PLACE IN RETURN Box to remove this checkout from your record. TO AVOID FINES return on or before date due. MTE DUE DATE DUE DATE DUE ABSTRACT HISTOCHEMICAL CHANGES IN RAT SKELETAL MUSCLE AFTER EXERCISE by V. Reggie Edgerton It has been clearly demonstrated that skeletal muscle fiber types can be reversed in such pathologic conditions as cross-innervation, reinnervation, tenotomy, and various myOpathies as well as in fetal maturation. There have been numerous reports suggesting a high correlation between the quantity and quality of mitochondria in an organ and the functional activity of that organ. it is known that oxidative as well as other enzymes are closely associated with the mitochondrial membranes. It has been found also that in severe physical training programs altera- tions in enzyme activity of skeletal muscle fibers will occur. Since the number and size of mitochondria and enzyme activity levels are characteristics which aid in typing muscle fibers, it seemed reasonable to suspect that the proportion of red and white fibers and/or metabolism of fibers should be related to the nature of the work load placed on a given muscle. Based on these assumptions this study was undertaken. V. Reggie Edgerton The left soleus, plantaris and gastrocnemius muscles of adult male white rats were studied histochemically using myosin adenosine triphosphatase (myosin ATPase), mitochond- rial a-glycerOphOSphate dehydrogenase (mito a-GPD), malate dehydrogenase (MDH), succinate dehydrogenase (SDH), reduced nicotinamide adenine dinucleotide diaphorase (NADH-D), and a modified trichrome stain. Twenty control animals were kept in sedentary cages. One group of twenty rats, housed in sedentary cages, were subjected to one 30—minute swimming period a day, six days a week for fifty-two days. A third group swam for two 30-minute periods per day for the same duration and was housed in cages equipped with voluntary activity wheels. During each swimming period a weight equalling three and four percent of the body weight was attached to the tip of the tail of the second and third group respectively. Approximately 350 muscle fibers of the soleus of each animal were rated as light or dark according to the intensity of myosin ATPase staining. Intensity of MDH and mito d-GPD staining demonstrated fibers rated as medium or dark. No white fibers were demonstrable in the rat soleus with MDH or d-GPD. With MDH and mito a-GPD the staining intensity of approximately 350 muscle fibers from each animal were rated as light, medium, or dark signifying white, intermediate, and red muscle fibers respectively in the plantaris. These V. Reggie Edgerton ratings were made without knowledge of the treatment groups from which the animal came. It can be concluded that an exercise program of low resistance and highly repetitive activity such as was used in this study will not alter the preportion of inter- mediate and red fibers of the soleus as demonstrated by myosin ATPase, MDH or mito a-GPD. No changes were found in the plantaris with myosin ATPase. However, the experimental treatments did result in an increase in the prOportion of red muscle fibers in the plantaris as demonstrated by mito d-GPD, MDH, SDH, NADH-D and a modified trichrome stain. This study supplies further evidence for the hypo- thesis that the nature of the metabolism of an individual muscle fiber is related to the nature of the work load placed on that muscle fiber. In view of recent investiga- tions of motor units, the results of this study are partially supported by the findings in an accompanying study on the anterior horn cells in which the anterior horn cells of the heavily exercised animals generally had higher enzyme activity than the controls. This study supplies more direct evidence supporting the hypothesis that muscle fiber types are mutable in a non pathologic state. HISTOCHEMICAL CHANGES IN RAT SKELETAL MUSCLE AFTER EXERCISE BY 45“ VI Reggie Edgerton A THESIS 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 1968 9 Copyright by V. REGGIE EDGERTON ‘ 1969 DEDICATION To Mother, Dad, Alma, Buggy, Jack and my wife, Lois ACKNOWLEDGMENTS My gratitude goes to Dr. Rex Carrow for the many suggestions, motivation and guidance which made this study possible; and to Drs. W. D. Van Huss and W. W. Heusner for their encouragement, interest and support. A thank you is in order to Bob Echt and LeRoy Gerchman for an intellectually stimulating working relationship. A debt of gratitude is due Trudy Van Huss for tolerating a demanding work pace during sacrifice procedures and Barbara Wheaton for her unending laboratory assistance throughout this study. My thanks also go to Dr. R. A. Fennell for catalyzing my histochemical interests as well as for his generous use of equipment and supplies. Dr. L. Guth and H. Yellin are due a word of appre- ciation for their scholarly Opinions, suggestions and interests. A word of appreciation goes to Dr. W. E. Cooper for use of his desk computer as well as his statistical suggestions. iv TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . LIST OF LIST OF Chapter I. II. III. TABLES O O O O O O O O O O O FIGURES O O O O O O O O I O 0 THE PROBLEM . . . . . . . . . Introduction . . . . . . Statement of the Problem . Importance of the Problem . The Meaning of a Muscle Fiber Interpretation and Limitations of chemistry in Skeletal Muscle Limitations of the Study . REVIEW OF RELATED LITERATURE Introduction . . . . . Morphology of Muscle Fibers Mechanical Characteristics of Fibers . . . . . . . Metabolism of Muscle Fibers Motor Units and Fiber Types Maturation of Muscle Fibers Response of Muscle Fibers in P Conditions . . . . . . . Atrophy O O O I O O O I O Tenotomy . . . . . . . . Type a Mu m o 0 th Denervation and Cross-Innervation My0pathies . . . . . . . Responses of Muscle Fibers to Low Resistent-Highly Repetitive ActiVity O O O O O O 0 RESEARCH METHOD . . . . . . . sample 0 O O O O I O O O O oooLQoooo Page iv vii ix ll 12 21 26 28 28 3O 35 37 46 46 Page Treatment Groups . . . . . . . . . . . . . 46 Sedentary-Control Group . . . . . . . . 46 Sedentary-Forced Group . . . . . . . . . 47 Voluntary-Forced Group . . . . . . . . . 47 Treatment Procedures . . . . . . . . . . . 47 Sacrifice Procedures . . . . . . . . . . . 49 Histochemical Procedures . . . . . . . . . 50 Methods of Tissue Analysis . . . . . . . . 51 Subjective Evaluation of a Large Num er of Individual Muscle Fibers . . . . . 51 Overall Subjective Evaluation of Tissue Sections . . . . . . . . . . . . . . . 52 Determination of Met bolic Patterns in Muscle Fibers . . . . . . . . . . . . 53 Statistical Methods . . . . . . . . . . 53 IV RESULTS AND DISCUSSIONS . . . . . . . . . . 56 Results 0 O O O O O O O O O O O O O O O O 56 SOleus O O O I O O I O O O O O O O O O C 56 Plantaris O O O O O O O O O O O O O O O 59 Discussion . . . . . . . . . . . . . . 70 Enzyme Patterns in the Plantaris and Gastrocnemius . . . . . . . . . . . . 78 Enzyme Patterns in the Soleus . . . Significance of Results in Light of Interdependence of Nerve and Muscle . 83 Significance of Results in Light of Being an Adaptive Mechanism to ExerCise O O O O O O O O O O O O O O O 88 V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS . . 92 sumary O O O I O O O O O O O O O O O O O 92 Conclusions . . . . . . . . . . . . . . . 94 Recommendations . . . . . . . . . . . . . 95 BIBLIOGRAPHY O I O O O O O O O O O O O O O O O O O O 107 APPENDICES . . . . . . . . . . . . . . . . . . . . . 122 vi Table 10 LIST OF TABLES Mean Percentages and Analysis of Muscle Fiber Types of the Soleus (Area I) as Determined by Myosin ATPase, Mito a-GPD and MDH . . . . . . . . . . . . . . . . . Chi—square Test for Treatment Effects on Overall Staining Intensity (MDH) of the SOleuS O O O O O O O O O O O O O O O Chi-square Test for Treatment Effects on Overall Staining Intensity (SDH) of the SOleus O O O O I I O O O I O O O I 0 Mean Percentages of White, Intermediate and Red Muscle Fibers from Areas II and III of the Plantaris . . . . . . . . . . . . Analysis of Mean Percentages of White, In- termediate and Red Muscle Fibers from Areas II and III of the Plantaris . . . . Chi-square Test for Treatment Effects on the Overall Number of Red Fibers (MDH) in Area II of the Plantaris . . . . . . . Chi-square Test for Treatment Effects on the Overall Number of Red Muscle Fibers (SDH) in Area II of the Plantaris . . . . Chi-square Test for Treatment Effects on the Overall Number of Red Muscle Fibers (Trichrome) in Area II of the Plantaris . Chi-square Test for Treatment Effects on the Overall Number of Red Muscle Fibers (MDH) in Area III of the Plantaris . . . . . . . . . . . . . . . . Mean Muscle Weights (Triceps Surae and Plantaris) and Body Weights (grams) and Percent Body Weights of the Three Treatment Groups . . . . . . . . . . . . vii Page 57 58 58 60 61 64 64 64 65 66 Table Page 11 Correlations of Muscle Weight to Body 6 weight 0 O O O O O O O I O O O I O O O I O 6 12 Consistency of Typing Muscle Fibers in Area III of the Plantaris . . ._. . . . . 68 13 Consistency of Selecting Similar Muscle Fiber POpulations and Typing Specific Muscle Fibers Similarly on Succeeding Trials in Area III of the Plantaris . . . 69 14 Fiber Type Patterns of the Plantaris and Gastrocnemius . . . . . . . . . . . . . . 80 81 15 Fiber Type Patterns of the Soleus . . . . . viii Figure 10 11 12 13 14 LIST OF FIGURES Diagram of Areas of Muscles Analyzed in Detail 0 O I O O O O O I I O O O I O O O 0 Variation in Concentration of Types of Fibers in the Medial Head of Gastro- cnemius (MDH) O O I O O O O O O O I O O O Enlarged Muscle Fibers from a Rat Drowned While SWimIning (NADH-D) o o o o o o o o o Enlarged Muscle Fibers from a Rat Drowned While Swimming (NADH-D) . . . . . . . . . Enlarged Muscle Fibers Demonstrated with MYOSin ATPase 0 O O O O O O O O O O O O 0 Muscle Fibers of Normal Size Demonstrated with Myosin ATPase . . . . . . . . . . . . Unusually Small Muscle Fibers of the Soleus of Exercised Rats (MDH) . . . . . . . . . Unusually Small Muscle Fibers of the Soleus of Exercised Rats (Trichrome) . . . . . . Demonstration of Fiber Types at a Higher Magnification . . . . . . . . . . . . . . Area II of the Plantaris of a Sedentary Rat (MDH) o o o o o o o o o o o o o o o 0 Area II of the Plantaris of an Exercised Rat (MDH) O I O I O O O O O O O O O I 0 0 Enzyme Patterns of the Plantaris With MYOSin ATPase 0 O O O O I O O I O O I O 0 Enzyme Patterns of the Plantaris with MDH . Enzyme Patterns of the Plantaris with Mito a-GPD o o o o o o o o o o o o o o o o o 0 ix Page 98 98 98 98 98 98 100 100 100 100 100 102 102 102 15 16 17 18 19 20 21 22 23 24 25 26 Enzyme Patterns of the Plantaris with MDH O I O O O I 0 I O O O O O O O 0 Enzyme Patterns of the Plantaris with NADH-D o o o o o o o o o o o o o o Myosin ATPase of the Soleus . . . . . MDH of the Soleus . . . . . . . . . . Mito a-GPD of the Soleus . . . . . . SDH of the Soleus . . . . . . . . . . NADH-D of the Soleus . . . . . . . . Enzyme Pattern of the M.Gastrocnemius (Myosin ATPase) . . . . . . . . . . Enzyme Pattern of the M.Gastrocnemius (MDH) o o o o o o o o o o o o o o 0 Enzyme Pattern of the M.Gastrocnemius (Mito G-GPD) o o o o o o o o o o 0 Enzyme Pattern of the M.Gastrocnemius (SDH) O O O O O O O O O O O O I O I Enzyme Pattern of the M.Gastrocnemius (Trichrome) . . . . . . . . . . . . Page 102 102 104 104 104 104 104 106 106 106 106 106 LIST OF APPENDICES Appendix Page A Analysis of Variance Tables . . . . . . . . . 122 B Raw Data of Fiber Types . . . . . . . . . . . 126 C Reliability Data for Plantaris LArea III) . . 139 D Raw Data of Body Weights and Muscle Weight. . 141 E Abbreviations . . . . . . . . . . . . . . . . 142 xi CHAPTER I THE PROBLEM Introduction There is an increasing awareness of the intra- cellular effects of exercise on skeletal muscle (97, 141, 76). This awareness is the result of investigators attempting to define adaptations which occur in chronically exercised animals (97, 141, 76). Exercise is also being used more frequently in basic research studying the rela- tive importance of various metabolic pathways in different muscle fiber types (8, 74, 75, 141, 172). For this purpose the histochemical and histological techniques employed in this investigation are especially approPriate for studying the chronic adaptations of muscle fibers to exercise. Statement of the Problem The purpose of this study was to determine the effects of two levels of physical activity on the histo- chemical and histological characteristics of the plantaris, soleus, and gastrocnemius musCle fibers of male white rats. Importance of the Problem The results of this investigation should provide additional insight into the functional significance of muscle fiber types and the effects of an extended swimming regimen on muscle fiber types. Swimming has been a common technique of exercising white rats (25, 70, 97, 158) primarily for lack of a more controlled method. This mode of exercise has been critic- ized as being too moderate an overload to induce enzyme changes in skeletal muscle and other tissue (97). Such a statement should not be made without examining.the proce- dures which investigators have employed to determine enzyme activity. Homogenates of skeletal muscle are used for enzyme studies (90, 104). However, it must be realized that skeletal muscle fibers are not homogeneous. Muscle fibers vary in functional and structural characteristics and the proportion of muscle fibers having a given charact- eristic vary intramuscularly as well as intermuscularly. Thus homogenate studies are suspect for such purposes. It seems mandatory that methods be employed which can localize enzymes within muscle fibers and which can be quantified to some degree. By the use of such methods, one is able to evade the invalid assumption that the enzyme activity of all muscle fibers are affected equally and similarly. Small overall enzyme changes could take place and appear insignificant unless fibers are assayed individually. A small, apparently insignificant, and generalized change may actually be large, significant and localized if fibers are observed individually rather than collectively. In view of the findings which convincingly illustrate the neural influences on skeletal muscle fiber kinetics and metabolism (4, 21, 22, 83, 173), it was hOped that this in- vestigation would shed more light on (a) the trophic inter- dependence of the nerve and skeletal muscle, (b) the significance of muscle fiber types, and (c) the mutability of types of muscle fibers in the process of adaptation to a chronic exercise program. The Meaning of a Muscle FiBer Type It is necessary to clarify the usage of the term, fiber types, in order for the reader to correctly interpret this author. The term, fiber type, is used in this paper for the sake of convenience, not accuracy. Observations made in this study and statements by other authors such as Romanul (141) leads this author to conclude that there are not two fiber types, three, or even eight fiber types, but there are a large number of fibers with varying degrees of kinetic and metabolic characteristics. When studying the metabolism of muscle fibers one should think in terms of a continuous spectrum which includes red muscle fibers on one end of the spectrum, perhaps intermediate fibers in the middle and white fibers on the Opposite end of this Spectrum. It cannot be assumed that fibers which were rated as intermediate in enzyme activity are intermediate in kinetic characteristics. Close (30) has reported that 27 out of 30 motor units of the soleus in the rat are slow kinetically and three of 30 are intermediate. This is the reverse of what is apparently found histochemically in the soleus. The fact that it is some of the intermediate fibers of the gastrocnemius and plantaris that show the least mito a-GPD and ATPase activity also suggest that something is perhaps peculiar about these fiber types. Further study of the kinetic pr0perties of intermediate fibers is needed to test this possibility. Since mitochondria can migrate intracellularly, although extremely limited in skeletal muscle (111) it would theoretically be possible for a fiber to accumulate mitochondrial aggregates peripherally as the biogenesis of these organelles occurred. In a subsequent phase of metabolic adaptation the peripheral mitochondria could perhaps become more evenly distributed intracellularly. Such a change in a fiber would be identified histochemically as a conversion of a red to an intermediate fiber. This would not be in keeping with the currently held concept that the red fiber would be at one end of the spectrum as men— tioned above, but would be intermediate in nature. Such a hypothesis is purely speculative, but could morphologically and metabolically account for the kinetic data of the soleus cited above. The division of muscle fibers into types is strictly arbitrary, adOpted solely for convenience. Such a phenomenon is suggested by the results of this study since there appears to be a shift of the fiber frequency toward the red end of the Spectrum. Thus, when a muscle fiber is said to be "redder" than another it is meant that this fiber would be rated nearer the red end of the fiber spectrum than the white. "Red" should be interpreted as a combination of metabolic, kinetic and structural characteristics, and not confined to color. The author also suggests that oxidative enzymes are the most reliable criteria used in this study for typing muscle fibers. Myosin ATPase perhaps yields a simple staining pattern, but the functional significance of the patterns are not well understood. By using the oxidative enzymes such as NADH-D, MDH, and SDH for typing fibers, it is likely that the number and size of mitochondria as well as the density of mitochondrial cristae are the critical criteria for fiber typing since these enzymes are closely associated with the mitochondrial membranes. Each of the dehydrogenase enzymes except mito d-GPD give identical patterns of enzyme activity, but not necessarily identical intensities. It should also be realized that this study involves only some of the histochemical characteristics and none of the quantitative chemical and kinetic characteristics that are associated with the various fiber types. However, the overall correlation of these various parameters is substan- tial in mammals. A brief summary of these characteristics has been reported (134). Interpretation and Limitations of Histochemistry in Skeletal Muscle Dehydrogenase enzyme activity is demonstrated by the reduction of a soluble tetrazolium salt (NBT) to a colored insoluble diformazan granule. The pattern of the formazan granules in the muscle fibers represents enzyme activity. Unreduced tetrazolium salts show a selective binding to the sarc0plasmic reticulum and mitochondria of skeletal muscle (18). The intermyofibrillar region stains clearly with NBT (18). This region is said to contain the aqueous com- ponent of the sarcoplasm and the membranous components (the sarc0plasmic reticulum and the mitochondria) as well as lipid droplets (18). NBT binds to a lipoprotein which mitochondrial membranes and sarc0p1asmic reticula include as part of their structure. The intermyofibrillar network of muscle fibers is illustrated in Fig. 10, 11 and 23. The red component of the modified trichrome stain used in this study has previously been reported to show a remarkable similarity with NBT staining as well as with sudan black B (54). Thus types of fibers can be identified with the trichrome stain as was done in some analyses of this study (Fig. 9 and 26). Brooke and Engel (18) con- cluded that considerable caution must be used in the inter- pretation of the staining patterns in the techniques designed to demonstrate the oxidative enzymes using NBT, iodonitrotetrazolium (INT) and tetranitro blue tetrazolium (TNBT) . Limitations of the Study L. 1. Exercise programs which involve swimming white rats are inherently limited by lack of control of the in- tensity of muscular activity. Only the duration of the exercise can be controlled. 