Basic Models ModelingResistance Training: An Updatefor Basic Scientists Interestedin Study Skeletal MuscleHypertrophyJASON CHOLEWA,1 LUCAS GUIMAR~AES-FERREIRA,2 TAMIRIS DA SILVA TEIXEIRA,3

MARSHALL ALAN NAIMO,4 XIA ZHI,5,6 RAFAELE BIS DAL PONTE DE SA,3 ALICE LODETTI,3

MAYARA QUADROS CARDOZO,3 AND NELO EIDY ZANCHI3*1Department of Kinesiology Recreation and Sport Studies, Coastal Carolina University, Conway, South Carolina2Laboratory of Experimental Physiology and Biochemistry, Center of Physical Education and Sports,

Federal University of Espirito Santo, Vitoria, Brazil3Postgraduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense, Criciuma, Brazil4Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia5Exercise Physiology and Biochemistry Laboratory, College of Physical Education, Jinggangshan University, Jinggangshan, PR, China6Exercise Physiology Laboratory, Department of Exercise Physiology, Beijing Sport University, Beijing, PR, China

Human muscle hypertrophy brought about by voluntary exercise in laboratorial conditions is the most common way to study resistanceexercise training, especially because of its reliability, stimulus control and easy application to resistance training exercise sessions at fitnesscenters. However, because of the complexity of blood factors and organs involved, invasive data is difficult to obtain in human exercisetraining studies due to the integration of several organs, including adipose tissue, liver, brain and skeletal muscle. In contrast, studyingskeletal muscle remodeling in animal models are easier to perform as the organs can be easily obtained after euthanasia; however, not allmodels of resistance training in animals displays a robust capacity to hypertrophy the desiredmuscle. Moreover, somemodels of resistancetraining rely on voluntary effort, which complicates the results observed when animal models are employed since voluntary capacity issomething theoretically impossible to measure in rodents. With this information in mind, we will review the modalities used to simulateresistance training in animals in order to present to investigators the benefits and risks of different animal models capable to provokeskeletal muscle hypertrophy. Our second objective is to help investigators analyze and select the experimental resistance training modelthat best promotes the research question and desired endpoints.J. Cell. Physiol. 229: 11481156, 2014. 2013 Wiley Periodicals, Inc.

In humans early training gains in muscle strength have beenregarded as the result of both neural and musculatureadaptations. Over the last half-decade several animal trainingmodels have been developed as a way to increase both forceoutput and mass (hypertrophy) in the exercised muscle.Contrary to the increases in maximal oxygen consumptionobserved in animals with aerobic training using a treadmill,measurements of maximal and submaximal force capacity invivo are complicated by several factors, including voluntarycapacity to perform resistance training, non-voluntaryelectrical-based training under anesthesia, surgicalmanipulation of muscles involved in the hypertrophic response,and the utilization of positive or negative reward to stimulatethe animals to perform the exercise. Thus, the greatestmotivation for an animal to produce maximal capacityvoluntary muscular force in classic operant models is via directelectrical stimulation to the brain, which is virtually impossibleto perform in subsequent experiments with the same animal(Olds and Milner, 1954).

Pain avoidance has been demonstrated to be a greaterstimulus than food or water reward (Miller, 1951). Accordingto Timson (1990), the animal will perform a task only until theeffort involved in the task performance exceeds its desire for

the stimulus. Thus, a model employing starvation as the mainstimulus will motivate the animal to exert only 5060% of itsmaximal voluntary capacity, which will then negatively affectmuscular hypertrophy capacity either due to lack of overloador nutrition. Therefore, we will first review animal modelsemploying non-voluntary maximal capacity force production asa way to induce hypertrophy, and then discuss new methodsinvolving voluntary models. A summary of results of themodelsreviewed is available in Tables I and II.

Dr. Jason Cholewa and Dr. Lucas Guimar~aes-Ferreira contributedequally to this work.

*Correspondence to: Nelo Eidy Zanchi, Av. Universitaria, 1105Bairro Universitario, C.P. 3167 | CEP 88806-000, Criciuma/SantaCatarina, Brazil. E-mail: neloz@ig.com.br

Manuscript Received: 12 December 2013Manuscript Accepted: 16 December 2013

Accepted manuscript online in Wiley Online Library(wileyonlinelibrary.com): 25 December 2013.DOI: 10.1002/jcp.24542

REVIEW ARTICLE 1148J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology

2 0 1 3 W I L E Y P E R I O D I C A L S, I N C.

