Key Words: muscle growth, muscle strength, overload, resistance training, skeletal muscle

Transcription

1 Animal Models for Inducing Muscle Hypertrophy: Are They Relevant for Clinical Applications in Humans? Dawn A. Lowe, PhD 1 Stephen E. Alway, PhD, FACSM 2 Journal of Orthopaedic & Sports Physical Therapy Muscle hypertrophy is an adaptive response to overload. Progressive resistance exercise (PRE) is thought to be among the best means to achieve hypertrophy in humans. While functional adaptations to PRE in muscles of humans are made in the clinic, it is difficult to evaluate hypertrophic responses and underlying mechanisms because the adaptations require many weeks or months before they become evident and there is a large variability in response to PRE among humans. In contrast, various animal models have been shown to induce rapid and extensive muscle hypertrophy and some models allow precise control of the exercise parameters. By examining the animal models of muscle hypertrophy and understanding the advantages and disadvantages of each, clinicians may be able to evaluate and use relevant data from these models to design new strategies for modification of PRE in humans. The purpose of this article is to review animal models that are currently used in basic research laboratories, discuss the hypertrophic and functional outcomes, and relate these to PRE used in the clinic. J Orthop Sports Phys Ther 2002;32: Key Words: muscle growth, muscle strength, overload, resistance training, skeletal muscle An increase in muscle strength is a desirable outcome of many rehabilitation therapies. Strength gains are often a result of muscle hypertrophy, and progressive resistance exercise (PRE) is the primary mode of producing muscle hypertrophy in the rehabilitation setting. Several recent reviews have detailed the specifics of PRE in terms of modality, specificity of training, and outcomes, 23,43,44,51,61,62,64 and PRE has been shown to be beneficial as a therapeutic intervention in osteoporosis, 46,71 stroke, 70 aging, 25,50 cardiovascular disease, 48,53 metabolic disease, 66 and muscular disease. 65 In this commentary, we will focus on the hypertrophic response of skeletal muscle to PRE. Physiological and cellular mechanisms of muscle hypertrophy have been reviewed extensively, 30,39,43,44 and molecular mechanisms that initiate and regulate muscle hypertrophy is a relatively new and rapidly expanding topic in the field of exercise science. 11,15,19,20,30 However, 1 Assistant professor and research associate, Department of Biochemistry, Molecular Biology, and Biophysics, and the Center on Aging, University of Minnesota, Minneapolis, MN. 2 Associate professor and director of graduate studies, Division of Exercise Physiology, Robert C. Byrd Health Science Center, West Virginia University, Morgantown, WV. Send correspondence to Dawn A. Lowe, University of Minnesota, BMBB, Jackson Hall 6-155, 321 Church Street, Minneapolis, MN dl@ddt.biochem.umn.edu while the functional outcomes of PRE have been identified largely from human studies, much of the data used in deriving cellular and molecular mechanisms of muscle hypertrophy are drawn from studies using laboratory animals. Several different animal models for inducing muscle hypertrophy are used, and some produce outcomes that are similar to those of PRE in humans. By fully understanding the various animal models used, the clinician will be able to interpret muscle hypertrophy research results generated using laboratory animals. This information can be used by the clinician to evaluate current PRE programs and perhaps design new or modified strategies for implementing PRE. The ultimate goal is to optimize PRE in humans to acquire better functional outcomes in the clinic. The purpose of this article is to review animal models that are currently used in basic science laboratories for investigating skeletal muscle hypertrophy. Examples of recent research using each model will be presented. These examples will not be an inclusive list for the use of each model or of the investigators who have used each model. The hypertrophic and functional outcomes induced by each model will be discussed in terms of the relevance to PRE in humans. 36 Journal of Orthopaedic & Sports Physical Therapy