2. The results presented herein for rats swimming thirty-minute bouts cannot be extrapolated to other forms of muscular exercise such as high-resistant, low-repetition programs. 3. Physiological and quantitative chemical para- meters collected simultaneously with the histochemical data would have given a more complete picture of the effects of the swimming program on the soleus, plantaris and gastro- cnemius muscles. 4. The methods employed for determining enzyme activity levels are quantitatively limited. CHAPTER II REVIEW OF RELATED LITERATURE Introduction The functional significance of fiber types in skeletal muscle has been studied for a number of years. However, many fundamental questions are yet to be answered. With a growing trend for investigators to work as teams and coordinate their ideas and skills in seeking answers to basic biological questions much progress has been made. If tissue samples of a muscle are studied histochemically, morphologically, physiologically, and by quantitative techniques simultaneously, a more complete picture of the functional capacity of this tissue can be determined. By attacking the basic questions related to the functional significance of fiber types in this manner, the mechanical, morphological and chemical characteristics of red and white muscles could be more thoroughly under- stood. Therefore, it is felt that the significance of this study will be better appreciated if the reader understands the background of work that has been accomplished in the various disciplines. In light of recent findings related to fiber types, the question is no longer whether muscle fiber types are mutable, but rather what conditions are necessary in order for muscle fiber types to be induced to change. A number of unnatural stimuli have been used in order to stimulate these changes in mature animals (1, 22, 163). A question which naturally arises is, will the pr0portion of red and white muscle fiber types vary also with the functional load placed on them. This question can be approached more intelligently by considering the results of previous studies which have attempted to shed light on the functional significance of fiber types. Morphology of Muscle Fibers Skeletal muscle fibers can be categorized into different fiber types morphologically. Various degrees of redness and paleness of different skeletal muscles have been a common observation for years, although the meaning of this was not recognized for some time (35, 36). On the cellular level, one can find a number of structural differences. Generally, the red muscle fibers have a smaller diameter than the white fibers. White muscle contains a relatively greater amount of connective tissues than red (10). The quantity and quality of mitochondria is one of the most critical morphologic differences between 10 types of fibers. Mitochondria are more numerous and larger in red muscle fibers than in white (65, 66, 157). Red muscle fiber mitochondria even have a more elaborate cris- tae system and may contain more intramitochondrial granules (111). Subsarcolemmal aggregations of mitochondria can readily be seen in red fibers. This is not the case with white fibers (65, 66). The extensiveness of the sarc0plasmic reticulum in different fibers also has been investigated. A difference apparently exists in that there is found a more elaborate sarc0plasmic reticulum in the white fibers (135). White fibers are the faster contracting fibers and the reticulum is thought to aid in conduction of impulses throughout the muscle fibers. Evidence to support this is convincing. Capillarization has been recognized since the late 1800's to be greater in "redder" muscles. However, it was not until 1965 that a reasonable hypothesis was preposed which specifically (92, 124, 142) related the number of capillaries associated with a muscle fiber to the metabol- ism of that muscle fiber. It has been shown that red fibers have a considerably greater number of capillaries, and that if the fiber type is changed by cross;innervation the capillarization changes prOportionately (144). Z-band thickness (66) and size of neuromuscular junctions (131) are examples of other morphologic differences 11 in red and white muscle which suggests functional differences of muscle fiber types. Mechanical Characteristics of Muscle Fibers Physiologists have been able to relate some of the morphological characteristics previously discussed with mechanical features of muscular contraction. Red muscle has greater contraction and one-half relaxation times than white muscle. Contraction time is commonly measured by determining the time elapse from initiation of contraction to the peak of contraction. One- half relaxation time is the time elapse from the peak of the contraction to one-half of the peak contraction. An explanation for the variation in speeds of con- traction probably 1ies in the sarc0p1asmic reticulum of red and white fibers. Calcium binding in red muscle is approx- imately one-third that of white muscle according to Harigaya et a1. (88). Loss and sequestration of calcium by the sarc0p1asmic reticulum are events closely associated with muscular contraction and relaxation. Harigaya et al. in 1968 found the yield of microsomes from red mhscle fibers to be one-half that of white muscle. This is in agreement with the electron microscopic results of Pelligrino and Franzini (135), but at odds with Shafiq et al. (149). Red muscle fibers are able to sustain a given ten- sion for a longer duration than white fibers. A lower 12 stimulation frequency is required in red muscle in order to produce tetany. It was from these findings that the theory arose that red fibers are tonic in nature and their function is to maintain posture. On the other hand, the white fibers are phasic in nature and are better adapted to quick forceful intermittent type contractions. Metabolism of Muscle Fibers The observation of variations in the metabolism of different muscle fibers is perhaps the most significant single factor in the interpretation of the true meaning of fiber types. Recognition of this characteristic has enabled investigators to categorize fibers into a number of types histochemically. Glycogen content in white fibers is found to be higher than in red fibers (8). Correspondingly phosphory- 1ase is also found to be higher in white fibers. Uridine diphosphoglucose (UDPG) synthetase, which catalyzes the synthesis of glycogen from glucose, has been found to be lower in white than red fibers (17, 154). Beatty et a1. has found that following two hours of incubation glycogen is depleted faster in white muscle and that lactate produc- tion is greater in white fibers (8). Alpha-GPD has been shown to be higher in white- muscle, both quantitatively and by histochemical techniques, than in red (12, 159). Mito a-GPD has been pr0posed to be 13 important as a shuttle mechanism (111, 115) in muscle by resupplying NAD. Therefore, it may be that this mechanism is more important in white muscle because it can help to maintain anaerobic glycolysis in white fibers. Aldolase has been demonstrated to be higher in broad fibers (white) by George (67). In general, glycolysis in white fibers is thought to be the predominant metabolic pathway which apparently is adequate to maintain the energy supply for this type of fiber. It has been demonstrated that a red muscle such as the soleus is dominated by an aerobic metabolism relying heavily on the citric acid cycle as a means of producing energy (39, 126, 127, 128). Enzymes which function in this pathway are found to have greater activity in red muscle fibers than white muscle fibers. For example, the soleus has lower phosphorylase but higher oxidative enzyme activity than a whiter muscle such as the lateral head of the gastrocnemius. Several authors have quantitatively shown that by comparison with white, the red fibers have relatively lower endogenous glycogen utilization (8, 38) and anaerobic glycolysis (8, 38, 128) as well as lower activities of LDH, NAD-linked d-GPD (12). Red muscle has been shown to have higher pyruvate oxidation (40), aceto— acetate uptake (8), acetoacetate conversion to B-hydroxy- butyrate (B-OHB), succinate oxidation (128) and oxygen consumption (8). 14 Bocek et a1. (16) have reported glucose uptake to be higher in red muscle than white. They also reported that synthesis of glucose into glycogen and breakdown into CO2 was higher and that the breakdown into lactate and pyruvate was lower in red than white muscle. Labelled glucose uptake into lactate and pyruvate is approximately one-half as great in red muscle as in white. Bocek et a1. (15) reported that a higher percent incorporation of label from glucose Cl4 into CO2 in red muscle can be accounted for by the higher specific activity of lactate and pyruvate presented to the citric acid cycle and cannot necessarily be interpreted as indicating a higher citric acid cycle activity in red than in white muscles. Bocek et al. reported a higher activity of UDPG- synthetase in red than white muscle (17). Consequently, they suggested that, glycogenolysis and glycolysis is greater in the red muscle than white. Incorporation of glucose Cl4 into glycogen with incubation also indicates a higher level of glycogenesis in red muscle (16). Bocek et al. (15), in a study of glycogen metabolism in red and white muscle, found that conversion of glucose to glycogen is more active in red than white muscle. It has been prOposed that the species of glycogen may vary in size since a higher amylo-(l,4 + 1,6) transglycosylase activity has been reported in red muscle fibers (15, 141). Bocek et a1. (15) suggested that this could explain a greater 15 glucose to glycogen activity in red muscle if branching is a limiting factor in glycogen synthesis of this type of muscle. The greater rate of conversion of glucose to gly— cogen in red fibers agrees with the histochemical findings of Hess and Pearse (93), Engel (53) and Saski and Takeuchi (146) in that UDPG-glycogen transferase has greater activ- ity in red muscle fiber as compared to white. Bocek et al. (15) also confirmed these findings in her laboratory using rats and rhesus monkeys. Bocek et al. (15) suggested that the greater synthesis of glucose into glycogen in red muscle may be due to a difference in hexose-phosphate pool sizes. Peter et al. (136) found greater hexokinase in red muscle than in white muscle in the guinea pig. This was interpreted as an indication of greater conversion of glu— cose to glycogen (136). This could also indicate that glycolysis is more active in red muscle than white. This finding is especially important since hexokinase is one of the pacemaker enzymes for glycolysis. Fahimi and Karnovsky (57) found glyceraldehyde-B- phosphate dehydrogenase (GAPD) and LDH to have similar activity in red and white fibers. Glucose and oxygen uptake have been reported to be higher in red muscle (8). However, the percent oxygen up- take by carbohydrate oxidation appeared similar for the different fiber types. 16 The presence of lactate dehydrogenase (LDH) isozymes is another metabolic characteristic of skeletal muscle which differentiates fiber types (33). Blanchaer et al. (11) reported Type I and II LDH isozymes in red muscle and Type IV and V LDH isozymes and sometimes Type III in white muscle. LDH I and II were found to be most active in tissue such as the heart, brain and kidney which have vigorous aerobic metabolism. LDH IV and V predominate in skeletal muscle and other tissue capable of anaerobic metabolism (64). There definitely are species differences in this isozyme pattern, but LDH IV and V account for most of the LDH activity in humans (45). Blanchaer et a1. (11) said that the activity of LDH I and II in human muscle, which is a mixture of red and white fibers, depends partially on the relative prOportion of fiber types in the sample examined. Muscle LDH maintains its activity in the presence of relatively high levels of pyruvate, whereas, heart LDH is strongly inhibited under the same conditions (11, 33). It appears that "white" muscles contain more muscle type LDH and less heart type LDH than red muscles. The electro- phoretic pattern of LDH isozymes of the soleus, for example, look much like that of the heart, suggesting that red skeletal muscle fibers metabolically parallel heart muscle more so than white skeletal muscle fibers. Garcia-Bufieul et a1. (64) have suggested a biological explanation for the 17 isozymes by saying that high levels of pyruvate favors oxidative decarboxylation and further breakdown of this substance by activating heart LDH (aerobic), thereby re- ducing production of lactate. Thus, a muscle like the soleus, a tonic muscle, could be supplied with a continuous source of energy. Muscle LDH on the other hand favors the formation of lactate (anaerobic) from high amounts of pyru- vate. This reaction may regenerate the NAD required for oxidation of glyceraldehyde 3-phosphate, thereby allowing anaerobic processes to maintain its rate. Vesell (161), however, has produced evidence contrary to the aerobic- anaerobic theory described above. Frequently biochemical discrepancies in results are found between quantitative chemical techniques and histochemical analyses of LDH (34, 141). Histochemically, LDH is commonly reported to be of higher intensity in red fibers but the converse is found quantitatively. It has been suggested that the discrepancy is in the histochemical procedure, since a diaphorase may be the limiting factor-- not the LDH (56, 57). Therefore, the procedure used commonly may be a test of diaphorase, instead of LDH. This could account for much of the confusion in the literature. Altera- tions have been made in this technique to account for this finding even though it is not frequently used. Romanul has simply suggested that the discrepancy is due to the peculiar nature of the different types of isozymes of LDH (141). 18 The significance of the pentose cycle in skeletal muscle is questionable but glucose-6-phosphate dehydrogenase and glucose-6-phosphogluconate dehydrogenase has been demon- strated in skeletal muscle (60, 72, 138, 155). A greater than normal activity Of these enzymes has been found in dystrOphic muscle (60). Anaerobic reactions of the pentose cycle involving transketolase and transaldolase have also been shown to Operate in muscle (98). Beatty et al. (9), under conditions specifically designed to demonstrate the pentose cycle activity, concluded that the pentose cycle of carbohydrate metabolism in skeletal muscle is present but insignificant in relation to the total metabolism of glucose..They stated, however, that the cycle's involvement may be significant in constantly active muscle. Present evidence indicates that fatty acids are substrates for muscle (26, 62). Dubowitz and Pearse (44) stated that the high activity Of oxidative enzymes in red muscle suggests intensive oxidation of fatty acids by this muscle via the citric acid cycle. Beatty et al. (8) found acetoacetic acid uptake to be 30-35 percent higher in red fibers. This supports the hypothesis of a higher oxidation activity in red muscle where mitochondria are numerous, since B-OHBD is reported to be to the respiratory enzyme assembly in the mitochondrial membrane (111, 170). Histochemically, B-OHBD has been found to have greater activity in red than white muscle (141). 