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Review Article
Basic Models Modeling Resistance Training: An Update for Basic
Scientists Interested in Study Skeletal Muscle Hypertrophy
Jason Cholewa2# Lucas Guimares-Ferreira3#, Tamiris da Silva
Teixeira1, Marshall Alan Naimo4, XIA
Zhi5,6, Rafaele Bis Dal Ponte de S1, Alice Lodetti1, Mayara
Quadros Cardozo1, Nelo Eidy Zanchi1*
1- Postgraduate Program in Health Sciences, Health Sciences
Unit, Universidade do Extremo Sul Catarinense, Cricima/SC,
Brazil.
2- Department of Kinesiology Recreation and Sport Studies,
Coastal Carolina University, Conway, SC, USA. 3- Laboratory of
Experimental Physiology and Biochemistry, Center of Physical
Education and Sports, Federal
University of Espirito Santo, Vitria/ES, Brazil. 4- Division of
Exercise Physiology, West Virginia University School of Medicine,
Morgantown, USA. 5- Exercise Physiology and Biochemistry
Laboratory, College of Physical Education, Jinggangshan
University,
Ji'an,Jiangxi, PR China. 6- Exercise Physiology Laboratory,
Department of Exercise Physiology, Beijing Sport University,
Beijing, PR China.
Running head: Basic models modeling resistance training
Keywords: resistance training, experimental models, skeletal muscle
hypertrophy # Dr. Jason Cholewa and Dr. Lucas Guimares-Ferreira
contributed equally * Corresponding author: Nelo Eidy Zanchi
Email:[email protected] Av. Universitria, 1105 - Bairro Universitrio
C.P. 3167 | CEP: 88806-000 Cricima / Santa Catarina Phone: +55 48
3431-2500 Fax: +55 48 3431-2750 This article has been accepted for
publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version
of Record. Please cite this article as doi: [10.1002/jcp.24542]
Received 12 December 2013; Revised 14 December 2013; Accepted 16
December 2013 Journal of Cellular Physiology 2013 Wiley
Periodicals, Inc.
DOI 10.1002/jcp.24542
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Abstract
Human muscle hypertrophy brought about by voluntary exercise in
laboratorial conditions is the most
common way to study resistance exercise training, especially
because of its reliability, stimulus
control and easy application to resistance training exercise
sessions at fitness centers. However,
because of the complexity of blood factors and organs involved,
invasive data is difficult to obtain in
human exercise training studies due to the integration of
several organs, including adipose tissue,
liver, brain and skeletal muscle. In contrast, studying skeletal
muscle remodeling in animal models
are easier to perform as the organs can be easily obtained after
euthanasia; however, not all models
of resistance training in animals displays a robust capacity to
hypertrophy the desired muscle.
Moreover, some models of resistance training rely on voluntary
effort, which complicates the results
observed when animal models are employed since voluntary
capacity is something theoretically
impossible to measure in rodents. With this information in mind,
we will review the modalities used to
simulate resistance training in animals in order to present to
investigators the benefits and risks of
different animal models capable to provoke skeletal muscle
hypertrophy. Our second objective is to
help investigators analyze and select the experimental
resistance training model that best promotes
the research question and desired endpoints.
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Introduction
In humans early training gains in muscle strength have been
regarded as the result of both neural
and musculature adaptations. Over the last half-decade several
animal training models have been
developed as a way to increase both force output and mass
(hypertrophy) in the exercised muscle.
Contrary to the increases in maximal oxygen consumption observed
in animals with aerobic training
using a treadmill, measurements of maximal and submaximal force
capacity in vivo are complicated
by several factors, including voluntary capacity to perform
resistance training, non-voluntary
electrical-based training under anesthesia, surgical
manipulation of muscles involved in the
hypertrophic response, and the utilization of positive or
negative reward to stimulate the animals to
perform the exercise. Thus, the greatest motivation for an
animal to produce maximal capacity
voluntary muscular force in classic operant models is via direct
electrical stimulation to the brain,
which is virtually impossible to perform in subsequent
experiments with the same animal (Olds and
Milner 1954).
Pain avoidance has been demonstrated to be a greater stimulus
than food or water reward (Miller
1951). According to Timson (1990), the animal will perform a
task only until the effort involved in the
task performance exceeds its desire for the stimulus. Thus, a
model employing starvation as the main
stimulus will motivate the animal to exert only 50-60% of its
maximal voluntary capacity, which will
then negatively affect muscular hypertrophy capacity either due
to lack of overload or nutrition.
Therefore, we will first review animal models employing
non-voluntary maximal capacity force
production as a way to induce hypertrophy, and then discuss new
methods involving voluntary
models. A summary of results of the models reviewed is available
in Tables 1 and 2.
Non voluntary non electric exercise-induced enlargement in
animal models.