Non-Voluntary Non-Electric Exercise-InducedEnlargement in Animal Models

One of the first methods to induce skeletal musclehypertrophy was developed by Thomsen and Luco (1944)whereby a passive stretch applied to immobilized joints placeslongitudinal 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 anelongation of the target muscle. The application of rapamycinwas demonstrated to robustly suppress this response,suggesting the mammalian target of rapamycin (mTOR)pathway is involved in the longitudinal hypertrophy induced byjoint immobilization. This model of overload may beappropriate to study skeletal muscle remodeling as a result ofstretch overload or joint immobilization; however, resistancetraining in humans requires dynamic tension generation,resulting in a force overload, and leading to the synthesis ofadditional sarcomeres in series. Therefore, future investigatorssought to develop methods that more closely modeledresistance training.

Goldberg (1968) developed an effective non-voluntary non-electrically stimulated model (Fig. 1A) to induce skeletal musclehypertrophy through synergistic ablation (surgical removal of asynergistic muscle, most often the gastrocnemius calcaneusportion, generating overload and muscle hypertrophy of thesoleus and plantaris muscle). Although the use of this model tomimic the effects of human strength training has been highlycriticized due to the surgical procedures (Taylor andWilkinson, 1986), McCarthy et al. (2011) demonstrated nodifferences in muscle hypertrophy between mice with geneticsatellite cell depletion and non-depleted controls with 2 weeksof synergistic ablation overload. Given the similar significantimprovements in muscle hypertrophy in both groups,synergistic ablation remains an effective method to studycellular signaling pathways leading to acute skeletal musclehypertrophy (Miyazaki and Esser, 2009).

On the other hand, because the targeted muscle is exposedto a static stimulus (the animals bodyweight) the increase inmuscle mass occurs most rapidly during the first week of theprotocol and appears to reach a plateau 2 weeks followingsurgery. Additionally, the animal is under constant overloadevery time it moves, compared to separate training sessionsused in human resistance training or other animal models.Thus, synergistic ablation cannot be used in long term studiesnor does it appear compatible with modeling the progressiveoverload or periodization phases and nutrition schedulesrequired in human resistance training to induce maximalchanges in hypertrophy and strength.

Tenotomy is a technique where the gastrocnemius tendon isdetached and the synergistic muscle is placed under increasedmuscle tension (Fig. 1A). Tenotomy appears less effective atinducing overload and the resultant musculature hypertrophyof the synergist (e.g., plantaris) when compared with surgicalablation (Timson, 1990). Although the reason for thedifference is not clear, it appears that the cut tendon is able toreattach when left intact within the muscle fascia. The critiquesof tenotomy are the same as those related to synergisticablation methods; however the magnitude of hypertrophy isless and the possibility of the gastrocnemius tendon reattachingthe calcaneus tendon.

The use of chronically restricted venous blood flowwas firstreported by Kawada and Ishii (2005) to induce skeletal musclehypertrophy in rats. This model does not involve exercise;rather, blood flow to the hind limbs is diminished via a surgicalintervention. Fourteen days following the operation theplantaris muscle increased in dry weight by 10% and theconcentration of myofibrillar protein increased by 23%.Additionally, levels of nitric oxide synthase and the muscleTA

BLE

I.Voluntary

musclehypertrophymodels

Author

Year

Technical

Size/height

Angulation

Sessions/weeks

Training

period

Series/day

Muscle

hypertrophy(%)

Yarasheski

etal.

1990

Progressive

liftwithloadsattached

tothetailusingamesh

40cm

90

5days/week

8weeks

20

Increase

inRFweight

Duncanet

al.

1998

Ladder

climbing

40cm

Vertical

4days/week

26weeks

1215

Increase

inEDLandSO

Lweights

relative

tobodymassandfibre

hypertrophy

Leeet

al.