19 A high lipid content has been reported in red fibers (both triglycerides and phospholipids) as well as a higher lipase activity (65, 66, 68, 69). Red Muscle of pigeon preferentially utilizes lipids for energy production during contraction (68). Apparently red muscle is better equipped for oxidative metabolism--particularly lipid utilization. George suggests that lipase activity is an index of the extent of fat utilization and the capacity of a muscle to sustain activity. Lipase activity is known to be in mito- chondria (111). It has been reported that voluntary muscles have a supply Of endogenous fatty acids available as substrate for in vitro respiration (23, 123). Wirsen stated in a paper (169) that he found differ- ent rates of labelled palmitic acid uptake in three fiber types. Though not stated Specifically, it is assumed that a greater uptake was Observed by the red muscle fibers. There is some evidence to suggest that mitochondria may be a major site of cellular fatty acid synthesis (111). This is in keeping with the hypothesis that red muscle fibers have a greater fatty acid uptake than white fibers since red fibers contain a considerably greater number of mitochondria. Romanul (141) showed that skeletal muscle fiber phosphorylase activity was inversely pr0portionate to esterase and to some extent also to that Of B-OHBD. 20 Cytochrome oxidase (Cyto O) and SDH were directly prOportional to the "redness" of fibers, but did not parallel esterase activity. This was Shown quantitatively as well by Dawson and Romanul (34)._ Thus Dawson and Romanul concluded that some skeletal muscle fibers derive their energy of contraction from the anaerobic breakdown of glyco- gen and others from the aerobic utilization of fatty acids. Muscle fibers with high lipid and general oxidative meta- bolism and low anaerobic glycolysis were found to be similar to cardiac muscle (141). Esterases and B-OHBD activity of red fibers were found to be the reciprocal Of enzyme activity in white fibers. But enzymes of the citric acid cycle have frequently been reported to be reciprocal to the white fibers. Romanul (141) suggested that the frequency of con- tractions of muscle fibers appear to relate better with their relative prOportion of carbohydrate and lipid meta- bolism than their general oxidative metabolism. In relating the metabolism Of a muscle fiber with the function of capillaries white muscle fibers predominantly utilize the relatively anaerobic glycolytic metabolic path- way. An adequate amount of substrate is stored in white fibers and oxygen is not as immediately essential as in red fibers. Romanul (142) has suggested that the critical function of the capillaries of white fibers is to remove metabolites, whereas capillaries associated with red fibers should have the function of supplying adequate quantities 21 of substrate and oxygen for aerobic metabolism. In agreement with this proposal, it has been repeatedly re- ported that red fibers are associated with a greater num- ber of capillaries than white fibers (25, 35, 92, 124). An extensive review Of muscle metabolism has been recently reported by Drummond (43). Motor Units and Fiber Types As a result of numerous findings indicating a strong neural influence on muscle fibers, motor units have drawn increasingly greater attention. COOper and Eccles (31) stated that individual mechanical responses of a muscle to repetitive stimulation of its nerve are fused into a smooth tetanic contraction at a rate which is determined by the contraction time Of the muscle. Muscles with short contrac- tion times fuse at a slower frequency of stimulation. McPhedran et al. (120) have stated that the same is true for motor units. At a stimulating frequency of 5-10 per second, the soleus develops a large percentage of its maxi- mum tension. The slow fusing motor units of the soleus, therefore, seem to have greater significance in light of the fact that this muscle maintains constant tone. There is a high correlation between conduction velocity and maximum tension (120). This suggests that larger nerve fibers innervate a greater number of muscle fibers. In turn, this accounts for a gerater maximal 22 tension when a fast conducting nerve fiber is stimulated. Eccles and Sherrington (50) proposed the idea that the size Of the motor unit can be estimated by the cross sectional area of the parent nerve fiber. The study of McPhedran et al. (120) supports this since they found high correlation between the tension of motor units and nerve fiber sizes. Henneman and Olson (92) reported a highly signifi- cant finding when they stated that the excitability of a motoneuron was inversely related to its size. Denny-Brown reported a Similar finding in 1929 (36). Thus, there is substantial evidence that the size of the neuron, to some degree, dictates the activity of that motor unit. The mechanical prOperties Of the motor unit suggest that it is a homogeneous entity with each of the muscle fibers quite similar. It appears that the motoneuron may specify in one way or another the kinetic prOperties of the muscle fibers associated with that motoneuron. Wuerker et al. (171) concluded that motor units could be identified in terms Of size, speed of contraction and conduction veloc- ity in the soleus. Henneman and Olson (92) and Close (30) suggested type A, B and C motor units existed, not simply A, B and C (white, intermediate and red respectively) muscle fibers. Nerve fibers were reported to be largest in the type A motoneuron and smallest in type C. Histochemical analysis 23 in this study led Henneman and Olson to the conclusion that the soleus fibers resembled more closely the type B fibers of a heterogeneous muscle, such as the gastrocnemius or plantaris, but did not contain as great an aggregation of subsarcolemmal mitochondria. The muscle fiber diameters of the soleus were found to be quite homogeneous as well. In the case Of the medial gastrocnemius, the dia— meter Of each axon may be related to the type and size of the muscle fibers it supplies as well as to the number of fibers. This relationship is not as constant in the soleus since the fiber sizes are so uniform in this muscle. The medial gastrocnemius is capable of six times more tension than the soleus even though it is only two or three times heavier (92). This may be the result Of the large pale fibers which are found in the medial gastrocnemius and are absent in the soleus. However, Brust (19) found that ten- sion per gram of tissue was greater for the soleus than the gastrocnemius in normal and dystrOphic mice. The medial gastrocnemius also has a greater poten- tial range of tension than the soleus. This probably can be accounted for by the greater heterogeneity of muscle fiber types in the gastrocnemius than in the soleus (92). The significance of morphological and functional interrelationships of a motor unit has more meaning when the size frequency distributions are studied. Axonal con- duction velocities graphically form a bimodal distribution 24 as does the axon size of nerve fibers. Muscle fibers have shown a similar distribution (73) although evidence contrary to a bimodal distribution has also been reported (150). Spinal ganglia size Show a similar bimodal distribution with the smaller cells having the greater enzyme activity (105). This is in agreement with the findings that smaller neurons have lower stimulating thresholds consequently higher metabolic activity. Size and relative metabolic rate need to be studied in motoneurons as was done in spinal ganglia. Thus, the morphological and functional character- istics are apparently as would be eXpected if in fact a "red" or "slow motor unit" has a relatively small moto- neuron with a low threshold, small axon which conducts slowly, innervates fewer, smaller, and metabolically more active muscle fibers that contract tonically. The soleus muscle would be a typical muscle fitting this characteri- zation. Hill has suggested that the muscle fiber can be adapted to save time or energy (94). Such a prOposal im— plies the existence Of slow and fast muscle fiber types or even motor unit types, which would result in a greater work efficiency in tonic and phasic types Of muscular activity. Types of motoneurons can be identified by their duration of after polarization following a spike potential (37). If after polarization is less than 100 msec. then the contraction times and 1/2 relaxation times will be 25 Shorter than in instances where the after polarization duration is greater than 150 msec. This also suggests the existence of different types of motoneurons. Reports on the physical and chemical interrelation- ships of red and white muscle have been remarkably compatible except for a study recently reported by Hall—Graggs (87). Investigating the cricothyroid and thyroarytenoid, he found the cricothyroid to be the slower of the two muscles. The histochemical profile, however, approximated that of the predominantly white tibialis anterior. The thyorarytenoid, on the other hand, had a homogeneous pOpulation of fibers with high SDH activity. He also reported a greater number of capillaries in the thyroarytenoid than the cricothyroid. Typically the thyroarytenoid would be classified as slow contracting but this muscle was found to have an extremely short contraction time. The author suggests, as Romanul (141) and Bocek et a1. (17), that muscle fibers may not be reciprocal, but complimentary, in nature as far as metabolic pathways are concerned. Evidence points strongly toward the hypothesis that the metabolism Of a muscle fiber is related to the frequency of the contractions which are chronically required of it as well as to the length of time for which they must be sus- tained. The extremely active fiber probably develOps the capability Of having available to it the services of anaerobic and aerobic metabolism to compliment one another-- 26 not one or the other. The study of Peter at al. (136) and Hall-Gregg (87) supports this hypothesis. Thus, it seems that the variation of metabolism in the various fiber types is one of degree more than one of different kinds of meta— bolism. If glycolysis were not as active in red fibers as in white fibers, then the substrate, acetyl CoA, would have to be derived from fats or proteins rather than from glu- cose or similar sugars. Such has been proposed (160). Maturation of Muscle Fibers At six weeks of age, the fast muscles of the normal cat attain adult speed. In the newborn all muscles are equally slow. Slow muscles also are said to quicken, but to a lesser extent over the first five weeks. Thereafter there is a progressive decrease in the quickening of slow muscles with adult values being reached after 16-20 weeks (20). After spinal cord transection differentiation of muscle fibers is unaffected; but nerve transection prevents differentiation from occurring. Degeneration of afferent fibers has no effect on differentiation. However, after cord transection the later stage of slowing was absent and in a few weeks the soleus and crureus became almost as fast as normal muscles (21). A later study (22) further illu- strated a neural influence on muscle since the effects Of cross-union were prevented by transection of the Spinal 27 cord one week after cross-union in four week-old kittens. Close has made Observations in the rat and mouse similar to those in the cat (27, 29, 30). Postnatal differentiation of muscle into slow and fast fibers has been reported in a number of animals includ- ing the rabbit (35), dog (106, cat (20), rat (27) and mouse (28). Beatty et a1. (10) pointed out that the degree of maturity at birth is highly variable from species to species. However, some correlation does exist between the degree Of differentiation of muscle fibers at birth and the general mobility of the animal (46). SDH activity was found to be lower in fetal and infant monkey muscles as compared to those of adults by Beatty et a1. (10). They also reported that SDH enzyme activity was higher in red than white muscle as early as 90 days fetal age in the rehsus monkey. Histochemical differentiation was feasible at 120 days with SDH. The same authors reported a greater amount of connective tissue in white (brachioradialis) than red (soleus) muscle in the adult rhesus. Dubowitz was able to differentiate fiber types histochemically at 20 weeks in the human fetus, and by 30 weeks the muscular pattern is much like that Of the adult (46). Fenichel (59) found light and dark myotubes in human fetuses at 8-10 weeks with ATPase reactions. At this stage, he said white fibers are more numerous and larger. At 20 28 weeks, the two types of fibers, as determined by myosin ATPase, were equal in number; but the red fibers were largest. In the chick (71) between the seventeenth and nineteenth day Of hatching, differentaition of three muscle fiber types was reported. Response of Muscle Fibers in Pathologic ConditiOns AtrOphy.--Fudema et a1. (63) found that by immobil- izing a cat's limb, a reduction of electrical activity of the muscles occurred. Fischer (61) reported that atrOphy as a result Of a cast on a limb in the extended position caused only one third as much loss of protein as denerva- tion atrOphy or atrOphy due to tenotomy. Protein concen- tration Of the gastrocnemius of the rabbit decreased by 14 percent during denervation atrOphy in about 25 days. Noncollagenous protein decreased by 13, 25 and 29 percent for immobilization, tenotomy, and denervation, respectively. Thus, the sarc0p1asmic and/or structural proteins may be altered in the various types of atrOphies. This suggests that the fiber type pattern of the involved muscles is probably affected. Bajusz found that in dener- vation atrOphy, some fibers atrOphy much more rapidly than do others within the same muscle (3). LDH isozymes following immobilization of the gastro- cnemius resulted in a decrease in the muscle type LDH isozyme (64). In the soleus there was a reduction in the 29 heart type LDH isozyme. This finding may be because the gastrocnemius normally contains predominantly muscle type LDH whereas the soleus contains predominantly heart type LDH . Tenotomy.--Vrbova (163) found electromyographic suppression in the tenotomized soleus, but not the tenoto- mized gastrocnemius Of a rat. McMinn and Vrbova (119) said that red type fibers are affected by such a treatment more so than white type fibers. A loss Of protein has been reported by Guth (80) following tenotomy. Degenerative changes in the tenotomized soleus can be prevented by simultaneously sectioning the spinal cord (164). It was therefore postulated that supraspinal impulses were responsible for the dramatic changes observed in the soleus. These results were hypothesized earlier by Hines (95). Electrical stimulation delays atrOphy after tenotomy only if it is done under conditions which result in the production Of effective tension (164). Thus, a tenotomized muscle will atrOphy even more rapidly with the Spinal cord intact than when it has been sectioned. Tenotomy Of one ankle of a rabbit leads to a shortening of the contraction time of the tenotomized soleus (163) with or without the spinal cord cut. In such a case postural tone, therefore, afferent impulses are decreased. 30 But Buller et a1. (22) stated that there was no support from their data for the hypothesis that tenotomy caused a conversion Of slow to fast muscle. They based this conclu- sion on the maximum rate of tension developed during iso- metric tetani and the tetanus twitch ratio of the tenotomized soleus. Engel et a1. (55) clearly illustrated preferential atrophy Of type I fibers (stain lightly with myosin ATPase) in the gastrocnemius following tenotomy, and a preferential atrOphy Of type II fibers (stain darkly with myosin ATPase) following denervation. Red muscle has been converted to pale muscle simply by changing the position of the tendon insertion Of the soleus, to that of the tibialis posterior in rabbits (1). Denervation and Cross-innervation.--According to Bajusz (3) denervation atrOphy Of white fibers is rapid, but red fiber changes in diameter are minimal. Romanul and Hogan (143) on the other hand found that the rate of atrOphy is similar in both red and white fibers. Romanul and Hogan said that the most rapid change in red fibers was a lower enzyme activity subsarcolemmally which charact- erizes a normal red muscle fiber. There was a tendency to the denervated white fibers to acquire intermediate fiber type characteristics. Romanul and Hogan reported acid phOSphatase markedly increased following denervation (143). A decrease in heart type LDH in the soleus and muscle type 31 LDH in the gastrocnemius was found by Garcia-Bufiuel et a1. (64) in immobilization; following denervation, the amount of muscle LDH tripled in the soleus but was reduced to one third of the normal level in the gastrocnemius (64). Romanul and Hogan (143) suggested that de-differentiation takes place in both types of muscle as a result Of nerve transection. Chemical differences between red and white fibers tend to disappear following denervation according to Gutmann (86). Guth and Watson (82) found that denervation of the plantaris caused its protein pattern to gradually resemble that of the soleus. Active and total phosphory- lase is maintained after denervation for five days but at ten days a reduction is noticeable (99). Myosin ATPase does not reveal such a labile picture. Denervation does not transform patterns Of myosin ATPase to those charact- eristics of slow muscle (6). Oppenheimer et al. (129) reported identical ATPase activity in dystrOphy even though there was a 50 percent reduction in myosin content. In denervated white muscle of the rat --but not the cat, sol— uble protein changes so that the electrOphoretic pattern resembles that of red muscle (83). Vrbova has suggested that the changes observed following denervation can possibly be explained by a lack of impulses, lack of mechanical work and/or lack of trOphic influences of the nerve on the muscle (164). 32 Following denervation, the mitochondrial count falls (32), there is a reduction of glycogen (112), reSpiratory enzymes (90, 100) and aldolase (90). Phosphorylase is in- creased (101, 118). Muscle fiber lipids are reduced by denervation (122). Each of these findings points to the instability of fiber types in denervation and suggests that these parameters could be altered in an intact neuromuscular system, perhaps by changing the metabolic load on a muscle. The mutability of muscle fibers has been more fully realized as a result of numerous cross-innervation and re- innervation studies. Buller et al., (21) postulated that changes in the speed of muscular contraction were produced by specific influences emanating from two types of alpha motoneurons that innervated fast and slow muscles. He also found that the slowing of the muscular contraction of a normal white muscle occurred when reinnervation was accom— plished. However, slowing of contraction times and relaxa- tion times occur simply with denervation, even in the soleus which is normally slow. It was suggested by the authors that initial slowing Of contraction times following denerva- tion may be due to vibratory stress Of fibrillation, but that slow contractions are later maintained by reinnervation from slow motoneurons. By excising the dorsal root ganglia, afferents were found not to affect differentiation of muscle fibers (51). In a young spinal cat, slow muscles will react in a manner 33 which is mechanically similar to that of fast muscle within a few weeks and will remain indefinitely in that state (20). The force-velocity relation of the flexor hallicis longus muscle may be altered by innervation with soleus motoneurons, but reinnervation Of the soleus by flexor hallicis longus motoneurons produces little or no change in soleus force-velocity ratios (22). In effect, these authors were saying that the conversion of white to red was more complete than from red to white muscle fibers. Romanul and Van Der Meulen (144) were not able to notice any differences in the rates of interconversion of red to white and white to red muscle fibers. However, following cross-innervation or reinnerva- tion (47, 48, 144, 173) of muscles, the histochemical pat- terns generally resemble those Of the muscle which the nerve originally innervated. The myoglobin content of red muscle diminishes slightly in red muscle and then increases slightly in white muscle following random reinnervation (121). Only some of the electrOphoretic protein bands are altered following cross-innervation (83). The electrOphoretic pattern of the cat was found to be unlike that of the rat. No changes were Observed in this pattern following cross-innervation. Since the histochemical profiles are apparently interconvertible following cross-innervation, the question posed by Guth et a1. arises. Is this change quantitative, 34 or is it a rearrangement of the intracellular distribution of the enzymes (83)? Such an explanation does not seem likely in light Of the findings of Karpati and Engel (103). They clearly demonstrated that red and white muscle fibers (Type I and II) are interconvertible following experimental reinnerva- tion. Fiber types were determined with myosin ATPase, a procedure which does not Show intracellular variation in normal or in experimentally reinnervated muscle fibers. Consequently, there is obviously a true conversion of fiber types as identified with myosin ATPase. Intracellular changes in localization of enzyme activity could not account for their results. Another point is clearly illustrated by Karpati and Engel's (103) study. For two reasons it convincingly demonstrates better than any previous study, the neural influence on muscle fiber types. (a) After reinnervation the ATPase pattern Of fibers is homogeneously grouped in- stead Of the normal random pattern. This phenomenon has been reported earlier (83, 173), but not as clearly as was demonstrated with ATPase by Karpati and Engel (103). Grouping of fiber types is thought to be the result of intramuscular terminal nerve fibers branching and rein- nervating neighboring muscle fibers that were denervated. Intramuscular nerve fiber branching has been ob- served by this author following trauma tO the tibial nerve 35 (52). Terminal nerve fiber branching would result in a grouping of Similar muscle fiber types Since the rein- nervated muscle fibers would in effect be innervated by a common motoneuron. (b) Conversion of muscle fiber types was demonstrated by myosin ATPase, an enzyme which is con- siderably more stable and constant under eXperimental conditions than many of the other commonly used techniques, viz., oxidative enzymes, phOSphorylase, etc. ATPase con- version suggests that changes occurred in the myosin molecule itself since myosin molecules are thought to differ in red and white muscle fibers (147, 148). There was an increase in muscle type LDH in the cross-innervated soleus measured quantitatively. Electro- phoretic patterns showed similar results. Histochemically, it was indicated that soleus conversion was more noticeable than conversion in the gastrocnemius (83) of the rat. On the other hand Prewitt and Salafsky (137) said that there is no conversion but a reduction in enzymes in cross-innervation. Dubowitz (48) suggested that only white fibers could be converted to red fibers. Myopathies.