimulated muscle and non-stimulated control muscle using
a Student's t-test. The level of significance was set at
P<0.05 in all cases. All data are presented as means ±
standard error of the mean (s.e.m.) with N=4.
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Discussion |
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).
Mitochondrial biogenesis is an integral component of the fast-to-slow fiber
type switch to support increased activity
(Hood et al., 2006). We began
this study by questioning whether phenotypic plasticity of muscle
mitochondria, intracellular lipid stores and excitation–contraction
coupling is a unique (i.e. derived) feature of mammals, or alternatively is a
`primitive' or ancestral trait of vertebrate skeletal muscle. In other words,
is vertebrate skeletal muscle inherently structurally responsive to activity
level or is the observed phenotypic plasticity a property that evolved,
perhaps subsequent to respiratory and cardiovascular systems capable of
supplying oxygen to a large mass of aerobic muscle fibers? If the evolution of
muscle plasticity required the prior evolution of endothermy or highly
developed aerobic metabolism, then birds may exhibit this trait as well. This
seems to be the case, based on data from chronic stretch overload
(Alway, 1994), cold exposure
(Barré et al., 1987;
Bénistant et al., 1998)
and treadmill exercise training
(Brackenbury and Holloway,
1991).
In order to investigate this question we chose an experimental paradigm
that was not intended to mimic, necessarily, the kinds of stimulation patterns
found in nature. Rather we employed chronic electrical stimulation, as it
circumvents any constraints on oxygen delivery that may limit the muscle's
responsiveness. Using this method, a single muscle can be targeted and the
need for a training regime eliminated. Thus, if the muscle were capable of
demonstrating shifts in contractile and metabolic properties, CES should be
the most powerful stimulus to induce that response. When this technique is
employed with various mammalian species, a profound alteration of skeletal
muscle phenotype is observed (Pette and
Vrbova, 1999). One example of CES in non-mammalian species is
provided by the experiments of Moorthy et al.
(Moorthy et al., 1981).
Although the time course of their experiments was very short, upregulation of
mitochondrial protein content and cytochrome c oxidase activity in
stimulated frog muscle supports the proposal that muscle plasticity is an
ancestral trait of tetrapods.
Our control values for mitochondrial volume density in un-stimulated muscle
are similar to those reported for hindlimb muscle of Cuban iguanas (2.94%;
Cyclura nubila) (Conley et al.,
1989) and from the `white' portion of the iliofibularis muscle
from Agama pallida (3.2%)
(Abu-Ghalyun, 1995),
Dipsosaurus dorsalis (3.8%)
(Gleeson et al., 1984) and
Varanus exanthematicus (2%)
(Mutungi, 1990). Thus even
though the animals in this study were relatively sedentary, the inactivity
imposed by laboratory housing did not appear to have an appreciable effect on
the ultrastructure of their white muscle. In these species, the `red' portion
of the muscle, containing more oxidative fiber types, was reported to possess
considerably higher mitochondrial volume density (7–12%), higher than we
found in the electrically stimulated iliofibularis in the present study
(Gleeson et al., 1984;
Mutungi, 1990;
Abu-Ghalyun, 1995). In the
present study, the lizards were very inactive, perhaps explaining why the
control iliofibularis muscle had slightly lower mitochondrial volume density
than previously reported, although this difference is small. As inactivity
favors a `fast' phenotype, this had to be overcome for CES to drive the
observed fast-to-slow skeletal muscle transition, demonstrating the strength
of the intervention. Additionally, in these studies we used the iliofibularis
muscle from the contralateral limb as a control muscle. It is possible that
circulating factors released from the stimulated limb could influence our
control muscle. However, we saw no evidence that this occurred and indeed, if
this is an important variable, it would serve only to reduce the magnitude of
the response, making our conclusions more robust.
The results of this study suggest that skeletal muscle mitochondria, SR
structure and lipid stores all respond to chronic electrical stimulation, even
in the Savannah monitor lizard, a vertebrate with limited aerobic capacity
(Wang and Hicks, 2004), and in
much the same way as they do in mammalian species. Higher mitochondrial volume
as well as lower sarcoplasmic reticulum volume reflects a shift toward slower,
more oxidative fibers. Our data thus support the idea that, in addition to
sharing common mechanisms of contraction and metabolism across vertebrate
taxa, another common feature of adult vertebrate skeletal muscle that may have
been set early in evolution is phenotypic plasticity.
In general the varanids have relatively more highly developed respiratory
and cardiovascular systems compared to other reptiles; they could be capable
of the levels of activity necessary to demonstrate this phenotypic plasticity.
One may wonder about the adaptive value of a trait in animals that appear
unlikely to exploit it. However, across evolutionary time scales, the capacity
of muscle to respond to altered activity demands may be a critical first step
towards adaptive change, leading to the wide variation in muscle structure and
function such as exists in the Iguanidae
(Bonine et al., 2001). Given
that muscle function is a critical component of the ability of animals to
forage, escape predation and seek mates, and is thus likely linked with
fitness (Irschick and Garland,
2001), muscle plasticity may have played a critical role in the
evolutionary diversification of many tetrapod lineages
(Losos et al., 2000).
Apparently an inherent property of vertebrate muscle is the ability to
acclimate to current use patterns.
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Acknowledgments |
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