The dynamic nature of the so-called “fiber types” of mammalian skeletal muscle

EXPERIMENTAL

The

NEUROLOGY

Dynamic

31,277-300

Nature of Mammalian LLOYD GUTH

Laboratory

(1971)

of the So-Called Skeletal AND HERBERT

“Fiber

Types”

Muscle YELLING

of Nerkropatlzolog~~ and ?ic~krroa~kato~lkisal Scicrtces, Natiorral Ikkstitzctes Diseases and Stroke, Natiotkal Institzktes of Health, Public Health Service, U. S. Departrne?kt of lisalth, Education, akrd IVclfare, Bethesda, Maryland 20014

of Newological

Received

February

22, 1971

In general, when mammalian muscles are stained by a histochemical procedure, three categories of muscle fibers become evident. However, careful analysis of individual fibers in serial sections stained histochemically for different enzymes reveals muscle fibers with diverse combinations of enzyme activities. Thus, the “metabolic profile” of individual muscle fibers is more varied than might be supposed and, on this basis, there are considerably more than three fiber types in mammalian skeletal muscle. To ascertain whether the histochemical characteristics of individual fibers undergo transformations in response to increased work, we excised several muscles of the posterior compartment of the rat’s hind leg, leaving only the soleus or plantaris muscles as the primary extensors of the ankle joint. Subsequently, alternate serial sections of each muscle were studied histochemically for actomyosin ATPase, intermyofibrillar ATPase, and succinic dehydrogenase activities. Within 2 weeks, profound changes in these enzymes and in the size of certain muscle fibers was observed: this remodelling of the muscle fibers continued throughout the 21-week duration of the study. We interpret these observations as indicating that muscle cells undergo continual alteration throughout life in adaptation to changing functional demands, and that the histochemitally demonstrable “fiber types” merely reflect each muscle fiber’s constitution at a given moment in time. We submit that a new, more flexible approach to fiber typing is needed to appreciate the dynamic nature of the muscle cell. Furthermore, the physiological factors regulating each enzyme must be evaluated in order to achieve a fuller understanding of the significance of the histochemical attributes of normal, exercised, or diseased muscle fibers. Introduction

The results of electronmicroscopical ( l&29), histochemical (16, 28, 41) , and electrophysiological (2, 4, 26) studies have revealed that muscle fibers and motor neurons can be grouped into three general categories. However, * The authors gratefully Brown and the histological

acknowledge the technical assistance assistance of Mrs. Janina D. Ziemnowicz.

of Mr.

William

C.

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FIRERS

there is reason to believe that the cIasjiiiCatio;l of mammaiian mn5cle fiber5 int3 three types is a misleading oversimplification (38). The most careful investigation of histochemical properties of individual muscle fibers was performed by Romanul (34) who incubated serial sections of muscles for several readily demonstrable enzymes. By evaluating various histochemical reactions in the same fiber, he was able to distinguish at least eight different classes of muscle fibers. The validity of this conclusion has been verified for normal (3s. 41) and reinnervated muscle (44). Furthermore, there is an additional objection to the current approach to muscie fiber ciassification, viz., the very term “fiber type” tends to mislead one into believing that there are stable, characteristic differences between these earions kinds of muscle cells. But, quite to the contrary, it is now known that these fiber types undergo rapid alterations in histochemical appearance as :t result of exercise (7, S, 23), and that even the intracellular distribution of the mitochonclria is altered sutkiently to produce a change in qualitatively determined fiber-type characteristics (7 ) Of course, these considerations have profound implications in the study of physiological alterations (e.g . hypertrophy) and pathological changes of muscle. To minimize the variability in histochemical appearance that CXI result from effects of use and disuse, the myofibrillar ATPase reaction has been recommended as being stable (9). For example, in a study of enforced esercise ii1 rats, fiber type a!teration s were observed when the muscles were incubated for succinic deyhdrogenase (SDH) activity but not when incubated for myofibrillar XTPase activity (7). However, certain recently clescribed modifications of the &\TPase procedure ( 16. 17) have rendered it more sensitive and capable of revealing quite subtle distinctions between fibers; we therefore undertook the present investigation to study and conpare the effects of experimentally induced hypertrophy on the ATPase and SDH reactions. We report that rapid and profound changes in activity an3 distribution of these enzymes occur during hypertrophy and, furthermore, that a broad spectrum of fibers, rather thx the generally accepted three fiber types, can be distinguished. As a result of these observations, we suggest t!lat an attempt must now be made to characterize and underst:und the FIG. 1. Variety of muscle f&ers of normal rat tibialis anterior after incubation iol i\TPase. A. alkaline preincubation for demonstrating actomyosin ATPase (Ah1 hTPase), X 123 ; B, acid preincubation for demonstrating intermyofibrillar ATPase ( IMF ATPase), X 123 ; C, “ATPase” reaction after alkaline preincubation, but wi;h .4DP as substrate, X123; D. same as C but section washed in three 5-min changes of 0.1 M Tris, pH 7.3 before incubation; E, alkaline preincubation (AM ATPase), X920. Note the punctate distribution of the reaction product which is myofibrillar in localization : F, acid preincubation (IMF ATPase), X920. Note the circular profiles of reaction product, which is indicative of an intermyofibrillar localization.