--The significance Of fiber types is of further importance since it has been suggested that some types may be more susceptible to specific pathologic con- ditions than others. Brust (19) says that dystrOphy reverses or partially inhibits the normal differentiation of skeletal muscle and that earlier forms of muscle are 36 more resistant to the disease than the later ones. His data suggests a greater prOportion Of red fibers in dys- trOphic mice than in normal ones. Similar findings were earlier published by Eberstein and Sandow (49). In central core disease, a lack Of differentiation into histochemically distinct types of muscle fibers has been found by Engel (53). He proposed the possibility of abnormal neural influences during embryonic differentiation of muscle fibers as an explanation for this disease. It has been suggested that metabolic disturbances of muscles in old age could be due to the decline of trOphic influences of nerve cells (175). Degenerative changes found in infantile-spinal muscular atrophy are noted in large motor cells of the spinal cord and brain stem. A decrease in the size of Type I fibers (red) was observed in all human cases. Type II (white) are changed only slightly or are unchanged (58). When hypertrOphy is noted, it is almost exclusively in Type I fibers. However, normal patterns Of staining for ATPase, a-GPD and other dehydrogenases are found in this disease (58). The ATPase pattern has been reported to be normal in muscular dystrOphy (2), but not LDH (42). Bajusz and Jasmin (2) noted in advanced muscular dystrOphy that the normal SDH difference between fibers disappeared histochemically as well as a reduced correla— tion between fiber size and enzyme activity. 37 From these findings it is Obvious that an intact neuromuscular system is not found in many so-called primary myOpathies. It is also apparent that the neural dependence Of the musculature can be reflected in the histochemical patterns of muscle fiber types. For an excellent description of the histochemistry of the various myOpathies one should refer to Engel (53, 55) or Bajusz (5). Responses Of Muscle Fiber to Low Resistant-Highly Mtitive Activity.--The mutability of muscle fiber types in several pathologic conditions can hardly be questioned, but little is known Of what happens in this regard follow- ing a long term physical training period. The results of numerous studies suggest that red and white muscle fiber tYpes may vary in proportion to the work load placed on the muscle fiber, which may in turn be partially dependent on the types of neurons from which its innervation comes. Whipple found a higher concentration of myoglobin in more active hunting dogs than in more sedentary dogs (168) . Active muscles were noted to have a darker red color than inactive muscles. Lawrie (109) found a high Correlation between the activity Of a muscle and its myo- glObin content. An 80 percent increase in myoglobin was found. in animals exercised chronically by Pattengale and HOlloszy (132) . Hearn reported no significant difference in Cyto O in the gastrocnemius of exercised rats (89) . 38 Elevated levels of phosphorylase and glycolytic enzymes in the gastrocnemius have also been Observed (78). The same authors noted no change in myosin content but did find an increase in ATP Splitting activity by myosin in rats trained by swimming. Gordon et al. (75) found a decreased concentration of sarc0p1asmic proteins in forcefully exercised rats, but an increased concentration of myofibrillar protein was Observed. The converse was found in rats exposed tO a low resistance, high-repetition type of activity (74). Helander (91) concluded that exercise increased myofibrillar protein. Restricted activity reduced myo- fibrillar protein with a concomitant relative increase in sarc0p1asmic proteins in calf muscles. Holloszy (97) confirmed Hearn's (89) finding that SDH levels in the gastrocnemius were not changed by a training program consisting of 30 minutes of daily swimming. In rats that were trained strenuously by running on a treadmill, Holloszy (97) found that the capacity of the mitochondrial fraction from the gastrocnemius to oxidize pyruvate doubled. SDH, NADH-dehydrogenaSe, NADH-cytochrome C reductase, succinate oxidase, and Cyto 0 per gram of muscle tissue doubled in these same animals. There was approximately a 60 percent increase in the total protein count of the mito- chondrial fraction. Holloszy suggested that the rate of 39 aerobic metabolism of pyruvate was not limited by the oxygen supply but by the capacity of mitochondria to oxidize pyruvate. Gould and Rawlinson (78) Observed no changes in levels of MDH and ATPase activity in rat skeletal muscle which had been exercised. Rawlinson and Gould (139) re- ported no changes in ATPase and creatine phosphatase in trained animals either at rest or immediately after swimm— ing for five minutes. Garcia-Bufiuel et a1. (64) reported an increase in muscle type LDH in the soleus from an immobilized or de- nervated leg. He revealed his appreciation of the neural influence on muscle metabolism by pointing out that such a finding could be due either to changes in the anterior horn cells or to an increase in work load. He suggested that the latter was a better explanation. However, it seems more likely that neither one could occur without the other since their degree of activity parallels one another. He found no significant changes in the gastrocnemius con- tralateral to the immobilized leg. It was hypothesized that the increased work load was basically for posture maintenance, since the animals were housed in small cages which restricted their mobility. If such is the case, he suggested that one would eXpect a change in the soleus but not the gastrocnemius (64). However, his findings can be interpreted in quite a different light. If elevated levels 40 of muscle type LDH in the soleus do occur, then the muscle would more closely resemble a white muscle (lateral gastro- cnemius) enzymatically. Red skeletal muscle has predomin— antly heart type LDH normally and white skeletal muscle has muscle type LDH. Consequently, one could infer that the increased overload could have been of a phasic as well as of a tonic nature. Kendrick-Jones (104) Observed an elevation Of creatine phosphokinase activity in rabbit muscles that had been exercised and a decrease when physical activity was restricted. * Lamb et al. (108) reported a greater glycogen concentration in white muscle than in red. They observed the super-compensation phenomenon after single and repeated exercise bouts and that glycogen was more depleted in the untrained than the trained animals following an exercise bout. Hexokinase activity was likewise significantly in- creased by single and repeated exercise bouts. Greater glycogen synthetase activity also'has been observed in the trained animals (108) than in nontrained. Yakovlev found an increase in myosin content and consequently ATPase activity in rats trained intensively (172). He also re- ported an increase in phosphorylase and glycolytic activity during acute muscle activity and during the process of training (172). 41 Exposure to high altitudes results in an increase in the size and number of the mitochondria of red fibers, whereas those of white fibers remain unaffected (165). Small intramitochondrial granules have been reported to undergo changes in number, diameter, and electron-Opacity with variations in the metabolic state of the tissue (111). HypertrOphied mitochondria as well as an increase in number and an elaboration of cristae within the mito- chondria have been observed in rats after treatment with thyroid hormones (41, 79). The greatest change was found perinuclearly and subsarcolemmally. Results of the studies cited above definitely suggest that some differential changes will take place enzymatically in an anoxic condition such as exercise. Investigations involving the mechanical properties of muscular contractions have aided in the attempt to develOp a sound eXplanation for the differences in function- ing motor units. It is well established that mammalian slow muscles are normally activated more continuously and at a lower frequency than fast muscles. Salmons and Vrbova (145). stimulated the pOpliteal nerve at a frequency Of lO/sec. from one to six weeks continuously. In all cases, the stimulated tibialis anterior was significantly Slower than its unstimulated counterpart. Contraction times and relaxa— tion times were prolonged by up to 140 percent. The authors (145) noted that these changes were sufficient to account 42 for the alterations in speeds of contraction which occur following cross-innervation Of slow and fast muscles (21). Consequently, skeletal muscle is capable of responding adaptively to the type of activity imposed upon it (145). In regard to red and white muscle, other parameters should be worth considering in trained animals. The effects of training on muscle weight, fiber size and number of fibers Obviously has not been resolved. Even the rela- tionship Of muscle weight to body weight has not been satisfactorily established. A correlation as high as .99 has been reported (81). Yet some investigators still question the existence of a significant correlation between these parameters. Holloszy (97) says that muscle weight roughly parallels body weights in both sedentary and exer- cised rats. Frequently muscle weight has been reported to have increased after chronic exercise (113, 140), but just as frequently one may find that the weight of the exercised muscle was unchanged (97) or even decreased (73). Goldspink (73) found that exercised muscles did not necessarily weigh more than the control muscles even though the fibers were larger. He interpreted this appar- ent discrepancy by presuming a reduction Of the extracellu- lar components of the exercised muscle. Goldspink also Observed that the number Of myofibrils per fiber increased in exercised (high resistant type) muscles Of mice. He 43 seemed to think that the size Of the myofibrils increased also, but he had inadequate data to support this. Carrow et a1. (25) noted an exercise-induced in- crease in the cross sectional area Of both red and white fibers in the gastrocnemius Of white rats, but the increase was more pronounced in the red than in the white area. The two exercise treatments were one 30-minute daily swim for 35 days and voluntary activity in a revolving drum attached to the cage of each rat. Man-i et al. (117) subjected 50 and 120 day Old rats to a treadmill training program. They found red muscle fibers to enlarge 110 percent and white fibers 21 percent in the 50 day Old trained rats. NO changes in prOportion of fiber types were found using sudan black B as a technique for identifying fiber types. NO training effects Of any kind were found in the adult rats (120 day Old). Exercised muscles of the rats weighed less than the non exercised as was found in this study as well as many others. They studied the tibialis anterior, soleus and gastrocnemius. Perhaps the diameter Of muscle fibers is a better assay for work load than muscle weight. But even investi- gations reporting such data reveal considerable discrepancies. Hypertrophy of muscle fibers has been reported numerous times and there seems little doubt that it does occur. Yet, some 44 investigators (91, 152, 156) have not been able to Obtain evidence Of enlargement in exercised animals. The type and duration Of training, as well as the particular animal being trained can account for some of the discrepancies. Reitsma (140) found so much variation in hypertrOphy that an Optimal training period for hyper- trOphy could not be determined.. A more plausible eXplana— tion appears to involve the differential responses of red and white muscles to different kinds of exercises. One type Of exercise may predominantly affect one fiber type, whereas another type Of exercise would produce changes in another type. Goldspink (73) suggested that hypertrophy after exercise was not due to a simultaneous increase in the diameter Of all fibers. Hypertrophy probably occurs in only some Of the fibers. This is likely if the reaction of a muscle to exercise occurs in motor units as was suggested by the earlier discussion of motor units and fiber types. It seems reasonable to suSpect that one type of fiber may become smaller and another type larger when a muscle is chronically overloaded, since strenuous exercise Often does not alter the ratio of body weight to muscle weight. Gordon's data supports such a hypothesis (76). Reitsma (140) found that some areas Of the plantaris hyper— trophied more than others. Greater changes were found in 45 the soleus than in the plantaris. If this proposed theory is proven correct, it will explain many of the conflicting results found in the literature. For example, the muscle itself, the area of a muscle, identification of fiber types and the nature of the training program could yield almost random results if the investigator was unaware of the differential Significance of muscle fiber types. CHAPTER III RESEARCH METHODS Sample Seventy-two, 100 day old, male, albino rats (Sprague-Dawley strain) were housed in sedentary cages for seven days to provide environmental adjustment period. Sixty of these animals then were selected on the basis of weight, eliminating the extremes. They were placed into twenty trios by weight and randomly assigned within trios to one of three treatment groups. The rats were initially placed in trios in order to reduce between-group variances in size of nerve fibers and anterior horn cells resulting from variations in body size. Fiber and cell sizes of the sciatic nerve and the spinal cord were parts of other pro- jects associated with this study. Treatment Groups The three treatment groups used in this investiga- tion were as follows: Sedentary-Control Group.--These rats were housed in sedentary cages (24 cm. long by 18 cm. wide by 18 cm. tall) for the duration of the exPeriment. They were 46 47 removed from their cages weekly for body weight determina- tions only. Sedentary-Forced Group.--This group of rats also was housed in sedentary cages. Each of these animals was forced to swim for one 30-minute period per day with a weight equal to 3 percent of its body weight attached to the tip Of its tail. Voluntary-Forced Group.--This group was housed in individual cages as previously described. However, each cage was equipped with a revolving drum 35 cm. in diameter and 13 cm. wide in which the rat could exercise at will. Each of these rats was forced to swim for two 30-minute periods per day with a weight equal to 4 percent of its body weight attached to the tip of its tail. Treatment Procedures TO facilitate sacrifice procedures animals from two trios began treatments on each of ten consecutive days at the beginning Of the experiment. This allowed the animals to be sacrificed in the same order on ten consecu- tive days when the experimental treatments were terminated. In this way, the duration of the treatments for all animals was held constant. Rats were swum in individual cylindrical tanks measuring eleven inches in diameter and having a depth of thirty inches. Following each swim, the rats were dried with a towel and returned to their respective cages. 48 The sedentary-forced group swam six days per week for 52 days between the hours Of 9:00 a.m. and 12:00 noon. The voluntary-forced group swam Six days a week for 52 days between the hours Of 9:00 a.m. and 12:00 noon and again between 5:00 p.m. and 8:00 p.m. The weights which were attached to the rats' tails were adjusted weekly according to their body weights. These weights were attached to the tips of the tails with miniature plastic Clothespins. Adhesive tape was inserted between the prongs of the Clothespins to reduce trauma. Placing the weight to the tip of the tail as Opposed to the base, necessitated them to consistently work harder than when the weight is placed at the base Of the tail, as has been done in this laboratory heretofore. For the first two days of the training period for each rat no weight was attached to the tail. If‘a rat remained submerged for ten seconds during a swim, he was removed from the tank, the weight on the tail was removed and the animal was given a brief rest before returning him to the tank to complete the swim without his tail weight. Allganimals were fed ad libitum with commercial block feed. Ambient air temperature in the animal quarters ranged from 21°C-25°C. The water temperature for the swimming program ranged from 32°C to 34°C.