2004

Ladder

climbingIGF-Iadenovirus

1m

85

Every

thirdday

8weeks

8climbsoruntilfailure

Increase

inFH

Lweight

Klitgaardet

al.

1988

Plantarflexionofanklejoint

Inthemorning,at

noon,

andin

theevening

(Monday

andTuesday,

Thursday

andFriday)

36weeks

30min

oftraining

threetimes

per

day

Increase

inSO

LandPLA

weights

Zanchiet

al.

2009

Plantarflexionofanklejoint

Threetimes/week

12weeks

16

Increase

inPLA

weight

Tam

akiet

al.

1999

Squat

Like

Exercise

45days/week

12weeks

6575%

1RM

HypertrophyofGASandPLA

andincrease

inthenumber

ofmuscle

fibers

DelaCruzet

al.

2012

Jumpin

aPVC

cylinder

containingwater

Every

2days

5weeks

15jumpsessions

Increase

inEDLandSO

LCSA

Wirth

etal.

2003

Operantconditioning(progressive

lift)

8weeks

1215

Increasedperform

ance

Hornberger

2004

Ladder

climbing(w

ithprogressive

load

attached

tothetail)

1.1m

80

8weeks

1Increase

inFH

Lweigthandtotaland

myofibrillarprotein

content

Schefferet

al.

2012

Ladder

climbing

43steps

Alternatedays

12weeks

315

Fluckey

etal.

2002

Flyw

heelresistance

training

4weeks

25

Attenuationofhindlim

bsuspension-induced

muscle

atrophyin

SOL

RF,rectusfemoris;EDL,extensordigitorum

longus;SO

L,soleus;FH

L,flexorhallucislongus;PLA

,plantaris;GAS,gastrocnem

ius;CSA

,cross-sectional area.

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insulin like growth factor-1 (IGF-1) also increased. It is difficultto speculate on the level of difficulty or safety of this model as adetailed description of the surgery is not completely available inthe literature; however, this model appears to be consistentsince Kawada and Ishii (2008) reproduced the results of thefirst study and also reported decrements in type Imuscle fibers.Although plantaris hypertrophy was modest compared tosynergist ablation, chronic blood flow restriction may be anovel model to study hypertrophy in animals.When translatingthe results to human training two questions arise: (1)What arethe effects of chronic blood flow restriction combined withmuscular tension? (2) Does blood flow restriction occurringfor longer than 2weeks compromise the health of the animal orresult in a plateau in muscle hypertrophy? Given thatintermittent blood flow restriction under low tensionphosphorylates P70S6K and muscular hypertrophy in humans(Fujita et al., 2007), answering these questions are essential toevaluating the ability to translate this model to humanresistance training.

Non-Voluntary, Electric Exercise-Induced Enlargementin Animal Models

Wong and Booth (1988) developed a novel non-voluntarymodel to load the hind limb and induce muscle hypertrophy. Inthis model the animal is anesthetized, the foot is attached to animmovable metal plate with adhesive tape, and muscularcontraction is stimulated electrically with joint of the animalstarting in a neutral position (Fig. 1E). The ability of this modelto induce hypertrophy and increased muscle fiber crosssectional area is inconsistent and produces only modestresults; however, using a modified model, Baar and Esser(1999) demonstrated P70S6K phosphorylation andpolyribosome formation, which indicates that the Wong andBooth model is capable of increasing protein synthesis.

Goldspink (1999) modified the protocol proposed byWongand Booth (1988) by loading the limb in a stretched position(elongation) and allowing for the electrical stimulus to induce adynamic contraction (Fig. 1I). This combined model resulted ina greater increase in protein synthesis compared to theelongation model or isometrically loaded models alone.Moreover, using the combination of elongation and dynamicoverloadGoldspink demonstrated the activation of a transcriptderived from the IGF-1 local to skeletal muscle, which has beenlabeled mechano growth factor (MGF). MGF presents an insertwith 52 base pairs in the E domain of the gene, which alters thereading frame of the 30 end, resulting in satellite cellproliferation/activation following muscle damage, ultimatelyleading to muscular repair and hypertrophy (Hill andGoldspink, 2003). This model allows the researcher to apply anidentical maximal pulse to generate maximal tetanic force, andthus eliminates the need to readjust the electrical stimuli.Although the combination of muscular elongation and non-

voluntary contraction may be viable in studying acute increasesin protein synthesis, electrical pulses under anesthesia aredifficult to perform, as is the ability to apply a consistent,progressive increase in electrical stimulation to match anincreased load required to induce hypertrophy.