280

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dynamics of the muscle fiber, and that the search for a nomenclature on simple, static, fiber-type characteristics be abandoned. Materials

and

281 based

Methods

Species Studied. Selected hind-limb muscles were removed from adult rats (Osborne-Mendel, 200 g), mice (Swiss, 25 g), guinea pigs (NIH, 600 g ) , rabbits (New Zealand, 3000 g) and cats (2500 g) that had been anesthetized with chloral hydrate or sodium pentobarbital. The muscles were dipped in talcum powder, frozen in liquid nitrogen, and transferred to a cryostat in which serial sections of 8~ thickness were prepared. Some sections were preincubated in acid or alkali (16) and stained for ATPase activity using a modification (17) of Padykula and Herman’s technique (31). Adjacent serial sections were stained for SDH using MTT (32). Exjwriwwh! Hypertroplzy. Thirty Osborne-Mendel female rats weighing 150 g were anesthetized with chloral hydrate (400 mg/kg, ip). One leg of each animal was shaved and scrubbed and a skin incision made from the popliteal fossa to the heel. In 15 animals the tendons of medial and lateral gastrocnemius and soleus muscles were transected near the heel and the muscles completely excised leaving the plantaris muscle intact. In the other 15 animals the plantaris and both gastrocnemii were removed leaving the soleus muscle as the only muscle in the posterior compartment. At intervals of 2-21 weeks the animals were anesthetized and the operated and contralateral control muscles were removed and processed as described for histochemical examination. To control for the effect of muscle and nerve trauma, in six animals the muscles were not excised, but the tendons of the gastrocnemii and either soleus (three rats) or plantaris (three rats) were cut, reflected, and sutured to the undersurface of the skin of the calf (to preclude tendon regeneration,). The muscles were examined histochemically 4 and 8 weeks postoperatively. Results

Modification of the ATPase Reaction by Altering the Incubation CondiWhen frozen sections of muscle are fixed in formalin and incubated for ATPase activity, fibers of three distinct intensities are revealed (16, 41). Preincubation of the fixed sections in alkali (pH 10.4 for 15 min) considerably enhances the distinction between the fibers (fig. 1A). An tions.

FIG. 2. AM ATPase (alkaline preincubation) and IMF ATPase (acid preincubation) reactions of tibialis anterior muscles of various species, X246. A, mouse AM ATPase; B, mouse, IMF ATPase; C, rabbit, AM ATPase ; D, rabbit, IMF ATPase; E, guinea pig, AM ATPase; F, guinea pig, IMF ATPase. See Table 1 for a summarized description of these staining reactions and the footnote to Table 1 for explanation of asterisks.