‘ 49 The animals were exposed to a twenty-four hour light cycle that was automatically controlled by an electr- ical timer which provided the rats living quarters with twelve hours of light and twelve hours without light daily. Sacrifice Procedures Each animal was anesthetized intraperitoneally with 2 cc. Of 1 percent Pentobarbital on the day following his final treatment. A section of the left sciatic nerve and soleus muscle was excised immediately distal to hip joint and embedded in Epon. This procedure was required for a related project involving nerve fiber sizes and electron microscopic Observations of soleus muscle fibers. Following nerve transection, the left gastrocnemius, soleus and plantaris were excised as a unit by transecting at the origins and insertion. These muscles were weighed as a single unit. A section about 1 cm. thick was ablated from the belly of the muscles and then placed on a freezing block with 5 percent gum tragacanth. The freezing block was quenched for approximately 10 seconds in isopentane which was cooled to a viscous fluid with liquid nitrogen. Tissue sections were cut at about 10u in thickness at a temperature of -20°C in an Ames Lab—Tek cryostat. The sections were briefly fan dried immediately after the Sections were placed on cover glasses. 50 Histochemical Procedures A modified Gomori trichrome (54) was used to investigate morphological detail of the fibers. SDH activity was studied using NBT [2,2'-di—p-nitr0phenyl- 5,5'-diphenyl-3,3'-(3,3'-dimethoxy-4,4'-diphenylene) ditetrazolium chloride] as an electron acceptor, as de- scribed by Barka and Anderson (7, 125). Myosin ATPase activity was investigated employing the techniques de- scribed by Padykula and Herman (130). The Wattenberg and Leong's (166) method for "menadione-linked" a-GPD (mito a-GPD) and the Novikoff et a1. procedure for NADH-D was used, each employing NBT as an electron acceptor (125). The procedure for malate dehydrogenase involved tissue incubation in a medium consisting of 1 ml. of 1.0 M malic acid, 10 mg. of NAD, 15 mg. of NBT and 10 m1. of 0.2 M Tris buffer at a pH of 7.4. The final pH of the incubating medium was adjusted to a pH between 7.2 and 7.4 with 10 N NaOH. Thirty minute incubation times were used for all enzyme histochemical procedures. Glycerin jelly was used as the mounting medium for all procedures except myosin ATPase. Permount was used for myosin ATPase. Control sections were incubated with each of the enzymes in order to determine the Specificity of the enzyme reactions. Controlled incubating solutions excluded 51 the substrate in some cases, while in others the coenzyme NAD was eliminated in those procedures which require it for adequate reactions to take place. Methods Of Tissue Analysis Subjective Evaluation Of a Large Number of Indivi- dual Muscle Fibers.--Since the variation in concentration of fiber types varies as much intramuscularly as it does intermuscularly, specific regions of the muscles had to be identified for analysis. The muscles and regions analyzed in this study include area I of the soleus and areas II and III of the plantaris, as illustrated in Fig. 1. This intramuscular and intermuscular variation can be seen easily in Fig. 2. From each animal, approximately 350 muscle fibers in the soleus and about 350 muscle fibers in each Of areas II and III Of the plantaris were rated. In the soleus muscle fibers were rated according to staining intensities as light or dark. Myosin ATPase, MDH and mito a-GPD activity determines the staining intensity in a muscle fiber. This same rating plan was used for myosin ATPase in the plantaris. Since light, intermediate and dark fiber types are evident in the plantaris with mito d-GPD and MDH the fibers of this muscle were rated as such in areas II and III. The fibers of area II were rated as light, intermediate or dark only with MDH, since this 52 region is homogeneous in regards to mito a-GPD and myosin ATPase. The pattern as well as intensity of enzyme activity served as a criterion for defining muscle fiber types. For example, electron micrographs have demonstrated that red muscle fibers have subsarcolemmal aggregations of numerous mitochondria. These mitochondria have more dense cristae than those found in white fibers. Peripherally located mitochondrial aggregations result in a dark subsarcolemmal staining pattern with oxidative enzymes. Also, such a fiber would stain more intensely throughout than would a white fiber. Many intermediate fibers were rated as such due to the presence of enzyme activity similar to that found in red fibers but without the dark subsarcolemmal staining. Consequently, enzyme activity in intermediate fibers is more homogeneous than that of red fibers, since it lacks the dark subsarcolemmal staining with oxidative enzymes. Overall Subjective Evaluation of Tissue SectionS.-- An overall subjective evaluation of the preeminence of specific types of fibers was made in area I of the soleus and areas II and III Of the plantaris from each animal. These evaluations were made without knowledge of the treatment group to which each animal belonged. This procedure was carried out using the following staining techniques: 53 Area I - MDH and SDH Area II - MDH, SDH and modified trichrome Area III - MDH Determination of Metabolic Patterns in Muscle Fibers.--Observations were made in the soleus, plantaris and gastrocnemius in order to define the activity of a series Of enzymes in each type of fiber. This was done by defining morphologic characteristics and enzyme patterns with myosin ATPase, NADH-D, MDH, SDH, mito a-GPD and a modified Gomori trichrome stain. Statistical Methods.--The percentages of the three types Of fibers in each area were analyzed using one-way analyses of variance (77). That is, mean percentage differences between the three treatment groups were exa— mined for red, intermediate, and white fibers with separate one-way analyses of variance. When the Erratio was signi- ficant, the Duncan multiple range test was used to deter— mine which means differed (14). When the E-ratio was not significant, the power of the test was calculated (77) in order to determine whether the null hypothesis should be accepted or whether judgment should reserved. Conclusions from this investigation must take into account the inherent dependence of the separate analyses for each type Of fiber. For example, determination of the percentages Of red fibers within an area of a muscle limits 54 the percentage of intermediate and white fibers. However, there was independence of data within each test. A contingency Chi-square (14) was used to analyze the presence Of interaction of the overall subjective evaluation Of enzyme activity in a specified area of a muscle and the group treatments. Each cell of the Chi- square tables contains the cumulative frequency of a specific rating Of muscles in each of the treatment groups. The .05 level of probability was chosen to deter- mine significance in the one-way analyses of variance and the contingency Chi-square. A .20 level of probability was chosen as an acceptable value for 8. Reliability of the procedures employed in rating fiber types was determined in two ways. Since considerable variation in the percentage of red, intermediate and white fibers exists intramuscularly, consistency of selecting a similar area for analysis from each animal was a source of variability. In addition, the consistency of typing a specific fiber, irregardless of the position of the fiber within a muscle, was also a source of variability. Due to the nature of the data, consistency in choice of an area and fiber typing consistency were deter- mined collectively by typing approximately 200 muscle fibers in area III about one month following the original evaluation. Variations of the percentages of each fiber 55 type, between the first and second evaluations are expressed as mean percent deviations. Consistency Of fiber typing is represented by the percent of the fibers typed similarly, irregardless Of muscle fiber type, on the two evaluations. CHAPTER IV RESULTS AND DISCUSSIONS Results For a large sample of muscle fibers, the prOportion of fiber types in the soleus and plantaris was determined by subjectively evaluating the enzyme activity and the pattern of that activity within each muscle fiber. This was done in Specified areas as described earlier. These same areas were also rated subjectively for overall enzyme activity as either high or low. Soleus.--Tab1e 1 gives the mean percentages of intermediate and red fibers of the soleus, as determined by myosin ATPase, mito a-GPD and MDH, for each of the three treatment groups. Statistical analysis of the soleus data showed that the mean percentages of intermediate fibers, as determined with the three enzymes mentioned above, did not differ significantly among any of the three groups of animals. Since the Type II error probability was 2.20 in each case, neither can the null hypothesis be accepted as true. Con- sequently, the results of the soleus are inconclusive. 56 57 .Oanmummoom mum3 mosam> m mo HO>OH wanmumooom omcflfiumumo Axes «0 sueflenmnoum ones. a no: u Hmnuwoc mocwm ow>uommn mm3 ucmEmmosw.mO£B .om. W Iowa on» soap umumonm ma Oshucm some now HOHHO HH mama m m: .mmmmu Honey mo mommusooumm come no moonoummmwo unmowmacmflm mHHMOHumwumum 30am on mummmoomc mo. W a mo msam> HOOADAHO oocfleuouoomum map Home magma menu SH mowuonrm owns» on» no mcoz« o.m.<.* SH. M 4 mm.H o.sa «.mm m.mH ~.Hm m.o~ a.me mos o.m.a.* OH. M . m¢.~ a.mH m.em «.ma G.Hm m.- n.5s moons one: U.m.¢«« OH. M « mm.o «.ma m.vm a.ma a.mm m.mH ~.mm Ommmad camomz mo. w_a Hounm muonam was com .ooEHousH pom .ooeuoucH com .OOEHODSH mahucm chmemmEOU H mama reomsumucH Mom coo: mo .noum Oflumurm Urromouom mlrooonom «Iraouucou mmsouw com3uom thumpcsao> rhumusoomm raumucoomm H » E .mnz was nmwra one: .ommmam Geno»: an owcwenmumo mm “H mmh030m .msoaom on» GH mm xumo HO named Hmcuwo mm Hoommm mumnflm ommsa«s .HO>OH mo. on» no ucooamwcmwm Owumurha A.om m.mH a.mm «.ma o.¢m a.me mmmmea memos: HHH when: .emsuoucH whens .omsumucH when: .eoeumucH Mm HO UGMtk HO UOM¥k HO UTMkt «.mm a.ma m.He M.Gm m.o~ a.me m.s~. a.me a.mm. mos HHH ~.- e.«~ a.mm m.- ~.o~ H.em m.o~ m.mH p.mm omens one: HHH p.ma ¢.m a.mm m.ea m.mH ~.os a.ms o.ea. a.me. was HH pom .omEumucH wuwnz omm .OOEHODCH muflnz com .OOEHODGH ouwnz meancm OOH< OOOHOEISHMDGOH0> U omouomrmumucmomm m Houucourhumucmomm 4 (U muumuqmfim was to HHH one HH mmoué Eoum mnwnwm OHOmsS com ocm mumwowfiumucH .Ouflnz mo mommucmouwm ammzrr.v OHQMB .oo>nwmmn me mammnuomhn Had: may MO OOGODQOOOO co psoEmosn .Ouomouosu .oowmmwuom uoc mo3 cm. W n no HO>OH omcfleuouoooum «as 61 .30. u 3 mcmOE msoum coo3umnrmoocmHOMMHo unmowmecmwm on mo mfimonuommn mo coauomnmm« v o.mv4 «Hoo.AmAmoo. mm.oa. mom was «goa.Am ~m.o .omEumucH was o.mA< «Hoo.Am ms.¢ae opens no: HHH «soa.Am me.a com omura one: ««OH.Am m>.H .ooeumucH nmwra one: ««OH.Am vv.o muflaz amwra one: HHH .«oa.Am em.e .6msumch mummea cwmomz HHH o.mA< .Hoo.Aono. em.e. com mos o.m.« «mmo.AmAmo. mm.m« .oosuoucH so: UAmA¢ «HooXm mméac muwsz was HH chmHHmmEOU Hounm H mama Owumurm mama Oaxaca mend coo: mo .noum masonw cmmzuom Honda I (I summonses may no HHH one HH means some mumnfim maomsz com ocm oumflomeuoucH .ouflcz mo mommucooumm cows mo mammamcdrr.m manna 62 Statistical evaluation of the dark fibers as determined by MDH shows the mean percentages of the vol- untary-forced and sedentary-forced groups to be similar but at the same time greater than the mean percentage of the sedentary-control rats. In area III no differences in the mean percentages of medium fibers, as determined with myosin ATPase, among the three groups was found. However, the 8 value for this analysis indicates that the hypothesis of equal means can- not be accepted. Similar results were found for light, medium and dark fibers as rated with mito a-GPD in area III. Analyses of the prOportion of light and dark fibers in area III show that the sedentary-forced and voluntary- forced groups are similar but differ significantly from the sedentary-control group. The apparently incomplete shift Of fibers with mito a-GPD in the Opposite direction from that of MDH lends further support for the hypothesis that predominant meta— bolic pathways in fibers are not completely reciprocal but can be supplementary in nature when increased metabolic demands are placed on a given muscle fiber. Observations by Hall-Graggs on the throat musculature support such a phenomenon (87). The results previously discussed further support the proposal that there is a tendency for the muscle fibers to shift toward the red end of the fiber type spectrum. 63 However, it should be pointed out that the trends are not as clearcut in area III as that found in area II of the plantaris. This could be due to a greater variance Of fiber sampling as well as variance in fiber typing since this part of the plantaris proved to be the most difficult to analyze. Overall subjective evaluations of the plantaris in areas II and III were made as was done in the soleus ana- lysis (Tables 6, 7, 8). The numerical values in Tables 6, 7, 8 and 9 represent the number Of animals from each group having a specified area of a muscle receiving an overall rating of either light or dark. A specified area of a muscle was rated high or low as to the prOportion of dark to light fibers in each muscle. Area II was analyzed using MDH, SDH and a trichrome stain as the criteria for muscle fiber typing. A significant contingency Chi-square was found between the treatment groups and the relative number of dark fibers in area II. This suggests that the animals exposed to greater amounts of exercise have a greater prOportion of red fibers, and conversely the more sedentary rats have a greater percentage of white fibers than the exercised. The same results were found in area III of the plantaris, but the evaluation was more difficult to make than in area II (Table 9). 64 Table 6.--Chi-square Test for Treatment Effects on the Overall Number Of Red Fibers (MDH) in Area II of the Plantaris A B C Group Sedentary-Control Sedentary-Forced VOluntary-Forced Light 15 12 8 Red 2 5 ll Chi-square Of 8.72 (S at .05 level) Table 7.--Chi-square Test for Treatment Effects on the Overall Number of Red Muscle Fibers (SDH) in Area II Of the Plantaris A B C Group Sedentary-Control Sedentary-Forced Voluntary-Forced Light 14 8 6 Red 5 8 13 Chi-square of 6.74 (S at .05 level) Table 8.--Chi-square Test for Treatment Effects on the Overall Number of Red Muscle Fibers (Trichrome) in Area II of the Plantaris A B C Group Sedentary-Control Sedentary-Forced Voluntary-Forced Light 15 9 8 Red 4 7 ll Chi-square of 5.39 (S at .05 level) 65 Table 9.--Chi-square Test for Treatment Effects on the Overall Number of Dark Muscle Fibers (MDH) in Area III Of the Plantaris A B C Group Sedentary-Control Sedentary-Forced Voluntary-Forced Light 15 9 9 Dark 1 6 9 Chi-square of 7.88 (S at .05 level) This was due to the fact that area II of the plantaris almost wholly consists of white fibers. There- fore, if an increase in the prOportion Of red fibers does occur, it should be easier to detect than in area III. Area III of the plantaris is quite mixed in fiber types having a large percentage of red and intermediate fibers in addition to white fibers (Table 5). Since area II contains few if any red fibers, minimal differentiation of fiber types was found with myosin ATPase and mito a-GPD. Consequently this area of the plantaris appears relatively homogeneous. Therefore, only MDH was used to differentiate a large number of fiber types in area II. The data in Table 10 shows the mean muscle weight (triceps surae and plantaris) and body weight Of Group A to be greater than Group B and the Group B weight to be greater than Group C. Completed F-table values are listed 66 in Appendices A-l4 to A-15. Table 11 shows the correlation Of muscle weight to body weight in each of the three groups as well as the total correlation for all three groups. Table 10.--Mean Muscle Weights (Triceps Surae and Plantaris) and Body Weights (grams) and Percent Body Weights of the Three Treatment Groups A B C Sedentary- Sedentary- VOluntary- Control Forced Forced Body Weight * 424.30 371.52 333.63 Muscle Weight ** 2.93 2.61 2.35 Percent Of Body Weight/ Muscle Weight 0.69 .70 .70 *A>B>C at .05 level. **A>B>C at .05 level. Table ll.--Corre1ations Of Muscle Weight to Body Weight A B C Sedentary- Sedentary- Voluntary- Group Control Forced Forced Total Correlation .66 .79 .55 .87 The sedentary-forced group had more difficulty in tolerat- ing the training program than the voluntary-forced group. 67 Four rats died during the experiment, each while swimming. Three Of these animals belonged to the sedentary- forced group. The fourth rat belong to the voluntary-forced group. During the experiment the medial gastrocnemius from two of the four animals that drowned during their daily 30- minute swim were examined histochemically. The muscle fibers appeared normal (Fig. 6) in every way except for a few red fibers that were extremely large and round or oval in shape instead Of the conventional angular-shaped muscle fibers (Fig. 3, 4, 5). An even more interesting observa- tion was the presence of what appeared to be a thick coiled membranous-like feature in some of the extremely large fibers (Fig. 3, 4). They were red type fibers in every case as typed with NADH-D, MDH and myosin ATPase. This Observation cannot be taken as conclusive evidence of an acute effect Of swimming since only two animals were studied under this condition. However, it does suggest that perhaps further investigations on the acute effects of exercise should be undertaken. Similar fibers as shown in Fig. 3 and 4 have been reported by Dubowitz (47) in muscular dystrOphy. The question Of consistency in the rating of fiber types was checked in area III of the plantaris with MDH and mito d-GPD. These enzymes in area III were chosen for reliability checks since it was apparent to the author that 68 this was the portion of the subjective analysis which was most questionable. Therefore, if adequate consistency was found here, then the remaining enzyme analyses should be adequately reliable. Table 12 includes the data which represents the reliability of typing a known fiber similarly on succeed- ing evaluations. Table 13 represents the consistency of determining the prOportion of red, intermediate and white muscle fibers on succeeding trials. This does not repre- sent evaluation Of a known muscle fiber, but an evaluation of a population of fibers in a general area. Therefore, variability between the original and final evaluation is due to typing error in addition to the variability in selecting the same fiber population as was typed originally. Table 12.