2 GENERAL ADVANTAGES OF ANIMAL MODELS A primary benefit of using an animal model in lieu of human PRE for studying muscle hypertrophy is the tight experimental control the investigator has when using animals. The exercise regimen can be precisely controlled while the environment and nutritional intake are regulated and virtually identical for each subject. Animals used in laboratory studies are more homogenous than human subjects and the studies can be randomized. These factors increase the sensitivity and reproducibility of the experimental outcomes. Another advantage of using laboratory animals is that following the experiment, animals can be sacrificed and muscles can be removed. Muscles can then be subjected to a variety of physiological, biochemical, histochemical, and molecular analyses in an attempt to understand mechanisms of hypertrophy. (Human muscle biopsies are often inadequate because a very small amount of tissue is obtained; thus, these biopsies are limiting with regard to sample size and may not represent adaptations that occur throughout the entire muscle.) The chief disadvantage of using animal models is the extent to which the results can be generalized to humans (ie, there is a potential threat to external validity). ANIMAL MODELS OF MUSCLE HYPERTROPHY Resistance Training in Conscious Animals Animal weightlifting models have been used for inducing muscle hypertrophy. An apparatus for rats has been designed and used by several laboratories to mimic the traditional squat exercise performed by humans. 24,42,63 Rats are operantly trained to stand upright and extend their hindlimbs and the weight lifted during the movement is increased throughout the training period by adding weight to a belt, vest, or shoulder harness. The challenge for the investigator in this model involves training the animal to do the desired movement. In some cases, the animal receives a food reward after completing the lift. In other cases, failure to lift the weight results in an electric shock. For example, Ho and colleagues 38 trained animals to respond to a visual stimulus; the TABLE 1. animals learned to perform the squat movement within a specified amount of time to avoid the application of a mild electrical shock through the floor of the cage. Similarly, rats have been motivated to do squat-like exercises by applying a mild current to the animal s tail 63 or by requiring them to perform the movement to acquire their daily food. 42 Exercise protocols have varied in this rat model but the typical outcome is 20% hypertrophy of the trained leg muscles (Table 1). For example, 8 weeks of 16 maximal-load squats/d, done 4 d/wk, resulted in a 12% increase in the mass of adductor longus muscles. 38 Twelve weeks of 15 sets of 15 squats/set at 65 75% of a single repetition max (RM), performed 4 5 d/wk, resulted in 20 30% hypertrophy of plantaris and gastrocnemius muscles. 63 Klitgaard 42 also found 30% increases in rat soleus and plantaris muscle masses following 36 weeks of a similar protocol. In that study, contractile properties were measured on isolated soleus and plantaris muscles from trained and untrained rats. Maximal isometric tetanic tension (the maximal amount of force produced during an isometric contraction) was 37 65% greater in the trained muscles demonstrating that a functional muscle hypertrophy had occurred. 42 Also, the amount of weight lifted by the rats approximately doubles following weeks of resistance training. 42,63 A second type of resistance training that has been used to induce hypertrophy of forelimb muscles in cats, mice, 31 hamsters, 40 and rats 69 is a weighted lever that is pulled down to obtain food. For example, Gonyea and colleagues trained cats to use their right paws to acquire food After 41 weeks of unilateral training, forelimb muscles of the right paw hypertrophied 7 34% and fiber diameters were 11% greater in the flexor carpi radialis muscle of the right paw compared to the left paw. 34 In a subsequent study, muscle function improvements were shown as maximal isometric tetanic tension of the trained muscles increased 30%. 33 While rodents will not voluntarily exercise without a food reward or mild electrical shock, large animals such as ponies and horses have an innate willingness Typical hypertrophic responses and functional outcomes obtained from animal muscle-hypertrophy models. Duration (weeks) Muscle hypertrophy (mass, % increase) Fiber hypertrophy (CSA, % increase) Force production (% increase) Resistance training a Electrical stimulation b Compensatory overload c Chronic stretch d CSA = cross-sectional area a 24,31,33-35,37,38,40,42,63,69 b 12,67,68,72-74 c 2,10,13,22,28 d 5 9,14,16 18,21,27,29,30,32,45,47,59,75 CLINICAL COMMENTARY J Orthop Sports Phys Ther Volume 32 Number 2 February