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253

identical reaction occurs when ,4TP is replaced as substrate by cytidine, guanosine, thymidine, or uridine triphosphate ; this is not unusual in view of the known lack of specificity of myosin ATPase to other nucleotide triphosphates (40). Although the diphosphate form of cytidine, guanosine, thymidine, and uridine gave no staining when used as substrate in place of .4TP, adenosine diphosphate (ADP) gave a reaction only slightly weaker than that obtained with ATP (Fig. 1C). The staining with ADP as substrate apparently results from the action of a soluble myokinase which catalyzes the conversion of ADP to ATP in muscle, since the ADP reaction, unlike the ATP one, was eliminated by washing the sections in dilute buffer solution (0.1 M Tris-HCL, pH 7.5) before incubation. When frozen sections are preincubated in acid (pH 4.35 for 5 min) rather than alkali and incubated with ATP as substrate, a more or less complete reversal of the staining pattern occurs (Fig. 1B). Washing the frozen sections prior to fixation and alkali preincubation does not alter the staining pattern and results in a picture identical to Fig. 1A. However, when washed frozen sections are preincubated in acid and incubated for ATPase, there is a striking change from the usual acid-preincubation staining pattern. The fibers which stain most weakly in unwashed preparations, stain very intensely in the washed ones, while the other fibers remain relatively unaltered. This observation, which we cannot esplain. led us to think that the alkali and acid preincubation procedures may result in the subsequent staining of different ATPase enzymes. The Cytological Distribution of the ATPasr Reaction Product. Sections that are stained for ATPase after fixation and preincubation in alkali clearly reveal a myofibrillar localization of the reaction product. When viewed under high magnification (Fig. lE), each myofibril is stained by reaction product and is surrounded by a relatively clear sarcoplasmic network. Sections that are preincubated in acid before incubation for ATPase show an entirely different localization of the reaction product. The intermyofibrillar spaces stain intensely and appear as very dark rings surrounding the less intensely stained myofibrils. We therefore believe that the alkali preincubation procedure results in a reaction that is relatively specific for actomyosin ATPase (AM ATPASE) ; the acid preincubation procedure, perhaps by inhibiting the myofibrillar enzymes more than the intermyofibrillar ones, results in a picture that is demonstrative of ATPases that are primarily intermyofibrillar (IMF ATPase).

FIG. 3. .4M ATPase and IMF ATPase reactions of limb muscles of various species, X246. A, rat, AM ATPase; B, rat, IMF ATPase; C, cat, AM ATPase: D, cat, IMF ATPase; E, man, AM ATPase; F, man, IMF ATPase. See Table 1 for a summarized description of these staining reactions and the footnote to Table 1 for explanation of asterisks.

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Species Differences Between Staining of Individual Fibers by the AM *4TPase and IMF ATPase Reactions. For purposes of clarity in exposition we must define certain abbreviations. AM ATPase (actomyosin ATPase) refers to the ATPase activity of sections that were fixed and preincubated in alkali. With this stain three fiber types occur, and in conformity with previous usage (46) the darkest staining (i.e., alkali-stable) fibers are designated as (Y, the weakest staining (alkali-labile) as /3, and the moderately staining fibers as &. The IMF ATPase refers to the ATPase of sections that were preincuhated in acid (S-10 min at pH 4.35) ; after this treatment the ATPase localization is primarily intermyofibrillar. Serial sections were stained for AM ATPase and IMF ATPase and individual fibers examined for the activity of both enzymes. Although both techniques reveal three fiber types in most fast muscles, the reactivity of the individual fiber for both enzymes is not identical in all species. The results are illustrated in Figs. 2 and 3 and are summarized in Table 1. For example, in all species the p fiber has the highest IMF ATPase activity, but in rat and mouse the (Y is generally lowest in IMF activity whereas in cat, guinea pig, rabbit. and man the o/3 is usually lowest in IMF ATPase activity. It is clear that there is no constant interspecies relationship between activities of these two kinds of ATPases. Relation betzuecn IMF ,4TPase and Succinic Dellydvogenasc ,4ctivity. Muscle fibers have been classified into three fiber types according to the distribution of reaction product after incubation for SDH activity. The A fiber (sometimes called white) has sparse, usually linearly arranged particles, the B fiber (sometimes called intermediate) has numerous, uniformly disseminated particles, and the C fiber (sometimes called red) has numerous particles localized predominantly beneath the sarcolemma (30, 41) Although fibers have been classified according to the intensity of the SDH reaction, this method seems inadvisable because the overall intensity of the B fiber may be less than (rat, mouse) or greater than (cat, man) that of the C fiber (Fig. 4, Table 2). In Fig. 4 are presented typical serial sections from rat and man that have been stained for SDH and IMF ATPase activity and the results are summarized in Table 2. From Table 2 it can be appreciated that there is a general interspecies relationship between the fiber types as determined by the SDH and IMF i*iTPase reactions, provided that the qualitative criteria are used for the SDH fiber classification. In contrast, the AM ATPase activity of muscle fibers cannot be correlated eiFIG. 4. Comparison of the succinic dehydrogenase and intermyofibrillar ATPase activities in serial sections of muscles from rat and man, X246. A, rat, SDH; B, rat, IMF ATPase; C, man, SDH; D, man, IMF ATPase. See Table 2 for a summarized description of these staining reactions and the footnote to Table 2 for explanation of asterisks.