One of the first methods to induce skeletal muscle hypertrophy
was developed by Thomsen and Luco
(1944) whereby a passive stretch applied to immobilized joints
places longitudinal tension upon the
muscle (Alway et al. 1989) (Fig. 1F). Utilizing this model of
overload Aoki et al. (2006) reported an
increase in sarcomeres in series leading to an elongation of the
target muscle. The application of
rapamycin was demonstrated to robustly suppress this response,
suggesting the mammalian target
of rapamycin (mTOR) pathway is involved in the longitudinal
hypertrophy induced by joint
immobilization. This model of overload may be appropriate to
study skeletal muscle remodeling as a
result of stretch overload or joint immobilization; however,
resistance training in humans requires
dynamic tension generation, resulting in a force overload, and
leading to the synthesis of additional
sarcomeres in series. Therefore, future investigators sought to
develop methods that more closely
modeled resistance training.
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Goldberg et al. (1968) developed an effective non-voluntary
non-electrically stimulated model (Fig.
1A) to induce skeletal muscle hypertrophy through synergistic
ablation (surgical removal of a
synergistic muscle, most often the gastrocnemius calcaneus
portion, generating overload and muscle
hypertrophy of the soleus and plantaris muscle). Although the
use of this model to mimic the effects
of human strength training has been highly criticized due to the
surgical procedures (Taylor and
Wilkinson 1986), McCarthy et al. (2011) demonstrated no
differences in muscle hypertrophy between
mice with genetic satellite cell depletion and non-depleted
controls with 2 weeks of synergistic
ablation overload. Given the similar significant improvements in
muscle hypertrophy in both groups,
synergistic ablation remains an effective method to study
cellular signaling pathways leading to acute
skeletal muscle hypertrophy (Miyazaki and Esser 2009).
On the other hand, because the targeted muscle is exposed to a
static stimulus (the animals
bodyweight) the increase in muscle mass occurs most rapidly
during the first week of the protocol
and appears to reach a plateau 2 weeks following surgery.
Additionally, the animal is under constant
overload every time it moves, compared to separate training
sessions used in human resistance
training or other animal models. Thus, synergistic ablation
cannot be used in long term studies nor
does it appear compatible with modeling the progressive overload
or periodization phases and
nutrition schedules required in human resistance training to
induce maximal changes in hypertrophy
and strength.
Tenotomy is a technique where the gastrocnemius tendon is
detached and the synergistic muscle is
placed under increased muscle tension (Fig. 1A). Tenotomy
appears less effective at inducing
overload and the resultant musculature hypertrophy of the
synergist (ex. plantaris) when compared
with surgical ablation (Timson 1990). Although the reason for
the difference is not clear, it appears
that the cut tendon is able to reattach when left intact within
the muscle fascia. The critiques of
tenotomy are the same as those related to synergistic ablation
methods; however the magnitude of
hypertrophy is less and the possibility of the gastrocnemius
tendon reattaching the calcaneus tendon.
The use of chronically restricted venous blood flow was first
reported by Kawada and Ishii (2005) to
induce skeletal muscle hypertrophy in rats. This model does not
involve exercise; rather, blood flow
to the hind limbs is diminished via a surgical intervention.
Fourteen days following the operation the
plantaris muscle increased in dry weight by 10% and the
concentration of myofibrillar protein
increased by 23%. Additionally, levels of nitric oxide synthase
and the muscle insulin like growth
factor-1 (IGF-1) also increased. It is difficult to speculate on
the level of difficulty or safety of this
model as a detailed description of the surgery is not completely
available in the literature; however,
this model appears to be consistent since Kawada and Ishii
(2008) reproduced the results of the first
study and also reported decrements in type I muscle fibers.
Although plantaris hypertrophy was
modest compared to synergist ablation, chronic blood flow
restriction may be a novel model to study
hypertrophy in animals. When translating the results to human
training two questions arise: 1) What
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are the effects of chronic blood flow restriction combined with
muscular tension? 2) Does blood flow
restriction occurring for longer than 2 weeks compromise the
health of the animal or result in a
plateau in muscle hypertrophy? Given that intermittent blood
flow restriction under low tension
phosphorylates P70S6K and muscular hypertrophy in humans (Fujita
et al. 2007), answering these
questions are essential to evaluating the ability to translate
this model to human resistance training.
Non voluntary, electric exercise-induced enlargement in animal
models.
Wong and Booth (Wong and Booth 1988) developed a novel non
voluntary model to load the hind
limb and induce muscle hypertrophy. In this model the animal is
anesthetized, the foot is attached to
an immovable metal plate with adhesive tape, and muscular
contraction is stimulated electrically with
joint of the animal starting in a neutral position (Fig. 1E).
The ability of this model to induce
hypertrophy and increased muscle fiber cross sectional area is
inconsistent and produces only
modest results; however, using a modified model, Baar and Esser
(1999) demonstrated P70S6K
phosphorylation and polyribosome formation, which indicates that
the Wong and Booth model is
capable of increasing protein synthesis.