3 to work hard. Heck and colleagues 37 described an unconventional resistance-training model where mature ponies carried sheets of lead over their saddle region while walking on a level treadmill at 1.9 m/s. The ponies trained 3 d/wk using a system where weights were made progressively heavier during the workout. The workout began with a 44.5-kg weight and was increased in 22.3-kg increments, so that by the end of the eight-week training protocol, the ponies were carrying between kg. Eight weeks of this resistance training resulted in increases in peak weight carried (260%) and total weight carried (1525%) during each workout. Forelimb girth increased 12% with a corresponding 19% increase in muscle diameter. The primary advantage of these resistance-training models in conscious animals is that they can be similar to human PRE in terms of experimental design (Table 2). The intensity, duration, and frequency of the exercise can mimic a typical prescription for human PRE. The magnitude and time course of muscle hypertrophy and strength gains in studies on animals are similar to those obtained with PRE in humans as well (Table 1). Therefore, resistance-training animal models are well suited to study hypertrophic and functional outcomes related to PRE. The model is problematic simply because many animals do not perform resistance exercises voluntarily. It is usually necessary to deprive animals of food or use an electrical shock to motivate them to perform the exercise. These motivators also cause stress, including hormonal fluctuations, which may confound results (Table 2). Another drawback of some of these models (eg, squat-like exercises and pony PRE) is the training is bilateral so muscles from experimental animals have to be compared to muscles from control, nonexercised animals. This is a particular problem in studies using a reward system because rodents will typically do the minimal amount of exercise needed to satisfy their food requirements or to avoid shock, or both, and in doing so they usually consume less food. As a result, these animals grow slowly or lose weight relative to their nonexercised counterparts that eat freely. The problem is frequently addressed by expressing muscle mass as a ratio to body weight (ie, muscle weight/ body weight), but this data expression is less preferable than comparing trained muscles with contralateral untrained muscles or from pair-fed control animals. Lastly, resistance training in animals results in much slower adaptations with generally lower degrees of hypertrophy relative to other animal models of hypertrophy. For example, approximately two years of resistance training was required to achieve a 20% increase in some forearm flexors of cats. 49 TABLE 2. Advantages and disadvantages of the different animal models used to study muscle hypertrophy. Resistance training Advantages Mimic PRE protocols Quantitative Similar hypertrophic and functional outcomes that are the most directly applicable to PRE Disadvantages Food deprivation or shock application for compliance in rodents Stress induced by forced exercise Reduced animal growth if food is withheld Labor intensive for researcher (months or years) Electrical stimulation Contralateral control muscle available Repeated anesthesia Quantitative and reproducible Size principal recruitment pattern not followed Independent of animal motivation and cooperation Maximal activation of all motor units Myosin isoform changes depending on frequency of stimulation Compensatory overload Contralateral control muscle available Chronic exercise Hands-off postsurgery Large and fast hypertrophic responses Surgical implication (infection, edema, inflammation, etc) Confounding factors one week Mechanism dissimilar to PRE? Chronic stretch Contralateral control muscle available Most studies are chronic exercise Nonsurgical and can study initiating events Mechanism dissimilar to PRE? Very large and fast hypertrophic responses Study hyperplasia (new fiber formation) Responses from intermittent (not chronic) protocols may be more similar to PRE PRE = progressive resistance exercise 38 J Orthop Sports Phys Ther Volume 32 Number 2 February 2002

4 Electrical Stimulation (Resistance Training in Unconscious Animals) A resistance-training program consisting of involuntary muscle contractions evoked by electrical stimulation results in muscle hypertrophy. Wong and Booth 74 described a model in which one hindlimb of an anesthetized rat contracts against a weighted pulley bar when subcutaneous electrodes inserted along the plantar flexor muscles are stimulated. Following 16 weeks of these maximal contractions (4 sets of 6 contractions/set performed every third day), the weight lifted by the trained muscles approximately doubled and gastrocnemius, plantaris, soleus, and tibialis anterior muscles hypertrophied 13 18%. The importance of the protocol design in resistance studies is illustrated by data reported in subsequent papers by the same researchers using the same model. 