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:neloz@ig.com.br 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

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.

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.

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

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.

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

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 (

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

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

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

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).

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.

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.

References

Alway SE, Winchester PK, Davis ME, Gonyea WJ. 1989. Regionalized adaptations and muscle fiber proliferation in stretch-induced enlargement. J Appl Physiol Bethesda Md 1985 66:771781.

Aoki MS, Miyabara EH, Soares AG, et al. 2006. mTOR pathway inhibition attenuates skeletal muscle growth induced by stretching. Cell Tissue Res 324:149156. doi: 10.1007/s00441-005-0081-4

Baar K, Esser K. 1999. Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am J Physiol 276:C120127.

Duncan ND, Williams DA, Lynch GS. 1998. Adaptations in rat skeletal muscle following long-term resistance exercise training. Eur J Appl Physiol 77:372378.

Fluckey JD, Dupont-Versteegden EE, Montague DC, et al. 2002. A rat resistance exercise regimen attenuates losses of musculoskeletal mass during hindlimb suspension. Acta Physiol Scand 176:293300. doi: 10.1046/j.1365-201X.2002.01040.x

Fujita S, Abe T, Drummond MJ, et al. 2007. Blood flow restriction during low-intensity resistance exercise increases S6K1 phosphorylation and muscle protein synthesis. J Appl Physiol Bethesda Md 1985 103:903910. doi: 10.1152/japplphysiol.00195.2007

Goldberg AL. 1968. Protein synthesis during work-induced growth of skeletal muscle. J Cell Biol 36:653658.

Goldspink G. 1999. Changes in muscle mass and phenotype and the expression of autocrine and systemic growth factors by muscle in response to stretch and overload. J Anat 194 ( Pt 3):323334.

Haddad F, Adams GR, Bodell PW, Baldwin KM. 2006. Isometric resistance exercise fails to counteract skeletal muscle atrophy processes during the initial stages of unloading. J Appl Physiol Bethesda Md 1985 100:433441. doi: 10.1152/japplphysiol.01203.2005

Hill M, Goldspink G. 2003. Expression and splicing of the insulin-like growth factor gene in rodent muscle is associated with muscle satellite (stem) cell activation following local tissue damage. J Physiol 549:409418. doi: 10.1113/jphysiol.2002.035832

Hornberger TA Jr, Farrar RP. 2004. Physiological hypertrophy of the FHL muscle following 8 weeks of progressive resistance exercise in the rat. Can J Appl Physiol Rev Can Physiol Applique 29:1631.

Kawada S, Ishii N. 2008. Changes in skeletal muscle size, fibre-type composition and capillary supply after chronic venous occlusion in rats. Acta Physiol Oxf Engl 192:541549. doi: 10.1111/j.1748-1716.2007.01761.x

Kawada S, Ishii N. 2005. Skeletal muscle hypertrophy after chronic restriction of venous blood flow in rats. Med Sci Sports Exerc 37:11441150.

Klitgaard H. 1988. A model for quantitative strength training of hindlimb muscles of the rat. J Appl Physiol Bethesda Md 1985 64:17401745.

Lee S, Barton ER, Sweeney HL, Farrar RP. 2004. Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J Appl Physiol Bethesda Md 1985 96:10971104. doi: 10.1152/japplphysiol.00479.2003

McCarthy JJ, Mula J, Miyazaki M, et al. 2011. Effective fiber hypertrophy in satellite cell-depleted skeletal muscle. Dev Camb Engl 138:36573666. doi: 10.1242/dev.068858

Miller, N. E. 1951. Learnable drives and rewards. In: Stevens, S. S. (ed) Handb. Exp. Psychol. Wiley, New York, pp 435472

Miyazaki M, Esser KA. 2009. Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals. J Appl Physiol Bethesda Md 1985 106:13671373. doi: 10.1152/japplphysiol.91355.2008

Miyazaki M, McCarthy JJ, Fedele MJ, Esser KA. 2011. Early activation of mTORC1 signalling in response to mechanical overload is independent of phosphoinositide 3-kinase/Akt signalling. J Physiol 589:18311846. doi: 10.1113/jphysiol.2011.205658

Nicastro H, Zanchi NE, da Luz CR, et al. 2012a. Effects of leucine supplementation and resistance exercise on dexamethasone-induced muscle atrophy and insulin resistance in rats. Nutr Burbank Los Angel Cty Calif 28:465471. doi: 10.1016/j.nut.2011.08.008

Nicastro H, Zanchi NE, da Luz CR, et al. 2012b. An experimental model for resistance exercise in rodents. J Biomed Biotechnol 2012:457065. doi: 10.1155/2012/457065

Olds J, Milner P. 1954. Positive reinforcement produced by electrical stimulation of septal area and other regions of rat brain. J Comp Physiol Psychol 47:419427.

Phillips SM. 2011. The science of muscle hypertrophy: making dietary protein count. Proc Nutr Soc 70:100103. doi: 10.1017/S002966511000399X

Scheffer DL, Silva LA, Tromm CB, et al. 2012. Impact of different resistance training protocols on muscular oxidative stress parameters. Appl Physiol Nutr Metab Physiol Applique Nutr Mtabolisme 37:12391246. doi: 10.1139/h2012-115

Tamaki T, Uchiyama S, Nakano S. 1992. A weight-lifting exercise model for inducing hypertrophy in the hindlimb muscles of rats. Med Sci Sports Exerc 24:881886.

Taylor NA, Wilkinson JG. 1986. Exercise-induced skeletal muscle growth. Hypertrophy or hyperplasia? Sports Med Auckl NZ 3:190200.

Thomsen P, Luco JV. 1944. Changes of weight and neuromuscular transmission in muscles of immobilized joints. J Neurophysiol 7:245251.

Timson BF. 1990. Evaluation of animal models for the study of exercise-induced muscle enlargement. J Appl Physiol Bethesda Md 1985 69:19351945.

Wadley GD. 2013. A role for reactive oxygen species in the regulation of skeletal muscle hypertrophy. Acta Physiol Oxf Engl 208:910. doi: 10.1111/apha.12078

Wirth O, Gregory EW, Cutlip RG, Miller GR. 2003. Control and quantitation of voluntary weight-lifting performance of rats. J Appl Physiol Bethesda Md 1985 95:402412. doi: 10.1152/japplphysiol.00919.2002

Wong TS, Booth FW. 1988. Skeletal muscle enlargement with weight-lifting exercise by rats. J Appl Physiol Bethesda Md 1985 65:950954.

Yarasheski KE, Lemon PW, Gilloteaux J. 1990. Effect of heavy-resistance exercise training on muscle fiber composition in young rats. J Appl Physiol Bethesda Md 1985 69:434437.

Zanchi NE, de Siqueira Filho MA, Lira FS, et al. 2009. Chronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3beta and p70S6K levels in rats. Eur J Appl Physiol 106:415423. doi: 10.1007/s00421-009-1033-6

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.

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.

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.

Figure 1