Godspink (1999) modified the protocol proposed by Wong and Booth
(1988) by loading the limb in a
stretched position (elongation) and allowing for the electrical
stimulus to induce a dynamic contraction
(Fig. 1I). This combined model resulted in a greater increase in
protein synthesis compared to the
elongation model or isometrically loaded models alone. Moreover,
using the combination of
elongation and dynamic overload Godspink demonstrated the
activation of a transcript derived from
the IGF-1 local to skeletal muscle, which has been labeled
mechano growth factor (MGF). MGF
presents an insert with 52 base pairs in the E domain of the
gene, which alters the reading frame of
the 3 end, resulting in satellite cell proliferation/activation
following muscle damage, ultimately
leading to muscular repair and hypertrophy (Hill and Goldspink
2003). This model allows the
researcher to apply an identical maximal pulse to generate
maximal tetanic force, and thus eliminates
the need to readjust the electrical stimuli. Although the
combination of muscular elongation and non-
voluntary contraction may be viable in studying acute increases
in protein synthesis, electrical pulses
under anesthesia are difficult to perform, as is the ability to
apply a consistent, progressive increase
in electrical stimulation to match an increased load required to
induce hypertrophy.
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Resistance training (RT) exercise under unloading conditions
Another interesting resistance training model was presented by
Haddad et al. (2006) whereby rats
were unloaded via hind limb suspension (HS) to induce muscular
atrophy for six days. Animals in the
resistance training group (HST) were trained every other day.
Briefly, animals were anesthetized and
stimulation electrodes consisting of Teflon-coated stainless
steel wire were introduced into the
subcutaneous region adjacent to the popliteal fossa via 22-gauge
hypodermic needles. Wire
placement was lateral and medial of the location of the sciatic
nerve allowing for field stimulation of
the nerve. The stimulation wires were then attached to the
output poles of a Grass stimulus isolation
unit interfaced with a Grass S8 stimulator. This allowed for the
delivery of current to the sciatic nerve
resulting in muscle contraction. The right leg was positioned in
a footplate attached to the shaft of a
Cambridge model H ergometer, adjusted to produce maximal
isometric tension. Each training bout
consisted of a series of four sets of contractions with 5 min of
recovery between sets. Each set
consisted of a series of 10 maximal isometric contractions
lasting 2 s each with 20 s of rest in
between contractions. Thus each training session lasted for 27
min, during which the muscle was
activated for a cumulative time of 80 s.
Compared with normal controls Haddad et al. (2006) reported the
gastrocnemius of the HS animals
decreased 20%. Although the RT program had a positive effect on
maintaining relative muscle weight
at a higher level compared with the HS group (8%), this response
may in part have been due to
edema, as total protein concentration was slightly lower (7%,)
in the HST compared with the HS
group. This response demonstrates the negative impact of
unloading on the hind limb musculature by
illustrating that the myofibril pool was indeed a primary target
of the atrophy response. The results of
this study suggest that the process of muscle atrophy is not
opposite of muscle hypertrophy, and
demonstrate the inability of isometric based RT to spare muscle
protein during unloading. Therefore,
although an isometric model of RT may be appropriate to induce
hypertrophy, researchers using
resistance training in animal models of diseases (i.e.
dexamethasone-induced diabetes) (Nicastro et
al. 2012a) should consider performing experimental pilot studies
with dynamic based contractions
prior to data collection.
On the other hand, Fluckey et al. (2002) demonstrated that
dynamic resistance training is capable of
preventing muscle wasting during unloading. In this model,
Fluckey et al. developed a modified
version of the human flywheel resistance exercise apparatus so
rats could be trained while in hind-
limb suspension. This poses a major advantage over the model
used in Haddad et al. as the animals
can be trained with dynamic resistance exercise independent of
gravity and without being removed
from the cage. Briefly, a rat is tethered via a leather and
velcro vest attached to a nylon cord and
spooled around an inertia wheel located on the outside of the
resistance exercise apparatus. The rat
is allowed to place its feet on a shock grid suspended at the
top of the apparatus (to accommodate
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the HS state) and an illumination bar capable is located in the
apparatus opposite to the shock grid.
The bar is then illuminated which results in a repetition by the
animal. The movement is similar to
squats as performed by humans, as extension occurs at the hip,
knee and ankle joints. When
required a shock is applied briefly (
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EDL and soleus muscles were greater in trained rats than control
rats. Despite an increased ability of
the rats to lift progressively heavier loads, this heavy
resistance training model did not induce gross
muscle hypertrophy nor did it increased the force-producing
capacity of the EDL or soleus muscles.
The discrepancy in results between Duncan et al. (1998) and
Yarasheski et al. (1990) was likely due
to the muscles sampled and measured. The soleus is predominantly
type I muscle fiber and likely did
not suffer enough overload to induce hypertrophy. We suggest
researchers using the ladder climb
model to study hypertrophy or molecular signaling in protein
synthesis evaluate samples from
muscles with a higher proportion of type II fibers, such as the
rectus femoris or gastrocnemius.