72,73 In the two latter studies, two bouts of exercise were performed each week for ten weeks but during each bout the muscles were stimulated to contract 192 times (compared with 24 contractions/ bout in the first study). The 192-contraction protocol resulted in no hypertrophy of gastrocnemius muscles, but a 30% hypertrophy of tibialis anterior muscles was induced. 72,73 Recently, Baar and Esser 12 reported on a modified weighted pulley bar rat model where an electrode is surgically implanted on the sciatic nerve of one hindlimb, proximal to the muscles to be studied. This was an improvement over the subcutaneous electrodes because stimulation at high frequencies through the nerve ensured that all motor units of all lower leg muscles were maximally activated during the contractions and the muscles to be studied were not physically damaged by the electrodes. Tibialis anterior and extensor digitorum longus muscles hypertrophied 14% following six weeks of 10 sets of 6 contractions/set done 2 d/wk. After verifying that the model produced muscle hypertrophy, the role of S6 protein kinase during the initiation of hypertrophy was investigated (S6 protein kinase is a protein that is involved in regulating protein synthesis in muscle). 12 The study by Baar and Esser is an excellent example of the use of an animal model for elucidating underlying molecular mechanisms of muscle hypertrophy. A stimulating electrode on the proximal sciatic nerve elicits contractions of all lower leg muscles; others have described methods for implanting electrodes on the more distal tibial and peroneal nerves of rat and mouse hindlimbs. 67,68 These preparations are advantageous because it is possible to stimulate specific muscle groups instead of the entire lower leg. While these preparations have not been employed to study muscle hypertrophy per se, the chronic implantation of electrodes on nerves of specific groups of muscles could be utilized in carefully controlled hypertrophy experiments. The major advantage of eliciting muscle contractions and ultimately muscle hypertrophy via electrical stimulation is the precise control that the investigator has in terms of the exercise design (Table 2). Training parameters such as stimulation frequency and duration, the percent of maximal muscle strength generated per contraction, the number of contractions, and rest periods between contractions can be carefully controlled. In addition, this model bypasses the animal s voluntary neural drive so hypertrophic and functional responses should be more predictable. To date, this model has been used minimally. Stimulating at high frequencies (eg, 200 Hz) essentially guarantees that all motor units within the innervated muscles will be activated. This may be considered an advantage because each contraction is identical (ie, it is maximal). However, it could also be considered a disadvantage because the size principle of motor recruitment is not followed; in other words, the contractions may be considered nonphysiological. Nevertheless, electrical stimulation is often used as an adjunctive modality during rehabilitation and results in adequate muscle responses. For rehabilitative purposes the order of motor unit recruitment may be less critical than the ability to stimulate the muscle fibers to respond to overload. In contrast, nonphysiological muscle recruitment by electrical stimulation may not be the best rehabilitative strategy for an athlete who desires to perform maximally on the field. For example, PRE is associated with a transition of fibers containing Type IIB myosin to Type IIA myosin. 60 In contrast, high frequencies of stimulation may do the opposite. For example, transformation of fibers containing Type IIA to Type IIB myosin in rat soleus muscle has been reported following electrical stimulation. 36 Depending upon the question, electrical stimulation in unconscious animals may or may not be applicable to clinical PRE therapies and hypertrophic outcomes. Compensatory Overload Models When synergistic muscles are rendered inactive, the remaining functional overloaded muscle must compensate for the loss and this induces muscle hypertrophy. There are three ways that compensatory overload has been accomplished in rodent hindlimb muscles tenotomy of synergists, synergist ablation, and synergist denervation. Severing the tendon of synergist muscles is referred to as tenotomy. Many studies in the 1960s and 1970s employed this method to induce muscle hypertrophy. For example, tenotomy of the gastrocnemius muscle for just five days resulted in a 40% increase in soleus muscle mass and a 20% increase in plantaris muscle mass. 28 However, after five days there was no further change in the masses. In CLINICAL COMMENTARY J Orthop Sports Phys Ther Volume 32 Number 2 February

