REVIEW ARTICLE
Journal of
Basic Models Modeling Resistance Training: An Update for Basic Scientists Interested in Study Skeletal Muscle Hypertrophy
Cellular Physiology
2 ~ JASON CHOLEWA,1 LUCAS GUIMARAES-FERREIRA, TAMIRIS DA SILVA TEIXEIRA,3 3 ALICE LODETTI,3 MARSHALL ALAN NAIMO,4 XIA ZHI,5,6 RAFAELE BIS DAL PONTE DE SA, MAYARA QUADROS CARDOZO,3 AND NELO EIDY ZANCHI3* 1
Department of Kinesiology Recreation and Sport Studies, Coastal Carolina University, Conway, South Carolina
2
Laboratory of Experimental Physiology and Biochemistry, Center of Physical Education and Sports, Federal University of Espirito Santo, Vitoria, Brazil
3
Postgraduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense, Criciuma, Brazil
4
Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia
5
Exercise Physiology and Biochemistry Laboratory, College of Physical Education, Jinggangshan University, Jinggangshan, PR, China
6
Exercise 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 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. J. Cell. Physiol. 229: 1148–1156, 2014. ß 2013 Wiley Periodicals, Inc.
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 ß 2 0 1 3 W I L E Y P E R I O D I C A L S, I N C.
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 I and II.
Dr. Jason Cholewa and Dr. Lucas Guimar~aes-Ferreira contributed equally to this work. *Correspondence to: Nelo Eidy Zanchi, Av. Universitaria, 1105 Bairro Universitario, C.P. 3167 | CEP 88806-000, Criciuma/Santa Catarina, Brazil. E-mail: [email protected] Manuscript Received: 12 December 2013 Manuscript Accepted: 16 December 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 25 December 2013. DOI: 10.1002/jcp.24542
1148
3–15 25 RF, rectus femoris; EDL, extensor digitorum longus; SOL, soleus; FHL, flexor hallucis longus; PLA, plantaris; GAS, gastrocnemius; CSA, cross-sectional area.
2012 2002 Scheffer et al. Fluckey et al.
JOURNAL OF CELLULAR PHYSIOLOGY
Non-Voluntary Non-Electric Exercise-Induced Enlargement in Animal Models
12 weeks 4 weeks Alternate days — — — 43 steps —
5 weeks 8 weeks 8 weeks Every 2 days — — — — 80˚ — — 1.1 m 2012 2003 2004 Dela Cruz et al. Wirth et al. Hornberger
Jump in a PVC cylinder containing water Operant conditioning (progressive lift) Ladder climbing (with progressive load attached to the tail) Ladder climbing Flywheel resistance training
— — — — 2009 1999 Zanchi et al. Tamaki et al.
Plantar flexion of ankle joint “Squat Like Exercise”
15 jump sessions 12–15 1
Increase in PLA weight Hypertrophy of GAS and PLA and increase in the number of muscle fibers Increase in EDL and SOL CSA Increased performance Increase in FHL weigth and total and myofibrillar protein content — Attenuation of hindlimb suspension-induced muscle atrophy in SOL 16 65–75% 1 RM 12 weeks 12 weeks
8 weeks 36 weeks
Every third day In the morning, at noon, and in the evening (Monday and Tuesday, Thursday and Friday) Three times/week 4–5 days/week 1m — 2004 1988 Lee et al. Klitgaard et al.
Ladder climbing þ IGF-I adenovirus Plantar flexion of ankle joint
85˚ —
12–15 26 weeks 4 days/week 1998 Duncan et al.
40 cm
Vertical
8 climbs or until failure 30 min of training three times per day
Increase in EDL and SOL weights relative to body mass and fibre hypertrophy Increase in FHL weight Increase in SOL and PLA weights
Increase in RF weight 8 weeks 5 days/week 90˚ 40 cm
Progressive lift with loads attached to the tail using a mesh Ladder climbing 1990 Yarasheski et al.
