~3- ‘ R " -.-ac . .In ' ,3‘2-7. '- .v' "11“ v: liLIQI . . v. 4- u.-y-- 0"-"n ~ 4 n-n‘aflsx: ' 1:“3. r. I}: may MRI-Shir?» State ‘ L U3. Sit-Sit? {o t: .m- arm-.‘V 0 "" w r - Ill-llll—I-lllifl" This is to certify that the thesis entitled SPECIFIC CHANGES IN A HISTOCHEMICAL PROFILE OF RAT HINDLIMB MUSCLE INDUCED BY TWO EXERCISE 7" REGIMENS presented by I ROLAND RICHARD ROY has been accepted towards fulfillment of the requirements for Ph . D . . HPR degree in Major professor Date February 25, I976 0-7 639 mum in: "ME 5 50"? BMW BMW INC. LIBRARY BMDERS ABSTRACT SPECIFIC CHANGES IN A HISTOCHEMICAL PROFILE OF RAT HINDLIMB MUSCLE INDUCED BY TWO EXERCISE REGIMENS By Roland Richard Roy This investigation was undertaken to determine the effects of eight weeks of sprint (SPT) or endurance (END) training on a histo- chemical profile of the various fiber types found in the hindlimbs of adult male albino rats (Sprague-Dawley strain). Two muscle areas were selected for study on the basis of homogeneity of fiber type: the central portion of the soleus which is composed primarily of slow— twitch oxidative (SO) fibers and the posterior part of the plantaris which consists mainly of fast-twitch glycolytic (FG) fibers with some fastvtwitch oxidative glycolytic (FOG) fibers interspersed. Histochem- ical profiles were determined using the reactions of adenosine triphos- phatase (ATPase 9.4) as an indicator of contractile speed, lactic dehydrogenase (LDH) to reflect lactate fermentation activity, succinic dehydrogenase (SDH) to indicate Krebs cycle activity, and Sudan Black B (SUD) and periodic acid-Schiff (PAS) to localize intracellular fat and glycogen respectively. Roland Richard Roy A histochemical photometer was used to obtain objective photo- metric evaluations in serial cross-sections for a group of 30 adjacent muscle fibers from each of the two muscle areas investigated. Chi-square analyses, within muscle areas for each stain, revealed significant (P<<.Ol) differences between distributions in all treatment comparisons except that for SDH in the plantaris. In general, the exercise-induced metabolic adaptations were similar in the SO soleus and FG-FOG plantaris areas. The SPT and END exercise regimens each produced a number of alter- ations in the histochemical profiles of the muscle cells. Both train- ing regimens resulted in decreased staining intensities for ATPase 9.4 and increased reactivities to SDH staining. The SPT program specifical- ly enhanced LDH and PAS staining reactions, whereas END training pro- duced a large group of fibers staining darkly with SUD. In effect, the END training program resulted in an increased aerobic capacity of the muscle cells while the SPT program enhanced both their aerobic and anaerobic metabolic capacities. SPECIFIC CHANGES IN A HISTOCHEMICAL PROFILE OF RAT HINDLIMB MUSCLE INDUCED BY TWO EXERCISE REGIMENS By Roland Richard Roy A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Health, Physical Education, and Recreation I976 DEDICATION To my wife, Sharon, and our moo boys, Stephen and Michael. ii ACKNOWLEDGEMENTS My sincerest appreciation belongs to my wife, Sharon, for her unselfishness, her patience, and her continued assistance, understand- ing and encouragement throughout my graduate program. A very special thank you is extended to Dr. N. w. Heusner for the continued guidance, counseling and assistance he provided me as my graduate advisor and doctoral committee chairman. Deep appreciation is given to the members of my doctoral commit- tee, Dr. w. w. Heusner, Dr. N. D. Van Huss, Dr. J. F. Taylor and Dr. R. Echt, for making the writing of this dissertation worthwhile and enjoy— able and for making the last five years a memorable learning experience. A special thank you is extended to Barbara Wheaton for her assistance in laboratory techniques, to Dr. T. B. Gilliam, Dr. A. T. Reed, and Dr. K. w. Ho for their thought provoking discussions, to Dr. G. Mikles for his continued support, and to Bonnie Smoak and Crystal Fountain for their assistance during the processing of the tissues. A very special thank you is offered to Ken Stephens and Marty Pomerantz for their constant friendship and concern, and for helping to maintain my morale in times of frustration. Gratitude is due to Dr. R. Carrow for the extensive use of the facilities of the Neuromuscular Research Laboratory, Department of Anatomy. TABLE OF CONTENTS CHAPTER Page LIST OF TABLES ........................................... vi LIST OF FIGURES .......................................... vii I. THE PROBLEM .............................................. l Statement of the Problem .............................. 3 Research Plan ......................................... 4 Rationale ............................................. 5 Significance of the Problem ........................... 6 Limitations of the Study .............................. 6 11. REVIEW OF RELATED LITERATURE ............................. 8 Fiber Types ........................................... 9 Metabolic Adaptations to Physical Training ............ 25 III. METHODS AND MATERIALS .................................... 49 Experimental Animals .................................. 49 Research Design and Treatment Groups .................. 50 Training Procedures .......................... . ........ 51 Animal Care ........................................... 53 Sacrifice Procedures .................................. 53 Histochemical Procedures .............................. 55 Muscle Areas .......................................... 58 Histochemical Evaluations ............................. 58 Analysis of Data ...................................... 59 IV. RESULTS AND DISCUSSION ................................... 6l Training Results ...................................... 61 Body and Muscle Weight Results at Sacrifice ........... 66 Histochemical Results ................................. 68 Discussion ............................................ 94 iv TABLE OF CONTENTS—~continued CHAPTER Page V. SUMMARY, CONCLUSIONS AND RECOMMENDATIONS .................. 98 Summary ................................................ 98 Conclusions ............................................ lOO Recommendations ........................................ lOO REFERENCES ....................................................... l02 APPENDICES A. Training Programs ......................................... l27 B. Basic Statistics for Training Data ........................ 129 LIST OF TABLES TABLE Page 1. Classification of Fiber Types Used by Histochemists ........ ll 2. Histochemical Metabolic Profile for the Three Fiber Types in Mammals ................................................. 12 3. Representative Values for Fiber Type Composition of Several Muscles Commonly Used in Histochemical, Biochemical and Physiological Investigations ............................... l7 4. Enzyme Activity Levels and Substrate Concentrations Deter- mined Biochemically in Muscle Homogenates of Predominately One Fiber Type ............................................. l8 5. Comparison of Animal Training Programs Used by Various Investigators .............................................. 27 6. Analysis of Variance for Overall Treatment Effects and Newman Keul's Tests of Paired Comparisons for Body Weight at Sacrifice and Absolute and Relative Muscle Heights ...... 67 7. Chi-square Analyses for Overall and Paired Treatment Effects on Frequency Distributions of Five Histochemical Stains in the Soleus Muscle ................................ 75 8. Chi-square Analyses for Overall and Paired Treatment Effects on Frequency Distributions of Five Histochemical Stains in the Plantaris Muscle ............................. 85 vi FIGURE 1. l0. LIST OF FIGURES Mean daily percent shock-free time (PSF) and percent expected meters (PEM) for CRw Sprint ...................... . Percent frequency distributions, by histochemical photometer values for muscle fibers ............................................. . Percent frequency distributions, by histochemical photometer values for fibers .................................................... . Percent frequency distributions, by histochemical photometer values for fibers .................................................... . Percent frequency distributions, by histochemical photometer values for fibers .................................................... . Percent frequency distributions, by histochemical photometer values for fibers .................................................... . Percent frequency distributions, by histochemical photometer values for muscle fibers ............................................. . Percent frequency distributions, by histochemical photometer values for muscle fibers ............................................. Percent frequency distributions, by histochemical photometer values for muscle fibers ............................................. vii . Mean daily percent shock-free time (PSF) and percent expected meters (PEM) for CRw Endurance ................... treatment groups, of ATP 9.4 in soleus treatment groups, of SDH in soleus muscle treatment groups, of LDH in soleus muscle treatment groups, of PAS in soleus muscle treatment groups, of SUD in soleus muscle treatment groups, of ATP 9.4 in plantaris treatment groups, of SDH in plantaris treatment groups, of LDH in plantaris Page 63 64 76 78 79 80 82 86 87 89 LIST OF FIGURES--continued FIGURE ll. l2. Page Percent frequency distributions, by treatment groups, of histochemical photometer values for PAS in plantaris muscle fibers ............................................. 90 Percent frequency distributions, by treatment groups, of histochemical photometer values for SUD in plantaris muscle fibers ............................................. 9l viii CHAPTER I THE PROBLEM Histochemical techniques are used to categorize skeletal muscle fibers according to various metabolic characteristics. In conjunction with biochemical, physiological and anatomical observations, histochem- ical profiles have helped to identify at least three major fiber type categories. Many systems of fiber-type classification have evolved, but the nomenclature of Peter et_al: (235) of fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG) and slow-twitch oxidative (SO) seems to be the most comprehensible and is supported in the cur- rent literature (39). Single-cell characterization can only be accomplished with histo- chemical and morphological techniques. No biochemical or physiological method has been perfected for determining individual fiber profiles. Biochemists and muscle physiologists usually depend upon histochemical analyses for selection of skeletal muscles, or parts of muscles, which are fairly homogeneous in composition. Recently, fiber populations of several mammalian muscles have been categorized according to their percentage of FOG, PG and SO fibers (4,24,65,88,ll7,173,235,26l,262). Fiber types, even in adult animals, are not immutable. There is evidence to indicate that muscle cells undergo continual alteration throughout life in adaptation to changing functional demands. For example, the metabolic profile of rat skeletal muscle can be modified in response to the functional overload induced by incapacitation of synergistic muscles (l35,259), by inactivity (l96,249), and by immobili- zation (3l,89,247,248). The nervous system plays a primary role in determining adaptive changes in the metabolic and physiological characteristics of skeletal muscle (59,l3l,l34,28l,296). Mutability of muscle fibers was first indicated in studies involving surgical alterations of the innervating nerves. Following denervation of mature fast and slow muscles, the enzymatic differences between muscle fibers gradually disappear. That is, the fibers lose their metabolic differentiation (l8,38,74,l51,l77, 27l). Twitch times of muscles composed mainly of F6 and FOG fibers are considerably lengthened, while muscles formed mainly by $0 fibers may show either a slight decrease in the speed of contraction or a slight increase (l38,l40,l93). When these muscles are reinnervated with their own nerves, there is no metabolic dedifferentiation (64,74, l7l,l72,253). However, cross—innervation of fast and slow muscles results in a shift of the energy metabolism of the muscle fibers (38, 4l,42,43,58,64,77,24l,252,253). Alterations of normal discharge patterns of the innervating nerve also produce significant changes. Fast muscles, electrically stimulated at normal rates of discharge for tonic fibers, become mark- edly slower in their contraction times (23l,239,255), are more resist- ant to fatigue (23l) and exhibit significant shifts in their enzyme aCtivities (90,231,249). Neural control of skeletal muscle differentiation seems to be regulated by the transport of specific neurotrophins from the neurons to the muscle via axoplasmic transport. Evidence for this transsynaptic transfer of specific neuroproteins has been accumulating (2,183,l84). Various regimens of physical training have produced specific changes in the fiber profiles of skeletal muscles. Prolonged programs of endurance exercise have resulted in significant increases in the aerobic metabolic capacity of all fiber types (155,158). Furthermore, increases in the percentages of SO and FOG fibers, especially in pre- dominately FG muscles, have been reported (2l,78,83,l94,l98,208). Exercise programs for laboratory animals that are solely dependent upon anaerobic metabolic processes are yet to be developed. However, studies utilizing training programs with relatively high anaerobic components have produced increases in anaerobic metabolic capacity and shifts in $0 muscles toward higher FOG fiber populations (l22,257,272). Isometric training has produced specific changes in enzyme activities and fiber type populations that are dependent upon the specifications of the training program used (96,97,l59,185,209,270,297). Statement 9f_the Problem In light of the evidence for mutability of muscle fibers, this study was undertaken to determine the effects of two very strenuous training programs on the histochemical profiles and distributions of various fiber types. Supplementary data were obtained on performance f . criteria, body weights, and muscle weights. Research Plan Normal adult male rats (Sprague-Dawley strain) were used as sub- jects. For each animal, a common area containing thirty adjacent fibers in each of the left soleus and plantaris muscles were studied. Several anatomical landmarks were used to help locate homologous areas in all tissue sections. The fibers selected were chosen as being typical of those in the central portion of the soleus and the medial posterior portion of the plantaris. The two training regimens were modifications of Controlled-Running Wheel routines previously reported from this laboratory (286, see Appendix A). The modified programs, an endurance running routine (END) and a Sprint running routine (SPT), represented attempts to stimulate selectively either aerobic or anaerobic metabolic processes in the experimental animals. At the termination of the study, the END animals were running continuously for one hour at the relatively slow speed of 36 m/min. The END program was expected to produce increases in aerobic metabolic capacity. The SPT program consisted of alternated work and rest periods. The animals ran at speeds of up to lO8 m/min, but the work periods were limited to l5 sec. Anaerobic metabolic pathways were expected to be taxed by the SPT program. The exercise treatments were administered five days per week for eight weeks. Histochemical profiles were determined using an adenosine tri- phosphatase (ATPase 9.4) reaction as an indicator of contractile speed. The lactate dehydrogenase (LDH) reaction was used to show lactate fermentation activity. The succinic dehydrogenase (SDH) reaction was selected to indicate tricarboxylic acid cycle activity. Localization of fat and glycogen as substrates was demonstrated by the use ofISudan Black B (SUD) and periodic acid-Schiff (PAS) stains respectively. Rationale Current literature has indicated that exercise consists of a con- tinuum of specific activities each of which elicits a specific response within the organism (29,99,l25,l26,127,128,202,242). The two training regimens used in this study were designed to provide functional over- loads of the aerobic and anaerobic ends of this continuum. The act of running in the rat involves plantar flexion of the foot. The soleus and plantaris muscles are both involved in plantar flexion and therefore were assumed to be highly active during the train- ing programs. The muscle areas were selected for homogeneity of fiber- type populations. The soleus in the rat has been reported to contain 84% SO, l6% FOG, and 0% FG fibers (4). The central portion of the soleus has been shown to be predominately SO (78). The posterior part of the rat plantaris has been observed to contain mainly FG fibers with some FOG fibers interspersed (78). It was postulated that the response of the different fiber types would be specific to the functional demands of the training programs. The selection of histochemical procedures was made to insure a reason- ably inclusive fiber profile. Enzymes involved in aerobiosis (SDH) and anaerobiosis (LDH) reflect different metabolic pathways. ATPase reaction indicates the contractile properties of the various fiber types. Substrate levels are indicated by PAS (glycogen) and SUD (fat). Significance gj_the Problem Metabolic fiber profiles have become valuable tools for assessing the functional state of individual muscle fibers. Specific adaptations in fiber metabolism have been shown to be induced by exercise and various surgical techniques and have been observed in numerous neuro- muscular disorders. The study of exercise-related alterations, by fiber types, may provide insight into the mechanisms of these metabolic adaptations. Limitations 9f_the_$tudy l. The results of this study are restricted to the soleus and plantaris muscles of normal male albino rats. 2. The training programs used may not have stimulated purely aerobic or anaerobic metabolic processes. 