286

GUTH

INTERSPECIES

COMPARISON

1 ATPASE

y

01 (dark) no (mod-rate) @ (light) LY (dark) a$ (moderate) /Y (light I

light moderate dark moderate light dar!c

and man

ATPase

ATPASE

INTERMYOFIBRILLAR FIBERS

Relative intensity with IMF ATpase reaction

” The relationships between AM ATPase and are those most commonly observed. However, examples of such deviants have been labeled with

ther with IMF tion (46).

AND

MUSCLE

Designation by intensity of AM ATPase r-action

and mouse

pig, cat, rabbit,

TABLE

OF INDIVIDUAL

Species

Guinea

YELLIN

OF ACTOMYOSIN

ACTIVITY

Rat

AND

(Table

IMF ATPase presented in this table deviations from this pattern occur; an asterisk in Figs. 2B al;d F al:d 3B.

1) or with the qualitative

SDH

classifica-

Intraspuies l/‘ariatioxs ilz ATPase Activities. It is difficult at times to classify fibers of a given muscle. For example, in Fig. 5, the fascicles in the lower portion of Fig 5A show only two fiber types according to the AM ATPase procedure whereas the corresponding fascicles of Fig. 5B reveal three fiber type; (according to IMF ATPase activity). The absenceof the third type in Fig jL4 is not the result of technical artifact, inasmuch as the upper portion 01 thi; figure reveals three types quite clearly. In the rat tibialis anterior, the a fibers are generally smaller than the a/3 fibers. If we subdivide the dark fibers of the fascic!es in the lower part of Fig. 5A on the basis of size, it is seen that the small fihers exhibit weak and the large fihers exhibit intermediate IMF ATPase inten-icy (as per Table 1). But

TABLE INTERSPECIES

COMPARISON ATPAW

Fiber type based on qualitative SDH criteria (distribution of reaction prsduct) A B C

2

OF SUCCINIC DEHYDKOGENASE ACTIVITY OF INDIVIDUAL MUSCLE

Jnt:nsity

of SDH

reaction

AND INTERMYOFIBRILLAK FIBERS a

Intensity

Cat and ma113

Rat and mouse

Cat and man

light dark moderate

light moderate dark

modcrate dark light

n The relationships between SDH and IMF most commonly observed. However, deviants deviations h?\-z b?en labeled with an asterisk

of IMF

ATPase Rat and mouse moderate dark light

ATPase presented in this table are those from this pattern occur; examples of such in Fig. 4B.

MUSCLE

FIBERS

287

closer inspection of Fig. 5B reveals not merely three types of fibers, but a gradation of reaction intensity. Thus, the CYand CY/~fibers can possess varying levels of the IMF ATPase activity and, therefore, the classification into three fiber types is an oversimplification. The necessity for a more dis-

FIG. 5. Serial sections of the tibialis anterior muscle of a normal rat incubated for AM ATPase (A) and IMF ATPase (B), X 123. Note that three fiber types are seen in the upper portion of A and only two in the lower portion of -4. However, the corresponding regions of B all possess three or more fiber types.