Resistance Training (RT) Exercise Under UnloadingConditions

Another interesting resistance training model was presentedby Haddad et al. (2006) whereby rats were unloaded via hindlimb suspension (HS) to induce muscular atrophy for 6 days.Animals in the resistance training group (HST) were trainedevery other day. Briefly, animals were anesthetized andstimulation electrodes consisting of Teflon-coated stainlesssteel wire were introduced into the subcutaneous regionadjacent to the popliteal fossa via 22-gauge hypodermicneedles. Wire placement was lateral and medial of the locationof the sciatic nerve allowing for field stimulation of the nerve.The stimulation wires were then attached to the output polesof a Grass stimulus isolation unit interfaced with a Grass S8stimulator. This allowed for the delivery of current to thesciatic nerve resulting in muscle contraction. The right leg waspositioned in a footplate attached to the shaft of a Cambridgemodel Hergometer, adjusted to produce maximal isometrictension. Each training bout consisted of a series of four sets ofcontractions with 5min of recovery between sets. Each setconsisted of a series of 10 maximal isometric contractionslasting 2 sec each with 20 sec of rest in between contractions.Thus each training session lasted for 27min, during which themuscle was activated for a cumulative time of 80 sec.

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 maintainingrelative muscle weight at a higher level compared with the HSgroup (8%), this response may in part have been due to edema,as total protein concentration was slightly lower (7%) in theHST compared with the HS group. This responsedemonstrates the negative impact of unloading on the hind limbmusculature by illustrating that the myofibril pool was indeed aprimary target of the atrophy response. The results of thisstudy suggest that the process of muscle atrophy is notopposite of muscle hypertrophy, and demonstrate the inabilityof isometric based RT to spare muscle protein duringunloading. Therefore, although an isometric model of RT maybe appropriate to induce hypertrophy, researchers usingresistance training in animal models of diseases (i.e.,dexamethasone-induced diabetes; Nicastro et al., 2012a)should consider performing experimental pilot studies withdynamic based contractions prior to data collection.

On the other hand, Fluckey et al. (2002) demonstrated thatdynamic resistance training is capable of preventing musclewasting during unloading. In this model, Fluckey et al.

TABLE II. 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 weightsGoldberg et al. 1975 Tenotomy of GAS 14 days Walk Increase in SOL weightWong and Booth 1988 Weight-lifting exercise 16 weeks Electric external load Increase in GAS we weight and

protein contentBaar and Esser 1999 Surgical implantation of electrodes

and electrical stimulation6 weeks Electric Increase in EDL and TA wet weights

Goldspink 1999 Stretch combined with eletrical stimulationof 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 weeks Venous occlusion Increases in PLA dry weight body weightand myofibrillar protein content

Haddad et al. 2006 Electrical stimulation 6 days Isometric contractions Attenuation of hindlimb suspension-inducedmuscle 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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Fig. 1. Progression of the development of resistance training models in Rodents (19682012). (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 ladderclimb; (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 theauthors.

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developed a modified version of the human flywheel resistanceexercise apparatus so rats could be trained while in hind-limbsuspension. This poses a major advantage over the model usedin Haddad et al. as the animals can be trained with dynamicresistance exercise independent of gravity and without beingremoved from the cage. Briefly, a rat is tethered via a leatherand velcro vest attached to a nylon cord and spooled around aninertia wheel located on the outside of the resistance exerciseapparatus. The rat is allowed to place its feet on a shock gridsuspended at the top of the apparatus (to accommodate the HSstate) and an illumination bar capable is located in the apparatusopposite to the shock grid. The bar is then illuminated whichresults in a repetition by the animal. Themovement is similar tosquats as performed by humans, as extension occurs at the hip,knee and ankle joints. When required a shock is applied briefly(

minor modifications to the protocols may greatly affect theresults such that the functionality of the model is reducedwhenmuscular hypertrophy is a major endpoint, thereby reducingthe ability to study the effects of genetic manipulation orergogenic aids.