3. Histochemical methods to evaluate precise quantitative enzyme con- centrations in individual muscle fibers are not available at the present time. 4. The limited number of histochemical techniques that were used cannot be expected to provide a complete picture of all exercise-related metabolic adaptations. 5. A control for the shock stimulus used to motivate the animals to run was not included in the investigation. However, previous experience in this laboratory suggests that the stimulus has no effect on histo- chemical or morphological parameters in the plantar flexor muscles. 6. Three sessions for the sectioning and staining of tissues were con- ducted. A single session included all animals from one treatment group. Intersession variability in staining reactions may have accounted for some of the histochemical differences observed. This confounding factor may be important especially for the highly pH sensitive ATPase reaction. CHAPTER II REVIEW OF RELATED LITERATURE Skeletal muscle fibers have been classified into three broad categories or fiber types according to their histochemical, morpho- logical, physiological and biochemical characteristics (48,207,235,238, 280). These characteristics are not fixed; individual fibers are known to be dynamic with regard to fiber type (60,l35). Various regi- mens of physical activity have produced marked changes in both metabolic and contractile profiles (l22,l56,l58,208,233). The direction and extent of enzymatic adaptations have been dependent on the specifica— tions of the training programs used. To facilitate a discussion of fiber-type mutability, the follow- ing review of literature is divided into two main sections with several subdivisions. A general description of the three fiber types will be presented under the first main heading. Histochemical and morphologic characteristics of single muscle fibers, biochemical correlates in muscles of nearly homogeneous fiber type, and physiological data uti- lizing whole muscle and single motor unit preparations will be dis- cussed. The second major part will deal with the adaptations of these fiber types to different exercise regimens. Histochemical and biochem- ical changes will be emphasized. Fiber Types Differences between individual muscle fibers can be seen best by histochemical and morphologic techniques. Biochemical methods are not available at present to establish single-fiber enzyme profiles, and physiological measures of contractile speed will differentiate only between fast-twitch and slow-twitch contractile elements. Histochemical Characteristics and Differences Enzyme histochemistry is a specialty that forms a connecting link between two methods of approaching the investigation of tissues: histology and biochemistry (171). When stained histochemically, indi- vidual muscle fibers show different degrees of coloration. These dif- ferences generally are thought to persist throughout the length of an individual fiber (66,72,92,98,273), but this hypothesis recently has been challenged (287). Serial cross-sections of muscle can be stained by different histochemical reactions to obtain metabolic profiles of individual fibers (93). It is important to note that relative degrees of fiber staining do not necessarily represent relative levels of enzyme activity (92). A histochemical reaction can only be indicative of the amount of accumulated end-product. Therefore, direct relative comparisons of histochemical staining intensities should be restricted to fibers in the same muscle of the same species (5,28,217,295). In the adult mammal, skeletal muscle fibers may be differentiated by a variety of histochemical techniques. Combinations of these stains plus biochemical reactions, physiological properties, and morphologic l0 characteristics have prompted investigators to categorize fibers accord- ing to several schema (see Table l). The descriptive taxonomy intro- duced by Peter gt_al, (235) of fast-twitch glycolytic (FG), fast-twitch oxidative glycolytic (FOG), and slow-twitch oxidative (SO) seems to have emerged as the most useful and has been adapted by a number of investigators. This classification system will be used throughout the current report. Table 2 summarizes the information that is now available concern- ing relative histochemical staining intensities of the three fiber types. Clearly, substrate levels in FG fibers are characterized by a high glycogen content as reflected by the PAS stain and by a low lipid content as reflected by the SUD stain. Aerobic capacity is assumed to be low since stains for localizing the activity of oxidative enzymes such as SDH, malate dehydrogenase, and NADH-diaphorase have minimal intensities and the myoglobin content is low. Anaerobic capacity is thought to be high because of the maximal staining reactions of anaero- bic enzymes such as M-lactate dehydrogenase, triosephosphate dehydro- genase, mitochondrial a-glycerophosphate dehydrogenase, and phosphoryl- ase. The high myosin ATPase reaction at pH 9.4 confirms the fast- twitch characteristic of F6 fibers. The histochemical profile for FOG fibers is quite different. These fibers are high in glycogen and lipids, have high to moderate reactions for most aerobic and anaerobic enzyme stains, and are fast contracting. SO fibers react strongly to most indicators of aerobic metabolism. These fibers exhibit the lowest glycogen content and only a moderate ll m—N mop mm_.mm_ .mmp.mmp.vm~.mmy.om_.o_ omm.mmm.¢op.wm m.m mxm.omm.m¢~.mm mmm.wmm .emm.¢mm.mpm.um.mom.onp .mF—.¢_F.mF—.o—P.Pop.oop .mm.mw.om.mm.mm.mm.mm._m vam.~m~.mm— me.mmp mmm._mp .owp.mup.muP.Nu_.mm_.mmp .mm.¢m.mm.mn.mm.nm.mm.mm mmm.mmm.mom .mom.nom.mpF.mop.mm.mm.v E=_emz zo_m a>wpac_xo ;m_I IUSL3p-zopm mu m .pUmELmu CH eupwzp-zo_m vmmcm m mpmwumsgmucH m HHH H Aomv m>wumquo eao_zb-zopm cam page m>woaewxo see: cuow3o-omaa cam gaowzp-pmaa eam-a u nmm ma HH HH woav owuxpooxpm m>wumuwxo guo_zu-pmau wows: “mam m>wpau_xo 304 cop_;p-omaa ap_;3 gup_3o-pmaa mowez-a < HH “wav owuxfiouz—m guowzo-pmaa mmocmgmwmm Empmxm cowumowmwmmmFu mpmwsmSUOpmw: ma com: mmazp cmnwu we mcowpmuwm_mmmpu ._ mfinm», l2 Table 2. Histochemical Metabolic Profile for the Three Fiber Types in Mammals Fiber Type FFast Fast Oxidative Slow Metabolic Glycolytic Glycolytic Oxidative Characteristics (FG) (FOG) (SO) References Myoglobin Content La H IIb l65,l66,25l,258 NADH-Diaphorase L H 1c 80,235 Glycogen Localization I-H H L ll6,2l4 Periodic acid—Schiff (PAS) H H L ll6,2l4,235 Phosphorylase H H L 80,82,2l4,235,293 Hexokinase L I H 233 Triosephosphate Dehydrogenase H L L 2l5 Lactate Dehydrogenase (M) H L—I L 2l4,215,2l8,233, (M—LDH) 234 Lactate Dehydrogenase (H) L H I 234,262,263,273 (H—LDH) Mitochondrial o—Glycerophosv H 1—H L 80,235,297 phate Dehydrogenase Succinic Dehydrogenase (SDH) L H I 3,80,95,l43,l44, l79,l80,2l2,2l5, 218,258,273,284, 293,297 Malate Dehydrogenase L H I 80,82,218,235 Lipid Localization L H IEH l09,l43 Sudan Black B (SUD) L H I l09,l43,262 Myosin Adenosine H H L 78,84,132,l33, Triphosphatase l79,293 Mitochondrial Adenosine L H I ll6,lBO Triphosphatase Myofibrillar Adenosine H L H 80,9l,95,253,273 Triphosphatase (ATPase) at pH 9.4 pH sensitivity of myo— fibrillar ATPase Formaldehyde sensitivity of myofibrillar ATPase Creatine Phosphokinase acid labile alkali stable sensitive H acid labile alkali stable stable acid stable 37,132,256,296 alkali labile l32,273 179,180 aL indicates a low staining reaction. b H indicates a high staining reaction. cI indicates a moderate staining reaction. l3 amount of lipid material. High myoglobin content and moderate to high reactions for oxidative enzymes are indicated. Stains for anaerobic enzyme activity are light. The reaction with myosin ATPase at pH 9.4 is low and reflects the slow-twitch characteristic of these fibers. Morphological Characteristics and Differences Morphological differences in skeletal muscle fiber types are found at both the gross and ultrastructural levels. Qualitative and quantitative disparities in cellular content and in the distribution and form of constituent organelles and inclusions are clearly evident. In addition, surrounding and associated tissues are quite variable among fiber types. Mitochondria.--One of the primary differences among fiber types is found in the number, form and distribution of mitochondria. All fiber types have mitochondria arranged in pairs opposite the I bands (ll2,219). However, the FOG fibers contain many large, interfibrillar mitochondria that are arranged in rows. These spherically-shaped organelles contain dense matrices with closely packed cristae (llO,lll, ll2,224,226,280). Subsarcolemmal and perinuclear aggregations of mito- chondria are typical of the FOG fiber (ll2,ll4,280). Sparsity and smallness of mitochondria distinguish the F6 fiber type from other types. Interfibrillar mitochondria are scarce and interfibrillar rows are absent. A few mitochondria may occupy perinu- clear regions. Subsarcolemmal organelles usually occur individually. Paired mitochondria at the 1 bands are present, but they are smaller and have fewer cristae and less dense matrices than do those of the FOG l4 fibers (ll2,224). Compared to those in FOG fibers, the mitochondria within the SO fiber are fewer, more pleomorphic and have less opaque matrices (280). Subsarcolemmal and perinuclear chains are present, but they are shorter and less conspicuous than in FOG fibers (2l9,265). Lipid and Glycogen Inclusions.--Lipids are numerous in both FOG and SO fibers but extremely rare in F6 muscle cells (280). A direct relationship seems to exist between mitochondrial density and trigly- ceride droplets (lO9). Abundant glycogen permeates the sarcoplasm of all fiber types but is most prominent in the I band region of PG fibers (280). This observ- ation may be related to the phasic nature of F6 fibers. Myofibrils.--FOG fibers generally have the smallest cross- sectional dimensions, and FG fibers have the largest. SO fibers are intermediate in size (68). The M line is more prominent in FOG and FG fibers than it is in $0 fibers (280). The width of the Z line, measured at comparable sarcomere lengths, usually is reported to be greatest in $0 fibers and smallest in F6 fibers (l12,209,254,280). Indications are that wide Z lines may be associated with tonic muscle contractions (280). However, recent work has determined that Z-line width is highly variable and may differ in the same fiber types of separate muscles of the same species (ll4,ll5) and in the same muscle between species (113). The significance of this finding is not yet clear. lS Sarcoplasmic Reticulum and Transverse System.--An extensive reticular network pervades the F6 fiber. This network consists primar- ily of longitudinal components at the A band, but it has numerous broad expansions and transversely or obliquely oriented components at the I band (280). A compact arrangement of broad parallel tubules is present at the H band (112). The reticulum in the FOG fiber consists of a plexus or fenestrated collar in the A band region between successive T tubules and a less extensive component at the I band (280). An elaborate network of narrow tubules is present at the H band. The sarcoplasmic reticulum of the SO fiber is less extensive than that of either the FOG or F6 fiber (30,280). The observation that fast- twitch fibers (FOG and FG) have a more extensive sarcoplasmic reticulum correlates well with their physiological characteristics (lO3). Neuromuscular Junction.--Obvious morphological differences in neuromuscular junctions exist between the three fiber types. The F6 nerve terminal is the largest and is characterized by many long thin branches which are relatively straight and have numerous small pearl- shaped swellings along their course (182,285). The profile of junction- al folds reveals increasing complexity as the folds extend towards the sarcoplasm (210,227). The site of contact has a large surface area (111) with deep wide folds (210). The small FOG nerve terminal possesses only a few short thick branches with more elongated swellings (285). The junctional folds have a relatively small number of branches which are shallow and flat (210). l6 The SD terminal possesses intermediate characteristics in terms of the number and size of terminal branches and swellings (285) and the size and form of junctional folds (210,227). Capillarity.