--Consistency Of Typing Muscle Fibers in Area III of the Plantaris Number of Number Typed Percent of Enzyme Fibers Typed as Original Consistency Myosin . ATPase 244 . 244 100 MDH 400 359 90 Mito a-GPD 378 358 95 69 Table l3.-—Consistency of Selecting Similar Muscle Fiber POpulations and Typing Specific Muscle Fibers Similarly on Succeeding Trials in Area III of the Plantaris Mean Percent Deviation Fiber Type White Intermed. Red Enzyme: MDH $3.1 17.5 15.9 - *Absolute Deviation -l.9 +3.8 —2.0 - *Mathematical Deviation $1.1 $3.7 13.9 - *Effective Deviation Mito a-GPD 14.5 15.2 17.7 - Absolute Deviation -2.3 -0.6 +2.9 - Mathematical Deviation 12.2 14.6 14.8 - Effective Deviation *See text for eXplanation (pp. 68-69). In Table 13 "absolute deviation" represents the average deviation between two analyses of the same muscles irregardless Of the direction of the deviation. The "mathematical deviation" was calculated taking into account the negative and positive deviation (mathematical summation). The "effective deviation" was derived by taking the abso- lute differences Of the two deviations described above. This parameter probably better represents the variation found in the present data. The effective deviation eliminates trends in the color estimation of staining intensity which may tend to shift the ratings of fibers toward the red or white end of 70 the fiber type Spectrum. For example, if the series of ratings of light fibers on the second analysis was con- sistently lower prOportionally to dark fibers than a higher prOportion of darker fibers would be the concomitant result on the second analysis. However, in such a case, the rela- tive proportion of all three fiber types would remain un- altered among the three treatment groups.' Since it is the comparison of relative proportions of fiber types, not the absolute prOportion of fiber types that is important for intergroup comparisons; it is the author's Opinion, that the effective deviation better represents the accuracy of data collection in this study. Discussion All data collected on the soleus indicates that one or even two one-half hour swimming periods per day for fifty-two days will not alter the prOportion of medium and dark fiber types. There are several plausible exPlanations for these results. The soleus Of a white rat contains no white :muscle fibers but only intermediate and red fibers (Fig. 17-21). It is perhaps reasonable to find no alteration in the prOportion of these fiber types in light of the find- ings Of the plantaris which demonstrated a shift toward a greater prOportion of red muscle fibers. In other words if? one interprets the "redness" of a fiber as its ability 71 to maintain tonicity then it would have to be assumed that these fibers had already adapted to a highly repetitive, low-resistant type of activity and that a relatively small increase in activity for a brief period each day would have no noticeable effect on these fiber types. A more strenuous training program possibly might reveal some adaptations in the soleus, assuming this muscle effectively contributes to the leg motions involved during swimming. Analysis of the soleus data suggests that if the differences found are of biological significance, then the power of the test was not adequate to make a final deci- sion of acceptance or rejection of the null hypothesis. Further calculations indicated that if mean group percent differences from the overall mean percent of approximately minus three, zero and plus three would have resulted, the test would have had sufficient power to justify a final decision. Consequently, if the differences observed are biologically significant, then the test was not powerful enough to detect such a difference (N must be larger). If differences at least as large as minus three, zero, and plus three percent are needed to be Of biological signifi- cance in the soleus, then the null hypothesis could be accepted in each case involving the soleus. Data from the plantaris are indicative of muscular adaptations which could occur when eXposed to a highly repetitive-low resistant training program. It appears that 72 any one or a combination of three things occurred in the plantaris which could account for an increase in the pro- portion of red fibers in the plantaris. One explanation would be a bifurcation of red muscle fiber types more than white fibers. The concept of splitting muscle fibers re- ferred to as numerical hypertrOphy by Reitsma (140) is not a well accepted phenomenon. Proof of such an occurrence cannot be supported by this author's findings. However, there is a meager suggestion Of some bifurcated fibers in the soleus Of exercised animals (Fig. 7, 8). This was not noticeable in the plantaris. If bifurcation of red or intermediate muscle fibers did occur in the soleus and not the plantaris then an alteration in the proportion of red fibers would be eXpected in the soleus, not the plantaris as was found in this study. Area II of the plantaris was not found to contain any suggestion of bifurcated fibers. However, this was the area which most Obviously had an increase in the pro- portion Of red or intermediate fibers (Fig. 10, 11). A second plausible explanation is that alterations in proportions Of fiber types is the result of white fibers acquiring the characteristics of red fibers. This seems to be a reasonable explanation in light of other related findings. Since the type of overload was of a low resistant and highly repetitive nature, it, perhaps, resembles the 73 work load of the soleus. Thus, it might be expected that the plantaris would take on the histochemical characteris- tics Of the soleus by becoming "redder" which probably means adapting for a greater capacity for tonic type con- tractions. Still a third eXplanation could be an intracellular rearrangement of enzyme activity instead of an increase in total enzyme activity. Such a mechanism has been suggested by Guth (83). It is unlikely that this could account for the change observed in light of a more recent report by Karpati and Engel (103). This hypothesis was discussed in detail in Chapter II. There have been a number of reports which point. out the correlation of the quantity and quality of mito- chondria and the metabolic activity of that cell. It is well established that oxidative enzymes such as SDH and MDH are clearly associated with the membranes of mitochon- dria. It has also been shown that activity of some enzymes can be increased in an intensive training program (97, 108, 136). Since the number and size of mitochondria as well as the density Of the mitochondrial cristae are some of the characteristics which aid in typing muscle fibers (66), it seems reasonable to suspect that the prOportion of red and white fibers could be modified by an alteration in the 74 mitochondrial pOpulation which in turn could alter the intensity of enzyme activity as demonstrated histochemically. In view of the metabolic peculiarities in the different fiber types, a change in fiber types suggests a change in metabolism Of individual fibers. This, perhaps, should not be uneXpected since some investigators have found a lowered respiratory quotient (R.Q.) in chronically trained men (107, 158). Although a gross measure of meta- bolism, a lowered R.Q. suggests that there is a general shift toward a more aerobic type Of metabolism with a greater capacity for utilizing fatty acids as substrates. It has become increasingly Obvious that red fibers have a greater capacity for oxidation of fatty acids than white fibers (26, 68). Red fibers contain a relatively large amount of triglycerides whereas white muscle fibers contain a very minimal amount (65, 66). Beatty et a1. (9) have also demonstrated greater acetoacetate uptake in red fibers. Therefore an increase in red fibers in a training program would be in agreement with other findings related to train- ing and fiber types. Glycolysis is generally accepted as the major metabolic pathway in white muscle fibers. This pathway has a reaction which yields reduced nicotinamide adenine dinucleotide (NADH) when glyceraldehyde phOSphate is oxidized. One prOposed mechanism for replenishing oxidized NAD so that glycolysis can be maintained is the reduction 75 of pyruvate to lactate. Other possibilities that have been proposed are the d-glycerOphosphate cycle and the direct oxidation Of NADH by mitochondria (110, 115). White muscle mitochondria has been reported to have a higher rate of oxygen consumption.*with a-glycerophosphate than with lactate, succinate, and NADH. On the other hand mitochon- dria from red muscle were less active with d-glycerophos- phate than with the other substrates (13). Blanchaer et a1. (13) suggested that in white muscle the a-glycerOphos- phate cycle is Operative, but in red muscle the direct oxidation of NADH may be more important than a-glycero- phosphate. Lundquist et al. (115) reported a 24 percent lower oxygen uptake in white than red muscle mitochondria with pyruvate + malate as substrate. With o-glyCerOphosphate the oxygen uptake almost doubled in white muscle mitochon- dria. Considering these results collectively, one can hypothesize an important role for mito a-GPD in white fibers. High mito d-GPD was found in white fibers in this study. Pearse found that mito a-GPD activity runs parallel with phosphorylase activity, unlike all other oxidative enzymes tested at that time (133). Pearse suggested that since the extramitochondrial a-GPD is strongest in smaller fibers (red) the concept Of an a- glycerOphosphate cycle is not fully supported. Engel (53) 76 reported extramitochondrial a-GPD activity to be moderate in all fiber types. Dubowitz and Pearse (44) also reported a reciprocal relationship Of phosphorylase and oxidative enzymes in skeletal muscle. This concept is not supported by the findings of Romanul (141) or by the results of this study. If Pearse's (133) finding, that phOSphorylase and mito a-GPD are directly related, is dependent on Dubowitz and Pearse's (44) earlier report that phosphorylase and oxida- tive enzyme activity is reciprocal, then it would be Romanul's results which would be in more agreement with this investigation. The results of this study indicate that mito a-GPD and oxidative enzyme activities are not recipro- cal. Therefore, if a direct relationship does exist between phOSphorylase and mito a-GPD, then phosphorylase cannot be assumed to be reciprocal to the oxidative enzymes. A lack of complete reciprocity of these enzymes was discussed earlier in this paper. Mito a-GPD and phos- phorylase parallel one another and oxidative enzymes and phOSphorylase are at least in part reciprocal. Therefore, the three patterns of enzyme activity as seen with mito a-GPD, MDH and myosin ATPase is sufficient to infer about the basic metabolic pathways predominant in skeletal muscle. Consequently, these three enzymes were chosen for extensive evaluation in this particular study. 77 Demonstration of fiber types with myosin ATPase is a procedure widely used and little understood. Its wide use can probably be accounted for because of its discreet division of fibers into a supposingly red or white fiber (Fig. 5, 6, 12, 17, 22). Intermediate type fibers are seemingly difficult or impossible to identify using this technique. Some investigators rely heavily on the myosin ATPase for fiber typing for histochemical analyses of myoPathies (55, 103). Engel has suggested that the two types Of fibers demonstrated by this technique represent the true or original fiber type (53). To change fiber types as demonstrated with this technique has proven difficult even with denervation and in myoPathies but not foreign reinnervation (103). Thus, it would be eXpected that exer- cise would not alter the myosin ATPase activity pattern. However, activation of myosin ATPase in normal muscle has been observed to be higher in white than red muscle (148, 149), thus corresponding to the histochemical results of this study. It is now well known that myosin ATPase is a complex enzyme. Myosin ATPase differs in its activity when exposed to varying concentrations of magnesium, calcium, ethylen- diaminetetraacetic acid (EDTA), as well as to solutions of varying pH values. The pattern of dark and light fibers as seen with one concentration of EDTA and specific pH will differ when 78 these ion concentrations and/or the pH of the incubating medium is varied. Thus, the significance Of a light and dark fiber can be interpreted simply as a fiber which has myosin ATPase that is activated or inhibited with the chemical characteristics Of that particular incubating medium. Enzyme Patterns in the Plantaris and Gastrocnemius.-- It is generally accepted that the myosin ATPase technique utilized in this study will illustrate dark and light fibers reciprocally to that demonstrated with oxidative enzymes such as MDH, SDH and NADH-D. This relationship has been found to be more complex than at first believed (Table 14). This investigation suggests that fibers which would be rated as intermediates with MDH, SDH or NADH-D would appear to be light using myosin ATPase as the criterion for a fiber type. The white and red fibers on the other hand would appear dark or as white fibers with myosin ATPase (Fig. 12-16 and 22-26). It should be noted that all of the fibers rated as intermediate in this study would not appear light with myosin ATPase and mito d-GPD. The detailed analyses Of the three Specified areas in- ‘volved classifying a given fiber as white, intermediate or red. As pointed out earlier, such a division is arbitrary Since there is basically a continuous spectrum of fiber ‘Qypes in the plantaris and gastrocnemius muscles. There- Ifibre, fibers rated as intermediate with oxidative enzymes 79 include some fibers without the characteristics which would cause it to stain lightly with myosin ATPase and mito d-GPD. Consequently, all fibers with low myosin ATPase and mito a-GPD activity would be intermediate if classified with MDH, SDH, NADH-D and/or a trichrome stain. But Of all the intermediate fibers, determined as such by MDH, SDH, NADH-D and/or trichrome, only some of them would be rated as low with myosin ATPase and mito a-GPD. A third pattern of enzyme activity in addition to that seen by myosin ATPase (Fig. 12 and 22) and an oxida- tive enzyme (Fig. 13, 23) has been seen with mito o-GPD by other investigators as well as by this author. Mito a-GPD has a pattern of enzyme activity in the gastrocnemius and plantaris which closely resembles myosin ATPase. However, mito a-GPD demonstrates more than the two fiber types seen with myosin ATPase. Fibers in the gastrocnemius and plan- taris which appear light with myosin ATPase will be light with mito a-GPD while the intermediate and dark fibers as seen with mito a-GPD will appear dark with myosin ATPase (Fig. 12-16 and 22-26). A modified Gomori trichrome stain can also be used to readily identify red fibers (Fig. 9, 26). Enzyme Patterns in the Soleus.--Un1ike the plantaris and gastrocnemius enzyme patterns in the soleus are apparently less complex. The soleus contains only inter- mediate and red fibers as illustrated in Fig. 17-21 and 80 Table 15. A fiber appearing light with myosin ATPase will appear intermediate with NADH-D, MDH, SDH and mito a-GPD. And a fiber appearing dark with myosin ATPase will stain dark with NADH-D, MDG and SDH. Mito d-GPD activity would be rated as moderate. This staining profile is remarkably constant in the soleus. For a summary of the histochemical enzyme activity Of different muscle fibers in the three muscles studied, see Tables 14 and 15. Table 14.--Fiber Type Patterns of the Plantaris and Gastro- cnemius. See Fig. 12-16 and Fig. 22-26 for Visual Verification Red Intermediate White Myosin ATPase high low ' high NADH-D ‘ high med low MDH high med low SDH high med low Mito a-GPD med low . high Trichrome high med low Relative enzyme activity among different fiber types in various muscles for a single enzyme can be compared with these histochemical staining techniques,but quantitative com- parisons of the relative importance of the different enzymes in Table 14 and 15 within a specific fiber cannot be made. 81 Table 15.--Fiber Type Patterns of the Soleus Red Intermediate Myosin ATPase high low NADH-Diaphorase high med MDH high med SDH high med Mito a-GPD med low Trichrome high med Kinetic and metabolic reciprocity of red and white skeletal muscle fibers is an accepted phenomenon by many investigators. But recent investigations lay stress on this idea. Romanul (141) identified eight types of fibers in skeletal muscle while pointing out that there was not a reciprocal relationship between phosphorylase activity and oxidative enzyme activity. He stated that esterase activity was reciprocal to phosphorylase and that oxidative enzymes were to some degree. It is also this author's Opinion that this reciprocity in histochemical enzymatic acitivy fre- quently reported is an inaccurate generalization since it is only the intermediate type fibers as demonstrated with NADH-D, MDH, SDH which revealed a characteristic reciprocal staining pattern with ATPase and mito o-GPD. 82 But the most impressive finding which strains the theory of reciprocity is the extremely fast muscle, thyroarytenoid, which looks like a $1 w muscle when characterized histochemically. All Of its fibers have high oxidative enzyme activity. The cricothyroid has the speed Of contraction and relaxation which would categorize it to be a normally fast muscle (87). Histochemically, it appears as such. Consequently, the thyroarytenoid is a muscle that is extremely fast contracting, which is also adapted to highly repetitive contractions. In other words this muscle has not made the choice as Hill put it "to conserve energy or time." It can contract extremely fast and is also highly resistant to "fatigue." Therefore, the prOposal that a fiber is slowly contracting and adapted to tonic contractions with the predominant metabolic pathways being the citric acid cycle and utilization of fatty acids is not true in many reSpects. A fiber can be fast contracting, adapted to phasic or per-7 haps even tonic contractions and its metabolism still be like that conventionally thought to be only in slow tonic muscle fibers. I Much could be learned of the reciprocal trophic influences Of the nerve and muscle by-future investigations which involve denervation, cross-innervation and develop- mental studies Of the throat muscles. 