Technical Year Author
TABLE I. Voluntary muscle hypertrophy models
Size/height
Angulation
Sessions/weeks
Training period
20
Series/day
Muscle hypertrophy (%)
BASIC MODELS MODELING RESISTANCE TRAINING
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 (1968) developed an effective non-voluntary nonelectrically 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 animal’s 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 (e.g., 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
1149
1150
CHOLEWA ET AL.
TABLE II. Involuntary muscle hypertrophy models Author
Year
Goldberg et al. Goldberg et al. Wong and Booth
1968 1975 1988
Baar and Esser
1999
Goldspink
1999
Kawada and Ishii Haddad et al.
Technical
Period
Estimulus
Surgical ablation of GAS Tenotomy of GAS Weight-lifting exercise
6 days 14 days 16 weeks
Walk Walk Electric þ external load
6 weeks
Electric
4 days
Electric
Increase in TA wet weight
2005
Surgical implantation of electrodes and electrical stimulation Stretch combined with eletrical stimulation of anterior tibialis muscle of adult rabbit Chronic restriction of blood flow to muscle
Increase in SOL and PLA weights Increase in SOL weight Increase in GAS we weight and protein content Increase in EDL and TA wet weights
Venous occlusion
2006
Electrical stimulation
Increases in PLA dry weight body weight and myofibrillar protein content Attenuation of hindlimb suspension-induced muscle atrophy in GAS (muscle weight)
2 weeks 6 days
Isometric contractions
Muscle hypertrophy (%)
EDL, extensor digitorum longus; SOL, soleus; TA, tibialis anterior; flexor hallucis longus; PLA, plantaris; GAS, gastrocnemius.
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 (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. Goldspink (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 Goldspink 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 30 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 nonJOURNAL OF CELLULAR PHYSIOLOGY
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 6 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 Hergometer, 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 sec each with 20 sec 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 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 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.
BASIC MODELS MODELING RESISTANCE TRAINING
Fig. 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.
JOURNAL OF CELLULAR PHYSIOLOGY
1151
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CHOLEWA 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 (
Journal of
Basic Models Modeling Resistance Training: An Update for Basic Scientists Interested in Study Skeletal Muscle Hypertrophy
Cellular Physiology
2 ~ JASON CHOLEWA,1 LUCAS GUIMARAES-FERREIRA, TAMIRIS DA SILVA TEIXEIRA,3 3 ALICE LODETTI,3 MARSHALL ALAN NAIMO,4 XIA ZHI,5,6 RAFAELE BIS DAL PONTE DE SA, MAYARA QUADROS CARDOZO,3 AND NELO EIDY ZANCHI3* 1
Department of Kinesiology Recreation and Sport Studies, Coastal Carolina University, Conway, South Carolina
2
Laboratory of Experimental Physiology and Biochemistry, Center of Physical Education and Sports, Federal University of Espirito Santo, Vitoria, Brazil
3
Postgraduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense, Criciuma, Brazil
4
Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia
5
Exercise Physiology and Biochemistry Laboratory, College of Physical Education, Jinggangshan University, Jinggangshan, PR, China
6
Exercise 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 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. J. Cell. Physiol. 229: 1148–1156, 2014. ß 2013 Wiley Periodicals, Inc.
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 ß 2 0 1 3 W I L E Y P E R I O D I C A L S, I N C.
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 I and II.
Dr. Jason Cholewa and Dr. Lucas Guimar~aes-Ferreira contributed equally to this work. *Correspondence to: Nelo Eidy Zanchi, Av. Universitaria, 1105 Bairro Universitario, C.P. 3167 | CEP 88806-000, Criciuma/Santa Catarina, Brazil. E-mail: [email protected] Manuscript Received: 12 December 2013 Manuscript Accepted: 16 December 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 25 December 2013. DOI: 10.1002/jcp.24542
1148
3–15 25 RF, rectus femoris; EDL, extensor digitorum longus; SOL, soleus; FHL, flexor hallucis longus; PLA, plantaris; GAS, gastrocnemius; CSA, cross-sectional area.