To monitor the variance in overload and work performedbetween groups we suggest measuring venous lactate andmodifying the load appropriately. Scheffer et al. (2012)demonstrated the effectiveness of this method to equalize theload between groups. Additionally, we suggest researchersusing this model to induce hypertrophymodify the length of theladder by reducing the number of steps the animal climbs andincreasing the load to more closely mimic human strengthtraining. As an example Scheffer et al. (2012) employed ahypertrophy protocol of 48 steps with 1.1 cm between steps.Although hypertrophy was not measured, a relationship existsbetween exercise-induced oxidative stress and musclehypertrophy (Wadley, 2013), suggesting that sets of lessrepetitions may be most effective in inducing hypertrophy.Additionally, this specific hypertrophy protocol on the laddermay be the most appropriate for evaluating satellite cellactivation and differentiation with resistance training.

Another animal model of voluntary resistance exercise wasproposed by Klitgaard (1988) rats were trained to perform aplantar extension in order to obtain a pellet of food (Fig. 1C).The original protocol was performed in 2-year-old rats andafter 36 weeks of training plantaris muscle mass increased 24%.On the other hand, utilizing the same protocol but in young ratsand for only 13 weeks we observed the plantaris musclehypertrophied by 13% (Zanchi et al., 2009). Our major findingusing this model was that the Atrogenes (MuRF-1 andAtrogin-1), ubiquitin ligases involved in muscle proteolysis bythe proteasome, decreased only in the trained group,demonstrating the ability of this model to modulate molecularsignaling. Since we did not measure the degree of muscleprotein synthesis or degradation in the isolated muscles, wecannot speculate on the ability of this model to impact proteinturnover as a whole. There are two factors to consider whenusing this model: (1) a longer training period is required forhypertrophy to occur when compared to the synergist ablationor the ladder model, and (2) this model uses starvation tomotivate the animals to perform the plantar extension. Thisstarvation period poses a major issue when studyingphysiological responses as it affects both voluntary work andnutrient status, and is also difficult to apply. Thus thetranslation of this model to humans must be interpreted withcaution, although several acute studies in humans areperformed under starvation conditions (Fujita et al., 2007).

Tamaki et al. (1992) described a weight lifting exercisemodel designed to induce muscle hypertrophy in the hind-limbby loading the animal with a canvas jacket attached to the torsoand requiring the animal to perform a squat like exercise(Fig. 1H). The main stimulus was provided by an electricstimulator linked to the tail of the animal so a punishmentstimuli was applied and the animals performed a squatlike exercise of progressively increasing loads within ahypertrophy range (6575% 1 Repetition Maximum, RM).Compared with 60min of treadmill sprints, acute squattraining resulted in an increase in plasma creatine kinase.When sprint and squat training was carried outfor 12 weeksat 45 days/week there was a 12% increase in the plantarismuscle compared with control animals receiving an electricstimulus; however, there were no significant differencescompared to the sprint training group. Although this modelcontains a similar biomechanical loading andmovement patternto human resistance training, its ability to overload the animalsand induce muscle hypertrophy is inferior to other voluntaryresistance training models, such as the ladder climb or foodmotivated plantar extension proposed by Klitgaard (1988).

Wirth et al. (2003) developed a revolutionary model whererats were operantly conditioned to perform a squat exercisevia both reward and punishment (Fig. 1K). Food was restrictedand rats were operantly conditioned with food rewards toenter a vertical tube, insert its head into a weighted ring (either70 or 700 g), lift the ring until its nose interrupted an infrareddetector, and then lower the ring. Load cells measured theexternal force generated, and displacement transducersmeasured the vertical displacement of the ring during eachlifting and lowering movement. The apparatus and trainingprocedures were computer automated. Peak force, velocity,work, and power were calculated for each movement. Rats inboth groups easily acquired the task after 1215 trainingsessions conducted 5 days/week. Themedian peak force, work,and power per lift for both concentric and eccentric weregreater for the 700 g group. Importantly, 8 weeks of lifting both70 g and 700 g five sessions per week increased plantaris, soleus,and gastrocnemius mass compared to sedentary controls;however, dry weight and muscular protein content was notmeasured, thus it is also possible these increasesmay have beenpartly the result of edema and/or inflammation. These resultsdemonstrate the utility of quantitating the biomechanics ofvolitional movements and suggest that the present modelcan establish and maintain controlled repetitive movementsnecessary 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 thistraining protocol, suggesting that this positive operant modelcombined with histological sampling is a valid protocol to studyresponses to resistance training.