This model of mesh scale was then modified into a second one
where Sprague-Dawley rats were
trained to climb a 1.1-m vertical (80 degree incline) ladder
with weights secured to their tail
(Hornberger and Farrar 2004). The rats were trained once every 3
days for 8 weeks. Each training
session consisted of 4-9 (6.02 +/- 0.23) climbs requiring 8-12
dynamic movements per climb. Based
on performance, the weight carried during each session was
progressively increased. Over the
course of 8 weeks, the maximal amount of weight the rats could
carry increased 287% and the
improved training performance was associated with a 23% absolute
increase in the weight of the
flexor hallucis longus (FHL), with a concomitant 24% increase in
both total and myofibrillar protein.
On the other hand, Scheffer et al. (2012) analyzed oxidative
stress in skeletal muscles using a similar
model of climb ladder (43 steps) in 4 different resistance
training protocols: Muscular resistance
training: RT consisted of climbing the ladder carrying a load of
10% of body weight, which was
progressively increased to 20%, 30%, 40%, and 50%, 3 to 6 sets
with 2-min breaks, and 1215
repetitions. Hypertrophy training: HT consisted of climbing the
ladder carrying a load of 25% of body
weight, which was progressively increased to 50%, 75% and 100%,
3 to 6 sets with a 2-min break
and 810 repetitions. Strength training: ST consisted of climbing
the ladder carrying a load of 25% of
body weight, which was progressively increased to 50%, 100%,
125%, 150%, 175%, and 200%, 3 to
6 sets with a 2-min break, and 35 repetitions (Fig. 3). After 12
weeks of training on alternate days,
body weight was not different amongst groups and the red portion
of the brachioradialis was removed
and oxidative parameters were assessed. Although muscle
hypertrophy was not measured, HT
caused an imbalance in oxidative parameters in favor of
pro-oxidants, leading to oxidative stress in
skeletal muscle.
In a related study Lee et al. (2004) tested whether adenoviral
administration of IGF-I (rats were
injected with recombinant AAV harboring rat IGF-I cDNA
(rAAVIGF-I) was capable to increase FHL
muscle mass. Using the ladder climb model (1-m ladder with 2-cm
grid steps and inclined at 85), 8
wks of resistance training, a 23.3% increase in muscle mass was
observed in the FHL (Fig. 1D). Viral
expression of IGF-I without resistance training produced a 14.8%
increase in mass and the combined
interventions produced a 31.8% increase in muscle mass.
Therefore, the combination of resistance
training and overexpression of IGF-I induced greater hypertrophy
than either treatment alone. These
results suggest that a combination of resistance training and
overexpression of IGF-I could be
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synergistic and can improve muscle hypertrophy through
adenoviral transfections. It must be
remembered that this original finding was revolutionary at that
time and generated a lot of knowledge
paving future research. Differences in muscle remodeling
following the same regimen used by Lee et
al. (Lee et al. 2004), Hornberger and Farrar (2004), Duncan et
al. (1998), and Yarasheski et al.
(1990) could be related to different muscles sampled in each
work, the ladder model, such as the
size and number of steps (which differed considerably amongst
different the 4 studies), and the
number of sessions per week, load progression, and volume in the
protocols. Thus, the ladder model
is a tool capable to induce positive adaptations in muscle
hypertrophy; however, minor modifications
to the protocols may greatly affect the results such that the
functionality of the model is reduced when
muscular hypertrophy is a major endpoint, thereby reducing the
ability to study the effects of genetic
manipulation or ergogenic aids.
To monitor the variance in overload and work performed between
groups we suggest measuring
venous lactate and modifying the load appropriately. Scheffer et
al. (2012) demonstrated the
effectiveness of this method to equalize the load between
groups. Additionally, we suggest
researchers using this model to induce hypertrophy modify the
length of the ladder by reducing the
number of steps the animal climbs and increasing the load to
more closely mimic human strength
training. As an example Scheffer et al. (2012) employed a
hypertrophy protocol of 48 steps with 1.1
cm between steps. Although hypertrophy was not measured, a
relationship exists between exercise-
induced oxidative stress and muscle hypertrophy (Wadley 2013),
suggesting that sets of less
repetitions may be most effective in inducing hypertrophy.
Additionally, this specific hypertrophy
protocol on the ladder may be the most appropriate for
evaluating satellite cell activation and
differentiation with resistance training.
Another animal model of voluntary resistance exercise was
proposed by Klitgaard et al (1988): rats
were trained to perform a plantar extension in order to obtain a
pellet of food (Fig. 1C). The original
protocol was performed in 2 year old rats and after 36 wks of
training plantaris muscle mass
increased 24%. On the other hand, utilizing the same protocol
but in young rats and for only 13 wks
we observed the plantaris muscle hypertrophied by 13% (Zanchi et
al. 2009). Our major finding using
this model was that the Atrogenes (MuRF-1 and Atrogin-1),
ubiquitin ligases involved in muscle
proteolysis by the proteasome, decreased only in the trained
group, demonstrating the ability of this
model to modulate molecular signaling. Since we didnt measure
the degree of muscle protein
synthesis or degradation in the isolated muscles, we cannot
speculate on the ability of this model to
impact protein turnover as a whole. There are two factors to
consider when using this model: 1) a longer training period is
required for hypertrophy to occur when compared to the synergist
ablation or the ladder
model, and 2) this model uses starvation to motivate the animals
to perform the plantar extension. This
starvation period poses a major issue when studying
physiological responses as it affects both
voluntary work and nutrient status, and is also difficult to
apply. Thus the translation of this model to
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humans must be interpreted with caution, although several acute
studies in humans are performed
under starvation conditions (Fujita et al. 2007).