5 general, tenotomy produces a large initial, apparent hypertrophic response (ie, increased muscle mass) but it is due largely to tissue inflammation from the surgery. A hypertrophic response several days posttenotomy often does not occur because the severed tendon reattaches. Synergist ablation (ie, complete removal of synergist muscles) avoids the complication of tendon reattachment. However, any hypertrophic responses during the first five days are still masked by inflammation caused by the surgical trauma. 10 Significant hypertrophy does occur later. For example, plantaris muscle mass increased 65% 9 12 weeks after ablation of gastrocnemius and soleus muscles. 13 Not only does muscle mass increase, so does fiber cross-sectional area and protein content, demonstrating that more than just inflammation, edema, or extracellular fluid accumulation occurs in the overloaded muscle. 2 This is an important consideration for determining true or functional muscle hypertrophy; that is, not only must muscle weight (muscle mass) increase so must the protein content of the muscle. Degens and colleagues 22 have obtained compensatory hypertrophy in rodent plantaris muscles by denervating synergist muscles. More specifically, the branches of the tibial nerve that innervate the gastrocnemius and soleus muscles were cut, but innervation to the ipsilateral plantaris muscle was kept intact. As a result, the plantaris muscle was overloaded during normal ambulation about the cage and the muscle hypertrophied 30 40% by 21 days postdenervation. The advantage of the denervation model is that there is less bleeding during the surgery, reduced inflammation following the surgery, and faster recovery compared to surgical ablation of synergists. The main advantage of the compensatory overload models is the large and relatively rapid hypertrophy that takes place (Table 1). Although it is not comparable to human PRE in the degree of muscle hypertrophy, it provides a distinctive opportunity to investigate phenomena that would be difficult to detect in models of modest muscle hypertrophy. For example, it is now well known that satellite cells play an important role in muscle remodeling and repair. However, because satellite cells make up less than 5% of the total muscle nuclei, 26,58 the likelihood of finding activated and proliferating satellite cells is low when the hypertrophic response is small. With the large and rapid hypertrophy that occurs from compensatory overload, a greater fraction of satellite cells are activated and thus can be more easily found and studied. It is unlikely that we would have fully appreciated the important roles that satellite cells play in skeletal muscle hypertrophy if the compensatory hypertrophy model of overload had not been used as a model for muscle hypertrophy. 1 3,52,54 57 On the negative side, the validity of any results obtained during about the first week of overload must be evaluated with caution due to surgical implications. For example, it has been reported that increased DNA content in hypertrophic muscles 2 12 days following ablation indicates the importance of satellite cells in the hypertrophic response. 1 However, it is probable that inflammatory cells such as macrophages had accumulated in the overloaded muscles during that time period and contributed significantly to the increased DNA. Chronic Stretch Models It is well known among therapists that casting a muscle in a lengthened position results in maintenance of muscle mass; whereas a muscle immobilized in a shortened position atrophies. Researchers have made similar observations in animals and have taken advantage of the simplicity of the stretch model to study hypertrophic adaptations in skeletal muscle. Birds and small mammals have been studied the most extensively. In general, hypertrophic results from the stretch of chicken and quail wing muscles 5,7,16,18,45,59 have been the same as those found in rabbit 21,30,75 and rat 27 hindlimb muscle stretch models. The functional and hypertrophic outcomes of the stretch model are well documented as described below but the mechanism underlying how stretch induces hypertrophy is not known (although it is likely a mechanoreceptor type of response). In birds, a weight corresponding to 5 10% of the animal s body weight is attached to one wing with the contralateral wing serving as an intraanimal control. 