--Tomanek t al. (280) recently found higher capillary to fiber ratios for SO and FOG fibers than for FG fibers in guinea pig soleus and vastus lateralis muscles. Other investigators have reported no differences in capillarity between fiber types (130,195). Due to the differences in staining and counting techniques used, the current results on capillary to fiber ratios are inconclusive. There is general agreement, however, that capillarization is directly related to the oxidative metabolism of the muscle fiber (54,130,160,161,l95,213, 244,252,253,280). Biochemical Characteristics and Differences Biochemical assays have been used to substantiate some of the inferences drawn from histochemical staining reactions. Since no bio- chemical technique has been devised to determine enzyme profiles in single muscle fibers, biochemists have used whole muscles or portions of muscles which have been identified histochemically as being rela- tively homogeneous (see Table 3). Table 4 summarizes some of the literature dealing with biochemical determinations in homogenates of predominately one fiber type. Fast—twitch glycolytic fibers are dependent chiefly upon anaerobic carbohydrate metabolism. These fibers have a high glycogen content and exhibit high levels of glycogenolytic (phosphorylase), glycolytic (phosphofructokinase, pyruvate kinase, glyceraldehyde 3-phosphate 17 Table 3. Representative Values for Fiber Type Composition of Several Muscles Commonly Used in Histochemical, Biochemical and Physiological Investigations Fiber Types Muscle Species FOG FG SO Reference Gastrocnemius rat 37 58 5 4 Superficial Vastus rat 0 100 0 11 Deep Vastus rat 70 O 30 ll Soleus rat 16 O 84 4 Extensor Digitorum Longus rat 59 38 3 4 Plantaris rat 53 41 6 4 Tibialis Anterior rat 66 32 4 Biceps Brachii rat central 61 23 16 297 peripheral 29 51 20 297 Rectus Femoris rat 54 42 4 4 Lateral Gastrocnemius guinea pig 32 56 12 4 Medial Gastrocnemius guinea pig 50 38 12 24 Red Vastus guinea pig 78 18 4 4 White Vastus guinea pig 23 77 O 4 Soleus guinea pig 0 0 100 4 Semimembranosus lesser bushbaby 33 66 1 4 Vastus Lateralis lesser bushbaby 13 87 O 4 Plantaris lesser bushbaby 3O 51 19 4 Soleus lesser bushbaby l3 0 87 4 Tibialis Anterior lesser bushbaby 45 43 12 4 18 Table 4. Enzyme Activity Levels and Substrate Concentrations Determined Biochemically in Muscle Homogenates of Predominately One Fiber Type Fiber Type Fast Fast Oxidative Slow Glycolytic Glycolytic Oxidative Metabolic Characteristics (FG) (FOG) (SO) References Myoglobin Content La Hb H 235 Cytochrome a L H IC 11,235 Cytochrome c L H L-I 11,235 Glycogen Content I-H H L 14,24,235,245 Glycogen Synthetase L H 168 Phosphorylase I-H H L l3,25,62,235,257 Hexokinase L I-H H 25,62,232,235 Phosphofructokinase H I 62,235,257 Triosephosphate dehydrogenase H L 25 Glyceraldehyde 3-phosphate H I L 235 dehydrogenase Pyruvate kinase H I L 235,257 Lactate dehydrogenase H I L 25,62,235 a-Glycerophosphate dehydrogenase H I-H L 25,62,235 Citrate Synthase L H I 11,25 Succinic dehydrogenase L H 1 235,257 Total Lipid Content L I H 103 Triglyceride Content L H L-I 14,245 Carnitine Palmityltransferase L H I-H 11,12 3-Hydroxyacyl CoA L H 25 3-Hydroxybutyrate dehydrogenase L I H 290 3-Ketoacid CoA-transferase L I H 290 Acetoacetyl-CoA thiolase L I H 290 Lipoprotein lipase L I H 32 Palmitate Oxidation L H I 11 Pyruvate Oxidation L H I 11 Myosin adenosine triphosphatase H H L 235,277 aL indicates a low enzyme activity. bH indicates a high enzyme activity. cI indicates a moderate enzyme activity. 19 dehydrogenase and triosephosphate dehydrogenase), and lactate fermenta- tion (lactate dehydrogenase) enzyme activities. High values of a-glycerophosphate dehydrogenase activity suggest an important role for the a-glycerophosphate shuttle system in the regeneration of NAD for glycolysis. Aerobic capacity is limited as is shown by low succinate dehydrogenase, citrate synthase, and cytochrome activities as well as low myoglobin content. Fat metabolism is relatively unimportant in these fibers. Low total lipid and triglyceride contents and low levels of activity of B-oxidation enzymes (B-3-hydroxyacy1 CoA and carnitine palmityl transferase) are found. Low lipoprotein lipase levels also suggest little dependence on exogeneous fat stores. Fast-twitch con- tractile characteristics are indicated by high levels of myosin adeno- sine triphosphatase (myosin ATPase). An opposite pattern of enzyme activities is found in the slow- twitch oxidative fibers. The fact that $0 fibers are slow contracting is shown by the low levels of myosin ATPase activity. Metabolically, these fibers appear to rely predominately on aerobic mechanisms. Total lipid content is high in these fibers, but it should be noted that tri- glyceride levels are relatively low. Intermediate to high activities of the enzymes of fatty acid oxidation and high levels of lipoprotein lipase activity indicate a heavy reliance on fat metabolism. High £3 oxidation levels substantiate this observation. Cytochrome and myo- globin levels are high as are the activities of the citric acid cycle enzymes. Glycogenolytic, lactate fermentation, and glycolytic enzyme activities are minimal and glycogen content is low. As expected, 20 hexokinase activity is an exception since it seems to vary directly with respiratory capacity (25,62). Fast-twitch oxidative glycolytic fibers appear to have the high- est capacity for aerobic metabolism. Succinic dehydrogenase and citrate synthase activities as well as cytochrome levels and myoglobin concen- trations are greatest in these fibers. Moderate total lipid and high triglyceride concentrations indicate a capacity for fat storage, and high activities of lipoprotein lipase and the fatty acid oxidation enzymes reflect high rates of fat metabolism. FOG fibers also are characterized by a moderate to high anaerobic capacity. They have the highest glycogen concentration with moderate activity levels of the glycolytic enzymes. Phosphorylase activity is high and a-glycerophos- phate dehydrogenase and lactate dehydrogenase activities are moderate. In summary, these fibers have adequate capacity for glycogenolysis, glycolysis and oxidative phosphorylation with a fast speed of contrac- tion. Physiologjcal Characteristics and Differences Histochemical and biochemical studies suggest the existence of marked differences in contractile characteristics between fiber types. These differences have considerable physiological importance. Fast- and Slow-twitch Characteristics.--It is now well-established that the histogenesis of striated muscle in mammals leads to the forma— tion of limb buds which at first are uniformly slow contracting (60,68, 207). Further differentiation into fast and slow muscles occurs later, but the developmental changes differ between muscles within the same 21 animal and in corresponding muscles between different species (40,56, 139,176). Differentiation in the rat appears to be brought about by a relative shortening of contraction time in potential fast muscles (e.g., extensor digitorum longus), there being little or no change in eventual slow muscles (e.g., soleus) (60). However, histochemical findings in the soleus muscles of the guinea pig, rabbit and cat reveal a mixed fiber pattern with a predominance of fibers having high ATPase activity (FOG and FG fibers) at birth, and many fibers having low ATPase activity (SO fibers) in adult animals (139,176,217). This slow- ing of contraction time in the soleus follows a different time course in each species and appears to be dependent upon the level of matura- tion at birth (139). Biochemical studies have shown that there are proportional changes in the intrinsic speed of contraction and the myosin ATPase level during ontogenetic differentiation of vertebrate fast and slow muscles (60). Barany and Close (17) and Barany (16) reported that specific activity of myosin ATPase is correlated with contraction time in adult muscle, and Guth and Samaha (132) demonstrated that actomyosin ATPase measured biochemically is correlated with the histochemical myo- fibrillar ATPase at pH 9.4. These observations have been substantiated by other investigators (24,88). Using the myosin ATPase reaction at pH 9.4, it has been shown that the SO fibers are slow contracting while both the FOG and FG fibers have fast contraction times (24,80,84,l32, 293). 22 Motor Unit Characteristics.--The contractile elements of skeletal muscle are organized into functional entities called "motor units". A motor unit consists of a group of muscle fibers and the single moto- neurone innervating them (76,264). Each motor unit appears to be homo- geneous with regard to muscle fiber type (33,48,90,l49,l99,292), and the fibers are scattered and intermingled with fibers of other motor units (33,45,90). The dynamic properties of motor units found in "slow" and “fast" muscles are quite different (57,60). There is evidence from animal studies that the size of the motor unit and its contractile properties are related in some way to the diameter of the innervating motor axon (1,148,149,199,292). However, this is not always the case (276,292). Alpha motor neurons have been divided into slow (S) and fast (F) types on the basis of distinctive twitch properties of the muscle fibers they innervate (199,292), but these neurons are indis- tinguishable in terms of their histochemical profiles since all are high in phosphorylase and low in SDH (50,51). Direct investigation of the histochemical, morphologic and physiologic characteristics of mammalian muscle fibers has become pos- sible using a variety of techniques that are based on the classical work of Kugelberg and Edstrom (90,187). These investigators developed a technique for the histochemical mapping of the muscle fibers belong- ing to a single motor unit using depletion of fiber glycogen following repetitive electrical stimulation. This technique permits the identifi- cation of stimulated fibers in PAS-stained sections as being unstained fibers outlined against the stained fibers of surrounding unstimulated 23 motor units. The process does not affect the staining properties of the stimulated fibers with other histochemical reactions and thus allows for fiber typing with serial sections. Burke gt 21, (44,47), using a modification of this technique, have presented evidence which suggests that motor units of the medial and lateral heads of the gastrocnemius of the cat may be classified into three nonoverlapping groups. These motor-unit groups are based on fatigue characteristics and contractile speed. The three groups are as follows: type FR, fast contracting and fatigue resistant; type FF, _fast contracting and fast fatiguing; and type S, slow contracting and fatigue resistant. It seems to be a reasonable extension of the exist- ing histochemical information to assume that the FF, FR, and S motor units contain FG, FOG and SO muscle fibers respectively. The histochemical data presented by Kugelberg (188,189) on rat hindlimb substantiates the ability to categorize motor units into three groups corresponding to muscle fiber types. In the anterior tibial muscle, Kugelberg (189, p. 9) identified a Type I motor unit that corre- sponds to the S group of Burke gt_al, (44), a Type IIA motor unit that corresponds to the FF group, and Types 118 and IIC that together corre- spond to the FR group. A similar histochemical profile in the soleus of the rat revealed motor units of only Type I or the S group and Types IIB and IIC or the FR group (188). It is of interest to note that indirect estimates of the per- centage of motor units belonging to each group in the medial gastroc- nemius of the cat have been reported by Burke and Tsairis (45) to be: 24 55% FF, 20% FR, and 25% S. The values compare favorably with those for the muscle fiber population that were determined histochemically by Ariano et_al, (4): 61% FG, 14% FOG, and 25% SO. Other data support these findings (49,201,275). It has been known for a long time that slow muscles are employed in slow contractions and in the maintenance of posture, whereas fast muscles are used primarily in quick phasic movements (68). This princi- ple should hold true for slow and fast motor units within any given muscle (47,220). In a recent study, Stephens and Stuart (275) observed the recruitment of motor units in the cat medial gastrocnemius in response to different intensities of electrical stimulation. At low contraction strengths, motor units which were largely fatigue resistant were stimulated; at high contraction strengths, motor units which were fast contracting and less fatigue resistant were recruited. The func- tional interpretation and importance of this dual role was emphasized. The medial gastrocnemius is a muscle which participates in a broad range of activities. Fatigue-resistant units (of the S and maybe FR groups) could be well-adapted to maintain long sustained contractions as needed in standing. Rapidly contracting units (of the FF and FR groups) may be required for phasic activities such as jumping and running. Animal exercise studies of motor unit recruitment have sup- ported the concept of task-specific recruitment patterns (86,87,117, 198). Fast— and slow-twitch units have been demonstrated by stimulating single motoneurones in man (267). Milner-Brown and co-workers (201,274) 25 provided direct evidence that human motor units of the first dorsal interosseous muscle of the hand are recruited during increasing volun- tary contraction in an orderly fashion. They also observed that the number of additional motor units recruited for a given increment in force declines sharply at high levels of voluntary force. This suggests that even though the high threshold units generate more tension, the contribution of recruitment to increases in voluntary force declines at higher force levels. Metabolic Adaptations t9_Physical Training The fact that muscle fiber types are mutable was first established in studies involving surgical alterations of motor nerves. Denervation (18,38,74,151,l77,l86,271), reinnervation (64,74,17l,l72,253), and cross-innervation (38,41,42,43,58,64,77,252,253) all have been shown to produce marked metabolic changes in muscle fibers. The obvious conclu- sion is that muscle fiber type is under neural control (131,281). This concept has been supported by direct stimulation of intact motor nerves (90,231,239,249,255). Evidence is accumulating that axoplasmic flow may be a regulating factor (2,183,184). Regardless of the nature of the control mechanism(s), alterations of nerve discharge patterns clearly produce significant changes in the metabolic characteristics of muscle fibers. It might be expected, therefore, that noninvasive physiological conditions which affect nerve function would produce similar changes in muscle fibers. Inactivity .(l96,249) and immobilization (31,89,248) are two such conditions that 26 have been shown to modify the metabolic profile of rat skeletal muscle. The effects of different regimens of exercise on fiber type have been studied in some detail (see Table 5). Endurance Training--Biochemical Alterations Prolonged programs of endurance training, performed regularly, have resulted in significant increases in the aerobic capacity of all fiber types (156,158). Myoglobin and Cytochrome Levels.--Myoglobin, which stores oxygen and enhances its rate of diffusion through the cell wall, has been shown to be increased by endurance exercise in mixed muscles of the rat (191,228) and in the FG portion of the vastus lateralis muscle of the lesser bushbaby (85). This rise may account for a portion of the in- crease in maximal oxygen uptake that occurs in response to prolonged endurance training (158). Cytochrome a (cytochrome oxidase) and cytochrome c (ferrocyto- chrome c-oxygen oxidoreductase) activities are elevated in endurance- trained rats (8,11,29,70,152,154,204,221,290), guinea pigs (23), and lesser bushbabies (85). All three fiber types are equally involved. The magnitude of the changes found in the rats was greatest, but this may be due to interspecies variations and/or differences in training programs. The question of the significance of the elevated cytochrome levels has been debated (23,106,236). After a 12-week treadmill pro- gram of running, untrained and trained guinea pigs were run to exhaus- .tion in a single bout of exercise (23,236). 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I- BE? flee 2 ”.8 RN .2 ea >923 mxee m>_» mop xowc_pew ece ccem: Iaummcou mm on II mcweewzm new own one m—F cLem: ece vacp—oo cps om comm; ea Ace seem c_e exee m)?» II II. m eee aces om x—Fe_mvcH Ieummceu mm om II mcwse_3m use «em meg m—p .Pe um xuvcppeu mcowueuvwvumem seemecc we Eecmoce mm_u c_e\c mmwmcmxu mm< co\ece mmeumem mucmcmema mceuemwmmm>c~ cevueceo Icmxm x_weo xuwoe~m> we meet a: pepuvcm peewuc_ce we cumcme emecwucouIIm m_eeh 32 different for the two groups. Yet, the trained animals had much higher levels of the cytochromes. The correlation coefficient between cyto- chrome c activity and running time to exhaustion was a low 0.37. These data indicate that cytochrome levels are not good indicators of aerobic capacity. However, Fitts et_al, (106) challenged this position. Rats run on a standard endurance treadmill program demonstrated significant correlations between cytochrome c, citrate synthase and respiratory capacity in the gastrocnemius muscle and the duration of a run to ex- haustion. Differences in training procedures seem to be the cause of this discrepancy. Glycogenolytic and Related Enzymes.