In 1992, Tamaki et al. (1992) described a weight lifting
exercise model designed to induce muscle
hypertrophy in the hind-limb by loading the animal with a canvas
jacket attached to the torso and
requiring the animal to perform a squat like exercise (Fig. 1H).
The main stimulus was provided by an
electric stimulator linked to the tail of the animal so a
punishment stimuli was applied and the animals
performed a squat like exercise of progressively increasing
loads within a hypertrophy range (65-
75% 1 Repetition Maximum - RM). Compared with 60 min of
treadmill sprints, acute squat training
resulted in an increase in plasma creatine kinase. When sprint
and squat training was carried outfor
12 weeks at 4-5 days/week there was a 12% increase in the
plantaris muscle compared with control
animals receiving an electric stimulus; however, there were no
significant differences compared to
the sprint training group. Although this model contains a
similar biomechanical loading and
movement pattern to human resistance training, its ability to
overload the animals and induce muscle
hypertrophy is inferior to other voluntary resistance training
models, such as the ladder climb or food
motivated plantar extension proposed by Klitgaard et al.
(1988).
In 2003, Wirth et al. (2003) developed a revolutionary model
where rats were operantly conditioned to
perform a squat exercise via both reward and punishment (Fig.
1K). Food was restricted and rats
were operantly conditioned with food rewards to enter a vertical
tube, insert its head into a weighted
ring (either 70 g or 700 g), lift the ring until its nose
interrupted an infrared detector, and then lower
the ring. Load cells measured the external force generated, and
displacement transducers measured
the vertical displacement of the ring during each lifting and
lowering movement. The apparatus and
training procedures were computer automated. Peak force,
velocity, work, and power were calculated
for each movement. Rats in both groups easily acquired the task
after 12-15 training sessions
conducted 5 days/wk. The median peak force, work, and power per
lift for both concentric and
eccentric were greater for the 700g group. Importantly, 8 weeks
of lifting both 70g and 700g 5
sessions per week increased plantaris, soleus, and gastrocnemius
mass compared to sedentary
controls; however, dry weight and muscular protein content was
not measured, thus it is also possible
these increases may have been partly the result of edema and/or
inflammation. These results
demonstrate the utility of quantitating the biomechanics of
volitional movements and suggest that the
present model can establish and maintain controlled repetitive
movements necessary for studying
injury and adaptation in muscles, tendon, and bone. Moreover,
contrary to Tamaki et al (1992) the
absolute weight of the rats was not decreased with this training
protocol, suggesting that this positive
operant model combined with histological sampling is a valid
protocol to study responses to
resistance training.
Given the potential of the models described by Wirth et al.
(2003) and Klitgaard et al. (1988), our
group (Nicastro et al. 2012b) proposed an equipment and system
of resistance exercise (RE), based
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on squat-type exercise for rodents, with the ability to more
precisely control the training variables
proposed in Wirth et al. (2003). In this model we developed an
operant conditioning system
composed of sound, scent, light, and feeding devices that
optimized resistance exercise performance
by the animal (Fig. 1L). With this system, it was not necessary
to tie the animal into the device or
impose chronic fasting or electric shock for the animal to
perform the task proposed (muscle
contraction). Furthermore, it was possible to perform muscle
function tests in vivo maximal voluntary
strength capacity (MVSC) within the context of the exercise
proposed and control variables such as
intensity (percent of MVSC or percent of body weight), volume
(sets and repetitions), rest intervals
between sets, and exercise session length. Importantly, sound
was the main stimulus given to the
animals as a way to optimize learning and reinforce exercise
training. Therefore, despite
experimental limitations, we believe that this RE apparatus is
closer to the physiological context
observed in humans. When testing the efficacy of this protocol
to counteract the effects of 7 days of
5mg/day dexamethasone (a diabetogenic and proteolytic catabolic
hormone) in a common model of
skeletal muscle atrophy, we observed that training attenuated
the loss of gross muscle mass and
increased plantaris mass when compared to controls.
Additionally, we observed an increase in
MVSC in trained animals, but not controls at the end of the
study (Nicastro et al. 2012a),
demonstrating the efficacy of this model to attenuate or even
prevent atrophy, and as a reliable
technique to study atrophic disease.
Variables to evaluate when selecting a resistance training
model
According to Timson (1990), when using animal models to evaluate
muscle enlargement produced by
strength training in humans, three factors must be considered:
1) Muscle recruitment and adaptations
in fiber characteristics; 2) magnitude of muscle enlargement.