83 Significance Of Results in Light of Interdependence of Nerve and Muscle.--There can be little doubt that there are neural influences which regulate, to some degree, the nature of metabolism which predomantes in a muscle fiber. Experimental procedures such as denervation, cross- innervation and daily stimulation of the peripheral nerves clearly illustrates the neural influence. Therefore, the question is not whether there are neural influences, but to what extent and through what mechanism do such phenomena take place. There have been suggestions in numerous reports that by cross-innervating a red and white muscle the transition is not complete. A summary Of such findings has been reported by Guth et a1. (83). The mechanism through which this neural influence is occurring has intrigued a number of investigators. There are at least two possible mechanisms which seem plausible. The pattern Of impulses or the total number of impulses per unit of time seems to be one enticing hypo- thesis. It is supported by findings Of Salmons and vrbova (145) who stimulated the popliteal nerve at a rate of lO/sec continuously from one to six weeks and found that the muscle contraction times following chronic stimulation increased about 140 percent as did the relaxation times. One question naturally arises from this study. Did the fibers change metabolically? 84 Henneman and Olson (92) have suggested that small tonic or red muscle fibers are innervated by small axons, which originate from small anterior horn cells, and that small anterior horn cells have lower threshOlds for activating action potentials. Hodes et al. (96) has re- ported an association of low thresholds with a slow con- duction velocity. Therefore, it was theorized by him that this muscle fiber contracts more or less continuously. Consequently the muscle fiber would have a relatively high concentration of myoglobin, its metabolism would be of an aerobic nature utilizing predominantly the citric acid cycle for adenosine triphosphate production. On the other hand large white muscle fibers are innervated by large anterior horn cells which have a high threshold of stimula- tion. Contractions of these fibers occur intermittently, having relatively long periods of inactivity at which times they can store glycogen. The glycogen can be called upon for immediate utilization when the muscle is stimulated to contract. Its metabolism is basically anaerobic in nature since glycolysis is preeminent in energy production. It is of interest to note that a bimodal frequency distribution has been found in spinal ganglionic neurons (105), nerve fibers (153), and muscle fibers (73), all of which support the neuron size, threshold of activation and relative activity hypothesis of neurons and muscle fibers. 85 The fact that the concentration of capillaries around red fibers is much greater than around white fibers teleologically supports the above hypothesis. More capillaries are probably needed by the red fibers since they must have a constant supply of substrates and oxygen. White fibers, on the other hand, have their substrates stored in the form of glycogen and their need for oxygen does not compare with the requirements of the red fibers. The capillaries would serve primarily to rid the white fibers of metabolites (142, 143, 144). Since the white fiber is intermittently activated it has time to synthesize glycogen between bursts of activity. Thus, from many viewpoints the prOposal that the quantity of impulses reaching a muscle fiber partially determines the metabolism and perhaps indirectly kinetic characteristics of contraction has some merit. Another mechanism for neural influences could be the "ooze" theory (174). This hypothesis suggests that substances from the axons ooze into the muscle fibers and effect its kinetic and metabolic characteristics. Vain attempts have been made to identify some of the chemical substances which could be candidates to account for this action. The "ooze" theory is supported indirectly by the finding resulting from transection of nerve fibers in the cat (22). But the most impressive study was performed by Gutmann et a1. (84, 85) where he stated that "metabolic 86 recovery processes (glycogen resynthesis) are controlled by a reflex mechanism and are dependent on the functional state of higher nerve centers and that the trOphic influence of the nervous system is exerted by the activation of meta- bolic recovery processes after functional activity." It has been demonstrated that impairment of glycogen synthesis occurs later after high section of n. ischiadicus, than after a section close to the muscle (84). Stimulating the nerve as well as general physiological activity, leads to transPort of substances along the axon into the muscle (167). Therefore, cutting the nerve so that a long nerve stump remains, apparently will enable a more complete resynthesis of glycogen following muscular stimulation since a longer nerve stump is available for transport. Length of the degenerating neuron also affects development of muscular fibrillation and sensitization (114). The two prOposed mechanisms of neural control do not have to be independent in their effect. It is con- ceivable that the number of impulses within an axon is directly proportional to the quantity of "ooze" into the muscle fiber. But in an experiment by VOdicka (162) re- synthesis of glycogen was dependent of the length of the nerve stump which was absent of neural impulses. With this background of neural influences on the skeletal muscle, the author is faced with proposing a mechanism which could explain an increase in the proportion 87 of red fibers in a relatively white muscle due to a chronic training program as was used in this study. It seems reasonable to prOpose that the plantaris of the heavily exercised animals were subjected to a greater number of impulses than the plantaris of sedentary animals. The moderately exercised plantaris of the rats which swam only once each day showed a moderate conversion of white to red muscle fibers. This finding supports the idea that the quantity of impulses may be proportional to the "redness" of the fiber. This does not discount the "ooze" hypothesis if the quantity of impulses and "ooze" vary proportionately. An accompanying study by Gerchman (70) on the anterior horn cells of these same animals perhaps lends some support to the suggestion that the effects of exercise on the prOportion of fiber types occured via the nervous system. The anterior horn cells of the most heavily exer- cised animals (Group C) showed greater enzyme activity than the horn cells of the sedentary animals (Group A). The so-called moderately exercised animals (Group B) showed less neuronal enzyme activity than the sedentary control rats (Group A). One explanation for this finding may be that Group B was subjected to an exercise program which could be likened to a series of acute exercise bouts in the life of a highly sedentary animal. It would then be possible 88 for a temporary depletion of some of the cytOplasm following an exercise period. Significance of Results in Light of Being an Adaptive Mechanism to Exercise.--Investigators have been able to identify a number of changes within a cell follow- ing a chronic training program which can account for improvement in that cell's capacity to function. Some of the more recent work by Gordon et a1. (74, 75, 76) suggests that the type of work performed as well as the amount of work in a training regimen dictates the adapted functional capacity of a cell. He found that highly repetitive and low-resistant type activity resulted in "sarc0p1asmic hypertrOphy" or an elevation of cyt0plasmic proteins but not structural proteins. Whereas the converse was found when the type of work was changed to one of high resistance and of low repetition. These findings suggest that a muscle will increase its capacity to maintain its metabolic activity by increasing its cytOplasmic proteins. In addi- tion it will also increase its capacity to work against greater loads with less repetition by increasing the structural proteins such as actin and myosin. Holloszy (97) has found elevated enzyme activities (SDH, NADH-dehydrogenase, NADH cytochrome C reductase, succinate oxidase, and Cyto O) in the gastrocnemius and soleus of rats that were run for several hours a day for several weeks. These elevated enzyme levels are metabolic 89 adaptations which likely enable rats to progressively improve in their running capacity. The body weight gain of the moderately and heavily exercised animals was less than in the sedentary control animals. The same was true for the muscle weight as well. This could be expected, however, due to the relatively high correlation of these two parameters (Table 11). A modifi- cation of body weight and muscle weight gain has been reported previously in exercised animals (117). This is particularly true in-an exercise involving a highly repe- titive type activity (swimming) as Opposed to a highly resistant type activity. Imms (102) reported similar findings in rats exPosed to various types of moderate stress (surgery, NaCl injections, etc.). He found food consumption to be identical in the stressed and control animals, but the body weight was significantly lower in the stressed animals. He concluded that the difference in body weight was due to an increased metabolic rate in the stressed rats (102). Carrow et a1. (25) have found lower body weights in exercised rats (swimming). Muscle fiber size was not an objective of this study since such an investigation should simultaneously take into account various types and degrees of activity as well as incorporating multiple criteria for identifying muscle fibers. 90 Cardiac hypertrOphy, as studied ultrastructurally, has been found to be accompanied with a quantitative in- crease in myosin molecules, not an alteration in the already existing myosin (24). The results presented in the present study give further evidence that cellular changes are specific to the nature and amount of work load placed on the muscles. Since these rats were eXposed to a low-resistant and highly repe- titive activity (swimming) they might be expected to have a greater proportion of red fibers in a white muscle follow- ing such a training program, if the assumption that red fibers are metabolically and kinetically better adapted for such activity is true, and if fiber types are mutable with an intact nerve supply. Several changes could have occurred within the muscle fibers exPosed to the exercise treatments previously described. Each of the fibers could have become slightly "redder." In such a case changes would be detectable with the technique employed here, in only the white fibers. If a red fiber would have been rated as such at one point, it would still be rated as red if it became slightly "redder." Therefore, the report on the fiber types of the soleus which indicated no changes cannot discount the possibility that some fibers did become "redder." 91 Another possible explanation which appears to have greater merit is that muscle fibers adapt individually or perhaps as a motor unit, but not as a whole muscle. Thus one is likely to make an incorrect assumption when he supposes that the changes in a muscle found after exercise will be homogeneous throughout all fibers. It is likely for this reason that many investigators have failed to identify adaptations in trained skeletal muscle unless the muscle had been heavily trained. This mode of thinking is clearly illustrated in the following statement by Holloszy, "Mild exercise such as used in previous studies, was found to have no effect on the level of SDH in muscle, suggest- ing that the failure of earlier studies to show an increase in respiratory enzyme activity resulted from the use of an insufficient exercise stimulus." It is likely that the insufficiency is in the analytical techniques employed, not the exercise stimulus. Negative results have been found probably because the investigators have failed to recognize the kinetic and metabolic heterogeniety within a single muscle (variation in muscle samples) as well as the assump- tion that any changes which do occur will be homogeneous throughout all muscle fibers, not within specific types of muscle fibers. CHAPTER V SUMMARY, CONCLUSIONS AND RECOMMENDATIONS Summary The left soleus, plantaris, and gastrocnemius of exercised, 100 day old male rats were studied histochemically using myosin ATPase, mito a-GPD, MDH, DPNH-D and a trichrome stain. Twenty control animals were kept in sedentary cages (Group A). Twenty more rats, housed in sedentary cages, were subjected to one 30—minute swimming period a day, six days a week, for fifty-two days. A weight approximating three percent of the body weight was attached to the tip of each of the rats' tails, (Group B). Each animal in the third group (voluntary-forced) swam for two 30-minute periods per day with approximately four percent of their body weight attached to the tip of its tail. The duration of the training program was fifty-two days. All animals were fed ad libitum with commercial block feed.. Ambient air temperature in the animal quarters ranged from 21°C-25°C. The water temperature for swimming ranged from 32°C-34°C. 92 93 The gastrocnemius, plantaris and soleus of the right leg was removed following i.p. anesthesia with pento- barbital. The tissue was frozen in isOpentane cooled with liquid nitrogen and sections were cut at lOu and treated with the various histochemical procedures mentioned above. Approximately 350 muscle fibers of the soleus and plantaris of each animal were rated as light or dark according to the staining intensity of myosin ATPase, MDH and mito a-GPD. No white fibers were found in the rat soleus with MDH or mito a-GPD. With MDH and mito a-GPD the staining intensity of approximately 350 muscle fibers from each animal were rated as light, medium, or dark signifying the presence of white, intermediate, and red muscle fibers respectively. All ratings were made without knowledge of the treatment group from which the animal came. It is concluded that a low-resistant and highly repetitive exercise program such as was used in this study will not alter the prOportion of intermediate and red muscle fibers of the soleus as demonstrated by myosin ATPase, MDH or mito a-GPD. No changes were found in the plantaris with myosin ATPase. However, the experimental treatments did result in an increase in the prOportion of red muscle fibers in the plantaris as demonstrated by MDH, mito a-GPD, SDH and a trichrome stain. 94 This study supplies further evidence for the hypothesis that the nature of the metabolism of an indivi— dual muscle fiber is related to the nature of the work load on that muscle fiber. This relationship in turn suggests that muscle fibers can be altered from one type to another without altering the nerve supply. The findings of an accompanying investigation on the anterior horn cells of these same animals found that the anterior horn cells of the voluntary—forced group generally had greater enzyme activity than either Group A or Group B. This finding is in accordance with the results found in the skeletal muscle if the hypothesis that the proportion of red fibers varies directly with the quantity of impulses in axons or enzyme activity of the anterior horn cells proves to be valid. This study supplies direct evidence supporting the hypothesis that the prOportion of different fiber types in a muscle is related to the nature of work load on that muscle. Consequently, it can be said that muscle fiber types are mutable in a non-pathologic state. Conclusions The results of this study have led to the following conclusions: 1. The prOportion of red and white muscle fibers varies tremendously intramuscularly and intermuscularly in the plantaris and gastrocnemius with the soleus being relatively homogeneous. 95 2. A continuous spectrum of fiber types exists in the plantaris, gastrocnemius and soleus, therefore, a separation of muscle fibers into two or three fiber types is arbitrary. 3. Exercise treatments as used in this study did not alter the prOportion of intermediate and red fibers in the soleus as determined by myosin ATPase, MDH, SDH, mito a-GPD, and a trichrome stain. 4. These same treatments did not alter the propor- tion of fiber types as identified by myosin ATPase in the plantaris. 5. An increase in the proportion of red fibers as identified by MDH, SDH, mito a-GPD and a trichrome stain was observed in the plantaris following a swimming program as used in this study. 6. The moderately exercised rats (sedentary-forced) were generally found to have a prOportion of red fiberS' which was between that found in the sedentary control and voluntary-forced group. This suggests that a direct correlation between the amount of chronic muscular activity and the relative prOportion of red fibers in that muscle. Recommendations l. A similar study should be done which involves greater intensities of exercise as well as various types of exercise. 96 2. Subjective ratings of fiber types should be replaced with photometric techniques which are now techn- ically available. 3. A physiological analysis of motor unit kinetics would aid in the interpretation of histochemical results. 4.- A quantitative chemical analysis of specific regions of the muscles to be studied should accompany the histochemical and physiological analyses. 5. Incorporation of electron microscopy will help in studying the structural characteristics of mitochondria which presumably accounted for some of the changes found in this study. 6. A similar analysis of the medial and lateral gastrocnemius of trained animals should be done. Figure 1. Figure 2. Figures 3 and 4. Figure 5. Figure 6. Plate 1 Outline of the musculature of the calf of a leg signifying the medial and lateral head of the gastrocnemius, the plantaris and the soleus. Detailed analyses of the proportion of various fiber types were done in areas identified as I, II and III. Low power of cross-section of medial gastrocnemius (MDH) illustrating intramuscular variation in "reddness." A small band of fibers are sectioned longitudinally and verifies the assumption that the fiber type ratings are constant throughout the length of the fiber (20X). Medial gastrocnemius (MDH) of a rat which drowned during one of the training sessions. Note a few of the unusually large muscle fibers. There appears to be a coiled membranous component con- tinuous with the periphery of the fibers which extends into the fiber centrally (145X). Medial gastrocnemius (Myosin ATPase). From the same animal as in Fig. 3 and 4. According to myosin ATPase the enlarged fibers would be classified as red or white, but not intermediate, since in the gastrocnemius and plantaris only the light fibers with myosin ATPase are intermediate with dehydrogenase enzyme activities, except mito a-GPD. No unusually large intermediate fibers were observed with myosin ATPase. (145X) Medial gastrocnemius (Myosin ATPase). Same muscle as in Fig. 3, 4 and 5 illustrating an area of normal size and distribution of muscle fiber types (145X). 