--In an early study small in- creases in phosphorylase activity were demonstrated in the biceps region of the hindlegs of rats swum for 15 weeks (129). Subsequent studies have verified that changes in phosphorylase levels do take place. Huston gt_al, (163) reported an increased activity in phos- phorylase in gastrocnemius homogenates of trained rats. Baldwin‘ gt_al, (13) found an increase in activity in the predominately SO soleus muscles of rats trained on a treadmill program. A decrease in the FOG deep quadriceps and no change in the FG superficial quadriceps also were reported. However, Edgerton gt_al, (85) trained lesser bushbabies on a treadmill for six months and found no change in phosphorylase activity in the mixed semimembranosus or the F6 vastus lateralis. It appears that phosphorylase activity may be affected mainly in the SO fibers which have the lowest initial values. 33 Increases in LDH activity in the biceps region of the hindlimbs of rats (243) and increases in aldolase activity in the gastrocnemius muscles of rats (146), swum from 5 to 15 weeks, were reported in early studies. These findings were surprising in view of the very light stress that a swimming program imposes on animals. Gollnick gt_al, (118,119) re- futed these findings when they found no change in the LDH activity of the gastrocnemius in rats swum for seven weeks. With prolonged running of rats on a treadmill, Baldwin §t_al, (13) and Holloszy gt_al, (155) found changes in the glycolytic enzymes that are fiber-type specific. Hexokinase activity increased greatly in FOG muscle (deep red quadriceps), less in SD muscle (soleus), and slightly in FG muscle (superficial white quadriceps). This enzyme was unique in that it was the only glycolytic enzyme to increase in all three fiber types. However, the finding was expected since hexokinase activity tends to vary directly with respiratory capacity (25,62). Increases in hexokinase activity were found in wholegastrocnemius homogenates of rats trained on a treadmill (163). Similar increases were reported in the red (FOG) and white (FG) portions of the vastus lateralis muscles of guinea pigs run on a treadmill (20,190,232). The changes were of the same magnitude in both parts of the vastus muscle. Phosphofructokinase, pyruvate kinase, and LDH all increased from 18 to 35% in the soleus and decreased approximately 20% in the red quadriceps of rats trained by endurance running on a treadmill (13). The only change in the white quadriceps was a 15% decrease in LDH. Molé et 31, (205), using the same training program, reported no 34 physiologically significant shifts of LDH isozyme patterns in the vari- ous fiber types. Edgerton gt_al, (85) also found no change in LDH activity in either the PG vastus lateralis or the mixed semimembranosus after six months of training. However, their results were from the lesser bushbaby and species specificity needs to be investigated. The glycerol phosphate shuttle is involved in unidirectional transport of reducing equivalents into the mitochondria of muscle cells. NAD-linked (cytoplasmic) and FP-linked (mitochondrial) a-glycerophos- phate dehydrogenases catalyze the first step of the reaction on either side of the mitochondrial membrane. This shuttle system is extremely important in the regeneration of NAD for glycolysis during anaerobic metabolism. The findings from exercise studies are inconclusive regard- ing the alterations that may occur in NAD-linked a-GPD activity. Baldwin et_al, (13) reported a significant increase in the SO soleus, a significant decrease in the FOG red quadriceps and no change in the F6 white quadriceps of the treadmill-trained rat. These results are consistent with the changes reported in glycolytic enzymes and phos- phorylase (13,155). However, other studies utilizing chronic activity of low (85,153) and high (272) intensity have shown no a-GPD effect in a variety of muscles and species (rat gastrocnemius and rectus femoris, and lesser bushbaby vastus lateralis and semimembranosus). Tricarboxylic Acid (TCA) Cycle Enzymes.--Ear1y work by Hearn and Wainio (145,146) indicated that changes in TCA intermediates might accompany endurance training. Moderate swimming programs resulted in increases in aldolase (146) and SDH (145) activities in the rat 35 gastrocnemius. Similarly, a moderate program of treadmill running in- creased SDH activity 30% in the rat gastrocnemius (121). More recent work has supported these results. The activity of citrate synthase, which catalyzes the primary rate-limiting step of the TCA cycle (192, p. 453), has been shown to increase two-fold in all types of muscle with prolonged endurance training (11,70,154,289,290). Other TCA cycle enzymes including NAD-linked mitochondrial isocitrate dehydrogenase (70,154,203), aconitase (157), and succinic dehydrogenase (70,152,154) have shown parallel two-fold increases in activity. Smaller significant rises have been reported for a-ketoglutarate dehy- drogenase (154) and malate dehydrogenase (70,154,205). The only excep- tion to increased TCA cycle enzyme activity with endurance training was reported by Edgerton §t_al, (85) who found no SDH change in the SO soleus of the lesser bushbaby. However, there was a 20% increase in the F6 part of the vastus lateralis muscle. A recent study by Benzi gt_al, (29) has given support to the con- cept of specificity of training effects. SDH, cytochrome c and cyto- chrome oxidase activity levels changed in relation to the daily workload and the total training time. More work in this area is needed. ATPase and Enzymes 9f_0xidative Phosphorylation.--The immediate source of energy required for muscular contraction is derived from the hydrolysis of ATP to ADP, a process that is catalyzed by adenosine tri- phosphatase. ATP stores are limited and must be replenished constantly by oxidative phosphorylation in the presence of oxygen. Under anaerobic conditions, phosphocreatine becomes a primary source of the high-energy 36 phosphate needed for ATP resynthesis. ATP-creatine transphosphorylase, now commonly known as creatine phosphokinase or creatine kinase, catalyzes the transfer of high-energy phosphate to ADP to form ATP. A secondary source of ATP regeneration is the myokinase reaction in which adenylate kinase catalyzes the transfer of high-energy phosphate from one molecule of ADP to another to form ATP plus AMP. Combined with glycolysis, these two reactions supply the needed ATP during anaerobic muscular contraction. Oscai and Holloszy (221) have shown that endurance training specifically increases oxidative phosphorylation without affecting the anaerobic ATP regenerating systems. Mitochondrial ATPase activity, used as a measure of mitochondrial coupling factor 1 (F1), increased two-fold in gastrocnemius muscle homogenates of endurance-trained rats. At the same time, the levels of mitochondrial and cytoplasmic adenylate kinase and creatine phosphokinase were unchanged. Myosin ATPase activity levels have been shown to be correlated with speed of muscle contraction (16,17), and the specific activity of myosin ATPase in "white" muscle was found to be two to three times greater than in "red" muscle (16). Several groups of investigators have studied the effects of prolonged endurance exercise on myosin ATPase activity with conflicting results. Early studies utilizing moderate swimming programs showed little or no change in homogenates of rat gastrocnemius muscle (147,243). Similar results were reported by Bagby gt_al, (10) for rats trained 11 weeks on a treadmill. Syrovy gt_al, (277) observed an increase in myosin ATPase activity in the 37 soleus of young rats swum for several weeks, but no changes in adult soleus or extensor digitorum longus muscles. Wilkerson and Evonuk (288) used mild and exhaustive programs of swimming for either 6 or 10 weeks. The rats trained for both durations of the exhaustive program demonstrated increased specific activities of myosin ATPase in gastro- cnemius homogenates. Recently, Baldwin gt_al, (15) investigated the adaptation of actomyosin ATPase in specific muscle fiber types to endurance running. Initial concentration levels were maintained after 18 weeks of training. Specific activity levels of actomyosin ATPase were increased in the SO soleus, decreased in the FOG red vastus lateralis, and unchanged in the FG white vastus lateralis. The reported changes paralleled earlier findings on glycogenolytic enzymes (13). Enzymes Involved jn_Fatty Acid and Ketone Metabolism.--Plasma free fatty acids (229) and plasma triglyceride fatty acids (107,164) have been shown to be important substrates for oxidation by skeletal muscle during exercise. Major increases in the levels of enzymes involved in the activation, transport and 8 oxidation of long-chain fatty acids (ll,69,71,108,157,203,204) and in the levels of enzymes involved in ketone oxidation (8,289,290,291) have supported these observations. Mole gt_al, (204) reported a doubling of palmityl CoA synthetase, carnitine palmityl transferase, and palmityl CoA dehydrogenase activi- ties in mixed muscle homogenates (quadriceps plus gastrocnemius) of rats trained on an endurance running program. The rates of palmitate oxidation by whole muscle homogenates and by mitochondrial fractions 38 from the leg muscles also were found to increase two-fold. Identical observations had been reported earlier for gastrocnemius homogenates (203). To determine which fiber types participate in the exercise-related increase in fat metabolism, Baldwin et_al, (ll) repeated the study of Molé using homogenates of the SO soleus, the FOG deep red quadriceps, and the superficial white quadriceps. The rate of palmitate oxidation and the activity level of carnitine palmityl transferase increased approximately two-fold in all three fiber types. Consequently, the relative capacities of the different fiber types for fat metabolism remained unchanged. Ketone oxidation also is affected by an endurance program (8,289, 290,291). Winder gt_al, (289,290) found a two-fold to three—fold in- crease in the rates of D-B—hydroxybutyrate and acetoacetate oxidation in gastrocnemius muscle homogenates under conditions of uncontrolled respiration. D-B-hydroxybutyrate dehydrogenase, 3-ketoacyl-COA trans- ferase and acetoacetyl-CoA thiolase, key enzymes in ketone metabolism, all increased significantly with training. Recently, Winder gt_al, (291) have shown that endurance training affects ketone metabolic path- ways in the three fiber types differently. The levels of 3-hydroxy- butyrate dehydrogenase activity increased slightly in FG, 2.6-fold in S0 and 6-fold in FOG fibers. Acetoacetyl-CoA thiolase activity in- creased approximately 40-45% in all fiber types, and 3vketo acid CoAv transferase activity increased 2-fold in FOG and FG muscle, but only 26% in SD muscle. This exercise-induced increase in the capacity of 39 skeletal muscle to oxidize ketones could play a major role in prevent- ing ketosis in the exercising animal (8,291). In a related study, Borensztajn et_al, (32) investigated the effects of prolonged endurance training on the activity of lipoprotein lipase in the same three muscles. This enzyme is responsible for the uptake of chylomicrons by skeletal muscle. Initial control measure- ments revealed the highest activities to be in $0 muscle. The soleus had activities which were 14 to 20 times greater than that in the FG white quadriceps and 2 times greater than that in the FOG red quadriceps. Twelve weeks of training resulted in a four-fold increase in lipoprotein lipase activity in the FOG muscle and two-fold increases in the S0 and FG muscles. The greater rise found in the FOG muscle may reflect selective recruitment of these fibers during treadmill running. In contrast to these findings, Askew gt_al, (7) found no significant change in lipoprotein lipase activity in the quadriceps muscles of rats trained for seven weeks on a treadmill. Endurance Trainingf-Histochemical Alterations Histochemical techniques have been used to determine muscle fiber metabolic profiles before and after specific training programs. Recruitment patterns induced by various work tasks and loads may be studied in this manner. Faulkner and co-workers (100,101,194,198) studied the effects of chronic exercise on the distribution of fiber types in hindlimb muscles of the guinea pig. The exercise regimen consisted of daily running on I a motor-driven treadmill at 0% grade with a maximum speed of 30 m/min. 40 The animals were exercised 30 to 45 min per day for eight weeks. SDH and myofibrillar ATPase were used to classify fiber types. In adult sedentary animals, the composition of the plantaris muscle was deterv mined to be 53% FG, 36% FOG, and 11% $0. The soleus was found to be 100% $0 and the psoas was 2% FG, 33% FOG, and 66% SD (198). These values agree closely with the findings of other investigators (4) and reflect three vastly different muscle fiber distributions. Training specifically affected the composition of the plantaris muscle but had no effect on the others. An increase in the proportion of FOG fibers, a decrease in the proportion of FG fibers, and no change in the propor- tion of SO fibers was reported for the plantaris. The results are consistent with those found in other studies of the effects of endur- ance exercise (21,83). Reversability of exercise-induced fiber changes in the guinea pig was observed with 16 weeks of detraining (101). Selective atrophy and degeneration of F6 fibers may have occurred, but the regression effect was attributed to a loss of mitochondrial density in FOG fibers which then were reclassified as FG fibers. It should be noted that the percentage of red fibers (presumably FOG) has been found to be significantly increased in the diaphragm of endurance—trained animals (194). The productive group of Edgerton, Barnard, Peter and their co- workers (20,21,22,23,24,78,79,80,81,82,83,84,85,86,87,88,89,102,103, 116,117,168,169,190,232,233,234,235,236,237) pioneered the early work of physiological, histochemical and biochemical correlational studies 41 and made many contributions to the concepts of mutability of fiber types and motor unit recruitment during exercise. Edgerton gt_al, (78) subjected male albino rats to a prolonged swimming program. No significant alterations in percentages of fiber types were found in the soleus. However, the plantaris muscles of the exercised animals had a greater proportion of fibers with high malate, SDH, and NAD—diaphorase staining reactions than did those of the seden- tary controls. These changes were observed in two areas of the plan- taris. One area had a mixed-fiber population and the other was com- posed predominately of FG fibers. No changes were found in the propor- tion of SO fibers with weak myosin ATPase reactions. Morphological changes also were investigated in these animals (81). Necrotic, angular and split fibers were observed in the soleus muscle but not in the gastrocnemius or plantaris muscles of all groups including the control group. The number of split fibers was the same for the three groups, but the total number of subfibers increased with the intensity of exercise. Split fibers have been reported in several other training (55,208) and surgically overloaded muscle studies (141, 142,283). In a series of classical papers Barnard g__gl, (21,22,23) reported the histochemical, biochemical and physiological changes induced by an endurance training program in guinea pig hindlimb muscles. After 18 weeks of training, the mitochondrial yield had significantly increased in the gastrocnemius and plantaris (21). Histochemical analysis (NADH- diaphorase) revealed a significant conversion of FG to FOG fibers in 42 the central "red" and peripheral "white" areas of the medial gastroc- nemius, The percentage of SO fibers did not change (21). Contractile properties as measured in the in situ gastrocnemius-plantaris muscle preparation revealed no exercise effect (22). Edgerton gt_al, (82) ran guinea pigs on a treadmill at 1.6 km/hr for 5 min, 10 min, or until exhaustion. With increasing durations of acute exercise, the percentage of fibers lacking phosphorylase activity increased. Selective depletion of phosphorylase content was found in the red fibers (presumably