Given the effects of varying models of
voluntary and non-voluntary resistance loading reported by our
team and others, we suggest six
other factors to consider: 3) The degree of nutrition required
for a positive reward; 4) Negative reward
(i.e. pain). 5) Time spent conditioning the animal to execute
the exercise; 6) Duration required to
obtain muscle remodeling; 7) Muscle voluntary capacity
percentage; 8) Resistance training under
atrophic or diseased conditions. 1- Muscle recruitment and
adaptations in fiber characteristics: With specific study questions
(i.e.: sarcopenia) type II muscle fiber hypertrophy is more
relevant that gross hypertrophy in
preventing the loss of muscle mass and function; however, not
all models of training are
capable of overloading all muscle fibers and thus eliciting a
substantial degree of hypertrophy
in muscles comprised of predominantly Type II fibers. As an
example, the plantaris is a mixed
fiber muscle and its hypertrophy through surgical ablation of
the gastrocnemius (a
predominantly type II muscle) may not be appropriate to study
the reversion of sarcopenia
compared to a squat based model (Nicastro et al. 2012b).
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2- Magnitude of muscle enlargement: Some exercise models are
difficult to perform and the resulting hypertrophy degree is very
poor when compared with others. For example the ladder
climb model is capable to generate hypertrophy in the muscles of
the lower limbs of the rat;
however, the hypertrophy of the FHL vary considerably in the
literature based upon ladder
length, load, and frequency (Duncan et al. 1998; Hornberger and
Farrar 2004). In contrast,
synergist ablation results in a robust hypertrophy of the FHL
(80% hypertrophy with synergistc
ablation in our group observed by Teixeira et al. 2013,
unpublished data), but not the thigh
muscles such as the rectus femoris. 3- The degree of nutrition
required for a positive reward: Klitgaard (1988) developed a model
whereby a collar and an acrylic cylinder where the rats where
trained to feed through
overextension of hindlimb (plantar extension) to take the pellet
and then feed. Utilizing this
model, our group (Zanchi et al. 2009) observed it required
approximately 24h of food
restriction to motivate the animals to perform the lift in order
to obtain a food pellet.
Additionally, the number of repetitions per day was very limited
(16 per day), although we
observed an increase of 13% of muscle mass (plantaris and
soleus) compared with paired
feeding control group. Thus, future investigations should
consider the effects of models that
require nutrition deprivation to perform exercise when major
endpoints include robust
increases in muscle hypertrophy. 4- Negative reward: It is well
known recognized that punishment is a stronger stimulus than reward
to induce rodents to perform resistance exercise (Zanchi et al.
2009). However,
sometimes this punishment stimuli is detrimental as the appetite
of the animals is reduced due
to the endocrine response involved in the fight or flight
(stress) reaction, thus impairing the
muscular and molecular adaptations to a pre-determined stimulus.
For example, Tamaki et al.
(1992) demonstrated increases in gastrocnemius and plantaris
mass with resistance training
compared to control groups following 12 weeks of training;
however, the resistance training
group lost approximately 200g of body mass. Thus, punishment in
this model influenced the
endocrine response and diminished the appetite of the trained
animals such that limb muscle
hypertrophy was likely compromised. 5- Time spent conditioning
the animal to execute the exercise: Through two different voluntary
exercise models, we observed that some rats are capable to learn
how to execute a task (resistance
training) very fast whereas others are not. Therefore, every
time we conducted a resistance training
protocol we selected for both control and intervention groups
only the animals capable to learn very
fast. Based on Klitgaard (1988) and Zanchi et al. (2009) and
modifying the model proposed by Wirth et
al (2003), we developed a computerized model using both reward
and punishment stimulus to induce
rodents to perform resistance exercise (hindlimb extension)
(Nicastro et al. 2012b). Based upon the
results from these models, we suggest researchers employing
models of voluntary maximal contraction
spend 14-16 unloaded training sessions spaced every day over two
weeks to condition the animals to
perform the resistance training protocol.
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6- Duration required to obtain muscle remodeling: In our first
model of food rewarded plantar extension (Zanchi et al. 2009), 3
months of training were required to increase plantaris mass
by 13%. Similarly, Tamaki et al (1992) spent 3 month to induce
relative bodyweight adjusted
(muscle weight/body weight) increases in gastrocnemius and
plantaris mass of 31.4% and
17.9%, respectively, and absolute increase of 12% in plantaris
mass when compared with
controls. In comparison, utilizing electrical simulation Baar
and Esser (1999) demonstrated
increases in tibialis anterior and EDL mass of 13.9 and 14%,
respectively, when performed
twice a week for 6 weeks. Further research is required to
elucidate the optimal combination of
frequency (sessions per week) and volume when designing animal
models leading to muscle
hypertrophy. Despite similar increases in absolute plantaris
weight, the rats in Tamaki (1992)
were trained 2-3 sessions per week more than those in our study.
These results are further
confounded by synergist ablation where the target muscle
(usually the plantaris) is chronically
under a load every time the rat moves (similar to a human
carrying extra bodyweight, not
taking part in resistance training). Thus, each model has
specific characteristics, and based
upon the load (light or heavy) and expected outcomes pilot
sessions should be conducted to
determine appropriate frequencies and study durations. 7- Muscle
Voluntary Capacity Percentage: It has been well described in the
literature that intensities in the range of 75-85% 1RM are ideal to
increase/hypertrophy the muscle mass.