Given the potential of the models described by Wirth et al.(2003) and Klitgaard (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 tomore precisely control the training variables proposed inWirth et al. (2003). In this model we developed an operantconditioning system composed of sound, scent, light, andfeeding devices that optimized resistance exercise perfor-mance by the animal (Fig. 1L). With this system, it was notnecessary to tie the animal into the device or impose chronicfasting or electric shock for the animal to perform the taskproposed (muscle contraction). Furthermore, it was possibleto perform muscle function tests in vivo maximal voluntarystrength capacity (MVSC) within the context of the exerciseproposed and control variables such as intensity (percent ofMVSC or percent of body weight), volume (sets andrepetitions), rest intervals between sets, and exercise sessionlength. Importantly, sound was the main stimulus given to theanimals as a way to optimize learning and reinforce exercisetraining. Therefore, despite experimental limitations, webelieve that this RE apparatus is closer to the physiologicalcontext observed in humans. When testing the efficacy of thisprotocol to counteract the effects of 7 days of 5mg/daydexamethasone (a diabetogenic and proteolytic catabolichormone) in a common model of skeletal muscle atrophy,we observed that training attenuated the loss of gross musclemass and increased plantarismass when compared to controls.Additionally, we observed an increase in MVSC in trainedanimals, but not controls at the end of the study (Nicastroet al., 2012a), demonstrating the efficacy of this model toattenuate or even prevent atrophy, and as a reliable techniqueto study atrophic disease.

Variables to Evaluate When Selecting a ResistanceTraining Model

According to Timson (1990), when using animal models toevaluate muscle enlargement produced by strength trainingin humans, three factors must be considered: (1) Muscle

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recruitment and adaptations in fiber characteristics; (2)magnitude of muscle enlargement. Given the effects of varyingmodels of voluntary and non-voluntary resistance loadingreported by our team and others, we suggest six other factorsto consider: (3) the degree of nutrition required for a positivereward; (4) negative reward (i.e., pain). (5) Time spentconditioning the animal to execute the exercise; (6) durationrequired to obtain muscle remodeling; (7) muscle voluntarycapacity percentage; (8) resistance training under atrophic ordiseased conditions.

1. Muscle recruitment and adaptations in fiber characteristics:With specific study questions (i.e., sarcopenia) type IImuscle fiber hypertrophy is more relevant that grosshypertrophy in preventing the loss of muscle mass andfunction; however, not all models of training are capable ofoverloading all muscle fibers and thus eliciting a substantialdegree of hypertrophy in muscles comprised of predomi-nantly Type II fibers. As an example, the plantaris is a mixedfiber muscle and its hypertrophy through surgical ablation ofthe gastrocnemius (a predominantly type II muscle) may notbe appropriate to study the reversion of sarcopeniacompared to a squat based model (Nicastro et al., 2012b).

2. Magnitude of muscle enlargement: Some exercise models aredifficult to perform and the resulting hypertrophy degree isvery poor when compared with others. For example, theladder climb model is capable to generate hypertrophy inthe muscles of the lower limbs of the rat; however, thehypertrophy of the FHL vary considerably in the literaturebased upon ladder length, load, and frequency (Duncanet 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 groupobserved by Teixeira et al., 2013, unpublished data), but notthe 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 acryliccylinder where the rats where trained to feed throughoverextension of hind-limb (plantar extension) to take thepellet and then feed. Utilizing this model, our group (Zanchiet al., 2009) observed it required approximately 24 h of foodrestriction to motivate the animals to perform the lift inorder to obtain a food pellet. Additionally, the number ofrepetitions per day was very limited (16 per day), althoughwe observed an increase of 13% of muscle mass (plantarisand soleus) compared with paired feeding control group.Thus, future investigations should consider the effects ofmodels that require nutrition deprivation to performexercise when major endpoints include robust increasesin muscle hypertrophy.