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criminating classification becomes even more apparent when one considers the changes in histochemical appearance of the hypertrophied muscles. Alterations in Histochekal Appearance After Hypertrophy. After removing all muscles of the posterior compartment of the rat’s leg except for the soleus, the fibers of the latter undergo a progression of changes and within 2 weeks a marked hypertrophy of the ,8 fibers has commenced. Although the soleus normally possesses only o( and ,8 fibers, it now shows many fibers of intermediate intensity. These changes in AM ATPase appear to be preceded by alterations in IMF ATPase. At 3 weeks postoperatively the vast majority of soleus fibers have been altered and exhibit high IMF ATPase activity whereas with the AM ATPase stain the transformation of fibers from LYto ,8 is still far from complete. (Fig. 6A, B). By 6 weeks the decrease in LYfibers is very appreciable and by 10 through 21 weeks the soleus muscle is composed entirely of /3 fibers (Fig. 6C, E) which have high IMF ATPase activity (Fig. 6D, F). With regard to SDH activity, by 10 weeks postoperatively, the soleus exhibits only B fibers (Fig. 10E) in comparison with the contralateral control which possesses B and C fibers (Fig. 1OD). The hypertrophied plantaris undergoes a corresponding sequence of changes. By 2 weeks postoperatively, the difference in AM ATPase staining intensity between (Y and a/3 fibers of the plantaris is reduced, and it becomes difficult to distinguish between fibers. Between 6 and 21 weeks postoperatively, further changes are apparent (Fig. 7&D). In comparison with the contralateral control, the p fibers increase in both number and size and there is a proportional decrease in the number of ap fibers. At 10 and 21 weeks postoperatively (Fig. S), the hypertrophied plantaris muscle exhibits only two fiber types when stained for AM ATPase activity. The pale fibers are obviously p, but the dark fibers camlot be subdivided into 01 and NP on basis of size or reaction intensity. However, serial sections incubated for IMF ATPase (Fig. 8D, 9B) or SDH (Fig. SC) activity demonstrate that this group is composed of A and C fibers and, therefore, that this altered muscle still retains three general categories of fibers. The inFIG. 6. Rat soleus muscles after unilateral excision of the gastrocnemius and plantaris muscles, X20. On the left side of each frame is the control soleus of the unoperated leg (cont.) and on the right side of each frame is the soleus of the operated leg (oper.) A, 3 weeks postoperative, AM ATPase; B, section adjacent to preceding, IMF ATPase; C, 10 weeks postoperative, AM ATPase; D, section adjacent to preceding, IMF ATPase; E, 21 weeks postoperative, AM ATPase; F, section adjacent to preceding, IMF ATPase. There is progressive decrease in AM ATPase intensity of the (Y (dark) fibers of the so!eus of the operated side. These same fibers show a corresponding progressive increase in IMF ATPase activity. There is also an appreciah!e. though less complete, loss of (Y fibers on the contralateral unoperated side.

MUSCLE

FIBERS

291

crease in number and size of the p fibers is first apparent in the deep regions of the plantaris muscle; this process subsequently extends superficially so that by 21 weeks postoperatively B fibers are found in the superficial fascicles (a region that is normally devoid of them). In SDH preparations of the hypertrophied plantaris, a progressive atrophy of A fibers and hypertrophy of B fibers is observed, so that by 21 weeks the A fibers are smaller, and the B fibers larger, than normal (Fig. IOA-C). At this stage, the superficial fascicles now contain many B fibers (a region that is normally composed of A and C fibers only) and the deep fascicles are composed almost exclusively of B fibers (whereas at 10 weeks postoperatively A, B, and C fibers were present) (Fig. 9C). It is noteworthy that the contralateral control muscles do not remain unaltered. Perhaps as a result of increased u:e forced upon the normal leg as a consequence of the operation, by 21 weeks the soleus exhibits fewer (Y fibers than normal (Fig. G’Z ). Furthermore, at 21 weeks the A fibers of the control plantaris (SDH stain) are darker and possess a more closely compacted and linearly disposed arrangement of the mitochondria than normal (Fig. lOA, B ) . Tenotomy of synergistic muscles produced changes similar to those resulting from escision of synergists. There was a decrease in (Y (C) fibers of the soleus muscles and a hypertrophy of the /? (B) fibers in so!eus and plantaris muscles. The changes in the plantaris were somewhat transitory, being more evident at 1 thzn at 8 weeks. Perhaps adhesions between the calcaneous and the tenotomizcd svnergists restored some measure of function to these muscles and thereby partially reversed the changes in the intact, iLGlateral plantaris. Thus, the effects of excision cannot he attributed entirely to operative trauma of muscle and nerve. Discussion

When actomyosin is biochemically iso!ated from fast muscles (composed mainly of CYand a/3 fibers), its ATPase activity is relatively acid-labile and alkali stabile, whereas the ATPase of slow muscle actomyosin (composed mainly of ,8 fibers) is relatively acid-stabile and alkali-labile (37, 39 ) . Although this relationship correlates with the histochemical .\TPase reaction of tissues preincubated in acid or alkali (16). a more careful examination FIG. 7. Rat plantaris muscles after uni!ateral excision of the gastrocnemics and soleus muscles, AM ATPase, X20. A, contralateral unoperated muscle, 6 weeks postoperative; B, operated plantaris, 6 weeks postoperative; C, contralateral unoperated plantaris, 21 weeks postoperative; D, operated plantaris, 21 weeks postoperative, The deeper part of the muscle which normally contains most of the p fibers is at the left. Note the progressive increase in /3 fibers in the plantaris of the operated side, Note in B the clusters of small dark muscle fibers which may represent fibers that have either split 1ong;tudinally or atrophied.