However, utilizing some models it is almost impossible to
predict those ideal ranges. For
example, utilizing the ladder model some investigators have
employed a maximum effort test
in the ladder model; however, the ladder contains 16-24 steps so
the rodent is performing, in
fact, a test of local muscular endurance (i.e.: a 16 RM test)
and not a true 1 RM. Predicting the
maximal voluntary power output of a rodent for a given stimulus
is also difficult and unreliable.
For example, when we tested the food-reward model developed by
Klitgaard (1988) the rats
were unable to perform more than 8 repetitions per workout
(Zanchi et al. 2009). A major
concern was whether the rat actually exerted maximal effort, or
if it was satisfied by a reduced
food intake. This issue was partially resolved in our more
recent model (Nicastro et al. 2012b)
as we observed the rats performed nearly 50 repetitions per
session. Moreover, we were able
to measure force developed through a computerized model and
compare that to MVSC to
ensure the animals were working the hypertrophy range. Thus, we
believe this voluntary
model allows the researcher to more precisely manipulate volume
and intensity in the study of
training induced muscular adaptations, specifically hypertrophy
and soft tissue remodeling. 8- Resistance training under atrophic
or diseased conditions: Several models of atrophy have been
described in the literature; the most commonly used mimics that of
spaceflight
(gravitational unloading) via hind limb suspension. Under this
condition of atrophy, isometric-
based resistance training models capable of producing robust
hypertrophy under gravitational
conditions have failed to induce hypertrophy or counteract
atrophy (Haddad et al., 2006). It is
possible that this resistance training model may have attenuated
the loss of muscle mass
-
under a gravitational model of atrophy (i.e.: administration of
rapamyacin). On the other hand,
it is well established in the literature that dynamic resistance
training plus nutritional support
(mainly proteins containing high biological value and leucine)
are capable of robustly activating
protein synthesis pathways (Phillips 2011) and counteracting
unloading-induced loss of
muscle mass as demonstrated by Fluckey et al. (2002). When
considering the catabolic state
as generated by glucocorticoids and diabetes mellitus, we
demonstrated that resistance
training is capable of counteracting the loss of muscle mass
(Nicastro et al. 2012a). Although
further research in humans is needed to describe the hormonal
milieu and muscle activation in
response to daily living activities in humans with atrophic
disease, our model provides
researchers with a greater degree of control over the variables
involved in resistance training,
and can be used to study the effects of resistance training on
atrophy during conditions of
gravitational unloading and glucocorticoid catabolism.
In example of how these factors may affect investigation
outcomes, Miyazaki et al. (2011) utilized the
synergistic ablation surgery to investigate the involvement of
ERK/MERK pathways and the well-
known Akt/mTOR/P70S6K pathways of protein synthesis initiation
in the muscle hypertrophy
phenomena. Using the synergist ablation model of muscle
hypertrophy, early and late periods of
muscle adaption were examined. Specifically, they measured these
adaptations in a model of form
follows signaling function observing plantaris hypertrophy
weight from day 0 to day 10 on a daily
basis. Miyazaki et al. (2011) demonstrated that Akt
phosphorylation (Ser 473 or Thr 308) was not
activated until days 2-3, whereas P70S6K (Ser 389, Thr 421/424)
and ribosomal protein (RPS6 Ser
235-236) where highly phosphorylated during the entire
hypertrophy process. Of note, this delay in
the classical Akt/mTOR activation was accompanied by a rapid and
prolonged MEK 1 and 2 (Ser
217/221) activation. The same pattern was observed in ERK 1 and
2 (Thr 221 and 224). Importantly,
this divergence utilizing a well-recognized animal muscle
hypertrophy model demonstrated parallel
pathways, one activated early and one later, that both
phosphorylated the RP6 protein culminating in
increased protein synthesis. In the early pathway ribosomal
kinases phosphorylated the RP6 in the
Ser 235/236 residue. Following the late pathways, RPS6 was
phosphorylated in the residues Ser
240/244. This study demonstrates the importance of matching
expected outcomes within the
research question to the resistance training model employed. If
Miyazaki et al. (2011) had utilized a
model requiring a longer duration of conditioning or training to
induce muscle remodeling (i.e.: the
squat model) these novel discoveries may not have been made.
Thus, investigators need to be
aware of these particular attributes to resistance training
models when investigating molecular
signaling pathways, ergogenic aids, and mechanical tensions and
muscle remodeling.
Conclusion
-
Many important molecular findings regulating skeletal muscle
remodeling have been made through
the use of diverse experimental resistance training models.
Although a number of models are
capable of inducing muscular hypertrophy, investigators should
consider several new variables when
selecting the most appropriate model to answer the research
question. For example, investigators
should be aware of the early and late signaling pathways leading
to hypertrophy, and chose a model
that activates the appropriate phase. In this same regard, if a
large degree of muscular remodeling is
required to answer a research question selecting a hypertrophy
model with a weak magnitude will
result in compromised findings.