97 Plate 1 98 Figure Figure Figure Figure Figure 7. 10. 11. Plate 2 Soleus (Mito a-GPD). Note the presence of unusually small fibers. Rat was exercised twice a day for more than seven weeks (400x). Soleus (Trichrome). Another example of small fibers in the same muscle as in Fig. 7 (410x). Lateral gastrocnemius (Trichrome). Note the variation of peripheral staining which aids in typing muscle fibers. The staining pattern approximates some lipid stains (650X). Plantaris, area II (MDH). A sedentary-control animal. Observe the predominance of white fibers (lGOX). Plantaris, area II (MDH). A heavily exercised animal (voluntary-forced). Note the greater prOportion of red and intermediate fibers as compared to Fig. 10 (160x). 99 Plate 2 100 Figures 12 to 16. Plate 3 Plantaris. Myosin ATPase, MDH, mito a-GPD, SDH and NADH-D, respectively (180x, 155x, 165X, 160x, 165x respectively). Light fibers with myosin ATPase (Fig. 12) appear intermediate in character with MDH (Fig. 13), SDH (Fig. 15), and NADH-D (Fig. 16). The light fibers with mito a-GPD correspond to light fibers with myosin ATPase. However, three fiber types are more evident with mito a-GPD in Fig. 13, 15 and 16. A relatively intense enzyme reaction without a dark subsarco- lemmal staining pattern characterizes the inter- mediate muscle fibers. The darkness of the staining depends to a large degree on the concen- tration of mitochondria and triglyceride drOplets (Fig. 13, 15 and 16) since NBT tends to bind with lipid components of a cell. Fiber 3 would be typed as intermediate, b’as red, and g as white. 101 102 Figures 17 to 21. Plate 4 Soleus. Myosin ATPase, MDH, mito a-GPD, SDH and NADH-D, respectively (all photographs l70X). In contrast to the patterns of enzyme activity in Fig. 12 to 16 and 22 to 26, the soleus enzyme patterns parallel one another. If a fiber is rated as dark with one enzyme, then it will be dark with the other enzymes mentioned above. The soleus contains only fibers classified as red or intermediate. 103 Plate 4 104 Figures 22 to 26. Plate 5 Medial gastrocnemius. Myosin ATPase, MDH, mito a-GPD, SDH and Trichrome respectively (l7OX, 160x, 160X, 160x, 160x respectively). Enzyme patterns as explained for the plantaris (Plate 3) hold true for the gastrocnemius as well. Again, note that it is the intermediate fiber as determined by MDH, SDH and Trichrome that appears lightest with myosin ATPase and mito a-GPD. The filamentous appearance of the fibers particularly with MDH represents membranous in- termyofibrillar staining. Fiber 3 would be typed as intermediate, b as red and c as white. Notice that the white fibers are generally the largest of the three fiber types identified in this study. 105 106 10 BIBLIOGRAPHY Bach, L. M. N. Conversion of red muscle to pale muscle. Proc. Soc. Exp. Biol. Med., 67:268-269, 1948. Bajusz, E. and Jasmin, G. Studies on the activity and distribution of oxidative and hydrolytic enzymes in the skeletal muscle of dystrOphic mice kept on various diets. Rev. Canad. Biol., 21:409-436, 1962. Bajusz, E. Red skeletal muscle fibers: Relative independence of neural control. Sci., 145:938-939, 1964. Bajusz, E. 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Normal muscle innervation: A predisposing factor in the expression of primary myopathies? Anat. Rec., 160:454-455, 1968. Zhenevskaya, R. P. Experimental histologic investiga- tion of striated muscle tissue. 21:457-470, 1962. Rev. Canad. Biol., APPENDIX A One-way Analysis of Variance Tables for Fiber Types, Muscle Weight, Body Weight and Treatment Groups 1. Intermediate Fibers of Soleus. Myosin ATPase Source of Variance Df Among levels 2 Within levels 29 Total 31 SS MS 31.00 15.50 1284.02 44.28 1315.02 Intermediate Fibers of Soleus. Mito a-GPD Source of Variance Df Among levels 2 Within levels 40 Total 42 SS MS 170.11 85.06 1366.28 34.16 1536.39 Intermediate Fibers of Soleus. MDH Source of Variance Df Among levels 2 Within levels 46 Total 48 SS MS 91.41 45.71 1513.30 32.90 1604.71 White Fibers of Area II of Plantaris. MDH Source of Variance Df Among levels 2 Within levels 50 Total 52 122 SS MS 980.37 490.19 2045.21 40.90 3025.58 0.35 2.49 1.39 11.98* 123 APPENDIX A (continued) One—way analysis of Variance Tables for Fiber Types, Muscle Weight, Body Weight and Treatment Groups 5. Intermediate Fibers of Area II of Plantaris. MDH Source of Variance Df Among levels 2 Within levels 50 Total 52 Red Fibers of Area II of Source of Variance Df Among levels 2 Within levels 50 Total 52 SS MS F 183.79 91.90 3.85* 1194.03 23.88 1377.82 Plantaris. MDH SS MS F 351.81 175.90 7.66* 1148.43 22.97 1500.24 Intermediate Fibers of Area III of the Plantaris-Myosin ATPase Source of Variance Df Among levels 2 Within levels 29 Total 31 SS MS F 72.70 36.35 1.54 683.42 23.57 756.12 Intermediate Fibers of Area III of the Plantaris. Mito a-GPD Source of Variance Df Among levels 2 Within levels 38 Total 40 SS MS P 215.79 107.90 1.79 2295.91 60.42 2511.70 Red Fibers of Area III of the Plantaris. Mito a-GPD Source of Variance Df Among levels 2 Within levels 38 Total 40 SS MS F 29.74 14.87 .44 1280.93 33.93 1310.67 124 APPENDIX A (continued) One-way Analysis of Variance Tables for Fiber Types, Muscle Weight, Body Weight and Treatment Groups 10. White Fibers of Area III of the Plantaris. Mito a-GPD Source of Variance Df SS MS F Among levels 2 317.72 158.86 1.73 Within levels 38 3496.15 92.00 Total 40 3813.87 11. White Fibers of Area III of Plantaris. MDH Source of Variance Df SS MS F Among levels 2 1633.25 816.63 14.76* Within levels 49 2710.38 55.31 Total 51 4343.64 12. Intermediate Fibers of Area III of Plantaris. MDH Source of Variance Df SS MS F Among levels 2 50.29 25.14 .916 Within levels 49 1345.17 27.45 Total 51 1395.46 13. Red Fibers of Area III of Plantaris. MDH Source of Variance Df SS MS F Among levels 2 1178.13 589.07 10.68* Within levels 49 2703.25 55.17 Total 51 3881.38 14. Muscle Weight and Treatment Groups Source of Variance Df SS MS F Among levels 2 3.36 1.68 56.00* Within levels 53 2.06 0.03 Total 55 5.42 Group A>B>C 125 APPENDIX A (continued) One-way Analysis of Variance Tables for Fiber Types, Muscle Weight, Body Weight and Treatment Groups 15. Body Weight and Treatment Groups Source of Variance Df SS MS F Among levels 2 80975.12 40487.50 40.30* Within levels 53 53234.87 1004.43 Total 55 134209.99 Group A>B>C U m c» N F4(3 w 3' APPENDIX B Raw Data of Fiber Types Legend Control group Moderately exercised group Severely exercised group Light staining intensity Medium staining intensity Dark staining intensity Number of muscle fibers typed Percentage of muscle fibers of a muscle of a particular type 126 127 H.¢H mh m.om mam m.ma m5 «.om mam m.oa hm m.mm ham o.vH wh o.wm wmw a.mH No m.am Ham N.mH Hm m.¢m mom m.oa mm «.mm mmu v.0m MOH m.mh No¢ o.va 5v a.mm mam EFEE n m m‘ m s.s~ mss m.ms sow «.ss sms m.ms mos m.ss oss m.~m ssm s.¢s as m.mm mos a.ms as ~.sm mes m.- ms ~.ss «pm m.~s ss m.sm mmm s.ws om a.mm ems G.~s mm s.hm mam s.s on m.~m mmm n m a m m m.vs cos «.mm mam a.mm ow «.mo mam ~.m mm m.om mam v.4s mm m.mm qsm ~.m~ oms a.ms mom m.~s om m.sm mam a.ms ms o.sm «as a.ms mm s.¢m mos a.mm was s.ss s64 ~.m~ as m.ss sow «.4 ms a.mm mam wumm mm: 6.5m mNm n m n» m a mummad :HthZIImsoHom Afiwficflunoov m xHQmem¢ v mm H Ha Nm ma mm ma om NN 5N m mv 0H 5v om MH m¢ mm mm mm mm on N o¢ hm hm «N vH HEB. U m 4 smassz sassqa 128 m.NN on m.bh NmN a.ms Hoa H.Hm mmv H.b NN a.mm omN N.om mm a.mh 5mm H.0H MN m.am mON a.mN mm v.oh mom N.0N hm a.mh vmm m.NH mm m.hm moN m.bN mm N.Nh mHN a.mN Hm a.mh mHN m.h va b.Nm 55H m.mH oHH m.om wmv n M Q m m.hN HmH ¢.Nh NNw a.mH em H.om mmN N.¢N mvH a.mb mwv m.mH hm h.mm NmN w.mH om ¢.om mNm NNH mw m.hh HmN b.0N wh m.mh mmN w.mH mm ¢.vm va m.HH hm N.mm Hom m.HN NMH a.mh 05v N.ha Nm m.Nm vwv v.m ma m.Ha «5H m.0H mm a.mm hmv m.0N ¢¢ h.mh mud n m n m m.sm mms m.ms was ~.- mm m.hs mam ~.- mos m.ss mum m.os mm m.mm mam a.ms 4m 0.4m s44 a.mm ems m.mm mom m.s~ mm «.ms ems m.ms mm m.sm mew M.GN mms a.ms omm m.sm oss m.mm mom s.ms mm m.om «mm m.s~ oss «.ms mam «.mm ems a.ms mmm a.ms so s.sm Nmm o.- as a.ms omm ~.ss om a.mm ass ¢.s~ Nos sump mmm. a m a m sumsssucoov m xsozmmma amouuuumsmsom s mm mm ss ms an es s mm as ms ow ms om om mm m mm as as mm am am cs «4 as en es mm as «s N mm as am ms 4m ms mm «m .summMMI o m a smassz smssqa 129 N.mH Nm m.¢m and a.ma hm m.om HH¢ m.N m b.5m mmm o.HN cad a.mh «aw N.hH Nm m.Nm HmN m.mN wh h.Mh NON a.mH Nm a.mm mme m.¢H hm N.mm mNm o.hN Aha o.m> vmm N.MH vm m.mw mmm o.¢N QNH a.mh 0mm m.mm wad p.mo mNN m.wH mm h.mm mmN H.HH mm a.mm me m.m 5N N.Hm HmN no no e.m~ ees e.os mam m.m~ oms s.es mem m.hs mss m.~m eem o.h~ es a.ms com m.es om s.mm emm e.m~ mm e.es som m.ms me s.em New s.~s me m.sm ssm o.es mm o.em mmm m.m~ oos s.es mmm m.ms me s.em ewe e.ss em e.~m mee m.ss sm s.mm mam a.ms me s.ee oem ~.es on a.me eme m.- se m.bw pww n m or m .pmscsucoov m xsozmmme N.HN hm m.mh me m.MN mHH a.mh mom m.mH om «.mm How m.HN No N.mb MNN m.ON NNH m.mh th a.mH mv m.Hw wad v.MN mad 0.05 55m m.mH Nm «.mm mmw o.NN MDH a.mb mom m.HN mm m.wh mON m.HN >5 v.mh omN a.mN OQN m.vh mam m.MN mm a.mh wan o.oN mo o.om th «.mH Ne a.mm wHN b.0H me m.mm va EFEE m m a m amouauumsmsom 130 m.o> mmN h.MN an m.em who a.ms HNH m.oh Nmm a.mN NmH >.om Haw m.ms mm c.5m NHN e.Ns om o.mm ome v.5H mm e.ew use p.ms up m.hm ooe h.NH mm m.om meN p.ma Hm H.em Nmm a.ma Nm N.mm Haw a.ma mm a.mw emm N.HN mo m.mm mae >.os om >.om NNm m.ma om e.om Nev m.mH mos H.om mmN a.ms Nb N.N> emm m.hN Nma m.hm hem a.ms we N.sm nee a.ms mo N.mm oee m.oa mm m.mm mmN m.eH me m.hh mmm e.NN mm h.em mme m.ma mm a.mm com a.ma we h.Hm mme m.ma mos a.mm hme a.ma em m.>h mum N.NN boa m.Nm moo m.> em m.sm mmm b.NH me .bhmm .an “haw emu bhbm mmm bhbm. wwu mnmw mwm mnmm mwl a m n m n m n m n m n m H m ¢ 0 cam m .< mmsouwlnHHH mflsmusmamlnommmad :Hmomz somscsucoov m xsozmmma H wa om he Nm me mm hm me HN 0 e ss mm mm mm mm ms we as so ms em em mm on oe em em es mw__mw m a smnssz smasqm Animal Number 34 20 48 14 44 42 12 37 30 26 13 10 27 15 52 59 APPENDIX B (continued) MDH--Plantaris II--Group A _a_ b— 91 75.2 69 81.2 109 79.0 118 76.1 101 82.1 126 86.9 135 80.4 104 71.2 126 80.3 106 80.3 136 73.1 100 66.7 103 68.2 136 79.1 143 77.7 161 73.5 158 75.6 183 71.5 175 68.6 2. _5__ 13 10.7 10.6 '16 11.6 18 11.6 18 14.6 9 6.2 22 13.1 30 20.5 19 12.1 10 7.6 24 12.9 25 16.7 26 17.2 25 14.5 25 13.6 33 15.1 26 12.4 57 22.3 57 22.3 131 a_ _b_ 17 14.0 7 8.2 13 9.4 19 12.3 4 3.3 10 6.9 11 6.5 12 8.2 12 7.6 16 12.1 26 14.0 25 16.7 22 14.5 11 6.4 16 8.7 25 11.4 25 12.0 16 6.3 23 9.0 132 APPENDIX B (continued) MDH--Plantaris II--Group B Animal B Number 1 2 3 _s_ _b___a_ _b__s_ _b__ 23 111 67.7 25 15.2 28 17.1 28 106 71.1 29 19.5 14 9.4 45 81 55.7 33 22.8 31 21.4 25 103 72.0 12 8.4 28 19.6 29 149 75.3 27 13.6 22 11.1 35 108 69.7 25 16.1 22 14.2 3 109 71.7 28 18.4 15 9.9 24 143 71.5 18 9.0 39 19.5 40 95 56.9 39 23.4 33 19.8 53 114 71.7 25 15.7 20 12.6 36 121 71.7 18 11.5 17 10.9 60 148 70.5 27 12.9 35 16.7 46 188 76.7 27 11.0 30 12.2 22 129 78.7 20 12.2 15 9.1 38 105 62.9 41 24.6 21 12.6 11 215 73.9 32 11.0 44 15.1 133 APPENDIX B (continued) MDH-~P1antaris II--Group C Animal Number _._ 31.2. _I_o__«:_ _b_ 21 85 75.2 15 13.3 13 11.5 5 146 69.5 29 13.8 35 16.7 51 110 74.3 12 8.1 26 17.6 39 119 74.4 35 21.9 6 3.8 31 95 65.5 35 24.1 15 10.3 41 98 64.9 36 23.8 17 11.3 17 104 65.0 28 17.5 28 17.5 56 127 66.8 31 16.3 32 16.8 57 100 67.6 29 19.6 19 12.8 2 112 61.2 29 15.8 42 23.0 33 130 71.0 25 13.7 28 15.3 49 128 68.4 34 18.2 25 15.4 32 131 64.2 29 14.2 44 21.6 47 102 52.3 31 15.9 62 31.8 9 112 64.7 36 20.8 25 14.5 50 171 71.8 49 20.6 18 7.6 16 117 60.9 43 22.4 32 16.7 1 140 48.4 88 30.4 61 21.1 Animal Number 34 54 14 44 42 37 30 26 13 10 27 15 52 59 134 APPENDIX B (continued) Mito a-GPD--Plantaris III-~Group A A I 2 .2. .12_ .1._b_ 2.32.. 67 21.5 47 15.1 197 63.3 139 33.4 74 17.8 203 48.8 129 28.5 103 22.8 220 48.7 76 15.3 94 19.0 326 65.7 83 21.8 96 25.3 201 52.9 124 28.8 85 19.7 222 51.5 67 18.4 75 20.6 222 61.0 51 11.7 126 29.0 258 59.3 68 17.6 60 15.5 258 66.8 46 21.3 51 23.6 119 55.1 63 14.8 128 30.1 235 55.2 19 7.7 37 V 15.0 190 77.2 71 15.0 93 19.7 308 65.3 76 23.1 78 23.7 175 53.2 74 16.3 81 17.9 298 65.8 124 17.2 127 17.7 468 65.1 Animal Number 28 45 25 29 24 40 36 60 46 22 38 11 Animal Number 51 39 43 31 57 49 32 47 50 16 135 APPENDIX B (continued) Mito a-GPD--P1antaris III--Group B B 1 2* .2. .E._ .2. .2_. .2. .E__ 66 26.7 42 17.0 139 56.3 74 21.8 84 24.8 181 53.4 67 12.4 170 31.4 305 56.3 77 18.0 159 37.1 192 44.9 44 12.5 47 13.4 261 74.1 106 26.2 92 22.8 206 51.0 46 15.1 73 23.9 186 61.0 38 15.9 43 18.0 158 66.1 57 21.3 54 20.1 157 58.6 78 20.3 75 19.5 231 60.2 65 24.1 39 14.4 166 61.5 168 27.5 188 30.8 254 41.6 Mito a-GPD--Plantaris III--Group C c 1 2 .11. .12.. .43. ..E.. .2. .2._ 116 37.8 70 22.8 121 39.4 56 '44.4 26 20.6 44 34.9 173 43.7 91 23.0 132 33.3 84 19.8 144 34.0 196 46.2 57 22.6 81 32.1 114 45.2 56 27.9 54 17.3 202 64.7 80 20.9 98 25.7 204 53.4 39 11.3 78 22.7 227 66.0 34 23.6 23 16.0 87 60.4 68 17.6 62 16.1 256 66.3 70 23.7 55 18.6 170 57.6 118 19.8 115 19.3 362 60.8 107 18.5 117 20.2 355 61.3 136 APPENDIX 8 (continued) MDH--P1antaris III--Group A Animal A s. Number 1 2 3 _g__9___a__2__a__b_ 6 252 50.7 77 25.5 168 33.8 34 144 45.7 59 18.7 112 35.6 54 210 56.1 65 17.4 99 26.5 4 229 41.2 137 24.6 190 34.2 14 338 51.7 138 21.1 178 27.2 44 39 65.8 6 10.0 15 25.0 42 258 54.1 68 14.3 151 31.7 12 411 53.0 209 28.0 138 19.0 8 349 57.1 82 13.4 180 29.5 37 257 53.4 89 18.5 135 28.1 30 341 56.7 99 16.4 162 26.9 26 263 48.6 101 18.3 177 32.7 13 337 51.5 100 15.3 217 33.2 10 191 74.6 30 11.7 35 13.7 27 268 54.8 73 14.9 148 30.3 15 259 48.4 131 24.5 145 27.1 52 275 62.6 81 18.5 83 18.9 59 197 47.1 117 28.0 104 24.9 4 400 50.8 162 20.6 226 28.7 137 APPENDIX B (continued) MDH--P1antaris III--Group B Animal 15 Number 1 2 3 .2. .b__ _2__b_ .a__2_ 28 225 45.0 111 22.2 164 32.8 45 184 34.8 116 22.0 228 43.2 25 357 49.4 103 14.3 262 36.3 29 171 46.6 52 14.2 144 39.2 35 275 46.8 135 23.0 178 30.3 3 305 55.8 96 17.6 146 26.7 24 348 45.5 181 23.7 235 30.8 40 115 33.4 88 25.6 141 41.0 53 153 42.1 99 27.3 111 30.6 36 155 38.7 88 21.9 158 39.4 60 190 39.4 142 29.5 150 31.1 46 113 33.5 78 23.1 146 43.3 22 339 52.2 115 17.7 195 30.0 38 205 46.4 61 13.8 176 39.8 11 176 32.8 90 16.8 270 50.4 138 APPENDIX B (continued) MDH--P1antaris III--Group C Animal C Number 1 ' 2 3 .3. .12... 3.2.. .2..P__ 21 297 56.8 87 16.6 139 26.6 5 225 47.4 86 18.1 164 34.5 51 180 43.3 116 27.9 120 28.8 39 56 44.4 26 20.6 44 34.9 43 156 34.8 48 10.7 244 54.5 31 145 . 49.0 34 11.5 117 39.5 17 328 42.2 146 18.8 304 39.1 56 275 53.0 113 21.8 131 25.2 57 89 28.0 91 28.6 138 43.4 2 141 38.0 66 17.8 164 44.2 33 130 40.1 40 12.3 154 47.5 49 176 35.7 89 18.1 228 46.2 32 182 36.4 75 15.0 243 48.6 47 124 31.2 71 17.8 203 51.0 9 303 40.3 173 23.0 275 36.0 50 265 47.7 163 29.3 128 23.0 16 384 46.9 200 24.4 235 28.7 1 214 36.4 143 24.3 231 39.3 139 a.mm m.sw m.m~ N.oN N.na m.mH H N.mm m.av m.eN m.om m.om m.hN Ha p.mo a.mm m.ma 5.5H m.eH N.>H v m.No m.om N.ba m.ma N.oN a.ma ms m.eh m.ao h.NH v.ea «.ma H.eN mm e.wm a.mw o.ON m.hH a.ma m.wH mm m.hm m.hm m.mH a.ma a.ma h.MN om m.Hh N.mm H.HH >.MN e.ha H.m~ Nm N.hm m.mo a.ma a.ma m.ea c.5a m a.mm N.ow H.5H m.mH m.ha m.oN NN a.mh m.mw e.HH p.ma m.ea o.ma ma m.hm v.om m.m~ o.oH «.ma o.mN he h.om a.mm w.Nm H.oN p.mN m.HN we N.mm N.>h a.mN a.ma m.mH n.> hN e.wm a.mm a.mN p.mN a.ma m.HH Nm a.mm a.mm w.mH a.ma a.ma a.ms om N.mm p.mm «.ma H.om e.ea m.ea ca wwomomm Hummmmso xomnoom Hmcwmflso xomsomm Hmcmmwuo mswnsm sumo mass msmnsm ssscms wuss muwnsm names mass sonssz usmoumm usmuuwm unmoumm Hmsflsé AHHH mmuflv mflsmuawam How amwla ouaz was was How mama huHHNQMNHmM U xHQmem¢ 140 N.Hm m.mm m.Hm m.eN m.bm «.mm s m.Ne e.om a.mN a.ms 5.0m a.mm Ha a.mN p.mN H.NN m.ON a.me m.om e m.mm p.mN o.eN e.eN N.Ne a.mv ma H.oe a.mm H.vN a.mH a.mm e.me mm N.mN m.e~ a.mN o.mN a.me H.5v mm H.em o.m~ N.mH m.mN b.om h.he om a.ma a.ma N.0N m.mH a.mm m.Nm mm e.em m.mm N.wN o.m~ e.mm m.oe m p.mm o.om m.HH h.ha m.sm N.Nm NN m.mN s.hN H.ha m.eN N.mm «.me ma p.mm o.Hm m.mm m.ha o.om N.Hm he e.mm m.mv a.mm A.MN v.am m.mm we m.mN m.om m.m~ m.eH m.om m.em 5N a.mm a.me m.mm a.ma h.am e.mm Nm H.sm a.sm m.m~ m.mN m.mm «.mm om xomnomm Hmcwmwuo wmmnoom HmsmmHMD xomnomm Hmswmwuo muonsm xnma mmNH unmask Eswpmz mmNa unmask usqu mama Hmnanz pcoouom unmouom ucoosom HMEHG¢ HHH mflhmuCMHm m0 an: semsasucoov o xsozmmma 141 hflflwt .-nummnr ss.m ems s se.m sms es ss.m ees om ss.m mss s es.s sms se ss.m ems ms ms.m oss me se.m sss ss me.m ees m ss.m ess se es.m ses ee ss.m mes ss ss.m mms se ss.m ess ss u- emsa .. ee ms.m ses se ee.m eos ss ee.m ems se eo.m ess e ms.m mes sm .z.z .s.m sunssz o sessca ee.m eee ss.m ess I- emsa .. ss.m eee me.m ems ee.m ses se.m sss me.m mes ss.m ees ee.m ses ss.m ess ee.m ems ss.m ees ee.m sse I- emsa I- ss.m ees ss.m mes ss.m eem me.m ees .. emsn .. .z.z .z.m m D XHQZfith Hmnfisz H.953 AmEMHm. muanoS whom mw.N mm.N mm.N Nm.N Nh.N mm.N Ho.m mo.m MN.m mm.N mm.N Nm.N Nm.N wo.m mm.N m5.N mv.m oo.m «o.m Mb.N .3.2 mam mmv QNV mNm mum HNv m.¢ haw Mbv mHv mN¢ NHv MN¢ omw m¢¢ ONv mmv mme Nme oov mm Nm mH 5N OH MH 0N on em NH Ne vv «H we ON em «m .s.m H0952 HMEqu B-OHBD BW CytoO EDTA GAPD INT LDH MDH Mito a-GPD MW Myosin ATPase NAD NADH NADH-D NBT NS R.Q. SDH TNBT UDPG APPENDIX E Abbreviations B-Hydroxybutyric acid dehydrogenase Body weight Cytochrome oxidase Ethylene diaminetetraacetic acid Glyceraldehyde-3-phosphate dehydrogenase Iodonitrotetrazolium Lactate dehydrogenase Malate dehydrogenase Mitochondrial a-glyceroPhosphate dehydro- genase Muscle weight Myosin adenine triphosphatase Nicotinamide adenine dinucleotide Reduced Nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide- diaphorase 2,2 -di-p-nitr0phenyl-5-5 -diphenyl- 3,3 -(3,3 -dimethoxy-4,4 -diphenylene) ditetiazolium chloride Non significant Respiratory quotient Significant statistically Succinate dehydrogenase Tetranitro blue tetrazolium salt Uridine diphosphate-glucose 142 TE UNIV. LIB "Ti‘flifuiifl‘fllzljulwmm WWI“ 7341‘