5,7,16,18,45,59 This unilateral model is advantageous because any systemic alteration (eg, hormonal changes) due to the experimental manipulation are common in the control and stretched muscles, so the effects of the mechanical overload can be distinguished. The use of young chickens is slightly disadvantageous because they continue to grow throughout the experimental period. Therefore, stretchinduced changes in muscle mass must be subtracted from the background of normally growing muscles. Quails on the other hand are fully mature (ie, fully grown) by six weeks of age, but because they are smaller, less muscle tissue can be obtained. Nevertheless, a weighted wing results in 100% hypertrophy of the muscles supporting the wing in only two weeks. Muscle mass increases of % after four to six weeks of constant stretch have also been reported. 7,8,17,59 Stretch induces hypertrophy in both fast and slow myosin-containing fibers 5,6,17 and it also produces a corresponding increase in muscle force production. 4 Limb immobilization (casting) by fixing a hindlimb joint in plantar flexion (to stretch the dorsiflexor muscles) or in dorsiflexion (to stretch the plantar flexor muscles) has been studied extensively 40 J Orthop Sports Phys Ther Volume 32 Number 2 February 2002

6 in rats and rabbits. For example, chronic immobilization of the rabbit ankle in plantar flexion resulted in a 20% increase in the dorsiflexor muscles after only three to four days. 29,32 Casting muscles in lengthened positions results in similar hypertrophic and functional changes seen in avian stretch and rodent ablation and tenotomy overload models. 75 It is interesting to note that studies have also shown that combinations of muscle stretch and muscle activity induced by electrical stimulation result in a 10% greater increase in muscle mass compared to stretch alone. 29 Most studies have used chronic stretch (ie, stretch for 24 h/d) to induce muscle hypertrophy. This stimulus is clearly not the same as PRE in humans where the hypertrophy stimulus occurs for only a few minutes each day. However, stretch of an intermittent nature has also proven to be a potent hypertrophic stimulus in the avian model. Stretch applied to one wing for 24 hours followed by hours of rest (unweighting) resulted in hypertrophy approaching 300% in just two weeks. 8,9 Nonetheless, the daily duration of stretch required to induce a hypertrophic change in avian skeletal muscle is likely to be much shorter. In fact, stretch durations of only 30 min/d resulted in nearly 50% of the mass increase that occurred with eight hours of stretch per day in chicken wing muscles. 14 Perhaps similar venues of short-duration stretch combined with PRE training in humans should be investigated to determine if stretch could amplify the normal training-induced adaptations during rehabilitative therapy. Taken together, the data from avian and mammalian stretch models demonstrate that chronic stretch induces extremely large and rapid increases in muscle mass. Because there is no surgical intervention with this hypertrophy model, data collected during the first few days, even during the first few minutes or hours of stretch, are not confounded by surgical trauma, edema, etc. Therefore, events that initiate the hypertrophic response can be studied. 47 Chronic stretch has also been shown to induce hyperplasia (new muscle fiber formation); 5,7,9,16,35 thus the stretch model is valuable for studying mechanisms of new fiber formation. It should be noted that evidence for hyperplasia exists from several other animal hypertrophy models (reviewed by Kelley 41 ) and also after PRE in humans. 6 SUMMARY Several animal models of muscle hypertrophy have been designed that mimic human PRE in terms of exercise prescription and type (eg, squat-like exercise performed by rodents). The hypertrophic and functional outcomes of those models are analogous to PRE in humans. Electrical stimulation is a similar model, the main difference is that the exercise is involuntary and can be more precisely controlled. The results obtained from electrical stimulation models are comparable to those obtained by PRE in animal models and humans. Rapid and large degrees of muscle hypertrophy are induced in compensatory overload and stretch models. Muscle masses double or even triple in days to weeks, enhancing investigators opportunities to study underlying mechanisms. Knowing the underlying mechanisms of muscle hypertrophy and how these animal models are used may provide clinicians the opportunity to design new strategies for optimizing PRE with the result of improving functional therapeutic outcomes. REFERENCES 1. Adams GR, Haddad F, Baldwin KM. Time course of changes in markers of myogenesis in overloaded rat skeletal muscles. J Appl Physiol. 1999;87: Adams GR, Haddad F. 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