A major issue facing clinic scientists investigating the
medicinal/therapeutic effects of resistance
training is that genetic modifications in animals that are
available to basic scientists are not applicable
to the general population. Consequently, a growing emphasis is
being placed on investigating the
efficacy and effectiveness of nutritional supplements and
ergogenic aids in conjunction with
resistance training. Although the discovery of new signaling
pathways has not suddenly changed or
advanced the methods people use to train, overtime these
discoveries have led to more effective
training methods, especially when resistance training is used as
physiotherapy. As the ability to more
accurately manipulate the variables involved in rodent
resistance training models (volume, intensity,
frequency, etc.) improve, scientists will be better able to
study the effects of distinct differences in
program design on molecular signaling and hypertrophy. These
improvements in model design and
the results obtained can then be used to improve the translation
between basic scientific discoveries,
clinical practices, and the application to human health and
performance.
-
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Figure legends
Figure 1: Progression of the Development of Resistance Training
Models in Rodents (1968-2012). A) Surgical ablation; B) Tenotomy;
C) Voluntary plantar extension; D) 85 weighted ladder climb; E)
non-voluntary hind-limb extension; F) Passive stretch; G) 90
weighted ladder climb; H) Electric stimulated squat; I) Modified
non-voluntary hind-limb extension; J) Modified flywheel with
hind-limb suspension; K) Operantly conditioned squat; L) Modified
operantly conditioned squat. M) Jumping submersed in water with
overload. Adapted by the authors.
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Table 1. Voluntary muscle hypertrophy models
Author Year Technical Size / Height Angulation Sessions / wk
Training period
Series / day Muscle hypertrophy
(%)
Yarasheski et al 1990 Progressive lift with loads attached to
the tail using a mesh
40 cm 90 Degrees 5 days / wk 8 wk 20 Increase in RF weight
Duncan et al. 1998 Ladder climbing 40 cm Vertical 4 days / week
26wk 12 to15 Increase in EDL and SOL
weights relative to body mass and fibre
hypertrophy
Lee et al. 2004 Ladder climbing + IGF-I adenovirus
1 m 85 Degrees Every third day 8 wk 8 climbs or until
failure
Increase in FHL weight
Klitgaard et al 1988 Plantar flexion of ankle joint - - In the
morning, at noon, and in the evening (Monday
and Tuesday, Thursday
and Friday)
36 wk 30 min of training 3
times per day
Increase in SOL and PLA
weights
Zanchi et al. 2009 Plantar flexion of ankle joint - - 3 Times /
Week 12 wk 16 Increase in PLA weight
-
Tamaki et al. 1999 "Squat Like Exercise" - - 4-5 Days / Week 12
wk 65-75 % 1 RM Hypertrophy of GAS and PLA and increase in the
number
of muscle fibers
Dela Cruz et al. 2012 Jump in a PVC cylinder containing
water
- - Every two days 5 wk 15 jump sessions
Increase in EDL and SOL
CSA
Wirth et al 2003 Operant conditioning (progressive lift)
- - - 8 wk 12 to 15 Increased performance
Hornberger, T.A 2004 Ladder climbing (with progressive load
attached to the tail)
1,1 m 80 Degrees - 8 wk 1 Increase in FHL weigth
and total and myofibrillar
protein content
Scheffer, et al. 2012 Ladder climbing 43 steps - Alternate days
12wk 3 to 15 -
Fluckey et ai 2002 Flywheel Resistance Training
- - - 4wk 25 Attenuation of hindlimb
suspension-induced muscle
atrophy in SOL
RF: rectus femoris; EDL: extensor digitorum longus; SOL: soleus;
FHL: flexor hallucis longus; PLA: plantaris; GAS: gastrocnemius;
CSA: cross-sectional rea.
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Table 2. Involuntary muscle hypertrophy models
Author Year Technical Period Estimulus Muscle hypertrophy
(%)
Goldberg et al. 1968 Surgical ablation of GAS 6 Days Walk
Increase in SOL and
PLA weights
Goldberg et al. 1975 Tenotomy of GAS 14 Days Walk Increase in
SOL weight
Wong and Booth 1988 Weight-lifting exercise 16 wk Electric +
external load
Increase in GAS we
weight and protein content
Baar and Esser 1999 Surgical implantation of electrodes and
electrical
stimulation
6 wk Electric Increase in EDL and TA wet weights
Goldspink 1999 Stretch Combined With Eletrical Stimulation of
Anterior Tibialis Muscle
of Adult Rabbit
4 days Electric Increase in TA wet weight
Kawada and Ishii 2005 Chronic restriction of blood flow to
muscle
2 wk Venous occlusion Increases in PLA dry
weight/ body weight and myofibrillar
protein content
Haddad et al 2006 Electrical stimulation 6 Days Isometric
contractions
Attenuation of hindlimb suspension-
induced muscle
atrophy in GAS
(muscle weight)
EDL: extensor digitorum longus; SOL: soleus; TA: tibialis
anterior; flexor hallucis longus; PLA: plantaris; GAS:
gastrocnemius.
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Figure 1