FOG) of the plantaris muscle. No consistent changes were found in the soleus. This finding reflects the homogeneous SO fiber population in the soleus (4) which shows negligible phosphoryl- ase activity even in controls. Edgerton gt_a1: (79,83) studied this depletion phenomenon further. Guinea pigs were trained on a progressive program of intermittent run- ning for 20 weeks. Indirect electrical stimulation caused total phos- phorylase to be selectively depleted in F6 fibers. The effect was less in trained (86%) than in untrained (97%) animals. The histochemical depletion of'phosphorylase was paralleled by glycogen depletion which was measured by spectrophotometric readings of PAS staining intensities. These PAS results support the findings of Kugelberg and Edstrom (187). Recently, Edgerton gt a1, (85,117) have attempted to extend their findings to a nonhuman primate, the lesser bushbaby. These animals were trained to run or jump on a motor-driven treadmill. After six months of endurance running, fewer glycogen-depleted fibers were found in the plantaris muscles of trained than untrained animals following 15 43 min of electrical stimulation (85). This finding reaffirms the train- ing-related resistance to fatigue reported for the guinea pig (83). The other biochemical, histochemical and physiological data also were in agreement with the results of previous work on guinea pigs (21,22,23). Endurance running produced increases in SDH and cytochrome a and c activities. Myoglobin content was enhanced. There was an increased proportion of FOG fibers, at the expense of F6 fibers, in the tibialis anterior but not in the soleus. Glycogenolytic enzyme concentrations and contractile properties were not altered. No significant changes in myosin nor actomyosin ATPase activities were found. In general, the results supported those of other histochemical (10) and biochemical (147,243) studies. A single 5-min to lS-min bout of running at 1.75 m/min or jumping at 2.4 to 2.9 m/min was used to determine the pattern of motor unit recruitment during specific types of exercise (117). Glycogen depletion was assessed by the PAS stain. FOG fibers were preferentially depleted in the vastus lateralis and gastrocnemius muscles after running. Jumping affected mainly the FG fibers in these two muscles. Both exer- cise regimens depleted the FOG fibers in the soleus. The findings sug- gest that the recruitment pattern of specific types of motor units is related to the nature of the specific movement being performed. Recent work has indicated that this also is the case in humans (124,125,126, 162). In a comprehensive investigation, Muller (208) attempted to deter- mine the temporal progress of mutability in muscle fibers. Young female 44 rats were exercised on a motor-driven treadmill six days a week for periods of 3, 6 and 12 weeks. At the end of the study, the mean fiber areas in the soleus, gastrocnemius and rectus femoris muscles of the exercised and control animals were not different. However, progressive splitting of S0 fibers was seen in the soleus muscles of the trained rats at 3 and 6 weeks. Fiber splitting was not evident in the control animals. This observation conflicts with that of Edgerton gt_al, (81) who noted that a minimal amount of splitting is to be expected even in untrained animals. Muller (208) also reported a significantly decreased percentage of fast-twitch fibers in the soleus muscle. This decrease presumably was caused by the transformation of FOG fibers to S0 fibers. Small but similar endurance-training effects were observed in the pre- dominately FOG and FG areas of the gastrocnemius and rectus femoris muscles. The general trend of adaptation was from FG to FOG to $0 fibers. The conversion of fast-twitch to slow-twitch fibers was not found in several earlier biochemical (147,243) and histochemical (10, 52,85) studies. However, recent evidence indicates that myosin ATPase activity may be altered in response to specific exercise regimens (15, 137). Sprint Training A program of sprint running could be expected to provide consider- able stimulation for the anaerobic metabolic mechanisms. Unfortunately, due to the inherent difficulties associated with training animals at high running velocities, relatively little work has been done to date . with this type of exercise. 45 Saubert et_al, (257) trained adult male rats on a treadmill at speeds of up to 80.5 m/min for 11 weeks. Glycogenolytic and glycolytic mechanisms were affected but only minimally. Phosphorylase activities were unchanged in the FG white portion of the gastrocnemius, the FOG red portion of the gastrocnemius, the red vastus, and the mixed rectus femoris muscles. The only change in phosphorylase activity was a 70% increase in the S0 soleus. Hexokinase activity increased 50% in both the mixed rectus femoris and the soleus muscles. No changes in phosphofructokinase, pyruvate kinase, triosephosphate dehydrogenase, or lactate dehydrogenase activi- ties were reported except in the soleus where there was a 35% increase in pyruvate kinase. In a parallel study by Staudte gt_al, (272), run- ning at 80 m/min produced a 17% increase in triosephosphate dehydro- genase activity of the rat soleus. The slight anaerobic adaptation of the soleus muscle was evident in the ATP regenerating system. Creatine phosphokinase activity in- creased 12% in the soleus but remained unchanged in the rectus femoris (272). Bagby gt_al, (10) also ran rats for 11 weeks at speeds up to 80.4 m/min and found myosin ATPase activity was unchanged in homogenates of the mixed gastrocnemius muscle. No alterations in the percentages of FG, FOG, or SO fiber types were observed. Possible explanations for these relatively small changes include: (a) the F6 and FOG fibers of the rat already may be equipped metabolic- ally to handle an anaerobic stress; or (b) the running speed of 80 m/min may not be fast enough to act as a pure anaerobic stimulus. The latter 46 hypothesis is supported by the fact that histochemical fiber typing techniques revealed a significant increase in the percentage of FOG fibers, with an accompanying decrease in FG fibers, for the white portion of the gastrocnemius (257). There was an accompanying shift towards an FOG fiber population in the soleus muscle. Fitts _t_al, (104) studied several histochemical parameters in the miniature pig following a sprint-running program known to have physiologically measurable training effects. No changes in fiber types were observed, and the investigators concluded that the histochemical techniques were not sensitive enough to distinguish metabolic adapta- tions. The effects of sprint training on aerobic metabolism are not clear at this time. No change in myoglobin content in any fiber type of sprint-trained miniature pigs was reported by Fitts gt_al, (105). Staudte gt_al, (272) found increases of 20% in citrate synthase activity in homogenates of both the mixed rectus femoris and the SO soleus muscles. However, no changes have been observed in a variety of muscles assayed for SDH activity (257,272). Although not enough information is available to draw firm conclusions, it appears that current sprint pro- grams for animals may have a substantial aerobic component. Isometric, Weight Lifting, and Miscellaneous Training Three parallel studies have been conducted to determine the ef- fects of an isometric training program on histochemical and biochemical profiles of exercised skeletal muscle (96,97,209). Male and female 47 rats were forced to climb a 60° incline and support a predetermined amount of weight until exhaustion (approximately 5 min). Activities of several enzymes of glycolysis, glycogen metabolism, fatty acid oxidation, lactate fermentation, and the ATP regeneration system were determined in homogenates of the rectus femoris and soleus muscles (96,97). Changes in anaerobic enzymes were evident. Creatine phosphokinase, glycogen phosphorylase, and triose phosphate activities increased in the rectus femoris and decreased in the soleus. Lactate dehydrogenase also decreased in the soleus. Contraction times became faster in the rectus femoris and slower in the soleus muscles of the females (96). Histochemical changes in the female rats were studied using SDH and myofibrillar ATPase to classify muscle fibers (209). The percentage of FOG fibers decreased at the cost of the FG fibers in rectus femoris but not in the soleus or the lateral head of the gastrocnemius. The percentage of SO fibers did not change significantly in any of the muscles studied. Together, these parallel studies would indicate that a rise in anaerobic capacity occurs in predominately FG-FOG muscle which is sub- jected to isometric training (96,97,209). This increased anaerobiosis may be modulated by a shift of FOG to FG fibers (209). A concomitant decrease in aerobic capacity might accompany such a shift (270). However, conflicting results have been reported by other investigators (159,185,297). 48 Howells and Goldspink (159) devised a counter-weighted basket which the animal had to pull down to obtain food. Hamsters subjected to this regimen for five weeks had increased SDH levels in the mixed biceps brachii, the slow soleus, and the fast extensor digitorum longus muscles. Similar increases in SDH values were reported by Zika §t_al, (297) in the biceps brachii of young rats subjected to tonic stress on a ladder for four to six months. Significantly elevated levels of a-glucanphosphorylase and nonspecific esterases also were found. There were no changes in LDH or mitochondrial a-glycerolphosphate dehydro- genase values. Kowalski §t_al, (185) trained adult female rats on a weight lift- ing program of vertical climbing with an attached load for six weeks. Six preselected regions of the quadriceps muscles were investigated histochemically. Weight lifting resulted in overall increases in SDH, phosphorylase and cytochrome oxidase in all six regions regardless of the fiber-type population. The increases in oxidative enzymes observed by Howells and Gold- spink (159), Zika gt__l, (297), and Kowalski et_al, (185) suggest that the various training programs used in these studies may have had a common aerobic component. CHAPTER III METHODS AND MATERIALS Gross measurements of total-body oxygen debt and oxygen uptake have been used to reflect human metabolic responses to physical activity. Exhaustive sprint running leads to an increased tolerance of oxygen debt which presumably reflects a greater capacity for the generation of muscular energy via anaerobic metabolism. Training regimens based on this type of running are characterized by maximal workloads and rela- tively short bouts of repeated exercise. In contrast, distance running is thought to be dependent chiefly upon oxidative muscle metabolism and tends to increase total-body oxygen uptake capacity. Moderate or light workloads and relatively long bouts of continuous exercise are typical of endurance training programs. This study was designed to investigate cellular-level alterations in two preselected areas of the plantar flexor muscles of the male albino rat following eight weeks of sprint and endurance training. Experimental Animals Forty-two normal male albino rats (Sprague-Dawley strain) were obtained from Hormone Assay, Inc., Chicago, Illinois. They were re- ceived at weekly intervals in three shipments of 15, 12, and 15 animals 49 50 respectively. Each shipment was designated as a separate treatment group. A standard period of 12 days was allowed for adjustment to laboratory conditions. The treatments were initiated when the animals were 84 days old. The application of selection criteria (to be dis- cussed later) reduced the final sample to a total of 27 animals. Research Desigg and Treatment Groups This study was conducted as a one-way design with three treatment groups of nine animals each. The duration of the treatment period was eight weeks. The three treatment groups were as follows. Control Group The 12 animals in the second shipment constituted the control (CON) group. These animals received no special treatment and were housed in individual sedentary cages (24 cm x 18 cm x 18 cm) during both the adjustment period and the treatment period. Sprint Group The sprint running (SPT) group was comprised of the 15 animals in the first shipment. Each of these animals was housed in an individual voluntary-activity cage (sedentary cage with access to a freely revolv- ing activity wheel) during the adjustment period and in an individual sedentary cage during the treatment period. The SPT animals were sub- jected to an interval training program of high-intensity sprint running (Appendix A). The workload of the SPT program was gradually increased until on the 27th day of training, and thereafter, the animals were 51 expected to complete six bouts of exercise with 2.5 min of inactivity between bouts. Each bout included five 15-sec work periods alternated with four 30-sec rest periods. During the work periods, the animals were required to run at the relatively fast speed of 108 m/min. Endurance Gregg The endurance running (END) group was composed of the 15 animals in the third shipment. These animals were housed under the same condi- tions as the SPT animals. The END animals were subjected to a demanding program of distance running (Appendix A). The workload was progressive- ly increased so that on the 30th day of training, and thereafter, the animals were expected to complete 60 minutes of continuous running at 36 m/min. Training Procedures The SPT and END groups were trained in a battery of individual controlled-running wheels (CRW). This apparatus has been described as: ... a unique animal-powered wheel which is capable of induc- ing small laboratory animals to participate in highly specific programs of controlled reproducible exercise. (286) Animals learn to run in the CRW by avoidance-response operant con- ditioning. A low-intensity controlled shock current, applied through alternating grids comprising the running surface, provides motivation for the animals to run. A light above the wheel signals the start of each work period. The animal is given a predetermined amount of time (acceleration time) to attain a prescribed running speed. If the 52 animal does not reach the prescribed speed by the end of the accelera- tion time, the light remains on and shock is applied. As soon as the animal reaches the prescribed speed, the light is extinguished and the shock is discontinued. If the animal responds to the light and attains the prescribed running speed during the acceleration time, the light is extinguished immediately and shock is avoided. If the animal fails to maintain the prescribed speed throughout the work period, the light- shock sequence is repeated. Most animals learn to react to the light stimulus after only a few days of training. A typical training session consists of alternated work and rest periods. The wheel is braked automatically during all rest periods to prevent spontaneous activity. The brake is released and the wheel is free to turn during work periods. Performance data are displayed for each animal in terms of the total meters run (TMR) and the cumulative duration of shock (C05). The TMR and the total expected meters (TEM) are used to calculate the per- centage of expected meters (PEM): PEM = 100 (TMR/TEM) PEM values are the chief criteria used to evaluate and compare training performances. A secondary criterion is provided by the percentage of shock-free time (PSF) which is calculated from the C05 and the total work time (TWT): PSF = 100 - 100 (CDS/TWT) In this study, all exercise treatments were administered once a day, Monday through Friday, between 12:30 p.m. and 5:30 p.m. 53 Animal Care All housing cages were steam-cleaned every two weeks. Standard procedures for daily CRW cleaning and maintenance were observed. The animals received food (Wayne Laboratory Blox) and water ad_ II'hitum.1 A relatively constant environment was maintained for the animals by daily handling as well as by temperature and humidity control. The animals were exposed to an automatically regulated daily sequence of twelve hours of light followed by twelve hours without light. Since the rat normally is a nocturnal animal, the light sequence was established so that the lights were off between 1:00 p.m. and 1:00 a.m. and on between 1:00 a.m. and 1:00 p.m. This lighting pattern altered the normal day-night schedule for the animals so that they were trained during the active phase of their diurnal cycle. Body weights of the SPT and END animals were recorded before and after each training session. The CON animals were weighed weekly. Sacrifice Procedures Anticipated limitations of time and personnel restricted the number of animals that could be handled at sacrifice to 12 in each 1The three groups of animals used in this study were the placebo groups for a larger diet-training investigation. Seven days a week, between 7 p.m. and 9 p.m., each animal was given approximately .1 cc of 5% sugar solution/100 gms body weight, by oral syringe. Administration of the placebo was begun the day prior to the initiation of treatments and was terminated the day prior to sacrifice. Since all of these animals received the same dietary treatment, the effect of the placebo can not be evaluated. However, the internal validity of this study could not have been affected. 