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of the histochemical preparations revealed that only the alkali pre incubation procedure results in a myofibrillar deposition of the reaction product. :jfter acid preincubation, the ATPase reaction product is predominantly intermyofibrillar in localization and, in this respect, it resembles the staining reaction obtained by the \Z’achsteill-Meisel lead-precipitation technique ( 11). We therefore have suggested that these two preincubation techniques reveal different ATPase enzymes ; the alkaline preincubation procedure demonstrates actomyosin ATPase activity and the acid preincubation demonstrates intermyofibrillar ATPase (s) . It should be emphasized, of course, that the intermyofibrillar region3 of the muscle fiber are composed of many elements (e.g., mitochondria, sarcoplamic reticulum, tubular systems, triads, etc.) each of which utilizes energy derived from hydrolysis of ATP. Consequently, numerous ATPases occur within the muscle cell, and there is no basis for assuming that the IMF ATPase reaction need reflect the activity of any single enzyme. Further studies on purified muscle fractions are needed to elucidate the variety of ATPases within the muscle cell. Although both AM ATPase and IMF ATPase procedures reveal three general classes of muscle fibers, there is no consistent relationship between activity of these enzymes in each muscle fiber. For example, the IMF and AM ATPase activities are inversely related in muscle fibers of rats and mice but not in those of man, guinea pig, rabbit, and cat. A similar interspecies discrepancy has previously been noted with respect to AM ATPase and SDH (46). In the cat, the CYfiber is an A and the a/? is a C fiber, whereas in the rat, the 01 fiber is a C and the a+3 an A fiber. A universal classification would be possible only if such discrepancies did not exist, but it would be naive to anticipate so simple a distribution of enzymes. Every enzyme subserves a different physiological role, and each one is regulated by various factors among which is the type of work that the muscle fiber must perform. For example, the myofibrillar ATPase is correlated with speed of contraction ( 1) and therefore reflects the frequency of impulses in a given burst, whereas SDH activity is probably related to the resistance to fatigue (2, 38) and therefore reflects the amount of use, i.e., the frequency of burst activity (tonic, infrequent phasic, frequent phasic, etc.) FIG. 8. Rat plantaris muscles 21 weeks after unilateral excision of the gastrocnemius and soleus muscles, X 175. A, contralateral unoperated muscle incubated for AM ATPase; B, section adjacent to preceding incubated for IMF ATPase; C, operated p!antaris incubated for AM ATPase: D, section adjacent to preceding incubated for IMF ATPase. With the AM ATPase procedure, three fiber types are seen in the control muscle (A) but only two in the operated muscle (C). With the IMF ATPase procedure, on the other hand, three or more fiber types are seen in both the control (B) and the operated (D) muscles.