4. Negative reward: It is well known recognized that punish-ment is a stronger stimulus than reward to induce rodentsto perform resistance exercise (Zanchi et al., 2009).However, sometimes this punishment stimuli is detrimentalas the appetite of the animals is reduced due to theendocrine response involved in the fight or flight (stress)reaction, thus impairing the muscular and molecularadaptations to a pre-determined stimulus. For example,Tamaki et al. (1992) demonstrated increases in gastrocne-mius and plantarismass with resistance training compared tocontrol groups following 12 weeks of training; however, theresistance training group lost approximately 200 g of bodymass. Thus, punishment in this model influenced theendocrine response and diminished the appetite of thetrained animals such that limbmuscle hypertrophywas likelycompromised.

5. Time spent conditioning the animal to execute the exercise:Through two different voluntary exercise models, weobserved that some rats are capable to learn how to

execute a task (resistance training) very fast whereas othersare not. Therefore, every time we conducted a resistancetraining protocol we selected for both control andintervention groups only the animals capable to learnvery 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 andpunishment stimulus to induce rodents to performresistance exercise (hindlimb extension; Nicastro et al.,2012b). Based upon the results from these models, wesuggest researchers employing models of voluntary maximalcontraction spend 1416 unloaded training sessions spacedevery day over 2 weeks to condition the animals to performthe resistance training protocol.

6. Duration required to obtain muscle remodeling: In our firstmodel of food rewarded plantar extension (Zanchiet al., 2009), 3 months of training were required to increaseplantarismass by 13%. Similarly, Tamaki et al. (1992) spent 3-month to induce relative bodyweight adjusted (muscleweight/body weight) increases in gastrocnemius andplantarismass of 31.4% and 17.9%, respectively, and absoluteincrease of 12% in plantaris mass when compared withcontrols. In comparison, utilizing electrical simulation Baarand Esser (1999) demonstrated increases in tibialis anteriorand EDL mass of 13.9 and 14%, respectively, whenperformed twice a week for 6 weeks. Further research isrequired to elucidate the optimal combination of frequency(sessions per week) and volume when designing animalmodels leading to muscle hypertrophy. Despite similarincreases in absolute plantaris weight, the rats in Tamakiet al. (1992) were trained two to three sessions per weekmore than those in our study. These results are furtherconfounded by synergist ablation where the target muscle(usually the plantaris) is chronically under a load every timethe rat moves (similar to a human carrying extra body-weight, not taking part in resistance training). Thus, eachmodel has specific characteristics, and based upon the load(light or heavy) and expected outcomes pilot sessionsshould be conducted to determine appropriate frequenciesand study durations.

7. Muscle Voluntary Capacity Percentage: It has been welldescribed in the literature that intensities in the range of7585% 1 RM are ideal to increase/hypertrophy the musclemass. However, utilizing some models it is almostimpossible to predict those ideal ranges. For example,utilizing the ladder model some investigators have employeda maximum effort test in the ladder model; however, theladder contains 1624 steps so the rodent is performing, infact, a test of local muscular endurance (i.e., a 16 RM test)and not a true 1 RM. Predicting the maximal voluntarypower output of a rodent for a given stimulus is also difficultand unreliable. For example, when we tested the food-reward model developed by Klitgaard (1988) the rats wereunable to perform more than eight repetitions per workout(Zanchi et al., 2009). A major concern was whether the ratactually exerted maximal effort, or if it was satisfied by areduced food intake. This issue was partially resolved in ourmore recent model (Nicastro et al., 2012b) as we observedthe rats performed nearly 50 repetitions per session.Moreover, we were able to measure force developedthrough a computerized model and compare that to MVSCto ensure the animals were working the hypertrophy range.Thus, we believe this voluntary model allows the researcherto more precisely manipulate volume and intensity in thestudy of training induced muscular adaptations, specificallyhypertrophy and soft tissue remodeling.