54 treatment group. Since one of the inherent purposes of the study was to compare various parameters in two groups of highly trained animals and a group of untrained animals, three extra rats originally were in- cluded in the SPT and END groups. Twelve animals were selected for sacrifice from each of these two groups on the basis of their health and their training performance throughout the treatment period. Only animals subjectively determined to be in good health were chosen. Because the training requirements were extremely vigorous, no absolute minimal performance criteria were established. However, individual daily records of PEM and PSF values were examined, and those animals making the best adaptations to the training regimens were selected for sacrifice. All 12 CON animals were judged to be healthy and were sacrificed. Three sacrifice periods of two-days duration (Monday and Tuesday) were established. All animals within a treatment group were killed during a single sacrifice period (i.e., six animals each day). The trained animals were killed either 72 or 96 hr after their last exer- cise bouts were completed. This procedure was followed to eliminate any transient effects of acute exercise. The animals were either 140 or 141 days old at sacrifice. 1 Final body weights were recorded immediately prior to sacrifice. Each animal was anesthetized by an interperitoneal injection (4 mg/lOO gnbody weight) of a 6.48% sodium pentobarbital (Halatal) solution. The right hindlimb was skinned and the superficial posterior crural muscles were exposed by reflecting the overlying tissue. The right triceps 55 surae (gastrocnemius and soleus) and plantaris muscles were removed as a block. Similar procedures were used on the left hindlimb except that the plantaris and soleus muscles were separated, individually weighed, and discarded. Upon removal, the right muscle block was rolled in talcum powder. The block was held with forceps, gently stretched to approximate its physiological length, and quick frozen in 2 methylbutane (isopentane). The isopentane had been precooled to a viscous fluid (-l40 to -160° C.) by liquid nitrogen. The frozen muscles were stored in aluminum 35-mm film containers at -20° C until sectioning and histochemical procedures could be initiated. Using precooled stainless steel knives, sandwich blocks approximately 10 to 15 mm thick were cut from the mid-portions of the frozen muscles. The sandwich blocks were oriented distal end up and frozen onto cork strips using 5% gum tragacanth. The cork strips were used to attach the muscle blocks onto cryostat chucks for section- ing. Fresh-frozen serial cross-sections, 10 micra thick, were cut using a rotary microtome-cryostat (International-Harris Microtome). Sections were picked up on cover glasses and fan-dried for at least one hour. Histochemical Procedures Succinic dehydrogenase (SDH) reactivity was used as an indicator of aerobic capacity and resistance to fatigue. In the Krebs cycle, succinate is oxidized to fumarate by SDH. The covalently bound flavin adenine nucleotide picks up the two hydrogens removed and transports them to the electron transport system. SDH is bound firmly to the 56 mitochondrial membrane, and thus it also is a good indicator of mito- chondrial distribution. In this study, SDH localization was demon- strated using nitro blue tetrazolium (NBT) as the electron acceptor. The method has been described by Barka and Anderson (19, p. 313). NBT yields a colored precipitate of diformazan when it is reduced and the formazan deposition observed with the light microscope indicates the localization of oxidative enzymes (216). A direct correlation between the qualitative histochemical classification by staining intensity for SDH and the quantitative measurements of SDH activity has been reported (27). Lactate dehydrogenase (LDH) reversibly oxidizes lactate to pyruvate in the last step of glycolysis. It is found in all cells which are capable of glycolysis. Five isozymes have been isolated bio- chemically each consisting of one or a combination of two polypeptide chains designated as M (muscle) or H (heart) (67). All LDH isozymes catalyze the same reaction but have different activity levels. LDH localization was determined in this study using NBT as the electron trap and nicotinamide adenine dinucleotide (NAD+) as the cofactor (230, p. 911). Staining intensity was assumed to be an indicator of lactate fermentation capacity. Because of the rather uniform intermyofibrillar network that is associated with this enzyme, it does not differentiate fiber types as well as do some other glycolytic enzymes. This lack of discriminative ability perhaps is due to the fact that LDH is a water soluble enzyme which may be present in the aqueous sarcoplasm (35). Myosin adenosine triphosphatase (ATPase) localization was investi- ‘ gated by the method of Padykula and Herman (223) as modified by Guth 57 and Samaha (132) and presented by Dubowitz and Brooke (75, p. 32). The reaction is one in which both the preincubation of the tissue section and the incubation in the ATP mixture are carried out at a pH of 9.4. Under these conditions, the reaction develops in the myofibrils, and the intermyofibrillar network seems to dissolve out of the tissue sec- tion at some stage during the reaction (75, p. 32). The localization of this ATPase in the myofibrils has been substantiated by selective extraction procedures (260). Myosin ATPase is an enzyme involved in the hydrolysis of ATP to ADP with the release of a high energy bond available for muscle contraction (192). A direct correlation between myosin ATPase activity and speed of muscle contraction has been demon- strated biochemically (16,17,60) and substantiated histochemically (24, 53,88,132). Fast-twitch fibers (FOG and FG) stain darkly and slow- twitch fibers (SO) stain lightly at a pH of 9.4. Glycogen localization was determined using the periodic acid- Schiff (PAS) reaction (197, p. 132). Previous studies have shown that spectrophotometric measures of glycogen content are correlated highly with PAS staining intensity in frozen tissue when the PAS response is evaluated either by microphotometric methods or by subjective ratings (117,188). Lipid localization was demonstrated using the Sudan Black 8 (SUD) method (197, p. 126). SUD, a colorant, is soluble in absolute alcohol and has a high affinity for fatty material. Harris' alum hematoxylin and eosin (H 8 E) was applied to the fresh-frozen sections to facilitate observations of morphological char- acteristics (197, p. 29). 58 Incubation times were varied according to the staining procedure. The mounting medium for the ATPase, SDH and LDH sections was glycerin- jelly. PAS, SUD and H & E sections were mounted in permount (Histoclad). Muscle Areas Histochemical evaluations were performed on two muscle areas in this study. These areas were selected to represent two different fiber populations. The central portion of the soleus (area 1) normally is composed primarily of SO fibers. Only a few FOG fibers are present. The posterior part of the plantaris (area 2) consists mainly of FG fibers with some FOG fibers interspersed (78). Histochemical Evaluations Each histochemical stain was evaluated objectively with the use of a Histochemical Photometer (HCP) at a magnification of 80X. The operator of the HCP is able to isolate a photometric beam on the center of a single muscle fiber in the projected image of a muscle cross- section. The photometer registers the percentage of light that is trans- mitted through the fiber on a scale from O to 100. The percentage of light transmitted is converted to percentage of light absorbed so that higher HCP readings reflect higher values of substrates and enzymes. Repeated measures of the same fibers on different days have shown the average percent error for the HCP to be :_O.3%. 59 Photometric evaluations of each stain were determined for a group of 30 adjacent muscle fibers1 from both muscle areas in each animal. The planned analysis of data imposed the requirement that histochemical values had to be taken on the same fibers in all serial cross-sections from a given animal. Fiber tracings of the SDH sections were matched with the projected images of the other sections to insure that this requirement was satisfied. Although 12 animals in each treatment group were sacrificed, identical fibers could be found throughout the serial cross-sections of only 9 animals per group. In each of the other cases, various artifacts prevented conclusive fiber identification in one or more of the sections. Consequently, the final sample was limited to a total of 27 animals. The HCP values obtained on cross sections from the control animals served as reference standards for the histochemical stains. All photo- metric determinations of each stain were performed at the same time without knowledge of the treatment groups. Analysis gf_0ata The body weights and the absolute and relative muscle weights were analyzed using a one-way fixed-effects analysis of variance routine on 1In a previous study, a sample of 30 fibers was calculated to be more than necessary and sufficient for a four-way (7x4x8x10) mixed- model nested analysis of variance that was run on HCP data when: (a) the probability of making a type I statistical error was limited to the .01 level, (b) the probability of making a type II statistical error was limited to the .05 level, (c) the minimal mean difference to be detected as significant was set at 0.5 standard deviations, and (d) a moderate variability between subgroup means was assumed. Consequently, standard laboratory protocol now is to take readings on 30 fibers in each muscle area of interest. 60 the Michigan State University Control Data 6500 Computer (CDC 6500). Newman-Keuls tests were used to evaluate differences between pairs of means whenever a significant (P g .05) F-ratio was obtained. The histochemical data for each stain were plotted by treatment group and muscle area. A Chi-square contingency analysis (ACT routine) was used to determine if there were any significant differences (P g .01) between frequency distributions for treatment groups within muscle areas. CHAPTER IV RESULTS AND DISCUSSION The material in this chapter is organized into four main sections. The first part deals with the training results from the Controlled- Running Wheel (CRW) programs and includes a summary of basic statistics for the percentage of body weight lost during the daily exercise periods, the environmental factors that operated during training, and the data obtained on the two performance criteria. Body and muscle weight results at sacrifice are given next. A major section is devoted to the histochemical data which are presented by muscle area. Finally, a discussion is offered that attempts to relate the present findings to those of other investigations reported in the literature. Training Results The sprint (SPT) and endurance (END) Controlled-Running Wheel (CRW) training programs are presented in Appendix A. These programs are modi- fied versions of standard regimens routinely used in the Human Energy Research Laboratory, Michigan State University, East Lansing, Michigan. The modifications were incorporated in an attempt to design strenuous exercise programs which would specifically stimulate anaerobic or aerobic metabolic processes in individual muscle cells. The performances 61 62 of the animals were evaluated using the percentage of expected meters (PEM) and the percentage of shock-free time (PSF) as criterion measures. The performance data for the SPT group are presented in Figure 1. Progressive increases in the required running velocity were made rapidly. From the beginning of the fourth week of training to the end of the program, the animals were expected to run at velocities ranging from 90 to 108 m/min (see Figure l and Appendix A, Table A-l). No com- parable exercise programs for small animals has been found in the literature. The results indicate the animals could not maintain the program requirements. PEM and PSF values fell to approximately 50 and 40 respectively during the last three weeks of training. Several pos- sible explanations could account for these relatively poor performance data. The required running velocities may have been too fast, but observations during the training sessions revealed that the animals were capable of sprinting at the desired speeds. Low PEM and PSF values might suggest that the animals responded to the unconditioned shock stimulus rather than to the conditioned light stimulus. Improper ini— tial training and defects in the CRW equipment could lead to such a learning problem, but the END animals learned to run under the same conditions and had no such difficulties (see Figure 2). A lack of con- trol of environmental factors affecting training performance might have accounted for these results. This is particularly true for air tempera- ture and percent humidity, but again the END data make this explanation improbable (see Appendix B). The most likely cause of the low PEM and PSF values is that the SPT regimen may have produced a state of 63 °‘—° VVBd P meamwm .5:QO >36 .8 22mg 8922 88QO Emoemn. eco €de mEP mmeuixogm Emecma 360 :82 (_II D (_l I‘Tl |+m . 8. cm omllTllmliTlmc. B m is}: ..w> m _ c. _ mJI _ n41 _ e _ n _ m _ . '— 23 .235 cc mm on mm em 9 o. m >40 .235 O 1 b _ F _ p _ p p _ L p _ _ — bP — _ p _ _ — r — b — _ F b h h — _ F — b P p h — II—IO I Le ON I L O¢ 0? rl l 00 IO 8 I: l J em I . . I ow H I. (o I... 8 I . . . . . I 2. j o o o o I. 8. I I om vl o i ON. I I om TI 1 co. I I oo. W3d 64 N mgemwm moz... Jm> _1 m _ c. _ e _ e _ a _ m _ m _ _ 13.2.45 0v mm on mm ON 0. O. 0 >40 2.4m» o cc_eh_.___e______ercF__c.__c_________c__70 e m ON T L cc 0? l 00 d e e, om T I. 00 H r o I. LON ON_1 10m OS I L 00. 65 overtraining. The data in Figure 1 support this hypothesis. Increases in the required velocity were expected repeatedly from the SPT animals before they were fully adapted to the previous velocity. The constant additional stress could have resulted in overtraining.1 The training data for the END group are shown in Figure 2. PEM values were 70 or higher on all but one day and averaged 81.3. PSF values were above 60 on all but three days. The mean PSF value was 68.4. These results indicate that the animals were able to maintain the daily requirements of the END program relatively well. The END animals ran at the relatively slow speed of 36 m/min. Periods of continuous running were progressively increased to 60 min at the end of five weeks of training and were maintained at this level for the remainder of the eight week program (see Appendix A, Table A-2). The single bout of exercise was determined subjectively to result in daily physical exhaustion of the animals. Repeated exposure to this level of stress could have resulted in a mild state of overtraining. 