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It is well-established that alterations in “muscle fiber types” occur after cross-reinnervation ( 15 ). The present study confirms that muscle responds not only to these specific neural influences, but to pattern of use as well (7, 18, 19, 24, 36, 42, 43). It remains to be elucidated whether these changes are brought about entirely by transformation of the muscle fibers or whether the formation of new fibers (21, 27) or longitudinal splitting of existing ones (6, 20, 33) contribute significantly to the remodelling process. In any event, after excising certain synergistic muscles of the rat’s hind limb, we observed a decrease in the a/? and an increase in the p fibers. We interpret these changes to be the result of an altered pattern of use, inasmuch as the nerves supplying these muscles were left intact. Of course, the excision or tenotomy of the synergistic muscles undoubtedly alters the sensory feedback and may influence other spinal mechanisms. If there is a preferential functional relationship between the muscle spindles and the motor neurons supplying adjacent muscle fibers (5, 45), the preferential increase in p fibers in the deep region of the plantaris could be attributed, at least in part, to altered spindle activity. In any event, we can conclude that altering the input to the motor neuron and the type of work that the muscle must do results in a change in enzymic activity of individual muscle fibers. Furthermore, the atrophy or hypertrophy of individual muscle fibers is a highly specific phenomenon. Denervation produces a selective atrophy of (Y or cy/3 fibers (10) and tenotomy causes selective atrophy of p’ fibers ( 10, 25). The process of hypertrophy is correspondingly selective (3, 14, 35). In the present study the plantaris hypertrophied grossly during the first few weeks after excision of synergistic muscles as has been reported (13). This gross hypertrophy subsequently diminished as the ap fibers became smaller while the /3 fibers continued to increase in size. Thus, hypertrophy can be a highly selective process with regard to individual fiber types, and it apparently represents a differential response to the pattern of stimulation. Once we acknowledge that the pattern of use influences the enzyme content of muscles it becomes easy to understand why there are interspecies discrepancies in enzymatic properties. Depending on the manner in which a muscle fiber is used, the (Y fiber can be a C fiber (as in the rat) or an A fiber (as in the cat). Since the posture and gait is quite different in these species, the discrepancies in fiber type characteristics are not surprising. It is, therefore, apparent that since characteristics of a given fiber type can FIG. 9. Serial sections of the hypertrophied rat plantaris muscle 10 weeks after excision of the gastrocnemius and soleus muscles, X2-16. A, AM ATPase; B, IMF ATPax; C, SDH. The two labeled (Y fibers are identical by the AM ATPase reaction but are distinctly different in IMF ATPase intensity and must be classified as C and ,4 according to the SDH reaction.

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change differentially in response to environmental demands! one cannot classify muscle fibers on a static absolute basis. In order to evaluate the properties of a given fiber, we nust understand precisely how various patterns of use influence each of its metabolically active constituents. For this purpose our knowledge is exceedingly fragmentary and more research is needed. For example, we know that running or swimming (endurance exercise) increases the amount of SDH (7, 22) and converts fibers from A to C (7) but has little or no effect on AM ATPase activity. On the other hand, it would appear that a more weight-bearing type of tonic exercise (such as resulted from the excision of synergistic muscles in the present study) results in an alteration of the type of AM ATPase as well as a change in the pattern of SDH in individual fibers. In all species that we have studied, there are two classes of high ATPase fibers. In the cat the (~/3 fiber is high in oxidative enzymes, glycogen and vascularity whereas the (Y fiber is low in these attributes. Recent studies have shown that, although both of these fibers are fast in contraction time, the a/3 fiber is resistant to fatigue while the LYis readilv fatigued (2). Thus, the oxidative activity, the vascularity, and the glycogen content appear to be related to fatiguability. Most of the past attempts to classify muscle fibers have been predicated on the assumption that a generalized nomenclature could be achieved if we could elucidate some stable biochemical property of the muscle fiber (9). We reject this approach not merely because we believe that no one histochemical attribute will prove sufficiently immutable, but because we feel that a truly meaningful classification will have to be dynamic and reflect the variety of responses of the muscle fiber (38). It seems apparent that plasticity of the muscle fiber is the rule rather than the exception, and this property of muscle has undoubtedly served in evolutionary development to provide for the greatest adaptability to environmental requirements. The path for further research is now clear: we must evaluate the factors regulating each metabolic system in order to understand the dynamics and significance of muscle fiber adaptations.

FIG. 10. Succinic dehydrogenase activity of rat muscles, X246. A, plantaris of a normal unoperated rat ; B, contralateral unoperated plantaris ; C, experimental plantaris of an operated rat 21 weeks after unilateral excision of gastrocnemius and soleus muscles; D, contralateral unoperated soleus, and E, experimental soleus 10 weeks after excision of gastrocnemius and plantaris muscles. The contralateral unoperated plantaris (B) shows a greater intensity of reaction, particularly with reference to the A fibers, than does the normal plantaris (A). The plantaris of the operated limb (C) shows profound hypertrophy of B fibers and marked atrophy of the A fibers. The contralateral unoperated soleus (D) possesses C fibers (circles) scattered among the B fibers, whereas the experimental soleus (E) is constituted uniformly of B fibers.

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AND

YELLIN

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