2012 2002 Scheffer et al. Fluckey et al.
JOURNAL OF CELLULAR PHYSIOLOGY
Non-Voluntary Non-Electric Exercise-Induced Enlargement in Animal Models
12 weeks 4 weeks Alternate days — — — 43 steps —
5 weeks 8 weeks 8 weeks Every 2 days — — — — 80˚ — — 1.1 m 2012 2003 2004 Dela Cruz et al. Wirth et al. Hornberger
Jump in a PVC cylinder containing water Operant conditioning (progressive lift) Ladder climbing (with progressive load attached to the tail) Ladder climbing Flywheel resistance training
— — — — 2009 1999 Zanchi et al. Tamaki et al.
Plantar flexion of ankle joint “Squat Like Exercise”
15 jump sessions 12–15 1
Increase in PLA weight Hypertrophy of GAS and PLA and increase in the number of muscle fibers Increase in EDL and SOL CSA Increased performance Increase in FHL weigth and total and myofibrillar protein content — Attenuation of hindlimb suspension-induced muscle atrophy in SOL 16 65–75% 1 RM 12 weeks 12 weeks
8 weeks 36 weeks
Every third day In the morning, at noon, and in the evening (Monday and Tuesday, Thursday and Friday) Three times/week 4–5 days/week 1m — 2004 1988 Lee et al. Klitgaard et al.
Ladder climbing þ IGF-I adenovirus Plantar flexion of ankle joint
85˚ —
12–15 26 weeks 4 days/week 1998 Duncan et al.
40 cm
Vertical
8 climbs or until failure 30 min of training three times per day
Increase in EDL and SOL weights relative to body mass and fibre hypertrophy Increase in FHL weight Increase in SOL and PLA weights
Increase in RF weight 8 weeks 5 days/week 90˚ 40 cm
Progressive lift with loads attached to the tail using a mesh Ladder climbing 1990 Yarasheski et al.
Technical Year Author
TABLE I. Voluntary muscle hypertrophy models
Size/height
Angulation
Sessions/weeks
Training period
20
Series/day
Muscle hypertrophy (%)
BASIC MODELS MODELING RESISTANCE TRAINING
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 (1968) developed an effective non-voluntary nonelectrically 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 animal’s 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 (e.g., 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
1149
1150
CHOLEWA ET AL.
TABLE II. Involuntary muscle hypertrophy models Author
Year
Goldberg et al. Goldberg et al. Wong and Booth
1968 1975 1988
Baar and Esser
1999
Goldspink
1999
Kawada and Ishii Haddad et al.
Technical
Period
Estimulus
Surgical ablation of GAS Tenotomy of GAS Weight-lifting exercise
6 days 14 days 16 weeks
Walk Walk Electric þ external load
6 weeks
Electric
4 days
Electric
Increase in TA wet weight
2005
Surgical implantation of electrodes and electrical stimulation Stretch combined with eletrical stimulation of anterior tibialis muscle of adult rabbit Chronic restriction of blood flow to muscle
Increase in SOL and PLA weights Increase in SOL weight Increase in GAS we weight and protein content Increase in EDL and TA wet weights
Venous occlusion
2006
Electrical stimulation
Increases in PLA dry weight body weight and myofibrillar protein content Attenuation of hindlimb suspension-induced muscle atrophy in GAS (muscle weight)
2 weeks 6 days
Isometric contractions
Muscle hypertrophy (%)
EDL, extensor digitorum longus; SOL, soleus; TA, tibialis anterior; flexor hallucis longus; PLA, plantaris; GAS, gastrocnemius.
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 (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. Goldspink (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 Goldspink 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 30 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 nonJOURNAL OF CELLULAR PHYSIOLOGY
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 6 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 Hergometer, 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 sec each with 20 sec 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 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 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.
BASIC MODELS MODELING RESISTANCE TRAINING
Fig. 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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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 (