0n the average, the rats lost 2.7% body weight during each training session (see Appendix B, Table B-2). Body weight data were used to award an unplanned recovery day on Wednesday of each of the last three weeks of training. The animals were run on the 39th and 40th days of the program, but the results were not recorded due to a technician error. 1Supplementary data on hindlimb bone weights of the animals (to be reported elsewhere) also suggest an overtraining phenomenon. The bones of the SPT group were approximately 40% lighter than those of the CON group. This observation was totally unexpected and does not agree with the results of previous work in which less strenuous training regimens were used (282). 66 The requirements of the END program appear to be similar to those of the training protocol used in the experiments conducted by Holloszy and a number of other investigators (see Table 4). In those studies animals ran continuously for periods of up to two hours at 31 m/min. The discrepancy in the duration of time the animals could run probably is due to the different modes of training. Holloszy and co-workers used a motor-driven treadmill, whereas the CRW used in the present study is animal-powered. The animals must displace the mass of the running wheel during the acceleration period and then maintain the rota- tion of the wheel at some required speed for the entire program. At any given running velocity, the CRW is a more demanding exercise module than the motor—driven treadmill. The metabolic changes produced by these two pieces of apparatus need not coincide. Body and Muscle Weight Results at_Sacrifice At the end of eight weeks of exercise, the trained animals were significantly smaller than the sedentary control animals (see Table 6). The difference in body weight between the SPT and END groups of animals was not statistically significant. Both trained groups were approxi- mately 20% lighter than the CON group. These results are in agreement with those of previous studies using the CRW (150,278) and support the general observation that strenuous exercise slows the usual gain in body weight seen in the male rat over time (21,63,100,152). The slower rate of weight gain is usually attributed to an increase in caloric expenditure associated with exercise and, in some instances, to a 67 Table 6. Analysis of variance for overall treatment effects and Newman Keul's tests of paired comparisons for body weight at sacri- fice and absolute and relative muscle weights. Newman Dependent Treatment Means F P Keul's Variable CON SPT END Value Value Test** Body Weight at 517.2 409.9 420.6 52.722 <0.0005* SPT = Sacrifice (g) END<:CON Absolute Soleus 0.200 0.160 0.164 6.801 0.005* SPT = Weight (g) END< CON Absolute Plantaris 0.501 0.365 0.456 20.744 <0.0005* SPT < Weight (g) ENDImp Po. mcu we cemwceeeeo pceewmecmwm an mcemweeeamu ucmEHemc» emcwee sm.Fm mm s~.Fm mm sm.me mm wm.om_ we mam sm.mom NF cm.mm up em.nom up cm.mom em m Hem ozm m> zoo Hem m> zoo mcemweeeeeo ucmEueme» ppeem>o Feewemcoepmw: AF emcwm Ce mceweeeecumwo xecmeemcm ce muumemm pcmEpemcp emcwee ece _Pecm>o com mmm>Fec< mceeemecu .m mpeee 76 .% FREQ. 16 1 q l4 - CONTROL a: l SPRINT ENDURANCE a 1 r 2‘ /\/\/\_ " /‘ A 0'1 rrttt rvrv‘lvvrt IUIIIYUIT IIUII'YIUI IIUU'IIII IIIIIUIIU II’IIIfiUY I'th 30 4O 50 60 7O 80 90 Histochemical Photometer Value (°/o Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for ATP 9.4 in Soleus Muscle Fibers (Area I) Figure 3 77 exercise-induced enzyme depletion, cannot be ruled out from the present data. Considering the time between the last exercise bout and sacrifice, however, such an effect seems to be unlikely. An apparent third group of fibers with relatively low values of ATPase 9.4 can be seen in all three treatment distributions. This ob- servation is interesting although it cannot be explained at the present time. The SDH distributions (Figure 4) were shifted to the right with both types of training. This adaptation would indicate that increases in aerobic metabolism took place. The shift is more apparent in the END group than in the SPT group. LOH is an index of lactate fermentation and is used as a marker for anaerobic metabolism. With training, LDH reactions in individual muscle cells generally were increased (Figure 5). The SPT training program produced the largest overall shift in HCP readings, whereas an additional effect of the END program was to decrease the range of values. Note, however, that the distribution for the END trained animals showed a small shift towards high LDH values. Evidence indicates that endurance exercise may alter the metabolic profile of red (SOvFOG) muscle to resemble cardiac muscle. That is, the red muscle may assume the ability to utilize lactate as an energy source (174). This phenom- enon may be reflected in the increased LDH activity in the S0 soleus. SPT training resulted in marked increases in glycogen stores as reflected by the PAS stain (Figure 6). This finding is consistent with the observed increase in LDH reactions for the SPT group since anaerobic 78 FREQ. CONTROL a l I SPRINT ab 1 1 12- q 10- 8" .1 6- .4 44 ’2‘ ENDURANCE ‘ 0 d l‘.“r"'fi vvvv'm' "V'I VVVVVVV 'V"' 50 60 7O 80 90 Histochemical Photometer Value (98 Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for SDH in Soleus Muscle Fibers (Area I) Figure 4 SPRINT CONT ROI... ONAO ENDURANCE anteater 7o FREQ. ONh 79 If F MM _A/‘ I I I I I I I I [S M r""'m' 'V'Vl‘jvv 'r'V'I'V' 'U'U'U'UVIIUUVI '''''''' l '''''''' ' VVVVVVV t'r‘ IO 20 '30 4O 5O 60 7O 80 Histochemical Photometer Value (“lo Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for LDH in Soleus Muscle Fibers (Area I) Figure 5 8O ‘79 FREQ. 12 IO CONTROL Gnome: SPRINT - ch Lkl l l l l L] I2] “3- 8- 6.. 4- EADURANCE q 2‘ d 0" Th! I I IIIIVTII UIVV‘IVIV‘ III—V'V‘VT TVUV'UIU I0 20 3O 4O ' 50 Histochemical Photometer Value (“lo Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for PAS in Soleus Muscle Fibers (Area I) Figure 6 81 metabolism is known to depend primarily on glycogen as a substrate (122). The PAS distribution was shifted only slightly to the right with END training. The small change implies a relatively trivial role of glyco- gen as a substrate in prolonged activity. Fat metabolism becomes increasingly important during physical activity of long duration (107,108,164). With END training, approxi- mately ten percent of the soleus muscle fibers demonstrated a high reac— tion to SUD staining (Figure 7). However, a large increase in the number of fibers exhibiting low HCP readings also was evident. The dis— tribution of values for the SPT group was shifted slightly to the left. Pictorial representations of the phenotype changes observed in the soleus muscle are shown in Plate 111. Plantaris Muscle (Area g).--The Chi-square analyses for differ- ences between frequency distributions of HCP readings in the plantaris muscle (Area 2) are summarized in Table 8. All but one of the compari- sons were significant at the .01 level. Nearly all of the plantaris muscle fibers in the CON animals had HCP values between 80 and 90 for ATPase 9.4 (Figure 8). Homogeneity of staining reaction was expected since this muscle area is composed prev dominately of fast contracting fibers. The frequency distributions of the two trained groups were shifted to the left and were widely dis- persed. The general pattern of training effects was the same as that seen in the soleus muscle. Significant increases in SDH staining intensity (Figure 9) were observed in both the SPT and the END groups. A similar pattern was 82 FREQ. CONTROL m l l SPRINT & J 1 10- q 8-I q 6-1 d 4-1 2 I Mk [TIUYIUII ' V'V""‘ 'V‘ """"" ""' v‘rtv‘vv 0 IO 20 30 40 50 Histochemical Photometer Value (% Light Absorbed) ENDURANCE Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for SUD in Soleus Muscle Fibers (Area I) Figure 7 83 PLATE III Representative phenotype changes in the histochemical profile of the soleus muscle (Area 1) following eight weeks of strenuous training. The first column (A, D, G, J and M) contains cross-sections from CON animals. The center column (B, E, H, K and N) contains sections from SPT animals. The last column (C, F, I, L and 0) contains sections from END animals. The sections are not serial. (X 33) A-C: G-I: J-L: M-O: ATPase 9.4 sections. Note the general decreases in staining intensity and the reduced number of dark staining fibers in the trained animals. SDH sections. There is an exercise-related increase in both the SO and the FOG fibers. This is particularly apparent in the END animal. LDH sections. The same general pattern is seen here as with SDH. However, in this case the SPT animal has the highest staining re— action. PAS sections. The SPT animal has a large number of fibers with high staining intensity. Reaction levels in the END animal are similar to those in the CON animal. SUD sections. There are many dark staining fibers in the END animal. PLATE III 85 .Pm>m_ _o. mco ac cemwceeeee pceewwwcowm .4. cm.on _m s_.oo_ Fm sm.ooF Fm «o.u- Ne oom cm.mom oF so.NFF op c_.m_e op np.mmo mm m Hem ozo m> zoo Fem m> zoo mcemwccQEeo ocmEuemce ppecm>o Fcu_emcoeomwz mcemwceQEeo ocmEpemcp emceee AN emc_o me mcewm:e_epm_o zecmeemco ce muomeoo pcmEuemce emcwee ece ppccm>o gem mmszec< mceeemI_co .o mpeee % FREQ. 24- IB-J CONTROL I Ie-I 12: Iol 8a 6.. SPRINT l 4. 2.. 0J I4- 12- IO- 8. 6- ENDURANCE l l 4.. 2... 0.. 86 I I I I I j A A I I I I I I 1 'vav vvvvvvvv vvvv'vvvw vvtvrw—v—vv Irvv'IYIT TV‘rq 50 60 7O 80 90 Histochemical Photometer Value (°/o Light Absorbed) Percent Frequency Distributions, by Treatment .Groups, of Histochemical Photometer Values for ATP 9.4 in Plantaris Muscle Fibers (Area 2) Figure 8 87 9G FREQ. CONTROL N 45 > O '> 'I 1 J SPRHWT b l ENDURANCE ONLO‘ r'U'IITIVI W‘V'YVI’I ViiT‘rm TttI'vvvv rfivlvvvv W vvvvvv vvvvi I 30 4O 50 60 7O 80 90 Histochemical Photometer Value (°/o Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for SDH in Plantaris Muscle Fibers (Area 2) Figure 9 88 found in the soleus muscle. Although the SPT program originally was de- signed specifically to stimulate anaerobic metabolic processes, it is clear that both training programs had large aerobic components. The range of HCP readings for LDH (Figure 10) was greater than for any other stain. This pattern was found in the soleus muscle also and suggests that the LDH reaction is quite variable within fiber types. Since the LDH stain used in this study is not isoenzyme specific, these diverse values were anticipated. M-type and H—type LDH could be expected to respond differently to the specific exercise programs. With SPT training, a general shift to the right was observed. The END running program resulted in a large number of fibers having low LDH reactions. SPT training caused a large increase in PAS staining intensity (Figure 11). This adaptation would indicate an enhanced ability of the fast contracting fibers to store glycogen after a program of strenuous running. END training also produced a shift to the right, but the change was much less pronounced than it was in the SPT animals. Both training programs caused the distribution of HCP readings for SUD to be shifted towards the middle and to be concentrated at the lower end of the continuum (Figure 12). A larger percentage of low SUD values were found in the END group than in the SPT group. Plate IV gives a pictorial representation of the phenotype changes observed in the plantaris muscle. (XMVTROL. SPRNVT ONO Groom EIEXNMANCE 96 FREQ. 89 AA” A A/ T I I I I I W “W MA A I__ I I I I I I I I f MAN/IA [IA/\jxv [IIITIIIII ITI‘TYTI IVIIIWTI Ilitmil UIUVIIUUT IIII‘IIII Ivtr'vltv rvvv'vw-vv Io 20 so 40 so so 70 so _90 Histochemical Photometer Value (% Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for LDH in Plantaris Muscle Fibers (Area 2) Figure 10 9O CONTROL I SPRINT J ENDURANCE ' Io I I N 0 [VIII "Ii'er' IIIIIIII IITVIVVVV VIVf' I0 20 3O Histochemical Photometer Value (% Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for PAS in Plantaris Muscle Fibers (Area 2.) Figure 11 91 CONTROL as I I l4-I I2- SPRINT 01 I 1 I2- ENDURANCE m J lO-i 8-1 4-1 2.. o: ' A 4-I 2.1 "I OJ 'UIII 'IfiF1‘1 I'I'I'UY' IIIrI‘V‘ fivv'vvr Io 20 so 40 so Histochemical Photometer Value (°/o Light Absorbed) Percent Frequency Distributions, by Treatment Groups, of Histochemical Photometer Values for SUD in Plantaris Muscle Fibers (Area 2) Figure 12 92 PLATE IV Representative phenotype changes in the histochemical profile of the plantaris muscle (Area 2) following eight weeks Of strenuous training. The first column (A, D, G, J and M) contains cross-sections from CON animals. The center column (B, E, H, K and N) contains sections from SPT animals. The last column (C, F, I, L and 0) contains sections from END animals. The sections are not serial. (X 33) A—C: ATPase 9.4 sections. A similar pattern to that found in the soleus is seen. Note the general decrease in staining intensity in both the SPT and END animals. D-F: SDH sections. An increased reaction is seen in both 50 and FOG fibers with training. The percentage of FOG fibers may be increased. G-I: LDH sections. Dark staining fibers are increased in the SPT animal and decreased in the END animal. J—L: PAS sections. Many fibers exhibit intermediate to dark reactions in the SPT animal. M-O: SUD sections. A moderate number of dark fibers can be seen in both of the trained animals. PLATE IV 94 Discussion The two muscle areas investigated have markedly different fiber- type populations. The central portion of the soleus muscle (Area 1) is composed primarily of SO fibers with some FOG fibers interspersed. The medial posterior portion of the plantaris muscle (Area 2) consists almost entirely of fast-twitch fibers, and a majority of these are FG. Serial cross-sections revealed typical patterns of relative staining intensities for the fiber types within each muscle area. However, different intensities of reaction to given staining procedures were obtained for the same fiber types in the two areas. Consequently, fiber-type comparisons between areas were not warranted. This observa- tion supports the results of previous investigations (185,278). The SPT and END exercise regimens each produced a number of alter- ations in the histochemical profiles of muscle cells that were training- program specific. In most cases, however, the differences between the SPT and END effects were in the magnitude rather than in the direction of the shifts. Surprisingly, most of the distribution changes were similar in both magnitude and direction in the two muscle areas. Some notable exceptions were observed. The tissue sections from both training groups had large increases in the number of fibers with low ATPase 9.4 values. This adaptation was seen in both the slow soleus and the fast plantaris areas. Since the intensity of histochemical staining at this pH has been shown to parallel speed of contraction and fatiguability, the results suggest an _increase in the number of fibers possessing relatively slow contractile 95 properties and high resistance to fatigue. The decreases in staining intensity could have resulted from an actual transformation of fiber types, a decreased reaction across the muscle cells of one or more fiber types, or some combination of these possibilities. The frequency distributions of the quantitative HCP readings for ATPase 9.4 show that the staining intensities within categorically determined groups of “dark" and "light" fibers are not homogeneous. There are gradations of both "dark" and “light". These gradations be- come very apparent in the graphs of the trained animals where the ranges of values are large. It is possible that an increased range of values reflects a progressive adaptive process in the contractile properties of the muscle.. Recent biochemical (15) and histochemical (208) studies have demonstrated exercise-induced changes in the ATPase activity of specific muscle fiber types. Baldwin gt_al, (15) reported significant changes in the specific activity