8. Resistance training under atrophic or diseased conditions:Several models of atrophy have been described in theliterature; the most commonly used mimics that of

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spaceflight (gravitational unloading) via hind limb suspen-sion. Under this condition of atrophy, isometric-basedresistance training models capable of producing robusthypertrophy under gravitational conditions have failed toinduce hypertrophy or counteract atrophy (Haddadet al., 2006). It is possible that this resistance trainingmodel may have attenuated the loss of muscle mass under agravitational model of atrophy (i.e., administration ofrapamyacin). On the other hand, it is well established inthe literature that dynamic resistance training plusnutritional support (mainly proteins containing high biologi-cal value and leucine) are capable of robustly activatingprotein synthesis pathways (Phillips, 2011) and counter-acting unloading-induced loss of muscle mass as demon-strated by Fluckey et al. (2002). When considering thecatabolic state as generated by glucocorticoids and diabetesmellitus, we demonstrated that resistance training is capableof counteracting the loss of muscle mass (Nicastroet al., 2012a). Although further research in humans isneeded to describe the hormonal milieu and muscleactivation in response to daily living activities in humanswith atrophic disease, our model provides researchers witha greater degree of control over the variables involved inresistance training, and can be used to study the effects ofresistance training on atrophy during conditions ofgravitational unloading and glucocorticoid catabolism.

In example of how these factors may affect investigationoutcomes, Miyazaki et al. (2011) utilized the synergisticablation surgery to investigate the involvement of ERK/MERKpathways and the well-known Akt/mTOR/P70S6K pathways ofprotein synthesis initiation in the muscle hypertrophyphenomena. Using the synergist ablation model of musclehypertrophy, early and late periods of muscle adaption wereexamined. Specifically, they measured these adaptations in amodel of form follows signaling function observing plantarishypertrophy 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 23, whereasP70S6K (Ser 389, Thr 421/424) and ribosomal protein (RPS6Ser 235236) where highly phosphorylated during the entirehypertrophy process. Of note, this delay in the classical Akt/mTOR activation was accompanied by a rapid and prolongedMEK 1 and 2 (Ser 217/221) activation. The same pattern wasobserved in ERK 1 and 2 (Thr 221 and 224). Importantly, thisdivergence utilizing a well-recognized animal musclehypertrophy model demonstrated parallel pathways, oneactivated early and one later, that both phosphorylated the RP6protein culminating in increased protein synthesis. In the earlypathway ribosomal kinases phosphorylated the RP6 in the Ser235/236 residue. Following the late pathways, RPS6 wasphosphorylated in the residues Ser 240/244. This studydemonstrates the importance of matching expected outcomeswithin the research question to the resistance training modelemployed. If Miyazaki et al. (2011) had utilized a modelrequiring a longer duration of conditioning or training to inducemuscle remodeling (i.e., the squat model) these noveldiscoveries may not have been made. Thus, investigators needto be aware of these particular attributes to resistance trainingmodels when investigating molecular signaling pathways,ergogenic aids, and mechanical tensions and muscleremodeling.

Conclusion

Many important molecular findings regulating skeletal muscleremodeling have been made through the use of diverseexperimental resistance training models. Although a number ofmodels are capable of inducing muscular hypertrophy,

investigators should consider several new variables whenselecting the most appropriate model to answer the researchquestion. For example, investigators should be aware of theearly and late signaling pathways leading to hypertrophy, andchose amodel that activates the appropriate phase. In this sameregard, if a large degree of muscular remodeling is required toanswer a research question selecting a hypertrophymodel witha weak magnitude will result in compromised findings.

A major issue facing clinic scientists investigating themedicinal/therapeutic effects of resistance training is thatgenetic modifications in animals that are available to basicscientists are not applicable to the general population.Consequently, a growing emphasis is being placed oninvestigating the efficacy and effectiveness of nutritionalsupplements and ergogenic aids in conjunction with resistancetraining. Although the discovery of new signaling pathways hasnot suddenly changed or advanced the methods people use totrain, overtime these discoveries have led to more effectivetraining methods, especially when resistance training is used asphysiotherapy. As the ability to more accurately manipulate thevariables involved in rodent resistance training models(volume, intensity, frequency, etc.) improve, scientists will bebetter able to study the effects of distinct differences inprogram design on molecular signaling and hypertrophy.These improvements in model design and the results obtainedcan then be used to improve the translation between basicscientific discoveries, clinical practices, and the application tohuman health and performance.

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