of actomyosin ATPase in rat skeletal muscle homogenates after 18 weeks of endurance running on a treadmill. Similarly, Muller (208) found a decrease in the percentage of fast- twitch fibers in the soleus muscles of rats run for 12 weeks on a tread- mill. The change in fiber-type composition was attributed to a trans- formation of fast-twitch fibers to slow-twitch fibers. Physiological studies involving motor unit composition and recruitment also support the present findings. Motor units composed of muscle fibers with light ATPase 9.4 staining characteristics have been shown to be fatigue resistant (44,46). These units are involved in 96 maintaining prolonged low levels of physical activity (117). The Ob- served decreases in ATPase 9.4 staining intensities found in this study suggest that both the END and the SPT training programs produced en- hanced capacity for aerobic work. A word of caution is appropriate regarding the evaluation and interpretation of ATPase findings. The ATPase reaction is highly pH sensitive. That sensitivity may have affected the results of the cur- rent study to some unknown degree. The three groups of animals used in the study were all involved in a larger diet-training experiment. The research design of the parent study required the tissues of the COM, SPT and END groups to be processed separately. The potential bias inherent in following such a protocol is obvious. However, the ATPase changes observed in this study were so striking that it appears they must have been due, at least in part, to the training programs. Histochemical and biochemical procedures for demonstrating SDH reactivity are used routinely to indicate the aerobic capacity of individual muscle cells (see Table 2). Several studies using low- intensity exercise as a stimulus have reported increases in the activi- ties of most tricarboxylic acid cycle enzymes (80,100,154,l94,198,297). In this study, SDH staining intensity was enhanced in both muscle areas by both training regimens. This finding is in agreement with the ATPase 9.4 results and suggests that the SPT program has an aerobic component which is at least as great as that of the END program. Metabolic adaptations specific to sprint-type training have recently been reported. Biochemical assays have shown a 70% increase 97 in phosphorylase activity (257), a 35% increase in pyruvate kinase activity (257), and a 17% increase in triosephosphate dehydrogenase activity (272) in soleus muscle homogenates of sprint-trained rats. Corresponding changes were not found in predominately FG, FOG or mixed muscles. In the present study, an increase in the glycogenolytic capacity of the muscle cells of the SPT animals was indicated by an increased number of high PAS and LDH readings. The shifts in distribu- tions were greater in the soleus than in the plantaris. Since FG and FOG fibers have been shown to have higher initial levels of glycogeno- lytic and glycolytic enzymes than do SO fibers (see Tables 2 and 4), these findings are consistent with current knowledge. CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summar This study was undertaken to determine the effects of two strenu- ous training regimens on a histochemical profile of various fiber types. Two muscle areas were selected for study on the basis of homogeneity of fiber type: the central portion of the soleus (a predominately SO area) and the posterior part of the plantaris (an FG-FOG area). Normal male adult rats (Sprague-Dawley strain) were used as subjects. The two train- ing regimens were modifications of Controlled-Running Wheel routines previously reported from this laboratory (286). The modified programs, an endurance running routine (END) and a sprint running routine (SPT), represented attempts to stimulate selectively either aerobic or anaero- bic metabolic processes in the experimental animals. Histochemical profiles were determined using the reactions of ATPase 9.4 as an indi- cator of contractile speed, LDH to reflect lactate fermentation activ- ity, SDH to indicate Krebs cycle activity and, SUD and PAS to localize intracellular fat and glycogen respectively. Forty-two animals were brought into the laboratory and randomly assigned to CON, SPT and END treatment groups. An eight-week treatment period began when the animals were 84 days of age. Selected animals 98 99 were sacrificed 72 to 96 hours after their last training session. Each histochemical stain was evaluated objectively with the use of a Histochemical Photometer (HCP). Photometric evaluations were determined in serial cross-sections for a group of 30 adjacent muscle fibers from each of the two muscle areas investigated. Selection criteria developed for training performance and staining characteristics resulted in a final frequency of nine animals per treatment group. The histochemical data for each stain were plotted by treatment group and muscle area and statistically analyzed for distribution dif- ferences using a Chi-square contingency analysis (ACT routine). All comparisons of frequency distributions by treatments were significant (P <.01) except that for SDH in the plantaris (Area 2). In most cases, the changes caused by training were similar in both magnitude and direction in the two muscle areas investigated. That is, the exercise—induced metabolic adaptations were similar in the S0 soleus and the FG-FOG plantaris areas. The SPT and END exercise regimens each produced a number of alter- ations in the histochemical profiles of the muscle cells. Both train- ing regimens resulted in decreased staining intensities for ATPase 9.4 and increased reactivities to SDH staining. The SPT program specifical- ly enhanced LDH and PAS staining reactions, whereas END training pro- duced a large group of fibers staining darkly with SUD. In effect, the END training program resulted in an increased aerobic capacity of the muscle cells while the SPT program enhanced both their aerobic and anaerobic metabolic capacities. 100 Conclusions The results of this study have led to the following conclusions: . A wide range of staining intensities for histochemically demonstrated metabolic markers can be found within each muscle fiber type. . The SPT and END training programs produced similar increases in the aerobic capacity of muscle cells in the two areas investigated. This adaptation was indicated by the increased staining reactions for SDH. . The contractile properties of the muscle cells involved were altered by both training regimens. This change was reflected by decreased reactivity to the ATPase 9.4 staining procedure. . The SPT training program resulted in specific anaerobic metabolic adaptations as indicated by the enhanced staining reactions to the LDH and PAS techniques. Recommendations . The present study should be repeated with the intersession staining factor eliminated. . In any follow-up study using the SPT program, additional anaerobic metabolic markers should be included. The response of enzymes such as phosphorylase and phosphofructokinase would be helpful in evalu- ating the metabolic adaptations to sprint training. . The specifications of the SPT program should be refined to produce as specific an anaerobic effect as possible. In addition, other high-intensity exercise regimens for animals should be developed. 101 . Correlative morphological, biochemical, histochemical and physio- logical studies are needed for complete muscle evaluations. . Power-type events for animals must be designed to add to the present knowledge of the metabolic adaptations resulting from activities across the endurance continuum. High-jumping and weight-lifting programs should be developed for this purpose. . Circulatory adjustments in skeletal muscle produced by anaerobic training should be investigated. . The metabolic adaptations in animals resulting from exercise should be substantiated in human subjects via muscle biopsy and energy metabolism studies. These results then should be extended to the applied clinical and training areas. . The effects of specific exercise regimens on the rate of flow and the composition of axoplasmic transport materials should be studied. In addition, the entire area of trophic relationships within the neuromuscular unit must be explored in relation to exercise. . 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Differences in histochemical attributes between diaphragm and hindlég muscles of the rat. Anat. Rec. 173:333. 1972. 126 296. Yellin. H. Limitations to the neuroregulation of enzymes in mammalian skeletal muscle. Anat. Rec. 182:479. 1975. 297. Zika,kL, Z. Lojda and M. Kucera. Activities of some oxidative and hydrolytic enzymes in musculus biceps brachii of rats after tonic stress. Histochemie. 35:153. 1973. APPENDICES Table A-1. Modified Eight Week Sprint Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels 127 APPENDIX A TRAINING PROGRAMS Total Ac- Time Time Total celer- Work Repeti- Be- of Total Work Day Day ation Time Rest tions No. tween Run Prog. Exp. Time ‘ of of Time (min: Time per of Bouts Shock Speed (min: Meters (sec) Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts (min) (ma) (m/min) sec) TEM TWT 0 4-T -2 3.0 40:00 10 l 1 5.0 0.0 27 40:00 --- --- 5=F -1 3.0 40:00 10 l l 5.0 0.0 27 40:00 --- --- 1 1=M 1 2.0 00:10 10 10 8 2.5 1.2 36 42:50 480 800 2=T 2 2.0 00:10 10 10 8 2.5 1.2 36 42:50 480 800 3=W 3 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 4=T 4 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 5=F 5 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 2 l=M 6 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 2=T 7 1.5 00:10 15 10 8 2.5 1.2 54 49:50 720 800 3=W 8 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 4=T 9 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 5=F 10 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 3 1=M 11 1.5 00:15 30 6 7 2.5 1.2 72 43:00 756 630 2=T 12 1.5 00:15 30 6 6 2.5 1.2 81 36:30 729 540 3=W 13 1.5 00:15 30 6 6 2.5 1.2 81 36:30 729 540 4=T 14 1.5 00:15 30 6 6 2.5 1.2 81 36:30 729 540 5=F 15 1.5 00:15 30 6 6 2.5 1.2 81 36:30 729 540 4 1=M 16 1.5 00:15 30 6 6 2.5 1.2 81 36:30 729 540 2=T 17 2.0 00:15 30 5 6 2.5 1.2 90 32:00 675 450 3=W 18 2.0 00:15 30 5 6 2.5 1.2 90 32:00 675 450 4=T 19 2.0 00:15 30 5 6 2.5 1.2 90 32:00 675 450 5=F 20 2.0 00:15 30 5 6 2.5 1.2 90 32:00 675 450 5 1=M 21 2.0 00:15 30 5 6 2.5 1.2 90 32:00 675 450 2=T 22 2.0 00:15 30 5 6 2.5 1.2 99 32:00 743 450 3=W 23 2.0 00:15 30 5 6 2.5 1.2 99 32:00 743 450 4=T 24 2.0 00:15 30 5 6 2.5 1.2 99 32:00 743 450 5=F 25 2.0 00:15 30 5 6 2.5 1.2 99 32:00 743 450 6 l=M 26 2.0 00:15 30 5 6 2.5 1.2 99 32:00 743 450 2=T 27 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 3=W 28 2.0 00:15 30 S 6 2.5 1.2 108 32:00 810 450 4=T 29 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 5=F 30 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 7 1=M 31 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 2=T 32 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 3=W 33 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 4=T 34 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 5=F 35 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 8 1=M 36 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 2=T 37 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 3=W 38 2.0 00:15 30 S 6 2.5 1.2 108 32:00 810 450 4=T 39 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 5=F 40 2.0 00:15 30 5 6 2.5 1.2 108 32:00 810 450 This training program is a modified version of a standard Sprague-Dawley strain (150,278). program designed using male rats of the All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the program. Failure to provide this adjustment period will impose a double learning situation on the animals and will seriously impair the effectiveness of the training program. 128 APPENDIX A--cantinued Table A-2. Modified Eight Week Endurance Training Program for Postpubertal and Adult Male Rats in Controlled-Running Wheels Total Ac- No. Par- Time Time Total celer- Work Repeti- of tial Be- of Total Work Day Day ation Time Rest tions Com- Bouts tween Run Prog. Exp. Time of of Time (min: Time per plate (min: Bouts Shock Speed (min: Meters (sec) ‘Wk. Wk. Tr. (sec) sec) (sec) Bout Bouts sec) (min) (ma) (m/min) sec) TEM TWT 0 4=T -2 3.0 40:00 10 1 5.0 0.0 27 40:00 -- --- 5=F -1 3.0 40:00 10 1 5.0 0.0 27 40:00 --- --- l 1=M 1 2.0 02:30 0 1 6 2.5 1.2 27 27:30 405 900 2=T 2 2.0 02:30 0 1 6 2.5 1.2 27 27:30 405 900 3=W 3 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 4=T 4 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 5=F 5 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 2 1=M 6 1.5 05:00 0 1 3 5.0 1.2 36 25:00 540 900 2=T 7 1.0 07:30 O l 2 5.0 1.2 36 20:00 540 900 3=W 8 1.0 07:30 0 1 2 2.5 1.2 36 17:30 540 900 4=T 9 1.0 07:30 0 1 2 1.0 1.2 36 16:00 540 900 5=F 10 1.0 15:00 O 1 l 0.0 1.2 36 15:00 540 900 3 1=M 11 1.0 15:00 0 1 1 05:00 1.0 1.2 36 21:00 720 1200 2=T 12 1.0 15:00 0 1 1 07:30 1.0 1.0 36 23:30 810 1350 3=H 13 1.0 15:00 0 1 1 10:00 1.0 1.0 36 26:00 900 1500 4=T 14 1.0 15:00 0 1 1 12:30 1.0 1.0 36 28:30 990 1650 5=F 15 1.0 15:00 0 1 2 1.0 1.0 36 31:00 1080 1800 4 1=M 16 1.0 15:00 0 l 2 05:00 1.0 1.0 36 37100 1260 2100 2=T 17 1.0 15:00 0 l 2 07:30 1.0 1.0 36 39130 1350 2250 3=W 18 1.0 15:00 0 1 2 10:00 1.0 1.0 36 42100 1440 2400 4=T 19 1.0 15:00 0 1 2 12:30 1.0 1.0 36 44:30 1530 2550 5=F 20 1.0 15:00 0 1 3 1.0 1.0 36 47:00 1620 2700 5 1=M 21 1.0 15:00 0 1 3 05:00 1.0 1.0 36 52:00 1800 3000 2=T 22 1.0 15:00 O l 3 07:30 1.0 1.0 36 54:30 1890 3150 3=W 23 1.0 15:00 0 l 3 10:00 1.0 1.0 36 57100 1980 3300 4=T 24 1.0 15:00 0 1 3 12:30 1.0 1.0 36 59:30 2070 3450 5=F 25 1.0 15:00 0 l 4 1.0 1.0 36 63:00 2160 3600 6 1=M 26 1.0 15:00 0 1 4 1.0 1.0 36 64:00 2160 3600 2=T 27 1.0 30:00 0 1 2 5.0 1.0 36 65:00 2160 3600 3=W 28 1.0 30:00 0 1 2 2.5 1.0 36 62:30 2160 3600 4=T 29 1.0 30:00 0 1 2 1.0 1.0 36 61:00 2160 3600 5=F 30 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 7 1=M 31 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 2=T 32 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 3=W 33 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 4=T 34 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 5=F 35 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 8 13M 36 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 2=T 37 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 3= 38 1.0 60:00 0 l 1 0.0 1.0 36 60:00 2160 3600 4=T 39 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 5=F 40 1.0 60:00 0 1 1 0.0 1.0 36 60:00 2160 3600 This training program is a modified version of a standard program designed using male rats of the Sprague-Dawley strain (150,278). , All animals should be exposed to a minimum of one week of voluntary running in a wheel prior to the start of the program. Failure to provide this adjustment period will impose a double learning situation in the animals and will seriously impair the effectiveness of the training program. 129 APPENDIX B BASIC STATISTICS FOR TRAINING DATA Table B-1. Basic Statistics for Percentage of Body Weight Loss, Environmental Factors and Performance Criteria for the Sprint Running Group Simple Correlations Percent Body a Standard Air Percent Bar. Weight Variable N Mean Deviation Temp. Humidity Press. Loss PEM Air Temp. (°F.) 340 73.1 4.6 Percent Humidity 340 38.6 12.3 .122 Bar. Press. (mm Hg) 340 740.7 4.2 - 255 -.717 Percent Body WgtLoss 340 1.7 .6 -.041 -.l82 .029 PEM 340 66.2 25.1 -.197 —.477 .266 .100 PSF 340 66.5 22.3 -.287 -.339 .155 .038 .840 aTotal training days for all animals. Table B-2. Basic Statistics for Percentage of Body Weight Loss, Environmental Factors and Performance Criteria for the Endurance Running Group Simple Correlation Percent Body a Standard Air Percent Bar. Weight Variable N Mean Deviation Temp. Humidity Press. Loss PEM Air Temp. (°F.) 314 73.9 4.0 Percent Humidity 314 47.1 10.6 .147 Bar. Press (mm Hg) 314 739.5 3.8 -.290 -.679 Percent Body Wgt Loss 314 2.7 1.0 .455 .131 -.l73 PEM 314 81.3 19.3 -.258 -.263 .323 -.060 PSF 314 68.4 19.4 -.282 -.120 .174 .759 .759 aTotal training days for all animals.